Nutritional and functional; evaluation of wheat flour cookies supplemented with gram flour

1. Nutritional and functional evaluation of wheat flour cookies
supplemented with gram flour
ALI A. YOUSAF1
, ANWAAR AHMED1
, ASIF AHMAD1
, TABASSUM HAMEED2
,
MUHAMMAD ATIF RANDHAWA3
, IMRAN HAYAT1
, & NAUMAN KHALID4
1
Department of Food Technology, PMAS-Arid Agriculture University, Rawalpindi, Pakistan,
2
National Agricultural Research Center, Islamabad, Pakistan, 3
National Institute of Food Science and Technology,
University of Agriculture, Faisalabad, Pakistan, and 4
Department of Global Agriculture,
Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan
Abstract
Protein-enriched cookies were prepared by supplementing gram flour into wheat flour at levels of 0%, 10%, 20%, 30%, 40% and
50% and analysed for physicochemical properties. The protein quality of the cookies was assessed by feeding gram flour-
supplemented cookies to albino rats for 10 days. The supplementation resulted in a significant increase in protein, fat, crude
fibre and ash contents of the cookies. The thickness and spread factor of cookies differ significantly while non-significant effect
was observed in the width of the cookies. The protein efficiency ratio, net protein utilization, biological value and true
digestibility differed significantly among diets containing cookies with gram flour fed to rats. Cookies with 30% substitution of
straight grade flour and gram flour produced acceptable cookies as compared to control. The cookies containing 40–50% gram
flour were best regarded as protein bioavailability for rats.
Keywords: biscuits, chickpea flour, protein enrichment, biological evaluation, malnutrition
Introduction
Bakery products of cereals are often the mainstay of
emergency feeding programmes designed to serve
large number of people. Due to the cheap nutritional
source of calories and protein, they are used among the
masses (Akhtar et al. 2009). However, the prolonged
consumption of diet consisting mainly of cereal grains
leads to multiple nutritional deficiencies, because
cereals fail to supply sufficient amounts of certain
essential nutrients such as amino acids, minerals and
vitamins (Rehman et al. 2001).
Malnutrition due to protein deficiency is an atrocious
dilemma among the masses whose diet is generally
based on cereals or other starchy foods (Barker 2002;
Reilly 2002). Feeding on protein-deficient diets can
lead towards many disorders such as breast cancer,
coloncancer,heartdiseaseandosteoporosis(Alametal.
2003; Bhan et al. 2003). Therefore, the utilization of
protein-enriched diet is important to fight against
infections and diseases as it facilitates production of
antibodies to activate our immune system (Friedman
1996; Alexander et al. 1998). The increased costs and
limited supplies of animal proteins have geared the
present-day research efforts towards the evaluation and
utilization of inexpensive locally available protein
sources such as high-protein oilseeds and legumes
(Enujiugha and Ayodele-Oni 2003). Grain legumes are
renowned as important sources of food and feed
proteins. In many countries, legume seeds are
considered as the distinguishing source of protein in
the diet (Marcello and Cristina 1997). Moreover,
consumption of legumes is also associated with
ISSN 0963-7486 print/ISSN 1465-3478 online q 2012 Informa UK, Ltd.
DOI: 10.3109/09637486.2012.694851
Correspondence: Nauman Khalid, Department of Global Agriculture, Graduate School of Agriculture and Life Sciences, The University of
Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel: þ 81-80-3385-0786. Fax: þ 81-3-5841-8883.
E-mail: nauman_khalid120@yahoo.com
International Journal of Food Sciences and Nutrition,
2012; Early Online: 1–6
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2. reduction in proinflammatory status and improvement
in some metabolic features (Hermsdorff et al. 2011).
Gram, also named as chickpea (Cicer arietinum L.),
is one of the oldest and the widely consumed legumes
in the world, particularly in tropical and subtropical
areas (Singh et al. 1991). The flour of decorticated
gram seeds is used in several dishes and as a
supplement in weaning food mixes, bread and biscuits
(Wood 2009). Gram contains about 23% protein, 64%
total carbohydrates (47% starch and 6% soluble
sugar), 5% fat, 6% crude fibre, 3% ash and high
mineral content (Champ 2001; Boye et al. 2010).
