Effects of pseudocereals, legumes and inulin addition on selected nutritional properties and glycemic index of whole grain wheat-based biscuits

Abstract

Based on the nutritionists‘ opinion that diet may have a preventive and protective role in many diseases, the possibilities of development of biscuits with lower glycemic index (GI) as functional cereal-based products were examined in this work. Results showed that, by substitution of 30% of white wheat flour with selected pseudocereals (buckwheat or amaranth flour) and legumes (soya or carob flour), it was possible to achieve better macronutritive quality of biscuits. Incorporation of selected flours resulted in a decrease of in vitro starch digestibility in terms of significant decrease of rapidly available glucose (9.6–47.6%), and rapidly digestible starch (9.8–50.4%). A significant increase in the resistant starch content was achieved by the implementation of pseudocereals (43.5–56.1%). Control biscuits, biscuits with added saccharose, and biscuits with buckwheat flour and without inulin, had medium GI (58.7–66.9), while samples with amaranth, soya or carob flour had low GI (44.9–52.5). None of the investigated biscuits had high GI. A very good correlation between the results obtained in vitro and GI values were determined indicating that the formulation of biscuits with lower starch digestibility represented a good approach in lowering GI of biscuits, and therefore increasing its functional properties.

Full text

Journal of Food and Nutrition Research (ISSN 1336-8672) Vol. 53, 2014, No. 2, pp. 152–161
152 © 2014 Národné poľnohospodárske a potravinárske centrum (Slovakia)
Biscuits are a very popular snack food used
worldwide across all population groups [1]. They
usually contain a high proportion of lipids and sac-
charose to achieve more appealing flavour, which
classifies them as foods whose regular consump-
tion represents a risk factor for obesity, and thus
an increased risk of developing type 2 diabetes,
high cholesterol and coronary heart disease [2]. In
the modern world dominated by highly processed
and nutritionally poor foods, there are efforts to
complement the diet with valuable nutrients by
so called functional products. Taking into account
trends in the food industry, as well as the recom-
mendations of nutritionists who are increasingly
pointing out at preventive and protective role of
diet in many diseases, the efforts of food technolo-
gists are focused in recent years to preparing bis-
cuits with functional properties. Generally, func-
tional foods are challenging the food industry in
technological as well as in economic terms since
studies have shown that most consumers rarely
waive taste for health and it is therefore impor-
tant to develop functional food products with re-
tained traditional organoleptic characteristics [3].
Hence, many studies have focused on the creation
of biscuits with enhanced nutritional characteris-
tics without disruption the biscuit quality, where-
by a number of improvements are based on the
replacement of certain amount of refined wheat
flour with various raw materials of high nutritional
value such as soya, buckwheat or amaranth flour
[4–6].
Buckwheat is a rich source of proteins, fibres,
antioxidants, minerals and starch that contains
substantial amounts of resistant starch, which may
contribute to lowering of glycemic index (GI) of
food [5]. Moreover, in the recent study [7] a de-
crease in GI was reported for bread made from
a mixture of buckwheat and wheat flours. This was
due to binding of fatty acids, rutin and epicate-
Effects of pseudocereals, legumes and inulin addition
on selected nutritional properties and glycemic index
of whole grain wheat-based biscuits
LOVORKA VUJIĆ – DUBRAVKA VITALI ČEPO – BLAŽENKA ŠEBEČIĆ – IRENA VEDRINA DRAGOJEVIĆ
SUMMARY
Based on the nutritionists‘ opinion that diet may have a preventive and protective role in many diseases, the possibili-
ties of development of biscuits with lower glycemic index (GI) as functional cereal-based products were examined in
this work. Results showed that, by substitution of 30% of white wheat flour with selected pseudocereals (buckwheat or
amaranth flour) and legumes (soya or carob flour), it was possible to achieve better macronutritive quality of biscuits.
Incorporation of selected flours resulted in a decrease of in vitro starch digestibility in terms of significant decrease of
rapidly available glucose (9.6–47.6%), and rapidly digestible starch (9.8–50.4%). A significant increase in the resist-
ant starch content was achieved by the implementation of pseudocereals (43.5–56.1%). Control biscuits, biscuits with
added saccharose, and biscuits with buckwheat flour and without inulin, had medium GI (58.7–66.9), while samples
with amaranth, soya or carob flour had low GI (44.9–52.5). None of the investigated biscuits had high GI. A very good
correlation between the results obtained in vitro and GI values were determined indicating that the formulation of
biscuits with lower starch digestibility represented a good approach in lowering GI of biscuits, and therefore increasing
its functional properties.
