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European Journal of Clinical Nutrition (2008) 62, 594–599

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ORIGINAL ARTICLE
The impact of freezing and toasting on the
glycaemic response of white bread
P Burton and HJ Lightowler

Nutrition and Food Science Group, School of Life Sciences, Oxford Brookes University, Oxford, UK

Objective: To investigate the impact of freezing and toasting on the glycaemic response of white bread.
Subjects/methods: Ten healthy subjects (three male, seven female), aged 22–59 years, recruited from Oxford Brookes
University and the local community. A homemade white bread and a commercial white bread were administered following four
different storage and preparation conditions: (1) fresh; (2) frozen and defrosted; (3) toasted; (4) toasted following freezing and
defrosting. They were administered randomized repeated measures design. Incremental blood glucose, peak glucose response,
2 h incremental area under the glucose response curve (IAUC).
Results: The different storage and preparation conditions resulted in lower blood glucose IAUC values compared to both types
of fresh white bread. In particular, compared to the fresh homemade bread (IAUC 259 mmol min/l), IAUC was significantly lower
when the bread was frozen and defrosted (179 mmol min/l, Po0.05), toasted (193 mmol min/l, Po0.01) and toasted following
freezing and defrosting (157 mmol min/l, Po0.01). Similarly, compared to the fresh commercial white bread (253 mmol min/l),
IAUC was significantly lower when the bread was toasted (183 mmol min/l, Po0.01) and frozen, defrosted and toasted
(187 mmol min/l, Po0.01).
Conclusions: All three procedures investigated, freezing and defrosting, toasting from fresh and toasting following freezing and
defrosting, favourably altered the glucose response of the breads. This is the first study known to the authors to show reductions
in glycaemic response as a result of changes in storage conditions and the preparation of white bread before consumption. In
addition, the study highlights a need to define and maintain storage conditions of white bread if used as a reference food in the
determination of the glycaemic index of foods.
European Journal of Clinical Nutrition (2008) 62, 594–599; doi:10.1038/sj.ejcn.1602746; published online 4 April 2007

Keywords: glycaemic response; bread; food storage; food preparation; blood glucose