Gram has consistently revealed hypocholesterolae-
mic effects as well as lower triglyceridaemia (Boualga
et al. 2009). The incorporation of protein-rich legume
flours such as gram flour in bakery products like bread
and biscuits can attain the goal of protein enrichment
(Patel and Rao 1996; Singh et al. 1996; Gandhi et al.
2001; Sharma and Chauhan 2002). Among processed
bakery items, biscuits grasp huge popularity in rural as
well as in urban areas among all the age groups
(Agrawal 1990). The high nutritive protein-enriched
cookies can be prepared from composite flours such as
wheat flour fortified with soy, cottonseed, pulses,
peanut, mustard or corn germ flour (Tsen et al. 2006).
The challenge of selecting the best-suited protein
source has geared bakery industry to explore such
ingredients which impart desirable nutritional and
functional characteristics to the baked products (Tyagi
et al. 2006). Keeping in view the importance of
protein-enriched diet, the present research was
conducted to prepare protein-enriched cookies by
using gram flour and to evaluate nutritional and
chemical properties of those protein-enriched cookies.
Materials and methods
Raw materials purchase and analysis
The raw materials such as straight grade flour (SGF),
gram flour, hydrogenated vegetable fat and all the
other ingredients for the preparation of cookies were
purchased from the local market. The SGF, gram flour
and composite flours were analysed for their proximate
composition according to their respective methods
described in AACC (2000). A profile of essential
amino acids of gram flour was also determined by
using an auto analytical apparatus according to the
method of Yu (1994). All reagents used were of
analytical grade (BDH Chemicals, Poole, UK).
Preparation of cookies
The gram flour was mixed with SGF at different levels
for the preparation of cookies. The cookies with 0%,
10%, 20%, 30%, 40% and 50% supplementation level
of gram flour were prepared according to the AACC
(2000) Method No. 10-50D with slight modifications.
In the recipe, supplemented flour of 100 g, sugar 45 g,
hydrogenated fat 50 g, dextrose solution 4.5 ml,
beaten egg 7.5 ml and baking powder 1 g were used
for making cookies.
Nutritional evaluation of cookies
Gram flour supplemented cookies and SGF cookies
were analysed for moisture content (Method No.
44-19), crude protein (Method No. 46-10), crude
fat (Method No. 30-10), crude fibre (Method No.
32-10.01) and mineral contents (Method No. 40-70)
according to their respective methods as described
in AACC (2000) with slight modifications where
necessary.
Physical evaluation of cookies
Physical characteristics of cookies (width, thickness
and spread factor) were determined according to
AACC (2000) Method No. 10-53.
Biological evaluation of cookies
The biological evaluation of gram flour supplemented
cookies was conducted at National Institute of Health
(Veterinary Division), Islamabad, by feeding different
diets to adult albino rats (Sprague-Dawley) of uniform
age. The feeding trials were conducted according to
the procedure adopted by Tetens et al. (1995) with
slight adjustments as suggested by Awan et al. (1995).
Thirty-two young albino rats were used for the
nutritional evaluation of the experimental diets. All the
diets were kept isonitrogenous and isocaloric. The rats
were fed on basal diet for a period of 1 week and
then randomly divided into eight groups comprising
of four rats each. All the experimental diets (Table I)
were randomly allotted to each group. All the groups
were fed ad libitum for a period of 10 days. Composite
Table I. Composition of various diets containing supplemented
cookies.
Diets
Ingredients T0 T1 T2 T3 T4 T5
Casein
(Tc)
Non-
protein
(Tnp)
Casein – – – – – – 6.0 –
Cookies 87.6 78.0 73.8 66.7 59.9 54.9 – –
Glucose 3.0 3.0 3.0 3.0 3.0 3.0 3.0 3.0
Vitamin premix 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Mineral premix 2.5 2.5 2.5 2.5 2.5 2.5 2.5 2.5
Corn oil 5.0 5.0 5.0 5.0 5.0 5.0 5.0 5.0
Corn starch 1.4 11 15.2 22.3 29.1 34.1 83.0 89.0
Total 100 100 100 100 100 100 100 100
Notes: T0, control (SGF cookies); T1, 10% gram flour containing
cookies; T2, 20% gram flour containing cookies; T3, 30% gram flour
containing cookies; T4, 40% gram flour containing cookies; T5, 50%
gramflourcontainingcookies;Tc,caseindiet;andTnp,non-proteindiet.
