TM

Food Science and Technology Bulletin

Functional Foods
Volume 1

CHIEF EDITOR

Professor Glenn R. Gibson

The University of Reading
Reading
UK

EDITORIAL ADVISORY BOARD 2005

Dr Edward Farnworth Dr Colette Shortt

Food Research and Development Centre Yakult UK
St. Hyacinthe London
Canada UK

Dr David P. Richardson Dr Tiina Mattila-Sandholm

dprnutrition Valio Ltd
Croydon Finland
UK
Food Science and Technology Bulletin: Functional Foods comprises a minimum of eight minireviews per
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ISSN 1476-2137
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Contents

1. Functional Foods, Blood Lipids and Coronary Heart Disease ............................... 1-1
1.1 Coronary Heart Disease and Risk Factors ....................................................................... 1-1
1.2 Effects of Probiotics on Blood Lipids: the Evidence ......................................................... 1-2
1.3 The Effect of Fructan-type Oligosaccharide Prebiotics on Lipid Metabolism in
Humans ............................................................................................................................ 1-5
1.4 The Effects of Synbiotics on Coronary Heart Disease ..................................................... 1-9
1.5 References ....................................................................................................................... 1-9

2. Gastrointestinal Infections and the Protective Role of Probiotics and

Prebiotics .................................................................................................................... 2-1
2.1 Introduction ...................................................................................................................... 2-1
2.2 Acute Pathogenesis in the Human Gut ............................................................................ 2-1
2.3 Chronic Gut Disorder ....................................................................................................... 2-2
2.4 Functions of the Normal Human Gut Microbiota .............................................................. 2-4
2.5 Probiotics and Prebiotics .................................................................................................. 2-5
2.6 Examples of Foodstuffs Containing Probiotics and Prebiotics ......................................... 2-7
2.7 Mechanisms of Effects of Probiotics and Prebiotics on Gastrointestinal Pathogens ....... 2-8
2.8 Conclusions ...................................................................................................................... 2-11
2.9 References ....................................................................................................................... 2-11

3. Functional Foods and Bowel Cancer ........................................................................ 3-1

3.1 Introduction ...................................................................................................................... 3-1
3.2 The Biology of Cancer ...................................................................................................... 3-1
3.3 Diet as a Risk Factor ........................................................................................................ 3-2
3.4 Functional Foods – Modifying the Intraluminal Environment ........................................... 3-2
3.5 Phytochemicals – Foodborne Anticarcinogens ................................................................ 3-4
3.6 Polyunsaturated Fatty Acids (PUFAs) .............................................................................. 3-6
3.7 Conclusion ....................................................................................................................... 3-7
3.8 References ....................................................................................................................... 3-7

4. Functional Foods: Dietary Intervention Strategies in Autistic Spectrum

Disorders ..................................................................................................................... 4-1
4.1 Introduction ...................................................................................................................... 4-1
4.2 Functional Foods .............................................................................................................. 4-1

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vi Contents

4.3 What Is Autism? ............................................................................................................... 4-2

4.4 Interventions in Autism: Gluten and Casein Removal ...................................................... 4-2
4.5 Interventions in Autism: Phenolic Food Components and Enzymes ................................ 4-3
4.6 Interventions in Autism: the Gut Microflora ...................................................................... 4-4
4.7 Conclusions ...................................................................................................................... 4-7
4.8 References ....................................................................................................................... 4-8

5. From Hypoallergenic Foods to Anti-allergenic Foods ............................................ 5-1

5.1 Introduction ...................................................................................................................... 5-1
5.2 Allergy, Atopy and the Origin of Allergic Disease ............................................................. 5-1
5.3 Normal Intestinal Microflora ............................................................................................. 5-4
5.4 Inflammation ..................................................................................................................... 5-5
5.5 Current Prevention Strategies and Treatment of Allergy ................................................. 5-6
5.6 Functional Foods .............................................................................................................. 5-6
5.7 Potential of Functional Foods for Allergic Individuals ....................................................... 5-8
5.8 Future Prospects .............................................................................................................. 5-8
5.9 Conclusions ...................................................................................................................... 5-8
5.10 Acknowledgements .......................................................................................................... 5-9
5.11 References ....................................................................................................................... 5-9

6. Canine Functional Foods ........................................................................................... 6-1

6.1 Introduction ...................................................................................................................... 6-1
6.2 Functional Food Types ..................................................................................................... 6-1
6.3 Colon-targeted Functional Foods ..................................................................................... 6-2
6.4 Conclusion ....................................................................................................................... 6-7
6.5 References ....................................................................................................................... 6-7

7. Perspectives on Foods for Specific Health Uses (FOSHU) ..................................... 7-1

7.1 Introduction ...................................................................................................................... 7-1
7.2 Drivers Behind FOSHU .................................................................................................... 7-1
7.3 How FOSHU Status Is Granted ....................................................................................... 7-2
7.4 Current Products .............................................................................................................. 7-3
7.5 Consumer Reaction ......................................................................................................... 7-6
7.6 Conclusions ...................................................................................................................... 7-7
7.7 References ....................................................................................................................... 7-8

8. Functional Foods in the USA: Emphasis on Probiotic Foods ................................ 8-1

8.1 Introduction ...................................................................................................................... 8-1
8.2 Probiotics – the Potential ................................................................................................. 8-2
8.3 Ensuring the Authenticity of Probiotic Products ............................................................... 8-3

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 Contents vii

8.4 Product Labelling Claims for Probiotic Products .............................................................. 8-6

8.5 Conclusions ...................................................................................................................... 8-8
8.6 References ....................................................................................................................... 8-8

9. Functional Foods in Infant Formulae ........................................................................ 9-1

9.1 Introduction ...................................................................................................................... 9-1
9.2 Neonatal Gut Microflora ................................................................................................... 9-2
9.3 The Gut Microflora of Breast-fed and Formula-fed Infants ............................................... 9-2
9.4 The Search for Breast Milk Substitutes ............................................................................ 9-3
9.5 Conclusions ...................................................................................................................... 9-9
9.6 References ....................................................................................................................... 9-10

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Functional foods, blood lipids and coronary heart disease

Kim G Jackson and Julie A Lovegrove

Hugh Sinclair Unit of Human Nutrition, School of Food Biosciences, The University of Reading, Whiteknights, PO Box 226,
Reading RG6 6AP, UK. Tel. 0118 378 6418. Fax 0118 378 0080.
E-mail food@reading.ac.uk

Abstract

For the past 20 years, the principal focus of public health strategies for reducing the risk of coronary heart
disease (CHD) has been aimed at lowering serum cholesterol levels. However, recent findings have highlighted
not only cholesterol but also triacylglycerol as a significant lipid risk factor for CHD. Dietary strategies which
are able to reduce these circulating lipid levels and which are able to offer long-term efficacy comparable with
effective drug treatments are currently being sought. One dietary strategy that may benefit the lipid profile
involves supplementation of the diet with prebiotics, probiotics and synbiotics; this method improves the health
of the host by supplementation with and/or fortification of certain health promoting bacteria present in the
gastrointestinal tract, by dietary means. Probiotics (live organisms) in the form of fermented milk products have
been shown to exert cholesterol lowering properties, whereas non-digestible fermentable carbohydrate
prebiotics have been shown to reduce triacylglycerol levels in animal studies. However, in human studies using
both prebiotics and probiotics, there have been inconsistent findings with respect to changes in lipid levels,
although, on the whole, there have been favourable outcomes.

Keywords: cholesterol, fermented milk products, fructans, glucose, insulin, prebiotics, probiotics, synbiotics,
triacylglycerols

Abbreviations: CHD, coronary heart disease; GIP, glucose-dependent insulinotrophic polypeptide; GLP-1,
glucagon-like peptide-1; HDL, high-density lipoprotein; LDL, low-density lipoprotein; OFS, oligofructose;
TAG, triacylglycerol; VLDL, very low-density lipoprotein

1. Coronary heart disease and risk factors
Diseases of the circulatory system account for an
appreciable proportion of total morbidity and mortality in
adults worldwide. Coronary heart disease (CHD) accounts
for around 115 000 deaths each year in the United Kingdom
alone (Department of Health 1999). The rates of mortality
due to CHD vary considerably throughout the world; for
example, in Finland, the annual incident rate has been
reported to be thirteen times higher than that reported in
Japan (Keys 1980). The incidence rate is reducing in most
countries, although in Eastern Europe and developing
countries, CHD is still increasing in incidence.
The underlying basis for clinical cardiovascular disease is
a combination of atherosclerosis and thrombosis
(Mangiapane and Salter 1999). Atherosclerosis is a
condition in which the arterial lining is thickened in certain
locations by raised plaques which occur as a result of the
excessive accumulation of modified lipids (predominantly
cholesterol), and of proliferation and migration of smooth
muscle cells from deeper layers within the arterial wall.
Formation of an atherosclerotic plaque can occlude one or
more arteries, mainly coronary and cerebral, resulting in
CHD or a stroke, respectively. In addition, a superimposed
thrombus or clot may further occlude the artery, with the
latter being an acute event which causes a myocardial
infarct (Mangiapane and Salter 1999).
CHD is a multifaceted condition which has no one single
cause. Potential risk factors are being identified
concurrently with progress in CHD research. The known
risk factors for development of CHD are shown in Table 1.
This review is mainly concerned with blood lipids and CHD
risk. Epidemiological studies examining CHD risks in
different populations have observed a positive correlation
between fasting cholesterol levels in individuals, especially
that of low-density lipoprotein (LDL) cholesterol, and the
development of CHD (Keys 1970). The role of cholesterol
reduction as a public health strategy in the primary
prevention of CHD was unequivocally supported by the
findings of the West Scotland Heart Study (Shepherd et al.
1995). However, in addition to plasma cholesterol (a classic
risk factor) other lipid risk factors have also been identified.
These include low levels of high-density lipoprotein (HDL)
Food Science and Technology Bulletin: Functional Foods 1 (1) 1–11
DOI: 10.1616/1476-2137.3965. Published 8 May 2002
ISSN 1476-2137 © IFIS Publishing 2002. All Rights Reserved
1-2 Functional foods, blood lipids and coronary heart disease K. G. Jackson and J. A. Lovegrove
Table 1. Risk factors for the development of coronary
heart disease
Unmodifiable Gender (male)
Increasing age
Genetic traits (including lipid metabolism
abnormalities)
Body build
Ethnic origin
Modifiable Cigarette smoking
Some hyperlipidaemias (increased plasma cholesterol
and triacylglycerol)
Low levels of high-density lipoprotein (HDL)
Obesity
Hypertension
Low physical activity
Increased thrombosis (ability to clot)
Stress
Alcohol consumption
Diseases Diabetes (glucose intolerance)
Geography Climate and season (cold weather)
Soft drinking water
cholesterol (Gensini et al. 1998), an elevated concentration
of triacylglycerol (TAG; a major fat in the blood) in both
the fasted and fed (postprandial) states (Austin, 1991; Karpe
et al. 1994; Wilson 1994) and insulin resistance (Reaven et
al. 1993; Haffner et al. 1999).
There is a substantial, diverse, and generally consistent
body of evidence linking diet and CHD. Current dietary
strategies for the prevention of CHD advocate adherence to
low-fat/low-saturated-fat diets (Department of Health
1994). Although there is no doubt that, under certain
experimental conditions, low-fat diets do offer an effective
means of reducing blood cholesterol, on a population basis
they appear to be less effective. This is largely due to poor
compliance attributed to the poor palatability and
acceptability of such diets to the consumer. Attempts have
therefore been made to identify other dietary components
which can address lipid abnormalities associated with CHD.
Recently, dietary intervention strategies based around
probiotics and prebiotics have been used with varying
degrees of success in lowering plasma cholesterol and TAG
levels.
2. Effects of probiotics on blood lipids: the evidence
A probiotic has been recently redefined as 'a live microbial
food ingredient that is beneficial to health' (Salminen et al.
1998). Since the early 1970s, research into probiotics and
CHD risk has increased dramatically, although to date the
data have been inconsistent and inconclusive (Table 2).
2.1 Evidence from the Maasi
Mann and Spoerry (1974) investigated the effect of an
exogenous surfactant material, known to be
hypercholesterolaemic, in 24 male Maasi warriors.
Although a significant fall (9.8%) in serum cholesterol was
reported, because of a number of confounding factors
including the introduction of an exercise programme,
coupled with an inability to control food intake and subject
weight gain, this study is now considered simply as a
curiosity and does not lend much to overall findings.
2.2 Evidence for the milk factor
As a follow-up to the Maasi trial, Mann (1977) fed a small
group (n = 4) of US volunteers 4 l of yoghurt
(microbiological activity unspecified) per day over a 12-day
period, and reported a significant fall of 37% in serum
cholesterol. When the intake of the yoghurt was reduced to
2 l per day, this hypocholesterolaemic effect was
maintained, although intake of 1 l per day resulted in a
return to baseline cholesterol levels. The rate of cholesterol
biosynthesis was monitored by measuring specific activity
of plasma digitonin-precipitated sterols, 2 h after a pulse of
[14C] acetate. A 28% fall in acetate incorporation was
reported 16 days after a 12 day ingestion of the high
yoghurt dose (4 l per day). To help explain the fall in serum
cholesterol, Mann proposed the presence of a milk factor
such as hydroxymethyl glutarate (an inhibitor of 3 hydroxy-
methyl-glutaryl CoA reductase which is an important
enzyme involved with cholesterol synthesis in the liver).
Drinking milk has also been shown to be associated with
reduced mortality from CHD. At least seven prospective
cohort studies have investigated the effect of milk
consumption on death from CHD. The body of evidence
suggests that drinking milk is not detrimental and may be
mildly protective against CHD (Ness et al. 2001).
Intervention studies on milk and plasma cholesterol levels
are mixed: some studies show beneficial reductions of LDL-
cholesterol after three daily servings of low-fat milk (Barr et
al. 2000; Obarzanek et al. 2001), while others do not
(Buonopane et al. 1992). The general consensus is that milk
consumption per se does not increase CHD risk, but neither
does it consistently reduce lipid risk factors.
Howard and Marks (1979) investigated candidates for the
milk factor and fed lactose (with or without Ca/Mg), cheese
whey or yoghurt to volunteers over a 2 wk period. The
yoghurt alone significantly reduced plasma cholesterol by
5.5%. However, this trial was also subject to the same
problems of lack of dietary control as the Mann and Spoerry
study, with substantial changes from the volunteers’
habitual diet resulting in a number of confounding factors.
Unfortunately, most of the early studies introduced
confounding factors due to a lack of control in the subjects'
diet. Hepner and colleagues (1979) performed a study
which attempted to control for such factors. This
investigation was a crossover study in which 720 ml of
yoghurt and 750 ml milk were given to the subjects over a
 Functional foods, blood lipids and coronary heart disease K. G. Jackson and J. A. Lovegrove 1-3

Table 2. Summary of human studies to evaluate the hypocholesterolaemic properties of fermented milk
products

Author Subjects (n) Product (vol/type) Duration Total cholesterol LDL cholesterol

Mann et al. (1974) 24M 8.3 l lacto/yoghurt 3 weeks -9.6% (P<0.001) NA

Mann (1977) 3M 1F 4 l WMY 12 days -16.8% (P<0.05) NA
Mann (1977) 3M 2F 2l SMY 12 days -23.2% (P<0.05) NA
Howard et al. (1979) 10 3l 3 weeks 5.5% (P<0.05) NA
Hepner et al. (1979) 6M 4F 720 ml (A) 4 weeks -5.4% (P<0.01) NA
Hepner et al. (1979) 5M 3F 720 ml (B) 4 weeks 8.9% (P<0.01) NA
Rossouw et al. (1979) 11M 2 l yoghurt 3 weeks +16% (P<0.01) +12% (P<0.001)
Thompson et al. (1982) 13 11 UPY 3 weeks NS NS
Bazzare et al. (1983) 5M 16F 550 g yoghurt 1 week -8.7% NA NA
Massay (1984) 30F 480 ml yoghurt 4 weeks NS NS

Jaspers et al. (1984) 10M 681 g yoghurt 2 weeks -11.6% transient (P<0.05) NS

McNamara et al. (1989) 18M 16 oz LFY 4 weeks NS NS

Agerbaek et al. (1995) 58M 200 ml UPY 6 weeks -6.1% (P<0.001) -9.8% (P<0.001)
-9% transient
Richelsen et al. (1996) 47M 43F 200 ml UPY 21 weeks NA
(P<0.05)
Sessions et al. (1997) 78M 76F 200 ml UPY 12 weeks NS NS
Bertolami et al. (1999) 11M 21F 200 ml UPY 8 weeks -5.3% (P<0.004) 6.2% (P<0.001)
De Roos et al. (1999) 78 500 ml UPY 6 weeks NS NS

Agerholm-Larsen et al. (2000) 20M 50F 450 ml UPY 8 weeks NS -8.4% (P<0.05)

F, female; M, male; WMY, whole milk yoghurt; UPY, unpasteurised yoghurt; SMY, skimmed milk yoghurt; PY, pasteurised yoghurt; LFY, low
fat yoghurt; NA, data not available; NS, not statistically significant.

Adapted from Taylor & Williams 1998

2.3 Probiotic effect on lipid parameters

4 wk period. Significant reductions in plasma cholesterol
were observed after the first week of both supplementation
periods (Hepner et al. 1979). The observation that
cholesterol levels could fall significantly after acceptance
onto a study has been well documented (Rossouw et al.
1981; Sessions et al. 1997). This is probably due to a
conscious or even subconscious modification of the diet by
the volunteers due to awareness of dietary assessment. In an
attempt to reduce this confounding factor, baseline run-in
periods are essential (Sessions et al. 1997). Of the early
negative studies that have been published, those of
Thompson et al. (1982), Massey (1984) and McNamara et
al. (1989) have incorporated a run-in period. The study of
McNamara et al. was one that was more carefully
controlled. They investigated effects of the ingestion of 480
ml unspecified yoghurt and reported no significant
cholesterol reduction. From the evidence presented above, it
can therefore be concluded that there is little evidence that
milk or fermented milk products affect serum lipid
parameters per se.
Hepner et al. (1979) assessed whether the presence of live
bacteria was important for reported effects of yoghurt on
lipid parameters. The aim of the study was to compare the
effects of 750 ml of pasteurised and unpasteurised yoghurt,
using milk as the placebo. After a 12 wk intervention
period, all treatments significantly reduced plasma
cholesterol, with milk resulting in a lesser reduction.
Unfortunately, the nutritional and microbiological content
of the products used was not reported which severely
hampers comparison with other study data. Thompson et al.
(1982) analysed a wide range of milk-based products
including milk fortified with Lactobacillus acidophilus (titre
1.3 x 107 counts/ml), buttermilk (a milk product fermented
with Streptococcus cremoris and Streptococcus lactis - titre
6.4 x 108 counts/ml) and a yoghurt (fermented with
Lactobacillus bulgaricus and Streptococcus thermophilus -
titre 1.2 x 109 counts/ml). A 1 l supplement was offered for
a 3 wk period, but no significant changes were reported in
serum total cholesterol, LDL or HDL. Another study (De
Roos et al. 1999) involving seventy-eight male and female
1-4 Functional foods, blood lipids and coronary heart disease K. G. Jackson and J. A. Lovegrove
participants (mean cholesterol 5.4 ± 0.7 mmol/l)
investigated the effect of consuming 500 ml of control
yoghurt or yoghurt enriched with Lactobacillus acidophilus
L-1 for a 6 wk period. No significant reduction in
cholesterol or LDL cholesterol was reported in this study.
The possible importance of variation in yoghurt cultures
stimulated Jasper et al. (1984) to assess the effect on plasma
lipid levels of consuming 681 g per day of different samples
of a non-fat unpasteurised yoghurt fermented with a 1:1
ratio of Lactobacillus bulgaricus and Streptococcus
thermophilus. Two yoghurt strains (CH-I and CH-II) were
taken for a 14 day period each, followed by a third strain
(SH-III) which was taken for 21 days. A 21 day 'washout
period' was permitted between each dietary intervention
period to re-establish normal serum cholesterol levels.
Significant falls in serum total cholesterol and LDL
cholesterol levels occurred after 1 wk with strain CH-II and
2 wk with strain SH-III. Such transient changes could be
explained either by effects of volunteers commencing the
study as discussed previously, or could be a true difference
between the efficacious properties of different strains and
indeed different types of probiotics.
A commercially available yoghurt, GAIO®, containing a
specific bacterial culture, CAUSIDO® (consisting of
Enterococcus faecium and Streptococcus thermophilus, and
has been shown to have hypocholesterolaemic properties
when tested on animals) has been studied, albeit with
inconsistent findings. Agerbaek et al. (1995) tested the
effect of GAIO® against an identical yoghurt that had been
chemically fermented with an organic acid (δ-gluco-
lactone). Fifty-eight middle-aged men with moderately
raised cholesterol levels (5.0-6.5 mmol/l) were fed 200 ml
per day of yoghurt for a 6-wk period. They observed a 9.8%
reduction in LDL cholesterol levels (P < 0.001) for the live
yoghurt group. Although this was a well-controlled study,
an unforeseen skew in randomisation resulted in a
significantly different baseline total and LDL cholesterol
levels in the two groups. The reduction in these parameters
observed in the live yoghurt group could be ascribed to a
regression towards the mean. Another study performed
using the same yoghurt and a similar design for a longer
period (6 months) was carried out in 87 men and women
aged between 50 and 70 years (Richelsen et al. 1996).
Although after 12 wk there was a significant drop in LDL
cholesterol levels in the group taking the active yoghurt,
these reductions were not sustained. This was partly
explained by a reduction in the titre of the yoghurt at 12 wk.
A more recent study investigated the same product at 200 g
per day for 8 wk with 32 patients who had mild to moderate
hypercholesterolaemia. The patients were asked to follow a
lipid-lowering diet for 8 wk and then were given the test or
control product for two 8 wk periods. The results from this
study showed a significant reduction (5.3% (P = 0.004) and
6.2% (P = 0.01), respectively) in total and LDL cholesterol
after consumption of the active product. However, the
authors did question whether or not an average reduction of
approximately 5% for total and LDL cholesterol was
clinically important (Bertolami et al. 1999). A more recent
publication investigated the effects of consumption of a
larger volume (450 ml per day) of GAIO® yoghurt
containing the CAUSIDO® culture on seventy overweight
and obese men and women for 8 wk. After adjustment for
changes in body weight, a significant reduction in LDL-
cholesterol (8.4%) was reported in the GAIO® group
(Agerholm-Larsen et al. 2000a).
However, not all studies have reported a significant
reduction in cholesterol after GAIO® ingestion. One study
reported by Sessions et al. (1997) was conducted in 160
middle-aged men and women with moderately raised
cholesterol. Volunteers consumed 200 ml per day of either
the active (containing a minimum titre of 1 x 106 counts/ml)
or chemically fermented yoghurt for a 12 wk period.
Stratified randomisation was used and the study was well
controlled. During the 2 wk run-in period, both groups
showed significant reductions in blood cholesterol (P <
0.05), but thereafter there were no further changes in either
group or between the groups at any of the time points.
A meta-analysis of short-term intervention studies using
GAIO® was published by Agerholm-Larsen et al. (2000b).
They concluded that consumption of the fermented yoghurt
produced 4 and 5% decreases in total and LDL cholesterol
respectively; however, long-term studies were required to
demonstrate sustained effects on blood lipids.
2.4 Possible mechanisms of action
It is important to highlight the fact that viable and
biologically active microorganisms are usually required at a
specific target site in the host. It is therefore essential that
the probiotics involved not only exert the characteristics that
are necessary to produce any desired biological effects, but
also have the required viability and are able to withstand the
host's natural barriers against ingested bacteria. The classic
yoghurt bacteria, Streptococcus thermophilus and
Lactobacillus bulgaricus are technologically effective, but
they do not reach the lower intestinal tract in a viable form.
Therefore, intrinsic microbiological properties, such as
tolerance to gastric acid, bile and pancreatic juice, are
important factors when probiotic organisms are considered
(Huis In't Veld and Shortt 1996).
The mechanism of action of probiotics on cholesterol
reduction is unclear, although a number of possible
mechanisms have been proposed. These include the
physiological actions of fermentation end products (short
chain fatty acids), deconjugation of bile acids (which could
reduce cholesterol by co-precipitation at acidic pH or by
increasing excretion of bile acids, thereby increasing the
amount of cholesterol required for de novo synthesis in the
liver, or a combination of both these mechanisms),
cholesterol assimilation, and cholesterol binding to bacterial
cell walls. The short chain fatty acids that are produced by
bacterial anaerobic breakdown of carbohydrate are acetic,
propionic and butyric acids. The physiological effects of
these are discussed in more detail in the prebiotic section
below.
 Functional foods, blood lipids and coronary heart disease K. G. Jackson and J. A. Lovegrove
It has been well documented that microbial metabolism of
bile acid is a peculiar probiotic effect involved in the
therapeutic role of some bacteria. The deconjugation
reaction is catalysed by a conjugated bile acid hydrolase
enzyme, which is produced exclusively by bacteria.
Deconjugation is widely found in many intestinal bacteria
including genera such as Enterococcus, Peptostreptococcus,
Bifidobacterium, Fusobacterium, Clostridium, Bacteroides
and Lactobacillus (Hylemond 1985). This reaction liberates
an amino acid moiety and a deconjugated bile acid, thereby
reducing cholesterol re-absorption by increasing faecal
excretion of the deconjugated bile acids. Many in vitro
studies have investigated the ability of various bacteria to
deconjugate a variety of different bile acids. For example,
Grill et al. (1995) reported Bifidobacterium longum as being
the most efficient bacterium when tested against six
different bile salts. Another study reported that
Lactobacillus species had varying abilities to deconjugate
glycocholate and taurocholate (Gilliland and Speck 1977).
Studies performed on in vitro responses are useful, although
in vivo studies in animals and humans are required to more
fully determine the contribution of bile acid deconjugation
to cholesterol reduction. Intervention studies on animals and
ileostomy patients have shown that oral administration of
certain bacterial species can lead to an increased excretion
of free and secondary bile salts (De Smet et al. 1998;
Marteau et al. 1995).
There is also in vitro evidence to support the hypothesis
that some bacteria can assimilate (take up) cholesterol. It
has been reported that Lactobacillus acidophilus (Gilliland
et al. 1985) and Bifidobacterium bifidum (Rasic et al. 1992)
have the ability to assimilate cholesterol during in vitro
studies, but only in the presence of bile salts and under
anaerobic conditions. However, despite such reports, there
is uncertainty about whether the bacteria are assimilating
cholesterol or whether cholesterol is co-precipitating with
the bile salts. Studies have been performed to address this
question; for example, Klaver and Van der Meer (1993)
concluded that removal of cholesterol from the medium in
which Lactobacillus acidophilus and Bifidobacterium were
growing was not due to assimilation, but due to bacterial
bile salt deconjugase activity. The same question was
addressed by Tahri et al. (1995) but with conflicting results:
they concluded that part of the removed cholesterol was
found in the cell extracts and that cholesterol assimilation
and bile acid deconjugase activity could occur
simultaneously.
Cholesterol binding to bacterial cell walls has also been
suggested as a possible mechanism for the
hypocholesterolaemic effects of probiotics. Hosono and
Tono-oka (1995) reported that Lactococcus lactis subsp.
lactis biovar. diacetylactis R-43 had the highest binding
capacity for cholesterol for a range of bacteria tested. It was
speculated that differences in binding of the bacteria were
due to chemical and structural properties of their cell walls,
and that even non-viable cells may have the ability to bind
cholesterol in the host intestinal tract.
1-5
The mechanism of action of probiotics on cholesterol
reduction could include one or all of the above mechanisms,
with an ability of different bacterial species to have varying
effects on cholesterol lowering. Moreover, inconsistencies
in the literature regarding beneficial effects of probiotics
could be due to a number of different factors. The period of
intervention, volume consumed and, more importantly, the
bacterial titre of the product are all important considerations
that may vary (Thompson et al. 1982; Richelsen et al.
1996). The baseline concentrations of cholesterol also affect
study outcomes, with hypercholesterolaemic groups
responding to probiotics in some (Agerbaek et al. 1995;
Agerholm-Larsen et al. 2000a) but not all (Sessions et al.
1997; De Roos et al. 1999) studies. The study outcome
depends also on the bacterial species used, its metabolic
capabilities, and viability throughout the gastrointestinal
tract (Jasper et al. 1984). Further research is required to
more fully elucidate the relative importance of such
parameters before the true beneficial effects and mechanism
of action of probiotics in relation to CHD can be
determined.
3. The effect of fructan-type oligosaccharide
prebiotics on lipid metabolism in humans
In recent years, there has been increasing interest in the
important nutritional roles of prebiotics as functional food
ingredients. A prebiotic is a selectively metabolised dietary
ingredient that stimulates 'beneficial' microorganisms in the
lower gut. This interest has been derived from animal
studies which have shown marked reductions in TAGs and,
to a lesser extent, cholesterol levels when diets containing
significant amounts of the prebiotic (oligofructose (OFS))
were fed. However, studies conducted in humans examining
the effects of prebiotics on plasma lipids levels have
generated inconsistent findings (Table 3). In individuals
with raised blood lipids, three studies have shown
significant decreases in fasting total and LDL cholesterol,
with no significant changes in TAG levels (Yamashita et al.
1984; Hidaka et al. 1991; Davidson et al. 1998), whereas
one recent study in type II diabetics did not reveal any
change in cholesterol levels (Alles et al. 1999). In
normolipidaemic subjects, only two studies involving
supplementation with inulin have demonstrated significant
changes in both fasting TAG and total cholesterol levels
(Canzi et al. 1995; Brighenti et al. 1999), with another
study showing a significant decrease in TAG levels only
(Causey et al. 2000). However, Luo et al. (1996) and
Pedersen et al. (1997) have recently reported no effects of
OFS or inulin treatment on plasma lipid levels in young
healthy subjects. In a group of middle-aged men and
women, lower plasma TAG levels were observed 8 wk after
inulin treatment compared to a placebo (Jackson et al.
1999). In this study, follow-up blood samples were taken 4
wk after completion of the supplementation period, by
which time concentrations of TAG had returned to baseline
values, thus supporting the conclusion that inulin feeding
1-6 Functional foods, blood lipids and coronary heart disease K. G. Jackson and J. A. Lovegrove

Table 3. Summary of human studies to examine the effects of fructan supplementation on blood lipids

Significant changes
Author Subjects Fructan Dose Study design Duration Vehicle
observed in:
Blood lipids Glucose

Yashashati 8M and 10F OFS 8g DB parallel 2 weeks packed coffee ↓ TC ↓ glucose

et al. 1984 Type II diabetes drink ↓ LDL-C
canned coffee jelly
Hidaka et 37 (M and F) OFS 8g DB parallel 5 weeks confectionery ↓ TC NS
al, 1991 hyperlipidaemic
Canzi et al. 12 M inulin 9g Sequential 4 weeks breakfast cereal ↓ TAG N/A
1996 normolipidaemic ↓ TC
Luo et al. 12 M OFS 20 g DB crossover 4 weeks 100 g biscuits NS NS
1996 normolipidaemic
Pedersen et 66 F inulin 14 g DB crossover 4 weeks 40 g margarine NS N/A
al. 1997 normolipidaemic
Davidson et 21 (M and F) inulin 18 g DB crossover 6 weeks chocolate ↓ LDL-C N/A
al. 1998 hyperlipidaemic bar/paste or coffee ↓ TC
sweetener
Alles et al. 9 M and 11 F OFS 15 g SB crossover 3 weeks supplement not NS NS
1999 Type II diabetes specified
Brighenti et 12 M inulin 9g Sequential 4 weeks breakfast cereal ↓ TC NS
al. 1999 normolipidaemic ↓ TAG
Jackson et 54 (M and F) inulin 10 g DB parallel 8 weeks powder added to TAG ↓ insulin
al. 1999 normolipidaemic food and drinks
Causey et 12 M inulin 20 g DB crossover 3 weeks low-fat ice-cream ↓ TAG NS
al. 2000 normolipidaemic

M, male; F, female; DB, double blind; N/A, not measured; NS, not significant; SB, single blind; TC, total cholesterol; LDL-C, LDL cholesterol;
OFS, oligofructose; TAG, triacylglycerol

may have been responsible for the decrease in TAG levels.
Raised postprandial TAG concentrations have been
recognised as a risk factor for CHD (Austin 1991). Data
from studies in rats have shown a 40% reduction in
postprandial TAG concentrations when diets containing
10% (w/v) OFS were fed (Kok et al. 1998). However, very
little information regarding the effects of prebiotics on
postprandial lipaemia in human subjects are available,
although one recent study in middle aged volunteers has
shown no effect of inulin treatment on postprandial TAGs
(Williams 1998).
The marked reduction in fasting lipid levels, notably
TAGs, which has been observed in animal studies has not
been consistently reproduced in humans. The amount of
fructans used in the human studies listed in Table 3 varies
between 9 and 20 g; this is small compared to that used in
animal trials (50-200 g OFS/kg rat chow; Roberfroid 1993)
and is equivalent to a dose in humans of approximately 50-
80 g OFS or inulin per day. The prebiotic nature of OFS and
inulin restricts its dosage in humans of up to 15 g per day
since doses greater than this cause gastrointestinal
symptoms such as stomach cramps, flatulence and diarrhoea
(Pedersen et al. 1997). It is not known whether, at the levels
used in human studies, significant effects would be
observed in animals (Delzenne et al. 1993).
The types of dietary vehicles used to increase amounts of
OFS or inulin in the diet differ. In the case of Luo et al.
(1996), 100 g of biscuits were eaten every day and for
Pedersen et al. (1997), 40 g of margarine was consumed
which may have contributed towards the negative findings
in blood lipids. In the case of Davidson et al. (1998),
significant changes in total and LDL cholesterol levels were
observed over 6 wk with inulin, as compared to a placebo
(sugar). The percentage change in each of the lipid
parameters was calculated over each of the 6-wk treatment
periods and, unexpectedly, there was an increase in total
cholesterol, LDL cholesterol and TAG during the placebo
phase. Non-significant falls in these variables were
observed during inulin treatment; therefore, when net
changes in the variables were calculated (change during
inulin minus that during the control treatment), there were
significant differences in total and LDL cholesterols
between the two treatments. The authors attributed the
increase in total and LDL cholesterol levels during the
placebo phase to be related to an increased intake of
saturated fatty acids in the chocolate products which were
used as two of the vehicles in the study (Davidson et al.
1998). In later studies, the use of inulin in its powdered
form enabled it to be added to many of the foods eaten in
the subjects' normal diet without any need for dietary advice
 Functional foods, blood lipids and coronary heart disease K. G. Jackson and J. A. Lovegrove
thus avoiding changes in body weight. Since inulin has
water binding properties, as a powder it can be added to
orange juice, tea, coffee, yoghurt and soup (Jackson et al.
1999).
The significant relationship between subjects' initial TAG
concentration and the percentage change in TAG levels over
the 8 wk study demonstrated by Jackson et al. (1999) lends
support to the hypothesis that the initial TAG levels could
be important in determining the degree of response to
inulin. The lack of response in some individuals may be as a
result of their being less responsive to inulin, variations in
their background diet or non-compliance with the study
protocol. Speculation as to possible reasons for the
variability in response would be aided by an improved
understanding of the mechanism of action of inulin on
plasma TAG levels.
The length of supplementation period used in the studies
summarised in Table 3 may be another factor for the
inconsistent findings in changes in TAG levels in human
subjects given supplements of inulin. The studies were
conducted over 2-8 wk, with significant effects occurring in
only three studies conducted over 3-4 wk (Brighenti et al.
1999; Canzi et al. 1996; Causey et al. 2000). The lack of
lipid lowering noted in the studies of Alles et al. (1999),
Pedersen et al. (1997) and Luo et al. (1996) may be due to
insufficient duration of supplementation in their chosen
subject groups. In the study of Jackson et al. (1999), a trend
for TAG reduction upon inulin treatment occurred
sometime between 4 and 8 wk; this may reflect the period
necessary for modification of the gut microflora. A 4 wk
wash out seemed to be sufficient for the TAG
concentrations to return to baseline values.
Whilst some of the studies to date support beneficial
effects of inulin on plasma TAG, the findings are by no
means consistent and more studies are required to provide
convincing evidence of the lipid lowering consequences of
prebiotic ingestion.
3.1 Mechanism of lipid lowering by inulin and
oligofructose
Prebiotics have been shown to be an ideal substrate for
health promoting bacteria in the colon, notably
bifidobacteria and lactobacilli (Gibson and McCartney
1998). During the fermentation process, a number of by-
products are produced, including gases (hydrogen sulphide,
carbon dioxide, hydrogen and methane), lactate and short
chain fatty acids (acetate, butyrate and propionate). The
short chain fatty acids acetate and propionate enter the
portal blood stream where they are utilised by the liver.
Acetate is converted to acetyl CoA in the liver and acts as a
lipogenic substrate for de novo lipogenesis, whereas
propionate has been reported to inhibit lipid synthesis
(Wolever et al. 1989; Demigne et al. 1995). Butyrate, on the
other hand, is taken up by the large intestinal cells
(colonocytes) and has been shown to protect against tumour
formation in the gut (Van Loo et al. 1999). The type of
1-7
short chain fatty acids which are produced during
fermentation is dependent on the gut microflora that are
stimulated by the prebiotic. Inulin, for example, has been
shown to increase both acetate and butyrate levels (Van Loo
et al. 1999).
Inulin and OFS have been extensively studied to
determine the mechanism of action of prebiotics in animals.
Early in vitro studies using isolated rat hepatocytes
suggested that the hypolipaemic action of OFS was
associated with an inhibition of de novo cholesterol
synthesis by propionate, following impairment of acetate
utilisation by the liver for de novo lipogenesis (Demigné et
al. 1995). This is in agreement with human studies where
rectal infusions of acetate and propionate resulted in the
latter being able to inhibit the incorporation of acetate into
TAGs released from the liver (Wolever et al. 1995).
Fiordaliso et al. (1995) demonstrated significant reductions
in plasma TAGs, phospholipids and cholesterol in
normolipidaemic rats fed a chow diet containing 10% (w/v)
OFS. The TAG-lowering effect was demonstrated after only
1 wk of OFS and was associated with a reduction in very
low density lipoprotein (VLDL) secretion. TAGs and
phospholipids are synthesised in the liver by esterification
of fatty acids and glycerol-3-phosphate before being made
available for assembly into VLDL, suggesting that the
hypolipidaemic effect of OFS may be occurring in the liver.
The reduction observed in cholesterol levels in the rats was
only demonstrated after long term feeding (16 wk) of OFS.
Recent evidence has suggested that the TAG-lowering
effect of OFS occurs via a reduction in VLDL TAG
secretion from the liver due to a reduction in the activity of
all lipogenic enzymes (acetyl-CoA carboxylase, fatty acid
synthase, malic enzyme, ATP citrate lyase and glucose-6-
phosphate dehydrogenase), and, in the case of fatty acid
synthase, via modification of lipogenic gene expression
(Delzenne and Kok 1998; Figure 1). However, further work
is required to determine the mechanisms whereby short
chain fatty acids lower cholesterol levels in humans.
3.2 The effect of prebiotics on glucose and insulin
levels
Very little is known about the effects of prebiotics on
fasting insulin and glucose levels in humans. Of the
supplementation studies that have been conducted in
humans, a significant reduction in glucose was observed in
subjects with type II diabetes (Yamashita et al. 1984) and a
trend for a reduction in glucose was observed in
hyperlipidaemic subjects (Hidaka et al. 1991) consuming
OFS. However, a recent study has reported no effect of OFS
on blood glucose levels in type II diabetics (Alles et al.
1999). A significant reduction in insulin levels was
observed in healthy middle-aged subjects with inulin,
although this was not accompanied by changes in plasma
glucose levels (Jackson et al. 1999). The effect of ingestion
of acute test meals containing OFS on blood glucose,
insulin and C-peptide levels in healthy adults showed a
1-8 Functional foods, blood lipids and coronary heart disease K. G. Jackson and J. A. Lovegrove
Figure 1. Hepatic fatty acid metabolism
trend for a lower glycaemic response and peak insulin
levels, following the OFS enriched meals (Rumessen et al.
1990).
It has been suggested that the mechanism of action of
prebiotics on lowering of glucose and insulin levels is
associated with short chain fatty acids, especially
propionate. A significant reduction in postprandial glucose
concentration was observed following both acute and
chronic intakes of propionate-enriched bread (Todesco et al.
1991). The effect of propionate intake on postprandial
insulin levels was not investigated. A recent animal study
has shown an attenuation of both postprandial insulin and
glucose levels following 4 wk of feeding with OFS. These
effects were attributed to the actions of OFS on the
secretion of the gut hormones glucose dependent
insulinotropic polypeptide (GIP) and glucagon-like peptide
1 (GLP-1) (Kok et al. 1998). These hormones are secreted
from the small intestine (GIP) and the terminal ileum and
colon (GLP-1), and contribute towards the secretion of
insulin following a meal in the presence of raised glucose
levels (Morgan 1998).
In summary, the mechanisms of action of prebiotics,
especially inulin and OFS, have been determined largely
from animal studies. Present data suggest inhibition of de
novo lipogenesis as the primary mode of action of prebiotics
in mediating their lipid lowering effects via down regulation
of the enzymes involved. If this is the case, more modest or
inconsistent effects might be expected in humans, in whom
de novo lipogenesis is extremely low or variable, depending
on their background diet. In animal studies, rats are fed a
diet that is low in fat and high in carbohydrate. Thus, de
novo lipogenesis is an upregulated pathway in these animals
for the synthesis of fatty acids. It is interesting to note that
when rats are fed OFS along with a high fat diet typical of
the Western-style diet, TAG levels are thought to be
decreased by a different mechanism involving enhanced
clearance of TAG-rich lipoproteins. An increased GIP
secretion in OFS treated rats was observed by Kok et al.
(1998) and this gut hormone has been shown to enhance the
activity of lipoprotein lipase, the principal enzyme involved
in the clearance of TAG-rich lipoproteins following fat
ingestion (Knapper et al. 1995). The release of GIP and
GLP-1 in the colon may act to mediate systemic effects of
prebiotics such as inulin and OFS, on blood lipid, insulin
and glucose levels. However, further work is required to
determine the metabolic pathways that are influenced by
prebiotics. Their effect on gastrointestinal kinetics such as
gastric emptying and modification of the levels of TAG-rich
lipoproteins and glucose in the circulation has recently been
proposed as a potential modulator of systemic effects
(Delzenne 1999). Therefore the design of future studies to
investigate the effect of prebiotics in humans should
consider the choice of subjects, length of supplementation
period and type of vehicle used to increase the intake of
prebiotics in the diet, as all of these variables may influence
the outcome of the study.
 Functional foods, blood lipids and coronary heart disease K. G. Jackson and J. A. Lovegrove
4. The effects of synbiotics on coronary heart
disease
The use of synbiotics as functional food ingredients is a new
and developing area; very few human studies have been
performed which look at their effect on risk factors for
CHD. A synbiotic is a combination of both probiotic
organisms and probiotic compounds. In one study, the effect
of a fermented milk product with and without the addition
of Lactobacillus acidophilus and fructooligosaccharides
was examined in healthy men (Schaafsma et al. 1998). The
design of the study was a randomised placebo controlled
crossover form, in which there were two treatment periods
of 3 wk, with a 1 wk washout period. The authors reported a
significant reduction in total and LDL cholesterol following
ingestion of the fermented milk product containing both the
probiotic and prebiotic, compared to the placebo fermented
milk. However, these data should be interpreted with
caution since there was an increase in cholesterol levels
during consumption of the placebo fermented milk product.
Other research has concentrated on the composition of the
gut microflora. In one study of healthy subjects, a fermented
milk product containing Bifidobacterium spp. with or
without 18 g of inulin was given daily for 12 days. The
authors concluded that administration of the fermented milk
product (probiotic) substantially increased the proportion of
bifidobacteria in the gut, but that this increase was not
enhanced with the addition of inulin. The composition of
the gut microflora was then assessed 2 wk after completion
of the supplementation period; it was found that subjects
who received the fermented milk product with inulin
maintained their gut bifidobacterial population compared to
subjects receiving the fermented milk product only.
Although a synergistic effect on the bifidobacterial
population in the gut was not observed with the synbiotic,
these results suggest that either there was better
implantation of the probiotic and/or there was a separate
prebiotic effect on bifidobacteria already present in the gut
(Bouhnik et al. 1996). The maintenance of high numbers of
bifidobacteria in the gut flora may be beneficial in terms of
maintaining healthy intestinal function, however, its effect
on blood lipid reduction remains to be determined. A more
recent study has shown that a lower dose of prebiotic (2.75
g) added to a Lactobacillus-fermented milk was able to
significantly increase numbers of bifidobacteria when fed
over a 7 wk period in healthy human subjects (Roberfroid
1998). If this effect was a result of the synbiotic product
used in the study, the use of lower doses of prebiotics in
synbiotic preparations will help to reduce the
gastrointestinal complaints observed with prebiotics alone
and will improve the acceptability of these types of products
by the general public.
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About the authors
Dr Kim G. Jackson and Dr Julie A. Lovegrove are Senior
Research Fellows working at the Hugh Sinclair Unit of
Human Nutrition, the University of Reading. Their research
interests are primarily concerned with the relationship
between diet and coronary heart disease, in particular lipid
metabolism. Recent work has concentrated on the effects of
different fatty acids (especially monounsaturated and long
chain omega-3 polyunsaturated fatty acids) as well as
prebiotics and probiotics, on lipid risk factors in a number
of population groups including post-menopausal women,
middle aged men and Sikh volunteers.
Gastrointestinal infections and the protective role of probiotics and
prebiotics

Glenn R Gibson and Robert A Rastall

Food Microbial Sciences Unit, School of Food Biosciences, The University of Reading, Whiteknights, Reading RG6 6AP,
UK. Tel. 44 (0)1189 357223. Fax 44 (0)1189 357222. E-mail g.r.gibson@reading.ac.uk

Abstract

Protection afforded by probiotic and prebiotic foods against gastrointestinal infections is reviewed. Aspects
considered include: acute pathogenesis in the gastrointestinal tract (Escherichia coli, Campylobacter,
Salmonella); chronic gut disorders in which microorganisms are believed to play a role (gastritis, peptic ulcer
and stomach carcinoma, irritable bowel syndrome, bowel cancer, ulcerative colitis, Crohn's disease,
pseudomembranous colitis); functions of the normal gut microflora; use of probiotics and prebiotics to modulate
the gastrointestinal microflora; foods containing probiotics and prebiotics (live yoghurts, fermented dairy
beverages, cheese, squeezable yoghurt, fromage frais, sweet chews, fruit juice, weaning foods, jelly, foods
containing fructooligosaccharides and other prebiotics, synbiotics); and mechanisms underlying the effects of
probiotics and prebiotics on gastrointestinal pathogens (direct inhibition, immunomodulation, competition,
adhesion, attenuation).

