ARTICLE IN PRESS

FOOD
MICROBIOLOGY
Food Microbiology 21 (2004) 119124

www.elsevier.nl/locate/jnlabr/yfmic

Short communication

Prebiotic effects of a wheat germ preparation in human

healthy subjects
D. Matteuzzia,*, E. Swennena, M. Rossia, T. Hartmanb, V. Lebetb
a

Department of Pharmaceutical Sciences, University of Bologna, Via Belmeloro, 6, Bologna 40126, Italy
b
Multiforsa AG, Hinterbergstrasse, 58, Steinhausen CH-6312, Switzerland
Received 12 July 2002; received in revised form 16 January 2003; accepted 26 January 2003

Abstract
A double-blind placebo-controlled study was performed to investigate the behaviour of different intestinal bacterial groups in 32
healthy subjects during treatment with the prebiotic wheat germ preparation ViogermsPB1.
Microbiological methods and uorescent in situ hybridization technique were used to identify the following bacterial groups:
coliforms, clostridia, bacteroides, lactobacilli and bidobacteria. After 20 days of supplementation of the product, the coliform
population and pH decreased signicantly. The number of lactobacilli and bidobacteria increased signicantly only in subjects with
low basal levels. No signicant changes were observed for the other bacterial groups and total bacteria did not increase. Treatment
with placebo did not induce any variation. These results showed that the product ViogermsPB1 possesses a prebiotic effect and has
a potential to improve hosts health.
r 2003 Elsevier Ltd. All rights reserved.
Keywords: Prebiotic effect; Wheat germ; Intestinal microbiota

1. Introduction
The large bowel of humans is colonized by a complex
microbial community, which deeply interacts with the
host. The concentration of microbes increases as
progression is made down the intestinal tract ranging
from values of about 104 g
1 in the small intestine to
106107 g
1 in the ileo-caecal region and 10111012 g
1 in
the colon. The intestinal microbiota includes hundreds
of bacterial species, mainly anaerobic, but only 3040
species account for 9598% of the micro-organisms in
the community (Savage, 1989). Concerning the different
intestinal bacterial groups, it is well known that
bidobacteria and lactobacilli can be used as probiotics,
i.e. live microbial food ingredients that are benecial to
health, while some others, like Escherichia coli and
proteolytic clostridia, are unfavourable for humans and
animals in terms of health and nutrition.
In the last decade many efforts were made in order to
increase in the colon the number and/or the activity of
the bacterial groups considered benecial for the host
*Corresponding author. Fax: +39-051-2099734.
E-mail address: matt@alma.unibo.it (D. Matteuzzi).
0740-0020/03/$ - see front matter r 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0740-0020(03)00009-1

and to decrease those considered as harmful. Prebiotics

are non-digestible oligosaccharides that reach the colon
in the intact state and stimulate growth of benecial
colonic bacteria (Gibson and Roberfroid, 1995). Their
use is considered a powerful tool to increase the number
or the activity of the two main health-promoting
bacterial groups, bidobacteria and lactobacilli (Tuohy
et al., 2001; Fukuda et al., 2002). Oligosaccharides
proposed as potential prebiotics include lactulose,
galacto-oligosaccharides, fructo-oligosaccharides (oligofructose and inulin), soybean oligosaccharides, etc.
(Cummings et al., 2001).
The wheat germ product ViogermsPB1 is a dietary
supplement that provides concentrated nutrients of high
biological value. Within the carbohydrate fraction, it
presents cell wall polysaccharides and rafnose, which
resist human digestion and reach the large bowel, where
they can affect the colonic microora. In fact, a recent
study demonstrated the bidogenic activity of ViogermsPB1 by comparing its in vitro fermentation
properties with those of other commercial prebiotics
(Arrigoni et al., 2002).
The objective of this study was to evaluate the
prebiotic activity of the product ViogermsPB1 in

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D. Matteuzzi et al. / Food Microbiology 21 (2004) 119124

healthy subjects, by analysis of the changes in the

intestinal microbiota. The concentration of relevant
intestinal bacterial group were enumerated by plate
counting and uorescent in situ hybridization (FISH)
technique (DeLong et al., 1989; Amann et al., 1995).