A.A. Yousaf et al.2
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3. weight of each group of rats was recorded daily with
electronic top load balance.
The excreta of each group of rat were also collected
on the separate filter paper sheet. Fresh and clean
water was provided all the times to each group in
separate calibrated glass bottles. At the end of 10 days,
the faecal material was collected and dried at 1058C
and stored in polyethylene bags for nitrogen esti-
mation. Then the rats were killed by chloroform
anaesthesia. The body and meat were dried at 1058C.
After full drying, all the material was ground through
electric grinder and stored in polyethylene bags for
nitrogen estimation. The nutritional parameters such
as feed consumption and body weight, protein
efficiency ratio (PER), true digestibility (TD), net
protein utilization (NPU) and biological value (BV)
were determined by adopting the method of Miller and
Bender (1955).
Statistical analysis
All experiments were run in triplicate and mean values
were calculated. The data obtained were analysed by
using STATISTIX 8.1 data analysing software and
interpreted according to Steel et al. (1997). The level
of significance was set at p values ,0.05.
Results
Chemical analyses of flours and cookies
The chemical compositions of SGF and gram flour
used for cookies preparation are shown in Table II.
Crude protein, crude fat, crude fibre and ash content
were found to be higher in gram flour in comparison
with SGF. The amino acid profile for gram flour is
exhibited in Table II, indicating higher concentration
of essential and limiting amino acids like lysine than
that of wheat flour.
A significant difference ( p , 0.05) was obtained
between control and gram flour supplemented cookies
for parameters regarding proximate composition
(Table III). Results of the chemical analysis of cookies
revealed that protein content of the cookies, prepared
from gram flour blends, was significantly higher
(p , 0.05) than the protein content of control cookies
(6.85%). With regard to the other chemical par-
ameters, it was observed that the addition of gram
flour resulted in an increase in ash content of cookies
up to 1.16% and crude fibre content up to 0.59%,
while crude fat content was found to be 28.52% (50%
of gram flour level). Cookies made with SGF
contained 2.21% moisture which was increased to
3.70% when wheat flour was substituted with 50%
gram flour. Supplementation of cookies with gram
flour also significantly (p , 0.05) increased the levels
of iron, manganese, magnesium, calcium, copper,
zinc, sodium and potassium to 33.45, 7.74, 300.80,
14.68, 2.91, 17.25, 1581.00 and 4074.90 mg/kg,
respectively (50% of gram flour level; Table IV).
Physical characteristics of cookies
The results of physical attributes of supplemented
cookies are illustrated in Table V. It was manifested
that there was no significant difference ( p . 0.05)
between the values obtained for the width of cookies
supplemented with gram flour and the control (100%
wheat flour) cookies; however, thickness and spread
factors were significantly (p , 0.05) higher among
different treatments.
Biological evaluation of protein quality
The results of feeding trials on rats, conducted for the
determination of protein quality of cookies, compared
Table II. Proximate composition and amino acid profile of SGF
and gram flour.
Parameters SGF ^ SD Gram flour ^ SD
Moisture (%) 15.43 ^ 0.27 9.45 ^ 0.19
Protein (%) 8.54 ^ 0.03 25.16 ^ 0.10
Crude fat (%) 0.96 ^ 0.08 4.69 ^ 0.06
Crude fibre (%) 0.02 ^ 0.42 1.17 ^ 0.51
Ash (%) 0.55 ^ 0.20 2.70 ^ 0.11
NFE (%) 74.50 ^ 0.27 56.83 ^ 0.23
Amino acid (g/100 gm)
Cysteine 0.32 ^ 0.03 0.73 ^ 0.47
Valine 0.63 ^ 0.34 3.42 ^ 0.44
Methionine 0.21 ^ 0.04 1.45 ^ 0.34
Phenylalanine 0.74 ^ 0.02 6.47 ^ 1.49
Threonine 0.42 ^ 0.08 2.88 ^ 1.22
Alanine 0.42 ^ 0.03 4.28 ^ 0.53
Isoleucine 0.55 ^ 0.05 3.69 ^ 0.35
Leucine 0.97 ^ 0.04 7.74 ^ 0.11
Lysine 0.26 ^ 0.02 11.68 ^ 0.15
Note: Means values are ^SD of three replicates.
