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https://doi.org/10.17113/ftb.60.04.22.7655 original scientific paper

Gluten-Free Crackers Based on Chickpea and Pumpkin Seed Press Cake Flour:
Nutritional, Functional and Sensory Properties

Running head: Utilization of Non-Conventional Flours in Formulation of Gluten-Free Crackers

Jelena Tomić1*, Dubravka Škrobot1, Ljiljana Popović2, Tamara Dapčević-Hadnađev1, Jelena

Čakarević2, Nikola Maravić1 and Miroslav Hadnađev1

1University of Novi Sad, Institute of Food Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia
2University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, 21000 Novi Sad, Serbia

Received: 17 January 2022

Accepted: 15 July 2022

SUMMARY
Research background. Despite the growing trend of the gluten-free market and the presence of
a wide range of gluten-free products, there are still some shortcomings of these products in terms of
nutritional and sensory quality. The commercially available gluten-free products are characterised as
products of inferior nutritional quality, particularly in terms of protein and dietary fibre content and with
high glycemic index. On the other hand, from a sensory point of view, gluten-free products usually show
inappropriate textural and mechanical properties, poor mouthfeel and flavour. This is a consequence of
the limiting choice of raw materials that mainly possess large share of carbohydrate components.
Experimental approach. Chickpea flour (ChF) and two types of pumpkin seed press cake flour
(virgin (VF) and cold pressed (CF)), at two substitution levels (20 and 35 %, m/m) were blend to produce
gluten-free crackers without the presence of conventional gluten-free starch-rich ingredients. This study

*Corresponding author:

Phone: +3814853780
E-mail: [email protected]

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aims to investigate the suitability of using these non-conventional flours on nutritional and physico-
chemical properties, sensory acceptability, antioxidant activity and glycemic index of crackers.
Results and conclusions. All produced crackers can bear a nutritional claims "high fibre", "source
of protein", "source of minerals". Replacing ChF with VF and CF increased protein and total phenolic
content and enhanced antioxidant activity. The selected combination of raw materials allows the
production of gluten-free crackers with a moderate glycemic index. Besides nutrient content, CF addition
increased overall sensory acceptability, noticeably improving taste and flavour scores compared to
Control and VF containing counterparts.
Novelty and scientific contribution. To the best of our knowledge, there is no study investigating
the use of chickpea and pumpkin seed press cake flour blend without using conventional gluten-free
flours and starches. The used non-conventional flours represent complementary raw materials in terms
of protein quality and valuable alternatives to produce nutrient dense, health promoting gluten-free
crackers with reduced glycemic response and acceptable sensory properties.

Keywords: gluten-free; chickpea flour; pumpkin seed press cake flour; glycemic index; total phenolic
content; protein content

INTRODUCTION
In the circumstances of constant population growth and modern diet trends, the food industry
faces great challenges in its desire to balance market dynamics and sustainable supply of various
nutritious foods. The baked products (bread, biscuits, and crackers) as the core of the consumer segment
in the food industry are particularly susceptible to modifications and constant development in order to
improve their nutritional composition without compromising consumer acceptability. In this regard, the
utilization of various food ingredients which will provide adequate nutritional effects becomes the priority
for both the food scientist and the food industry.
For the new food ingredients, especially in the case of gluten-free products, it is desirable to
provide dual benefit of enhancing nutritional and retaining or improving technological quality of final
products. How gluten-free products are recognized as products with inferior nutritional quality, many
studies with different technological and compositional approaches were devoted to the fortification of this
category of food (1–4).

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The lower dietary fibre and complex carbohydrates contents along with higher glycemic index (GI)
are considered as main drawbacks of gluten-free diet (5). In addition, a lower protein content compared
to their gluten-containing counterparts has also been reported (6,7).
A compromise between the health benefits of a gluten-free diet and its nutritionally balanced
profile and technological quality is very difficult to achieve. The reason for this is certainly the limiting
choice in terms of raw materials that would provide the functional properties characteristic of gluten. By
reviewing the literature data, but also by looking at commercially available gluten-free products, it is
evident that rice and corn flour, as well as starches of different origins, are the most common ingredients
of gluten-free products (5,8). Their selection is based primarily on the fact that they are of neutral taste
and acceptable colour, good digestibility and hypoallergenic properties (9,10). On the other hand, as a
consequence of the large share of carbohydrate components in these raw materials, gluten-free products
are usually characterized by poorer sensory quality, especially in terms of textural and mechanical
properties (crumbling texture, poor mouthfeel and flavour) (11). Taking into consideration all mentioned
shortcomings of gluten free products, reformulation of food by introducing wide range of unconventional
gluten free flours become a common practice. Evidence about considerable amounts of nutrient-rich
ingredients found in chickpea has focused a lot of attention on the matter of redesigning conventional
foods containing this raw material, mainly in the form of protein flour (1,12). Chickpea is a valuable source
of proteins with a good amino acid profile (high lysine), complex carbohydrates (dietary fibres, resistant
starch, and oligosaccharides), important vitamins and minerals (B-vitamins, folates, and iron), as well as
antioxidants and polyphenols (13). Additionally, the benefit of usage of chickpea flour as an alternative
raw material in gluten-free product is reflected through the slow release of glucose from starch which
contributes to the lower glycemic index (2). In the view of the environmental sustainability and
minimisation of food waste, agro industrial by-products present a real impetus for the development of
additional health promoting ingredients. Pumpkin seed press cake, a by-product of oil production, is food
resources that offer various health benefits. In addition to fibre, pumpkin seed press cake is a source of
proteins, essential fatty acids, polyunsaturated fatty acids, omega 3, 6 and 9 fatty acids, as well as
significant amounts of vitamins and minerals (14–16). Regarding the protein quality, this raw material has
a suitable amino acid profile. Compared to soya bean meal it contains higher levels of most essential
amino acids (except lysine) (17).
From nutrition point of view, chickpea and pumpkin seed press cake flours are complementary
raw materials in terms of protein quality. Most studies that have investigated these raw materials were