2. Materials and methods

2.1. Composition of ViogermsPB1
ViogermsPB1 was prepared by Multiforsa AG,
Steinhausen, Switzerland, by coldpression of wheat
germ. Hundred grams of the product contain 30 g of
proteins, 6.9 g of oil, 54.1 g of total carbohydrates, 4.5 g
of minerals and are a good source of vitamins and trace
elements. The main components of the carbohydrate
fraction, expressed as percentage of total sugars, are:
starch 30.6, sucrose 26.0, dietary bre (cellulose,
hemicellulose and pentosans) 21.1 and rafnose 17.5.
Rafnose and dietary bre are not digested in the small
intestine and can behave as prebiotics, having marked
effects on faecal microora.
2.2. Selection of volunteers and study design
A double-blind placebo-controlled trial was carried
out in two different centers (University of Bologna, Italy
and Multiforsa AG, Steinhausen, Switzerland), where
equal experimental conditions were strictly maintained.
The study population was composed of 32 healthy adult
volunteers (22 male and 10 female), free of antibiotic
treatment for at least 3 months. Subjects consumed their
usual diets without any extra supplement of prebiotics,
avoiding fermented dairy products containing bidobacteria or lactobacilli. These limitations were observed
since 10 days before the treatment until the end of a 10
days wash-out period. For 20 days they consumed 10 g
of prebiotic product (ViogermsPB1) or 10 g of placebo
(milled bread crumbles) once a day.
2.3. Microbiological analyses
2.3.1. Bacterial enumeration and identification by plate
counting
Faecal samples from all 32 volonteers were collected
in sterile plastic vessels immediately after defecation at
the end of each period of the trial (T0 =start of the
treatment period, T20 =end of the treatment period,
T30 =end of the wash-out period), stored at
20 C and
analysed within 10 days. All analyses and preparations
were done in an anaerobic cabinet (Anaerobic System,
Mod. 2028, Forma Scientic Co., Marietta, OH, USA).
Specimens were homogenized and serially diluted
with prereduced half-strength Wilkins Chalgren anaerobic broth (Oxoid, Basingstoke, UK). From each of the

dilutions, 0.1 ml was plated in triplicate onto selective

media: MacConkey Agar (Merck, Darmstadt, Germany) for coliforms, OPSP Agar (Oxoid, Basingstoke,
UK) for Clostridium perfringens, LAMVAB Agar
(Hartemink et al., 1997) for lactobacilli and RB Agar
(Hartemink et al., 1996) for bidobacteria. All media
were kept X24 h in the anaerobic chamber before being
used. MacConkey agar plates were incubated aerobically at 37 C for 24 h, all other media were incubated
anaerobically at 37 C for 4872 h.
Representative colonies of each selective medium were
identied at genus level by standard bacteriological
procedures, such as Gram stain reaction, colonial and
cellular morphology, biochemical reactions, API System
(BioMerieux, Lyon, France). After identication, colonies were counted; counts from triplicate plates were
averaged. The lowest limit of detection was 1000 microorganisms g
1 of faeces.
2.3.2. Bacterial enumeration by FISH technique
Six groups of micro-organisms were directly studied
by FISH technique in faecal samples of 18 volunteers
treated with ViogermsPB1 or placebo, randomly
selected. For this purpose, ready to use commercial kits
(Microscreen B.V., Microbial Diagnostics, Groningen,
The Netherlands) specic for the following bacterial
groups, were used: Lactobacillus group (Lactobacillus
10-ME-H006), Bifidobacterium genus (Bifidobacterium
10-ME-H001), Clostridium coccoides group (C. coccoides 10-ME-H011), C. butyricum group (C. butyricum
10-ME-H009), Bacteroides group (Bacteroides/Prevotella 10-ME-H008) and E. coli (E. coli 10-ME-H004).
The slides were evaluated with a Nikon Eclipse E-600
epiuorescence microscope equipped with a mercury arc
lamp (Nikon, HBO, 100W) and the FITC specic lter
Nikon BA 520.
2.3.3. Enumeration of total bacteria
For analysis of total microbial community of the
faecal sample, the uorescence assay LIVE/DEAD
BacLight Bacterial Viability Kit (Molecular Probes,
Leiden, The Netherlands) was used. Faecal slurries at
10% were made in saline, vigorously homogenized with
glass spherules, then centrifuged at 50g for 5 min. The
supernatant was diluted 1:100 in saline and the cells
were washed twice in a microfuge to remove signicant
traces of interfering components. To enumerate the total
bacteria as dead cells, the nal pellet was resuspended in
70% isopropyl alcohol and the suspension was incubated for 1 h at room temperature, mixing every 15 min.
The cells (400 ml) were washed twice with saline and
resuspended in the same volume. 0.6 ml of component B
(SYTO 9 1.67 mm, propidium iodide 18.3 mm) was
added to the suspension. The sample was mixed and
incubated at room temperature in the dark for 15 min. A
known volume of the stained suspension was diluted