Keywords: diseases, human physiology, pathogens, food poisoning, gastrointestinal tract, prebiotics, probiotics

1. Introduction 2. Acute pathogenesis in the human gut

The incidence and frequency of microbial pathogenesis,
especially that caused by poor food hygiene, continues to
increase and imposes an enormous medical, social and
economic burden. Currently, microbiological food safety
has attracted a high consumer, industrial and research
profile, although it is important that more effective
measures are needed to combat gut infections. A great deal
of effort has been expended in the tracking and monitoring
of individual pathogens in the food chain, helping to build
physiological information that can help to predict infection
and virulence. Moreover, improved hygiene standards, the
training of food handling personnel and guidelines such as
HACCP (Hazard Analysis and Critical Control Point) are all
geared towards reducing the risk of microbial transmission
from farm to fork. Nevertheless, gut infection still occurs
and it is worth considering that fortification of the
indigenous microflora through dietary supplements may
improve the resistance to gastrointestinal infection. This can
be achieved through use of probiotics, prebiotics or both
(synbiotics) that are designed to increase the lactic acid
producing flora in the gut. Similarly, this approach may be
feasible for treating chronic gut diseases that have a
suspected microbial aetiology.
Food Science and Technology Bulletin: Functional Foods 1 (2) 1–17
DOI: 10.1616/1476-2137.3664. Published 6 January 2003
ISSN 1476-2137 © IFIS Publishing 2003. All Rights Reserved
Whilst viruses probably cause most cases of gastroenteritis
in children and adults, it is bacteria that are most
problematic in terms of food safety. So called 'food
poisoning' may be caused by eating foods containing either
pathogenic microorganisms or their toxins. Clinical features
of acute gastroenteritis vary greatly and can range from
mild to life threatening. Symptoms may include vomiting,
nausea, abdominal cramps and/or diarrhoea, depending on
the causative agents (Hui et al. 1994). A microbiological
analysis is usually carried out to confirm the diagnosis.
Bacteria present the main concern because they are
ubiquitous and are therefore more likely to contaminate
food. Moreover, the food environment can provide a good
matrix and substrate supply for growth. Bacteria also
proliferate rapidly, although the minimum level for
detection varies significantly. For example, shigellae
produce a very powerful toxin able to produce
gastroenteritis from as low as 10 bacterial cells, while it
may require over one million Clostridium perfringens cells
to cause a similar infection. Common sources of bacterial
contamination in foods are shown in Table 1.
Many bacteria capable of causing food poisoning reside
in the gut flora of humans and animals. As such, there is a
risk of infection in meat and poultry, particularly at the time
of slaughter. However, it is important to realise that poor
slaughter practices may lead to infection, even in the
cleanest of slaughterhouses. Similarly, contamination of
2-2 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall
Table 1. List of principal bacterial causes of food
poisoning
Bacterial species Foods that may be commonly
contaminated
Bacillus cereus Cooked rice, meat
Campylobacter spp. Poultry, unpasteurised milk
Clostridium botulinum Fish, meat
Clostridium perfringens Cooked meat, poultry
Escherichia coli Meat, raw milk
Listeria monocytogenes Pate, soft cheeses
Salmonella spp. Meat, poultry, eggs
Shigella spp. Eggs, salads
Staphylococcus aureus Ham, poultry, dairy products
Yersinia enterocolitica Milk, poultry
dairy produce may occur through the use of non-pasteurised
milk, while vegetables may be exposed to risk if animal
manure is used as fertiliser.
Whilst infection via the food chain to the home is
possible by various means, several steps may be taken to
minimise the risk. However, the home also poses a potential
site for further transmission of infectious bacterial agents,
and it is here that the consumer can exert the most impact.
Bacterial growth in foods can be affected by temperature,
acidity and moisture content. In the kitchen, this is readily
controlled by adequate cooking of food, with temperatures
of around 100°C being lethal for vegetative bacterial cells.
However, some bacteria (e.g. clostridia, bacilli) are able to
produce spores that are more resistant to such conditions. It
is therefore imperative that the cooking process is thorough,
with correct handling and storage also adding significantly
towards the maintenance of hygienic processes.
In recent years, several bacteria have gained increased
notoriety through food poisoning outbreaks:
2.1 Escherichia coli
Most strains of Escherichia coli are harmless commensal
organisms that are resident in the gut. However, some
strains can exhibit pathogenic traits through invasive (via
intestinal cells) and/or toxigenic means. Verocytotoxin-
producing E. coli cells (e.g. strain O157:H7) are especially
significant as they produce a severe form of food poisoning
characterised by the occurrence of bloody diarrhoea and
complications such as kidney failure (Bell and Kyriakides
1998; Sussman 1997; Stewart and Flint 1999). The
bacterium was widely identified in the 1980s, with an
outbreak in Scotland in 1996 being significant in terms of
the number of fatalities (21 elderly people) that occurred.
This devastating episode of food poisoning caused more
deaths from a single source than that of any other food
poisoning incident. Farm animals are thought to be a major
reservoir for the organism, which is believed to have
acquired its toxic trait through plasmid-borne transfer, from
species of Shigella.
2.2 Campylobacter
Species of bacteria that belong to the Campylobacter group
are responsible for most reported cases of food poisoning in
the United Kingdom. Around 60 000 cases are reported per
annum (Macfarlane and Gibson 1995). However, as with all
other foodborne bacterial causes of gastroenteritis, the real
incidence is probably at least 10-fold higher. It is thought
that the primary source of infection is poultry, with most
chickens thought to be carriers. Interestingly, the bacterium
does not cause any symptoms in poultry. Adequate cooking
procedures eliminate this pathogen effectively.
2.3 Salmonella
This group consists of thousands of different serotypes and
has been a primary focus for food poisoning. Most episodes
have been related to eggs and poultry. As such, antibiotic
treatment for poultry flocks has been permitted, and
attention has also focused on reduction of contamination in
feeds (ICMSF 1996; Hui et al. 1994).
2.4 Other bacteria
Other predominant sources of food poisoning have included
sporadic outbreaks involving Listeria monocytogenes
(Gibson and Macfarlane 1995). This organism may be
spread from person to person, although foods such as pâtés
and soft cheeses have also been implicated. The bacterium
is especially resistant to cool temperatures and, because of
its ability to cross the placenta, is a particular risk for
pregnant women and their foetuses.
Lower incidences of food poisoning have been observed
with species of clostridia, bacilli and staphylococci (ICMSF
1996).
3. Chronic gut disorder
Microorganisms are also thought to be responsible for many
chronic and debilitating disorders of the gut, some of which
can be fatal. Specific examples are as follows:
3.1 Gastritis, peptic ulcer, stomach carcinoma
Helicobacter pylori is probably the most intensively studied
microorganism of recent times (Clayton and Mobley 1997).
Realisation of the epidemiological links between H. pylori
carriage and various gastrointestinal complaints have
markedly altered the way in which diseases of the stomach
and duodenum are viewed. It is now accepted that H. pylori
infection causes chronic active gastritis (Type B) and is the
main cause of this condition. It is also evident that the
bacterium has a strong association with duodenal ulceration
and even stomach cancer. Of course, the implications of
identifying a transmissible agent in a form of cancer has
enormous potential for both preventative (development of
appropriate vaccines) and treatment (efficient antimicrobial
therapy) aspects of tumorigenesis. Whilst the virulence of
H. pylori has not been adequately identified, it is likely to
 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall
be at least partly due to an over production of ammonia,
which is toxic (Rathbone and Heatley 1992). Despite
extensive research, the precise route by which H. pylori is
transmitted remains undefined. Its isolation from faeces
(Thomas et al. 1992; Kelly et al. 1995; Gibson et al. 1995b)
and occurrence in dental plaque (Desai et al. 1991) suggest
that the faecal-oral route may be one mechanism. In the
stomach, the organism is afforded physical protection from
the adverse effects of gastric acid by virtue of its location in
the mucosal layer.
3.2 Irritable bowel syndrome
Irritable bowel syndrome (IBS) probably occupies more
medical consultation time than any other disorder. It is said
to affect up to 20% of the UK population, with women
suffering the highest incidence. IBS is characterised by
irregular bowel movements and general malaise (King et al.
1998; Farthing 1999). Whilst certain hypotheses have linked
stress with IBS, there is stronger evidence that gut
dysfunction is involved. In particular, it is thought that the
microbiota of the large intestine plays a key role in the onset
and maintenance of IBS. Attention has been drawn to the
relationship between carriage of yeasts such as Candida
spp. and symptoms associated with the disorder (Balsari et
al. 1982; Middleton et al. 1992), and indeed some of the
antimicrobial compounds (e.g. nystatin) used to treat yeast
infections (e.g. Candida induced recurrent thrush) may
encourage the onset of IBS.
3.3 Bowel cancer
Colon cancer is a severe cause of morbidity and mortality in
Western populations, with the large intestine being the
second most common site for tumours (van Tassell et al.
1990). There is a postulated link between gut microflora
activities and production of carcinogens from dietary
residues. Putative carcinogens may be derived from
bacterial activities and include nitrosamines, heterocyclic
amines, 2-amino-3-methylimidazo[4,5-F]quinoline (IQ),
fecapentaenes, etc. (Gibson and Macfarlane 1994).
Moreover, the metabolism of lipid precursors may produce
diacyglycerols which can then act as tumour promoters
(Morotomi et al. 1990) and are therefore problematic for
sufferers of polyps.
3.4 Ulcerative colitis
Sulphate-reducing bacteria (SRB) have a significant
economic relevance in the oil industry since the corrosive
nature of their metabolic end products (sulphides) has a
debilitating effect on pipelines, metal tanks, etc. They have
been most frequently isolated from anaerobic marine
sediments where a plentiful supply of sulphate exists in
seawater. Bacterial dissimilatory sulphate reduction
comprises the main route of organic carbon turnover in such
anaerobic ecosystems (Gibson 1990; Barton 1995).
2-3
It has also been established that SRB are important
inhabitants of the human large intestine (Gibson et al. 1990,
1993; Willis et al. 1996). In healthy individuals, they
compete with methanogens and acetogens for gaseous
hydrogen produced during the fermentation process (Gibson
et al. 1988a, 1998b, 1998c). In this context, they help to
reduce gas distension in the gut (Gibson et al. 1990).
However, their end product (hydrogen sulphide) is both
odiferous and extremely toxic. The main genus present is
Desulfovibrio which is a Gram negative, highly motile rod
found in the faecal specimens of about 50% of the healthy
adult population (Willis et al. 1997). They are also
recoverable from the oral cavity (Willis et al. 1995, 1999).
Importantly, sufferers of the chronic intestinal disease
ulcerative colitis (UC) have a universal carriage of
desulfovibrios (Gibson et al. 1991). UC is a severe chronic
inflammatory disease of the human large intestine and is
almost exclusively found in western populations, where it
affects about 10 in 100 000 persons. Onset most frequently
occurs between the ages of 20 and 35 years; although the
disorder cannot be cured by current drug therapy, it can be
managed through the use of anti-inflammatory
pharmaceutical products. Symptoms of UC include frequent
passage of watery stools containing blood, together with
intestinal pain. In the severest cases, surgery is required.
The aetiological factor leading to inflammation of the
large intestine has never been elucidated. Implications from
studies with germ-free animals and the fact that the disease
is confined to the colon (which is the most heavily
colonised region of the human body) has led to the
assumption that bacteria are involved (Levitt et al. 1995;
Campiere and Gionchetti 2001).
The feeding of sulphated polymers to conventional
laboratory animals results in colitis-like lesions; this is not
the case for germ-free counterparts, indicating that specific
microbial populations able to ferment such polymers are
required (Marcus and Watt 1969; Araki et al. 2001; Hans et
al. 2001). The hypothesis is that sulphatase-producing gut
bacteria, such as the bacteroides, are able to liberate
sulphate in a free form. This is then taken up by
dissimilatory SRB like Desulfovibrio spp. which produce
toxic sulphides.
Isolates of SRB from a colitic gut generate much higher
quantities of sulphides in vitro than equivalent strains taken
from a healthy colon. Moreover, in UC, desulfovibrios are
able to adapt to certain clinical manifestations of the disease
such as a rapid transit of gut contents and low substrate
availability (Gibson et al. 1991). Further evidence has also
shown that the bacteria are able to attach to, and then
invade, colonocytes, thereby causing extensive cell
destruction (Willis 1997).
It is, therefore, a sound hypothesis that SRB may be
aetiological 'triggers' for UC in humans (Jorgensen and
Mortensen 2001). Determination of a specific causative
agent has great medical, social and scientific interest, and
current available evidence indicates that such sulphate
reducers have a predominant role. The principle species
2-4 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall
resident in the human gut is Desulfovibrio desulfuricans.
3.5 Crohn's disease
Crohn's disease (CD) is a second predominant form of
inflammatory bowel disease. In contrast to UC, which is
confined to the large intestine, CD can affect any area of the
digestive tract from mouth to anus (van Heel et al. 2001).
Because CD can affect areas of the gut that are not heavily
colonised by bacteria, the evidence for a microbial aetiology
is less convincing than for UC. However, where the
bacterial count is low, evidence has accumulated for species
of mycobacteria as specific causative agents in children and
adults (Chiodini et al. 1984; Sutton et al. 2000; Hulten et al.
2001).
3.6 Pseudomembranous colitis
This is caused by the organism Clostridium difficile. Under
normal circumstances, C. difficile is repressed by the
activities of the normal gut flora. However, upon exposure
to antibiotics this suppressant activity is compromised.
Almost all antibiotics have been postulated to affect this
suppression, although those most frequently implicated in
antibiotic associated diarrhoea are ampicilllin,
cephalosporin and clindamycin (Bartlett et al. 1978;
McFarland 1998). After the barrier effects of the gut flora
are lost, C. difficile is able to produce two types of toxin.
These then contribute towards formation of a
pseudomembrane composed of mucin, fibrin and epithelial
cells, which can eventually occlude the gut lumen (Bartlett
1983; McFarland et al. 1999). Conventional therapy is
administered through antibiotics (e.g. vancomycin) directed
against C. difficile, and resins which bind the toxins.
3.7 Others
3.7.1 Pneumatosis cystoides intestinalis
This is a rare disorder affecting gas metabolism which is
characterised by gas-loaded cysts in the bowel wall. It is
thought to be due to an absence of bacteria capable of
metabolising hydrogen, which is produced during the
fermentation process (Christl et al. 1992, 1993).
3.7.2 Food allergy/intolerance
The gut is the first point of contact for all foods.
Microorganisms are able to metabolise many xenobiotic
compounds, although if this is compromised, food
intolerance or even food allergy may result (Apostolou et al.
2001).
3.7.3 Translocation
Under conditions of extreme trauma such as intensive
surgery, bacteria may translocate (migrate) from the gut
which is made more 'leaky' by the extreme stress induced in
the patient (Hartung et al. 1997).
4. Functions of the normal human gut microbiota
The bacterial microbiota in the human large intestine is
thought to comprise around 95% of the total cells in the
body, representing 1012 cells/g dry weight faeces. The vast
majority of these bacteria reside in the large intestine, where
slow transit time, availability of nutrients and pH are
favourable for microbial growth. Through the activities of
the resident microflora, the colon plays a major role in host
nutrition and welfare (Cummings and Macfarlane 1991).
Dietary modulation of the human gut flora can be of great
benefit to health and, in recent years, the functional food
concept has moved away from mineral and vitamin
supplementation towards a situation whereby improved gut
function is a major driving force (Gibson and Williams
2000).
The gastrointestinal tract is a sterile environment at birth
and bacterial colonisation begins during parturition (from
the maternal faecal or vaginal flora and/or the environment).
The first bacteria colonising the colon are facultatively
anaerobic strains such as Escherichia coli and streptococci.
These first colonisers metabolise any traces of oxygen in the
gut, thereby reducing the environment to one that is strongly
anaerobic. The bacterial species that subsequently colonise
the gut are largely depend upon the feeding profile of the
infant. Breast-fed infants develop a different gut flora to
those fed on formula feeds. In the breast-fed infant,
bifidobacteria are the predominant genera, whereas in
infants given formula feeds, a more complex, adult-like gut
flora results, with clostridia, bacteroides, bifidobacteria and
streptococci being present in large numbers (Stark and Lee
1982; Yuhara et al. 1983; Benno et al. 1984; Mevissen-
Verhage et al. 1987; Yoshioka et al. 1991; Langhendries et
al. 1995; Kleessen et al. 1995; Harmsen et al. 2000). A
major reason for these differences is that breast milk
contains a 'bifidus' factor, a glycoprotein containing
glucose, galactose, fructose and N-acetyl glucosamine
which stimulates growth of bifidobacteria (Cooperstock and
Zedd 1983). Breast-fed infants generally have fewer
gastrointestinal problems than their formula-fed
counterparts and this may be attributed to the powerful anti-
pathogen effects exerted by bifidobacteria (see later). The
final phase of microflora acquisition occurs at weaning
when a complex microflora develops.
The resident gut microbiota ferment substances that
cannot be digested by the host in the small gut, including
resistant starch, non-digestible carbohydrates,
oligosaccharides, proteins, and mucins (Gibson and
Macfarlane 1995). The two main types of fermentation that
are carried out in the gut are saccharolytic and proteolytic.
Saccharolytic fermentation is more favourable than
proteolytic fermentation due to the end products that are
formed, with saccharolytic fermentation forming mainly
short chain fatty acids such as acetate, propionate and
butyrate. All such elements contribute towards the host's
daily energy requirements. Acetate is metabolised in
systemic areas such as muscle, while propionate is
transported to the liver and used to generate ATP. Butyrate
 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall
is an important source of energy for colonocytes and has
anti tumour properties (Cummings et al. 1990; Demigne et
al. 1999). Conversely, the end products of proteolytic
fermentation include nitrogenous metabolites (e.g. phenolic
compounds, amines and ammonia), some of which are
carcinogens. The proximal gut (right side) is essentially a
site of saccharolytic fermentation, whereas the more distal
portion of the gut (left side) is an area in which proteolytic
fermentation occurs to a greater extent than in the proximal
bowel (Macfarlane et al. 1992). This is probably one reason
why many gastrointestinal disorders (including colon
cancer, ulcerative colitis) occur distally.
Principal genera of gut bacteria include Bacteroides,
Bifidobacterium, Clostridium, Fusobacterium,
Eubacterium, Lactobacillus, Peptococcus, anaerobic
Streptococcus, Peptostreptococcus, Ruminococcus,
Veillonella, enterococci, coliforms, methanogens,
dissimilatory sulphate-reducing bacteria and acetogens.
Depending on their physiological capabilities, it is possible
to categorise components of the gut microbiota on the basis
of whether they exert potentially pathogenic or health
promoting aspects. For example, lactic acid producing
genera such as bifidobacteria or lactobacilli have a long
standing 'health image' and belong to the latter category
(Gibson and Roberfroid 1995; Kaptan 2000). As the gut
flora composition responds primarily to the supply of
substrate for its growth, it makes sense that such bacterial
populations are the principal targets for dietary modulation.
Thus, development of both probiotics and prebiotics relies
on fortification of the lactate flora of the human or animal
gut (Hamilton-Miller 2001; Mahoney et al. 2001; Reid et al.
2001).
5. Probiotics and prebiotics
The most frequently used dietary method of influencing the
gut flora composition is that of probiotics, in which live
microbial additions are made to appropriate food vehicles
(Fuller 1989, 1992, 1997; Fuller and Gibson 1997; Gibson
and Angus 2000; Naidu et al. 1999; Tannock 1999; Ziemer
and Gibson 1998). The first recorded intake of deliberate
bacterial consumption goes back over 2000 years. However,
the concept was given a scientific basis by the work of
Metchnikoff at the Pasteur Institute (Metchnikof 1907). He
hypothesised that longevity in Bulgarian peasants was
associated with their elevated intake of 'soured milks', i.e.
dairy based drinks containing live bacteria, now recognised
as probiotics. Some selection criteria for probiotics are
shown in Table 2.
Important findings that helped the development of
probiotics included the observation that faeces contained
'protective' factors that could help promote resistance to
infections by bacteria such as salmonellae and C. difficile
(Gibson and Fuller 1998). Such protection could not be
replicated in germ free animals. One of the most popular
definitions of probiotics is that of Fuller (1989), who stated
that a probiotic was 'a live microbial feed supplement which
2-5
Table 2. Common selection criteria for probiotics
Exertion of a beneficial effect on the consumer
Non-pathogenic and non-toxic (including an inability to transfer
antimicrobial resistance)
Good viability
Ability to survive in the gastrointestinal tract
Acceptable sensory properties
Genetic stability under industrial conditions
Long shelf-life and ability to remain viable during storage and use
beneficially affects the host animal by improving its
intestinal microbial balance.' A recent formal definition of
probiotics was agreed by a working party of European
scientists and is given as 'a live microbial feed supplement
that is beneficial to health' (Salminen et al. 1998). This
emphasised the importance of definitive improvements in
health as well as the possibility that probiotics could have
systemic effects outside the gut (e.g. vagina, skin and
mouth).
Over the years, many species of microorganisms have
been used in the development of probiotics. They consist
mainly of lactic acid bacteria (lactobacilli, streptococci,
enterococci, lactococci, bifidobacteria) but also Bacillus
spp. and fungi such as Saccharomyces spp. and Aspergillus
spp. The commonest probiotics belong to the genera
Lactobacillus (e.g. L. casei, L. acidophilus, L. rhamnosus,
L. johnsonii and L. reuteri) and Bifidobacterium (e.g. B.
bifidum, B. longum and B. breve). New probiotic strains are
also constantly being isolated (Hyoung et al. 2001;
O'Sullivan 2001; Savova et al. 1999).
Probiotic trials should use the best methodologies
available although difficulties lie in the recovery of fed
strains from faeces. In addition, it appears that a significant
proportion of the human gut microbiota is 'non-culturable.'
As such, there has been a recent move away from cultural
procedures coupled with phenotypic assessments of culture
identity towards more sophisticated molecular procedures
(Harmsen et al. 1999; Satokari et al. 2001). These include
nucleic acid fingerprinting studies for reliable identification,
as well as development of genetic probing systems, so that
predominant components of the gut flora can be quantified
in a culture independent manner (Kleessen et al. 2001;
Tuohy et al. 2001a, 2001b).
An alternative, or additional, approach is the prebiotic
concept. This term was first coined in the mid 1990s and
takes the view that, since probiotic organisms are
indigenous to the gut, a rational approach towards
increasing their numbers would be to supply non-viable
food ingredients (e.g. carbohydrates) that are selectively
metabolised in the lower gut (Gibson and Roberfroid 1995,
1999). A prebiotic can be described as ‘a non-digestible
food ingredient that beneficially affects the host by
selectively stimulating the growth and/or activity of one or a
limited number of bacteria in the colon, and can improve
host health.’ Thus, the prebiotic approach advocates the
2-6 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall
administration of non-viable entities (Crittenden 1999;
Playne and Crittenden 1996). To be an effective prebiotic, a
compound should satisfy the following requirements:
• It should not be hydrolysed or absorbed in the upper
part of the gastrointestinal tract
• It should be selectively fermented such that the
composition of the intestinal microbiota is altered to
become more beneficial to health.
As the majority of bacteria resident in the gut microbiota are
present in the colon, prebiotics are usually directed towards
lower gut bacterial populations. The first stage in the
development of a new prebiotic is to make sure that it
reaches its target intact. Pure culture work can then
determine whether probiotic bacteria can ferment these
compounds. More detailed fermentation work such as
mixed batch culture and continuous culture using chemostat
or 'gut models' is then performed to identify the selectivity
of the substrate (Macfarlane et al. 1998). If the compound
shows a prebiotic effect in vitro, it must be confirmed in
vivo, either using animal models or in human clinical trials.
Modern molecular techniques are now being employed in
the validation of prebiotics. The use of such methods gives
a much clearer picture of the gut microbiota and the effects
of prebiotics. As mentioned previously, traditional methods
of bacterial enumeration such as the use of selective agars
are being replaced with molecular techniques directed
towards determing prebiotic functionality. One such method
is fluorescent in situ hybridisation (FISH), using specific
rRNA probes (Harmsen et al. 1999). Other methods include
denaturing gradient gel electrophoresis (DGGE),
temperature gradient gel electrophoresis, ribotyping, and
rRNA community analysis. Such methods enable analysis
of faecal microflora at least to the genus level (Collins and
Gibson 1999; McCartney et al. 1996; O'Sullivan 1999;
Vaughan et al. 1999; Wilson and Blitchington 1996; Suau et
al. 1999).
Dietary carbohydrates, such as fibre, are candidate
prebiotics; however, oligosaccharides are the most
promising compounds. In particular, the ingestion of
fructooligosaccharides has been shown to stimulate
bifidobacteria in the lower gut. As prebiotics exploit non-
viable food ingredients, their applicability in diets is wide
ranging. Virtually any food that already has a carbohydrate
content is suitable for prebiotic fortification; examples
include beverages, fermented milks, health/sports drinks,
infant formulas, weaning foods, cereals, bread,
confectionery, cakes, biscuits, spreads, sauces, pastries,
savoury products and snack bars. It is likely that, given the
ease of use of prebiotics and their proven efficacy at gut
flora modulation, many new products in these sectors will
arise in the forthcoming years.
A further approach is the use of synbiotics, in which
probiotics and prebiotics are combined (Gibson and
Roberfroid 1995). Here, survival of the live microbial
addition in the gastrointestinal tract would be enhanced by
coupling it with a selective growth substrate.
The prebiotic activity of fructose-containing
oligosaccharides has been confirmed in both laboratory and
human trials (Bouhnik et al. 1996; Buddington et al. 1996;
Djouzi and Andrieux 1997; Gibson et al. 1995a; Gibson and
Wang 1994a, 1994b, 1994c; Kleessen et al. 1997, 2001;
Wang and Gibson 1993; Williams et al. 1994). This is
because such carbohydrates have a specific colonic
fermentation directed towards bifidobacteria - recognised to
have a number of health promoting properties (Kaptan
2000). Bifidobacteria are able to break down and utilise
fructooligosaccharides due to their possession of a β-
fructofuranosidase enzyme, providing a competitive
advantage in a mixed microbial culture environment like the
human gut (Imamura et al. 1994). Whilst many bacteria are
able to metabolise fructooligosaccharides in pure cultures,
bifidobacteria seem to have a selective advantage in more
complex ecosystems. Both the inulin type
fructooligosaccharides and the smaller oligomer versions
(oligofructose) are efficient prebiotics.
Galactooligosaccharides (GOS) are another class of
prebiotics that are manufactured and marketed in Europe as
well as in Japan. These consist of a lactose core with one or
more galactosyl residues linked via ß1→3, ß1→4 and
ß1→6 bonds (Ito et al. 1990, 1993; Teuri et al. 1998; Gopal
et al. 2001). They have found application in infant formulas
as they are naturally present in human milk.
A different class of oligosaccharides used as prebiotics in
Japan are those isolated from soybean whey. These soybean
oligosaccharides (SOS) are composed of galactosyl residues
joined via α1→6 linkages to a sucrose core (Hayawaka et
al. 1990; Wada et al. 1992).
Glucooligosaccharides can also act as prebiotics (Djouzi
et al. 1995; Valette et al. 1993). Isomaltooligosaccharides
(IMO) are comprised of glucosyl residues linked by α1→6
bonds (Kaneko et al. 1994; Olano-Martin et al. 2000) and
are only partially prebiotic since they are metabolised by
humans. They are, however, very slowly metabolised and
most isomaltooligosaccharides in the diet would pass
through to the colon.
Xylooligosaccharides (XOS) are also used as prebiotics in
Japan (Okazaki et al. 1990). These consist of xylosyl
residues linked by ß1→4 linkages and are much more acid
stable than other prebiotics. For this reason, they have found
application in soft drinks, which tend to be acidic.
Finally, our own studies at the University of Reading
have demonstrated the prebiotic nature of gentio-
oligosaccharides (Rycroft et al. 2001). In addition, further
studies will no doubt yield efficacious prebiotics that may
be derived through enzymatic generation and/or hydrolysis
and will include 'designer forms' that have multiple
biological activities (Rastall 2001).
In Europe, three carbohydrates have been proven to be
prebiotics in that they have been tested in human trials using
robust methodologies, and elicit a selective increase in
bifidobacteria and/or lactobacilli (Gibson et al. 2000).
 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall
These are the fructooligosaccharides,
galactooligosaccharides and lactulose, which is a synthetic
disaccharide manufactured from lactose (Ballongue et al.
1997; Terada et al. 1992). In Japan, there is a longer list of
accepted prebiotics and these are now also being tested for
the European market. Examples are the oligosaccharides
mentioned above as well as lactosucrose (Fujita et al. 1995;
Terada et al. 1993).
6. Examples of foodstuffs containing probiotics and
prebiotics
The incorporation of probiotics, prebiotics and synbiotics
into foods is a fast moving area. Many novel formulations
are about to be launched and the functional food market has
recently seen a large impetus towards gut microflora
modulation. Many different food vehicles exist, the most
popular being fermented milks, although tablets, sprays,
pills, capsules and liquid suspensions are all used (Klont
and Mannion 2000; Salminen 2001). The Japanese
situation, not surprisingly, is more advanced than elsewhere
but the progression in Europe has also been quick,
especially in Scandinavia where extremely encouraging data
for protection against childhood infection and atophy have
arisen (Isolauri et al. 1999).
There are now many examples of foods that contain both
probiotics and prebiotics.
6.1 Live yoghurts
Also called bio, active and bifidus. Manufacturers such as
Nestlé, St. Ivel, Danone, Onken and Vifit, include
probiotics in yoghurt. Similarly, supermarkets also have
their own versions. In order to be a probiotic form, such
products should differ from conventional yoghurt in that
strains are added, or used in manufacture, in addition to
standard starter species such as Lactobacillus delbrueckii
subsp. bulgaricus, or Streptococcus thermophilus.
6.2 Fermented dairy drinks
These liquid products allow incorporation of high microbial
numbers (up to 109 per ml). Examples are Yakult, Danone's
Actimel, Muller's Vitality, St. Ivel's Shape drink and
Nestlé's LC1Go.
6.3 Freeze-dried supplements
Freeze drying of microbial cultures allows extremely high
numbers to be incorporated into capsules, pills, sprays, etc.
Examples in the UK are Multibionta (for humans - this
contains Lactobacillus acidophilus, Bifidobacterium
bifidum and Bifidobacterium longum as well as the RDA of
all vitamins and an 'enteric coating' system that guarantees
delivery to the lower bowel) and Protexin (for animals -
containing a total of seven lactic acid producing bacteria).
Several other products exist, especially in the USA, where
yeast as well as bacteria are popular probiotics.
2-7
6.4 Cheese
An example of a probiotic cheese is Gefilus which contains
Lactobacillus GG.
6.5 Squeezable yoghurt
Available in tubes, e.g. Yo Squeez for children contains six
different types of probiotic organism.
6.6 Fromage frais
Some products contain live bifidobacteria and/or lactobacilli
e.g. Yoplait. New developments also exploit prebiotics.
6.7 Sweet chews
'Probiotica' lemon flavoured chewable supplement contains
Lactobacillus reuteri.
6.8 Fruit juice
A widely used form is Proviva which contains Lactobacillus
plantarum 299V.
6.9 Infant weaning foods
In an attempt to more closely mimic some of the effects of
breast milk, manufacturers are altering the form of their
products to include probiotics (e.g. a bifidobacterium called
Bb12 is popular in Asia) or adding oligosaccharide
prebiotics. These products aim to exert typical probiotic
effects such as enhanced resistance to gut pathogenesis, as
well as improving transit time and consistency of stools.
6.10 Jelly
In Japan, probiotics are added to many different foods
including yoghurt flavoured jelly.
6.11 Fructooligosaccharides (FOS)
FOS are obtainable in powdered form and can be
manufactured from sucrose (e.g. Actilight, Neosugar) or
extracted as inulin from chicory roots (Raftiline, Raftilose,
Synergy). They are available as supplements or can be
added to an increasing number of foods. The Belgian
company Orafti has been responsible for many innovative
novel food ingredients based around a chicory root extract.
One product, Aviva (from Novartis), is available as either
biscuits or a powdered chocolate drink and delivers about
8g/day FOS to the gut where it stimulates probiotic
bifidobacteria.
6.12 Other prebiotics
These may include lactulose, trans galactooligosaccharides
(both common in Europe), xylooligosaccharides,
maltooligosaccharides, soybean oligosaccharides,
lactosucrose and glucooligosaccharides. A wide variety of
foods (including biscuits, breads, ice cream, vinegar,
2-8 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall
chewing gum, snack bars, chocolate, soft drinks, salad
dressing, cereals, health drinks, weaning foods, and sauces)
have all been fortified for the Japanese market. Designer
forms which offer multiple biological effects are now being
manufactured and tested (Gibson et al. 2000).
6.13 Synbiotics
Examples of these products, which include both prebiotics
and probiotics, include yoghurts such as B2, Actilife,
Symbalance and Lacto Pro.
Probiotics and prebiotics are also used in animal feeds for
domestic pets such as cats and dogs. In addition, they are
available for non-oral use i.e. as suppositories, panty liners,
hospital tube feeds, creams and sprays.
The commercial success of probiotics and prebiotics is
large (the former alone is thought to generate over 1 billion
Euro per annum in Europe). It is crucial, however, that such
success should be sustained through independent scientific
validation which will underpin any health benefit with
realistic and proven mechanisms of effect. Moreover, if one
particular probiotic or prebiotic is seen to elicit a positive
effect on health then this cannot necessarily be transposed
to all other products. The most successful products will
have a wealth of scientific data to support any claims that
are made, and such claims should only be levelled against
the product tested, not those which are 'similar' in nature.
Some considerations are as follows:
• Modern and reliable technologies such as post
genomic approaches should be used for identifying
microbial changes as well as individual genotypes
(Vaughan et al. 1999).
• Well controlled in vivo trials should be carried out in
a blind coded fashion, with an appropriate placebo.
• Replication of effects should be sought in more than
one study centre, with reporting of results taking
place in stringent peer reviewed publications.
• The realistic health outcomes should be identified,
with mechanistic explanations being formulated for
any effects that may occur.
• Accurate labelling should be carried out; some
product labels do not accurately reflect the
composition and numbers of the probiotics contained
therein, while some manufacturers do not describe
their 'probiotic' contents. For prebiotics, the active
ingredient often contains impurities that should not
be present in the most efficacious products.
7. Mechanisms of effects of probiotics and
prebiotics on gastrointestinal pathogens
Evidence suggests that increased levels of probiotics in the
human gut, whether encouraged through use of prebiotics or
live microbes in the diet, can have a variety of positive
influences (Fooks et al. 1999; Lee et al. 1999; Mattila-
Sandholm et al. 1999; Salminen and Playne 2001;
Simmering and Blaut 2001; Wanke 2001; Zubillaga et al.
2001). These include a reduction in serum cholesterol,
improved hormonal status, vitamin synthesis, protection
from bladder cancer, treatment of recurrent thrush and
prevention of liver failure (which can often lead to
encephalopathy). These benefits have been tested in a
laboratory environment or using animal models, although
the applicability of such results to humans is still unproven.
Nevertheless, the area of gut flora modulation holds great
promise in tackling gastrointestinal pathogenesis ascribed to
microbial aetiologies (Andersson et al. 2001; de Groote
2000). Using this approach, the acute and chronic disorders
mentioned earlier may be prevented or managed.
Research investigating the link between probiotics,
prebiotics and gastrointestinal pathogenesis is progressing at
a rapid pace and, because robust tools are now in place to
monitor the gut flora during health and disease, such
research is increasingly being more mechanistically driven
(Vaughan et al. 1999). A number of plausible mechanisms
in which both probiotics and prebiotics can resist
pathogenic activity are being identified (Isolauri et al.
1999). These are outlined below.
7.1 Direct inhibition of pathogens
There are a number of end-products that probiotics excrete
that can inhibit pathogens. The most obvious of these are
strong acids such as acetate and lactate. However, there is
also evidence that some species of lactobacilli,
bifidobacteria, lactococci and pediococci are able to
produce antimicrobial compounds with powerful effects that
are separate from acid formation (Anand et al. 1985; Araya-
Kojima et al. 1995; Fujiwara et al. 1987; Gibson and Wang
1994b; Kim et al. 2000; Ko and Ahn 2000; Kot et al. 2001;
Tellez et al. 2001). Recent evidence has shown that
Bifidobacterium infantis is an especially potent inhibitor of
E. coli O157 and Salmonella typhimurium, leading towards
use of the probiotic in infant weaning foods (Yusof et al.
2001). Lactic acid bacteria are also used as biopreservatives
because of their wide spectrum of antimicrobial actions
(Fragoso-Sousa and Fernandez-Riusech 2000). It has also
been suggested that certain plant derived substances,
including prebiotics, have antagonistic activities against gut
pathogens (Jeon et al. 2001; Lee et al. 2000a).
7.2 Immunomodulation
All bacteria are antigenic and therefore capable of
producing an immune response. Antibody generation is
conventionally targeted against the original stimulating
strain. However, the stimulation of a non specific immune
response increases cytokine activity and the levels of
circulating T cells (Perdigon and Alverez 1992); this in turn
may have potent effects upon pathogens. The immune
effects of probiotics have been reviewed by Wold (2001).
The attractiveness of determining the immomodulatory
effects of ingested living organisms, or the prebiotics that
 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall 2-9

Figure 1. Outline of microbial pathogenesis in the human gut. Bacterial pathogens need to persist for long enough in the gut
to cause damage. This is usually achieved by attachment to specific receptor sites on the gut wall, through bacterial
fimbriae. Commonly, the attachment sites are oligosaccharides. The microbe(s) may then excrete toxin(s) which can also
attach onto the mucosal layer. The bacterium may then invade gut cells and/or the toxin may interfere with normal cellular
function. The outcome is cell death and an inflammatory response.