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related to their valorisation and potential as gluten-free substitutes of conventional gluten-free flours in
the production of bakery products. However, there are no published data on the use of chickpea and
pumpkin seed press cake flour blend in gluten free bakery products processing without the presence of
conventional gluten-free flours and starches. Therefore, the objective of this study was to investigate the
possibility of using aforementioned raw materials as base ingredients for production of sensory
acceptable and nutritionally improved gluten-free crackers with respect to content of protein and dietary
fibre. Additionally, fatty acid content, glycemic index, polyphenolic content and antioxidant activity were
determined and discussed. Moreover, the impact of pumpkin seed press cake flour treatment history on
the quality of obtained crackers was evaluated.

MATERIALS AND METHODS

Raw materials
Chickpea flour (ChF) was locally produced and obtained from agricultural household "Mirilov",
Bačko Gradište, Serbia. Chickpea flour had moisture, protein, fat, ash and carbohydrates (calculated by
difference) contents of 9.27, 21.52, 4.78, 3.29 and 61.14 g/100 g, respectively.
Samples of hull-less pumpkin (Cucurbita pepo L) seed press cake, a by-product of the pumpkin
cold pressed and virgin pressing process, were obtained from a “Suncokret d.o.o”, Hajdukovo, Subotica,
that uses traditional mechanical oil pressing technology. The pumpkin seeds used for the production of
virgin oil, prior oil extraction went through thermal processing. The obtained press cake samples were
grinded to obtain flour. The roasted pumpkin seed press cake flour (VF) had moisture, protein, fat, ash
and carbohydrates (calculated by difference) contents of 2.70, 56.16, 13.92, 8.59 and 18.63 g/100 g,
respectively while the cold pressed pumpkin seed cake flour (CF) had moisture, protein, fat, ash and
carbohydrates (calculated by difference) contents of 8.27, 56.49, 16.79, 7.57 and 10.88 g/100 g,
respectively. Vegetable fat (palm oil based, melting range temperature 34-37 °C) was obtained from oil
factory “Dijamant” (Zrenjanin, Serbia). Salt, sodium bicarbonate, ammonium bicarbonate and powdered
sugar were purchased in a local food store.

Preparation of gluten-free cracker

Five different formulations of gluten-free crackers were produced under the same processing
conditions by mixing chickpea flour with virgin (VF) or cold press (CF) pumpkin seed press cake flour in
different ratio (Table 1). The other ingredients were used in the same proportions. Chickpea flour,

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pumpkin seed press cake flour, salt and baking powder were mixed together. The dough was prepared
using the following procedure: vegetable fat was mixed with soy lecithin for 2 min, and then water was
added and thoroughly blended to obtain homogenous mixture. Finally, all powdery ingredients were
added together and mixed for additional 3 min. The dough was laminated immediately after preparation
on pilot scale dough laminator (Thiene, Italy) to the desired thickness (b=3.0 mm). Cracker samples were
shaped using a circular cutter (Ø45 mm) and baked at 190 °C for 11 min in a laboratory oven (MIWE
gusto® CS, Arnstein, Germany). The crackers were produced in four batches where each batch yielded
20 crackers. The obtained gluten-free crackers were left to cool down at room temperature for 1 h and
then they were hermetically stored for analysis.

Proximate composition
Proximate composition of raw materials and crackers including protein (Method No. 920.87), fat
(Method No. 922.06), ash (Method No. 923.03) and moisture contents (Method No. 925.09) was
determined by AOAC standard methods of analysis (18). Total dietary fibre (TDF) content of the obtained
crackers was determined using of the Megazyme International total DF assay kit (adopted from AACC
method 32–07 and AOAC method 985.29) (18,19). Available carbohydrate was obtained by difference,
by subtracting the sum of grams of water, protein, fat, ash and dietary fibre from a 100 g basis mass. The
total energy was calculated using the European Regulation No. 1169/2011 (20).

Mineral content
Mineral content (Zn, Fe, Mg, K, Na, Ca) was determined by atomic absorption spectrophotometry
(Method No. 984.27) on a Varian Spectra AA 10 (Varian Techtron Pty Ltd., Mulgvare Victoria, Australia)
(19).