Table III. Proximate composition of gram flour supplemented cookies.
Treatments Moisture (%) Protein (%) Crude fat (%) Crude fibre (%) Ash (%) NFE (%)
T0 (control) 2.21 ^ 0.13c
6.85 ^ 0.01f
26.70 ^ 0.01f
0.03 ^ 0.01e
0.64 ^ 0.02f
64.40 ^ 0.11a
T1 (10% gram flour) 2.65 ^ 0.11b
7.69 ^ 0.02e
27.07 ^ 0.10e
0.06 ^ 0.02e
0.69 ^ 0.01e
61.85 ^ 0.05b
T2 (20% gram flour) 2.82 ^ 0.10b
8.13 ^ 0.02d
27.38 ^ 0.08d
0.28 ^ 0.01d
0.84 ^ 0.02d
60.56 ^ 0.06c
T3 (30% gram flour) 3.31 ^ 0.33a
8.99 ^ 0.02c
27.73 ^ 0.06c
0.32 ^ 0.02c
0.94 ^ 0.02c
58.72 ^ 0.28d
T4 (40% gram flour) 3.54 ^ 0.09a
10.02 ^ 0.03b
27.98 ^ 0.01b
0.39 ^ 0.01b
0.07 ^ 0.01b
57.00 ^ 0.07e
T5 (50% gram flour) 3.70 ^ 0.08a
10.93 ^ 0.04a
28.52 ^ 0.16a
0.59 ^ 0.02a
1.16 ^ 0.02a
55.10 ^ 0.17f
Notes: Means (^SD) carrying similar alphabets in a column do not differ significantly (p , 0.05); NFE, nitrogen-free extract.
Wheat flour cookies supplemented with gram flour 3
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4. with casein, are presented in Table VI. These results
provide the report of an improvement in the nutritive
value of cookies as a result of gram flour addition. Rats
fed on control diet had the lowest body weight gain,
significantly different from the gram flour sup-
plemented diets and the casein diet. Daily feed intakes
were also significantly different among rat groups
(Table VI). The diets formulated from the cookies
supplemented with gram flour at different levels gave
significantly higher values for BV, TD, NPU and PER
than the control diet containing SGF-based cookies.
Discussion
Previous studies revealed gram flour to be a rich source
of proteins, minerals and vitamins (Sotelo et al. 1987;
Saleh and El-Adawy 2006). The amino acid content in
this study proved the fact that the quality of gram flour
protein is much superior to SGF protein, being high in
lysine amino acid, gram flour is considered best for
complementary utilization with cereal proteins (Saleh
and El-Adawy 2006; Li et al. 2008). All the cookies
supplemented with gram flour were found to be
nutritious on the basis of chemical parameters. Singh
et al. (1991), Somchai et al. (1998) and Tsen et al.
(2006) reported that gram flour can easily be used to
increase the protein content in various types of food
products. The average daily protein intake for a
normal human individual should be 56–80 g/day
(0.8 g/kg body weight/day), while for infants it should
be 2–3 g/kg body weight and children require extra
4–5 g proteins daily for their healthy growth and
development (Awan 2007). This fact suggests that
cookies supplemented with gram flour may be useful
as food supplements for the alleviation of protein
malnutrition in vulnerable groups.
The moisture content of cookies showed significant
difference of up to 30% substitution of gram flour in
T3 while beyond which the difference in moisture
contents of cookies became non-significant ( p . 0.05)
among treatments T3, T4 and T5. The increase in
moisture content of cookies can be attributed to
relatively higher amount of fibre in gram flour than
SGF. Fibre has strong affinity for water, and products
containing fibre will bind larger quantities of water
(Susan and Yada 2010). The results regarding the
proximate analysis of cookies were in line with El-Hag
TableIV.Mineralcompositionofgramfloursupplementedcookies.