Keywords
in vitro starch digestibility; glycemic index; biscuits
Lovorka Vujić, Dubravka Vitali Čepo, Blaženka Šebečić, Irena Vedrina Dragojević, Department of Food Chemistry, Faculty
of Pharmacy and Biochemistry, University of Zagreb, Ante Kovačića 1, 10000 Zagreb, Croatia.
Correspondence author:
Lovorka Vujić, tel.: +385 1 4612690, e-mail: lvujic@pharma.hr

Nutritional properties and glycemic index of whole grain wheat based biscuits
153
final product in terms of increasing the contents of
dietary fibre, slowly digestible and resistant starch,
as well as proteins. The present work represents
an attempt to decrease the GI of this frequently
consumed snack.
MATERIALS AND METHODS
Preparation of biscuits
Seven types of biscuits were prepared under
laboratory conditions in Technological develop-
ment department in Kraš, confectionary factory in
Zagreb, Croatia, in three series, in order to ensure
the reliability of the obtained data. For biscuits
preparation, whole grain and white wheat flour
(Farina, Granolio, Zagreb, Croatia) were used as
basic flours. Buckwheat flour (Do-It, Barneveld,
Netherland), amaranth flour (Do-It), full fat soya
flour (Biovega, Zagreb, Croatia) and carob flour
(Šafram, Zagreb, Croatia) as substitute flours,
were incorporated into biscuit by replacing the
equivalent amount of white wheat flour in the bis-
cuit formulation. All flours were purchased at the
local market in Croatia.
Control biscuits were prepared with 949 g
whole grain wheat flour, 633 g white wheat flour,
274 g vegetable fat without trans fatty acids (K3-Z,
Zvijezda, Zagreb, Croatia), 259 g inulin (Beneo,
Tienen, Belgium), 87 g skimmed milk powder
(Lactoprot, Kaltenkirchen, Germany), 20 g ammo-
nium bicarbonate, 20 g salt (Solana Pag, Pag,
Croatia), 7 g vanilla flavour (Symrise, Holz minden,
Germany), 249 g isomalt (Beneo) and 0.3 g sucra-
lose (McNeil Nutritionals, Fort Washing ton, Penn-
sylvania, USA).
In four biscuit formulations, a definite amount
(480 g) of white wheat flour was replaced by dif-
ferent above mentioned substitute flour; one for-
mulation was prepared using powdered saccharose
(249 g) instead of saccharose substitute (isomalt)
and artificial sweetener (sucralose), and one was
prepared without inulin. Composition of the ex-
perimental biscuits is presented in Tab. 1.
Other ingredients were added in the same
amounts as in control biscuits. Water was added
as needed depending on the type of flour used.
Resulting dough was laminated on the machine
roller up to 0.5 cm thickness and followed by hand
shaping of biscuits using moulds 4 cm in diameter.
Biscuits were baked at 175 °C for 17–19 min, de-
pending on the raw material used and the amount
of water added. After baking, biscuits were cooled
and, before mincing, samples for GI determina-
tion were separated.
chin-dimethylhallate, contained in buckwheat, to
wheat flour starch.
Amaranth is another pseudocereal with pro-
tein content higher than wheat, with well balanced
amino acid composition. It is also a rich source of
dietary fibre, lipids and minerals [8]. According
to study of KIM et al. [9], amaranth grain could
be helpful in reduction of elevated serum glucose
levels and preventing diabetic complications.
Legumes (soya and carob) are low glycemic
foods, being excellent sources of dietary fibre, va-
riety of micronutrients and phytochemicals. Soya
contains high quality proteins as well as isofla-
vones with well documented health benefits [10].
Carob has high content of carbohydrates, appreci-
able content of proteins, low content of lipids, and
has antioxidant properties due to the high content
of condensed tannins [11].
Regarding the growing epidemic of obesity and
type 2 diabetes worldwide, more attention is paid
to creating lower GI products, since numerous
studies have demonstrated positive effects of low
glycemic diet on health. Hence, MARAGONI and
POLI [12] added specific pro prietary fibre mix to
the biscuit formulation thus significantly reducing
GI. JENKINS et al. [13] concluded that highly vis-
cous fibre blend (PGX; InovoBiologic, Calgary,
Canada) incorporated to a starchy snack (biscuit)
reduced the GI by 74% in healthy, and by 63% in
diabetic consumers.
Starch can be found in various physical and
chemical forms in foods, which affect the rate and
the extent of digestion and, consequently, its nu-
tritional and health properties. When the rate of
starch digestion is decreased, postprandial glucose
and insulin responses are reduced or delayed [14].