Fatty acid composition

The procedure published in Pojić et al. (21) was used to determine fatty acid profile. Total lipids
were extracted with a chloroform-methanol solution (2:1 ratio of chloroform to methanol) and the obtained
extracts were dried by vacuum evaporation (40 °C). The residue obtained after evaporation of solvent
under steam of nitrogen was weighed. The extracted lipids were transformed in fatty acid methyl esters
using 14 % boron (III)-fluoride in methanol. The samples were analysed by GC Agilent 7890A (Flame-
ionization detector, auto injection module for liquid samples, fused silica capillary column DB WAX 30 m,

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0.25 mm, 0.50 µm) (Agilent Technologies, Santa Clara, USA). As a carrier gas, helium was used with
purity over 99.9997 vol. % and flow rate of 1.26 ml/min. Identification of fatty acids was done by comparing
of retention times with standards from Supelco 37 Component Fatty Acid Methyl Ester Mix (Sigma-
Aldrich, St. Louis, MO, USA). All analyses were performed in duplicates and the results were expressed
as a percentage of each fatty acid in total fatty acids.

Total phenolic content

Total phenolics from the samples were extracted by the method of Świeca (22). Briefly, crackers
were ground (1.0 g) and mixed with 20 mL of PBS (phosphate buffered saline, pH=7.4) for 1 h at room
temperature. The procedure was repeated twice. The extracts were centrifuged at 9200 xg (Sorvall® RC-
5B Refrigerated Superspeed Centrifuge, Du Pont Instruments, Wilmington, USA) for 15 min and the
resulting supernatants were pooled and stored at 4 °C.
Total phenolic content of PBS extracts was determined spectrophotometrically by using Folin‐
Ciocalteu's reagent method described by Singleton et al. (23). The extract (0.1 mL) was diluted with
distilled water (7.9 mL). Folin‐Ciocalteu's reagent (0.5 mL) and 20 % sodium carbonate solution (1.5 mL)
were added at the reaction mixture. The mixture was allowed to stand for 60 min and the absorbance at
750 nm was measured (T80 UV–Vis Spectrophotometer; PG Instruments, Lutterworth, UK). Gallic acid
was used as a standard and results were expressed as Gallic acid equivalents (GAE) (mg GAE per g of
sample).

Antioxidant activity
The radical scavenging activity was determined by the ABTS scavenging activity assay described
by Popović et al. (24), with some modifications. Briefly, an aliquot of 30 μL of the PBS extract was mixed
with 3 mL of a daily prepared ABTS solution (A=0.7±0.02). The absorbance was measured at 734 nm
(T80 UV–Vis Spectrophotometer; PG Instruments, Lutterworth, UK), after 10 min. The activity was
expressed as Trolox equivalent in mM per g of sample.

In vitro starch digestion rate and predicted glycemic index

Predicted glycemic index of crackers was determined by method of Molinari et al. (25), with some
modifications. The ground crackers (200 mg) were mixed with 20 mL HCl-KCl buffer (0.01 M, pH=1.5)
and 0.2 mL of pepsin solution (1 g in 10 mL HCl-KCl buffer). The mixture was incubated for 60 min in a

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shaking water bath at 40 °C. After pepsin digestion, the total sample volume was adjusted to 60 mL with
phosphate buffer (0.2 M, pH=6.9). Then, the 10 mg of α-amylase were added and incubated for 3 h in a
shaking water bath at 37 °C. Aliquots (1 mL) at 0, 10, 20, 30, 60, 90,120, 150 and 180 min were obtained
from each sample and incubated at 100 °C for 5 min to inactivate the enzyme. After incubation time, 250
µL of each supernatant was taken to a volume of 750 µL with sodium acetate buffer (0.2 M, pH=4.75).
Subsequently, 30 µ of amyloglucosidase was added and incubated at 60 °C for 45 min with constant
stirring. Glucose content was measured by the GOPOD kit (Megazyme, Wicklow, Ireland).
The hydrolysis curve of samples was expressed by concentration of glucose measured in
samples during the time. The hydrolysis index (HI) was calculated as the ratio between the areas under
the hydrolysis curve (0–180 min) of the experimental samples and the area of reference sample (white
bread). The predicted glycemic index (pGI) was calculated using the equation (Eq. 1) proposed by Goñi
et al. (26):
pGI = 39.71 + 0.549 ∙ HI /1/

Physical properties
Eccentricity, spread factor and puffiness
Physical parameters including mass (g), diameters of baked cracker (d1 and d2, perpendicular to
each other), thicknesses of the cracker before (b1) and after baking (b2) were measured by using Vernier
calliper using 10 crackers taken randomly from the batch of crackers. The eccentricity was calculated as
the ratio between d1 and d2. Spread factor was calculated as the ratio between average diameter and
average thickness (3). Sample puffiness (P) was calculated as shown in Eq. 2:
𝑏𝑏2 −𝑏𝑏1
% 𝑃𝑃 = � 𝑏𝑏1
�∙ 100 /2/

Textural measurements
The crackers hardness and fracturability were measured by using TA-XT2 Texture Analyser
(Stable Micro System, Godalming, United Kingdom) equipped with a 30 kg load cell and three-point
bending rig (HDP/3PB). The two adjustable supports of the rig base plate were placed at 30 mm distance
apart so as to support the sample. The upper blade was positioned to be equidistant from the two lower
supports. During measurements, the upper blade descended at a speed of 1mm/s until a 50 g contact
force was detected and then travelled a distance of 5 mm through the cracker at a speed of 3.0 mm/s.

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Measurements were performed 24 hours after baking in six replicates per batch at ambient temperature
(25±1 °C).