Figure 2. The concept of decoy oligosaccharides. Decoy oligosaccharides mimic common oligosaccharide receptor sites on
the gut wall. Instead of attachment to the mucosal surface, the bacteria or toxins attach to the decoy molecules, leaving the
gut cells free of infection.
2 - 10 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall

Figure 3. Conceptual diagram to demonstrate how prebiotic and anti-adhesive linkages may be combined within the same
molecule. Such a product could be termed a designer probiotic which offers multiple biological activities.

induce them, is that the molecules are quantifiable and are 7.5 Attenuation
therefore useful biomarkers of effect (Kitazawa et al. 2001).
Experiments in animals (Pestka et al. 2001), children The prebiotic concept may be extrapolated further by
(Hatakka et al. 2001) and the elderly (Gill and Rutherford considering an attenuation of virulence in certain foodborne
2001) have recently been reported. Inactivated probiotics pathogens. For example, the plant-derived carbohydrate
are also capable of eliciting an immune response cellobiose is able to suppress the pathogenicity of Listeria
(Ouwehand et al. 2000). monocytogenes by down-regulation of its virulence factors
(Park and Kroll 1993). This organism is avirulent in its
natural habitat of soil, where it is exposed to rotting
7.3 Competition
vegetation derived from cellobiose. In the human body, an
The large intestine receives a ready supply of substrates for absence of cellobiose may allow the virulence factors to be
bacterial growth from dietary residues as well as from expressed, and it is possible that further incorporation of
indigenous sources (Gibson and Roberfroid 1999). On this disaccharide to foods susceptible to listerial infection
occasion, these may only be available in limited quantities. could reduce this virulence.
Elevated numbers of probiotics in the gut may help to
repress pathogen survival by effective competition for Table 3. Examples of bacteria which use
growth substrates. Many probiotics have also been shown to oligosaccharide receptors
attach to intestinal cells (Blum et al. 1999; Forestier et al. Actinomyces naeslundii Neisseria gonorrhoea
2001; Haller et al. 2001; Kankaanpaa et al. 2001; Lee et al.
Bordetella pertussis Propionibacterium granulosum
2000b; Ouwehand et al. 2001; Todoriki et al. 2001;
Tuomola et al. 2000), which may help to 'block' Chlamydia pneumoniae Proteus mirabilis
colonisation sites that may otherwise be occupied by Citrobacter freundii Pseudomonas aeruginosa
pathogens. Enterobacter aerogenes Salmonella Typhimurium
Escherichia coli Serratia marcescens
7.4 Anti-adhesive prebiotics
Haemophilus influenzae Shigella dysenteriae
Many pathogens utilise oligosaccharide receptor sites as
attachment sites prior to elaboration of a virulence Haemophilus parainfluenzae Staphylococcus aureus
mechanism (Table 3). Oligosaccharides in foods that mimic Helicobacter pylori Streptococcus mutans
these receptor sites offer the possibility of 'decoying' the Mycobacterium tuberculosis Streptococcus pneumoniae
pathogen away from the gut wall. This is explained
Mycoplasma pneumoniae Vibrio cholerae
conceptually in Figures 1-3. Such molecules are currently
being synthesised at the University of Reading, where they
are also being combined with other compounds which have
recognised prebiotic traits.
 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall
8. Conclusions
The area of functional foods concerned with the gut
microbiota and its activities are currently of huge market
and biological interest. In recent years, reliable molecular
tools have been produced to help determine and quantify the
gut flora in response to dietary intervention. This has helped
to underpin potential health effects of probiotics and
prebiotics with mechanistic explanations, producing a
degree of scientific rigour that allows success to be
sustained. With the increasing cost of medicinal expenses
and the fact that many therapies for gut problems are
ineffective, the area is now moving into one of medical
application, as well as prophylactic aspects. The latter will
continue, especially as gut diseases are often unpredictable.
For the former to be realised, it is important that scientific
principles rather than commercial gain drives progress.
Although at present the medical profession is not
completely supportive of probiotic and prebiotic concepts,
an ongoing critical mass of expertise and rational
assessments of functionality may mean that this standpoint
may change in future.
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hybridisation (FISH). Anaerobe- in press.
Tuohy, K.M., Kolida, S., Lustenberger, A. and Gibson, G.R.
2001b.The prebiotic effects of biscuits containing partially
hydrolyzed guar gum and fructooligosaccharides - A human
volunteer study. British Journal of Nutrition - in press.
Tuomola, E.M., Ouwehand, A.C. and Salminen, S.J. 2000.
Chemical, physical and enzymatic pre-treatments of probiotic
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75-81.
Valette, P., Pelenc,V., Djouzi, Z., Andrieux, C., Paul, F., Monsan,
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Molecular approaches to study probiotic bacteria. Trends in
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2 - 16 Gastrointestinal infections and the protective role of probiotics and prebiotics G.R. Gibson and R.A. Rastall

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Functional foods and bowel cancer

Ian T Johnson
Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK

Abstract

Colorectal carcinoma is the second or third most frequent cause of death from cancer in western industrialised
societies, but it is rare in many developing countries. Though diet may cause up to 80% of colorectal cancers in
the west, the precise risk factors and mechanisms of action are unknown. Designing functional foods to reduce
the risk of colorectal cancer is difficult, but the protective effects of diets rich in plant foods, and the likely
importance of the colonic microflora in pathogenesis of the disease, suggest fruitful strategies. Although
definitive proof of the benefits is difficult to establish, epidemiological evidence continues to support increased
consumption of dietary fibre. Recent studies on butyrate will encourage the development of new functional
food products. The use of live bacterial cultures (probiotics) and selective fermentable substrates (prebiotics) is
another approach to the manipulation of the colonic environment; the benefits of these formulations in relation
to colorectal cancer are unproven, although several possible mechanisms of action are now under investigation.
Finally, foodborne anticarcinogens, including phytochemicals and certain polyunsaturated fatty acids, may have
benefits that could be exploited in functional products. New research tools, biomarkers and advances in
epithelial cell biology will underpin the development of functional foods in the near future. However, any new
product must undergo a thorough assessment of both benefits and possible risks before it can be marketed.

Keywords: bowel cancer, dietary fibre, butyrate, phytochemicals, PUFA, probiotics, prebiotics

1. Introduction 2. The biology of cancer

There were 28 900 cases of colorectal cancer diagnosed in
the UK in 1997, a statistic that makes this disease the
second most common cancer in British women, and the
third most common in men (Quinn et al. 2001). In their
classic epidemiological survey of cancer and its causes,
Doll and Peto (1981) concluded that as many as 35% of all
human cancers were caused by adverse effects of diet.
However, the statistical uncertainty associated with this
estimate was very high, and the authors were not able to
offer any detailed explanation of the mechanisms involved.
More recent reviews have reached similar conclusions (Doll
1996; Willett 2001). Cancer is an immensely complex
disease, reflecting multiple failures in mechanisms
governing cell proliferation, differentiation and death.
Recent discoveries in cell and molecular biology, coupled
with powerful new genomic techniques, are providing
insights into the mechanisms of carcinogenesis. These
advances promise to shed new light on the role of diet in the
disease process. In this article, the biology of cancer will be
briefly discussed, followed by an exploration of the
evidence for diet as a risk factor for colorectal carcinoma.
Some assessment of the potential role of functional foods in
the prevention of the disease will also be given.
Food Science and Technology Bulletin: Functional Foods 1 (3) 1–10
DOI: 10.1616/1476-2137.11615. Published 22 January 2003
ISSN 1476-2134 © IFIS Publishing 2003. All Rights Reserved
Any focal accumulation of cells beyond the numbers
required for the development, repair or function of a tissue
is termed a tumour. The cells of complex organisms differ
enormously in size, functional specialisation and rate of
division, yet all carry an identical set of genetic
instructions. The subset of genes that are actually expressed
by any given cell type at any particular time determines the
phenotype – those characteristics that enable it to exist in
harmony with other cells within a tissue. The transition
from a normal tissue to a tumour is thought to begin with
genetic damage affecting one or more genes controlling the
rate at which cells divide, differentiate or die, or the
efficiency with which DNA damage is repaired (Anderson
et al. 1991). The precise nature of these genetic
abnormalities differs from one form of cancer to another,
and often between tumours of the same general type. The
crucial factor is that they confer a selective advantage on the
cell and its progeny, enabling them to survive and grow
more rapidly than normal neighbouring cells (Bodmer 1994;
Bodmer and Tomlinson 1996). Mutations of this type that
are inherited though the germ-line are known to contribute
to a number of familial cancer syndromes, including about
10–20% of colorectal cancer cases (Lynch and Lynch
1998). However, most of the genetic abnormalities
detectable in the more common, sporadic forms of cancer
are somatic mutations acquired during adulthood (Fearon
and Vogelstein 1990).
3-2 Functional foods and bowel cancer I.T. Johnson
Colorectal cancer develops through a well-defined
sequence of pathological stages, the so-called adenoma-
carcinoma sequence (Hill et al. 1978). Adenomatous
polyps are commonly found in the colons of middle-aged
and elderly individuals in populations that have a high
incidence of colorectal carcinoma. A small proportion of
these benign lesions make the transition to malignancy, and
early screening and removal of polyps is a valuable
preventative strategy. By studying polyps at all stages of
development, it has been possible to identify the crucial
genes that are associated with the disease process (Fearon
and Vogelstein 1990). Germline mutations of the APC gene
are the cause of familial adenomatous polyposis, a
congenital condition causing hundreds of polyps to form in
the colon during childhood, leading inevitably to cancer in
early adulthood (Fearnhead et al. 2001). Over 80% of
sporadic colorectal tumours carry somatic APC mutations,
which are amongst the earliest mutations to appear (Powell
et al. 1992), and are thought to cause an early deregulation
of normal cell turnover in the crypt. One example is the K-
ras gene, which codes for a protein regulating cell
proliferation, and is mutated and hence abnormally
expressed early in the development of approximately 40%
of human colorectal carcinomas (Bos et al. 1987). Another
example is the p53 gene (Donehower and Bradley 1993),
normal expression of which gives rise to a protein that
functions as a regulator of cell proliferation, and as a
mediator of programmed cell death or apoptosis. This is
one of the most important of the body’s defence
mechanisms against precancerous cells. The absence of
normal p53 allows cells bearing other mutations to continue
dividing and avoid apoptosis.
3. Diet as a risk factor
Although epidemiological evidence for the role of diet in
the pathogenesis of colorectal carcinoma is incontrovertible,
the underlying mechanisms remain poorly defined. There is
evidence for a relatively weak adverse effect of meat
consumption, and more particularly processed meat
products (Norat and Riboli 2001). Of more relevance in the
context of functional foods is the strong protective effect of
diets rich in vegetables (Riboli and Norat 2001) and dietary
fibre (Bingham 1996; Bingham 2000). However, no
conclusive case can yet be made for any particular food-
borne carcinogens on the one hand, nor for any single
protective factor derived from plant foods on the other. In
these circumstances, it is no simple matter to design
functional foods for the prevention of bowel cancer. There
are, however, promising new lines of evidence, relating to
the impact of food constituents and bacterial metabolites on
epithelial cell physiology, which seem likely to strengthen
existing dietary advice and open up entirely new strategies
over the next few years.
4. Functional foods - modifying the intraluminal
environment
Previously, what could be called 'classical' nutritional
science has been concerned mainly with the discovery of
essential nutrients, understanding their functions, and
defining the quantities required to meet the metabolic
requirements of individuals. In recent years, however, it has
become generally accepted that many dietary components
exert effects over and above the prevention of nutritional
deficiency disorders. Such components are able to modulate
specific physiological mechanisms in ways that optimise
health and reduce the risk of both acute and chronic
diseases. Dietary fibre was one of the first dietary
components of this type to receive serious scientific
attention, and high fibre breakfast cereals were amongst the
first 'functional foods' to enter the marketplace. In recent
years, there has been an enormous growth of interest in
functional foods. It has been pointed out recently that in
view of the sheer breadth of mechanisms covered by the
general functional food concept, it is more appropriate to
provide a working definition of the term, rather than a rigid
one, which would inevitably fail to include some legitimate
examples. According to Diplock et al. (1999), a food can
be regarded as functional 'if it is satisfactorily demonstrated
to affect beneficially one or more target functions in the
body, beyond adequate nutritional effects, in a way that is
relevant to either an improved state of health and well-being
and/or reduction of risk of disease'. Such a food may be
'natural', processed or manufactured, but it must remain a
food; the definition does not extent to pharmaceutical
products. Many functional foods are designed to modify the
microflora of the large intestine, and thereby influence the
immunology or epithelial physiology of the large bowel.
The normal human colon is thought to contain around
200 g of semi-liquid material (Cummings et al. 1990), over
half of which is composed of bacterial cells; these are so
numerous as to account for 90% of the total number of cells
within the human body. Most individuals are typically
thought to harbour at least 400 different viable bacterial
species. The largest groups present are Gram-negative
anaerobes of the genus Bacteroides, and Gram-positive
organisms comprising a number of different genera
including Bifidobacterium, Lactobacillus and Clostridium
(Gibson and Roberfroid 1995). The constant supply of
water, mucus and digestive residues entering from the small
intestine provides a nutrient-rich aqueous medium, ideally
suited for bacterial proliferation and metabolism. The
functions of the colon are to recover water, energy in the
form of short chain fatty acids derived from bacterial
fermentation, and some nutrients (Cummings and
Macfarlane 1997). Storage times for faecal material vary
considerably, and healthy subjects pass stools anywhere
between once or twice a day, to once every 2–3 days.
 Functional foods and bowel cancer I.T. Johnson
4.1 The fibre hypothesis
Plant foods contain many complex polysaccharides other
than starch, most of which are associated with the structural
components of plant cell walls. The fact that these
molecules resist digestion by pancreatic enzymes of the
upper alimentary tract, and therefore enter the colon and
form a substrate for bacterial fermentation, was first
recognised by a group of mostly British nutritionists who
coined the term dietary fibre in the early 1970s (Trowell et
al. 1976). The significance of fibre in relation to colon
cancer was particularly emphasised by Burkitt (Burkitt
1971a; Burkitt 1971b), who based his hypothesis on the
concept of faecal bulk. His field observations in Africa,
where cancer and other chronic bowel diseases are rare,
suggested that, in populations consuming traditional rural
diets rich in vegetables and cereal foods, the average
person’s stools are both more bulky and frequent than those
of individuals living in the industrialised west (Burkitt
1973). Burkitt argued that in industrialised societies, a low
volume of colonic contents and infrequent bowel
movements caused prolonged exposure of the colonic
epithelial cells to faecal mutagens. In contrast, fibre-rich
diets contain cell wall polysaccharides that are readily
fermented by the microflora and are converted into bacterial
mass. Lignified tissues, such as wheat bran, resist
fermentation but help to retain faecal water (Eastwood and
Morris 1991). The mildly laxative effects of dietary fibre
are now well recognised, and numerous human intervention
trials have confirmed that dietary supplements containing
fibre can increase the volume of faecal material and reduce
the colonic transit time (Smith et al. 1981).
It is well established that human faecal water is
mutagenic (Venturi et al. 1997) and a number of known
carcinogens including heterocyclic aromatic amines and N-
nitroso compounds are present at low concentrations in the
faecal stream (Bingham et al. 1996; Hill and Fernandez
1992). Furthermore, bile acids may cause chronic irritation
in the colon and hence act as endogenous tumour promoters
(Narisawa et al. 1975; Narisawa et al. 1974). Whilst it has
not been conclusively established that such factors cause
colorectal cancer in humans, a high consumption of fibre
will certainly tend to reduce their concentrations in the
colon. The plausibility of the original faecal bulking
hypothesis has been strengthened by epidemiological
studies showing a statistically significant inverse
relationship between average stool weight and risk of bowel
cancer across a range of populations (Bingham 1996;
Cummings et al. 1992). This hypothesis continues to
provide the rationale for Dietary Reference Values for
dietary fibre in the UK, which recommend that adults
consume 18 g of fibre (non-starch polysaccharides) per day.
Cereals based on wheat bran were marketed for their
roughage and mildly laxative properties well before
consumers were exposed to the concept of dietary fibre.
Bran-based cereal products can therefore be regarded as
amongst the earliest and most successful functional foods.
Although the fibre hypothesis is difficult to test
3-3
conclusively, it is probably the most widely recognised
strategy for avoiding colorectal cancer, and consumption of
high fibre products is consistent with official dietary
guidelines. There is undoubtedly scope for further
development of products, although the biological effects of
dietary polysaccharides cannot be deduced simply from a
single analytical value for dietary fibre. The physical,
chemical and biological characteristics of complex
polysaccharides are still not completely understood
(Johnson 1993; Johnson 1999) and unconventional sources
of these materials may have unexpected effects on colonic
epithelial physiology. Until more is known about their
effects on proliferating crypt epithelial cells,
polysaccharides from unconventional sources need to be
treated with caution (Wasan and Goodlad 1996).
4.2 Butyrate
In recent years, the fibre hypothesis has acquired new
impetus with an improved understanding of cancer biology.
Although the factors that initiate and drive the adenoma-
carcinoma sequence remain poorly understood, one
possibility is that intraluminal conditions favouring
increased cell division and suppressing apoptosis are
important (Kinzler and Vogelstein 1996). Apoptosis is
thought to be relevant at all stages of the adenoma-
carcinoma sequence because the speed at which tumours
grow depends upon their relative rates of cell production
and death (Tomlinson and Bodmer 1995). Any shift in this
balance from apoptosis in favour of mitosis would increase
the rate of growth, and hence the risk of progression to a
carcinoma. One of the main products of bacterial
fermentation of carbohydrate in the colonic lumen is
butyrate, a short chain fatty acid (SCFA; Cummings and
Macfarlane 1997). This fascinating molecule is essential to
the maintenance of a healthy colon (Roediger 1990), and
exerts a range of effects on tumour cells in vitro, stimulating
differentiation, suppressing cell division and inducing
apoptosis (Avivi-Green et al. 2000; Buommino et al. 2000;
Chai et al. 2000). Hague et al. (1993) showed that tumour
cell lines established from human adenomas and carcinomas
would undergo increased apoptosis in the presence of
butyrate at concentrations close to those that occur in the
human colon. There is, therefore, much interest in the
possibility that increased production of butyrate may be an
important factor underlying the protective effects of high
fibre diets (Crew et al. 2000; Johnson 1995).
The advent of the butyrate hypothesis has shifted
attention from sparingly fermented forms of fibre, which
provide bulk and retain water, to resistant starch (Ring et al.
1988) and readily fermentable, soluble polysaccharides such
as pectin and β-glucan (Lund and Johnson 1991). However,
it is difficult to manipulate the SCFA content of the
colorectal lumen with any degree of precision. In one
study, Kashtan et al. (1992) used dietary supplements of oat
bran (which is rich in β-glucan) and wheat bran (which
contains mainly insoluble non-starch polysaccharides) to
explore the effects of soluble dietary fibre on faecal SCFAs
3-4 Functional foods and bowel cancer I.T. Johnson
and mucosal markers of crypt cell proliferation in a group of
human volunteers. Consumption of oat bran (16.4 g/day)
was associated with a significant decrease in faecal pH
compared to the wheat bran group. However, SCFA and
butyrate levels were not raised in the oat bran group, and
there was no significant effect of either supplement on the
proliferation of crypt epithelial cells in rectal biopsies
obtained before and after the dietary intervention. More
recently, Bonithon-Kopp et al. (2000) conducted a dietary
intervention trial using isphagula husk, a complex semi-
soluble material used clinically as a laxative (Marlett et al.
2000). Patients with a history of adenomatous polyps
received a supplement of 3.5 g per day for 3 years to
determine the effect on adenoma recurrence. The outcome
was a modest but statistically significant increase in the risk
of adenoma recurrence in the isphagula-supplemented
group. Although the supplement used in this study was not,
strictly speaking, a food product, it serves to illustrate the
care needed in the development of functional foods using
non-conventional sources of fibre (Goodlad 2001).
4.3 Modulating the colorectal microflora
The colonic microflora helps to create faecal bulk and
provides SCFAs including butyrate; however, certain
bacterial species may exert adverse effects on the intestinal
mucosa. This concept underlies the long-standing search
for dietary strategies to reduce the numbers of supposedly
harmful bacteria in the colonic microflora, and replace them
with beneficial species. Interest in the use of edible
bacterial cultures to manipulate the human colonic
microflora goes back at least as far as the beginning of the
20th century. Metchnikoff (1907) proposed that fermented
milk could be used as a source of beneficial lactobacilli, and
that regular consumption of yoghurt would improve health
and prolong life. Functional food products designed using
modern derivations of this concept continue to stimulate
interest amongst consumers and food manufacturers.
The term probiotic refers to any live microbial feed
supplement that exerts a beneficial effect on the host by
modifying the balance of the intestinal microflora (Fuller
1989). The difficulty with this approach is that in order to
establish themselves in the colon, live bacteria must first
survive their passage through the highly acidic environment
of the stomach and then traverse the small intestine before
coming into competition with indigenous bacteria. To
overcome this problem, two other approaches have been
developed. Prebiotics are ‘non-digestible food ingredients
that beneficially affect the host by selectively stimulating
the growth and/or activity of one or a limited number of
bacteria in the colon’ (Gibson and Roberfroid 1995). In
practice, prebiotics are almost invariably non-absorbable
carbohydrates, but they range from relatively small
molecules, including sugar alcohols and oligosaccharides, to
various forms of resistant starch (Gibson 1999). When a
probiotic microorganism is combined in a food supplement
with a prebiotic favouring its growth in the colon, it is
called a synbiotic (Rolfe 2000).
One of the most frustrating aspects of research on
colorectal carcinogenesis is the fact that, although a number
of putative faecal carcinogens have been identified, none
has been proven to cause human disease. However, one
strong candidate for a role in the pathogenesis of human
colorectal cancer is the heterocyclic aromatic amine, 2-
amino-3-methylimidazo[4,5-f]quinoline (IQ). This
compound is a pyrolysis product, first isolated from broiled
fish, and later shown to be present in a variety of cooked
meat products (Sugimura et al. 1990). Administration of IQ
causes tumours in a number of different organs in
laboratory rodents, including the colon. It is tempting to
postulate that the epidemiological evidence for an increased
risk of colonic cancer associated with high meat intakes is
caused by chronic exposure to IQ and similar heterocyclic
aromatic amines. However, the dose needed to induce
cancer in rodents is much higher than that found in humans,
and the issue remains open. Nevertheless, there have been
numerous studies on colorectal carcinogenesis induced by
IQ in rats. Interest in the possibility of modifying the
colorectal microflora to counter the effects of faecal
carcinogens has been stimulated by evidence that dietary
exposure to freeze-dried cultures of the bacterium
Bifidobacterium longum substantially reduces the
effectiveness of IQ in rodent models (Reddy 1998; Reddy
and Rivenson 1993; Singh et al. 1997).
How might microorganisms like bifidobacteria and
lactobacilli protect against colorectal carcinogenesis? One
possibility is that dietary carcinogens are absorbed and
metabolised in the liver by phase II enzymes and converted
to conjugated forms such as glucuronides, which are soluble
and non-carcinogenic. The major route for excretion of
these compounds is by secretion into the bile, and then into
the faecal stream via the intestinal lumen. However, many
colorectal bacteria express β-glucuronidase enzymes that
can deconjugate and hence reactivate intraluminal
carcinogens. Bifidobacteria have a low β-glucuronidase
activity, and their introduction into the faecal stream is
thought to reduce overall activity (Rowland et al. 1998).
Another possibility gaining credence is that certain bacteria,
including lactobacilli and bifidobacteria, may detoxify
carcinogens by binding them and reducing their availability
for interaction with mucosal cells (Knasmuller et al. 2001).
It is to be hoped that further research in this area will
substantiate the beneficial effects of manipulating the
colonic microflora, and underpin the development of new
pro- and prebiotics.
5. Phytochemicals – foodborne anticarcinogens
Besides fibre, diets rich in fruits and vegetables contain a
complex mixture of plant secondary metabolites now
widely known as phytochemicals. Many of these
compounds provide the characteristic colours and flavours
of fruits, seeds and flowers, and some are thought to
function as natural pesticides. Whether by accident or
adaptation, phytochemicals are often biologically active,
and some are toxic. Surprisingly perhaps, the quantities of
 Functional foods and bowel cancer I.T. Johnson
potentially toxic and carcinogenic organic molecules
derived from plant foods in the human diet far exceed those
of synthetic contaminants (Ames and Gold
1990). Humans have probably become adapted to the
presence of a diverse cocktail of dietary phytochemicals by
constant exposure to plants throughout evolutionary
history. Indeed, hunter-gatherer societies consume
considerably greater quantities of plant foods, and from a
much broader range of plant genera and species, than are
used in modern agriculture (Johnson 1997). Interest in
phytochemicals began when it was realised that many are
potent antioxidants. More recently, it has been recognised
that phytochemicals can exert a host of other biological
effects which may modulate cellular functions in various
target tissues (Johnson et al. 1994). Many phytochemicals
are poorly absorbed from the diet, but for that very reason
may attain high concentrations in the alimentary tract and
exert beneficial effects on sites of potential neoplasia
including the oropharyngeal tissues, oesophagus, stomach
and large intestine (Gee and Johnson 2001; Halliwell et al.
2000). Several groups of phytochemicals are currently
under investigation to determine their possible role as
protective factors against colorectal carcinogenesis.
Amongst the most important of these are the polyphenols
and the glucosinolates.
5.1 Polyphenols
Plant foods are particularly rich in polyphenols, phenolic
compounds containing one or more aromatic rings. This
diverse range of molecules adds colour to plants, provides
robust structural components for cell walls and is often the
source of flavour and aroma in foods and beverages. One
particularly large and important class of phenolic
compounds is the flavonoids, all of which consist of two
aromatic rings linked by a central heterocyclic oxygenated
group. Amongst the most common flavonoids in the diet
are the flavonols quercetin, myricetin and kaempferol,
found mostly as water soluble glycosides in fruits,
vegetables, tea and wine (Formica and Regelson 1995). At
one time, it was thought the flavonols were an essential
dietary factor contributing to the maintenance of capilliary
permeability (Rusznyàk and Szent-Györgi 1936). This has
not been substantiated, although recent interest in dietary
antioxidants and metabolically active phytochemicals has
focused renewed attention on the possible beneficial effects
of flavonoids (Hollman et al. 1996).
Many flavonols are very effective antioxidants. They are
absorbed to a limited extent from foods, and are present in
plasma as glucuronides, some of which retain their
antioxidant properties (Plumb et al. 1999). These
compounds may therefore protect against cardiovascular
disease by reducing the oxidation of low-density
lipoproteins (Manach et al. 1995; Miranda et al. 2000).
There is some epidemiological evidence for this, but their
effects on the overall antioxidant capacity of the plasma
remains to be established. Flavonoids and other phenolic
substances may also exert local anticarcinogenic effects in
3-5
the intestine (Gee and Johnson 2001; Halliwell et al. 2000).
In addition to acting as intraluminal antioxidants, they may
induce phase II xenobiotic metabolising enzymes, suppress
the production of biologically active prostaglandins by
inhibiting the arachidonic acid cascade, and suppress
mitosis by inhibiting intracellular protein kinases (Gee and
Johnson 2001). Like many phytochemicals, quercetin
aglycone is mutagenic in vitro (MacGregor and Jurd 1978)
and has previously been suspected to be a natural
carcinogen (MacGregor 1984). However, the compound
can also be shown to protect against colorectal
carcinogenesis in some models. The possible role of
quercetin and other flavonoids as protective factors against
alimentary cancers is currently the subject of a European
research programme (Williamson 2000).
5.2 Glucosinolate breakdown products
The glucosinolates comprise more than 100 organic sulphur
compounds found in plants of the order Capparalles, which
contains many domestic brassica species used as vegetables
and condiments. All the glucosinolates contain a β-D-
thioglucose group and a sulphonated oxime moiety bearing
a variable side-chain derived from various different amino
acids (Mithen et al. 2000). The glucosinolates remain
compartmentalised within the intact plant tissues until they
are damaged in some way. Once released, the enzyme
myrosinase, which is always present in glucosinolate
bearing plants, hydrolyses the compounds and releases an
unstable intermediate, which rearranges to form a variety of
nitriles, thiocyanates and isothiocyanates. The
isothiocyanates are formed under the conditions of food
preparation and digestion, and they account for much of the
aroma of brassica vegetables, as well as the hot and bitter
flavours of mustard (Brassica nigra), and radishes
(Raphanus sativus; Fenwick et al. 1983).
A number of studies have established that isothiocyanates
are absorbed from foods, and that they block the mutagenic
effects of human carcinogens by modulating the activities of
phase I and phase II biotransformation enzymes, including
glutathione-S-transferase and UDP-glucuronyl transferase
(Hecht 1999). A high consumption of brassica vegetables is
protective against cancers of the lung and alimentary tract
(van Poppel et al. 1999), and there is growing evidence that
consumption of brassica vegetables can modulate the
activity of phase II enzyme activity in humans (Nijhoff et
al. 1995a; Nijhoff et al. 1995b; van Poppel et al. 1999). In
the case of colorectal cancer, the beneficial effects of
brassica vegetables may also be partly due to their effects
on colorectal crypt cell proliferation and apoptosis (Mithen
et al. 2000).
It has been demonstrated that exposure of human cancer
cells to benzyl and phenethyl isothiocyanates leads to
growth arrest and apoptosis (Yu et al. 1996; Yu et al.
1998). Musk and colleagues showed that allyl
isothiocyanate (Musk and Johnson 1993), phenethyl
isothiocyanate and benzyl isothiocyanate (Musk et al. 1995)
caused selective growth inhibition of human colorectal
3-6 Functional foods and bowel cancer I.T. Johnson
cancer cells. In an animal model, a diet enriched with the
glucosinolate sinigrin induced an increased level of
apoptosis in the colorectal crypts of rats after treatment with
the specific colorectal carcinogen 1,2-dimethylhydrazine
(DMH), and inhibited the formation of precancerous
lesions (Smith et al. 1998). These potentially protective
effects were induced by sinigrin at levels well in excess of
human exposure, but a juice derived from uncooked
Brussels sprout tissue exerted much the same effects as allyl
isothiocyanate on HT29 cells in vitro, and markedly
increased crypt cell apoptosis after treatment with DMH in
the animal model (Smith et al. 2000). It remains to be seen
whether glucosinolate breakdown products derived from the
diet can suppress the cell cycle and induce apoptosis in the
human bowel. For further information on European funded
research in this area, see Johnson (2001).
London et al. (2000) have recently described the
relationship between the concentration of isothiocyanate
metabolites in urine and subsequent risk of lung cancer in a
large cohort of Chinese men. Deletion-polymorphisms
were present for the GSTM1 and GSTT1 genes; these code
for two different forms of glutathione-S-transferase (GST),
one of the phase II enzymes which detoxify a host of natural
and synthetic chemicals, including the isothiocyanates. It
was clear from the study that individuals whose urine
contained detectable levels of isothiocyanate metabolites,
and therefore were likely to have consumed large quantities
of brassica vegetables, were at a reduced risk of cancer
compared to controls. However, the protective effect was
confined to subjects who lacked either GSTM1 or GSTT1,
and strongest in those with deletion of both GSTM1 and
GSTT1. GST enzymes play a major role in the metabolism
of both environmental mutagens and isothiocyanates. These
observations suggest a very complex interplay between
environmental factors, genotype, and risk of cancer, and
point to the possibility of targeting future functional foods
towards individuals who may be particularly receptive
because of their genetic makeup.
There is particular interest in the United States in the
possibility of using broccoli sprouts as a protective
functional food product (Shapiro et al. 2001). In this
context, it is interesting to note that, although GSTM1 null
individuals show no particular difference in risk of
colorectal cancer compared to those with the positive
genotype when diet is not taken into account (Gertig et al.
1998), a high consumption of broccoli has been reported to
confer protection against colon cancer, specifically in those
with the null genotype (Lin et al. 1998). Brassica vegetable
varieties can be bred to contain high levels of
glucosinolates, and both processing and storage can be
optimised for the retention of glucosinolates in the finished
product. There may be considerable scope for exploiting
these approaches in the future, but the development of new
products will need to be coupled with careful clinical
studies to ensure both safety and efficacy.
6. Polyunsaturated fatty acids (PUFAs)
Dietary fat consists mainly of triglycerides that are
hydrolysed to their component fatty acids during digestion,
and then re-esterified prior to transport and metabolism or
storage in adipose tissue. Fats from marine organisms are
rich in polyunsaturated fatty acids (PUFAs) of the n-3
series, including eicosapentaenoic acid (EPA) and
docosahexaenoic acid (DHA), both of which can be
absorbed from foods and incorporated into cells, where they
function as structural components of membranes, and as
metabolic substrates for the production of intracellular
signalling molecules. EPA and DHA are rapidly
incorporated into the phospholipid pool of human colorectal
epithelial cells. There is epidemiological evidence
suggesting that diets high in PUFAs of marine origin are
associated with a reduced risk of colorectal cancer (Schloss
et al. 1997). Moreover, oral supplementation with fish oil
can apparently normalise the spatial distribution of
colorectal crypt cell mitosis in human subjects (Anti et al.
1994).
In animal models of carcinogenesis, PUFAs of the n-6
series, such as linoleic acid, tend to promote the induction
of tumours more than saturated fats. Monounsaturated fatty
acids are largely without effect, and n-3 PUFAs have been
found to be protective (Fay et al. 1997). Dietary long chain
fatty acids exert important effects on the proliferation and
apoptosis of crypt epithelial cells in animal models in vivo.
In the rat, replacement of corn oil with fish oil in a semi-
synthetic powdered diet led to a reduction in crypt cell
mitosis (Pell et al. 1994). Latham et al. (1999) studied the
effects of dietary supplementation with PUFAs on the
induction of aberrant crypt foci (ACF) following treatment
with DMH. Rats were fed a basal diet containing corn oil
prior to treatment. Immediately after the DMH injections,
the groups were subdivided and half were transferred to
diets in which the corn oil was replaced with fish oil. In the
rats fed fish oil, the wave of apoptosis induced by treatment
with the carcinogen was approximately doubled in
comparison to the rats remaining on corn oil; crypt cell
mitosis was also significantly reduced in rats fed fish oil.
These effects were associated with a reduced number of
precancerous lesions after 18 weeks.
These observations are generally consistent with the
observed effects of PUFAs on tumour cells in vitro. It has
been clear for some time that certain essential fatty acids,
notably γ-linolenic acid, arachidonic acid, EPA and DHA
are selectively toxic to tumour cells. Das (1999)
demonstrated that the inhibition of tumour cell proliferation
in the presence of these PUFAs was caused by selective
cytotoxicity, and that cell death was blocked by
antioxidants, enhanced by pro-oxidants and proportional to
the degree of peroxidation induced in the cells. More recent
studies have confirmed these findings, extended them to
other cell lines and shown that apoptosis is the mechanism
of cell death (Finstad et al. 1998a; Finstad et al. 1998b;
Hawkins et al. 1998; Ramesh and Das 1998).
 Functional foods and bowel cancer I.T. Johnson
These various types of evidence suggest that dietary fatty
acids play a potentially important role in the regulation of
cellular proliferation and death in the colon. If these effects
can be shown to be beneficial, functional food products,
such as spreads and oils, which are rich in n-3 PUFAs,
might well be developed for use in this context. However,
the promising clinical experiments of Anti and colleagues
(Anti et al. 1994; Anti et al. 1992) carried out in the early
nineties do not appear to have been followed up and
confirmed. Further clinical research employing well
validated biomarkers will be necessary before it becomes
clear whether functional foods based on n-3 PUFA have a
protective role to play against colorectal cancer in the
general population.
7. Conclusion
Despite the continuing uncertainties concerning the role of
diet in relation to colorectal cancer, there are several reasons
to be optimistic about the role of functional foods in this
context. The first stems from the fact that high fibre cereals
have a long history of commercial success. Despite the
widespread publicity given to unsuccessful intervention
studies with fibre, epidemiological research continues to
show that high intakes of fibre are protective. There seems
little reason to deviate from current nutritional advice,
which is to increase fibre intake by eating a variety of foods
in which it occurs as a natural component. High fibre cereal
products have a role to play, and if the butyrate hypothesis
is substantiated, the development of new products based on
fermentable fibre may be justified, although this will require
a careful analysis of risk and benefit. Many of the
functional foods currently on the market are fermented dairy
foods based on the principles of pre- and probiotics. Such
products are widely accepted and enjoyed by consumers,
but further research is needed to substantiate their value in
relation to a wide range of health issues, including
colorectal cancer. Finally, there appears to be considerable
scope for the exploitation of foodborne anticarcinogens,
including lipids and phytochemicals. Here again,
considerable research effort will be needed to ensure
efficacy and to avoid unexpected hazards. The rapid
advances in the molecular biology of cancer, coupled with
new techniques for the analysis of gene expression, seem
likely to provide a range of new research tools and
biomarkers of disease progression which can be used to
accelerate the pace of research, and underpin the
developments of new dietary strategies for disease
prevention.
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Functional foods: dietary intervention strategies in autistic spectrum
disorders

Max Bingham
Food Microbial Sciences Unit, School of Food Biosciences, The University of Reading, Whiteknights, Reading RG6 6JD,
UK .

Abstract

Interest in the role of diet in human health and disease has grown significantly over the past few years and has
led to a rapid growth in the market for functional foods. However, one much less researched area is that of the
role of diet in autism and autistic spectrum disorders. In reviewing this area, the question of whether there is
any potential for dietary interventions is considered along with a detailed discussion of the possible roles the
human gut microflora might play in the development and symptomology of autism and autistic spectrum
disorders. It has been known for a number of years that the concepts of probiotics, prebiotics and synbiotics run
concurrently with the concept of a healthy gut. Many anecdotal reports and, to a lesser extent, scientific reports
suggest that many autistic subjects suffer from severe gastrointestinal problems. The issue of whether
probiotics, prebiotics and synbiotics as functional food ingredients can be used in the relief of some of the
symptoms of autism and autistic spectrum disorders is discussed.