Diets rich in slowly digested carbohydrates may
protect against chronic disorders and/or diseases
such as central obesity, hyperlipidemia and cancer
[15]. Various factors affect starch digestibility such
as nature of starch, protein and lipid interactions,
presence of antinutrients and enzyme inhibitors,
as well as food processing conditions [16]. This
suggests that, by careful selection of raw materials,
these parameters can be altered and also, conse-
quently, the GI of the final product. Additionally,
glycemia can be further reduced by replacing sac-
charose with indigestible carbohydrates such as
sugar alcohols [17].
The goal of this work was to examine the pos-
sibilities of improving the traditional biscuits for-
mulation by introduction of novel raw materials,
such as pseudocereals, legumes or inulin. The
proposed modifications are aimed primarily to re-
duce GI, while maintaining or enhancing the nu-
tritional quality and functional properties of the

Vujić, L. et al. J. Food Nutr. Res., 53, 2014, pp. 152–161
154
Chemical analysis of biscuits
Moisture content was determined according to
AACC 44-15A method, ash content according to
AACC 08-01, proteins according to AACC 46-12
[18] using multiplying the nitrogen content by
factor 6.25, and total lipids according to AOAC
920.39C method [19]. Total dietary fibre (TDF)
content was assessed by enzymatic–gravimetric
AOAC 991.43 method as the sum of the soluble
(SDF) and insoluble dietary fibre (IDF) fractions
[19]. The protein, ash, lipid and moisture contents
were subtracted from the total weight and the dif-
ference was considered as total carbohydrates.
Available carbohydrates were calculated as a dif-
ference between total carbohydrates and the sum
of total dietary fibre, isomalt and inulin.
In order to calculate the energy value of the in-
vestigated biscuits, Atwater general factor system
was used [20]. Conversion factors that were used
were 17 kJ·g-1 for proteins and available carbohy-
drates, 37 kJ·g-1 for lipids and 8 kJ·g-1 for dietary
fibre. Energy values used for isomalt and inulin
were 9 kJ·g-1 [21] and 6.3 kJ·g-1 [22], respectively.
Energy value (EV) in kilojoules was calculated
according to the following equation:
(1)
where P is content of proteins, L is content of
lipids, avCHO is content of available carbohy-
drates, F is content of fibre, IS is content of iso-
malt and IN is content of inulin (all in grams).
In vitro protein and starch digestibility
In vitro protein digestibility of the biscuits
was determined by enzymatic method with pep-
sin (P7000, Sigma-Aldrich, Steinham, Germany)
and pancreatin (P1750, Sigma-Aldrich) according
to KUMAGAI et al. [23]. In vitro starch digestibil-
ity was determined according to the procedure of
ENGLYST et al. [14]. The analysis was carried out
under controlled enzymatic hydrolysis with inver-
tase (I4504, Sigma-Aldrich), pancreatin (P7545,
Sigma-Aldrich) and amyloglucosidase (A3042,
Sigma-Aldrich) at 37 °C in capped tubes immersed
in a shaker water bath, followed by measurement
of released glucose by GOD-PAP (glucose oxi-
dase/peroxidase) method [24] using a diagnostic
kit (Dijag nostika, Sisak, Croatia). The value G20
(rapid ly available glucose) was obtained by meas-
uring the release of glucose after 20 min of enzy-
matic incubation, while G120 was estimated after
further 100 min of incubation.
The total glucose value (TG) was obtained by
treating the sample with heat-stable -amylase
(A3306, Sigma-Aldrich) in boiling water, followed
by immediate cooling, treatment with potassium
hydroxide (7 mol·l-1) at 0 °C, and followed by com-
plete enzymatic hydrolysis with amyloglucosidase.
Free glucose (FG) was determined by treat-
ing the sample with invertase in acetate buffer at
100 °C (water bath) for 30 min. Simultaneous tests
were run in the same manner with standard glu-
cose and blank.
Rapidly digestible starch (RDS), slowly digest-
ible starch (SDS), resistant starch (RS) and total
starch (TS) were calculated according to following
equations [14]:
(2)
(3)
(4)
(5)
Glycemic index determination
Ten healthy volunteers (five men and five
women) aged from 21 to 43, with an average body
mass index of (22.36 ± 1.12) kg·m-2, were included
in GI testing. The volunteers were non-smokers
Tab. 1. Composition of the experimental biscuits.