Colour measurements
The colour of the crackers top surface was measured 24 hours after baking using a Chroma meter
Minolta CR-400 (Konica Minolta Co., Osaka, Japan). The results were expressed as L*
(lightness/darkness), a* (redness/greenness), b* (yellowness/blueness).
The influence of different type of pumpkin seed press cake flour incorporation on the total colour
difference (∆E) between control and the pumpkin flour containing crackers was measured according to
the following the Eq. 3:

∆𝐸𝐸 = �∆𝐿𝐿∗ 2 + ∆𝑎𝑎∗ 2 + ∆𝑏𝑏 ∗ 2 /3/

where ΔL*, Δa* and Δb* indicate the difference in L*, a* and b* values measured for crackers with virgin
and cold press seed press cake flour, respectively.
Colour measurements were taken on 5 randomly selected crackers per sample batch.

Sensory evaluation
Sensory analysis of crackers was performed by semi-trained sensory panellists (28 female and
12 male, 27–50 years of age). All panellists were recruited from a staff working at the Institute of Food
Technology in Novi Sad. Panellists evaluated liking of the crackers colour, taste, flavour, texture and
overall liking on a 9-point structured hedonic scale where 1 indicates extreme disliking and 9 indicates
extreme liking. Panellists worked in a sensory laboratory in individually sensory booths. They received
two crackers per sample one at a time, in closed odourless plastic containers at ambient temperature
(25±1 °C) labelled with three randomly chosen digit numbers and drinking water for palate cleansing.
Before analysis all panellists received written information about the study and they signed informed
consent to participate. The study was approved by the Ethics Committee of the Institute of Food
Technology in Novi Sad, University of Novi Sad, Novi Sad, Serbia (Ref. No. 175/I/13-3).

Statistical analysis
All measurements were performed at least in triplicates if not stated differently. The means of
replicates for analysed parameters were statistically processed by using the software package XLSTAT

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2019.4.2. (27). Analysis of variance (ANOVA) and Fisher’s least significant differences test (p<0.05) were
used to determine significance of differences between sample means.

RESULTS AND DISCUSSION

Proximate analyses of crackers
Proximate composition of crackers is presented in Table 2. Changes in the chemical composition
are in function of the gradual substitution of chickpea flour with pumpkin seed cake flours. Although tested
samples showed significant differences in moisture content, the values were below 6 % which could be
considered favourable for quality and stability of the produced cracker (28). The increase in the ratio of
virgin (VF) or cold press (CF) pumpkin seed press cake flour influenced the higher content of proteins,
fats and ash whereas total carbohydrates content decreased (p<0.05). The highest values of proteins
and fats were observed for crackers with 35 % cold press (CF) pumpkin seed press cake flour. According
to Regulation (EC) No. 1924/2006 (29), a claim that a food is "source of protein" may only be made where
at least 12 % of the energy value of the food is provided by protein. According to this Regulation, the
control cracker and crackers with 20 % of VF and CF can be labelled as protein source since 13.94 %
(Control), 18.76 % (VF) and 18.91 % (CF) of their energy value is provided by protein. In the case of
crackers with higher level of VF and CF, they may bear a claim "high protein" as their proteins provide
22.74 % (VF) and 23.98 % (CF) of the energy value. The values of total dietary fibres slightly varied
between samples, and ranged from 9.17 % (20CFC) to 10.22 % (35VFC). Since all cracker samples
contained more than 6 g of fibre per 100 g, they also can be labelled as "high in fibre" (29).
Table 2

Mineral content of crackers

Potassium was the most abundant macroelement in all cracker samples, followed by calcium and
magnesium (Table 2). In comparison with gluten free crackers enriched with hemp flour and
decaffeinated green tea leaves (30), crackers produced in this study had higher content of all tested
minerals except Ca. Missbach et al. (31) found that potassium content was significantly lower in gluten
free snacks than in their gluten counterparts (190.4±160 mg/100 g compared to 247.5±130 mg/100 g),
while in this study potassium content was in range from 926 to 1007 mg/100 g. Substitution of chickpea
flour with VF and CF significantly (p<0.05) altered the content of minerals. With an increase in the amount
of pumpkin seed cake flours the content of almost all minerals progressively rose. The exception is Ca

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content where the highest content was recorded in the control sample. To declare product as a product
with a significant amount of certain minerals, it must contain 15 % of Dietary Reference Values (DRV) in
100 g of product (32). Based on the report of European Food Safety Authority (2017) (32), all samples
are significant source of Zn, Fe, Mg and K. Bearing in mind that gluten-free products have low amount of
minerals, which can be harmful for patients suffering from celiac disease (33), the selection of used raw
materials for crackers production is justified.

Fatty acid composition of crackers

The analysis of fatty acid profile has shown that the dominant fatty acids in all the samples were
palmitic acid (C16:0), oleic acid (C18:1n9c) and linoleic acid (C18:2n6c) (Table 2). The content of linoleic
acid was increased as a consequence of increasing the amount of pumpkin seed cake flours. In the case
of alpha linolenic acid, the presence of CF in the cracker formulation affected its reduction. According to
WHO/FAO in balanced died ratio of PUFA/SFA should been over 0.4. In all samples ratio were over 0.4,
which indicates that choice of ingredients contributes to favourable fatty acid composition. Considering
the MUFA intake, the Adult Treatment Panel III (2004) (34) recommended up to 20 % of energy intake
as MUFA. Regarding the obtained results for fatty acid composition, the content of MUFA is in accordance
with proposed recommendation.