Introduction Recent data support the preventive potential of a low-GI


diet against the development of type 2 diabetes and
The glycaemic index (GI), first introduced in 1981 (Jenkins cardiovascular disease (Salmeron et al., 1997a, 1997b; Frost
et al., 1981), is a classification of the blood glucose raising et al., 1999). There is also an interest in the potential of low-
potential of carbohydrate foods. It is defined as the GI diets in body weight management, with studies showing
incremental area under the blood glucose curve (IAUC) of that low-GI foods, or lowering the GI of a food, may reduce
a 50 g available carbohydrate portion of a test food expressed hunger and result in a lower energy intake (Ludwig, 2000;
as a percentage of the response to 50 g available carbohydrate Warren et al., 2003).
of a reference food taken by the same subject, on a different As consumer interest in GI has grown in recent years, a
day (FAO/WHO, 1998). major challenge for the food industry is to develop and
produce foods of low glycaemic response. In the UK, bread
Correspondence: Dr H Lightowler, Nutrition and Food Science Group, School
forms the most important staple amongst starch foods and is
of Biological and Molecular Sciences, Oxford Brookes University, Gipsy Lane
Campus, Headington, Oxford OX3 0BP, UK. one of the largest sectors in the food industry, producing
E-mail: hlightowler@brookes.ac.uk almost 12 million loaves and packs every day (Federation of
Contributors: PB planned and designed the study and was responsible for the Bakers, 2005). Moreover, white bread sales represent over
collection of data and writing the manuscript. HJL advised on the study design
70% of all bread sales in the UK. In 2004–2005, the
and contributed to the preparation of the manuscript.
Received 3 July 2006; revised 14 February 2007; accepted 19 February 2007; household consumption of white bread was almost 400 g
published online 4 April 2007 per person per week compared to 120 and 45 g per person per
Glycaemic response of white bread
P Burton et al
595
week for wholemeal and brown bread, respectively (Depart- Table 1 Characteristics of study population
ment for Environment, Food and Rural Affairs, 2006).
Mean7s.d.
In the UK, the baking industry predominantly uses the
Chorleywood Bread Process (CBP), which is responsible for Age (years) 45.4714.3
over 80% of bread produced. The CBP, developed in 1961, is Height (m) 1.7370.97
a mechanical dough development process involving inten- Weight (kg) 71.3710.1
BMI (kg/m2) 23.972.1
sive mixing, the use of oxidants, a short fermentation process
and the use of a pan for baking. In addition, the process has Abbreviations: BMI, body mass index; s.d., standard deviation.
an important impact in the UK as it enables the use of low-
protein home-produced wheat (Belderok, 2000). However, the
result of developments in the bread-making process is a community. To participate, subjects were required to be
highly favoured, but medium- to high-GI white bread. between 18 and 59 years of age, with a body mass index
In the UK, an increase in time pressure in family life has (BMI) o30 kg/m2. Interested subjects were asked to complete
meant that infrequent, bulk shopping and freezing are a health screening questionnaire to check against ill health,
widespread. Consequently, the consumption of bread that including clinically abnormal glucose metabolism (fasting
has been defrosted following freezing is commonplace. In blood glucose 46.1 mmol/l) and any medical conditions or
addition, consumption of bread in the form of toast, mainly medications that might affect glucose regulation, gastric
at breakfast, is also customary. emptying, body weight, appetite or energy expenditure.
Although some food processes, for example gelatinization, Anthropometric measurements were made in the fasting
may lead to high-GI foods (Granfeldt et al., 2000), some food state, using standardized methods, on the morning of the
manufacturing and handling may have an effect on the first test. Height was recorded to the nearest centimetre
degree of starch retrogradation, involving a rearrangement using a stadiometer (Seca Ltd, UK), with subjects standing
or realignment of initially heated starch molecules (Berry, erect and without shoes. Body weight was recorded to the
1986; Lang and Vitapole, 2004). Moreover, retrograded nearest 0.1 kg using the Tanita BC-418 MA (Tanita UK Ltd),
starch has been shown to reduce glycaemic response and with subjects wearing light clothing and no shoes. BMI was
there is evidence of this happening under conditions of calculated using the standard formula weight (kg)/height
cooling (Larsen et al., 2000; Frei et al., 2003). Retrogradation (m)2. Characteristics of the subjects are shown in Table 1.
is greater where the degree of gelatinization is more Ethical approval for the study was obtained from the
complete, facilitated by higher temperature and greater University Research and Ethics Committee at Oxford
water availability or with greater mixing, for example in Brookes University. Subjects were given full details of the
the formation of bread dough. Furthermore, it has also been study protocol and the opportunity to ask questions. All
shown that overall starch recrystallization reaches a max- subjects gave written informed consent before participation.
imum at around 41C and below this temperature further
starch recrystallization is minimal (Lang and Vitapole, 2004;
Goesaert et al., 2005). Importantly, the process of freezing Study protocol
and defrosting bread will involve bread passing through this The method used to measure glycaemic response and to
point of maximal recrystallization and retrogradation twice, calculate the GI value was in line with procedures recom-
once while cooling and again during defrosting. mended by the FAO/WHO (1998). In addition, on the day
Relatively small differences in the glycaemic response of preceding a test, subjects were asked to restrict their intake of
regularly consumed starch foods have shown beneficial alcohol and caffeine-containing drinks and to refrain from
effects on health, including reduced cardiovascular disease intense physical activity (e.g., long periods at the gym,
risk and glycaemic control (Frost et al., 1998; Brand-Miller et al., excessive swimming, running, aerobics). To minimize the
2003). Thus, investigations into ways of reducing the glycaemic possible influence of the second meal effect, subjects were
response to white bread are of important application. In the asked to refrain from eating an extra-large evening meal or
light of the above, the aim of the present study was to have an unusually high or low food intake throughout the
determine whether the glycaemic response of white bread could day preceding a test (Wolever, 1990). Where possible,
be changed by everyday storage and preparation conditions subjects ate a similar meal type on the evening before
such as procedures of freezing, defrosting and toasting. testing; however subjects were asked to avoid consuming
pulses for this meal to avoid the effects of colonic fermenta-
tion on postprandial glycaemic (Macintosh et al., 2003). All
Subjects and methods foods were tested in subjects after a 12 h overnight fast.
Test breads were administered to subjects in a randomized,
Subjects repeated measures design, with each subject acting as his/her
Ten healthy subjects (three male and seven female) were own control. Subjects travelled to the laboratory by car or
recruited to participate in the study. Subjects were staff and public transport and rested for 10 min before the test
students from Oxford Brookes University and from the wider commenced. Test breads were consumed first thing in the