Sample
Whole grain
wheat flour
White
wheat flour Raw material Inulin Isomalt/Sucralose Saccharose
[g]
Control sample 949 633 – 259 249/0.3 –
With saccharose 949 633 – 259 – 249
With buckwheat flour 949 153 480 (buckwheat flour) 259 249/0.3 –
With amaranth flour 949 153 480 (amaranth flour) 259 249/0.3 –
With carob flour 949 153 480 (carob flour) 259 249/0.3 –
With soya flour 949 153 480 (soya flour) 259 249/0.3 –
Without inulin 949 633 – – 249/0.3 –

Nutritional properties and glycemic index of whole grain wheat based biscuits
155
and had no history of metabolic disease, as deter-
mined by a questionnaire prior to recruitment. All
subjects gave written consent prior to inclusion in
the study after being given a detailed description
of the study protocol and had opportunity to ask
questions. The participants were asked to main-
tain standard diet and to refrain from heavy physi-
cal activity as well as consumption of alcoholic and
caffeinated drinks the day prior to the test. The
study protocol was approved by the Ethical Com-
mittee of the Croatian Institute for Transfusion
Medicine and Committee on Ethics of the Experi-
mental Work of the Faculty of Pharmacy and Bio-
chemistry in Zagreb, Croatia.
The protocol procedure was in agreement with
the recommendations of FAO/WHO [25] and
BROUNS et al. [17]. Each participant tested seven
different types of biscuits once in a random order,
while the reference food was tested twice. Tests
were performed during morning after a 12 h over-
night fast, with a gap of at least two days between
measurements. Each volunteer ate a biscuit por-
tion containing 50 g of available carbohydrates
within 10 min with 250 ml of tap water. Reference
food (anhydrous glucose) was prepared by dissolv-
ing 50 g of pure glucose in 250 ml of water. Ca-
pillary finger-prick blood samples were taken from
participants at 0, 15, 30, 45, 60, 90 and 120 min
after the beginning of the test, or after reference
food intake (time 0 min). The blood glucose con-
centration was measured using Contour automatic
glucose meter (Bayer HealthCare, Leverkusen,
Germany), whose accuracy was verified using high,
low and normal test solutions (Bayer Consumer
Care, Basel, Switzerland).
Statistical analysis
One-way ANOVA was used to investigate dif-
ferences between three examined series of biscuits.
Since no significant differences were observed
(p > 0.05), all results were presented as means of
series of biscuits ± standard deviation. Depending
on the applied method, analyses were conducted
in duplicates or triplicates within one series of bis-
cuits. One-way ANOVA and post-hoc Bonferroni
test were applied to study the differences between
different biscuit formulations, the significance
being determined at the level of p < 0.05.
For GI calculation, the incremental area under
curve (iAUC) of blood glucose response, ignoring
the area beneath the baseline, was calculated geo-
metrically [25]. GI of biscuits was calculated as the
ratio between mean iAUC for the biscuit tested
and mean iAUC of the reference food (glucose so-
lution). The GI results are expressed as mean ±
standard error (SEM). Correlation coefficients
(r) between GI, different starch and dietary fibre
fractions as well as protein and lipid contents were
also calculated.
All statistical analyses were performed using
GraphPad Prism 3 software (GraphPad Software,
San Diego, California, USA).
RESULTS AND DISCUSSION
Macronutritive and nutritional compositions
of investigated biscuits are shown in Tab. 2. It can
be seen from presented data that the addition of
flours of different origins to the control biscuits re-
sulted in significant improvement of certain nutri-
tive parameters.
Total protein and lipid contents were the high-
est in biscuits enriched with soya flour, as was
expected, since soya flour is rich in these com-
ponents. Although soybeans are high in lipids,
which are traditionally an undesirable ingredient
in human diet, majority of them are unsaturated
fatty acids with beneficial health effects [10]. Total
dietary fibre content was significantly increased in
biscuits enriched with buckwheat, amaranth and
soya flours, with the strongest effect achieved by
the addition of carob flour (an increase of 96%
compared to the control biscuits) solely as the re-
sult of high insoluble fibre content. Since all inves-
tigated biscuits contained more than 6 g of fibre
per 100 g dry matter, they can be classified as food
rich in fibre according to EU regulation 1924/2006
[26].
In vitro protein digestibility is an important
factor when assessing the nutritional quality of
a food product. The obtained results showed that
addition of all substitute flours to the control bis-
cuits decreased protein digestibility of the final
product, which is consistent with literature data
for products of similar type [27, 28]. The observed
results may be explained by the fact that high
levels of dietary fibre (biscuits with carob flour)
had a negative effect on protein digestibility due
to possible complex formation between the fibre
and the proteins, as reported BILGIÇLI et al. [29],
or by high levels of anti-nutritive components such
as trypsin inhibitor present in soybean [30] and
tannins present in carob [11]. Buckwheat contains
antinutrients such as the protease inhibitor and
tannins [31].