Total phenolic content (TPC) and antioxidant activity

Polyphenols, secondary plant metabolites, could possess different biological activities such as
ability to inhibit reactive oxygen and nitrogen species, oxidative enzymes, to activate antioxidant enzymes
and could act as free radical scavengers (35). Values of TPC were in the range from 0.72 mg GAE/g for
Control sample to 1.06 mg GAE/g for sample 35CFC (Table 3). The pumpkin oil cake incorporated
crackers showed significant (p<0.05) increase in the TPC.
The antioxidant activity of the formulated crackers determined by ABTS assay revealed a similar
trend to that found in the Folin-Ciocalteu assay (Table 3). The incorporation of pumpkin seed press cake
flours resulted in higher values of antioxidant activity ranging from 11.5 mM TE/g to 17.0 mM TE/g. The
higher values of antioxidant activity in the 35CFC might resulted from the initial presence of phenolic
compounds in the cold pressed cake, while in the virgin pressed cake the amount of phenolic compound,
which are known to be thermolabile components, can be reduced due to the thermal treatment of the
seeds before oil extraction. In this respect, the pumpkin seed press cake flour inclusion in cracker

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formulation enhanced their antioxidant potential which could be beneficial for stability against oxidative
damage during storage.
Table 3

Glycemic index of crackers

Compared to their gluten containing counterparts, gluten free baked products are often
considered as products with high glycemic index. This is due to the prevalent of carbohydrate
components of commonly used gluten free flours such as rice and maize flour. Moreover, there is a
theoretical presumption that in the absence of gluten protein network that usually surrounds the starch
granules, the starch granules become more susceptible to action of amylolytic enzymes which
additionally contribute to the higher glycemic index (36,37). Considering the complex matrix
characteristics of the bakery products, in addition to above, glycemic index are highly influenced by
several factors such as type of starch (the amylose/amylopectin ratio), the physical entrapment of starch
molecules within food, food formulations and processing, and by the present of other ingredients, such
as sugars, fat, protein, dietary fiber and anti-nutrient content (5,36,38,39). Accordingly, it is of great
importance to introduce raw materials with potential of reducing glycemic index in gluten free products
manufacturing. The starch hydrolysis curves during the second intestinal phase of in vitro digestion of all
produced crackers along with white bread are presented in Fig. 1. The results of hydrolysis index (HI),
calculated from the starch hydrolysis curves, and the corresponding predicted glycemic index (pGI) are
shown in Table 3. Glucose concentration increased during in vitro digestion for all samples, presenting a
sharper growth during the first 30 min. As expected, the maximum glucose concentration was recorded
in white bread which served as a benchmark while all created crackers had significantly lower glucose
concentration where maximum occurred at 60 min and remained almost at the same level during the all
examined digestion time. According to available literature data, the predicted glycemic index for gluten
free cookies and crackers with addition of pulses flours ranged from low (<55) to intermediate (55–70)
(5,40). The control cracker presented a glycemic index of 67.8±0.3 while substitution of chickpea flour by
pumpkin seed press cake flours at both levels (20 and 35 %) significantly (p<0.05) reduced cracker
glycemic index to approximately 60. The results obtained in this study showed that the selected
combination of raw materials used in this study is feasible to obtain gluten-free crackers with a moderate
glycemic index (41) where the use of flour from pumpkin seed cakes allows a further reduction in the
predicted glycemic index.

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Fig. 1

Physical properties of crackers

Eccentricity, spread factor and puffiness
Physical properties of crackers are summarized in Table 4. There were no significant differences
between eccentricity and spread factor of samples. However, an increase in spread factor values is
observed with substitution of chickpea flour with VF and CF. Compared to the Control sample, crackers
substituted with VF and CF have higher mass values. Furthermore, addition of VF and CF affected the
puffiness of samples. Significant dimensional changes are typical in the formulation of cookies with wheat
flour and composite blends as well as in gluten free formulations for cookies based on rice or maize flours
as a consequence of incorporation of high protein raw materials (15,42). Since the all tested samples
had a high protein and fibre content it can be assumed that used raw materials caused a higher amount
of water retention in the dough system, which limited the free water and thus did not affect significant
changes in dimensional parameters. This is in accordance with the research of Mancebo et al. (43),
where presence of flours with higher protein content in gluten free formulations had no significant
influence on spread ratio of cookies. Although a certain increase in cookies diameter and spread ratio is
desirable for better quality (42), the advantage of the uniform cookies dimensions could be reflected in
the simplified control of the products packaging.
Table 4

Colour of crackers
Crackers containing pumpkin seed press cake flour, regardless of type, possessed unusual colour
for this type of product, as can be observed from the Fig. S1. Regarding the lightness parameter L*, a
reduction with increasing VF and CF concentration can be observed (Table 4). Parameters a* and b*
have also been reduced, indicating an increase in green colour and decrease in the yellowness of
biscuits. The green colour of pumpkin seed press cake flours derives from protochlorophyll found in
chlorenchyma, the dark green layer around cotyledons of pumpkin cultivar Cucurbita pepo (44). The
results showed that VF containing crackers were more red and less yellow in comparison to CF containing
crackers. This could be related to the higher pigment degradation with the seed thermal treatment before
oil extraction and later crackers baking process. The total colour difference (ΔE) was highly influenced
by the amount of pumpkin seed press cake flours.