TreatmentsFe(mg/kg)Mn(mg/kg)Mg(mg/kg)Ca(mg/kg)Cu(mg/kg)Zn(mg/kg)Na(mg/kg)K(mg/kg)
T0(control)15.85^0.34b
3.39^0.99e
143.40^6.30f
9.12^0.07c
1.40^0.61c
12.80^0.02b
1207.00^2.40d
1589.50^3.60f
T1(10%gramflour)16.30^0.33b
3.82^0.64e
162.60^2.50e
10.59^0.25b,c
1.51^0.28c
13.90^0.29b
1292.00^3.10c
2080.80^4.70e
T2(20%gramflour)19.75^0.09b
4.76^0.35d
199.40^6.70d
10.93^0.16b
1.63^1.27b,c
16.00^0.21a
1411.00^6.80b
2687.70^8.10d
T3(30%gramflour)21.83^0.19b
5.59^0.78c
227.50^8.30c
11.87^0.27b
1.95^1.48b
16.25^0.75a
1504.00^3.20a
3005.60^2.90c
T4(40%gramflour)29.50^0.60a
6.89^3.50b
260.50^3.20b
14.03^0.05a
2.83^0.0.99a,b
17.10^1.43a
1513.00^7.40a
3728.10^5.30b
T5(50%gramflour)33.45^0.51a
7.74^6.10a
300.80^4.20a
14.68^0.15a
2.91^0.55a
17.25^0.18a
1581.00^4.30a
4074.90^2.10a
Note:Means(^SD)carryingsimilaralphabetsinacolumndonotdiffersignificantly(p,0.05).
Table V. Effect of gram flour supplementation on physical
properties of cookies.
Treatments Thickness (mm) Width (mm) Spread factor
T0 (control) 34.90 ^ 0.07c
278.50 ^ 0.01a
79.80 ^ 0.12a
T1 (10% gram flour) 35.00 ^ 1.56c
279.00 ^ 0.14a
79.69 ^ 1.22a
T2 (20% gram flour) 38.00 ^ 0.07b
275.50 ^ 0.14a
72.55 ^ 2.89b
T3 (30% gram flour) 39.50 ^ 0.21a,b
279.50 ^ 0.04a
70.67 ^ 0.10b
T4 (40% gram flour) 39.80 ^ 0.07a,b
273.50 ^ 0.04a
68.80 ^ 0.43b,c
T5 (50% gram flour) 41.00 ^ 0.01a
273.00 ^ 0.14a
66.63 ^ 2.30c
Note: Means (^SD) carrying similar alphabets in a column do not
differ significantly (p , 0.05).
A.A. Yousaf et al.4
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5. and El-Tinay (2001), Rababah et al. (2006), Gomez
et al. (2008) and Wood (2009). Mineral contents
in cookies increased with an increase of gram flour
in SGF which is due to the high mineral content
found in gram as compared to wheat (Ibanez et al.
1998; El-Adawy 2002; Vitali et al. 2008). The results
regarding the mineral contents of cookies are in close
agreement with the findings of Khan et al. (1995) and
Shirani and Ganesharanee (2009) who have reported
a proportionate increase in the mineral content of
baked and extruded products made with different
proportion of gram flour.
The results regarding the physical evaluation of
cookies prepared from different levels of supplemen-
tation with gram flour are in conformity with Rababah
et al. (2006) who reported a decrease in the spread
ratio as the concentration of gram flour increased in
cookies. The decrease in spread ratio of supplemented
biscuits might be due to the availability of more
hydrophilic sites that compete for limited free water in
biscuit dough prepared from high-protein flours
(Khan et al. 1995). Hooda and Jood (2005) also
reported that the spread factor of biscuits was
decreased by increasing the supplementation levels of
fenugreek flour in wheat flour.