Keywords: autism, human gut microflora, functional foods, probiotics, prebiotics, Candida, clostridia, sulphate
reducing bacteria, indoyl acryloglycine (IAG), gluten, casein

1. Introduction approach is required to help autistic subjects lead a more

Autism and related autistic spectrum disorders remain one
of a small number of conditions that can only properly be
described in terms of symptoms rather than cause and/or
treatment. Since symptomology can vary considerably
between subjects, many different theories relating to the
causes of autism, as well as its treatment now exist.
However, in many cases, these theories and treatments
remain unproven or speculative and this has led to
confusion in the nature of associated research and literature
arising from it. From a parent’s or carer’s viewpoint, it
must remain a daunting prospect to consider treatments and
advice, particularly when the scientific community cannot
agree on specific approaches and the orthodox medical
establishment is reluctant to endorse unproven avenues of
relevance.
One particular aspect of autism that both parents and
professionals seem to agree on is that autistic subjects often
tend to suffer from severe dietary or gastrointestinal
problems. As always, the variety of problems reported
remains individual and heterogeneous. Many research
groups are now considering the impact of diet on the
symptoms of autism and are proposing dietary interventions
and regimes in an effort to alleviate this. However, a
widely accepted notion between all groups concerned is that
diet alone will not 'cure' autism; rather, a multidimensional
comfortable life.
2. Functional foods
Interest in diet and food from a wider perspective has grown
considerably in the past 10 years. Consumer demand for
more information about what goes into food, how it is
produced, and what it can do has forced industry and
government to provide such information. Awareness and
knowledge of food issues has thus considerably increased.
One outcome of this improved awareness has been the
development of so called ‘functional’ foods. These are
foods that claim to have some sort of benefit to the
consumer in terms of health and disease protection, which is
over and above their normal nutritional value. Health
claims on foods are strictly regulated within Europe and, as
such, rigorous scientific research and testing is often
required before any claims are allowed on food products.
A concurrent increase in our food knowledge has thus
occurred. From an industrial point of view, such products
remain very attractive to develop, market and sell, since a
'value added' price can often be charged. If demand is
sufficient, profits can therefore be very rewarding.
Food Science and Technology Bulletin: Functional Foods 1 (4) 1–11
DOI: 10.1616/1476-2137.12102. Published 15 May 2003
ISSN 1476-2137 © IFIS Publishing 2003. All Rights Reserved
4-2 Functional foods: dietary intervention strategies in autistic spectrum disorders M. Bingham
Many food products are currently available that are
claiming some sort of functionality and health benefit.
Some have been available for many years and are household
brands. Other products are newer to the market and have
been designed specifically for claiming increased
functionality. Typical examples of the latter include
products that claim to reduce cholesterol, reduce the risk of
coronary heart disease or improve large bowel function.
Other examples of products claiming functionality include
some that have been available for many years. Various
cereal brands, dairy products, sauce brands and even
chocolate brands have started to claim functionality in one
form or another - all designed to help improve sales of
individual food products. We know that many of these
claims are based on recent scientific research and that much
of this is industrially funded or performed 'in house' by the
companies themselves.
Many products are now available that claim functionality
in terms of affecting or improving gut function. Examples
include probiotics, which are live microbial feed
supplements that beneficially affect the host by improving
its intestinal microbial balance (Fuller 1989). Many brands
are now available and each have specific strains of bacteria
as their functional components. Bifidobacterium and
Lactobacillus spp. are typical ingredients. Less widely
available are products that include prebiotics. These are
non-digestible food ingredients that beneficially affect the
health of the host by selectively stimulating the growth
and/or activity of one or a limited number of bacteria in the
colon (Gibson and Roberfroid 1995). Again, the variety of
prebiotic ingredients included in products varies and it is
also recognised that functionality varies. Other products
that have functional properties aimed at improving gut
function include ingredients such as specific carbohydrates,
fatty acids, amino acids, vitamins, minerals, or
phytochemicals. Functional claims for these products do
vary considerably, however.
Interest in the diets of more compromised individuals has
grown recently and autism is no exception. A number of
theories exist suggesting that certain dietary factors are
important considerations for the treatment of autism. The
question is now being raised as to whether optimising diet
for autistic subjects is a possibility or even a necessity and
whether functional foods can play any part, or be developed
such that they can help the relief of autistic symptoms.
3. What is autism?
The term autism is usually associated with the syndrome
first described by Kanner (1943). In more recent years,
specific criteria have been set out to aid in the diagnosis of
the disorder (Schopler and Mesibov 1988), with the autism
diagnostic interview (ADI; Le Couteur et al. 1989; Lord et
al. 1994) becoming the standardised diagnostic parental
interview, and the autism diagnostic observational schedule
(ADOS; Lord et al. 1989; Di Lavore et al. 1995) becoming
the accepted observational measure. Autism typically
develops early in childhood, although causality and
treatment are often hotly debated. Symptoms may include
hyperactivity, loss of eye contact, decreased vocalization
(i.e. loss of language), repetitive and stereotyped behaviours
(e.g. hand flapping, twirling and tapping), poor academic
performance and other similar social deficits. Other similar
disorders exist and are often collectively termed autistic or
autism spectrum disorders (ASDs). These include
Asperger’s Syndrome, attention deficit hyperactivity
disorder (ADHD), pervasive developmental disorder (PDD)
and many others, where symptoms are similar to autism but
specific differences are demonstrated.
4. Interventions in autism: gluten and casein
removal
Ever since Panskepp (1979) noticed the similarities between
the behavioural effects of animals on opioids such as
morphine and the symptoms of autism, much research has
been carried out on the hypothesis of opioid excess.
Reichelt et al. (1981) produced evidence of abnormal
peptides in the urine of autistic people. Shattock et al.
(1990) replicated these findings and found elevated levels of
substances with properties similar to those expected from
opioid peptides. It was proposed that these were a result of
the incomplete breakdown of certain proteins, since the
levels found were far too large to be of central nervous
system origin. Peptides with opioid activity have been
identified from both gluten and casein digests (Chiba et al.
1989; Meisel 2001; Fukudome and Yoshikawa 1992). It
has been suggested that gastrointestinal permeability may
be higher in autistic than in non-autistic subjects and that
this may be responsible for increased levels of peptides in
the urine of autistic subjects (D'Eufemia et al. 1996). A
theoretical model has been suggested in which autism is a
consequence of exogenous peptide action and thus affects
neurotransmission within the central nervous system. A
direct or indirect mechanism could also be responsible
(Shattock et al. 2001). Numerous studies have established
elevated urinary excretion of peptides derived from certain
proteins in milk and wheat in children with autism and
adults with schizophrenia. Furthermore, recent studies have
also concluded that autistic children who follow a gluten-
and casein-free diet develop significantly better than control
subjects over a one year period (Knivsberg et al. 2002).
4.1 Approaches for the removal of gluten and casein
from the diet
The actual removal of gluten and casein from the diet is not
a simple procedure. Gliadin, for example, is a protein
fraction of gluten found in all grains other than rice or
maize. Wheat, rye, oats, barley and buckwheat are used in
the manufacture of a variety of foods, beverages and
confections. Clearly, products such as bread and dairy
products must be removed, since a high proportion of their
structures, textures and nutritional qualities are inherently
linked with gluten and casein proteins; these may be present
as basic food ingredients or as derivatives when food is
 Functional foods: dietary intervention strategies in autistic spectrum disorders M. Bingham
processed or prepared. Thickening agents, for instance,
commonly utilise a gluten-containing product, while casein
is found at high levels in dairy products such as milk,
cheese, yoghurts and butters. Highly processed foods often
use milk powders and derivatives in a variety of ways.
Chocolate, cakes, and other confections are good examples.
The careful reading of labels on food packaging is very
important in these circumstances. Many forms of
information resources exist for the parent wishing to
implement a gluten- or casein-free diet for their children,
with some being more useful than others. Such outlets may
take the form of books, popular magazines, television
programmes and Internet sites. Careful consideration of the
content of these information sources must be taken,
however, since these diets can be very restrictive. Products
must be carefully considered when they are bought, and
nutritional adequacy must be at the forefront of every
purchase decision taken by the parent or carer. Of
particular concern are casein sources, since they are often
recommended sources for many essential vitamins and
minerals (particularly calcium). Indeed, Cornish (1998)
found that 13 out of 17 children diagnosed with autism
consumed 'excessive' amounts of milk, giving a calcium
intake of levels above 200% of reference nutrient intake
(RNI) levels. This study also found that many of the
children studied had nutrient intakes below RNI levels (and
in some cases below lower reference nutrient intake (LRNI)
levels). Nutrients included vitamins C, D, B6, niacin and
riboflavin, while minerals included calcium, zinc and iron.
Crucially though, the study also considered eating habits
and found that diets were 'prescriptive', with preferences
being specific for a variety of factors including dry/wet
form, colour and shape, and even brand packaging. Food
refusal and introduction of new foods were cited as
particular problems by parents, in connection with their
attempts to provide a balanced nutritional intake for their
autistic children. Further studies have indicated that the
implementation of a gluten- and casein-free diet in autistic
spectrum disorders had no significant effect on nutritional
intake in terms of macro and micronutrient intake when
compared to a control group of children with autistic
spectrum disorders who were not on a gluten- and casein-
free diet. These studies also indicated that the exclusion of
gluten and casein did not significantly improve their
nutrient intake either (Cornish 2002).
Removal of gluten- and/or casein-containing products
from the diet will require the active participation of all those
concerned with the child's well being. It is conceivable that
tests and interventions will be confounded by a well
meaning relative who ignores parental instruction, or by
schools or therapists who view the regime as pointless.
Parents often report that the normal diet of children with
this condition, prior to introduction of any dietary
intervention, is very restricted (Cornish 2002) and consists
almost entirely of wheat and dairy products in every
conceivable combination (Whitely et al. 1999). However,
Knivsberg et al. (2001) have recently examined studies
4-3
from the past 12 years that considered changes in autistic
characteristics following removal of gluten and casein from
the diet. All but one study reported beneficial results that
included reduction in autistic behaviour, increased social
behaviour and communication skills, and reappearance of
autistic characteristics after the diet had been broken.
In considering the applicability of functional foods to this
particular issue, we can take advice from approaches used in
coeliac disease treatment. This treatment is primarily based
on the removal of gluten from the diet, on the basis of
severe reactions exhibited by sufferers upon ingestion of
gluten products. Many products are now available from
supermarkets that are specifically labelled as 'gluten-free'.
This includes supermarket own brands and, more recently,
branded products. Much press space has also been given to
describing symptoms of wheat intolerance (even if not
supported by any sound scientific evidence); this has also
popularised products with wheat replacement factors (e.g.
xanthum and guar gums and the use of flours that do not
contain gluten). These products include gluten free breads,
flours, pasta, biscuits and cakes - all would normally
contain gluten, but have been reformulated to be gluten-
free.
Whether gluten-free foods can be considered as
functional foods is debatable, since they are products that
have had a component removed or not included and not
ones that have had a functional ingredient added. However,
the applicability of gluten- and casein-free diets in the
context of autism must be considered. Presumably, if
parents have problems with feeding autistic subjects gluten-
and/ or casein-free diets, products could be developed to
help replace the 'offending' components in much the same
way that products have been developed for other gluten-free
diets. The opportunity may exist for developing products
that would appeal to children, replacing the gluten and
casein components with other nutrients that would
otherwise be missing from the diet (Cornish 1998). Issues
such as nutritional quality and sensory perception would
have to be considered in developing such products, and
price factors may also be important since many formulated
gluten-free products currently come with a substantial price
tag. It appears, therefore, that the application of
functionality to foods specifically designed to be gluten-
and casein-free for autistic children may be possible,
although many nutritional and food design issues would
have to be overcome.
5. Interventions in autism: phenolic food
components and enzymes
Anecdotal reports (from parents) have suggested that
consumption of particular foods by autistic individuals
appears to result in the regression of certain autistic
symptoms, such as skills in social interaction and
communication. Possible foods include citrus fruits, apple
juice, chocolate and soya - all foods with specific phenolic
food components such as flavonoids, catecholamines,
4-4 Functional foods: dietary intervention strategies in autistic spectrum disorders M. Bingham
isoflavonoids and anthocyanins. A key role of the
sulphation pathways that take place in the liver and intestine
is the detoxification of these components. Waring et al.
(1997) measured the sulphation capacity of autistic children
against control subjects, using paracetamol as an in vivo
probe drug. Excretion of the sulphate conjugate was
significantly lower in the autistic group, as were plasma
levels of inorganic sulphate and activity of platelet
phenolsulphotransferase. They concluded that in certain
autistic subjects, there may be a decreased capacity to
detoxify endogenous and exogenous phenols and amines via
sulphation. Later studies extended and confirmed this
finding (Alberti et al. 1999; Waring and Klovrza 2000).
The role of sulphate in autism has only recently been
considered but may be pivotal to understanding the
condition. On balance, it appears that sub-optimal
sulphotransferase activities, as demonstrated in previous
studies (Waring et al. 1997; Waring and Klovrza 2000;
Alberti et al. 1999), are a consequence of low plasma
sulphate levels rather than a deficit in the actual enzyme.
Relatively little sulphate is absorbed from the diet, although
sulphites from food preservatives may contribute
substantially. Sulphate is predominately generated in vivo
from sulphur-containing amino acids, such as cysteine, by
the process of sulphoxidation (McFadden 1996). Anecdotal
reports have suggested that the addition of Epsom salts
(magnesium sulphate) trans-dermally may help in
improving these particular symptoms of autism. Likewise,
the removal of foods from the diet containing phenolic
components has also been advocated on the premise that
these components deplete plasma sulphate levels as a direct
consequence of their metabolism. Overall, the approach is
based on the hypothesis that non-detoxified components of
the diet (in particular phenolic components) have a range of
biological effects in vivo that ultimately affect neurological
functioning in autism. It is interesting to note that much
research is currently being undertaken in terms of functional
food development on the various activities of phenolic
components such as their antioxidant, anti-carcinogenic and
anti-oestrogenic effects. Whilst these reports remain
anecdotal and the efficacy of the proposed interventions are
not proven, this recent insight into the role of phenolic
based components, so often cited as having positive health
effects, may alter considerations for the design of functional
foods using these components in the future.
6. Interventions in autism: the gut microflora
6.1 Yeast metabolites in the urine of autistic children
In normal conditions, yeasts constitute only a very small
proportion of the human gut microflora (Holzapfel et al.
1998); this is possibly due to suppression of yeast growth
by competing bacterial species or immune functions.
However, gastrointestinal fungal overgrowth has been
proposed as contributing to the development of some of the
symptoms of autism. A leading proponent of this theory
has been Dr William Shaw of the Great Plains Laboratory in
the USA. However, the peer reviewed literature on this
topic is currently limited and based on indirect evidence.
It has been shown previously that children exhibiting
autistic features show increased excretion of arabinose as
well as analogues of Krebs cycle metabolites (including
tartaric acid; Shaw et al. 1995). Using gas
chromatography/mass spectrometry (GC/MS) to test for
urinary metabolites, it was found that two siblings with a
variant of autism associated with skeletal weakness excreted
extremely high values of tartaric acid and arabinose; this
was potentially due to an overgrowth of Candida
albicans. Both the muscle weakness and cognitive
abnormalities were reported to respond to treatment with an
anti-candidal therapy which also reduced tartaric acid
concentrations significantly. It is not clear at present
whether these data can be extrapolated to wider autistic
spectrum disorders and thus anti-candidal therapy cannot be
regarded as an evidence based treatment. Meanwhile, many
reports have suggested that the onset of autism may be
related to the occurrence in children of the middle ear
infection otitis media (Kontstantareas and Homatidis 1987).
It is common to treat otitis media with some sort of broad-
spectrum antibiotic. Intestinal overgrowth of yeast and
certain anaerobic bacteria are a well documented outcome
of the administration of broad-spectrum antibiotics
(Kennedy and Volz 1983; Danna et al. 1991; Ostfeld et al.
1977; Kinsman et al. 1989; Van der Waaij 1987; Samonis et
al. 1993, 1994a, 1994b).
It has also been shown that C. albicans produces
gliotoxins (Shah and Larsen, 1991, 1992) and
immunotoxins (Podzorski et al. 1989; Witkin 1985), which
may further impair the immune system. This would have
relevance in terms of promoting yeast growth and
increasing the chances of additional infections from bacteria
which would again result in antibiotic usage. Indeed, recent
research (Payne et al. 2003) has been able to demonstrate in
vitro the value of the normal gut microflora in the protection
against growth of C. albicans in the gut. In this study C.
albicans was not observed in the presence of an established
microflora. However, the addition of the antibiotic
tetracycline to the medium was followed by substantial
growth of Candida albicans. Subsequently, a probiotic
organism, Lactobacillus plantarum LPK, was added and
cell counts of C. albicans, whilst not completely eradicated,
were markedly reduced. As the authors suggest, this may
indicate a compromised physiological function on the part
of C. albicans in the presence of L. plantarum. Whilst the
role for Candida species in autism has yet to be
substantially proven, this is the first indication that a
specific probiotic strain may be used to target C. albicans
specifically; this observation may prove useful in the dietary
management of autistic symptoms in the future.
6.2 Clostridia and autism
Bolte (1998) outlined the possibility of a subacute, chronic
tetanus infection of the gut as the underlying cause for
symptoms of autism observed in some individuals. Here it
 Functional foods: dietary intervention strategies in autistic spectrum disorders M. Bingham
is postulated that a percentage of individuals with autism
have a history of extensive antibiotic use. As above, it is
known that oral antibiotics significantly disrupt protective
gut microflora, creating a favourable environment for
colonisation by opportunistic pathogens. Clostridium tetani
is a ubiquitous anaerobic bacillus that is known to produce a
potent neurotoxin. The normal binding site for tetanus
neurotoxin is the spinal cord, although the vagus nerve is
capable of transporting tetanus neurotoxin, thus providing a
route of ascent from the intestinal tract to the central
nervous system and bypassing the spinal cord. The result
would be that typical symptoms of a tetanus infection would
not be evident. Once in the brain, the tetanus neurotoxin
disrupts the release of neurotransmitters, and this may
explain the wide variety of behavioural deficits apparent in
autism. Bolte (1998) presents evidence of laboratory
animals exhibiting many of these behaviours after being
injected in the brain with tetanus neurotoxin, and also of
autistic children showing a significant reduction in
sterotyped behaviour following treatment with
antimicrobials which are effective against intestinal
clostridia.
In a pilot study of oral vancomycin therapy in autistic
children, for example, significant cognitive improvement
was reported following vancomycin treatment. Formal
psychological assessment and study of video recordings
were used to rate the children; approximately a week after
the course of antibiotics had finished, significant regression
was observed in autistic symptoms (Sandler et al. 2000). In
a more recent study, the number of clostridial species found
in the stools of autistic children and control children were
compared (Finegold et al. 2002). In all, the autistic children
had eight species of clostridia not found in the controls,
whereas the controls only yielded three species not found in
the autistic subjects. Overall counts of clostridia and
Ruminococcus spp. were higher in the stools of autistic
subjects; upon identification, it was found that one or more
of the species occurring only in the autistic children were
toxin producers (Finegold et al. 2002). Clearly, a treatment
to reduce the numbers of clostridia in the gut might help to
repress the effects of this inhabitation. However, clostridia
are spore forming, which means that recolonisation is
possible even after treatment. The relevance of probiotic
and prebiotic treatments has not been researched, yet appear
to be reasonable approaches in aiding suppression of
clostridial species in the gut.
6.3 Human gut flora and gluten and casein
intolerance in autistic children
Yeasts, including C. albicans, are known to secrete a
number of enzymes. These may include phospholipase,
which can break down phospholipids, and proteases such as
secretory aspartate protease, which metabolises proteins.
These enzymes may partially digest the gut membranes and
lining itself. Furthermore, it is known that the mycelium
and chlamydospores of yeasts are capable of tissue invasion
4-5
(Cole et al. 1988, 1990). It is likely that such factors could
increase the permeability of the gut, which has important
consequences in terms of food absorption and digestion. In
terms of autism symptoms, this may be highly relevant. It
has been shown that incompletely broken down portions of
gluten and casein may be crossing the gut into the blood and
having an opioid effect in autistic children, with the
resulting autistic symptoms being a consequence of this
opioid action (Reichelt et al. 1981; Shattock et al. 1990).
While many mechanisms have been suggested for this
incomplete breakdown, it seems key that a yeast and/or
clostridial overgrowth might affect this in some way.
6.4 Indolyl acryloyglycine (IAG)
Indolyl acryloyglycine (IAG) is a metabolic product of the
amino acid tryptophan and can be found in trace amounts in
the urine of apparently normal healthy individuals. It was
first described in the urine of a patient with a light sensitive
dermatitis (Kimmig et al. 1958). A number of groups have
described its presence in the urine in patients exhibiting a
variety of dietary disorders and each proposed that the
presence of unusual bacteria in the gut might be responsible
(Jepson 1966; Banwell and Crawford 1963; Marklova
1999). More recently, the work of Mills et al. (1998) and
Anderson et al. (2002) has shown the presence of IAG in
the urine of a number of subjects diagnosed with autistic
spectrum disorders.
Under normal conditions, tryptophan is catabolised to
indole pyruvate and indole acetate and can be detected in
the urine of normal subjects (Moore et al. 1987). Elsden et
al. (1976) discussed the end-products of metabolism of
aromatic amino acids including tryptophan by Clostridium
spp. Smith and Macfarlane (1997) carried out similar
studies in anaerobic batch cultures and were able to
investigate the effects of pH and carbohydrate availability
on the production of toxic metabolites. It has been
proposed that under certain abnormal gut conditions,
anaerobic coliforms can deaminate tryptophan to indole
proprionic acid, which is subsequently absorbed and
oxidised to indole acrylic acid and conjugated in the liver to
IAG. This is subsequently excreted in the urine (Loo and
Woolf 1957; Shaw et al. 1960). It has been proposed that
increased levels of tryptophan in the diet and/or the
presence of unusual bacteria in the large intestine may
contribute to this unusual metabolic state (Smith et al.
1968). Szeinberg et al. (1965) reported the disappearance
of IAG from the urine of a patient who had previously
excreted large amounts of the compound after
administration of the antibiotic neomycin. Following
completion of the course of antibiotics, IAG reappeared in
the urine. The authors concluded that the production of
IAG was likely to be mediated by ‘unusual’ gut bacteria and
that these bacterial species were probably passed between
family members of the patient. Moreover, elevated blood
levels of serotonin (5-hydroxytryptamine), a tryptophan
metabolite, have been noted in 30-40% of autistic children,
although there is no clear evidence currently to suggest that
4-6 Functional foods: dietary intervention strategies in autistic spectrum disorders M. Bingham
such an elevation plays a role in the disease (McDougle et
al. 1993). Bacterial action is not responsible for the
formation of serotonin from tryptophan; however, the
activities of bacteria related to other indole derivatives
makes this a particularly interesting area, particularly when
serotonin function is involved in social interaction,
obsessive compulsive activity and aggressive/impulsive
phenomena.
The presence of IAG in the urine of autistic subjects may
be significant and might provide some guidance on unusual
metabolites found in urine samples. Shattock and Savery
(1997) have roughly estimated that about 75% of subjects
with autism have IAG in their urine. Interestingly, the same
authors have also estimated that about 50% of fathers,
mothers and siblings show unusually high levels of IAG.
Although these appear to be very rough estimates of IAG
incidence, it may suggest that, among other things, an
unusual bacterial metabolism may be in action. Together
with the evidence provided by Szeinberg et al. (1965), it
suggests that we now need to consider identification of a
species and strain of gut bacteria, or a set of gut conditions,
that could contribute to the presence of IAG in the urine of
subjects with autism and other conditions, as well as
immediate relatives. Shattock and Savery (1997) have
suggested that IAG represents a detoxified version of a
parent compound that may affect the permeability of
membranes throughout the body. This is significant since it
is recognised that many autistic subjects are affected by
opiate substrates (derived from gluten and/ or casein) and
other gut metabolites (from aromatic amino acids such as
tryptophan) because of problems with the permeability of
membranes in the gastrointestinal tract and other organs
(Panskepp 1979). Using this knowledge, it may be possible
to manage the appearance of IAG and other unusual
metabolites in the urine of affected subjects and affect a
consequential improvement in symptomology.
It is currently too early to say whether functional foods
would help in the management of IAG production in
affected subjects. Since we now suspect that an unusual
bacterial metabolism may be at work, a possible dietary
intervention may include the use of probiotics, prebiotics or
synbiotics. These approaches have been described as one of
the most promising areas for the development of functional
foods (Salminen et al. 1998) and may have a significant
application in the management of IAG production in autistic
subjects. Correction of any unusual bacterial metabolism
by the replacement of these species with more beneficial
bacteria, such as lactobacilli and bifidobacteria may help
reduce IAG production. Indeed, a recent in vitro study
examined the effects of fructooligosaccharides (FOS, a type
of prebiotic) on the conversion of tryptophan to skatole and
indole metabolites by a mixed gut microbial population
from pigs. Significantly, the peak values of indole 3-acetic
acid (a microbial precursor of IAG) decreased with the
addition of FOS. Meanwhile, the viable counts of
bifidobacteria increased, whilst those of clostridia and
Escherichia coli decreased (Xu et al. 2002). Whilst this
effect still needs to be shown using mixed microbial gut
populations of human origin, as well as in vivo, this data
provides an interesting insight into the use of dietary
management techniques such as prebiotics in the control of
apparent autistic biomarkers (IAG).
6.5 Sulphate-reducing bacteria
Sulphate-reducing bacteria are strictly anaerobic and can be
spherical, ovoid, rod-shaped, spiral or vibrioid in shape.
They measure 0.4–3.0 mm in length and can occur singly,
in pairs or sometimes as aggregates. This group of bacteria
has the capacity to reduce sulphate to hydrogen sulphide
(H2S) and can metabolise both hydrogen (from fermentation
of other bacteria) and sulphate (from dietary sources).
Products are sulphite and/or H2S, the latter of which is a
toxic gas with a characteristic smell of rotten eggs.
Sulphation problems in autism have been proposed for
many years now. Waring (2001) has shown that around
95% of autistic children have serum sulphate levels that are
about 15% of that found in control subjects. This is seen as
significant since sulphation is required in the inactivation of
certain neurotransmitters in the brain involved in the
modulation of mood and behaviour. Reduced sulphation
also affects mucin proteins that line the gastrointestinal tract
and this finding is linked with increased gut permeability
and inflammatory bowel disease.
The important question why autistic children tend to
exhibit such low levels of serum sulphate remains,
however. Waring (2001) outlined the possibility that
cytokines, which are peptides produced in inflammatory
processes, may be responsible. It was found that autistic
children often have high cytokine levels, and this would
have the indirect effect of greatly reducing the production of
sulphate.
This also raises the possibility that sulphate-reducing
bacteria in the gut have a role in this process and possibly
contribute to the low levels of serum sulphate found in
autistic individuals. These bacteria would have the capacity
to reduce levels of sulphate in the gut by metabolising it to
H2S and/or sulphite, particularly if their population levels
were abnormally high. We know that, in vitro, a number of
species can desulphate mucins (Roberton et al. 1999) and
that colonic bacteria can produce sulphide which is a
neurotoxin (Kilburn 1997). However, no research has been
completed on whether sulphate-reducing bacteria in the gut
have a role to play in this respect and thus the evidence
remains speculative. However, it seems inevitable that
these bacteria would have some role to play in this process
and may therefore be of importance in the development of
autistic symptoms.
6.6 A role for probiotics, prebiotics and synbiotics?
The concept of a healthy microflora is not a new one and
probably originates with Metchnikoff at the turn of the 20th
century. Some gut bacteria are thought to be beneficial to
health, whilst others may be more harmful. It is a readily
 Functional foods: dietary intervention strategies in autistic spectrum disorders M. Bingham
accepted fact that some bacteria can be harmful through the
production of toxins causing diarrhea, mucosal invasion,
and activation of carcinogens. Potential health-promoting
bacteria are thought to principally include the bifidobacteria
and lactobacilli. Suggested reasons for this include the fact
that these two groups do not include any significant
pathogenic species and their dominance in the faeces of
breast-fed infants implies that such bacteria provide
protection against infection in the gut. Meanwhile, in
adults, they may be the principal species responsible for
efficient barrier function and for stimulating healthy
immune function. However, the human gut microflora is a
complex community of interacting organisms and its
functions are a consequence of the combined activities of
these components. Manipulation of the human gut
microflora offers the potential to improve host health
through a variety of mechanisms.
In terms of functional food ingredients, the concepts of
probiotics and prebiotics have been of interest to a number
of research groups in recent years – these have variously
been defined. For the purposes of human nutrition, the
definition by Fuller (1989) is considered useful and states
that probiotics are live microbial feed supplements which
beneficially affect the host. Prebiotics, meanwhile, are non-
digestible food ingredients that beneficially affect the host
by selectively stimulating the growth and/or activity of one
or a limited number of colonic bacteria, which have the
potential to improve host health (Gibson and Roberfroid
1995). Another approach is the use of synbiotics. These
are mixtures of probiotics and prebiotics that beneficially
affect the host by improving the survival and implantation
of live microbial dietary supplements in the gastrointestinal
tract by stimulating the growth and/or activating the
metabolism of one or a limited number of health-promoting
bacteria and thus improving host welfare (Gibson and
Roberfroid 1995).
Guidance on the usage of probiotics, prebiotics and
synbiotics can be taken from an examination of previous
clinical studies. In terms of probiotics, the most common
bacterial genera used are lactobacilli and bifidobacteria. A
number of health-related effects of probiotic use have been
documented. These include an alleviation of lactose
intolerance (Sanders 1993; Kolars et al. 1984; Marteau et
al. 1990), immune enhancement (Kaila et al. 1992;
Schiffrin et al. 1995; Marteau et al. 1997a, 1997b), a
decrease in faecal mutagenicity and bacterial enzyme
activity (Goldin and Gorbach 1984; Goldin et al. 1992; Ling
et al. 1994; Morotomi 1996) and a shortening of the
duration of rotavirus diarrhoea (Guarino et al. 1997; Raza et
al. 1995; Pant et al. 1996).
A number of different studies have demonstrated the
effectiveness of prebiotics in vivo. Fructooligosaccharides
(FOS) have been shown to selectively stimulate
bifidobacteria and are considered important prebiotic
substrates. An early general observation was made that a
greater bifidogenic effect can be achieved per dose in
individuals with a low initial bifidobacterial count (107/g of
4-7
faeces) as compared to those with higher initial counts
(109.5/g of faeces) (Hidaka et al. 1986). Furthermore, a
negative correlation between bifidobacteria and Clostridium
perfringens was observed (Wang and Gibson 1993; Gibson
and Wang 1994c). A large variation between subjects and
the effects of dosage on microbial populations in the gut has
been demonstrated (Hidaka et al. 1986; Williams et al.
1994; Buddington et al. 1996). Importantly, when FOS
administration ceases, it has been observed that there was a
decrease in bifidobacteria (Tuohy et al. 1991c; Buddington
et al. 1996). Human trials with FOS and inulin (a type of
FOS), including those following a controlled diet, have
demonstrated a prebiotic activity of these substances
(Gibson et al. 1995; Buddington et al. 1996; Kleessen et al.
1997). In vitro studies on FOS demonstrated the selective
stimulation of bifidobacteria and the inhibition of
Bacteroides, clostridia and coliforms (Gibson and Wang
1994a, 1994b).
Other prebiotic substrates include galactooligosaccharides
(GOS) (Bouhnik et al. 1997; Ito et al. 1990, 1993; Tanaka
et al. 1983), soybean oligosaccharides (Tamura 1983;
Hayakawa et al. 1990; Saito et al. 1992; Gibson et al.
2000), lactosucrose (Tamura 1983; Fujita et al. 1991),
isomaltooligosaccharides (Kohmoto et al. 1988; Olano-
Martin et al. 2000) and lactulose (Terada et al. 1992;
Fadden and Owen 1992). Human studies have shown the
prebiotic effect of FOS and lactulose (Tuohy et al. 2001a,
2001b, 2001c). Prebiotic substrates exhibit individual
benefits and drawbacks; however, recent advances in
biotechnology offer the chance to deliberately manufacture
multifunctional prebiotics. Examples might include forms
that have anti-adhesive properties against common
foodborne pathogens, types that persist to the large gut (the
main site for colonic disorders) and prebiotics that target
individual species rather than genera of gut bacteria (Gibson
et al. 2000).
Since there is now some evidence to suggest that autistic
subjects suffer from often acute gastrointestinal problems
and possibly a disturbed gut microflora, the concepts of
probiotics, prebiotics and synbiotics as functional food
ingredients appear to be applicable in autistic spectrum
disorders. It is clear that these ingredients may be able to
play a role in restoring the balance of the gut microflora in
autistic subjects. If it is the case that certain toxin-
producing gut microflora components are responsible for
some autistic symptoms, it may then become possible to
manage these through the use of probiotic, prebiotic and
synbiotic food ingredients.
7. Conclusions
As for the general role of functional foods in the
management of autistic symptoms, it is too early to tell
whether specific products can be developed for use by
autistic subjects. Many theoretical areas have been
discussed and reviewed in this and other papers and it seems
that we are currently in a state of rapid learning in terms of
4-8 Functional foods: dietary intervention strategies in autistic spectrum disorders M. Bingham
diet and autism. We have known for around two decades
that dietary management in terms of removal of offending
food constituents can bring transient improvements in
autistic symptomology. The last few years have seen a
surge in interest in functional foods, and many research
groups are now interested and actively researching the role
of diet in health and disease. One possible opportunity for
using functional foods in autism seems to lie in the field of
probiotics, prebiotics and synbiotics. As mentioned earlier,
one of the most promising areas for the development of
functional foods lies in the modification of the activity of
the gastrointestinal tract by their use. The gut is an obvious
target for the development of functional foods since it acts
as the interface between the diet and the metabolic events
that sustain life. Since there is significant interest in the role
of diet in autism, the role for functional foods and more
particularly probiotics, prebiotics and synbiotics in the
management of autism appears to be an important area for
future research. Much more fundamental work needs to be
completed in terms of gut microflora, immune and mucosal
function and digestive physiology in relation to the autistic
state. Work has already begun at the University of Reading
to explore these fundamental relationships between human
gut microflora and autism.
While it is clear that abnormal metabolites from the
human gut microflora may contribute to autistic symptoms,
it is certain that many other systems and pathways are
involved; it is possible that many are functioning
simultaneously but at varying levels between individuals.
This may give rise to the differences in symptoms exhibited
by sufferers, but may also explain the similarity of autism
and other chronic behavioural disorders such as Aspergers
Syndrome, PDD, ADHD, ADD and Rett syndrome. While
other pathways may exist, research into the role of human
gut microflora and the full identification of species involved
in autistic spectrum disorders may allow for more directed
treatments. Although these will not cure the disorder,
modifications of gut microflora function might significantly
improve symptoms.
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From hypoallergenic foods to anti-allergenic foods

Arthur Ouwehand*, Pirkka Kirjavainen, Kirsi Laiho and Samuli Rautava

Functional Foods Forum, University of Turku, FIN-20014 Turku, Finland. Tel. +358-2-3336894. Fax +358-2-3336884. E-
mail arthur.ouwehand@utu.fi

*Corresponding author

Abstract

Allergic diseases have become more prevalent in developed countries over the last few years, particularly with
changes in lifestyles. Improved sanitation, vaccination and the consumption of microbe-free foods seen today
are considered to be among the major causes for the increase in prevalence of allergic diseases. Indeed, the
intestinal microbiota plays a major role in the development and maturation of the immune system and, thereby,
in the aetiology of allergic disease. It has been proposed that selected functional foods may modulate the
composition of the intestinal microbiota in order to provide the immune system with appropriate microbial
stimulation. This overview describes the different forms of allergic diseases and their possible causes. The
relationship between allergy and the intestinal microflora is discussed, together with the potential of probiotics,
prebiotics and other functional ingredients to treat or prevent allergies.

Keywords: allergy, atopy, functional foods, probiotic, prebiotic, intestinal microflora

1. Introduction Atopy is defined as a personal or familial tendency to

Consumers have long been aware that food plays a role in
health and wellbeing, with phrases such as ‘you are what
you eat’ being common in everyday speech. However, the
concept has been neglected over the past few decades by
health professionals and researchers, but is now being
rediscovered, as indicated by increasing interest in
functional foods. The importance of food in health is clear
in the case of food allergies. Diet modification has,
therefore, been a means of relieving food allergy symptoms
for a long time. Recently, novel means of dietary treatment
and/or prevention of allergy have been explored
successfully and may provide alternative means for coping
with these diseases. The current review will, therefore,
focus on the possibilities and future prospects for using
functional foods in the treatment and prevention of allergic
diseases.
2. Allergy, atopy and the origin of allergic disease
2.1 Allergy, atopy, and the allergic type of immune
responsiveness
Allergy has recently been defined as a hypersensitivity
reaction initiated by immunological mechanisms, which
may or may not be mediated through antigen-specific IgE
antibodies (Johansson et al. 2001). The process of IgE
antibodies being produced against an environmental antigen
is referred to as sensitisation. IgE-mediated disorders may
further be divided into atopic and non-atopic allergies.
produce IgE antibodies in response to allergens and to
develop symptoms such as asthma, rhinoconjunctivitis, or
eczema/dermatitis (Johansson et al. 2001). There is a
marked co-morbidity in atopic diseases (Leynaert et al.
2000; Terreehorst et al. 2002); furthermore, different
manifestations of atopy tend to follow an age-related
pattern, the so-called allergic march (Wahn 2000). In non-
atopic disease, patients do not have elevated total serum IgE
and show negative skin prick tests, although it has been
suggested that such subjects may produce IgE locally and
against currently unknown allergens (Humbert et al. 1999).
T helper lymphocytes appear to be central in the
pathogenesis of both IgE and non-IgE associated allergic
disease (Romagnani 2000). T helper (Th) cells are divided
into Th1 and Th2 subgroups, based on cytokine production
patterns, as shown in Figure 1 (Mosmann et al. 1986; Del
Prete et al. 1991). Naïve Th0 cells develop into Th1, Th2,
Th3 or T regulatory (Tr1) cells under the influence of
different cytokines present. Th1 cells produce
predominantly interferon (IFN)-γ and interleukin (IL)-2 and
play a central role in immune defence against intracellular
pathogens. Their development is stimulated by IL-12 which
is produced by dentritic cells and macrophages. Th2 cells
produce IL-4, IL-5 and IL-13 and are implicated in response
Food Science and Technology Bulletin: Functional Foods 1 (5) 1–12
DOI: 10.1616/1476-2137.12408. Published 25 July 2003
ISSN 1476-2137 © IFIS Publishing 2003. All Rights Reserved
5-2 From hypoallergenic foods to anti-allergenic foods A.C. Ouwehand, P. Kirjavainen, K. Laiho and S. Rautava

Figure 1. Balance between T-helper (Th)1 and Th2 cytokine production patterns. Naïve Th0 cells develop into Th1, Th2,
Th3 or T regulatory (Tr1) cells under the influence of different cytokines present. Th1 cells produce predominantly
interferon (IFN)-γ and interleukin (IL)-2 and play a central role in immune defence against intracellular pathogens. Their
development is stimulated by IL-12 which is produced by dentritic cells and macrophages. Th2 cells produce IL-4, IL-5 and
IL-13 and are implicated in response to helminthic infections. In human disease, the involvement of the Th2-type immune
response is well-established in allergic and atopic conditions, with IL-4 and IL-5 being major triggers for production of
immunoglobulin (IgE) and eosinophils, respectively. Th2 responses are counter-regulated by Th1 cytokines. Recently, novel
gut-originating immunomodulatory T cell subgroups, namely Th3 cells, which secrete transforming growth factor (TGF)-β,
and Tr1 cells, which secrete IL-10 have been described. TGF-β suppresses mainly the Th2 responses, while IL-10
stimulates Th2 responses. However, IL-10 also has strong anti-inflammatory properties and is consequently thought to
prevent allergic sensitisation and responses despite its Th2 stimulating effects.