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Hardness and fracturability of crackers

Textural properties of crackers expressed as hardness (maximum peak force recorded from
force/distance curve) and fracturability (peak distance which represents the distance the cracker will
deform before breaking) are listed in Table 4. According to obtained results partial replacement of
chickpea flour with CF did not influence cracker texture although there was significant difference in
crackers composition in terms of protein and starch content (Table 2). The similar hardness and
fracturability of Control and CFC crackers were probably influenced with the combined effect of
gelatinized starch granules embedded in the protein matrix, where the former were more dominant in
Control sample while the latter contributed to texture formation in CFC samples. On the contrary,
incorporation of VF led to an increase in cracker hardness and decrease in its fracturability. Unlike CF,
VF passed through thermal treatment during oil extraction which influenced protein denaturation to higher
degree and consequently led to higher water retention in VFC cracker dough (45). Higher content of
retained water in VFC formulations in comparison to CFC produced stiffer doughs and subsequently
harder crackers (46). However, no significant difference in crackers hardness and fracturability was
observed with the increase in both VF and CF replacement level.

Sensory analysis (overall liking, colour, taste, texture and flavour) of crackers
Results of sensory study showed that all sensory scores for crackers were in the range from like
slightly to like very much (Fig. 2). Replacement of chickpea flour with VF slightly decreased while
replacement with CF significantly increased overall liking of crackers, especially in terms of taste and
flavour. Cracker 35CFC was superior in terms of all analysed sensory properties, followed by the 20CFC.
Cracker 35VFC was the least acceptable. In their study, Kaur and Sharma (47) showed that cookies
supplemented with raw pumpkin seed flour were more acceptable in comparison with the cookies
supplemented with roasted pumpkin seed flour which is in agreement with the results presented in this
study. Although colour differences between samples were clearly visible, the colour liking between
samples was not significant (p<0.05), except between samples 35VFC and 35CFC (p>0.05). Liking
scores were in the range from “slight liking” (5.80) for 35VFC to “like very much” (7.50) for 35CFC. Han
et al. (1) studied the effect of various pulse flours and fractions (desi chickpea, green lentil, red lentil,
pinto bean, navy bean, and yellow pea) on development of gluten-free pulse-based cracker snacks, and
similar to our findings, they found no statistical difference in colour acceptability scores that were in the
range from 6.0 (for crackers containing navy bean flour) to 6.7 (for crackers containing pea protein

13
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isolate). Moreover, they concluded that within the evaluated range of samples, the colour was not barrier
to product acceptability.
Fig. 2
CONCLUSIONS
The present study revealed that the combination of chickpea and pumpkin seed press cake flours
could be successfully utilised in gluten-free crackers production without using conventional gluten-free
starch-rich ingredients. This selected raw materials had a multiple benefits which are reflected in
increased nutritional quality of final product, including increased protein and dietary fibre content,
improved mineral and fatty-acid profile, enhanced total phenolic content and antioxidant activity
compared to the control sample. The selected combination of raw materials used in this study allows the
production of gluten-free crackers with a moderate glycemic index. Moreover, it was shown that cold
press pumpkin seed cake would perform better than virgin pumpkin seed cake in terms of product texture
and overall acceptability. Further research should be focused on the crackers shelf life assessment but
also on the safety aspects of this novel food product.

ACKNOWLEDGEMENTS
This work was supported by the Ministry of Education, Science and Technological Development,
Republic of Serbia (Contract No. 451-03-68/2022-14/200222).

CONFLICT OF INTEREST
The manuscript is original, and it strictly followed all ethical procedures. No part of the manuscript
has been published before, nor is any part of it under consideration for publication in another journal. The
authors declare that there is not any conflict of interest.

SUPPLEMENTARY MATERIAL
All supplementary materials are available at: www.ftb.com.hr.

AUTHORS' CONTRIBUTION
Jelena Tomić was in charge of the conceptualization, formal analysis, writing the original draft
and investigation. Dubravka Škrobot was in charge of the conceptualization, formal analysis, writing the
original draft. Ljiljana Popović was in charge of the investigation, writing the original draft. Tamara

14
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will undergo copyediting and typesetting before its final form for publication. We are providing this version as a
service to our readers. The published version will differ from this one as a result of linguistic and technical corrections
and layout editing.

Dapčević Hadnađev was in charge of the supervision and writing the original draft. Jelena Čakarević was
in charge of investigation and writing the original draft. Nikola Maravić was in charge of the formal analysis
and investigation. Miroslav Hadnađev was in charge of the resources, supervision and project
administration.