The findings regarding the biological evaluation of
cookies revealed that nutritive value of SGF was
improved with the supplementation of gram flour. The
relatively higher essential amino acid balance and
mineral contents in gram flour supplemented cookies
seem to favour these diets to give better weight gain than
the control diet. The best response in terms of biological
evaluation was obtained by addition of 30–50% gram
flour to SGF for the preparation of cookies. The
improvement in all biological parameters of rats fed on
gram flour supplemented cookies is certainly due to the
higher nutritional value of gram proteins and good
quality of other constituents such as minerals and
vitamins (Amjad et al. 2006) than straight grade wheat
flour. On the other hand, the rats fed on control diet
containing 100% SGF gave poor protein quality indices,
such as low NPU (%), BV (%) and TD (%).
In this study, it was also found that the cookies
supplemented with gram flour at the 50% level were
nutritionally at par with diets based on casein. The
above results may provide the proof of an improve-
ment in nutritive value of cookies as a result of gram
flour addition. The findings of the BVs were in
accordance with those of Abid et al. (1991) and Bhatty
et al. (2000) who reported that protein supplemented
diet significantly improved BV and promoted growth
in rats.
Conclusion
Keeping in view all the above results, it could be
concluded that the addition of gram flour in the range
of 20–30% results in favourable baked products
(cookies). These combinations also improved the
physicochemical properties of cookies. The results of
this study suggested a great potential of gram flour in
baking industry to provide new value-added nutritious
products for consumers especially in developing and
resource poor countries.
Declarations of interest: The authors would like to
thank all colleagues who contributed to this study. The
quality of this research work was greatly enhanced by
the gracious assistance of Mr Nouman Siddique,
Mr Amir Mumtaz, Mr Naeem and Mrs Saeeda Raza
(NARC). We are grateful to Dr Hussain (NIH) for his
collaboration during preliminary investigations and
biological analysis. The authors report no conflict of
interest. The authors alone are responsible for the
content and writing of the paper.
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Table VI. Protein quality parameters of diets containing gram supplemented cookies fed to rats.
Treatments
Weight gain
(g/group/10 days)
Feed intake
(g/rat/10 days) PER
TD
(%)
BV
(%)
NPU
(%) FER
T0 (control) 40.16 ^ 1.14f
154.00 ^ 0.11d
1.080 ^ 0.01d
67.810 ^ 0.51 g
61.48 ^ 0.86d
41.680 ^ 1.73 g
0.261 ^ 0.01e
T1 (10% gram flour) 45.60 ^ 4.24e
164.00 ^ 0.57c
1.159 ^ 0.02c
71.765 ^ 0.49f
63.76 ^ 0.78c
45.760 ^ 0.65f
0.280 ^ 0.01d
T2 (20% gram flour) 50.00 ^ 3.39d
167.40 ^ 0.55c
1.240 ^ 0.02b
74.030 ^ 0.37e
64.84 ^ 0.14b,c
48.610 ^ 0.52e
0.290 ^ 0.02c
T3 (30% gram flour) 52.70 ^ 1.41c
175.00 ^ 0.14b
1.255 ^ 0.01b,c
79.060 ^ 0.06d
65.67 ^ 0.22b
51.265 ^ 0.32d
0.301 ^ 0.01b,c
T4 (40% gram flour) 54.85 ^ 2.12b
180.50 ^ 0.78a,b
1.266 ^ 0.04a,b,c
82.175 ^ 0.05c
70.01 ^ 0.35a
57.650 ^ 0.39c
0.304 ^ 0.01a,b,c
T5 (50% gram flour) 56.22 ^ 4.30a,b
182.90 ^ 0.20a
1.281 ^ 0.01a,b
87.710 ^ 0.62b
70.02 ^ 0.71a
61.415 ^ 0.20b
0.307 ^ 0.02a,b
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183.50 ^ 0.08a
1.285 ^ 0.02a
91.730 ^ 0.51a
70.16 ^ 0.63a
64.215 ^ 1.16a
0.308 ^ 0.01a
Tnp 213.00 ^ 1.14 g
135.50 ^ 1.41e
– – – – –
Notes: Means (^SD) carrying similar alphabets in a column do not differ significantly (p , 0.05); PER, protein efficiency ratio; TD, true
digestibility; BV, biological value; NPU, net protein utilization; FER, feed efficiency ratio.
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