European Journal of Clinical Nutrition


Glycaemic response of white bread
P Burton et al
596
Table 2 Nutritional composition (per 100 g) of test breads, using frozen state does not occur (Gray and Bemiller, 2003). Bread
manufacturers’ data was toasted on the morning following baking or purchase
Test bread Available Protein Fat Dietary fibreb Sodium and, where applicable, following defrosting overnight at
carbohydratea room temperature. For the duration of the study, a toaster
was standardized to medium setting, ensuring consistent
Homemade bread 43.9 8.6 1.7 1.9 0.6 moderate toasting.
Commercial bread 40.5 9.2 4.5 3.1 0.4
a
Calculated according to FAO/WHO (1998).
b
AOAC International methodology. Blood glucose measurements
Finger-prick blood samples were taken for capillary blood
morning (i.e., as breakfasts) and were compared with a glucose analysis. Recent reports suggest that capillary blood
reference food (glucose) and were tested in equivalent sampling is preferred for reliable GI testing (FAO/WHO,
amounts (50 g) of available carbohydrate. Available carbo- 1998; Wolever and Mehling, 2003). To establish blood
hydrate was estimated according to the FAO/WHO procedure glucose stability at the start of the blood glucose response
(total carbohydrate minus dietary fibre), using calculated data curve, fasting blood samples were taken at 5 and 0 min
of macronutrient content (Table 2). Thus, in this context, before consumption of the food and the baseline value taken
dietary fibre was defined as unavailable carbohydrate. as a mean of these two values. The reference food/test bread
As blood glucose responses vary within subjects from was consumed immediately after this and further blood
day to day, the reference food (glucose) was tested 3 times samples were taken at 15, 30, 45, 60, 90 and 120 min after
in each subject. Thus, subjects tested each test bread once starting to eat.
and the reference food 3 times in random order on Blood was obtained by finger prick using the Glucolet 2
separate days, with at least a one-day gap between measure- multi-patient lancing system (Bayer HealthCare, Newbury,
ments to minimize carry over effects. All test breads and the UK). Where necessary, before a finger prick, subjects were
reference food were served with 200 ml water. A further encouraged to warm their hand under running warm water
200 ml water was given during the subsequent 2 h. Subjects to increase blood flow. Fingers were not squeezed to extract
were asked to eat the test breakfast within a 10–12 min blood from the fingertip as this may dilute with plasma.
period to reduce the influence of chewing on particle size Blood glucose was measured using Ascensia Contour auto-
(Hoebler et al. 1998). matic blood glucose meters (Bayer HealthCare). The blood
glucose meters were calibrated daily using control solutions
from the manufacturer and were also regularly calibrated
Test breads against a clinical dry chemistry analyser (Reflotron Plus,
Two white breads, one homemade bread prepared using a Roche, Welwyn Garden City, UK) and the HemoCue Glucose
standard home bread-making machine (Breadman Pro, 201 þ analyser (HemoCue Ltd, Dronfield, UK). Using the
Russell Hobbs, Manchester, UK) and one commercially Bland–Altman analyses, there was a very strong correlation
available bread (Hovis Classic, British Bakeries Ltd, UK), (r ¼ 0.980, P o0.001) and good agreement (mean difference
were tested for glycaemic response. The homemade white 0.2 mmol, 95% CI 0.3 to 0.2, limits of agreement 0.80
bread was made from 500 g white wheat flour (Carr’s white and 0.32) between blood glucose measurements for a
strong bread flour; a high-protein wheat that does not random selection of 140 blood samples simultaneously
contain enzymes, improvers or bleach), 8 g NaCl, 6 g sugar, measured using the Ascensia Contour and the HemoCue
287 ml water, 6 g butter, 7 g skimmed milk powder and 8 g Glucose 201 þ analyser.
dehydrated yeast without additives (Fermipan, DSM Bakery
Ingredients, Holland). Details of the macronutrient, dietary
fibre and sodium content of the two test breads are given in Statistical analysis
Table 2. Parallel investigations of homemade and commer- Statistical analysis was performed using the Statistical
cial breads allowed investigation of any modulation, for Package for the Social Sciences (SPSS version 11.0. 1,
example by additives, in commercial breads not found in the Chicago, IL, USA). Data are presented as means and standard
homemade bread. deviations. Before statistical analysis, the normality of the
Both breads were tested following four different storage data was tested using the Shapiro–Wilk statistic. The
and preparation conditions: (1) fresh; (2) frozen and Pearson’s correlation coefficient and the method of Bland
defrosted; (3) fresh and toasted; (4) toasted following and Altman (1986) were used to examine the correlation and
freezing and defrosting. Fresh bread was tested on the agreement between the automatic analyser and the Hemo-
morning following baking or purchase. Frozen bread was Cue Glucose 201 þ analyser. Levels of intra-individual
defrosted overnight at room temperature before consump- variation of the three reference (glucose) tests were
tion the following morning. Breads were frozen between 2 assessed by determining the coefficient of variation
and 7 days; it has been suggested that further retrogradation (CV% ¼ 100  standard deviation/mean). Repeated measures
during freezing following temperature reduction to the analysis of variance, with Bonferroni’s correction, was used