ORCID ID
J. Tomić https://orcid.org/0000-0002-8674-6932
D. Škrobot https://orcid.org/0000-0003-3292-8738
Lj. Popović https://orcid.org/0000-0001-9264-285X
T. Dapčević-Hadnađev https://orcid.org/0000-0001-6222-2889
J. Čakarević https://orcid.org/0000-0002-8446-1722
N. Maravić https://orcid.org/0000-0001-9926-7802
M. Hadnađev https://orcid.org/0000-0002-0055-5642

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Table 1. Formulation of crackers

Virgin Cold press

pumpkin pumpkin
Sample/raw Chickpea Vegetable Soy lecithin Baking
seed press seed press Salt w/% Water w/%
material flour w/% fat w/% w/% powder w/%
cake flour cake flour
(VF) w/% (CF) w/%

Control 100 - - 25 2.5 0.5 0.5 40

20VFC 80 20 - 25 2.5 0.5 0.5 40

35VFC 65 35 - 25 2.5 0.5 0.5 40

20CFC 80 - 20 25 2.5 0.5 0.5 40

35CFC 65 - 35 25 2.5 0.5 0.5 40

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Table 2. Proximate composition, mineral content and fatty acid composition of crackers

Parameters Control 20VFC 35VFC 20CFC 35CFC

Proximat composition w/%

Moisture (4.62±0.06)d (5.12±0.04)b (3.99±0.03)e (5.61±0.06)a (5.00±0.03)c

Carbohydrates (42.2±0.2)a (35.2±0.4)b (28.8±0.0)c (34.9±0.2)b (26.0±0.4)d

Crude Fat (24.3±0.1)d (24.8±0.1)c (26.1±0.1)b (24.9±0.1)c (26.8±0.1)a

Protein (16.5±0.1)d (22.2±0.1)c (27.3±0.0)b (22.3±0.1)c (28.7±0.1)a

Total dietary
(9.95±0.13)a (9.61±0.10)b (10.2±0.2)a (9.17±0.07)c (9.42±0.17)bc
fibre

Ash (2.47±0.03)e (3.06±0.01)d (3.67±0.03)b (3.14±0.03)c (3.99±0.02)a

Energy/(kcal/kJ) 473/1976 472/1971 479/2000 471/1967 479/1999

Mineral content w/(mg/100g)

Zn (3.00±0.04)d (4.76±0.08)c (5.90±0.06)b (4.91±0.05)c (6.17±0.13)a

Fe (4.02±0.09)d (4.67±0.11)b (5.51±0.06)ab (5.21±0.13)b (5.79±0.24)a

Mg (103±0)e (170±4)d (221±6)b (181±3)c (265±6)a

K (926±17)c (932±1)bc (952±9)b (986±11)a (1007±5)a

Na (810±16)c (904±2)b (1034±3)a (827±1)c (832±15)c

Ca (32.4±0.5)a (20.5±1.0)b (17.5±0.0)c (20.4±0.5)b (16.0±0.2)c

Fatty acids composition w/%

Lauric acid
(C12:0) (0.18±0,02)ab (0.18±0.01)ab (0.17±0.02)ab (0.16±0.01)b (0.19±0.01)a

Myristic acid
(C14:0) (0.79±0.01)a (0.77±0.01)bc (0.75±0.01)cd (0.74±0.01)d (0.77±0.01)b

Palmitic acid
(C16:0) (36.8±0.1)a (35.6±0.0)c (34.9±0.0)e (36.0±0.0)b (35.5±0.0)d

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Parameters Control 20VFC 35VFC 20CFC 35CFC

Palmitoleic acid
(C16:1) (0.18±0.01)a (0.17±0.02)a (0.17±0.01)a (0.16±0.01)a (0.14±0.01)b

Stearic acid
(C18:0) (4.21±0.02)c (4.55±0.03)b (4.72±0.02)a (4.56±0.01)b (4.74±0.02)a

Oleic acid
(C18:1n9c) (38.3±0.1)c (38.6±0.1)ab (38.7±0.1)a (38.5±0.1)abc (38.4±0.1)bc

Linoleic acid
(C18:2n6c) (18.0±0.1)d (18.6±0.1)b (18.9±0.1)a (18.5±0.0)c (18.9±0.1)a

Arachidic acid
(C20:0) (0.44±0.01)b (0.44±0.01)b (0.52±0.02)a (0.46±0.01)b (0.46±0.01)b

α-Linolenic acid
(C18:3n3) (0.77±0.03)a (0.74±0.01)a (0.74±0.01)a (0.68±0.02)b (0.67±0.01)b

Eicosanoic acid
(C20:1n9) (0.26±0.01)bc (0.33±0.01)a (0.26±0.01)b (0.24±0.02)c (0.25±0.00)bc

Total SFA 42.5 41.6 41.1 41.9 41.7

Total MUFA 38.8 39.1 39.2 38.9 38.8

Total PUFA 18.8 19.4 19.7 19.1 19.5

PUFA/SFA 0.44 0.47 0.48 0.46 0.47

Control=chickpea crackers, 20VFC and 35VFC=crackers in which 20 % or 35 % of chickpea flour was replaced with
seed press cake flour remaining after Virgin pumpkin oil extraction, 20CFC and 35CFC=cookies in which chickpea flour
was replaced with seed press cake flour remaining after cold pressed oil extraction, SFA=saturated fatty acids,
MUFA=monounsaturated fatty acids, PUFA=polyunsaturated fatty acids. Values are presented as mean (n=3) ±
standard deviations. Means in the same row with different superscript are statistically different (p ≤ 0.05) according to
Fisher (LSD) test

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Please note that this is an unedited version of the manuscript that has been accepted for publication. This
version will undergo copyediting and typesetting before its final form for publication. We are providing this
version as a service to our readers. The published version will differ from this one as a result of linguistic
and technical corrections and layout editing.