Key: + modulation

— modulation
 From hypoallergenic foods to anti-allergenic foods A.C. Ouwehand, P. Kirjavainen, K. Laiho and S. Rautava
to helminthic infections. In human disease, the involvement
of the Th2-type immune response is well-established in
allergic and atopic conditions (Romagnani 2000), with IL-4
and IL-5 being major triggers for production of IgE and
eosinophils, respectively (Jabara et al. 1998; Pene et al.
1988). Th2 responses are counter-regulated by Th1
cytokines. Recently, novel gut-originating
immunomodulatory T cell subgroups, namely Th3 cells,
which secrete transforming growth factor (TGF)-β (Fukaura
et al. 1996), and Tr1 cells, which secrete IL-10 (Groux et al.
1997) have been described. TGF-β suppresses mainly the
Th2 responses (Lúdviksson et al. 2000), while IL-10
stimulates Th-2 responses. However, IL-10 also has strong
anti-inflammatory properties and is consequently thought to
prevent allergic sensitisation and responses (Wills-Karp et
al. 2001) despite its Th2 stimulating effects.
2.2 Atopic eczema/dermatitis
Atopic eczema or dermatitis is a chronic, relapsing clinical
skin condition characterised by dry pruritic skin lesions with
a typical age-specific distribution on the face, neck,
extremities and limbs (Hanifin 1991). Atopic eczema may
begin in infancy and often constitutes the first manifestation
of atopic disease. In infancy, atopic eczema may be a
symptom of food allergy (Sicherer and Sampson 1999).
Exposure to allergens also leads to aggravation of eczema in
allergic individuals later in life. The acute phase of atopic
eczema is accordingly characterised by Th2-type immune
responsiveness, whereas in chronic eczema the skin lesions
are classified by the Th1 response (Wollenberg and Bieber
2000). Thus, both antigen-IgE mediated and cellular
immune mechanisms are involved in the pathogenesis of the
disorder.
2.3 Asthma
Asthma is an inflammatory disease of the airways
characterised by bronchial hyper-responsiveness and a
variable degree of airway obstruction (Busse and Lemanske
2001). The T cells encountered in asthmatic inflammation
are predominantly of the Th2-type and typically involve
eosinophils (Busse 2001). Allergic mechanisms play an
important role in the pathogenesis of asthma, and allergy is
a major risk factor (Holt et al. 1999). It is well documented
that exposure to allergens and IgE mediated reactions are
associated with aggravation of asthmatic symptoms. The
role of sensitisation in the development of asthma, however,
is less clear (Lau et al. 2000).
2.4 Nasal and ocular allergic disease
The symptoms of allergic rhinoconjunctivitis, which include
swelling of the nasal mucosa and/or the conjunctivae,
watery discharge and sneezing, occur typically after
exposure to aeroallergens such as pollen and animal dander.
Pollen-induced symptoms are sometimes referred to as
seasonal rhinoconjunctivitis or, in lay terms, as hay fever.
5-3
The condition appears to most often be IgE-mediated, and
the inflammation on the nasal mucosa resembles that of the
bronchial epithelium of asthmatics (Durham 1998).
2.5 Food allergy
Food allergy, which is defined as an immunologically-
mediated hypersensitivity reaction to food, may manifest
itself by producing symptoms in various organs. The
immunological basis of food allergy reactions may be
exclusively IgE-mediated (immediate reactions), partially
IgE-mediated, or exclusively cell-mediated (non-IgE-
mediated) (Sampson 1999, 2001). The most frequent
manifestations include gastrointestinal (e.g. vomiting,
diarrhoea, constipation, abdominal pain), cutaneous (e.g.
urticaria, atopic eczema) and respiratory (e.g. itching,
swelling, wheezing) symptoms, although systemic
anaphylaxis may also occur (Sicherer 2002). Food allergy is
most prevalent in infants and children (approximately 6%
are afflicted). The most common allergens from which
humans suffer early in life include cows milk, eggs, wheat
and peanuts. Children tend to become tolerant to these
foodstuffs with age; however, 2% of the adult population
continues to suffer from some form of food allergy.
2.6 The aetiology of atopy and allergic disease
The epidemiological relationships between allergen
exposure, sensitisation, and clinical allergy have been
extensively studied, although the causal roles of exposure
and sensitisation in the development of disease remain less
understood (Isolauri et al. 2002). Without exposure,
sensitisation does not occur. Exposure can take place in
utero, as has been observed for tobacco smoke (von Mutius
2002). After birth, the main routes of exposure are via the
gut, through food, and by means of inhalation, although
exposure via dermal contact can also take place. The factor
that these exposure routes have in common is that they
affect the mucosal lymphoid tissue and thus also have an
effect on areas other than the site of exposure (Lloyd 2003).
The relationship between exposure and sensitisation does
not always appear to be linear but, in fact, may be bell-
shaped as with e.g. cat hairs (Platts-Mills et al. 2001).
Furthermore, there is epidemiological evidence indicating
that a pet in the family might actually protect the infant
from becoming atopic (Nafstad et al. 2001), even though
contradictory data also exist (Jaakkola et al. 2002). It may
be argued that prolonged sensitisation might be the result of
deviant, atopic type immune responsiveness, and not the
cause of it. In addition to these obvious allergens, some
allergens may be encountered in more unexpected places,
e.g. casein has been found to be a common allergen in
house dust (Mäkinen-Kiljunen et al. 2002). Such ‘hidden’
allergens may make avoidance difficult.
The immunological Th1/Th2 dichotomy offers a useful,
yet simplistic, paradigm for investigating the development
of allergies and atopy. Foetal and neonatal immune
responses are physiologically Th2-skewed (Piccinni et al.
5-4 From hypoallergenic foods to anti-allergenic foods A.C. Ouwehand, P. Kirjavainen, K. Laiho and S. Rautava
1998), and sensitisation to dietary antigens early in life is
common (Bergmann et al. 1994). In healthy infants, an age-
dependent decline in Th2 responsiveness takes place, but a
converse pattern has been observed in infants who go on to
develop atopy (Prescott et al. 1999). Thus, it may be that the
development of atopic disease could be the result of
defective anti-allergenic immune responses that counter-
regulate pro-allergenic Th2 responses. The so-called
hygiene hypothesis of allergy, first proposed by Strachan
(1989), suggests that reduced exposure to microbial stimuli
early in life is causally related to the development of
allergy. There are epidemiological data indicating an
inverse relationship between infectious disease early in life
and the prevalence of atopy (Matricardi et al. 2000; Kilpi et
al. 2002); it is assumed that contact with pathogens
stimulates the immune system towards Th1-type responses,
which effectively redirects the original Th2-skewed
responsiveness. Recently, growing emphasis has focused on
the intestine as an immunological organ with its own
distinct features, namely tolerogenic Th3 and Tr1 cells
(Isolauri et al. 2002). It has been pointed out that the
indigenous intestinal microbiota are a major source of
microbial contact (Monteleone and MacDonald 2001).
Furthermore, there are data which show that the close
interaction between the intestinal microbiota and the
intestinal immune system in healthy individuals results in
tolerogenic immune responses. Taken together, the hygiene
hypothesis of allergy may then be expanded to emphasise
the importance of intestinal microbiota and the gut immune
system, which may, as a consequence, surpass the Th1/Th2
paradigm of allergic disease (Rautava and Isolauri 2002).
3. Normal intestinal microflora
3.1 Development of the normal intestinal microbiota
In utero, the gastrointestinal tract of the foetus is sterile, but
upon birth it soon becomes colonised by microbes from the
birth canal and the environment. The first microbes to
colonise are facultative anaerobes. Within a few days, the
redox potential in the latter part of the small intestine and in
the colon has reduced sufficiently to allow obligate
anaerobes to dominate (Benno and Mitsuoka 1986). In
breast-fed infants, bifidobacteria have traditionally been the
predominant members of the colonic microbiota, while their
levels are low in formula-fed infants. However, due to
current hygienic obstetric practices, the colonisation level of
bifidobacteria in breast-fed infants has been reduced and
improvements in infant formulas have led to higher levels
of bifidobacteria in formula fed infants (Harmsen et al.
2000). Upon the introduction of solid food, the intestinal
microbiota starts to change and resembles that of an adult
by the age of 2 years. Throughout adult life, the overall
composition of the intestinal microbiota does not change
significantly. However, at old age the number of
bifidobacteria starts to decline (Mitsuoka et al. 1974).
Western dietary habits, which involve low intakes of
fermentable oligosaccharides, have led to relatively low
levels of bifidobacteria and increased levels of clostridia in
the gut (Benno et al. 1989). To counteract this, functional
foods containing probiotics and/or prebiotics have been
introduced onto the market.
3.2 Aberrancies in the composition of the gut
microbiota associated with allergies
The predominant site for host-microbe interactions is in the
gut; thus, it has been suggested that composition of the gut
microbiota may be the key determinant in whether or not an
atopic genotype will be fully expressed, which further
affects the development of allergic diseases. Therefore,
determining the characteristics of the microbiota in the gut
of allergic and healthy subjects may help in understanding
the aetiology of allergic diseases, and may point to potential
microbial targets for a probiotic or prebiotic therapy.
Moreover, assessing the properties of key microbes may
reveal direct immunological treatment targets, and thereby
the immunomodulatory properties that a probiotic strain
should have.
As illustrated in Table 1, differences have been observed
in the composition of the faecal microbiota of allergic
children compared to similarly aged healthy children.
However, from these observations it is not clear whether
these differences are the cause or the result of allergy. To
resolve this matter, various prospective studies have been
performed (Björkstén et al. 2001, Kalliomäki et al.2001a).
Recent studies have already demonstrated differences in
faecal microbiota prior to the beginning of the expression of
atopy and allergic diseases, suggesting that these differences
may not be the result of the disease. In a study by our group,
some of the predominant microbial genera were enumerated
with fluorescently labelled 16S rRNA-specific
oligonucleotide probes (Kalliomäki et al. 2001a). Neonatal
faecal microbiota preceding the expression of atopy were
characterised by the presence of lower numbers of
bifidobacteria and higher numbers of clostridia than were
found in the faeces of infants who did not develop atopy
(Kalliomäki et al. 2001a). Björkstén and co-workers (2001)
demonstrated similar trends using culture based methods.
They found that fewer bifidobacteria and enterococci and
more lactobacilli were present in the neonatal faecal
microbiota of 2-year-old infants with atopic eczema (n =
18), than in the microbiota of symptomless infants. Later
during the first year of the study, there was also a higher
prevalence of Staphylococcus aureus and clostridia, and
lower numbers of Bacteroides, in the faecal microbiota of
infants who exhibited atopy at 2 years of age than in healthy
2-year-old subjects.
 From hypoallergenic foods to anti-allergenic foods A.C. Ouwehand, P. Kirjavainen, K. Laiho and S. Rautava
Table 1. Differences in the faecal microbiota
composition of allergic children compared to healthy
age matched children
Differences
Microbiota component in allergic Reference
‡
vs. healthy
Lactobacilli Ð Björksten et al. 1999
Coliforms Ï Björksten et al. 1999
Staphylococcus aureus Ï Björksten et al. 1999
Bacteroides Ð Björksten et al. 1999
Klebsiella Ï Kirjavainen et al. 2001
Streptococcus Ð Kirjavainen et al. 2001
Bifidobacteria* Ð Kirjavainen et al. 2001
Bifidobacterium adolescentis Ï Ouwehand et al. 2001
Bifidobacterium bifidum Ð Ouwehand et al. 2001
Gram positives Ð Kirjavainen et al. 2001
‡
Ð lower in allergic than in healthy children; Ï higher in allergic than in
healthy children
*In infants with gastrointestinal symptoms
3.3 Th1/Th2 balance
Bacteria encountered after birth are thought to be a major
trigger for the shift away from the predominant Th2
response, as there appears to be a universal tendency for
bacteria to stimulate Th cell differentiation into Th1 effector
cells. However, the responses triggered by bacteria may be
variable, e.g. an in vitro study suggests that Gram-positive
bacteria tend to be more potent inducers of IL-12 than their
Gram-negative counterparts, which were shown to induce
more IL-10 (Hessle et al. 2000). The induction of IL-12 and
IL-10 production in vitro has been also been shown to vary
between different Lactobacillus strains (von der Weid et al.
2001; Christensen et al. 2002). IL-12 is produced, for
example, by dendritic cells and macrophages, and is a key
cytokine promoting Th cell differentiation into Th1 cells
(Figure 1).
3.4 Formation of oral tolerance
The importance of microbial stimuli for normal formation
of oral tolerance is best demonstrated in a study by Sudo
and co-workers (1997) in which it was shown that Th2-
mediated humoral responses, but not Th1-mediated
responses, of germ-free mice were not susceptible to the
induction of oral tolerance; however, after the mice were
colonised with a single Bifidobacterium infantis strain,
tolerance was inducible. This may reflect microbial
induction of cytokine production promoting active
suppression and is supported by the observation that
treatment of allergic infants with bifidobacteria resulted in
increased serum concentrations of TGF-β. In addition, an in
vitro study showed that different Lactobacillus strains
5-5
provided different degrees of stimulus for the formation of
low proliferating Th cell populations producing TGF-β and
IL-10 (Von der Weid 2001).
4. Inflammation
As described above, there is a universal tendency to mount
Th1-mediated pro-inflammatory responses to Gram positive
bacteria, in particular. Such reactions would be wasteful and
detrimental if they were mounted against commensal gut
microbes. Therefore, the immune system has adapted and
recognises and tolerates the harmless components of the gut
microbiota. At the same time, the immune system monitors
composition of the intestinal microbiota via active uptake,
and mounts appropriate responses when potentially
dangerous organisms are encountered (Nagler-Anderson
2001). Moreover, pathogens may have a dual role with
regard to allergic inflammation. As they do not induce
tolerance, they may have greater potential to stimulate
immunity away from Th2-type responses than commensal
bacteria, towards which tolerance already exists
(Kirjavainen et al. 1999; Neish et al. 2000). Conversely, by
triggering inflammatory responses or by formation of
toxins, such bacteria may increase gut permeability and thus
exposure to potential allergens, as well as the risk for
allergic sensitisation (Lapa e Silva et al. 2000; Neish et al.
2000). Restraining potentially inflammatory microbiota
could, therefore, provide a potential target for probiotic
therapy (Kirjavainen et al. 1999; Madsen et al. 1999).
Indeed, in our recent study, it was shown that the allergic
inflammation-alleviating effects of bifidobacterial
supplementation during weaning were accompanied by
restraining the numbers of Bacteroides and Escherichia coli
microbiota, which correlated directly with total serum IgE
concentrations during breast-feeding (Kirjavainen et al.
2002).
Bifidobacteria and lactobacilli are antagonistic against
many pathogens due to competitive exclusion, production of
antimicrobial components (Lievin et al. 2000; Gibson and
Wang 1994) and stimulation of IgA production (Kaila et al.
1992; Majamaa et al. 1995). The latter effect can also
reduce intestinal inflammation by enhancing the exclusion
of allergens and reducing the risk of hypersensitivity
reactions to such allergens in already sensitised subjects
(Takahashi et al. 1998; Isolauri et al. 1993). As discussed
above, specific microbes may also reduce the wide range of
inflammatory responses by stimulating production of IL-10
and TGF-β and, therefore, antigen non-specific bystander
suppression (He et al. 2000; Pessi et al. 2000; Isolauri et al.
2000).
The degradation process is another defining parameter
with regard to inflammatory responsiveness mounted
towards dietary antigens (Barone et al. 2000; Isolauri et al.
1993). Most antigens encountered are already processed
when they contact the mucosal surface. Proteases produced
by the intestinal microbiota contribute to the processing of
food antigens in the gut and modify their immunogenicity.
5-6 From hypoallergenic foods to anti-allergenic foods A.C. Ouwehand, P. Kirjavainen, K. Laiho and S. Rautava
Such activity may also be utilised in probiotic therapy. This
has been demonstrated by the finding that purified casein
up-regulates IL-4 and IFN-γ production in infants allergic to
cows’ milk; however, caseins degraded by enzymes derived
from a probiotic bacterium, Lactobacillus GG, were shown
to further down-regulate IL-4 production, with no effect on
IFN-γ release (Sütas et al. 1996).
5. Current prevention strategies and treatment of
allergy
The continuing rise in prevalence of allergies and atopic
disease in the industrialised world has emphasised the
importance of primary prevention (Wahn and von Mutius
2001). As sensitisation has already begun in utero and the
allergic march may begin in early infancy, it is clear that
preventive measures should ideally be taken either
prenatally or during the first months of life. In support of
this concept, the indigenous intestinal microbiota is formed
and the original Th2-skewed immunity is redirected towards
a tolerogenic mode during this period. However, no specific
means of primary prevention are currently available.
Although allergen avoidance has been studied and practised
extensively, the results are far from conclusive and its
usefulness in prevention is uncertain (Schoetzau et al.
2001). This may be explained by our incomplete
understanding of the role of exposure and sensitisation in
the development of allergic disease. Epidemiological
studies have shown that non-smoking by carers and
exclusive breastfeeding during the first months of life have
both emerged as protective factors which may be beneficial
(Kulig et al. 1999; Gdalevich et al. 2001a, 2001b). Hence,
effective, safe and inexpensive preventive means are needed
to counter the epidemic of allergic disease.
Allergen avoidance is the cornerstone of treatment of
allergic disease (Kay 2001). In some cases, particularly with
food allergens, this may be relatively easily achieved.
However, food allergies may be debilitating socially and,
especially in infants and children with allergies to basic
foodstuffs, attention must be paid to the nutritional
consequences of a strict elimination diet. In this respect,
nutritionally valid hypoallergenic formulas are
commercially available for infants with cow’s milk allergy.
In addition to ‘traditional’ hypoallergenic foods, genetic
manipulation has made it possible to produce
hypoallergenic rice by blocking the production of selected
allergenic proteins (Nakamura and Matsuda 1996),
indicating that functional foods may also have a role here.
In certain cases, allergen avoidance may be virtually
impossible, as is the case with, for example, pollen allergy.
In such instances, symptom relief and suppression of
allergic inflammation are accomplished pharmacologically
with, for example, antihistamines.
6. Functional foods
6.1 What are functional foods?
The main function of food is to provide nutrition, i.e. to be a
source of energy and nutrients. In this sense, all foods may
be described as functional. There is, therefore, a need to
clearly define functional foods, in order to distinguish them
from regular foods. A food can be regarded as being
functional if it satisfactorily affects one or more target
functions in the body in a beneficial manner, which is
beyond adequate nutrition and results in a state of wellbeing
and good health or serves to reduce the risk of a disease
(Diplock et al. 1999). Thus, a functional food is a food from
which a harmful component, e.g. an allergen, has been
removed, or a beneficial component has been added
(Salminen et al. 2001). As an area of research, the field of
functional foods is still relatively new, having begun in the
early 1980s in Japan, and then spreading to Europe and the
US. One could argue that fortified foods such as salt that
has been supplemented with iodine, or wheat flour that has
been supplemented with iron are the precursors of
functional foods. However, these foods are intended to
treat/prevent deficiencies and not to target a specific
biological function in the body.
The four main types of functional foods that will be
discussed here are probiotics, prebiotics, fatty acids and
antioxidants. The first two specifically target the
composition and activity of the intestinal microflora, while
the latter two directly influence metabolism.
6.2 Probiotics
A common method of preventing allergy or reducing its
symptoms is the avoidance of the allergen concerned.
However, this approach has yielded only limited success
(Isolauri 1995). Instead of removing allergens from foods
and the environment, an alternative approach is to adapt the
immune system and to provide it with an appropriate
stimulus, since a lack of microbial stimulation of the
immune system during infancy has been suggested to cause
allergic disease, as already discussed above (Strachan
1989). For obvious reasons, appropriate levels of hygiene
must be maintained and vaccinations should not be
abandoned; however, an alternative source of microbes
could be provided by probiotics (Cross and Gill 2001;
Kirjavainen et al. 2002). Probiotics have been defined in
many ways (Salminen et al. 1999; Schrezenmeir and de
Vrese 2001) but can probably be best considered as
microbes that have a beneficial effect on the health of the
consumer (Fuller 1989). The most common probiotics are
lactobacilli and bifidobacteria, although members of other
genera are used as well. As will be discussed below,
selected lactobacilli (Majamaa and Isolauri 1997) and
bifidobacteria (Isolauri et al. 2000) have been shown to aid
in the management of atopic disease and to reduce the
incidence of atopic disease in at-risk infants (Kalliomäki et
al. 2001b), thus providing a prophylactic effect.
 From hypoallergenic foods to anti-allergenic foods A.C. Ouwehand, P. Kirjavainen, K. Laiho and S. Rautava
6.3 Prebiotics
Prebiotics are non-digestible food ingredients that
beneficially affect the host by selectively stimulating the
growth and/or activity of one or a limited number of
bacteria in the colon, and thus improving host health
(Gibson and Roberfroid 1995). Dietary fibre is sometimes
erroneously considered to be a probiotic; although both are
non-digestible, dietary fibre will not necessarily have a
beneficial effect on the activity or composition of specific
members of the intestinal microbiota. The efficacy of
prebiotics is less well established than that of probiotics,
with the best established effect being an increase in the
numbers of endogenous bifidobacteria or lactobacilli. This
change in microflora composition and activity may lead to a
modulation of the immune response; however, this has
received little attention so far (Roberfroid 2000). Animal
studies suggest that selected prebiotics such as
fructooligosaccharides (Field et al. 1999), arabinogalactan
(Grieshop et al. 2002), mannan, pectin and chitosan (Lim et
al. 1996) may alter the immune response. The level of IgA
secretion can be enhanced by prebiotics (Lim et al. 1996)
and the number of Peyer’s patches increased. (Pierre et al.
1997). Preliminary data from human studies suggests that
fructooligosaccharides may reduce the inflammatory
responses (Guigoz et al. 2002). However, these
observations require further investigation in order to be
substantiated. In addition, it is not known to what extent this
prebiotic-induced immune modulation can contribute to the
treatment or prevention of allergies.
6.4 Do nutrients have a role, other than nourishment,
in allergic disease?
Recent studies on the immunomodulatory properties of fatty
acids and antioxidant properties of certain nutrients
including ascorbic acid, α-tocopherol, β-carotene, selenium
and zinc, suggest that the role of these nutrients in the onset
and management of allergic disease is more extensive than
previously anticipated. In terms of allergic disease, foods
and nutrients, particularly proteins, have previously been
considered only as a source of antigens that may cause
allergic sensitisation and the symptoms of allergic disease.
Instead, via their immunomodulatory and antioxidative
properties, certain nutrients may manifest protective
properties by silencing or preventing allergic inflammation.
Furthermore, certain nutrients, including glutamine, may
protect the largest defensive organ of the human body,
namely the gastrointestinal tract, by its ability to maintain
and repair gut mucosa (Duggan et al. 2002).
Dietary antioxidants, as well as cellular enzyme-based
antioxidants, may counteract oxidative stress resulting from
allergic inflammation and balance the impaired homeostasis
of oxygen radicals (Greene 1999; Omata et al. 2001).
Antioxidants may thus be important in facilitating the
ability of an individual to restrain the inflammatory
response, although the role of antioxidants in the prevention
of allergic disease currently remains obscure. Some
5-7
evidence has been accumulated to suggest that antioxidant
deficiencies may be associated with the symptoms of
allergic disease. Low intake of antioxidants has been
associated with bronchial reactivity and the risk of asthma
(Soutar et al. 1997) and low concentrations of plasma
antioxidants (β-carotene, ascorbate and α-tocopherol) have
been recorded in wheezing illness (Bodner et al. 1999).
It is acknowledged that fatty acids have properties that
may contribute to cellular immunomodulation.
Polyunsaturated n-6 series fatty acids derived from dietary
linoleic acid (18:2, n-6) result in the production of
eicosanoids, which are considered to have proinflammatory
properties, whilst n-3 series fatty acids derived from dietary
α-linolenic acid (18:3, n-3) appear to have anti-
inflammatory properties (Sellmayer and Koletzko 1999).
Due to the typically higher dietary intake of linoleic acid
than α-linolenic acid in developed countries, it is thought
that metabolism of linoleic acid predominates. Since dietary
fatty acid composition, in turn, influences cell fatty acid
composition (Whelan 1996), it could be suggested that an
unbalanced dietary fatty acid intake, particularly between
series n-6 and n-3 fatty acids, may result in the modulation
of inflammatory responses that may be significant for the
development of allergic disease. Indeed, an abnormal
cellular fatty acid composition has been observed in atopic
patients (Biagi et al. 1993; Leichsenring et al. 1995; Yu et
al. 1998). Whether the observed alterations in the fatty acid
composition of cells in such patients result from a primary
defect that contributes to the onset of atopic disease or is a
consequence of atopic disease itself, is currently poorly
understood. However, it may be argued that the fatty acid
composition of the diet may impact upon the increased
prevalence of allergic disease.
6.5 Combinations of functional ingredients
The interactions between probiotics and other functional
food ingredients are largely unknown, with synbiotics
(combinations of probiotics and prebiotics) mainly being
investigated. However, the extent to which the
immunomodulating activity of a synbiotic preparation
distinguishes itself from a probiotic or a prebiotic is not
known, although it could be anticipated that the
performance of probiotics may be improved by the addition
of prebiotics, since certain prebiotics have been shown to
improve the survival of probiotics (Bezkorovainy 2001).
However, is not the case for all synbiotics (Alander et al.
2001).
Antioxidants such as ascorbic acid, do not affect the
survival and performance of probiotics (Dave and Shah
1997). Interestingly, some strains of Lactococcus,
Lactobacillus and Bifidobacterium have their own
antioxidant capacities (Lin and Yen 1999; Stecchini et al.
2001). Several strains of lactobacilli, streptococci,
lactococci and propionibacteria have also been observed to
increase levels of conjugated linoleic acid (Lin et al. 1999),
which has been reported to promote good health. However,
a combination of probiotics and certain polyunsaturated
5-8 From hypoallergenic foods to anti-allergenic foods A.C. Ouwehand, P. Kirjavainen, K. Laiho and S. Rautava
fatty acids has been suggested to be detrimental to the
particular probiotic by altering its viability and adhesion
properties (Kankaanpää et al. 2001). These reports indicate
that properties of new functional food formulations need to
be carefully assessed to ensure efficacy of the ingredients in
combination with other components.
7. Potential of functional foods for allergic
individuals
Probiotics are thought to exert their beneficial effects by
normalising increased intestinal permeability, improving
intestinal barrier function, restoring a healthy composition
of gut microbiota, degrading antigenic structures in the gut
lumen, and providing the intestinal immune system with
tolerogenic stimuli (Rautava and Isolauri 2002). All of these
factors have been implicated in the aetiology and
pathogenesis of atopy and allergic disease, and thus offer a
number of potential targets for functional foods and, more
specifically, for probiotic therapy for prevention and
treatment of these conditions. As aberrations in the
composition of the gut microbiota appear to precede the
development of atopic disease (as discussed above),
alteration of the gut microecology is likely to have an effect
on disease development. Results from a double-blind,
placebo-controlled trial evaluating the effect of probiotics in
prevention of atopic disease offer corroboration for this
concept. Administering Lactobacillus GG prenatally and
during the first months of life to high-risk infants resulted in
a significant reduction in the prevalence of atopic eczema at
the age of two years (Kalliomäki et al. 2001b).
Interestingly, no significant reduction in atopic sensitisation
was observed in the study, and it may be argued, therefore,
that the protective mechanism of probiotics in this case is
not associated with atopic sensitisation. However,
Lactobacillus casei Shirota has been demonstrated to
suppress IgE responses and systemic anaphylaxis in a
murine model of food allergy (Shida et al. 2002). Further
elucidation on the subject was obtained from a recent study
in a subgroup of mother-infant pairs in whom the duration
of breastfeeding exceeded three months. Maternal intake of
probiotics resulted in a more than two-fold concentration of
anti-allergenic TGF-β in breastmilk (Rautava et al. 2002). A
previous study has indicated that a low concentration of
TGF-β in maternal milk is associated with higher risk of
atopic disease in the infant (Kalliomäki et al. 1999).
In a randomised, double-blind study, extensively
hydrolysed whey formula supplemented with
Bifidobacterium lactis Bb12 or Lactobacillus GG both
resulted in more effective eczema alleviation, compared to
unsupplemented formula in infants with atopic eczema
(Isolauri et al. 2000). Furthermore, in infants with atopic
eczema and cow’s milk allergy, a hypoallergenic formula
supplemented with Lactobacillus GG, but not
unsupplemented formula, was associated with symptom
relief and, perhaps more importantly, with suppression of
intestinal inflammation (Majamaa and Isolauri 1997).
However, administrating Lactobacillus GG to individuals
suffering from pollen allergy offered no benefit (Helin et al.
2002). In this study, the subjects were teenagers and young
adults. It may be argued that probiotics exert their effect(s)
on the developing immune system at an early age and have
less potential to exert their effect(s) on fully established
atopic disease in older individuals.
The probiotic-supplemented formulas used in the studies
cited above might be regarded as prototype anti-allergenic
functional foods for allergic individuals. It must be
emphasised, though, that atopic diseases are a
heterogeneous group of disorders and that the probiotic
performance of different strains of bacteria varies.
8. Future prospects
The development of functional foods for allergic subjects
requires new criteria for the selection of appropriate
ingredients. In order that probiotics provide a safe means of
ensuring sufficient microbial stimulation of the immune
system, specific strains specially selected for this particular
use may be required. In addition, genetically modified
probiotics evincing improved or added functional properties
could provide an important new possibility. Such strains
could be engineered to produce anti-inflammatory cytokines
(Steidler et al. 1998). New designer prebiotics aimed at
specific species of the intestinal microflora, which have
additional functions such as inhibition of pathogen binding
sites, may prove useful in future treatments. However, our
knowledge on the cascade of events consisting of exposure,
sensitisation, and hypersensitivity is by no means complete.
A more profound understanding of the complex nature of
atopy and atopic disease is needed, as it is likely that there
are distinct etiological factors underlying the heterogeneous
manifestations of the disorder. It would thus appear
simplistic and erroneous to assume that one mode of
prevention or treatment would be sufficient for the plethora
of allergic diseases. Therefore, rigorous scientific effort is
required to elucidate the characteristics of distinct probiotic
strains in different manifestations of atopic and food
hypersensitivity disorders, as there appears to be no
universal solution to these complex phenomena. This will
lead to further development of foods that may treat and
prevent allergic disease. Instead of hypoallergenic foods,
foods are likely to be developed that provide the necessary
and safe modulation of the immune system.
9. Conclusions
Due, at least in part, to changes in western lifestyles,
allergic diseases are increasing. In particular, probiotics
have been shown to be promising in treating and preventing
allergic diseases, while other functional foods may provide
similar benefits. However, rigorous scientific effort is
needed to elucidate potential targets and appropriate
components for anti-allergic therapy using functional foods,
as it is conceivable that distinct products will be needed to
treat different manifestations of atopic disease in different
 From hypoallergenic foods to anti-allergenic foods A.C. Ouwehand, P. Kirjavainen, K. Laiho and S. Rautava
age groups.
10. Acknowledgements
Financial support for much of the work discussed here was
obtained from the Academy of Finland.
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Canine functional foods

George Tzortzis*, Glenn R Gibson and Robert A Rastall

School of Food Biosciences, The University of Reading, PO Box 226, Whiteknights, Reading RG6 6AP, UK.
Email G.Tzortzis@Reading.ac.uk

*Corresponding author

Abstract

The most recent research in food bioscience supports the hypothesis that diet, in addition to providing enough
nutrients to meet the body’s requirements, also controls and modulates various functions in the body and, in
doing so, contributes to the good health of the host. An important component of this hypothesis is the concept of
functional food which, in recent years, has become very popular. Because of its resident microbiota, the
mammalian colon is one of the body’s most metabolically active organs, where undigested food materials are
predominantly fermented by gut bacteria. The use of diet to fortify gut flora components is an important aspect
of the functional food concept, especially since the nature of gut bacterial fermentation may have different
health consequences. This review provides an overview of how dietary supplementation of dietary fibre,
probiotics, prebiotics, or synbiotics may contribute toward nutritional modulation of canine gut microbiology.

Keywords: canine, dietary fibre, probiotic, prebiotic, synbiotic

1. Introduction act in different ways. One attempt to categorise them is

During the twentieth century, nutritional standards, dietary
guidelines and food information guides have been
established mainly with the aim of preventing specific
deficiencies as well as supporting body growth,
maintenance and development. Advice has also been given
to eliminate or reduce certain ingredients, such as fat, sugar
and salt in the diet in order to lower cholesterol levels,
decrease calorific intake and protect against hypertension,
respectively. More recently, however, the focus has shifted
to the achievement of a balanced diet and maximisation of
both life expectancy and quality by identifying food
ingredients which improve the capacity to resist disease and
enhance health (Roberfroid 2000). This is the concept
surrounding functional foods, which are defined as ‘a
dietary component that may exert physiological effects on
the consumer which may eventually lead towards justifiable
health claims’ (Roberfroid 1996).
There has been great interest in using such approaches,
which were largely developed for humans and livestock, in
pet food applications, particularly those intended for dogs.
The fact that a dog’s diet can be maintained and controlled
makes domestic animals ideal for the application of
functional foods in their diet.
described below:
• Cholesterol reducing compounds
• Antioxidants
• Phytochemicals
• Dietary fibre
• Probiotics, prebiotics and synbiotics.
Several of these ingredients are already being manufactured
or researched within the pet food industry.
2.1 Cholesterol reducing compounds
Omega-3-fatty acids are believed to inhibit the absorption of
cholesterol and are thought to be a protective factor against
cardiovascular disease. Billman et al. (1994, 1999) reported
a protective effect of ω3 fatty acids against exercise- and
ischemia-induced ventricular fibrillation in dogs known to
be susceptible to sudden death. Consumption of diets
enriched with ω3 fatty acids has also been shown to
decrease the inflammatory response in dogs (Albina et al.
1993; Vaughn et al. 1994).