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D. Matteuzzi et al. / Food Microbiology 21 (2004) 119124

9.0

ViogermPBI

Placebo

8.8
8.6
8.4
8.2
pH

and ltered with a polycarbonate membrane lter. The

number of total bacteria was calculated by the following
formula: mean number of stained cells per eld of
view  total number of elds per effective lter surface 
dilution factor.
The slides were evaluated with a Nikon Eclipse E-600
epiuorescence microscope equipped with a mercury arc
lamp (Nikon, HBO, 100 W) and the specic lter Nikon
BA 590.

121

**

8.0
7.8
7.6
7.4

2.3.4. PCR detection of bifidobacterium

In order to conrm the identication of bidobacteria, 3050 colonies randomly selected from the highest
dilution of RB agar plates were amplied using the 16S
rDNA primer set Bif164/Bif662 specic for the genus
Bifidobacterium, under the conditions reported by Kok
et al. (1996).
2.4. Evaluation of faecal pH
During the prebiotic administration trial, faecal pH
was determined at T0 ; T20 and T30 directly in the faecal
sample diluted 1:10 with distilled water using a pHmeter (Radiometer, Copenhagen, Denmark).
2.5. Statistical analysis
Data were expressed as mean log10 number colony
forming units (cfu) g
1 of faeces (dry weight)7standard
deviation (s.d.).
Statistical analysis was performed using paired
Students t-test, to compare pretreatment, post-treatment and wash-out faecal concentration of the different
bacterial groups examined and faecal pH.
Statistical signicant differences were considered vs.
the basal concentration for the value of Po0:05:

3. Results
All the 32 subjects completed the study. There were
no reports of atulence or of problems with the
palatability of the product. Variations of the faecal pH
in 23 healthy subjects treated with ViogermsPB1 and in
nine volunteers administered with placebo are reported
in Fig. 1. Prebiotic consumption induced after 20 days a
signicant decrease of 0.4 pH units. The successive
wash-out period of 10 days allowed a gradual return of
the pH value to the initial level. In placebo-treated
subjects, no signicant differences in pH value were
detected at any time-point examined.
Titer variations of several intestinal bacterial groups
during the prebiotic administration were evaluated by
classical culture methods and FISH techniques. The
microbiological data, obtained using selective media for
enumeration of the viable cfu, and the genetic-based

7.2
7.0
T0

T20

T30

** P < 0.01

Fig. 1. pH values of faecal samples of volunteers during ViogermsPB1 treatment or placebo.