European Journal of Clinical Nutrition


Glycaemic response of white bread
P Burton et al
597
to compare glycaemic response between test breads stored Figure 2 shows the incremental blood glucose response
and processed in different ways. Statistical significance was curves for the commercial white bread. Peak rise in blood
set at Po0.05. glucose was significantly different across the storage and
preparation conditions (F ¼ 7.425, P ¼ 0.001). Peak rise in
blood glucose for commercial fresh toasted bread and
Results commercial bread that had been toasted following freezing
and defrosting was significantly lower than fresh commercial
The mean intra-individual variation in glycaemic response bread (P ¼ 0.024 and P ¼ 0.018, respectively) and commercial
to the three reference tests in the subjects was 28% CV. This bread that had been frozen and defrosted (P ¼ 0.020 and
value is consistent with previously reported data in normal P ¼ 0.006, respectively).
subjects (Wolever, 2006). There were significant differences in IAUC (F ¼ 6.105,
Figure 1 shows the incremental blood glucose response P ¼ 0.003) between the storage and preparation conditions
curves for the homemade white bread. There was no overall for the commercial bread (Table 3). IAUC values for fresh
effect of storage and preparation on the peak rise in blood toasted bread and bread that had been frozen, defrosted and
glucose response (P ¼ 0.64) in the homemade bread. toasted were significantly lower than for fresh bread
Table 3 shows the IAUC across the different storage and (P ¼ 0.001 and P ¼ 0.026, respectively). The IAUC value for
preparation conditions for the homemade white bread. fresh toasted bread was also significantly lower compared to
Significant differences in IAUC (F ¼ 10.503, Po0.001) were bread that had been frozen and defrosted (P ¼ 0.046).
seen. Compared to the fresh homemade bread, IAUC was
significantly lower for homemade frozen and defrosted bread
(P ¼ 0.010), homemade fresh toasted bread (P ¼ 0.007) and Discussion
homemade bread that had been toasted following freezing
and defrosting (P ¼ 0.001). The current study investigated the impact of storage
conditions and food preparation on the glycaemic response
to white bread. This is the first study known to the authors to
show reductions in glycaemic response as a result of changes
in storage conditions and the preparation of white bread
before consumption. All three procedures investigated, that
is freezing and defrosting, toasting from fresh and toasting
following freezing and defrosting, led to a reduced glycaemic
response and reduction in IAUC values.
The influence of food processing and cooking on glycae-
mic response is well documented (Brand et al., 1985; Bjorck
et al., 1994). Treatments incorporating the generation of
forces such as shearing, compression and extreme heat
treatment increase gelatinization, which results in the
breakdown of the starch granule. Thus, many processing
conditions lead to an increased susceptibility of the starch
Figure 1 Blood glucose response curve for homemade bread:
Glucose (*); fresh (J); frozen, defrosted (K); fresh, toasted (&);
frozen, defrosted, toasted (’).