Bearing in mind that the nature of starch and
consequently its digestibility has a fundamental
role in the rate of its absorption, an attempt to re-
duce starch digestibility and, consequently, glucose
response in vivo was made by introducing differ-
ent raw materials to biscuit formulation. As pre-

Vujić, L. et al. J. Food Nutr. Res., 53, 2014, pp. 152–161
156
sented in Tab. 3, significant differences were found
regarding the contents of rapidly available glucose
(RAG), RDS, SDS and TS.
It is noticeable that incorporation of substitute
flours resulted in a decrease of RAG and RDS con-
tent in relation to control sample, and the strong-
est impact was achieved by addition of amaranth
flour (24.9% and 26.6%, respectively), carob flour
(35.9% and 50.4%, respectively) and soya flour
(47.6% and 50.1%, respectively). Obtained data
are consistent with the fact that legumes (soya and
carob) have a higher amylose/amylopectin ratio
in starch granules compared to cereals, and are
therefore less susceptible to amylolysis. This is be-
cause amylose molecules contain more hydrogen
bonds making them less accessible to digestive
enzymes action than amylopectin mole cules with
a branchy structure [32]. Additionally, carob and
soya contain high amounts of dietary fibre, which
may affect starch digestibility. Furthermore, as
previous ly noted, soya contains high protein and
lipid amounts that have a protective effect against
amylolytic activity, diminishing the level of enzy-
matic hydrolysis by reducing the available surface
area through blocking the adsorption sites [33].
Also, carob is rich in tannins with antinutritive
effect [11]. Although amaranth contains relatively
high amounts of starch, small size of starch gran-
ules and low amylose content [8], i.e. factors that
increase starch digestibility, amaranth flour supple-
mentation of the control formulation resulted in its
reduction. The obtained results can be explained
by the relatively high protein, lipid and dietary fi-
bre contents, as well as low level milling used for
amaranth flour.
Statistically significant increase in RAG content
in relation to the control sample was determined
in samples with saccharose (17.4%) and without
inulin (22.0%). In this concern it should be noted
that inulin removal from the control formulation
had a more significant influence on the above men-
tioned parameter than the addition of saccharose.
Also, a significant increase in RDS values was ob-
served only in biscuit without inulin (25.4%), partly
as a result of a higher total starch content.
SDS is the starch fraction completely digested
in the small intestine but, as opposed to RAG, at
a lower rate. Modification of the control formu-
lation resulted in significantly reduced SDS con-
tent in all samples enriched with substitute flours,
partially due to the reduction of the total starch
content. Indeed, the greatest impact on SDS de-
crease was found in biscuits supplemented with
carob flour (19.8%) and with soya flour (25.0%),
which can be explained by the significant reduc-
tion in TS content in those samples (37.9% and
Tab. 2. Chemical and nutritional properties of biscuits.
Sample Proteins
[g]
Lipids
[g]
Ash
[g]
Total
carbohydrates
[g]
Total
dietary fibre
[g]
Insoluble
dietary fibre
[g]
Soluble
dietary fibre
[g]
In vitro protein
digestibility
[g]
Energy
value
[kJ]
Control sample 9.69 ± 0.13 a12.36 ± 0.18 a1.84 ± 0.01 a76.11 ± 0.06 a8.07 ± 0.40 a4.38 ± 0.14 a3.69 ± 0.27 ae 79.38 ± 2.43 a1 644
With saccharose 9.50 ± 0.06 a12.39 ± 0.19 a1.75 ± 0.04 b76.36 ± 0.28 ad 7.53 ± 0.29 b4.01 ± 0.10 a3.52 ± 0.25 a78.26 ± 3.78 a1 732
With buckwheat flour 10.24 ± 0.09
b12.25 ± 0.17 a2.06 ± 0.03 c75.45 ± 0.23 b9.81 ± 0.32 c5.27 ± 0.32 b4.54 ± 0.13 b69.70 ± 2.33 b1 623
With amaranth flour 10.97 ± 0.19
c13.54 ± 0.58 b2.30 ± 0.06 d73.19 ± 0.49 c9.95 ± 0.16 c5.89 ± 0.05 c4.06 ± 0.17 c72.50 ± 3.37 bd 1 648
With carob flour 9.04 ± 0.08
d11.96 ± 0.27 a2.26 ± 0.01 d76.74 ± 0.33 d15.82 ± 0.56 d12.86 ± 0.53 d2.96 ± 0.27 d51.97 ± 3.33 c1 564
With soya flour 16.66 ± 0.19
b17.61 ± 0.61 c2.80 ± 0.07 e62.92 ± 0.75 e11.31 ± 0.13 e7.35 ± 0.04 e3.96 ± 0.14 ec 70.00 ± 3.47 b1 711
Without inulin 10.44 ± 0.03 e13.05 ± 0.28 b 1.81 ± 0.01
ab 74.70 ± 0.25 f6.37 ± 0.41 f4.80 ± 0.21 f1.57 ± 0.20 f75.57 ± 1.74 ad 1 744
The results are reported as means of three investigated series of biscuits ± standard deviation, per 100 g on dry basis. Data in the same column marked with the same letter are the part of
the same statistical group (p > 0.05).