culture-independent FISH methodology, were consistent. The accuracy of the rst approach is limited by the
selectivity of the media: the more selective the media, the
higher the risk that microorganisms belonging to the
specic group will not grow. On the other hand, FISH
analysis can be limited by unspecic binding of probes
with 16S rRNA of other faecal bacteria and efcacy of
probe access to the bacterial target.
As shown in Table 1, faecal concentrations of
coliforms, enumerated by both culture method and
FISH technique, decreased signicantly after 20 days of
prebiotic treatment, compared to their baseline level
evaluated at time T0 (Po0:05). However, 10 days after
terminating the treatment, concentration of this bacterial group returned to the basal level. During the whole
trial, no signicant changes in faecal coliforms were
registered for the placebo group.
At a rst analysis, no signicant variations in number
of faecal C. perfringens, lactobacilli and bidobacteria,
evaluated by culture method, were found in prebiotictreated group or in placebo. Similar data have been
obtained by FISH technique even if the number of
faecal bidobacteria and lactobacilli obtained on
selective plates were signicantly lower (Po0:01) than
those obtained by FISH. It is conceivable that this
difference is due to the fact that FISH can enumerate
culturable, unculturable, living and dead bacteria.
Bacteroides community, evaluated by FISH, did not
show any signicant shift in response to consumption of
ViogermsPB1.
However, a consistent decrease in standard deviation
registered for the bidobacteria number in volunteers
treated with the prebiotic product, by using both
analysis methods, could be observed.
A more detailed evaluation of the microbiological
data obtained during the prebiotic treatment allowed to
identify a different gut microora modication behaviour in subjects depending on their initial level of
lactobacilli and bidobacteria. As reported in Fig. 2,

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122

Table 1
Enumeration by plate culture method and FISH technique of coliforms, clostridia, lactobacilli, bidobacteria and bacteroides in faecal samples of
volunteers during ViogermsPB1 treatment or placebo
C. perfringens

Coliforms
s

Viogerm PB1 Placebo

(n 23)
(n 9)
Mean bacterial count (log10 cfu g
1
T0
6.3371.33
T20
5.7071.60n
6.4571.46
T30

faecal dry weight7s.d.)

6.7271.63
4.8471.59
6.7171.11
4.9071.31
6.4471.32
4.6071.60

Viogerm PB1 Placebo

(n 23)
(n 9)

ViogermsPB1 Placebo
(n 23)
(n 9)

4.7171.22
5.0471.17
4.9671.24

8.1871.50
8.5170.66
8.3971.27

4.7871.41
4.6771.45
4.6771.22

Viogerm PB1 Placebo

(n 12)
(n 6)

Viogerm PB1 Placebo

(n 12)
(n 6)

ViogermsPB1 Placebo
(n 12)
(n 6)

8.0270.89
7.8370.79
7.5870.96

9.0470.96
9.4570.46
9.3770.39

9.9370.65
10.1070.53
10.1370.52

7.9370.94
7.8670.96
7.6470.97

9.1270.89
9.2870.75
9.1770.68

Bacteroides group
s

9.9970.75
10.0970.63
9.9570.65

C. butyricum group

Viogerm PB1 Placebo

(n 12)
(n 6)

Viogerm PB1 Placebo

(n 12)
(n 6)

ViogermsPB1 Placebo
(n 12)
(n 6)

7.0671.32
6.3171.64
7.3470.98

9.7770.78
9.7470.86
9.7370.70

9.5170.79
9.2370.81
9.2670.75

7.2371.21
7.3170.87
7.3871.03

7.4971.79
7.5271.67
7.4471.79

C. coccoides group

E. coli

T0
T20
T30

5.2071.04
5.3570.88
5.2770.88

Bifidobacterium spp.

Bidobacteria

Viogerm PB1 Placebo

(n 23)
(n 9)

Lactobacillus spp.

FISH technique
T0
T20
T30

Lactobacilli

9.5471.04
9.8370.88
9.8271.11

9.4370.80
9.2970.85
9.2270.71

ViogermsPB1 Placebo
(n 12)
(n 6)
Total bacteria
T0
T20
T30
n

12.0370.27
11.9870.50
12.0170.39

Po0:05:

10.00
9.00
Log10 CFU/g dry feces

11.9750.31
12.0670.12
12.0470.28

bifidobacteria
low
high

lactobacilli
high

low

8.00
7.00
6.00
*

5.00
4.00
3.00

T0

T20

T30

* P < 0.05

Fig. 2. Variation of bidobacteria and lactobacilli in faecal samples of

volunteers during ViogermsPB1 treatment considering their initial
concentration.