2. Functional food types

There is a huge range of foods that can be labelled as
functional, which claim to have various types of benefit and Food Science and Technology Bulletin: Functional Foods 1 (6) 1–10
DOI: 10.1616/1476-2137.12654. Published 5 November 2003
ISSN 1476-2137 © IFIS Publishing 2003. All Rights Reserved
6-2 Canine functional foods G. Tzortzis, G. R. Gibson and R. A. Rastall
2.2 Antioxidants
Oxidation and the concomitant production of free radicals
are an integral part of central metabolism. However, free
radical imbalance can lead to lipid peroxidation, cell death
and genetic damage as a result of the extremely reactive
nature of free radicals (Halliwell 1997). Plant and animal
cells deploy antioxidant compounds to trap or quench free
radicals as a defence mechanism against such damaging
reactions. Dietary antioxidants include vitamin E, carotene,
vitamin C and certain phytochemicals. Dietary
supplementation with antioxidants has been reported to
protect against DNA damage, leading to improved
immunological performance (Heaton et al. 2002), retarded
the ageing (Gaal et al. 1996) and increased reproductive
function (Weng et al. 2000) in dogs.
2.3 Phytochemicals
Plant foods are rich in micronutrients but also contain a
variety of biologically active, non-nutritive secondary
metabolites such as glucosinolates and phenolic compounds
(flavonoids) which are very effective antioxidants. Raw
onion (Briggs et al. 2001) and grape juice (Osman et al.
1998) are reported to contain such flavonoids which have
been shown to inhibit platelet aggregation and therefore
protect against development of coronary artery disease and
acute occlusive thrombosis in dogs.
2.4 Dietary fibre
Dietary fibre has been found to participate in the regulation
of gastrointestinal (GI) motility, as well as influence
glucose and lipid metabolism (Cherbut et al. 1995;
Schweizer and Edwards 1992). It is also involved in
promoting faecal output, stimulating gut bacterial metabolic
activity, and contributing towards maintaining the
equilibrium of the colon ecosystem (Cherbut et al. 1995;
Schweizer and Edwards 1992). In this respect, dietary fibre
fits the definition of a functional food by the fact that it can
affect one or more targeted functions in the body in a
positive manner (Diplock et al. 1999).
2.5 Probiotics, prebiotics and synbiotics
Probiotics had originally been developed as feed
supplements to improve breeding performance of farm
animals (Lestradet 1994) and therefore may be of benefit to
dogs as external factors are known to affect their intestinal
microflora (Biourge et al. 1998). The prebiotic approach
targets selected indigenous probiotic bacteria, mainly
bifidobacteria and lactobacilli, using non-viable food
ingredients, while synbiotics exploit the synergistic effect of
probiotics and prebiotics.
3. Colon-targeted functional foods
The sector of the functional food market that has attracted
the most interest from the pet food industry in recent year is
foods aimed at improving gut health. The microbial
community inhabiting the GI tract is characterised by its
high population density, species diversity, and complexity
of interactions that take place within the microbial
ecosystem. This complex microbial flora makes the gut the
most metabolically adaptable and renewable organ of the
body and plays a vital role in the normal nutritional,
physiological, immunological and protective functions of
the host animal (Mackie et al. 1997).
3.1 The canine gut microflora
Few papers have been published on the normal canine
intestinal microflora (Balish et al. 1977; Benno and
Mitsuoka 1989; Benno et al. 1992). Most of the reported
studies have been carried out using Beagles as experimental
models, mainly due to their medium size, good
temperament and the fact that their GI tract and associated
organs are similar to larger breeds such as Labradors
(Andersen 1970). These reports demonstrated that canine
intestinal microflora is diverse and consists of various
aerobic, facultative, and anaerobic bacteria, as well as
several types of yeasts and other fungi (Zentek 2000).
Research (Willard et al. 1994; Terada et al. 1992) utilising
German Shepherds, suggests that the bacterial genera
detected showed little to no difference between dog breeds,
with the exception of clostridia being slightly elevated in
German Shepherds. It has also been reported that numbers
of some bacterial species such as Bifidobacterium spp. can
change drastically from one group of animals to another,
while numerical differences based upon age of animal were
limited to some Bacteroides spp. Other reports (Benno and
Mitsuoka 1989, 1992) have shown an effect of age of
animal on faecal microbial profiles. Lactobacilli,
Bacteroides, peptostreptococci and bifidobacteria counts
were lower, while clostridia and bacilli numbers were
elevated in older compared to younger dogs. Recent work
(Simpson et al. 2002) reported total anaerobic bacterial
counts per gram of faeces averaging 4x1010, while total
aerobes numbered 2x109. The major bacterial groups
included Bacteroides, fusobacteria, lactobacilli and
streptococci. The enterococci, clostridia, bifidobacteria and
eubacteria, on the other hand, were less predominant,
occurring in numbers of 107/g faeces.
A major problem with all of these studies, however, is
that they were based on cultivation-dependent enumeration
with biochemical characterisation techniques. This approach
fails to reflect much of the diversity present in faecal
samples, as selective media, particularly those designed for
strict anaerobes, show extremely poor selectivity with the
microorganisms found in the canine GI tract (Greetham et
al. 2002). Using a molecular DNA fingerprinting technique
(Denaturing Gradient Gel Electrophoresis, DGGE),
Simpson et al. (2002) reported a tendency for dog’s gut
microflora to cluster together according to age (young or
old) and size (large or small). They concluded that, based on
DGGE analysis of faecal bacterial profiles, individual dogs
 Canine functional foods G. Tzortzis, G. R. Gibson and R. A. Rastall
had their own unique and stable characteristic banding
pattern. Although differences could be detected by the
presence or absence of bands as well as the intensity of the
bands, there were common, possibly dominant bacterial
species present in more than one individual.
The bacteria in the canine GI tract are not evenly
distributed; their diversity and numerical importance vary in
the different sections of the GI tract, where pathogenic,
benign and beneficial microbial species (e.g. bifidobacteria,
lactobacilli) are to be found, forming a closely integrated
ecological unit with the host. The proximal human colon is
comparatively nutrient-rich and bacterial growth rates are
high in this region. In contrast, the distal colon is nutrient-
limited, as the readily available carbohydrates substrates
have already been utilised. The shorter overall length of the
colon in cats and dogs, in relation to body size, compared to
humans, suggests there may be less distinction between
different regions of the large intestine and that gradients
from proximal to distal colon may not be so large (Burger
1993).
3.2 Fermentation processes in the canine gut
Because of the species diversity and metabolic capabilities
of the microflora, fermentation in the gut is a complex
process, where the metabolic end-products exerted by one
species serve as a growth substrate for another (Hudson and
Marsh 1995). Most of our knowledge of such processes is
derived from human studies. Growth substrates for colonic
bacteria include proteins, amino acids, bacterial secretions,
lysis products and mucins, although the principal substrates
are carbohydrates (Cummings and Macfarlane 1991). In the
human diet, these carbohydrates consist mainly of starches
and dietary fibre, together with oligosaccharides that have
escaped digestion in the upper GI tract. Such materials are
initially degraded by bacterial endo-glycosidases, proteases
and amino-peptidases to form smaller oligomers and their
sugar and amino acid components. These intermediates are
then fermented to short chain fatty acids (SCFA), organic
acids, gases and other end-products by colonic bacteria
(Gibson and Macfarlane 1995). The end-products of
microbial fermentation and digestion in the canine colon,
with respect to SCFA production (Banta et al. 1979), have
been established as acetate, propionate and butyrate,
representing 63, 26 and 11% of SCFA, respectively
(Swanson et al. 2002). SCFAs have been reported to
stimulate in vivo the propulsive motility in the ileum of the
dog (Kamath et al. 1992; Fich et al. 1989) but apparently
not in the canine colon (Flourie et al. 1989). Recent studies
(McManus et al. 2002) suggest that endogenous SCFA may
contribute more to the overall muscular tone of the colon.
Canine enterocytes and colonocytes readily oxidise
butyrate, propionate, glucose and glutamine (Drackley et al.
1998). Luminal butyrate is a potential energy source for
enterocytes and colonocytes since it is not affected by the
presence of glutamine, β-hydroxybutyrate, acetate or
propionate (Drackley et al. 1998), therefore a diet
promoting butyrate production is advantageous to dogs.
6-3
Malabsorbed fermentable material is converted to
hydrogen by multiple species of colonic bacteria
(Macfarlane and Cummings 1991). This hydrogen can be
absorbed, excreted in the breath, passed through the rectum,
or utilised by other bacterial species (Gibson et al. 1991),
affecting the environment or the pathological condition of
the colon (Levitt et al. 1995).
Among possible end-products, those resulting from
saccharolytic gut fermentation can be seen as benign or
health promoting, while metabolites resulting from protein
and amino acid fermentation, such as amines, ammonia and
phenolic compounds, can be harmful (Macfarlane et al.
1986; Smith and Macfarlane 1996). Under certain
circumstances (antimicrobial intake, stress, poor diet), the
microbial balance can be perturbed, resulting in the
production of undesirable fermentation metabolites such as
phenolic compounds, toxins, etc (Bengmark 1998); this can
result in acute or chronic gut problems.
Since the colonic microbiota is susceptible to
manipulation through diet (Salminen et al. 1998; Amtsberg
et al. 1980), the aim of colonic functional foods is to
maintain or improve the GI microflora composition and
activities. Dietary components used in this sense have
included dietary fibre, probiotics, prebiotics and synbiotics.
3.3 Dietary fibre
The beneficial effect of dietary fibre emerged in the 1970s
from medical workers studying the relationship between the
human diet and the incidence of chronic disease, with
particular reference to the role of polysaccharides in the diet
(Walker 1974; Burkitt and Trowell 1975).
In the past, all types of fibre were assumed to have a
similar effect on the GI tract, with emphasis being given to
increased dietary fibre intake. The re-classification of fibre
on the basis of solubility and fermentability has changed
opinions on this subject. Soluble fibre has high water-
holding capacity, forms gels, increases luminal viscosity
and is easily degraded by colonic bacteria, resulting in
significant production of short chain fatty acids (Davidson
and McDonald 1998). On the other hand, insoluble fibre has
little water content, is only partially degraded by colonic
bacteria and increases faecal bulk (Davidson and McDonald
1998). Because fibre sources contain different insoluble:
soluble fibre ratios, their rate and extent of fermentation are
different resulting in different positive effects to the host.
The fermentative capacity of the canine gut is generally
thought to be limited because of the anatomical features
(e.g. a small caecum, an unsacculated large intestine) of the
GI tract as well as the carnivorous nature of the canine diet
in the wild. Domesticated canine species, however, often
consume diets containing ≥35% starch (Fahey et al. 1990a).
Research has indicated that up to approximately 63% of
fibre can be digested by dogs, depending on its source
(Fahey et al. 1990b, 1992). Sunvolt et al. (1995) evaluated
the influence of dietary fibre source in canine diets on the
fermentative activity of their faecal microflora. Dogs were
fed a diet containing either a fermentable fibre (citrus pulp)
6-4 Canine functional foods G. Tzortzis, G. R. Gibson and R. A. Rastall
or a non-fermentable fibre (Solka Floc®); faecal samples
from each dog were used to inoculate selected fibrous
substrates in order to determine organic matter
disappearance (OMD) and SCFA production. The
fermentable fibre showed higher OMD and faster
fermentation, calculated as acetate to propionate ratio in
vivo, while in vitro, increased fermentative activity of the
intestinal microflora was characterised by a wide diversity
in fermentation patterns among substrates.
Swanson et al. (2001) studied fermentability of fruit and
vegetable fibre by canine microflora in vitro using faecal
samples. They also reported a wide variation in OMD,
SCFA and gas production, which were related to the
insoluble:soluble fibre ratio in the samples, with OMD,
SCFA and gas production increasing as the
insoluble:soluble fibre ratio decreased.
The influence of fibre fermentability on nutrient
disappearance at the terminal ileum and through the
digestive tract was evaluated in dogs surgically fitted with
ileal T-cannulas (Silvio et al. 2000). Dogs were fed two
types of fibre, a slowly fermented fibre (cellulose) and a
soluble, rapidly fermented fibre (pectin) in different
increments. The study showed that, by increasing
fermentable fibre, the digestion of dry matter (DM) and the
energy available increased, although crude protein
digestibility was adversely affected. The reduced crude
protein digestibility was attributed to larger microbial
protein excretion as a result of greater fibre fermentation. A
combination of fermentable and non-fermentable dietary
fibres could be used by the dog’s bacterial flora without any
extreme reduction in nutrient availability.
High fibre diets have also been found to influence
glycaemic control in dogs with diabetes mellitus (Graham et
al. 2002). The condition affects 0.5–1.5% of the canine
population (Mattheeuws et al. 1984), and almost all cases
have an insulin-dependent form of the disease (Nelson
1995). Switching to a high fibre diet was associated with
significant reduction of postprandial and 24 h plasma
glucose concentration. Plasma concentrations of
fructosamine, glycated haemoglobin, free glycerol and
cholesterol were also reduced, resulting in a significant
improvement of the dog’s activity.
Field et al. (1999) demonstrated that consumption of diets
differing in the amount of fermentable fibre changed the
proportion of T cells in canine gut-associated lymphoid
tissue (GALT), resulting in a higher response to mitogens in
vitro. The mechanism in which fibre type affects GALT is
not known, but could be related to the effect of fermentable
fibre on gut permeability, intestinal hormone production or
the production of fermentation products by the gut
microflora (Tappenden et al. 1996; Massimino et al. 1998).
Generally, dietary fibre is believed to be among the more
prominent colonic stimuli for cellular proliferation or
differentiation, resulting in morphological and metabolic
changes within the canine GI tract (Hallman et al. 1995). A
more direct approach for shifting the intestinal microbial
balance towards improved host health is by the use of
probiotics in the diet.
3.4 Probiotics
A probiotic is defined as ‘a live microbial supplement
which beneficially affects the host animal by improving its
intestinal microbial balance’ (Fuller 1989) and is usually a
species of lactic acid bacterium such as Lactobacillus or
Bifidobacterium. Health claims attributed to the
consumption of probiotics have been reported for a long
time. Metchnikoff (1907) claimed that the intake of yoghurt
containing lactobacilli resulted in a reduction of toxin-
producing bacteria in the gut and thus an increase in the
longevity of the (human). The mechanisms by which
probiotics improve host health are not completely
understood, but they could be due to several factors
including production of weak acids, antimicrobial
compounds or digestive enzymes, stimulatory effects on the
immune system, or the occupation of potential colonisation
sites (Fuller 1992).
The theoretical basis for the selection of probiotic
microorganisms includes safety, functional and
technological aspects. The safety of probiotic strains is of
prime importance and can be assessed by several
approaches (Salminen et al. 1996):
• Studies on the intrinsic properties of the strain: strain
should be isolated from the same species as that
intended for use and should not having a history of
pathogenesis or be associated with GI
disorders/diseases (Lee and Salminen 1995)
• Studies on the pharmacokinetics of the strains: include
survival through the GI tract, activity in the intestine,
e.g. lack of bile salts deconjugation in the small bowel,
and dose response effects (Marteau et al. 1995)
• Studies on the interactions of the strain and the host:
the probiotic strain should not carry any transmissible
antibiotic resistance genes (Donohue and Salminen
1996).
Functionality is very important when selecting probiotic
strains so that they can survive passage through the upper
GI tract and be viable and active at their sites of action.
Therefore, probiotic strains should be:
• Tolerant to acids and gastric juices in order to survive
passage through the stomach
• Tolerant to bile to survive passage through the small
bowel
• Able to adhere to epithelial surfaces and persist in the
GI tract
• Able to stimulate immunity but not show pro-
inflammatory effects
• Antagonistic against pathogens
• Non-mutagenic and non-carcinogenic.
 Canine functional foods G. Tzortzis, G. R. Gibson and R. A. Rastall
While numerous publications show that probiotics are
active in the gut after ingestion (Bartram et al. 1994; Ling et
al. 1994), some researchers have questioned such claims
and the beneficial effects that administered probiotics are
said to confer on their host (O’Sullivan et al. 1992). This is
mainly due to the fact that before reaching the colon, they
have to overcome two serious obstacles, namely gastric
acidity and bile salts. Furthermore, the colon, with 1010
microorganisms/g of dry matter, is a very stable and finely
tuned ecosystem (Mitsuoka 1992), in which the
establishment of ingested probiotics is likely to be very
difficult (Bouhnik et al. 1992). It has been estimated that the
survival rate of bifidobacteria fed to humans in fermented
milk is 25% (Pochart et al. 1992), with a figure of 10%
being suggested for lactobacilli (Marteau et al. 1997).
For many probiotic health effects, adhesion to the
intestinal mucosa is of primary importance so that the
probiotic bacteria may colonise and exert their effects on
other microorganisms. Barrow et al. (1980) demonstrated
that adhesion of lactobacilli to the cells of the host
organisms is species specific. However, Rinkinen et al.
(2000) studied the adhesive ability of eight strains of lactic
acid bacteria (four intended for human use, two for animal
use and two isolated from dogs) to canine small intestinal
mucus. Among these strains, the best adhesive ability in
vitro was observed for the strains used for humans, although
pre-treatment with canine jejunal chyme reduced the
adhesion of all the strains tested.
Even if a probiotic strain fulfills the necessary safety and
functional criteria, some technological aspects need to be
considered during their selection. Viability during
processing and stability in the probiotic product itself during
storage are of higher importance, although some non-viable
probiotics can have beneficial effects regarding immunity
and carcinogen binding in the host (Salminen et al. 1999).
Application of probiotics to companion animals to date
has been limited to herbal remedies. Royal Canin® have
tested the survival of Paciflor®, a patented strain of
Bacillus, marketed as a feed additive in animal nutrition
(Lestradet 1995), in a complete canine diet (Biourge et al.
1998). The study demonstrated that the addition of Bacillus
CIP 5832 in its sporulated form to a dry dog food was
feasible and that Bacillus CIP 5832 could survive and
germinate in the GI tract of dogs, although no health
benefits were measured. Addition of Enterococcus faecium
to dry dog food demonstrated an increase of the
enterococcal numbers in the ileum chyme and faeces during
the supplementation period (Zentek et al. 1998). Ileum
lactate levels were increased, while pH decreased for the
dogs ingesting the supplemented diet, although no effects
could be seen in the faeces (Zentek et al. 1998).
Commercial probiotic supplements used in dog foods
include Canine Gardion®, Cernivet® LBC SF68,
Protexin® and Eagle Pack®. All of them contain mainly
mixtures of lactic acid bacteria, although Aspergillus oryzae
(Eagle Pack®, Protexin®) and Candida pintolepesii
(Protexin®) can also be found.
6-5
The bacterial strains included in these preparations do not
appear to have been isolated from dogs, and often the same
supplement is recommended for dogs, cats, birds, horses
and fish; this is contrary to one of the selection criteria for
development of efficacious probiotics (Huis in’t Veld and
Shortt 1996).
An alternative approach for microfloral management is
the use of prebiotics, which are directed towards indigenous
gut bacterial genera and so selectively stimulate their
growth and beneficial effect.
3.5 Prebiotics
A prebiotic, as defined by Gibson and Roberfroid (1995), is
‘a non digestible food ingredient that beneficially affects the
host by selectively stimulating the growth and/or activity of
one or a limited number of bacteria in the colon that can
improve the host health’. In order for an ingredient to be
considered a prebiotic, it must:
• Neither be hydrolysed nor absorbed in the upper part of
the GI tract
• Have a selective fermentation such that the composition
of the large intestinal microbiota is altered towards a
healthier composition.
Although any dietary material that enters the large intestine
can be considered as a candidate prebiotic (e.g. resistant
starch, dietary fibre, proteins and lipids), in practice
prebiotics are comprised of ‘short chain carbohydrates, with
a degree of polymerization (DP) of two or more, soluble in
80% ethanol and not susceptible to digestion by pancreatic
and brush-border enzymes’ (Quigley et al. 1999).
Prebiotics have been shown to be a source of SCFA, both
in vitro and in vivo (Gibson et al. 1995; Cummings 1995;
Luo et al. 1996), although no particular distinguishing
pattern of production has yet been identified. The beneficial
effects of prebiotics also include: alteration in microbial
enzyme activity (e.g. fructooligosaccharides (FOS) cause a
decrease in β-glucuronidase and glycocholic acid
hydroxylase in humans in vivo (McBain and Macfarlane
1997)); decreased human lipogenic enzyme gene expression
(Delzenne and Kok 2001); increased neutrophil activity in
dogs (O’Carra 1997); resistance to pathogenic Escherichia
coli and Salmonella spp. by their action as receptor
analogues for Type-1 fimbriae (Oyofo et al. 1989; Spring et
al. 2000); and stimulation of absorption of several minerals,
depending on the type of carbohydrate and the ingested
dose (Glore et al. 1994; Jackson et al. 1999). They are,
however, a major source of hydrogen production in the gut,
which for some people is the major hindrance to their
consumption. On the other hand, findings from in vitro
fermentation studies suggest that this gas production differs
for carbohydrates of differing chain length and
monosaccharide composition (Christl et al. 1992), with
longer molecules being fermented more slowly and
resulting in less hydrogen excretion. Rycroft et al. (2001)
6-6 Canine functional foods G. Tzortzis, G. R. Gibson and R. A. Rastall
found that prebiotic oligosaccharides varied widely in their
production of gas when using human faecal samples.
Fructans resulted in the highest gas evolution, with
galactooligosaccharides and isomaltooligosaccharides
producing much less gas at the same dosage.
Oligosaccharides can be produced by various means such
as polysaccharide hydrolysis or by enzymatic synthesis of
lower molecular weight sugars (Crittenden and Playne
1996; Gibson et al. 2000). Various oligosaccharides have
been tested for their prebiotic properties by in vitro
methods, in animal models and in human clinical trials.
Examples of prebiotic compounds include FOS,
galactooligosaccharides (GOS), lactulose, lactosucrose
(LS), isomaltooligosaccharides (IMO), soyabean
oligosaccharides (SOS), and xylooligosaccharides (XOS;
Crittenden and Playne 1996).
Few published reports exist concerning the effects of
oligosaccharides on canine or feline colonic bacterial
populations, with most studies concentrating on FOS.
Terada et al. (1992) reported the positive effect of dietary
LS on faecal flora and colonic metabolites of dogs. A
significant increase in the bifidobacteria population was
accompanied by a decrease in levels of clostridia, especially
Clostridium perfringens. Faecal ammonia, phenol, indole
and skatole also decreased significantly after 14 days of LS
consumption. Willard et al. (1994) reported on the effect of
1% FOS supplementation of diets on small intestinal
bacterial populations in dogs diagnosed with bacterial
overgrowth. Small intestinal bacterial overgrowth in dogs is
attributed to recognisable causes of poor GI motility,
retention of food in the GI tract, and decreased gastric
acidity, although in some animals it may be idiopathic
(Banwell 1981). The results showed that consumption of
oligosaccharides decreased both aerobic and facultative
anaerobic bacterial numbers in fluid from the duodenum
and proximal part of the jejunum as well as in the duodenal
mucosa following an adaptation period of 50 days (Willard
et al. 1994), suggesting a positive health response to such
supplementation. Buddington and Sunvold (1998) studied
the effect of FOS added to diets containing either cellulose
or beet pulp on adult beagles. Population numbers of
Enterobacteriaceae and clostridia decreased, while
lactobacilli sustained higher populations. In a study
reviewed by Willard et al. (2000), effects of feeding FOS to
dogs on counts of bifidobacteria, lactobacilli, clostridia,
Bacteroides spp. and E. coli were investigated in faeces.
The results showed an inconsistency in isolating
bifidobacteria and lactobacilli from the canine faeces, while
clostridia and E. coli numbers were not affected. Only
Bacteroides spp. showed a significant difference in
population numbers, with a decrease being observed when
FOS were supplemented after a period during which dogs
were fed a normal diet. Based on studies with human faeces,
one should expect an increase in bifidobacterial counts with
the addition of FOS, mainly because all bifidobacteria
species grow well on FOS, with B. pseudolongum, B.
infantis and B. catenulatum recording the highest specific
growth rates (Wang and Gibson 1993). However, 70.7% of
the bifidobacterial isolates from canine faecal samples are
B. adolescentis (Mitsuoka and Kaneuchi 1977), the species
that showed the lowest specific growth rate on FOS (Wang
and Gibson 1993). Therefore, addition of FOS in canine
diets may not result in such significant increases in
bifidobacteria as is seen in other animals.
Strickling et al. (2000) carried out experiments to
evaluate the effects of oligosaccharide supplementation of
canine diets on dry matter, nitrogen, ammonia, volatile fatty
acids and bacterial counts in dogs surgically fitted with ileal
T-cannulas. The differences observed upon addition of
5g/kg FOS, mannanoligosaccharides (MOS) or XOS were
limited to the production of butyrate and propionate.
Butyrate production was higher in the control diet, with
MOS supplementation showing the lowest butyrate
production; this may be attributed to the decreased numbers
of clostridia counts in the presence of MOS. Propionate
levels tended to be higher in diets supplemented with MOS
than those supplemented with FOS and XOS.
Vickers et al. (2001) compared the fermentation
characteristics of FOS, four inulin products varying in their
degree of polymerisation, and MOS to beet pulp (as a
positive control) and wood cellulose (as a negative control)
using dog faeces in vitro. Total production of SCFA, as well
as the individual production of acetate, propionate, lactate
and butyrate, was higher for the FOS and inulin products
throughout the fermentation time. The acetate to propionate
ratio, as measured after 6 h of fermentation, decreased for
the FOS and inulin products, whereas it increased for MOS,
beet pulp and cellulose, suggesting that as fermentation time
increases, FOS and inulin products are degraded more
rapidly (Van Soest and Part 1987). Inulin fermentations
resulted in slightly higher levels of butyrate than FOS
fermentation. In vivo (Swanson et al. 2002), dogs surgically
fitted with ileal cannulas were fed a dry diet supplemented
with FOS, MOS or FOS + MOS to measure nutrient
digestibility, microbial populations, immune characteristics,
faecal SCFA and ammonia concentrations. Nutrient
digestibility did not differ significantly (P > 0.05), although
in the diets supplemented with MOS only, dogs tended to
have lower ileal dry matter and organic matter
digestibilities. Bifidobacteria, clostridia and E. coli were not
different among treatments, but total aerobes were
decreased in the MOS supplemented dogs; total anaerobes
were lower in the FOS + MOS supplemented dogs. Dogs
supplemented with MOS tended to have higher
concentrations of faecal lactobacilli and were also likely to
have an enhanced immune system with increased IgA and
lymphocyte concentrations (percentage of white blood
cells). Contrary to in vitro experiments (Vickers et al.
2001), no differences in faecal SCFA concentrations were
observed among treatments, with values lying within the
normal range for dogs.
 Canine functional foods G. Tzortzis, G. R. Gibson and R. A. Rastall
3.6 Synbiotics
There is great potential for exploiting the synergy between
prebiotic and probiotic ingredients for increasing the
potency of functional foods; this concept is currently
finding its way in the human functional food market
(Bielecka et al. 2002). A synbiotic is defined as ‘a mixture
of probiotics and prebiotics that beneficially affects the host
by improving the survival and implantation of live
microbial dietary supplement in the GI tract’ (Gibson and
Roberfroid 1995). It is based on the idea that both the
structure of the carbohydrate and the bacterial species
present in the ecosystem are important factors for the
management of gut microflora, and aims to enhance the
probiotic survival in the hostile environment of the colon by
offering an available selective substrate. An example
synbiotic is the mixture of a bifidobacterium with
fructooligosaccharides (Crittenden et al. 2001).
HEALTHY BREATH® contains a mixture of lactobacilli
(L. acidophilus, L. casei, L. fermentum, L. plantarum) and
Streptococcus faecium, as well as a prebiotic (FOS) to
provide a substrate for the probiotic strains. However, the
ability of those strains to grow well on FOS has not been
conclusively established.
A very interesting possibility is to use glycotechnology to
design and synthesise synbiotics which consist of an
efficient probiotic and a prebiotic tailored towards that
specific organism. This approach takes into consideration
the glycosidase specificity of the probiotic microorganism,
of which the level of expression and linkage pattern
specificity will probably be unique. This glycosidase profile
can then be used to design an oligosaccharide mixture,
tailored towards that particular probiotic, which will contain
monosaccharides in linkages susceptible to faster hydrolysis
by the glycosidases of this microorganism (Rabiu et al.
2001). In our laboratory (Tzortzis et al. unpublished data)
we have developed the first canine synbiotic consisting of a
prebiotic synthesised by enzymes extracted from the strain
used as the probiotic. The in vitro experiments have shown
that the prebiotic properties (i.e. increase in bifidobacteria
and lactobacilli; decrease in clostridia and E. coli) of the
synthesised oligosaccharide were further magnified by the
addition of the probiotic.
4. Conclusion
The first generation of functional foods was developed
through the necessity to supplement the diet with vitamins
and minerals. Most recent thinking and product
development is focused on GI activity and microbial
interaction. Colonic functional foods for humans have
become accepted in prevention and management of disease
by exerting their direct physiological effects on the GI tract
as well as on metabolic activities. Although there are, in
principle, no major effects associated with manipulation of
the gut microflora, one should consider that disturbance of
the balance between Gram-positive and Gram-negative
bacteria may result in metabolic D-lactate acidosis as a
6-7
consequence of bacterial carbohydrate metabolism
(Stolberg et al. 1982; Oh et al. 1979). A Gram-positive
overgrowth may cause an immunopathological process due
to the presence of peptidoglycans in the cell walls of Gram-
positive bacteria that cannot be broken down by mammals
lacking the appropriate polypeptides (Mills 1989; Kohasi et
al. 1985).
Even though many health benefits are associated with the
use of probiotics and prebiotics, research into the
mechanisms by which these ingredients exert their effects is
still at an early stage. Most of the studies associated with
modulation of the canine gut flora can be criticised by
methodological weaknesses, providing very interesting in
vitro results which cannot be proven in vivo. To improve
this situation, well-validated biomarkers of physiological
function and health end points are crucial to demonstrate
that colonic functional foods are truly effective. Moreover,
scientific work aimed at understanding the health-promoting
effects of these functional food ingredients at cellular level
should be considered crucial for securing future
development of these products. In this respect, new
advances in molecular biology that allow highly
sophisticated tracking of changed in the gut microbiota in
response to dietary manipulation are extremely helpful.
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Perspectives on foods for specific health uses (FOSHU)

Colette Shortt
Yakult UK, 12—16 Telford Way, Westway Estate, Acton, London W3 7XS, UK.

Abstract

The Japanese system of government-approved functional foods which are labelled as 'foods for specific health
use' (FOSHU) is discussed with reference to: characteristics of FOSHU; driving forces behind the development
of the FOSHU/functional food category; the process by which FOSHU status is granted; current FOSHU
products (health claim areas, types of products that are FOSHU approved in Japan, recent FOSHU
introductions); consumer response to FOSHU (consumer awareness in Japan); and the market for FOSHU in
Japan.

Keywords: functional foods, labelling, markets, health claims, Japan

1. Introduction increases in health problems unless measures are taken. One

The ‘Tokutei Hokenyo Shokuhin’ or ‘foods for specified
health use’ market (FOSHU; Table 1) is currently the fastest
growing segment of the nutritional market in Japan.
Swinbanks and O'Brien (1993) heralded the start of this
market activity in an article published in the journal Nature
entitled 'Japan explores the boundary between food and
medicine', in which they reported on the first FOSHU
approved food, hypoallergenic rice. Today, the
FOSHU/functional food market is well established in Japan
and recent retail market estimates suggest it is worth some
412 billion yen (Japanscan 2002).
Table 1. Characteristics of foods for specified health
uses (FOSHU)
Approved by government
Processed with active ingredients which ‘regulate the physical condition’
Efficacy and safety have been verified scientifically
May bear a health claim and FOSHU logo on label
2. Drivers behind FOSHU
One of the main driving forces behind the development of
the FOSHU/functional food category has been the growing
number of elderly people in Japan. The country has one of
the most rapidly ageing populations in the world due mainly
to a decreasing birth rate, an increase in longevity, and a
low level of immigration. Estimates suggest that over 28%
of Japanese people will be 65 or over by the year 2020 if
current trends continue (United Nations 2003). The
Japanese Government has concluded from these
demographic trends that the capacity of the health care
system will be placed under pressure to cope with expected
such measure is development of the FOSHU market, which
focuses on foods that target improvement of specific health
conditions (Table 2).
Table 2. Targets for FOSHU
General foods Healthy individuals
FOSHU Individuals with sub-optimal health
Drugs/medical foods Individuals who are ill
Recently, many authors have reviewed the policy
development that led to the establishment of the FOSHU
category (Euromonitor International 2000; FOSHU 2003;
Japanscan 2003). In 1984, a study group in the Japanese
Ministry of Education started to systematically examine
foods with functional benefits; a round table was then
established in 1988 to further elaborate on the topic. By
1990, the Committee of the Japanese Ministry of Health and
Welfare (MHW) was discussing legislation relating to
functional foods. Under the Nutrition Improvement Law of
1991, the MHW, now known as the Ministry of Health,
Labour and Welfare (MHLW), began to legislate for the
labelling of health claims on five groups of functional foods
entitled “Foods For Special Dietary Use”. These included:
• Foods for special diets
• Foods for pregnant or breast-feeding women
• Foods for infants
• Foods for elderly people
• Foods for specified health use (FOSHU).
Food Science and Technology Bulletin: Functional Foods 1 (7) 1–8
DOI: 10.1616/1476-2137.12686. Published 13 July 2004
ISSN 1476-2137 © IFIS Publishing 2004. All Rights Reserved
7-2 Perspectives on foods for specific health uses C. Shortt
Figure 1. Flow chart outlining approval process for FOSHU.
In addition to establishing the FOSHU category, this legal
framework was specifically aimed at prohibiting misleading
and ill-defined health claims. The first two foods were
approved for sale in June 1993 and today over 300 products
have been approved. In 2001, a new system regulating
functional foods was established, with FOSHU and foods
with ‘nutrient function claims’ now being included in a new
category called ‘foods with health claims’ (Arai et al.
2002). In addition, another recent modification stipulates
that the physiological functions and safety of the foods
concerned need to be assessed in humans. It is worth noting
that despite the FOSHU regulatory system being in place, it
is a voluntary system and a wide range of non-FOSHU
functional foods are marketed in Japan simply as health
foods. This is considered a weakness in the system.
3. How FOSHU status is granted
The process for obtaining FOSHU approval is outlined in
Figure 1 and typically takes about a year to complete. The
main items required to complete a FOSHU application are
as follows:
• Name of the product
• Expiration or validity date
• Reason for seeking permission
• Health claim (label on the product)
• List of ingredients and composition as percentages
• Details of the manufacturing process
• Certificate of nutrient and energy analysis
• Explanation of how the product contributes to dietary
improvement and the maintenance/ enhancement of
health of the entire population
• Recommended daily intake
• Instructions for preparation, storage or intake of the
product.
In addition, a sample of the product packaging with labels
and claims must be submitted, together with the following:
• Documentation that demonstrates clinical and
nutritional evidence relating to the health claim of the
product or its functional components
• Data supporting the recommended level of intake,
stability and safety of the product or its functional
components
• Documentation relating to the physicochemical
properties of the product and the analytical methods
employed
• Results of the quantitative and qualitative analyses of
its functional components as nutrient constituents of the
food, and the analytical methods used
• A copy of the applicant’s organizational statutes or
certificate of contribution
• Description of the production method and factory
equipment used, together with details of quality control
systems.
 Perspectives on foods for specific health uses C. Shortt 7-3

Table 3. Examples of health claims that have been

approved on yoghurt/lactic acid bacteria containing 300
products. 1993
Meiji milk products (Bulgarian yoghurt) 250
1994
Due to the effects of Lactobacillus LB 81, this yoghurt regulates the 1995
balance of gut bacteria, thus maintaining good intestinal conditions. 200
1996
Yakult 150
Due to the effects of Lactobacillus casei strain Shirota, which can reach 1997
the intestine alive, this product contributes to good intestinal health by 100 1998
increasing beneficial bacteria, decreasing harmful bacteria and improving
the intestinal environment. 1999
50
Morinaga milk industry (Bifidus yoghurt) 2000
This yoghurt contains live bifidobacteria (Bifidobacterium longum 0 2001
BB536). It helps to increase intestinal bifidobacteria, improve the FOSHU products approved
intestinal environment and regulate intestinal conditions.
Takanashi milk products (Onaka-He-GG)
Due to the effects of Lactobacillus GG, which can reach the intestine
Figure 2. FOSHU products approved by year.
alive, this yoghurt increases beneficial bacteria and decreases harmful
bacteria. It improves the intestinal environment and regulates gut 4. Current products
conditions.
The FOSHU system is unique in that it focuses on specific
If the applicant is not the manufacturer of the product, a products that are allowed to make approved health claims
copy of the consignment contract must also be submitted. (Heller et al. 1999). An example is ‘Nisshin Healthy
FOSHU legislation has made it possible to make health Balance Healthy Risetta’ from Nisshin Oillio. This edible
claims on the basis of a scientific dossier after approval by oil received FOSHU approval in December 2002, claiming
the MHLW. The products may bear the authorized health that ‘this oil contains medium-chain fatty acids, which are
claim and carry the FOSHU logo on the packaging. scarcely laid down in the body as fat. This oil is
Examples of authorized claims are shown in Table 3. In the recommended for people beginning to be concerned about
early days of FOSHU development, submissions to FOSHU body fat and obesity; it should be used in place of the usual
were limited because of the legislative requirements, edible oil.’ There is also an obligatory caution associated
although more recently uptake has increased (Figure 2). with the product that states ‘copious consumption will not
cure disease nor will it promote health’. The FOSHU
products can often be divided into discrete health claim
areas that involve the management of gastrointestinal

Table 4. FOSHU by health claim*

Number of products approved/(on the
Health claims Functional component
market)
Oligosaccharide 47 (30)

Improve gastrointestinal conditions Lactic acid bacteria 35 (18)

Dietary fibre 31 (15)
Peptide 8 (3)
Helpful for people concerned about high Dietary fibre 5 (2)
cholesterol and/or neutral fat
Others 8 (1)
Suitable for people with mild Glycoside, peptide
hypertension 7 (5)

Calcium citrate malate

Improve mineral absorption Casein phosphopeptide

8 (6)
Haem iron
Suitable for people concerned about blood Dietary fibre 2 (2)
glucose level
Non-cariogenic Sugar alcohol and/or green tea 5 (2)
polyphenols
*as of September 1999
7-4 Perspectives on foods for specific health uses C. Shortt

Table 5. Types of products that are FOSHU approved in Japan

Product category Number of products Approx. % approved

Beverage 61 33
Beverage powder 23 -
Carbonated drink 6 -
Jelly drink 4 -
Powdered jelly drink 1 -
Fermented milk 39 23
Lactic acid bacteria beverage 25 -
Frozen fermented milk 1 -
Milk drink 1 -
Soya milk 4 2
Table sugar 22 8
Cereal 6 2
Instant gruel 1 0.3
Instant noodle 5 4
Snack noodle 5 -
Others 0.7
Grain 1 -
Granulated powder 1 -
Cooking oil 10 5
Margarine 1 -
Seasoning vinegar 2 -
Powdered soap 13 5
Sausage 6 6
Ham 1 -
Hamburger 2 -
Meat ball 2 -
Fried meat 1 -
Tofu 1 -
Nattou 3 -
Candy 5 12
Tablet goody 4 -
Cookie 7 -
Biscuit 2 -
Chocolate 3 -
Pudding 1 -
Naradecoco 1 -
Aloe soaked in syrup 1 -
Powdered jelly 1 -
Gum 8 -
 Perspectives on foods for specific health uses C. Shortt 7-5

Table 6. Recent FOSHU introductions

Claim area Product details

Control of intestines Como, Croissants with lactosucrose
Control of intestines Fukushima Nyugyo, drinking yoghurts with Bifidobacterium
Control of intestines Yakult, Drinking yoghurt with Bifidobacterium breve
Control of intestines Nissin Food Products, Breakfast cereal with psyllium
Control of intestines Koiwai Nyugyo, Drinking yoghurt with yeast dietary fibre
Cholesterol related Meiji Seika, Dried soups with soy protein
Cholesterol related Drinking yoghurt with soy milk/protein
Blood sugar related Raikusutakagi, Tofu with indigestible dextrin
Blood sugar related Matsutani Chemical Industries, Soup with indigestible dextrin
Cholesterol related Nippon Rama Proactive, with phytosterol esters
Dental related Lotte Xylitol Gum Cool Herb with xylitol, palatinit, fukuronori extract & Ca diphosphate
Dietary fibre Guar gum, partial hydrolysate, indigestible dextrin
Gut health Lactic acid bacteria (LAB) (Bifidobacterium lactis, Lactobacillus acidophilus, L.heviticus).
Hypertension Casein dodecapeptide
Dietary fibre Ikuni Shokuhin Kogyo, Agar noodles with dietary fibre
Dietary fibre Ito En, Soft drink with indigestible dextrin
Dietary fibre Nippon Meat Packers, Meat products with indigestible dextrin
Dietary fibre Nippon Runa, Yoghurt with indigestible dextrin
Improve action of intestine Calpis Co., LAB drinks with L. acidophilus CK92 & L.helveticus CK60
Cholesterol Marudai Foods, Meat balls with soy protein
Dental health Warner-Lambert, Chewing gum with CPP-ACP
Dental health Tablet with CPP-ACP, Warner-Lambert (First official FOSHU approved tablet)
Dental health Lotte, Chewing gum with xylitol, reduced palatinose, fukuronori extract & Ca diphosphate
Dietary fibre Sanwa Kagaku, Soft drink with indigestible dextrin
Cholesterol and intestines Kaigen Soft drinks with LMW sodium alginate
Cholesterol and intestines Nisshin Pharmaceutical Powdered soft drink with psyllium husk fibre
Intestinal health Morinaga Milk Yoghurt with B. Longum BB536
Intestinal health UCC Ueshima Coffee Powdered soft drink with Lactosucrose
Intestinal health Meiji Milk Products Milk drink with propionic acid bacteria
Bone health Fujicco KK Soft drink with soy isoflavones
Cholesterol Taisho Pharmaceutical Soft drinks with LMW sodium alginate
Rise in serum neutral fats after meals Morinaga & Co.Soft drink with globin protein hydrolyzate
Bone health Fujicco Soy milk with soy isoflavones
Dental health Warner-Lambert Recaldent gum
Intestinal health Seasoning vinegar with galactooligosaccharides
High blood pressure Soft drink with katsuobushi oligopeptide. Bears a warning
Intestinal health Nissin Yoke , LAB drink with L.casei NY1301
Intestinal health Nihon Seibutsu Kakagu and Nissin Sugar Manufacturing, Table top galactooligosaccharides
Intestinal health and cholesterol Brewery, Powdered soft drink with psyllium husk dietary fibre
Intestinal health Yakult Honsha, Soft drink with galactooligosaccharide and polydextrose
Sugar absorption Nippon Supplement, Soft drink with extract of touchi
7-6 Perspectives on foods for specific health uses C. Shortt

Claim area Product details

Bone health Natto rich in Vitamin K2 (has Bacillus subtilis type OUV23481)
Cholesterol Ajinomoto, Edible soy oil Kenko Sarara with phytosterols
Bone health Milo with fructo-oligosaccharides
Bone health Snow Brand Milk Products, Soft drink with milk basic protein
Cholesterol - special approval category Benecol with phytostannol ester
Cholesterol Kibun Shokuhin, Fish paste with chitosan
Dietary fibre Iho Shokuhin Kogyo, Agar jelly powder with dietary fibre
Dietary fibre Kobayashi Phramaceutical, Dietary fibre food with indigestible dextrin
Dietary fibre Kelloggs Bran Flakes with wheat bran fibre
Dietary fibre Asahi Shokuhin Kogyo, Cracottes with wheat bran dietary fibre
Intestinal health Snow Brand Milk, Yoghurt with prebiotics (gasseri and bifidus)
Bone health Nestle, Milo and Milo C both with fructo oligosaccharide
Rise in serum neutral fats after meals Yakult Honsha Soft drink with globin protein hydrolysate
Digestion/absorption of CHO – blood sugar levels Miki Corp, Powdered soup with wheat albumin
Bone health Fujicco, Soft drink with soy isoflavones
Intestinal health Shinyaku, Juice of young leaves of wheat with indigestible dextrin dietary fibre

health, cholesterol/fat, hypertension and mineral
metabolism (Table 4). To date, over 300 products have been
approved as FOSHU; this is continuing to expand year on
year. Details of FOSHU products are available from
Japanscan (Japanscan 2003). Examples of types of products
and details of specific products and associated claims that
have recently been approved are outlined in Tables 5 and 6.
It is worth noting that some FOSHU products are also sold
outside the Japanese market, e.g. Yakult, the fermented milk
drink, is consumed by 16 million people outside Japan;
Kao’s cooking oil and Calpis’ Ameal S, a product
containing lactotripeptide which targets high blood
pressure, are also successful outside Japan (FOSHU 2003).
5. Consumer reaction
In Japan, as in other countries, the majority of consumers
tend to be resistant to dietary change (Childs 1996; de
Almeida et al. 1997). Indeed, even when consumers
understand the concept behind a food product, they may not
see the relevance of the food to themselves. It has been
estimated that the number of consumers that are ‘health
uninvolved’ far exceeds those that are ‘health aware or
active’. Such 'health uninvolved' consumers are unlikely to
adopt FOSHU products, even if the benefits of including
such foods in the diet have been substantiated and
communicated effectively. Thus, even if a FOSHU product
is addressing a relevant ‘physiological need’, a proportion
of potential consumers will not pick-up on the message
unless communication strategies are specifically developed
to address these issues of resistance and relevance (Shortt
2000).
Awareness and purchase data relating to FOSHU vary
widely. In 1999, the advertising company Dentsu
Incorporated conducted random interviews regarding
FOSHU awareness among 630 subjects aged between 15
and 59 years in the Tokyo district (Tanaka 2000). The study
showed that overall awareness of FOSHU in this sample
group was 25% (Table 7). Similarly, a survey involving 500
subjects between the ages of 34 and 64 years who purchased
FOSHU products was conducted by the Japan 21 Health
Promotion Forum in 2001. They concluded that 28% of the
women and 21% of the men were aware that they had
bought FOSHU products. Although there is evidence to
suggest that many purchase FOSHU by chance, data also
show that repeat purchase of FOSHU appears to be high.
The Japan 21 study showed that 92% of men and 84% of
the women had purchased FOSHU products previously. The
top products bought were lactic acid bacteria drinks,
yoghurt and drinkable yoghurt, natto (fermented soybeans),
dietary fibre drinks, and mineral fortified drinks (Japanscan
2002). Interestingly, a survey by the Japan Health Food and
Nutrition Association (JHFNFA) showed that 65% of
people aged 35–55 years were aware of FOSHU products
and that 22% of those surveyed buy FOSHU products
regularly (Japanscan 2002).
There is an ever-increasing myriad of channels through
which information about diet and health foods is conveyed
to consumers in Japan, such as billboards, magazines,
television/radio broadcasts, newspapers, food packages,
health professionals, pharmacies, supermarkets, and
industry leaflets. The Dentsu data (Table 7) highlight that
specific channels such as the product itself, together with
newspapers and television, are clearly the most frequently
cited sources of FOSHU information by consumers. While,
as expected, the media (e.g. television, magazines and
newspapers) represents a major channel of information
dissemination, it is important to note that other channels
also contribute significantly towards awareness of FOSHU,
for example the food package itself, as well as health
 Perspectives on foods for specific health uses C. Shortt 7-7