Nutritional properties and glycemic index of whole grain wheat based biscuits
157
39.5%, respec tively). In biscuits supplemented
with pseudo cereals (buckwheat and amaranth
flours), SDS decrease occurred partially due to
the increase in RS content. This is consistent with
the results of RAGAEE et al. [34], who determined
in vitro starch digestibility in bread with 30% of
white wheat flour replaced with various whole-
grain flours.
In comparison to other food rich in starch, in-
cluding biscuits [35, 36], the investigated samples
had lower RAG and RDS contents, and higher
SDS and RS contents, as a result of several fac-
tors. Namely, the basis of formulation of the in-
vestigated biscuits was whole grain wheat flour,
i.e. low grade milled flour, where starch granules
are trapped within the cell wall, which slows down
their degradation. Additionally, degradation of the
starch granules by grinding increases the sensitiv-
ity to enzymatic degradation [37]. The same effect
has extended milling since smaller particles have
a larger surface area available for amylolytic ac-
tivity [38]. Another fact that might contribute to
this effect was the high dietary fibre content and
baking under low moisture conditions, which re-
duce starch gelatinization resulting in partially
intact starch granules that are less sensitive to the
amylolytic enzymes [36].
Glycemic index of biscuits
The primary aim of this study was to examine
the impact of introducing different raw materials
on the postpradial glycemia of whole grain wheat-
based biscuits. The obtained results show that the
highest glucose response in volunteers was ob-
served 30–45 min after consumption of the test
food (Fig. 1).
The highest increase in blood glucose concen-
tration was observed after consumption of biscuits
Tab. 3. Content of different carbohydrates fractions in experimental biscuits.
Sample
Rapidly available
glucose
Rapidly digestible
starch
Slowly digestible
starch Resistant starch Total starch
[g·kg-1]
Control sample 289.20 ± 4.37
a250.72 ± 4.07 a157.46 ± 9.70 a35.56 ± 8.04 a443.74 ± 18.69 a
With saccharose 339.60 ± 3.99
b251.74 ± 4.16 a162.88 ± 10.26 a34.73 ± 6.72 ad 449.34 ± 12.06 a
With buckwheat flour 261.38 ± 10.78
c226.22 ± 9.69 b132.24 ± 11.66 bc 55.51 ± 1.40 b413.97 ± 4.02 b
With amaranth flour 217.32 ± 4.29
d184.12 ± 3.80 c137.81 ± 8.67 bd 51.02 ± 0.79 b372.95 ± 4.57 c
With carob flour 185.47 ± 3.57
e124.37 ± 3.72 d126.28 ± 9.34 bc 25.10 ± 2.25 c275.75 ± 12.14 d
With soya flour 151.68 ± 5.25
f125.16 ± 4.63 d118.08 ± 13.42 c25.08 ± 5.54 c268.32 ± 9.35 d
Without inulin 352.71 ± 9.27
g314.25 ± 8.30 e151.10 ± 10.63 ad 27.35 ± 6.86 dc 492.70 ± 11.02 e
The results are reported as means of three investigated series of biscuits ± standard deviation. Data in the same column marked
with the same letter are the part of the same statistical group (p > 0.05); values are expressed per kilogram on dry basis.
Fig. 1. Glycemic response to biscuits.
Values are expressed as mean ± standard error (n = 10).

Vujić, L. et al. J. Food Nutr. Res., 53, 2014, pp. 152–161
158
with saccharose (after 30 min) although, compared
to the reference sample, this difference was not
statistically significant (p > 0.05). Consumption of
biscuits with soya flour resulted in the lowest levels
of blood glucose, which proved to be statistically
significant compared to the sample with saccha-
rose (after 30 min and 45 min), and statistically
significant in comparison with the biscuit without
inulin (after 45 min). Given that food can be clas-
sified according to its glycemic effect [39] as those
that have a high ( 70), medium (56–69) and low
GI ( 55) compared to the standard (glucose solu-
tion, GI = 100), the investigated samples were cat-
egorized in the following way: control biscuits, bis-
cuits with saccharose, buckwheat flour and without
inulin were medium GI products, while biscuits
prepared with amaranth, soya or carob flours
had low GI. None of the investigated biscuits had
high GI. Although a wide range of GI values was
observed for the investigated samples (Tab. 4),
the differences were not statistically significant
(p > 0.05) due to a large dissipation among the
results. The obtained results indicate that by intro-
ducing pseudocereals (buckwheat and amaranth
flour) and legumes (carob and soya flour) instead
of white wheat flour, a decrease in blood glucose
response can be achieved.