volunteers with bidobacteria counts o8 log10 cfu g
1

dry faeces at the beginning of the trial showed a
signicant increase of 1 log unit in Bifidobacterium

population after 20 days of prebiotic treatment. In these

subjects, after the end of the treatment with ViogermsPB1, bidobacterial concentration slowly returned to its initial level. On the contrary, in
volunteers with initial bidobacterial concentration
>8.5 log10 cfu g
1 dry faeces, Bifidobacterium group
did not evidence any signicant change after prebiotic
treatment, neither after wash-out period.
Analogously, mean concentration of lactobacilli in
subjects with initial Lactobacillus values o4.5
log10 cfu g
1 dry faeces increased signicantly after 20
days of prebiotic treatment and remained at this high
level even after 10 days of wash out. In subjects with an
initial lactobacilli titer >5 log10 cfu g
1 dry faeces, no
signicant variation was never observed during the
study. No signicant correspondence was found among
subjects low in bidobacteria and those low in
Lactobacillus.
Concomitantly, consumption of ViogermsPB1
seemed to act selectively, decreasing coliforms and
stimulating growth of lactobacilli and bidobacteria,
whereas the effect on total bacteria was inconsistent.

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4. Discussion
A number of studies have shown that feeding
prebiotics to humans or animals alters the composition
of the faecal ora, increases absorption of calcium and
magnesium (Ohta et al., 1995), decreases plasma
triglycerides (Fiordaliso et al., 1995) and impedes the
development of chemically induced preneoplastic lesions
in the colon (Reddy et al., 1997).
Human feeding trials have been performed with
prebiotics in order to increase endogenous probiotic
microora. Most prebiotics that have been studied to
date are non-digestible oligosaccharides. Some galactooligosaccharides such as rafnose are metabolized by
bidobacteria and lactic acid bacteria, which possess
alfa-galactosidase. Tortuero et al. (1997) showed that
rafnose is a very effective substrate for fermentation in
the large intestine of rats and that diets containing
rafnose decrease faecal pH, increase total volatile fatty
acids and the concentration of lactobacilli.
The purpose of the present study was to evaluate if
ViogermsPB1, rich in rafnose and cell wall polysaccharides, may have a potential use in fortifying the
endogenous microora. Our study indicates that the
ingestion of this commercial wheat germ preparation
produces a signicant decrease of pH. It is conceivable
that this effect is due to the increase in the caecal pool of
short-chain fatty acids. Lower caecal pH values are
believed to prevent the growth of pH sensitive pathogens, such as E. coli and Salmonella and to increase
mineral uptake. Furthermore, short-chain fatty acids are
absorbed by colonic enterocytes, then used as energy
source (Clausen and Mortensen, 1994) or eventually
metabolized by the liver, affecting certain hepatic
metabolic pathways (Remesy et al., 1995).
The prebiotic efcacy of ViogermsPB1 was conrmed also by the signicant decrease in coliforms
concentration. This microbial group is associated to
detrimental enzymic activities, such as b-glycosidase, bglucuronidase, azoreductase, nitroreductase, leading to
changes in toxicity of ingested or endogenous substances
(Hill, 1995).
As far as bidobacteria and lactobacilli are concerned, no signicant increase was observed but the
overall decrease in pH suggested an increase in
fermentative activity. It is well known that the initial
levels of bidobacteria and lactobacilli inuence the
extent of elevation of these bacterial groups (Roberfroid
et al., 1997; Conway, 2001). Our results conrmed this
statement, presenting a signicant increase of bidobacteria and lactobacilli after consumption of ViogermsPB1, in subjects with initial levels o8 log10 and
o4.5 log10 cfu g
1 dry faeces, respectively. According to
Rao (1999) the doseeffect relationship between the
ingestion of the prebiotic and faecal bidobacteria
should be considered in terms of the initial levels.

123

In conclusion, our study conrms that the consumption of ViogermsPB1, a highly nutritious wheat germ
preparation, has a prebiotic effect. In fact, it is rich in
rafnose and other undigestable polysaccharides, which
are available for microbial fermentation and modify the
colonic microora by lowering some Gram-negative
bacteria, such as coliforms, and increasing potentially
health-promoting bacteria, such as bidobacteria and
lactobacilli. Furthermore, its selective effect did not
change the number of total bacteria.

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