Table 3 IAUC (mmol  min/l) for homemade bread and commercial


white bread with different storage and preparation conditions

Test food Homemade bread Commercial bread


(mean7s.d.) (mean7s.d.)

Glucose 2917100a 2917100a


Fresh 2597103a 2537106a,b
Frozen, defrosted 179774b 217799b,c
Fresh, toasted 193779b 183796d
Frozen, defrosted, toasted 157785c 187795c,d

Abbreviation: IAUC, incremental area under the glucose response curve; s.d.,
standard deviation. Figure 2 Blood glucose response curve for commercial white
Values in the same column with different letters are significantly different, bread: Glucose (*); fresh (J); frozen, defrosted (K); fresh, toasted
Po0.05. (&); frozen, defrosted, toasted (’).

European Journal of Clinical Nutrition


Glycaemic response of white bread
P Burton et al
598
granule to enzymatic salivary and pancreatic amylases recommended that future studies include such measure-
following consumption, resulting in greater availability of ments, including resistant and retrograded starch (Englyst
glucose for absorption and increased glycaemic response. et al., 1992; McCleary and Monaghan, 2002), both before
Examples of such treatments are flour milling, intensive and after storage and preparation conditions. In addition,
mixing in bread dough formation, as in the CBP, and bread other methods have been proposed to classify the glycaemic
baking, which may explain the relatively highGI value of impact of foods based on total carbohydrate rather than
white bread. available carbohydrate, such as relative glycaemic effect
Retrograded starch constitutes RS3, a form of resistant (Brouns et al., 2005). However, further research is needed on
starch, and there is evidence of the formation of RS3 during the use of alternative indices to measure the glycaemic
the processes of cooling and freezing (Hoebler et al., 1999; response of foods.
Goesaert et al., 2005). There has been an increasing interest In conclusion, this is the first study known to the authors
in resistant starch in recent years and positive health benefits to show reductions in glycaemic response following different
have been demonstrated, mediated through effects on colonic storage and preparation conditions of white bread and
fermentation and on both postprandial glucose and lipid highlights the need to define and maintain storage condi-
metabolism (Robertson et al., 2003; Higgins et al., 2004). tions of food products when the glycaemic response of foods
An important difference in the constituents of the home- is determined. Relatively small differences in the glycaemic
made and commercial white breads in this study was the response of regularly consumed starch foods have been
presence of dough conditioners and improvers. These shown previously to have beneficial effects on health (Frost
ingredients are widely used in commercial bread and are et al., 1998; Brand-Miller et al., 2003). Thus, considering the
designed to optimize dough formation and quality, reduce high consumption of white bread, the identification of ways
staling rate and maintain water retention during baking to reduce the glycaemic response of white bread and other
(Goesaert et al., 2005). However, water content and activity foods is important in the prevention and management of
within the dough facilitates the retrogradation process. chronic disease.
Moreover, amylopectin retrogradation, together with moist-
ure transfer between bread components, may be reduced
by the use of dough improvers (Baik et al., 2003). This may
Acknowledgements
explain smaller reductions in glycaemic response within the
commercial bread, compared to changes within the home-
Sponsorship: This study was supported by a Biotechnology
made bread.
and Biological Sciences Research Council studentship.
The implications of this study are twofold. First, it is
suggested that simple household methods, such as freezing,
defrosting and toasting, may alter the glycaemic response to
white bread, thus white bread need not always be a highGI
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