In order to establish the relationship between
in vitro starch digestibility and in vivo glucose re-
sponse after consumption of the investigated bis-
cuits, GI values were correlated with the amount
of starch fractions contained in 50 g of available
carbohydrates, as the amount containing servings
used to determine GI (Tab. 4). Although in vitro
methods cannot completely imitate in vivo condi-
tions, such as degree of gastric emptying or the
influence of intestinal hormones, a very good
correlation between the results obtained in vitro
method and GI values was determined (Tab. 5).
Correlation between GI and RAG values
proved to be the most significant (p = 0.01)
among the tested starch fractions, which is con-
sistent with literature [14]. Additionally, there
was a strong negative correlation between the
SDS and GI values (p = 0.04) indicating a reduc-
ing effect of SDS on in vivo glucose response.
The obtained results suggest that creating low
RAG/RDS and high SDS foods, is an effective ap-
proach for lowering GI. The analysis of the rela-
tionship between macro nutrient content and GI
values (Tab. 5) showed a significant negative cor-
relation between the protein content (p = 0.08)
and lipids (p = 0.05), which confirms the above
mentioned allegations about the impact of these
components on the digestibility of starch and, con-
sequently, GI. These findings are in accordance
with the results of GARSETTI et al. [40] who inves-
tigated the relationship between GI of plain sweet
biscuits and in vitro starch digestibility, and found
similar negative correlation between GI and pro-
tein and lipid contents, as well as between GI and
SDS content. Also, we established a very strong
negative correlation between GI and TDF con-
tent (r = –0.95; p = 0.00) and its fractions (SDF,
r = –0.73; p = 0.07 and IDF, r = –0.87; p = 0.01).
Similar results were obtained by WOLEVER [41],
who examin ed the relationship between dietary
fibre content and GI of different foods and estab-
lished a significant negative relationship with total
fibre and its insoluble fraction (p < 0.05), but not
with the soluble fibre content. Correlation with
SDF observed in this study can be explained by
the fact that the investigated samples were pro-
duced under strictly controlled conditions, had
Tab. 4. Portion size, rapidly available glucose, starch fractions and glycemic index of the examined biscuits.
Sample
Portion
size
Rapidly
available
glucose
Rapidly
digestible
starch
Slowly
digestible
starch
Resistant
starch Total starch Glycemic
index
[g]
Control sample 105.0 30.4 26.3 16.5 3.7 46.6 60.1 ± 4.8
a
With saccharose 85.6 29.1 21.5 13.9 3.0 38.5 65.8 ± 5.6
a
With buckwheat flour 110.5 28.9 25.0 14.6 6.1 45.8 58.7 ± 6.2
a
With amaranth flour 116.7 25.4 21.5 16.1 6.0 43.5 52.5 ± 5.9
a
With carob flour 123.4 22.9 15.3 15.6 3.1 34.0 46.8 ± 6.0
a
With soy flour 160.2 24.3 20.1 18.9 4.0 43.0 44.9 ± 5.3
a
Without inulin 93.8 33.1 29.5 14.2 2.6 46.2 66.9 ± 4.6
a
Rapidly available glucose and starch fractions are expressed as grams per serving containing 50 g of available carbohydrates.
Glycemic index is expressed as mean ± standard error (n = 10), data in the column marked with the same letter are the part
of the same statistical group (p > 0.05);

Nutritional properties and glycemic index of whole grain wheat based biscuits
159
similar formulation and same processing condi-
tions. Therefore, the sole impact of SDF could be
noticed.
JENKINS et al. [42] examined the interac-
tion between starch and proteins in whole grain
wheat bread and found that the removal of gluten
from wheat flour increased the amylolytic diges-
tion in vitro and increased the in vivo gly cemic
response. This was explained by formation of
a gluten-protein complex that interfered with the
amylase action. Similar results were obtained by
BERTI et al. [43] who examined the same para-
meters in pasta and bread, with and without glu-
ten. These authors also observed effects of the
technological processing on in vitro starch digest-
ibility. On the other hand, PACKER et al. [44] ex-
plained the phenomenon solely by differences in
the manu facturing process.