Table 7. FOSHU consumer awareness in the Tokyo

district (Tanaka 2000)
Awareness of FOSHU products %
Overall consumer awareness 25
Sources of increased awareness
Information on product labels 34
Newspapers 25
TV 19
Magazines 15
Physician or pharmacy 6
Purchase of FOSHU products
By chance 41
Because of health claim 40
Opinions on FOSHU products
Provide more information 38
Provide appealing health claims 35
They are convenient 25
Advertise FOSHU system 22
professionals. This underscores the importance of the
FOSHU logo and the health claim that appears on the label
(Figure 3). The Dentsu data also demonstrate that
displaying the health claim is an important stimulus to
purchasing the product, and that the demand for further
information on FOSHU is high among consumers. This
request for information on products which have such
complex messages is not unexpected. Although Japanese
consumers are interested in their health, there are many
reasons why they might have difficulty understanding the
concepts behind FOSHU products. Japanese consumers are
subjected to many dietary recommendations and are
exposed to a plethora of health messages that they are often
not able to integrate meaningfully into their daily diet. They
often have pre-existing ideas that prevent them from
believing, and therefore understanding, the health claims
that appear on FOSHU products. It is often difficult to
model or visualize the phenomenon or process on which the
health benefit is based, for example reduction of cholesterol
or blood pressure. Translating emerging scientific findings
into everyday language for consumers without loss of
accuracy is challenging. Thus, health claims and graphical
logos are critical in bridging the information gap in a
situation where the dietary health message is often complex.
There is some evidence to suggest that FOSHU-approved
foods are purchased by consumers in general rather than by
those with specific health complaints. Moreover, it is too
soon to analyse the impact that FOSHU products have had
on general public health, although there is an indication that
positive effects may be observed. Epidemiological data
from a Japanese study suggests that habitual intake of lactic
acid bacteria reduces the risk of bladder cancer (Ohashi
2002).
Figure 3. FOSHU product with packaging details.
6. Conclusions
The rapidly expanding FOSHU market in Japan is a
successful one, as evidenced by its retail value (Table 8).
The most prominent FOSHU foods are aimed at digestive or
gut-health benefits and, to date, this has been the most
important category from a product launch and sales
perspective. Indeed, gastrointestinal health-orientated
products accounted for 60% of FOSHU approvals at the end
of 2001 (Arai et al. 2002). The JHFNFA survey of its
members in 2001 showed that the retail value of FOSHU
products almost doubled between 1999 and 2001. In
addition, it showed that the retail value of lactic acid
bacteria-containing products was 317 billion yen in 2001,
with market share being 77% in 2001 compared to 82% in
1999 (Japanscan 2002).
Table 8. FOSHU market value in 2001
Category Value (billion Yen)
Lactic acid bacteria 317
Dental caries related 19
Blood sugar related 18
Lipid metabolism 15
Dietary fibre 13
Minerals 11
Blood pressure related 10
Oligosaccharides 6
Cholesterol related 3
Total 412
It is clear that while FOSHU products address dietary
problems such as provision of calcium, further products are
warranted that specifically target the major health concerns
of the Japanese in order to impact significantly on public
health. Furthermore, concerns have been raised regarding
the limited lifespan of many FOSHU products.
The introduction of FOSHU in Japan has provided a
strong and clear impetus to the Japanese food industry for
developing food products with added benefits over and
above traditional nutritional benefits. Retail sales also
suggest that the consumer is responding to this innovative
7-8 Perspectives on foods for specific health uses C. Shortt
science-based food category by purchasing FOSHU
products and requesting more information. However, it is
too soon to determine whether this category is having any
impact on the key issues of public health concern in Japan
such as diabetes, hypertension, obesity, hyperlipidaemia,
cerebrovascular disease and cancer (Japan Public Health
Association 2001). Nevertheless, it is clear that the FOSHU
model is being observed and embraced by industry,
regulators and academia in many parts of the world, from
the US to China (Bailey 2000; ILSI Japan 2000).
7. References
Anon. 2002. Legal affairs. Japanscan 19 (12): 32.
Arai, S., Morinaga, Y., Yoshikawa, T., Ichiishi, E., Kiso, Y.,
Yamazaki, M., Morotomi, M., Shimizu, M., Kuwata, T. and
Kaminogawa, S. 2002. Recent trends in functional food science
and the industry in Japan. Bioscience, Biotechnology and
Biochemistry 66: 2017-2029.
Bailey, R. 2001. Japanese functional foods regulations: a system
for other countries to consider? Agro Food Industry High-Tech
12: 39-41.
Childs, N. 1996. Functional foods and the food industry:
developing the incentive to innovate. Presented at the Annual
ILSI Meeting, Jan 22-24.
de Almeida, M., Graca, P., Lappalainen, R., Giachetti, I., Kafatos,
A., de Winter, A. and Kearney, J. 1997. European Journal
of Clinical Nutrition 51: S16.
Euromonitor International. 2000. A world survey. Euromonitor,
London.
Heller, I., Taniguchi Y. and Lobstein, T. 1999. Functional foods:
public health boon or 21st Century quackery? CSPI Reports:
International. Available at:
http://www.cspinet.org/reports/functional_foods/
ILSI Japan. 2000. Health claims on functional foods - proposals
on scientific substantiation and regulatory systems. ILSI Japan.
Japan Public Health Association. 2002. Measures against lifestyle
related diseases. Public Health of Japan 2002. Available at:
http://www.jpha.or.jp/jpha/english/apha/chapter_2.html
Ohashi, Y. 2002. Lactic acid bacteria and cancer: epidemiological
perspective. British Journal of Nutrition 88: 121.
Shortt, C. 2000. Communicating the benefits of functional foods to
the consumer In: Functional foods 11 Claims and Evidence, pp
70-75, Royal Society of Chemistry, Cambridge
Swinbanks, D. and O'Brien, J. 1993. Japan explores the boundary
between food and medicine. Nature 364: 180.
Tanaka, R. 2000. Positively leveraging nutrition on the product
label In: Proceedings of the International Dairy Federation
World Dairy Summit, Dresden, September 16-20.
United Nations. 2003. World population prospects: the 2002
revision population database. Available at:
http://esa.un.org/unpp/
Yamaguchi, P. FOSHU 2003. Paul Yamaguchi & Associates Inc.
Functional foods in the USA: emphasis on probiotic foods
†
Mary Ellen Sanders* and James T Heimbach

*Dairy and Food Culture Technologies, 7119 S. Glencoe Ct., Centennial, CO 80122-2526, USA. Tel. 303-793-9974. Email
MES@mesanders.com. Website http://www.mesanders.com
†
JHEIMBACH LLC, 4530 Broad Branch Road, NW, Washington, DC 20008, USA. Tel. 202-237-8406. Fax 202-478-0986
fax. Email Jim@jheimbach.com. Website http://www.jheimbach.com

Abstract

The role that functional foods might play in enhancing human health is continuing to develop. Comprehensive
approaches, which will enable scientists both to understand the total human physiological response to diet
(through application of human genomics and metabolomics) and evaluate health maintenance rather than
disease incidence, will advance this field greatly. In the United States, functional foods remain of interest to
consumers, although there is currently no legal definition of a functional food. One functional ingredient,
probiotics, shows much promise as a health-promoting addition to foods, or as a basis for dietary supplements.
A growth industry in the United States, the probiotics industry must develop a proactive policy in order to
bolster consumer confidence in commercial probiotic products. Many publications suggest that commercial
probiotic products do not comply with label claims. This article seeks to examine probiotics as functional food
ingredients and the regulatory framework for labelling and describing the benefits of functional foods, as well
as dietary supplements.

Keywords: probiotic, functional food, regulatory, dietary supplement, Lactobacillus, Bifidobacterium

1. Introduction matters such as food consumption patterns, food synergy,

Quit worrying about your health. It'll go away.
American humorist, Robert Orben.
There is a fundamental awareness among all cultures that
what we eat matters. Unfortunately, the dietary advice
received by modern Americans is anything but clear, since
emerging science is reported in the media as established
fact and public health policy is frequently influenced by
politics (Taubes 2001). ‘A consistent feature of news
about food...has been a lack of context in the reporting of
food news, an absence of perspective necessary for
consumers to actually make use of the information’, states
a report by the International Food Information Council
(IFIC) regarding US food news coverage (IFIC 2003).
In all fairness, however, making general dietary
recommendations to the public today is a difficult
undertaking, with the focus for nutrition having shifted
from concentration on single nutrient deficiency diseases
to sustaining health and preventing chronic disease.
German et al. (2003) state that ‘the importance of diet to
health has become even more obvious with the realization
that many of life’s modern diseases are the results of
subtle but chronic metabolic imbalances related in part to
diet’. The question of optimal diet is complicated by
interactions between food components, and the impact of
sustaining dietary patterns over the long term. The effect
of interventions for all metabolic functions, and not for
just a single physiological parameter, is rarely understood.
Finally, of paramount importance is the genetic and
physiological status of the individual. These issues
converge when attempting to assist the public on decisions
regarding healthy food choices.
The value of understanding an individual’s response to a
particular dietary intervention can be clearly illustrated with
diets that are low in saturated fat. Such diets have been
recommended as part of US public health policy in order
(HHS 2004) to reduce the risk of cardiovascular disease
(CVD). However, studies have shown that for a significant
portion of the public, such a diet can lead to worsening of
serum lipid profiles and hence increasing the risk of CVD
(Krauss 2001a, 2001b).
Embedded in the discussion of diet and health is the
concept of functional foods, frequently defined as foods that
offer health benefits beyond basic nutrition. Although the
term functional foods has no legal definition in the US, it
has not deterred the public’s interest in the topic. For
Food Science and Technology Bulletin: Functional Foods 1 (8) 1–10
DOI: 10.1616/1476-2137.13616. Published 2 November 2004
ISSN 1476-2137 © IFIS Publishing 2004. All Rights Reserved
8-2 Functional foods in the USA M.E. Sanders and J.T. Heimbach
example, US food news coverage in 2003, as reported in the
fifth annual ‘Food for Thought’ survey carried out by IFIC
(IFIC 2003) indicated that, although obesity and weight
management stories were most numerous, 11% of all 2003
US food news topics covered functional foods.
The existence of such a term suggests that there are easily
defined groupings of foods which can be labelled as
functional or non-functional; this is clearly not the case.
However, for the sake of this general discussion, we will
posit that such distinct groupings exist. A wide range of
compounds naturally present in some foods (which can be
added to others) has been identified as having compelling
effects on health status. Compounds such as fibres,
prebiotics, fatty acids, flavonoids, plant stanols and sterols,
bioactive peptides, and carotenoids are widely known. One
area which is growing in scientific substantiation,
commercial potential and public awareness is probiotics.
Probiotics have been defined as ‘live microorganisms which
when administered in adequate amounts confer a health
benefit on the host’ (FAO 2001). The potential, as well as
challenges associated with this category of functional food
ingredients in the US is the focus of this article.
2. Probiotics – the potential
The evaluation of the role that probiotics may play in
human health has been the subject of research for decades.
There is no shortage of reviews written on the topic, which
focus on the effect of probiotics on regulation of immune
function and prevention of infection (Bengmark 2003;
Tamboli et al. 2003; Gill 2003). Compelling effects in well-
controlled studies document the impact of probiotics on
reducing conditions such as inflammatory bowel disease
(Gionchetti 2003), infectious diarrhoea in infants (Van Niel
et al. 2002), onset of atopic dermatitis in high-risk infants
(Kalliomaki et al. 2001, 2003), and control of symptoms
associated with lactose intolerance (de Vrese et al. 2001).
Less dramatic, albeit statistically significant, results have
shown the ability of certain probiotic bacteria to decrease
the incidence of dental caries (Nase et al. 2001), as well as
antibiotic use (Hatakka et al. 2001), respiratory infections
(Hatakka et al. 2001) and antibiotic-associated diarrhoea
(Cremonini et al. 2002). It must be remembered that the
benefits reported should be considered specific to the
strain(s) used and the levels of viable microbes consumed.
Some animal studies have characterized potentially
beneficial activities from killed microbes (Rachmilewitz et
al. 2004), although, by definition, these are not probiotics.
The opportunity for probiotics to be genetically modified
for a specific function is another area of active research
(Steidler 2003).
A welcome improvement in probiotic research is that the
rigour of published trials has improved with the application
of placebo-controlled, randomized studies, using both well-
defined and well-described products. The impact of the
disciplines of genomics (the study of the genetic
complement of both the human host and the microbes they
harbour) and metabolomics (the study of metabolic activity
in response to different conditions of both the human host
and the microbes they harbour) on probiotic research is also
evolving (German et al. 2003; Rastall et al., submitted).
Clearly, there is an advantage from an efficacy and safety
perspective to better define the total human physiological
response to any dietary intervention, including probiotics.
The large array of beneficial microbe-associated
characteristics attributed to the normal gut microbes
emphasizes the value of understanding the human response
to an externally applied microbe. Native gut microbes have
been shown to be involved in immune system development
and expression, epithelial cell behaviour, pathogen
resistance and barrier effects (Mai and Morris 2004; Hooper
et al. 2003). How does applying high levels of an
autochthonous microbe to the already colonized human GI
tract affect these attributes? For example, the effect of
probiotics on immune function will be better understood
when whole patterns of gene expression response are
determined. It is clear that understanding the host-microbe
interaction is at the heart of understanding probiotic impact
(Hooper et al. 2003); such an understanding is possible
today by use of animal models. Furthermore, genetic
techniques such as terminal restriction fragment length
polymorphism (TRF), which enables examination of the
impact of probiotics on specific native species of the
bacterial community, provides more insight into probiotic
effects. With gut microbe-related diseases such as
inflammatory bowel disease, antibiotic-associated
diarrhoea, or perhaps irritable bowel syndrome, in which no
one pathogen has been identified to account for the
pathology of each condition, a systems approach to the
impact of probiotics on gut microbiota will undoubtedly
prove more fruitful.
We are at a time in history when many microbe-
influenced situations are considered high priority with
regard to public health policy. Circumstances such as the
emergence of antibiotic-resistant bacteria, highly virulent
foodborne pathogens and viral infections, together with
rapid increases in inflammatory and allergic diseases, and
the prevalence of ageing populations desiring to live longer
healthier lives, have converged. Emerging science suggests
that probiotics may play a role in managing such situations
(Reid et al. 2003), with the potential of this field being
reflected by a growing worldwide market for probiotics.
Although the US lags behind Europe and Asia in embracing
the concept of probiotics, this situation may be starting to
change. A survey of US media stories on functional foods
indicated that 4% of the stories in 2003 that dealt with
functional foods focused on probiotics/prebiotics (IFIC
2003). This suggests a rise in awareness in the US of
probiotics and prebiotics, as similar surveys conducted in
1999 and 2001 did not report any stories on this topic.
However, the survey showed that improved gastrointestinal
health, which is often associated with probiotics, was not
featured in the top ten functional foods-related stories
reported in the media.
The status of probiotic research is likely to have much in
 Functional foods in the USA M.E. Sanders and J.T. Heimbach
common with that of other functional food ingredients.
Research substantiating the physiological benefits of
functional food ingredients is often funded by companies
which stand to gain from positive results. Unbiased research
generated in multiple, independent laboratories provides
stronger support for efficacy. Other challenges include the
need to better define mechanisms of action and to formulate
products with the most efficacious and stable components.
In this way, a final product may be produced which has an
acceptable taste and price, is convenient for consumers, and
communicates product advantages to them in a fashion that
is truthful, does not mislead and complies with regulatory
constraints.
The challenge also exists to understand the impact of
normal gut microbes and exogenously applied live microbes
to the maintenance of health in humans. Food companies
which mostly fund applied, efficacy-based research on
probiotics, are interested more in enhancing health than
preventing disease; this is reflected by the fact that there are
no approved probiotic drugs for human use in the US.
However, measurement of health remains a challenge in a
research system dominated by a disease-based paradigm
(German et al. 2003).
3. Ensuring the authenticity of probiotic products
In the US, consumers may choose from a plethora of dietary
supplements, yoghurts and other dairy products which claim
to deliver probiotics. However, in the absence of any
unbiased assessment of the quality of these products,
consumers have no rational basis for their choice of
probiotic product. Useful information would include third
party verification of stated levels and types of viable
probiotics until the end of the product shelf life.
Furthermore, a physiologically meaningful level of
probiotics as determined by human studies should be
delivered by the product. To address the concerns relating
to such commercial probiotic products, some researchers in
the scientific community have conducted product surveys.
The number of publications regarding the content of
commercial probiotic products is growing. Accurate
labelling of the content of a probiotic product is essential;
without it, the safety and efficacy of a product cannot be
determined. A summary of studies addressing the levels of
probiotic bacteria in commercial products (foods and
supplements) is shown in Table 1. Generalizations from the
studies listed in this table are complicated, since methods
differ substantially among papers. However, it is
noteworthy that these studies all arrive at the same
conclusion – that the actual delivery of probiotic bacteria in
many commercial probiotic products does not match the
content claim information on their labels with regard to the
level and type of probiotics contained. This situation
consequently leads to an erosion of confidence in probiotics
by health care professionals and consumers.
Conclusions from product evaluations, however, should
not be accepted without scrutiny. For example, Huff (2004)
reported that none of ten Lactobacillus products purchased
8-3
in lower British Columbia, Canada, met label claims.
Methods used were semi-quantitative, with counts estimated
using streak plating from growth on blood agar. Although
the authors indicate that this method provided ‘rough
quantitation’, no comment is made on the range of error
common to this technique. Enumeration of probiotic
lactobacilli is usually undertaken using a quantitative
dilution to extinction approach, followed by spread plating
in duplicate on the surface of MRS agar; plates with 30–300
well distributed colonies are then counted. Although blood
agar is considered to be a non-selective medium in the
clinical laboratory and is no doubt of use for identifying the
presence of lactobacilli associated with bacteraemia, it is
not the growth medium of choice for those lactobacilli
which have been selected as probiotics. Furthermore, no
control strains of lactobacilli were plated using this
procedure to ensure the validity of the methods used.
Although Lactobacillus was not isolated from any of the ten
products in this study, this is as likely to be due to use of
inadequate methods as poor probiotic product quality.
Methodological problems were also apparent in a study
conducted by Berman and Spicer (2003). These authors
evaluated 20 dried supplements containing lactobacilli; only
one product was found to contain all the microbes listed on
the label. Again, a streak plating method was used to
generate single colony isolates, which were picked and
identified. Enumeration of colonies was not conducted.
Again, no positive controls were evaluated to ensure that
the different types of microbes present could be recovered
using the methods described which probably isolated only
the dominant microbes present in the probiotic blends.
Although it is likely that there are many probiotic
products on the market that do not meet label claims,
research that investigates compliance with such label claims
must be based on methods which provide reliable results.
Indeed, researchers provide a disservice to the probiotics
industry when responsibly formulated products are judged
to be inadequate by use of flawed methodology.
Although it is not true that US probiotic products are
‘unregulated’, it is true that no government authority
currently tests or approves such products. Current US food
and dietary supplement law stipulates that companies must
label products in a truthful and not in a misleading fashion
(see 21 USC 343(a)(1)). It is therefore incumbent on
industry to adopt procedures which address the issue of
eroding consumer confidence in probiotic products.
One option is for companies to submit their products to an
independent laboratory for evaluation. This would provide a
measure of assurance to consumers that the products are
formulated as labelled. Alternatively, efforts are being made
to establish meaningful standards or guidelines for probiotic
products, as spearheaded by industry, regulatory and
scientific groups worldwide (Table 2). Clearly, there is a
need for standardized and validated analytical methods for
determining the content of probiotic products.
An example of the possible confusion regarding labelling
of probiotic products can be seen in yoghurt, which is the
Table 1a. Studies of the content of commercial probiotic products available to consumers
Probiotic product tested
Lactobacillus supplements,
some blended with
bifidobacteria
Dried Lactobacillus
acidophilus supplements
Probiotic supplements
Probiotic supplements
Dry, liquid or dairy products
claiming to contain L.
acidophilus
Probiotic supplements
Probiotic supplements or
food products
No. of Country/region of Methods used Results of analysis References
products origin of product
tested
20 - 16S rDNA sequencing; 1 out of 20 samples contained microbes that were consistent with product Berman and Spicer 2003
no enumeration of levels label.
8 - Plate counts; carbohydrate ≤105 lactobacilli/g in 4 products; 3 products contained L. acidophilus. Brennan et al. 1983
fermentation
15 - Selective plating; carbohydrate 8 products were accurately labelled with regard to their microbial content; Canganella et al. 1997
fermentation 1 product was labelled with the correct amount of bacteria contained in the
product.
9 South Africa Selective plate count; DGGE 3 out of 9 products contained the correct amount of bacteria, as listed on Elliot and Teversham 2004
the label.
13 - Selective plate count with oxgall 3 products contained L. acidophilus; 6 contained bile-tolerant lactobacilli Gilliland and Speck 1977
>106/g or ml.
13 UK Selective plating; API rapid ID kits 2 out of 13 products met label claims for species present and quantity of Hamilton-Miller et al.
microbes contained; 8 out of 13 products were >1 log below the label 1996
claim for microbial counts.
52 API rapid ID kits; selective plating 4 out of 11 yoghurt labels declared specific microbes in product, others Hamilton-Miller et al.
provided only general descriptions; no mislabelling found in yoghurts; 12 1999
out of 29 UK probiotic supplements were accurate in terms of labelling of
microbial content and quantities.
Table 1b. Studies of the content of commercial probiotic products available to consumers (continued)
Probiotic product No. of products Country/region of Methods used Results of analysis References
tested tested origin of product
Lactobacillus 10 Canada Semi-quantitative streak method 0 out of 10 supplements matched label specifications. Huff 2004
supplements on blood agar
Probiotic full- and 7 (3 of which also Australia Selective plate count; tested L. acidophilus levels varied widely between products (<103–108/g); 1 out Micanel et al. 1997
reduced-fat yoghurts contained stability during 6 weeks storage of 4 brands had <103/g bifidobacteria.
with L. acidophilus bifidobacteria)

Probiotic yoghurts 50 Australia Selective plate count >106 L. acidophilus in 24% of products; >106 bifidobacteria in 14% of Rybka and Fleet 1997
with bifidobacteria products.
and
L. acidophilus

Probiotic food 10 (4 dairy, 1 juice, 5 DNA-based, culture 4 did not contain all labelled species. Temmerman et al. 2003
products dried) independent analysis; DGGE;
culture enrichment

Probiotic products 55 (30 dried Europe Selective plate counts 11 out of 30 supplements contained no detectable microbes; 6 out of 55 Temmerman et al. 2003
supplements, 25 products were accurately labelled.
dairy products)
Supplements 5 Selective plate counts 3 out of 5 supplements met the label claim for species contained; 2 out of 5 Weese 2002
products met the label claim for microbe level.

Dairy products 6 Carbohydrate fermentation 3 out of 6 products were correctly labelled with regard to their Yaeshima et al. 1996
study; microtitre bifidobacteria content.
colorimetric DNA hybridization
8-6 Functional foods in the USA M.E. Sanders and J.T. Heimbach

main probiotic food in the US. It is estimated that about
80% of the approximately $3 billion of yoghurt sold in the
US each year contains additional bacteria (as well as the
required Streptococcus thermophilus and Lactobacillus
bulgaricus starter bacteria). These additional bacteria are
added for their beneficial health effects and consist
primarily of L. acidophilus, but may also be bifidobacteria
and some other lactobacilli. Although yoghurt labels
disclose the presence of these adjunct microbes, there is
essentially no information on the levels, strains or, in the
case of Bifidobacterium, even species contained in such
products. Furthermore, there is little data on product labels
regarding the substantiated benefits imparted by the
microbes present. In some cases, information relating to the
benefits of live cultures can be found on company web sites,
although the specific information that consumers need is
often lacking. Consumers are therefore unable to derive
answers for simple questions which will enable them to
assess what benefits might be derived by them or their
family members from consumption of a particular yoghurt.
Consumers specifically looking for a source of probiotic
bacteria may consequently turn to dietary supplements over
foods as their preferred probiotic source. Probiotic
supplement products, in general, provide more information
to consumers about levels, types of microbes, strain
designations and benefits than probiotic-containing foods.
However, not all this information has either been
substantiated or is accurate.

Table 2. Organizations involved in attempting to establish standards for probiotic bacteria in commercial products
Organization
Food Agriculture Organization (FAO)/
World Health Organization (WHO)
International Dairy Federation
European Food and Feed Culture
Association
Codex Standard for Fermented Milks
(Codex Stan 243-2003)
National Yogurt Association
Website address
http://www.fao.org
http://www.fil-idf.org
http://www.effca.com
http://www.codexalimentarius.net
http://www.aboutyogurt.com
Region of Action
impact
Worldwide Developed guidelines for the evaluation of probiotics in
foods.
Worldwide Has begun working on methods to determine certain
functional and safety properties outlined in the FAO
guidelines for the evaluation of probiotics in food.
Europe Developed guidelines for use of probiotics in foods.
Worldwide Among other composition stipulations, this standard
specifies minimum numbers of characterizing and
additional labelled microbes in yoghurt, acidophilus milk,
kefir, kumys and other fermented milks.
USA Petition under consideration by the FDA which would
change the standard of identity of yoghurt, including the
requirement of minimum levels of live cultures in
yoghurt, but not specifically levels for any additional
probiotic cultures.

International Scientific Association for http://www.isapp.net Worldwide Industry Advisory Committee and Board of Directors to
Probiotics and Prebiotics consider method validation and establishment of
laboratory sites to assess microbiological content of
probiotic products.

4. Product labelling claims for probiotic products
Since the status of probiotic products as functional foods
derives from their ability to provide health benefits, it is
clearly necessary that such benefits should be conveyed to
the consumer or potential consumer. There are three routes
by which food companies can provide such information to
consumers: food labelling; advertising; and through third
parties. The claims communicated via these routes may be
of three types: disease claims; health claims; or
structure/function claims. These types of claims are
discussed in detail below.
4.1 Prohibited disease claims
In the US, health benefit claims for food products and
dietary supplements are tightly controlled by US Food and
Drug Administration (FDA) regulations which prohibit any
statement that an average consumer might understand as
indicating that the product is useful in the cure, prevention,
treatment, mitigation, or diagnosis of a disease or disorder
(see 21 USC 321(g)(1)(B)). Such claims are regarded as
disease claims, or drug claims, and unless the product
concerned has a New Drug Application (NDA) on file with
the FDA, the claim is deemed to be illegal. The truth or
falsity of the claim, and the amount of substantiation
available to support the claim, are irrelevant; by making
such a claim, the product is considered to be a drug and,
without a valid NDA, it is an illegal drug.
This limitation puts out of bounds any claims with regard
to several benefits that have been demonstrated or
suggested in probiotic research studies, such as reduction of
the risk of relapse in inflammatory bowel conditions,
treatment of infectious diarrhoea in infants, and prevention
of atopic dermatitis. Furthermore, the FDA has taken the
position that statements are also classed as drug claims if
 Functional foods in the USA M.E. Sanders and J.T. Heimbach
they suggest utility of the product as an adjunct to therapy,
or to treat, prevent, or mitigate against adverse events
associated with a therapy for a disease, if such adverse
events constitute diseases (see 21 CFR 101.93(g)(2)(vii and
ix)). Thus, for example, a claim that a product may be
useful in controlling antibiotic-induced diarrhoea would
likely be regarded by FDA as an illegal drug claim.
The US Federal Trade Commission (FTC) does not
operate under the same laws as the FDA, and consequently
advertising is regulated differently from food labelling.
Incidentally, ‘food labelling’ includes not only the food
label itself, but also any materials such as brochures, shelf
placards, and the like displayed or distributed in association
with the product (FTC 2001). The FTC requires only that
advertising claims be true, not misleading, and adequately
substantiated. In theory, the FTC could allow advertising
that included disease prevention or treatment claims for a
probiotic-containing food; however, it requires that the
adequacy of substantiation for claims must be evaluated
based on the potential consequences of conflicting claims,
as judged by experts in the field. In this case, such experts
would be scientists at the FDA, and the substantiation
required would be at least equal to that needed to have the
product approved as a drug. Furthermore, the FDA would
likely take the advertising claims themselves as
demonstrating an intention to position the product as a drug
and take measures against it on that basis (FTC 2001).
A third route of providing information to consumers
about probiotic products is available. It is possible for a
company to sponsor seminars to which physicians in private
practice have been invited. At these seminars, it is
permitted to present and discuss the science supporting the
use of specific probiotic microorganisms in the prevention
or clinical management of disease. The physicians in turn
may provide direct counselling to their patients, including
suggesting the use of probiotic food products.
4.2 Health claims
The Nutrition Labelling and Education Act (NLEA) of 1990
established one class of claims, health claims, as being a
specific exception to the prohibition against disease-related
claims for food products or ingredients (see 21 USC
343(r)). Health claims may only be used in labelling of a
food or dietary supplement after they have been reviewed
and authorized by the US FDA. The law defines a health
claim as a statement that ‘characterizes the relationship of
any nutrient or other substance in a food to a disease or
health-related condition’ (from 21 USC 343(r)(1)(B)).
However, the FDA’s implementation of the law is more
restrictive, taking the position that the only type of
relationship that is appropriate for such health claims is a
reduction in the risk of contracting a disease or health-
related condition by a member of the currently healthy
population. Thus, for instance, the FDA is unlikely to
authorize any health claims relating to reduction of the
likelihood, frequency or severity of recurrence of episodes
such as those characterizing Crohn’s disease, irritable bowel
8-7
syndrome, or gastro-oesophageal reflux disease. Nor, of
course, will it accept any claims of treatment or mitigation
of these diseases as legitimate health claims.
Use of such health claims may be possible for probiotic
products, assuming that adequate substantiation of the claim
is available. If a probiotic product can be shown to lessen
the risk of developing gastro-oesophageal reflux disease, for
example (rather than mitigate recurrences), this could be
used as a possible health claim. Similarly, reduction in the
risk of developing colon cancer, if adequately demonstrated,
is a feasible health claim, as is reduction in the likelihood of
developing certain allergies.
The burden of proof that is required for the FDA to
authorize a health claim is a high one: there must be
significant scientific agreement regarding the validity of the
claim. According to the FDA (FDA/CFSAN 1999) ,
meeting this level of significant scientific agreement
requires a sufficient body of evidence that shows
consistency across different studies and researchers, thus
ensuring that:
• A change in the dietary intake of the substance will
result in a change in a disease endpoint
• The validity of the relationship is not likely to be
reversed by new and evolving science, although the
exact nature of the relationship may need to be refined.
Because this required level of proof is so high, many
individuals have argued that American consumers are
deprived of useful information that could be provided by
health claims that include appropriate disclaimers disclosing
the lack of certainty of the scientific evidence available to
support the claims.
This point of view found voice in a 1999 decision by the
US Court of Appeals for the DC Circuit, Pearson versus
Shalala (see 164 F.3d 650 (DC Cir. 1999)) in which the
court determined that under the First Amendment, the FDA
could not prohibit health claims unless they were inherently
incapable of being rendered non-misleading. The court
indicated that ‘inherently misleading’ claims might be those
that have less evidence supporting them than evidence
against them.
In response to this judgment, the FDA introduced an
interim policy in 2003 of permitting qualified health claims
— claims supported by the weight of the available evidence
or, in a later formulation based on another adverse court
decision, claims supported by credible evidence, but do not
meet the standard of significant scientific agreement. These
claims are authorized by the FDA only with appropriate
qualifiers regarding the strength of the scientific evidence,
ranging from ‘there is little scientific evidence supporting
this claim’ to ‘although there is scientific evidence
supporting this claim, the evidence is not conclusive’. A
number of qualified health claims have been approved
under this interim policy, although to date none have been
approved for probiotics (US FDA/CFSAN 2002).
The type of claim eligible for consideration as a qualified
8-8 Functional foods in the USA M.E. Sanders and J.T. Heimbach
health claim is the same as for unqualified health claims, i.e.
it must deal with reduction in the risk of a disease or health-
related condition among the healthy population. Cure,
mitigation, and treatment claims remain illegal drug claims;
this cannot be remedied by any form of disclaimer or
qualification.
4.3 Structure/function claims
The benefits of a functional food product or dietary
supplement may be also communicated to consumers in the
form of structure/function claims, which are statements
describing the effect of the food or ingredient on the
structure or function of the body, or which describe the
mechanism by which the effect is produced (US
FDA/CFSAN 2004) While a structure/function claim may
not state or imply any effect on a disease or disorder, this
limitation is not as restrictive as it may at first appear.
Structure/function claims often promise to assist in
maintaining positive health that is the opposite of a disease
or disorder. For example, a claim to cure or mitigate
osteoarthritis is a drug claim; a claim to reduce the risk of
developing arthritis would be a health claim; and a claim to
help maintain healthy joints is a structure/function
claim. The distinctions between different types of claims
can be extremely small. For example, maintenance of
sexual potency is a drug claim because impotence is
considered a disease, but maintenance of sexual
performance is an acceptable structure/function claim.
The only regulatory requirement for a structure/function
claim is that the claim be true and not misleading; however,
the FDA has never expanded on what this means in terms of
the substantiation required. Generally, most observers agree
that the standard is set lower for structure/function claims
than for even qualified health claims. One clear difference
is in the body of research that must be available since
structure/function claims may be based on mechanistic and
other animal studies, in vitro testing, epidemiological data
and ethnomedical experience, and a variety of types of
human data. In evaluating the evidence base for health
claims, on the other hand, the FDA regards only human
studies as fundamental data; indeed, a health claim petition
is unlikely to be approved if it lacks multiple well-executed
randomized controlled intervention trials or prospective
observational cohort studies with consistent findings
between studies.
Listed below are several claims that might be
contemplated for a probiotic that impacts immune function:
• Helps maintain a healthy immune system
• Strengthens the body’s natural defences
• Promotes production of cytokines
• Improves the body’s immune response to attack
• May help protect your family against colds
• Helps provide protection against infection
• Reduces the severity of viral diarrhoea.
The first two of these, if supported with appropriate animal
and/or mechanistic data, are likely acceptable
structure/function claims since they describe the effect of
the product on a function of the body. The following two
are also probably acceptable structure/function claims, if
supported by appropriate mechanistic data, because they
describe the mechanism by which the product operates to
produce a beneficial effect. The next two statements (or
revisions of them) might form an acceptable basis for health
claims, but there would clearly have to be a number of well
designed and well executed clinical studies, in a variety of
populations and with a variety of strains of target bacteria
and viruses, showing both statistically and physiologically
meaningful reductions in risk. The final statement is a drug
claim and would not be approved under any circumstances.
5. Conclusions
Probiotics are a growing component of the functional foods
industry in the US. The need for research to confirm
physiological benefits, identify mechanisms of action and
develop the technology to improve delivery of products that
are attractive to the consumer is paramount. Although the
broad impact of microbes on human physiology ensures that
there is no shortage of potential targets for investigation into
probiotic-mediated effects, the probiotics industry must be
careful to recognize the importance of truthful
communication about these effects to both consumers and
health care professionals in order to assure growth, rather
than disillusionment, in the budding US probiotics industry.
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8 - 10 Functional foods in the USA M.E. Sanders and J.T. Heimbach

evaluation of a microplate hybridization method. International

Journal of Food Microbiology 30(3):303-313.

About the authors

Mary Ellen Sanders is a consultant in the area of probiotic

microbiology, providing services such as support for new
product development, coordination of efficacy
substantiation efforts, GRAS assessment, and assisting with
communications efforts on probiotics. She has worked with
FAO/WHO, the International Dairy Federation, and the
Federal Drug Administration on pressing issues in the
science of probiotics. She currently serves as President of
the International Scientific Association for Probiotics and
Prebiotics.

James Heimbach is President of JHeimbach LLC Food and

Nutrition Consulting. He has national and international
experience in a broad range of issues regarding food and
nutrition policy, as well as food consumption behaviour,
assessment of dietary intakes of nutrients, food additives
and contaminants, safety evaluation of food and dietary
supplement ingredients (including GRAS determinations
and NDI notifications) and food regulation.
Functional foods in infant formulae

Wolfram M Brück
bdw Consulting, Kattowitzer Str. 34, 51065 Cologne, Germany. Tel. 49 (0)221 698585. E-mail w_bruck@hotmail.com

Abstract

Diet, together with other environmental factors, plays an important role in health and disease. Enhancing infant
formulae to mimic the health-promoting benefits of breast milk has been of significant interest in recent years.
Differences in the gut microflora of breast-fed and formula-fed infants have been reported, which may explain
disparities in infection rates between these two groups. Various functional foods such as probiotics,
oligosaccharides and proteins, when added to infant formulae, could beneficially modify the composition of the
gut microflora in formula-fed infants. Although most of the products considered for addition to infant formulae
have been developed and commonly used in the adult diet, their safety and understanding as an infant food
supplement is not fully understood. The aim of this review is to describe the current knowledge of functional
food research and discuss their impact on the composition of the infant gastrointestinal microflora.