Table 3. Total phenolic content, antioxidant activity, hydrolysis index and predicted glycemic
index of crackers

Parameters Control 20VFC 35VFC 20CFC 35CFC

Total antioxidant
activity
(c(Trolox)/m(sa (7.96±0.35)d (12.0±0.2)b (11.9±0.2)b (11.5±0.3)b (17.0±0.4)a
mple))/(mM/g)

Total phenolic
compounds
(0.72±0.00)d (0.84±0.00)bc (0.82±0.03)c (0.88±0.01)b (1.06±0.00)a
w(galic
acid)/(mg/g)

Hydrolysis index (51.2±0.1)a (38.8±0.3)d (36.8±0.1)e (40.1±0.2)b (39.5±0.4)c

Predicted
(67.8±0.1)a (61.0±0.6)b (59.9±0.1)c (61.7±0.5)b (61.4±0.3)b
glycemic index
Control=chickpea crackers, 20VFC and 35VFC=crackers in which 20 % or 35 % of chickpea flour was
replaced with seed press cake flour remaining after Virgin pumpkin oil extraction, 20CFC and
35CFC=cookies in which chickpea flour was replaced with seed press cake flour remaining after cold
pressed oil extraction. Values are presented as mean (n=3) ± standard deviations. Means in the same row
with different superscript are statistically different (p ≤ 0.05) according to Fisher (LSD) test

24
Food Technology and Biotechnology 60 (4) 2022 www.ftb.com.hr

Please note that this is an unedited version of the manuscript that has been accepted for publication. This
version will undergo copyediting and typesetting before its final form for publication. We are providing this
version as a service to our readers. The published version will differ from this one as a result of linguistic
and technical corrections and layout editing.

Table 4. Physical properties of crackers

Parameters Control 20VFC 35VFC 20CFC 35CFC

Mass/g (4.85±0.05)b (4.89±0.06)ab (4.98±0.08)a (4.98±0.10)a (4.95±0.08)ab

Eccentricity/% (1.00±0.01)a (0.99±0.00)a (0.99±0.01)a (1.00±0.01)a (0.99±0.00)a

Spread factor/% (9.92±0.23)a (10.1±0.2)a (10.1±0.1)a (9.94±0.10)a (9.98±0.06)a

Puffiness/% (28.9±3.4)a (25.2±4.5)ab (23.4±3.4)ab (21.6±8.0)b (23.8±3.8)ab

Colour properties

L* (69.0±0.3)a (50.5±0.7)d (46.2±0.5)e (57.1±0.7)b (51.4±0.9)c

a* (5.18±0.15)a (4.18±0.21)b (4.38±0.15)b (1.90±0.23)c (1.18±0.25)c

b* (34.6±0.3)a (19.1±0.7)c (15.1±0.6)d (23.9±1.0)b (18.7±0.8)c

ΔE (24.2±1.0)b (30.0±0.8)a (16.4±1.2)c (23.9±1.2)b

Textural properties

Hardness/kg (2.79±0.67)ab (3.30±0.51)ab (3.46±0.44)a (2.65±0.51)b (2.96±0.48)ab

Fracturability/mm (2.15±0.31)a (1.04±0.10)b (0.95±0.09)b (2.22±0.22)a (2.02±0.25)a

Control=chickpea crackers, 20VFC and 35VFC=crackers in which 20 % or 35 % of chickpea flour was replaced with
seed press cake flour remaining after Virgin pumpkin oil extraction, 20CFC and 35CFC=cookies in which chickpea flour
was replaced with seed press cake flour remaining after cold pressed oil extraction, L*=lightness/darkness,
a*=redness/greenness, b*=yellowness/blueness, ΔE=total colour difference. Values are presented as mean (n=3) ±
standard deviations. Means in the same row with different superscript are statistically different (p ≤ 0.05) according to
Fisher (LSD) test

25
Food Technology and Biotechnology 60 (4) 2022 www.ftb.com.hr

Please note that this is an unedited version of the manuscript that has been accepted for publication. This
version will undergo copyediting and typesetting before its final form for publication. We are providing this
version as a service to our readers. The published version will differ from this one as a result of linguistic
and technical corrections and layout editing.

Fig. 1. Glucose concentration as a function of time during the second intestinal phase, relevant to
examined crackers

Fig. 2. Sensory evaluation scores for overall liking, liking of colour, taste, texture and flavour of
gluten-free crackers based on chickpea (Control), chickpea and virgin pumpkin seed press cake

26
Food Technology and Biotechnology 60 (4) 2022 www.ftb.com.hr

Please note that this is an unedited version of the manuscript that has been accepted for publication. This
version will undergo copyediting and typesetting before its final form for publication. We are providing this
version as a service to our readers. The published version will differ from this one as a result of linguistic
and technical corrections and layout editing.

flour blend (20VFC and 35VFC) and chickpea and cold pressed pumpkin seed press cake flour
blend (20CFC and 35CFC)

Fig. S1. Appearance of the gluten-free crackers based on chickpea (Control), chickpea and virgin
pumpkin seed press cake flour blend (20VFC and 35VFC) and chickpea and cold pressed
pumpkin seed press cake flour blend (20CFC and 35CFC)

27