In our study, gluten was not removed but glu-
ten-free flours were added at a rate of 30% in-
stead of refined white wheat flour. In spite of this,
no negative effects of dilution of gluten on the
observed parameters were noticed. Moreover, the
introduction of selected flours to control formula-
tion resulted in a decrease of in vitro starch digest-
ibility, as well as in vivo glucose response with the
highest impact observed for biscuits enriched with
soya flour (by 25.3% lower GI values compared to
the control sample). These findings are consistent
with the results of BLAIR et al. [45] suggesting that
food based on soya has low or medium GI values,
and is suitable for the regulation of blood glucose
and insulin levels. Such statements were con-
firmed in the study of SUGIYAMA et al. [46], where
the addition of grounded soybean into mixed meal
foods resulted in a significant (p < 0.001) decrease
of GI values.
In the present study, implementation of ama-
ranth flour into the control formulation led to the
reduction of GI by 12.6%, as opposed to findings
of CHATURVEDI et al. [47] who observed positive
dose-dependent relationship between the ama-
ranth flour added in the diet and GI values. Re-
sults were explained by physical properties of
amaranth starch (lower viscosity, high amylopectin
content, etc.). Taking into account that the strong
causal relationships were established between GI
values and macronutrient content in the examined
samples (Tab. 5), as well as lower in vitro starch
digestibility in biscuits enriched with amaranth, it
could be concluded that quoted macronutrients
and coarsely milled amaranth flour had a crucial
impact on the glucose response in vivo.
The buckwheat flour incorporation into the
biscuit formulation resulted in a slightly lower GI
value (by 2.3%), which was not statistically signifi-
cant. However, this effect suggests that the addi-
tion of buckwheat flour in larger quantities may
result in lower GI values of the final product. This
was previously indicated by ŠKRABANJA et al. [48],
who implemented 50% of buckwheat groats into
wheat-based bread and achieved a significant re-
duction in postprandial glucose and insulin levels,
compared with reference white wheat bread.
Inulin removal from the control biscuits result-
ed in an increase in GI (by 11.3%). The observed
effect was stronger in comparison to GI of sac-
charose-containing sample, where the presence of
saccharose instead of isomalt caused an increase
in GI by 9.5%. This observation indicates a strong
impact of inulin on the reduction of in vivo glu-
cose response. It can be explained by the fact that
inulin is highly hydrophilic and absorbs the water
present, thus inhibiting starch gelatinization and
digestion [49]. The obtained results are consistent
with those of BRENNAN et al. [50], who added up
to 10% of inulin in the wheat flour pasta recipe
and lowered starch digestibility in proportion to
the amount of the added inulin.
CONCLUSION
In the new millennium, the food industry is
faced with the challenge of formulating novel
products with optimal nutritional properties that
may have beneficial influence on human health.
The present study investigated the impact of
Tab. 5. Correlations between rapidly available glucose, different starch fractions
and macronutrient contents with glycemic index values.
RAG RDS SDS RS TS Proteins Lipids SDF IDF TDF
r0.87 0.68 –0.78 –0.30 0.30 –0.70 –0.76 –0.73 –0.87 –0.95
p0.01* 0.09 ** 0.04 * 0.52 0.52 0.08 ** 0.05 * 0.07 ** 0.01 * 0.00 *
* – correlation significant at level p < 0.05, ** – correlation significant at level p < 0.1.
RAG – rapidly available glucose, RDS – rapidly digestible starch, SDS – slowly digestible starch, RS – resistant starch, TS – total
starch, SDF – soluble dietary fibre, IDF – insoluble dietary fibre, TDF – total dietary fibre.
RAG, starch fractions and macronutrient contents are expressed as grams per serving containing 50 g of available carbohydrates.

Vujić, L. et al. J. Food Nutr. Res., 53, 2014, pp. 152–161
160
an addition of selected raw materials on in vitro
starch digestibility as well as on in vivo glucose
response. Although in vitro methods cannot com-
pletely imitate conditions of digestion in the hu-
man body, a very good correlation between these
two approaches was observed. It suggested that
creation of biscuits with a low content of rapidly
available glucose and rapidly digestible starch, and
consequently with a high content of slowly digest-
ible starch, represents an effective way to lowering
the GI of this popular food. Therefore, in the era
of increasing demands for products with health-
enhancing effects, the obtained results could help
the confectionery industry to offer new products as
a part of a balanced diet, in particular for diabetics
and persons with impaired glucose tolerance.
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Received 16 July 2013; 1st revised 23 September 2013;
accepted 30 October 2013; published online 5 May 2014.