Keywords: functional foods, infant formula, colonic microflora, dietary modulation

1. Introduction relevant to either the state of well-being and health or the

Functional foods, including whole, fortified, enriched, or
enhanced foods, have evolved beyond the simple treatment
of malnutrition and deficiency syndromes, to the actual
reduction of disease risk (Hasler 2002; German et al.
1999). It is now widely recognized that food supplements
may influence the gastrointestinal tract and therefore host
defence (Cunningham-Rundles and Lin 1998).
Attitudes towards functional foods differ between
countries. Asia, particularly China and Japan, were the first
to explore the boundaries between the medical and food
sciences, and targeted a wide variety of conditions (Arai et
al. 2001). In Japan, where 90% of infant formulae contain
prebiotics, the world’s first policy permitting the
commercialization of several functional foods was initiated
in 1991 (Ghisolfi 2003; Arai 2002). In the West, the
introduction of functional foods, such as prebiotics, to
infant formulae is a much more recent event, primarily due
to vague legislation, or lack of appropriate policies
(Edwards and Parrett 2003). In the USA, functional foods
are broadly defined as neutraceuticals and focus on the
reduction of cancer and coronary heart disease (Milner
2000). US FDA regulations remain confusing and, under
current regulations, functional foods can be placed into a
number of different regulatory categories. However, many
companies choose to market functional foods based on the
US Dietary Supplement Health and Education Act
(DSHEA) of 1994, which enables such companies to claim
health benefits for their functional food products without
linking them to a specific disease (Hasler 2002). In the
European Union, functional foods have been defined as a
food that contains a 'component that benefits one or a
limited number of functions in the body in a targeted way
reduction of the risk of disease'. (Roberfroid 2001).
The critical stages of gut microflora development occur
after birth and during weaning. Its is assumed that the adult
gut microflora is established during this critical period
(Edwards and Parrett 2002). Therefore, optimizing the
microflora of infants through the addition of functional
foods such as probiotics and prebiotics among others can
have long-term benefits. Infant formulae on the market
today should aim to provide the best alternative to breast
milk for infants of women who are unable to continue
breast-feeding. The aim is to mimic the functional outcome
of the breast-fed infant (i.e. growth and development), and
not to duplicate the composition of human milk (Agostoni
and Haschke 2003). Manipulation of the infant gut
microflora may allow the promotion of a more beneficial
flora which could reduce gastrointestinal problems, allergies
and various other debilitating conditions and chronic
diseases of adulthood such as heart disease and colon
cancer. However, in order to predict the long term
consequences of supplementing infant feeds, it is crucial to
fully understand the bacterial colonization process and
metabolic activity of the flora (Edwards and Parrett 2002).
This review will outline infant intestinal microbiology and
discuss the current knowledge of functional food research
and the effects of such foods on the composition of the
infant gastrointestinal microflora.
Food Science and Technology Bulletin: Functional Foods 1 (9) 1–14
DOI: 10.1616/1476-2137.13734. Published 30 November 2004
ISSN 1476-2137 © IFIS Publishing 2004. All Rights Reserved
9-2 Functional foods in infant formulae W.M. Brück
2. Neonatal gut microflora
During birth, the gut of the sterile neonate rapidly becomes
colonized by bacteria, which are provided as an inoculum
during the delivery process (von Sonnenborn et al. 1990;
Escherich 1885). However, it may take several years to
establish a gut microbial ecosystem which is similar to that
of adults (Adlerberth 1998). It is generally believed that
establishment of the intestinal microflora begins after the
rupture of the foetal membranes following delivery. At this
time, and providing the foetus is delivered through the birth
canal, the inoculum mainly consists of the mother’s vaginal
and faecal flora, as well as environmental microorganisms
(von Sonnenborn et al. 1990). One study showed that the
percentage of Escherichia coli in the oronasal cavity of
infants increased during longer deliveries (Cornelison et al.
1946). However, microflora transmission rates between
mother and infant do vary between studies. Bettelheim and
Lennox-King (1976) reported that, out of 28 mother and
infant pairs, 22 infants yielded the same E. coli serotypes as
their mothers. However, other studies reported transmission
rates as low as 25%, which may be attributed to different
laboratory identification procedures of E. coli and varying
hygienic measures applied during delivery (Gothefores et
al. 1976; Gareu et al. 1959). In contrast, delivery by
Caesarean section is thought to provide the neonate with an
inoculum derived principally from the environment since it
is unlikely that they would come into contact with the
mother’s faecal or vaginal flora during delivery.
Transmission of bacteria to neonates may also occur via
nurses’ hands as previously demonstrated (Hall et al. 1990;
Bettelheim and Lennox-King 1976). Additionally, one
study has also shown viable microbes present in gastric
aspirates of newborn infants delivered by Caesarean section
with intact membranes (Mims et al. 1972). In this study, 3
out of 15 babies delivered by Caesarean had Staphylococcus
epidermidis present in gastric aspirates. The reason for this
is uncertain and could possibly be due to contamination
during the collection procedure.
Generally, in infants delivered vaginally, the neonate is
colonized during the first two days of life by nutritionally
undemanding, fairly aerotolerant organisms such as
enterobacteria, staphylococci and streptococci (Balmer and
Wharton 1989; Mata and Urrutia 1971). The more
fastidious and anaerobic bacteria to which the infant is
exposed may not be able to establish themselves in the
neonatal intestine at this time due to a positive oxidation-
reduction potential in the gut (Stark and Lee 1982).
Lactobacilli present in the maternal vaginal flora did not
colonize the intestines of any babies in a study involving
five mother and infant pairs (Tannock et al. 1990). Once
the facultative bacteria have increased in numbers, they
consume oxygen; this means that the positive redox
potential (Eh) and relatively high oxygen content rapidly
declines until a typical redox potential of –150mV is
achieved (von Sonnenborn et al. 1990). Thus, within the
first week of life, facultative and obligate anaerobes such as
bifidobacteria, bacteroides, and lactobacilli also become
established, which then start to proliferate (Mitsuoka
1995). When populations of anaerobes expand, numbers of
facultative anaerobes may decline, possibly because of a
limitation of the availability of nutrients in a dense
anaerobic environment (Freter 1992). However, relatively
high numbers of facultative bacteria may still persist
through the first few months, or even years, of life
(Adlerberth 1998).
3. The gut microflora of breast-fed and formula-fed
infants
After initial bacterial colonization, differences in the
composition of the intestinal microflora are thought to be
largely dependent on the nature of the diet (Fuller and
Gibson 1997). It is believed that breast-fed infants
commonly acquire an intestinal flora with lower population
levels of enterobacteria and higher levels of bifidobacteria
and lactobacilli, as first described by Tissier (1905). The
faeces of infants that are not breast-fed, on the other hand, is
thought to contain a mixture of microbes, with enterococci
and bacteroides being the predominant organisms (Goldman
2000). Differences in the development of the intestinal
microflora of breast-fed and formula-fed infants usually
means that a lower risk of intestinal infections is often
displayed in the former. Additionally, maternal immunity is
transferred to the breast-fed infant, further helping to protect
it from disease, while enhancing the immature immune
system. Stephens et al. (1980) showed that as little as 5 ml
of colostrum/kg birth weight is sufficient to prevent
enteropathogenic infections. More interesting was the
observation that enteropathogenic E. coli (EPEC) and
Salmonella spp. occurred in the faeces of some breast-fed
infants without causing any symptoms. Additionally,
human milk may also have other beneficial systemic effects
by reducing the risk of septicaemia and meningitis in the
newborn infant (Gothefors et al. 1975).
It is commonly believed that, in the breast-fed infant,
bifidobacteria outnumber other microorganisms by a factor
of at least 100 (Gibson and Roberfroid 1995). This is due in
part to a reduced buffering capacity caused by a low protein
and mineral content in mother’s milk as compared to
unadapted cow’s milk formulae (Kleessen et al. 1995).
This allows acidic metabolic end products to accumulate in
the breast-fed infant gut, thereby allowing bifidobacteria to
grow, inhibiting more acid-sensitive organisms such as
enterobacteria, clostridia, and the bacteroides (Bullen and
Tearle 1976; Gothefors et al. 1975). The faecal pH of
breast-fed infants was found to be between 5 and 6, whereas
bottle-fed infants had a faecal pH in the range of 8–9
(Bullen et al. 1976). Additionally, N-acetylglucosamine,
glucose, galactose, fructose and lactoferrin are factors
present in breast milk that facilitate bifidobacterial growth
(Gibson and McCartney 1998). In formula-fed infants, the
gut microflora is thought to greater resemble that of an adult
in its complexity. Bullen et al. (1977) found that high
bifidobacterial counts were common in formula-fed infants
 Functional foods in infant formulae W.M. Brück
in only the first week, though after that time no species
predominated. The flora was also found to produce acid,
although this is not permitted to accumulate because of the
high buffering capacity of cow’s milk formula (Kleessen et
al. 1995). The more alkaline conditions of the gut do not
select for acid-resistant organisms; therefore, slow growing
bifidobacteria are not able to compete with faster growing
and potentially pathogenic organisms such as enterobacteria
(Roberts 1986). Based on these data, exclusive breast-
feeding, especially in developing countries, is essential to
protect the infant from a large variety of infections such as
diarrhoea, septicaemia and urinary tract infections, thus
reducing infant mortality (Adlerberth 1998).
Table 1. Factors influencing the development of the
intestinal microflora in infants
Vaginal vs Caesarean delivery
Environment during birth, home vs hospital
Hygiene measures (use of antiseptics during prenatal period)
Environmental contaminants - maternity wards
Mother's faecal, vaginal and skin microflora
Developmental stage of the gastrointestinal tract (premature vs full terms
infants)
Breast- vs formula- fed
Type of formula used
4. The search for breast milk substitutes
Just four decades ago, it was believed that human milk
supplied only nutrients to the infant, although there was
strong evidence to the contrary (Goldman 2000). The
contemporary view, however, has been considerably
expanded beyond this belief on the basis of the following
discoveries:
• A wide spectrum of bioactive agents exist in human
milk
• There are potential targets in the gastrointestinal tract
for these agents
• Breast-feeding modifies some functions of the
gastrointestinal tract
• Breast-feeding modifies the gastrointestinal microflora
to protect and benefit the host.
Thus, human milk is considered a species-specific complete
food and can be defined as the best functional food for
infants (Langhendries 2002; Mountzouris et al. 2002).
During the first few months of life, maternal milk is
considered to be the best source of nutrition and well-being
for the newborn infant (Räihä 1985). At the beginning of
lactation, infants receive colostrum, which is rich in
immunoglobulins, hormones and growth factors, and may
provide immune defence against gastrointestinal diseases.
These bioactive agents modulate organs in a specific
9-3
manner and influence the development and growth of the
infant (Oddy 2002; Velonà et al. 1999). As lactation
progresses, major milk proteins (such as caseins, β-
lactoglobulin and α-lactalbumin) increase in the postpartum
mature milk to provide stronger nutritional support
(Schanbacher et al. 1998). Apart from being nutritious,
human milk also contains a myriad of compounds with
bioactive and immunological roles. Immunoglobulin
(secretory) sIgA, peptide and non-peptide hormones,
growth factors, lipids and enzymes such as lysozyme are
some such active components; these are individual to each
mother and offer protection to the infant (Koldovsky and
Thornburg 1987).
Whenever breast-feeding is impossible or unavailable in
adequate amounts, infant formulae may still provide a safe
and nutritious alternative for growth and development.
However, such formulae cannot replicate all the nutritious,
bioactive and immunomodulatory properties of breast milk
since they may require the various additional factors found
in breast milk in order to give similar, synergistic effects
currently not seen in formulae (Wharton et al. 1994). Based
on these facts, formula manufacturers are keen to produce a
product that more closely simulates the qualities of breast-
milk through the addition of functional ingredients to
formulae (Roberts 1986).
Modern infant formulae have been improved to mimic
breast milk and so have lower amounts of protein and
electrolytes compared to unmodified cow’s milk. Some
have casein as the predominant protein while others contain
demineralized whey protein. They are also supplemented
with vitamins A, B group, C, D, E and K. However, even
with these advances, it is difficult to mimic the qualitative
aspects of breast milk.
More recently, the inclusion of mucins, glycoproteins,
glycolipids, glycosaminoglycans, nucleotides and
oligosaccharides in formulae has been investigated
(Morrow 2003; Peterson et al. 1998). Of these,
oligosaccharides have recently been of particular interest
due to their resistance to enzymatic breakdown in the upper
gastrointestinal tract, their bifidogenic effects within the
large intestine, and their use as prebiotics in adult foods
(Crittenden and Playne 1996). Human milk contains more
than 130 different oligosaccharides of varying chain length.
They are considered to be the most abundant sugars after
lactose and are largely based on five monosaccharide
residues including sialic acid, N-acetylglucosamine, fucose,
glucose and galactose. Oligosaccharides also affect growth
of the intestinal flora In particular, N-acetylglucosamine-
containing oligosaccharides, together with lactose, were
shown to stimulate growth of Bifidobacterium bifidum
(Mountzouris et al. 2002). The structure of
oligosaccharides may simulate the conformation of bacterial
receptors on epithelial cells. Oligosaccharides may
therefore act as ‘decoy’ receptors for intestinal bacteria,
thereby decreasing or preventing the attachment of
undesirable microorganisms to the intestinal tract (Newburg
1997). However, Brand-Miller et al. (1998) demonstrated
9-4 Functional foods in infant formulae W.M. Brück
that even though total oligosaccharide concentration
remained stable, the relative concentrations of individual
oligosaccharides varied throughout lactation.
Supplementation of formula, on the other hand, may be able
to provide the infant with a continuing supply of specific
oligosaccharides that have previously exhibited beneficial
effects such as selective fermentation.
4.1 Prebiotics
A prebiotic is a non-digestible food ingredient that
beneficially affects the host by selectively stimulating the
growth and/or the activity of one or a limited number of
bacteria in the colon (Gibson and Roberfroid 1995). In
order to be considered a prebiotic, a food component must
fulfil the following criteria (Roberfroid 2001):
• Stability under acidic conditions in the stomach and the
small intestine
• Resistance to enzymatic digestion
• Capacity for hydrolysis and fermentation by colonic
bacteria
• Ability to selectively stimulate growth of a limited
number of colonic microorganisms.
The concept of prebiotics is based on the hypothesis that the
bacteria of the human colon may be classed as those that are
either beneficial or detrimental to human health (Gibson
and Roberfroid 1995). Thus, lactic acid bacteria and
bifidobacteria are the main targets for a prebiotic approach
because of their associated beneficial functions in the gut
and their stimulation of the immune system (Collins and
Gibson 1999; Fuller and Gibson 1997).
Any dietary material that enters the large intestine is a
candidate prebiotic (Collins and Gibson 1999). This
includes carbohydrates such as resistant starch and dietary
fibre, as well as proteins and lipids (Ziemer and Gibson
1998). However, in reality, most prebiotics are confined to
non-digestible oligosaccharides, many of which seem to
achieve a degree of selective bacterial growth, a criterion
required for a candidate prebiotic (Miniello et al. 2003).
Future investigations of prebiotic properties may
nevertheless focus on the preventative effects of prebiotic
compounds against the colonization and proliferation of
detrimental microorganisms. A study on the ability of
cellobiose to attenuate virulence in Listeria monocytogenes
has shown promising evidence of the prebiotic potential in
the prevention against infection (Collins and Gibson 1999).
Oligosaccharides are sugars consisting of approximately
2–20 saccharide units, i.e. they are short-chain
polysaccharides (Cummings et al. 2001). Some occur
naturally in several foods such as leeks, asparagus, chicory,
garlic, artichokes, onions, wheat and oats, as well as
soybeans (Roberfroid et al. 1998; Ziemer and Gibson 1998;
Bach Knudsen and Hessov 1995; van Loo et al. 1995;
Gibson and Wang 1994; Saito et al. 1992).
Oligosaccharides can be commercially produced through
the hydrolysis of polysaccharides (e.g. dietary fibre, starch)
or through catabolic enzymatic reactions from lower
molecular weight sugars (Crittenden and Playne 1996).
Major food-grade oligosaccharides in commercial
production that can act as prebiotics by selectively
stimulating the growth of bifidobacteria, and, to a lesser
extend, lactobacilli, include (Rastall and Maitin 2002;
Playne and Crittenden 1996; Gibson et al. 1995):
• Lactulose
• Galacto-oligosaccharides (GOS)
• Fructo-oligosaccharides (FOS)
• Isomalto-oligosaccharides
• Palatinose oligosaccharides
• Soybean-oligosaccharides
• Lactosucrose
• Gentio-oligosaccharides
• Xylo-oligosaccharides (XOS).
Even though the amount of infant research on prebiotics is
limited, there are a number of studies demonstrating the
beneficial effects of oligosaccharides on the human adult
intestinal microflora which can be correlated to infant
feeding strategies. Of the large number of prebiotic
substances, only four prebiotic supplemented infant
formulae have been studied.
In a study of 271 full term infants, supplementing infant
formula with GOS at a concentration of 2.4 g/l resulted in
significant stimulation of the growth of bifidobacteria and
lactobacilli in the intestine compared to negative controls.
No significant difference was found between groups fed
GOS supplemented formula and breast-fed infants (Xiao-
ming et al. 2004). In a randomized trial with healthy full
term infants, formulae supplemented with GOS showed a
10-fold increase of bifidobacteria compared to formula
supplemented with lactose (Napoli et al. 2003).
Bifidobacterial ranges became comparable to the reference
group of breast-fed infants. Also, faecal pH after 21 days of
feeding was significantly lower in the GOS group that in the
control group while lactate concentrations increased 4-fold.
The addition of GOS and inulin to cow’s milk based
infant formula has also been shown to significantly
stimulate the growth of bifidobacteria and lactobacilli
(Vandenplas 2002). Similar results were found in more
recent studies with infants fed cow’s milk formulae
supplemented with a combination of FOS and GOS (Boehm
et al. 2004; Moro et al. 2003). The prebiotic, dose-
dependent effect of this mixture was tested in full term and
pre-term infants by measuring faecal flora. Conventional
plating methods and DNA fluorescent in situ hybridization
(FISH) were used simultaneously to analyse the
composition of the infant microflora. In both studies,
bifidobacteria were significantly increased compared to
unsupplemented formula. At a concentration of 8g
oligosaccharides/l formula, numbers of bifidobacteria in
formulae were similar to those of breast-fed controls
 Functional foods in infant formulae W.M. Brück
(Boehm et al. 2004). In both studies, stool consistency and
faecal pH were also positively affected.
Gibson and Roberfroid (1995) carried out a volunteer trial
with adult subjects on strictly controlled diets supplemented
with 15 g/day of FOS. Sucrose was used as the control and
faecal samples were processed, in a blind manner, within 30
min of passage. Most importantly, the study used follow-up
phenotypic characterization techniques to fully identify the
microflora that developed during feeding regimes. These
studies showed that the intake of FOS significantly
modified the composition of the faecal microflora by
stimulating the growth of bifidobacteria, which, after 2
weeks of the feeding period, became by far the numerically
predominant bacterial group. Additionally, infant formulae
supplemented with FOS significantly reduced counts of
bacteroides, fusobacteria and clostridial populations. These
effects lasted for as long as the prebiotic was consumed.
However, at the end of a 2 week follow up on the controlled
diet, the faecal flora of all volunteers still had higher
bifidobacterial numbers than when recruitment started.
Formulae supplemented with FOS only are not currently
used commercially, although some clinical studies of
formulae containing FOS obtained by the hydrolysis of
inulin exist. FOS-supplemented formula was fed to
newborns over a period lasting from 2 weeks to 8 months.
Surprisingly, no bifidogenic effect was demonstrated in any
study (Ghisolfi 2003).
Despite these results, other oligosaccharides such as
lactulose, XOS and soybean oligosaccharides still require
further research. In particular, human volunteer trials are
needed to confirm their effects in vivo on the consumer,
especially in infants (Roberfroid 1997, 2001; Steer et al.
2000). Ballongue et al. (1997) carried out an adult
volunteer trial to confirm the prebiotic nature of lactulose.
The authors found significant increases in bifidobacteria,
lactobacilli and streptococci, whilst bacteroides, clostridia,
coliforms and eubacteria all decreased during the test
period. These results would suggest a prebiotic activity. To
examine infant formula supplemented with lactulose,
Baskaran et al. (1999) fed groups of 21 day old rats a diet
containing lactulose at 0.5% for 4 weeks. The gain in body
weight of rats fed a lactulose-supplemented diet were
similar to that of rats fed a lactulose-free or a skim milk
powder diet. This suggests that supplementation of
lactulose in infant food formula does not affect either
protein quality or overall acceptability by human subjects
and thus infant trials would be needed to confirm their
prebiotic effects in vivo.
It is likely that, in a similar way to adults, inclusion of
dietary oligosaccharides in formulae may benefit formula-
fed infants by acting as ‘dietary fibre’ ingredients
(Flickinger et al. 2000). Several oligosaccharides
encourage an infant intestinal microflora rich in
bifidobacteria and lactobacilli and simultaneously reduce
coliforms, clostridia and bacteroides by acting as receptor
homologues (Steer et al. 2000; Kawakami 1997; Coppa et
al. 1990). Data also suggest that oligofructose will enhance
9-5
populations of bifidobacteria and prevent colonic epithelial
mucosa atrophy in neonates (Howard et al. 1995).
However, compared to adults, the infant faecal flora has a
lower fermentation capacity for oligosaccharides and other
complex carbohydrates (Parrett and Edwards 1997; Parrett
et al. 1997). In dose dependency studies, a softening of
stools, a decrease in faecal pH and an increase in the
frequency of defecation was observed in infants (Ghisolfi
2003). Thus, even though prebiotic-supplemented formulae
do not seem to have any adverse effects on intestinal transit,
nitrogen balance, amino acid metabolism, mineral
bioavailability or water balance, careful dose assessments
are warranted to prevent carbohydrate overload which can
result in undesirable gastrointestinal symptoms such as
diarrhoea (Cummings et al. 2001; Livesey 2001; Marteau
and Flourie 2001). On the whole, it is likely that inclusion
of dietary prebiotics in moderate amounts may benefit
formula-fed infants by establishing a bifido-rich
microflora. The prebiotic carbohydrate could be added in
addition to existing lactose concentrations since formulae
do not otherwise contain the human milk oligosaccharides
which play a role as prebiotics in breast-fed infants
(Mountzouris et al. 2002; Gnoth et al. 2000).
4.2 Probiotics
Another functional food recently considered for inclusion in
formula feeds are probiotics. The term ‘probiotic’ derives
from the Greek meaning ‘for life’ and was originally used
to describe organic and inorganic supplements necessary to
restore health of patients suffering from malnutrition caused
by eating too many highly refined foods (Hamilton-Miller
et al. 2003). Later definitions specified probiotics as
‘organisms and substances which contribute to intestinal
microbial balance’ (Parker 1974). Fuller (1989) provided
further refinement of this hypothesis by pointing out the
importance of incorporating live microbial supplements and
thus defined probiotics as ‘a live microbial feed supplement
which beneficially affects the host animal by improving its
intestinal microbial balance.’ This latter version is now the
most widely used definition and has gained widespread
acceptance (Simmering and Blaut 2001; Roberfroid 2001;
Bengmark 2000; Steer et al. 2000 and others).
Recent research has been directed towards the use of
intestinal isolates of bacteria as probiotics which might be
regarded as ‘protective’ against gastrointestinal infections,
cancer, inflammatory bowel disease, intestinal dysfunctions
and lactose intolerance (Marteau and Boutron-Ruault 2002;
Bengmark 2000). Over the years, many species of
microorganisms have been used, mainly lactic acid bacteria
(lactobacilli, streptococci, enterococci, lactococci,
bifidobacteria; Fuller and Gibson 1997). However, strains
from a diverse choice of genera such as Bacillus,
Leuconostoc, Pediococcus and Streptococcus, as well as
fungi such as Saccharomyces spp. and Aspergillus spp.,
have also been used as probiotics in both animal and human
nutrition (Fuller 1989). Nonetheless, the most common
probiotic microorganisms are Lactobacillus (i.e. L.
9-6 Functional foods in infant formulae W.M. Brück
acidophilus, L. casei, L. plantarum, L. delbrueckii subsp.
bulgaricus, L. reuteri, L. brevis, L. cellobiosus, L.
fermentum), Bifidobacterium (i.e. B. bifidum, B.
adolescentis, B. infantis, B. animalis, B. longum, B.
thermophilum), and Gram positive cocci (i.e. Lactocoocus
lactis subsp. cremoris, Streptococcus salivarius subsp.
thermophilus, Enterococcus faecium, Staphylococcus
diacetylactis, S. intermedius; Collins and Gibson 1999).
The primary reason for the significant trend towards using
mainly bifidobacteria and lactobacilli as probiotics is that
both are not disease causing organisms (Kullisaar et al.
2002; Dunne et al. 1999).
Lactobacilli have long been associated with health foods
and have been used in yoghurt and cheese production, as
well as probiotics (Fuller and Gibson 1997). Bifidobacteria
have similar qualities to lactobacilli and were originally
classified in this genus (Gyorgi et al. 1954). Tissier (1905)
first reported the beneficial role of bifidobacteria (then part
of the lactobacilli) to human hosts by noting their benefits
to breast-fed infants.
Besides their apparent inability to cause pathogenesis, the
lactic acid microflora of the human gastrointestinal tract is
thought to play a significant beneficial role for the host,
from improved resistance to pathogen colonization to
vitamin B synthesis (bifidobacteria; Isolauri et al. 1999;
Geis 1989). There are a number of possible mechanisms for
this:
• Metabolic end products such as acids excreted by these
microorganisms may lower the gut pH to levels below
those at which pathogens are able to effectively
compete (Gibson and Wang 1994)
• Competitive effects resulting from lactic acid bacterial
occupation of the colonization sites normally occupied
by pathogens (Gibson and Wang 1994)
• Direct antagonism through natural excretion of
antimicrobial compounds (Geis 1989)
• Competition between lactic acid bacteria and pathogens
for nutrients (Gibson and Wang 1994)
• Stimulation of the immune response by lactic acid
bacteria so that pathogens are inhibited (Perdigon and
Alvarez 1992).
With regard to bifidobacteria, studies have indicated that
some species are able to exert direct antimicrobial effects on
various Gram-positive and Gram-negative intestinal
pathogens including salmonellae, campylobacters and E.
coli, known as the ‘Bifidobacterium barrier’ (Fooks et al.
1999; Gibson and Wang 1994).
Despite the widespread use of probiotics, this approach
has a number of associated difficulties and continues to
attract controversy in the scientific community (Dunne et al.
1999; Berg 1998). The key problems are related to the fact
that the bacteria used are usually anaerobic and sensitive to
extremes of temperature. In addition, other problems
relating to viability and stability of probiotics may arise,
either during use or under storage, and thus the probiotic
may not reach its final destination in the lower gut intact
(Fuller and Gibson 1998). B. bifidum, B. breve, B. infantis
and B. longum have been introduced into the gut and their
generation times and acid production trends studied.
Results suggest that growth characteristics of bifidobacteria
in infant formula is species-specific and formula-
dependent. Growth is optimal in formulae based on milk
rather than soy (Dubey and Mistry 1996).
Various claims have been made regarding the beneficial
effects of probiotics on gastrointestinal infections.
Controlled randomized studies on diarrhoea in both adults
and infants have shown that probiotics are beneficial in
preventing diarrhoea and that they survive in sufficient
numbers to affect gut metabolism (Bezkorovainy 2001).
Typical causal agents identified in studies examining the
influence of probiotics on the incidence of diarrhoea
included Salmonella, Escherichia coli, Shigella,
Clostridium perfringens, Campylobacter jejuni,
Helicobacter pylori and Yersinia enterocolitica (Isolauri et
al. 1999). These pathogens generally either colonize and
grow within the gastrointestinal tract and then invade host
tissue, or secrete exotoxins which disrupt the normal
function of the intestinal mucosa, causing nausea, vomiting
and diarrhoea (Salyers and Whitt 1994). The gut microflora
itself usually acts as a barrier against potential pathogens;
however, probiotics may aid this resistance to pathogen
colonization, thereby warding off infections more
effectively (Isolauri et al. 1999). This was shown by the
introduction of two non-pathogenic, antibiotic-susceptible
E. coli strains into the guts of premature infants. This
probiotic treatment was successful in significantly reducing
the number of antibiotic-resistant enteropathogens through
antagonistic colonization mechanisms (Lari et al. 1990).
Probiotics are also being used in experimental formulae
in attempts to promote health in human infants.
Supplementation has shown that some probiotic strains do
persist in the infant gut while lowering stool pH
(Langhendries et al. 1995; Miller et al. 1993; Bennet et al.
1992; Fuller 1991). B. lactis strain Bb12, when added to
acidified infant formula showed some protective effect
against acute diarrhoea in healthy infants younger than 8
months living in residential care centres (Chouraqui et al.
2004). Altogether, the risk of developing diarrhoea was
reduced by a factor of 1.9 in infants given the experimental
formula, compared to infants fed conventional formula. In
another study, Lactobacillus GG was administered to
prematurely delivered infants and, although the intestine
was colonized by Lactobacillus GG, there were no clinical
benefits observed (Miller et al. 1993). A possible
explanation for this lack of effect might be that <10% of the
administered probiotic generally survived upper
gastrointestinal transit. However, it was also suggested that
the presence of milk proteins markedly improved probiotic
tolerance to gastric transit by up to 100% and that the
presence of mucin and milk proteins exerted a protective
effect during upper gastrointestinal transit (Mountzouris et
al. 2002). On the other hand, when Lactobacillus GG was
 Functional foods in infant formulae W.M. Brück
administered in fermented milk or as a freeze dried powder
in older infants (4–45 months), the duration of diarrhoea
was shortened without any mucosal disruption when
compared to controls (Isolauri et al. 1991). Similar results
were observed when formulae were supplemented with B.
bifidum and S. thermophilus, which led to significant
reduction of acute diarrhoea and rotavirus shedding
(Saavedra et al. 1994). Altogether, the treatment and
prevention of rotavirus-induced diarrhoea is one of the best
documented health effects of probiotics (Salminen et al.
1998).
4.3 Milk proteins
Recommendations for the milk protein composition of
infant formulae are based on its composition in human
milk. Ranges of 1.7–4.5 g/100 kcal in the USA and 1.2–1.9
g/100 ml in Europe are generally considered a safe and
acceptable level (Järvenpää et al. 1982); however, protein
absorption can be considerably higher after acute
gastroenteritis (Holm et al. 1992). In human milk, the
protein ratio of whey to casein is 60:40 (Lien 2003), while
mature cow’s milk has a higher casein composition
(Hambræus et al. 1978). Whey-predominant infant
formulae are made by mixing a cow’s milk base with
demineralized whey protein in order to match the whey
ratio of human milk (Räihä 1985).
Protein-derived bioactive components have multiple
functions. They play a vital role in infant growth and
development, act as immunomodulatory agents and
antimicrobials, provide fundamental amino acids and aid
nutrient assimilation, while stimulating growth of beneficial
microorganisms in the gut (Liepke et al. 2002; Tomé and
Debabbi 1998; Schanbacher et al. 1998). Milk protein
derived peptides may also function as exogenous regulatory
substances (Meisel 1998). Changes in milk protein quantity
and quality facilitate the balance of blood amino acids
(which may impact on neurotransmitter function during
early stages of brain development); reducing the total
protein intake could also be important for the prevention of
weight problems in adults. However, hydrolysed proteins
are important in the prevention of atopic disorders and
certain milk proteins have been shown to have antibacterial
activity. Many trials have been published so far with short-
term assessments, most of them with positive findings
(Agostoni and Haschke 2003).
In a French study, the potential of a new formula
containing active metabolites of whey retentate from
fermented milk has been examined (Yazourh et al. 2000).
Metabolites obtained by the fermentation of milk with B.
breve and S. thermophilus appear to produce a prebiotic
effect on the intestinal microflora. As with oligosaccharide
prebiotics, analysis showed that bifidobacterial counts were
increased in supplemented formula compared to the control,
and that counts of C. perfringens and Bacteroides fragilis
were reduced (Meddah et al. 2001). Susceptibility to
heating and oxidation suggested that an enzyme or protein
9-7
might play a role in the induced microbial changes (Mullie
et al. 2002).
The whey fraction of human and cow’s milk contains
proteins such as α-lactalbumin, β-lactoglobulin (cow’s
milk) and lactoferrin; these have been shown to exert
antimicrobial functions and bifidogenic properties
(Pelligrini et al. 1999; Pihlanto-Leppälä et al. 1999;
Schanbacher et al. 1998; Petschow and Talbott 1991).
Similarly, some proteins and peptides of the casein fraction
can have an effect on the intestinal microflora through the
regulation of gut motility, antibacterial action and
bifidogenic properties (Schanbacher et al. 1998).
Simultaneously, human milk proteins and peptides may also
provide an environment highly stimulating for the growth of
bifidobacteria (Liepke et al. 2002). The addition of the two
milk proteins α-lactalbumin and glycomacropeptide has
been of particular interest, even though no published data
exists to date on their effects on infant microflora in vivo.
α-Lactalbumin is a major whey protein found in human
breast-milk and is of considerable nutritional value to the
infant as it is one of the two subunits of lactose synthase
(Xue et al. 2001) and is therefore required for lactose
synthesis. It is a small acidic Ca2+ binding protein which
facilitates the absorption of essential minerals while
providing a well-balanced supply of essential amino acids
to the growing infant (Lönnerdal and Lien 2003; Kuhn et al.
1980). In transgenic mice expressing 5–15 times more α-
lactalbumin in their milk than control mice, pups showed a
4% increase in growth after 10 days of lactation, which
resulted from increased milk production, as estimated using
the weigh-suckle-weigh technique (Boston et al. 2001). In
comparison, Stinnakre et al. (1994) created α-lactalbumin-
deficient mice by disrupting its gene using homologous
recombination in embryonic stem cells. The resulting
homozygous mice were all viable and fertile although
females were unable to feed their offspring since they
produced a highly viscous milk rich in fat and protein but
devoid of any α-lactalbumin; as a consequence, this milk
proved too thick for the pups to suckle. A 1.7Å resolution
crystallographic analysis of α-lactalbumin showed that its
three dimensional structure is similar to that of a hen egg
white (type-c) lysozyme, which suggests that they share a
common ancestral gene (Xue et al. 2001).
In vitro, the testing of α-lactalbumin and lactoferrin
purified from cow’s milk showed that they were potent
growth promoters for bifidobacteria, showing greatest
activity for B. infantis, B. breve, and two strains of B.
bifidum (Petschow and Talbott 1991). In contrast, Pihlanto-
Leppälä et al. (1999) demonstrated that α-lactalbumin,
hydrolysed with pepsin or trypsin, lowered the metabolic
activity of E. coli JM103 to just 21% of normal activity
after 6 h incubation; the undigested protein did not inhibit
bacterial growth or metabolism. The bacteriostatic effect of
the α-lactalbumin hydrolysates occurred at a higher
concentration (25 mg x ml-1) than that observed with the
lactoferrin hydrolysate (10 mg x ml-1) (Saito et al. 1991).
However, the study was carried out under optimum
9-8 Functional foods in infant formulae W.M. Brück
conditions of growth for E. coli, which does not necessarily
represent the environment of the colon. Pelligrini et al.
(1999) found that proteolytic digestion of α-lactalbumin by
trypsin and chymotrypsin yielded three peptide fragments
with bactericidal properties against Gram-positive bacteria
and a limited action against Gram-negative organisms.
Hence, it may be assumed that digestion by endopeptidases
gives α-lactalbumin some antimicrobial function.
The advantage of α-lactalbumin-supplemented formulae
was shown by Heine et al. (1996). In a crossover study
with standard formula and two low protein formulae
containing low and high concentrations of α-lactalbumin,
infants receiving low to high concentrations of α-
lactalbumin had serum tryptophan concentrations as high as
those of breast fed infants. In a multi-centre trial, the
influence of α-lactalbumin enriched, low protein formula on
growth and serum protein status in infants was
investigated. The data confirmed that normal growth and
protein status was maintained (Lien et al. 2004), which
suggests that development of a low-protein formula
enriched with α-lactalbumin would yield a formula similar
in composition to human milk.
The biological activity of glycomacropeptide (GMP)
derived from bovine κ-casein has been the focus of much
interest in recent years and a variety of physiological
functions have since been attributed to it (Brody 2000). In
cow’s milk, GMP bears saccharides of various lengths
attached to a single peptide chain (Otani et al. 1995), as
detailed below:
• Monosaccharide: GalNAc-O-R
• Disaccharide: Galβ1 → 3GalNAc-O-R
• Trisaccharide: NeuNAcα2 → 3Galβ1 → 3GalNAc-O-R
• Trisaccharide: Galβ1 → 3NeuNAcα2 → 6GalNAc-O-R
• Tetrasaccharide: NeuNAcα2 → 3Galβ1 →
3NeuNAcα2 → 6GalNAc-O-R
where Gal = galactose; GalNAc = N-acetylgalactosamine;
NeuNAc = N-acetylneuraminic acid (Saito and Itoh 1992;
Saito et al. 1981; Doi et al. 1979).
GMP activity has been attributed to both the polypeptide
portion of the molecule and the carbohydrate chain
containing the sialic acid derivative N-acetylneuraminic
acid (NeuNAc; Fiat and Jolles 1989).
Cholera toxin produced by Vibrio cholerae causes
gastroenteric disorders such as diarrhoea. Inhibition by κ-
casein GMP of cholera toxin-derived morphological
changes of Chinese hamster ovary (CHO-K1) cells has been
investigated. Kawasaki et al. (1992) demonstrated that
concentrations as little as 20ppm of GMP resulted in about
70–80% inhibition of such changes. At a concentration of
100ppm, GMP resulted in almost complete rounding of
CHO-K1 cells, indicating that GMP had bound to the
cholera toxin, leaving cells intact. Enzymatic digestion of
GMP with sialidase, which hydrolyses sialic acids, lowered
the effectiveness of inhibition, while removal of NeuNAc
resulted in a complete loss of GMP inhibitory activity.
However, inhibitory activity still remained, to a certain
extent, after GMP hydrolysis with proteinases. This finding
implied that GMP interacted with the cholera toxin through
NeuNAc, resulting in inhibition of the binding ability of
cholera toxin to CHO-K1 cells (Kawasaki et al. 1992).
Similar inhibitions were obtained against E. coli heat labile
enterotoxins LT-I and LT-II (associated with colonization
factor antigen CFA7I and CFA/II, respectively) using the
CHO-K1 model (Kawasaki et al. 1992). Additionally, in
vivo studies using a mouse model were carried out and
showed that feeding of κ-casein GMP resulted in 100%
protection against cholera toxin and 80% against LT-I.
However, binding of E. coli was not inhibited.
Neeser et al. (1994) investigated the effect of milk
components on the prevention of dental caries. GMP plus
disaccharide II prevented haemagglutination by
Streptococcus mutans, S. sanguis and Actinomyces viscosus,
while binding to a tooth model was prevented only for S.
sobrinus and S. sanguis. Proof of GMP binding to S.
sanguis was obtained by electron microscopy (Neeser et al.
1995). Stromquist et al. (1995) showed that when sections
of formalin-fixed, paraffin-embedded stomach tissue were
incubated with purified human κ-casein, adhesion of
bacteria to the tissue was inhibited. This disappeared when
oxidation by metaperiodate occurred, suggesting that the
glycan part of the protein was responsible for an adhesive
effect.
NeuNAc-containing substances such as GMP are also
believed to possess growth-promoting effects on
bifidobacteria (Gyorgy et al. 1954). Petschow and Talbott
(1991) reported that growth promoting activity for some
bifidus species was present in ultrafiltered cow’s milk
permeate as well as retentate, leading to the assumption that
the amino acid portion was critical for bifidus growth, since
oligomerization prevented GMP from passing through the
ultrafiltration membrane. Azuma et al. (1985), however,
reported that GMP derived from human milk κ-casein was
effective as bifidus growth-promoting factor even at
concentrations as low as 5 x 10-2 µg/l. In vitro, GMP
strongly promoted the growth of B. breve, B. bifidum and B.
infantis (Idota et al. 1994).
In vitro studies on the influence of GMP and α-
lactalbumin on gut microflora were confirmed in several
recent studies using a two-stage continuous culture system
containing fresh faecal specimens of infants aged 1, 3 and 6
months. In vessels containing breast-milk, α-lactalbumin
and GMP-supplemented formula, counts of Bacteroides,
clostridia and E. coli were significantly decreased, while
bifidobacteria remained stable. This showed that while a
bifidogenic effect was not observed, α-lactalbumin and
GMP did have an inhibiting effect on the potentially
pathogenic gut microflora (Brück et al. 2002). In an in vitro
infection study, similar results were observed, while
enteropathogenic E. coli counts decreased in vessels
containing α-lactalbumin and breast milk. Salmonella also
decreased significantly in all vessels containing breast milk,
 Functional foods in infant formulae W.M. Brück
α-lactalbumin and GMP (Brück et al. 2003a). The
influence of α-lactalbumin and GMP on the gut microflora
was further examined in Rhesus monkeys challenged with
108 cfu enteropathogenic E. coli. While monkeys fed an α-
lactalbumin and GMP-free control formula developed acute
diarrhoea, those fed breast milk and formula supplemented
with α-lactalbumin had no diarrhoea. Infant monkeys fed
GMP-supplemented formulae also developed diarrhoea,
even though episodes were of shorter duration and
associated symptoms such as fever and dehydration were
less severe (Brück et al. 2003b).
4.4 Nucleotides
Supplementation of infant formula and follow-on formula
with nucleotides is permitted by the European Union and is
regarded as a significant improvement in the continuing
development of formula feeds (Schlimme 2000; Tanaka et
al. 1996). Nucleotides are low-molecular-weight non-
protein nitrogen components that form the building blocks
of nucleic acids and play major roles in multiple
biochemical processes such as iron absorption, lipid
metabolism, gastrointestinal development, and hepatic
function (Schlimme 2000; Cosgrove 1998). They are
naturally present in high concentrations in breast milk in
both free and bound forms. Nucleotides have been shown
to affect in vitro epithelial cells from the foetal upper
intestine by modulation of cell proliferation, differentiation
and apoptosis (Kit et al. 1999). Since they were first
recognized in breast milk in 1960, 13 acid-soluble
nucleotides have been identified (Carver 1999).
Animal studies indicate that exogenous nucleotides may
have beneficial gastrointestinal and immunological effects,
especially during times of rapid development (Agget et al.
2003). Van Buren et al. (1985) demonstrated that mice fed
diets containing nucleotides were less susceptible to
Candida albicans infections. Survival rates from sepsis
caused by S. aureus were also increased in mice fed a diet
supplemented with nucleotides (Kulkarni et al. 1986).
Some studies with human infants, while conflicting with
others, have confirmed these findings and reported
beneficial effects of nucleotides on gut microflora,
diarrhoea and immune function (Yu 2002). It was shown
that the effect of nucleotide-supplemented formulae on the
gut microflora was characterized by a higher percentage of
bifidobacteria and a lower percentage of enterobacteria, as
compared to unsupplemented formulae, resulting in faecal
microbial patterns similar to the stools of breast-fed infants
(Mountzouris et al. 2002; Yu 2002). Significant differences
in the percentage populations of bifidobacteria, lactobacilli,
and enterobacteria were found when comparing groups fed
infant formula supplemented with nucleotides, and non-
supplemented groups. However, infants fed human milk
still had significantly higher counts of bifidobacteria when
compared to either group (Gil et al. 1986). However, some
studies have shown negative effects associated with
nucleotide supplementation of infant formulae (Balmer et
al. 1994). Thirty-two infants were fed infant formula
9-9
supplemented with nucleotides from birth; compared to the
breast-fed and non-nucleotide supplemented controls, the
study groups had E. coli as the dominant organism in their
faecal flora.
A comparable conflict can be found in studies examining
the effect of nucleotides on diarrhoea episodes. In a trial by
Brunser et al. (1994), no significant differences in either the
nucleotide-supplemented or control groups on the number
or duration of recurring diarrhoea episodes was found.
However, the number of initial episodes was significantly
lower in the nucleotide-supplemented group. In another
study, no significant differences in populations of
enteropathogens or severity of infections was observed in
healthy full term infants that were either breast-fed or fed
standard formula supplemented with 33 mg nucleotides/l of
standard formula (Carver et al. 1991). More recently, a 12
month multi-centre study on 336 infants aged between 8–28
weeks showed that those fed nucleotide-supplemented
formulae had a significantly lower risk of diarrhoea than
infants fed a control formula (Similac). Significantly higher
levels of IgAs were also observed in the same groups (Kuo-
Inn et al. 2000). Navarro et al. (1999) examined the effect
of nucleotides on immunoglobulin development in pre-term
infants. While IgG levels fell progressively during the first
3 months of life, IgM levels increased in pre-term infants
fed a formula supplemented with nucleotides when
compared to infants fed a control formula.
These results suggest that the mechanism by which
nucleotides affect the maturation of the infant gut and
immune system is not fully understood. Even though
significant advances have been made in the understanding
of the effects of dietary nucleotides, many questions are still
left unanswered.
5. Conclusions
The gut microflora of breast-fed and formula-fed infants
differs significantly. While breast-fed infants harbour a
predominantly benign microflora consisting principally of
bifidobacteria, formula-fed infants have a more complex
flora which includes facultative anaerobes, bacteroides and
clostridia. It is possible that the gut microflora offers
protection from disease and infection and there is evidence
that nutrition and food supplements may influence the
gastrointestinal tract and therefore host defence.
Supplementation of infant formulae with one or a
combination of functional food ingredients may promote
improvement of health and influence the general
development of the infant. However, despite recent
important advances, the available data concerning the
introduction of functional foods into the infant diet is too
fragmentary to establish their immediate and long-term
efficacy and safety. Thus, recommendation of a specific
product requires meticulous analysis of all issues at hand. It
is vital to understand all implications when introducing
fermentable components into the diet of the young. Further
in vivo and in vitro investigations are necessary to
9 - 10 Functional foods in infant formulae W.M. Brück
understand the complete potential of such food as additions
to infant feeds.
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