Annals of Agricultural Sciences xxx (xxxx) xxx–xxx

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Impact of chickpea as prebiotic, antioxidant and thickener agent of stirred

bio-yoghurt
⁎
Hend Husseina, Sameh Awada, , Ibrahim El-Sayedb, Amel Ibrahima
a
Dairy Microorganisms and Cheese Research Laboratory (DMCR), Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University, Egypt
b
Department of Dairy Science and Technology, Faculty of Agriculture, Alexandria University, Egypt

A R T I C LE I N FO A B S T R A C T

Keywords: The present study aimed to investigate the effect of chickpea as a prebiotic, antioxidant and thickener agent on
Prebiotics the probiotic viability and physicochemical properties of stirred bio-yoghurt. Stirred bio-yoghurts were made
Probiotic using probiotic cultures (Bifidobacterium bifidum and a mixture of Bifidobacterium animalis subsp. lactis and
Stirred bio-yoghurt Lactobacillus acidophilus) with chickpea flour at different concentrations (1, 2 and 3%). Physicochemical, anti-
Chickpea
oxidant, rheological, microbiological and sensorial properties of stirred bio-yoghurt were evaluated during
Antioxidant activity
storage. The obtained results showed that the chickpea flour stimulated the growth of probiotic bacteria. The
bacterial counts in bio-yoghurt made using Bifidobacterium bifidum were log 8.28, 8.12, 8.04 and 7.32 CFUg−1
with 3, 2, 1% chickpea flour and control respectively. Stirred bio-yoghurt supplemented with chickpea flour
received high values of antioxidant capacity (91, 83 and 76.6%) at concentrations of 3%, 2% and 1% chickpea
flour respectively, while control received low value (31.12%). The viscosity of stirred bio-yoghurt was enhanced
by chickpea supplementation. The sensory evaluation revealed that samples with 1 and 2% chickpea flour were
preferred by panelists in texture and flavour properties. The obtained results concluded that the chickpea flour
could replace the skimmed milk powder at concentrations of 1 or 2% to enhance the quality of stirred bio-
yoghurt and reduce the production cost.

1. Introduction effects, cholesterol reduction and immune system stimulation (Shah

Recently, consumers attach great importance to the nutritional and
healthy properties of food. They demand food not only for eating but
also for preventing nutrition-related diseases. Functional food is one of
the foods that introduce the best choice for these consumer demands. It
contains potentially beneficial components found naturally in foods or
added to them as functional ingredients and have a major role in health-
enhancing (Granato et al., 2010). Functional ingredients have physio-
logical functional such as antioxidant activity, cholesterol reduction,
immunomodulation and lowering of blood pressure (Hasani et al.,
2017). Examples of functional foods include dairy products supple-
mented with probiotics, prebiotics and enriched with vitamins or con-
taining omega-3 fatty acid, antioxidants and phytochemicals (Ismail
et al., 2017). One way in which dairy products are modified to become
functional is by incorporating probiotics (Kumar et al. (2015).
Fermented milk considers as a functional dairy product due to its
therapeutic, nutritional, and probiotic effects. Also, fermented milk has
some physiological benefits such as antimicrobial activity, anticancer
and Champagne, 2016). Yoghurt is a popular type of fermented dairy
products manufactured from the fermentation of milk by yoghurt
starter culture (Christopher et al., 2010). There is a trend towards in-
creasing the health benefits of yoghurt by adding probiotics to it and
the resulting product is called bio-yoghurt (Santivarangkna, 2016). Bio-
yoghurt is produced using a yoghurt starter culture along with other
probiotics strains such as “lactobacilli and bifidobacteria” (Kee et al.,
2012). Prebiotic was added during bio-yoghurt manufacturing for
conferring the advantageous health benefits to the host (Roy et al.,
2015).
Several studies have focused on consumption of dairy products
fortified with prebiotics such as lentil flour, soy flour, rice and partially
hydrolysed guar gum to produce functional yoghurt (Kumari et al.,
2015; Mudgil et al., 2016; Zare et al., 2012).
Chickpeas (Cicer arietinum L.), like other legumes, are an excellent
source of carbohydrates, protein, dietary fibre, vitamins, and minerals.
Compared with the other pulses, chickpea provides a higher value of
total carbohydrate (Jukanti et al., 2012). Chickpea is considered as

⁎
Corresponding author at: Dairy Microorganisms and Cheese Research Laboratory (DMCR), Department of Dairy Science & Technology, Faculty of Agriculture (El-
Shatby), Alexandria University, 21545, Egypt.
E-mail address: sameh.awad@alexu.edu.eg (S. Awad).

https://doi.org/10.1016/j.aoas.2020.03.001
Received 26 October 2019; Received in revised form 29 February 2020; Accepted 2 March 2020
0570-1783/ 2020 Production and hosting by Elsevier B.V. on behalf of Faculty of Agriculture, Ain Shams University. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Hend Hussein, et al., Annals of Agricultural Sciences, https://doi.org/10.1016/j.aoas.2020.03.001
H. Hussein, et al.
healthy and nutrient-dense food, providing rich content of protein
especially in essential amino acids, fibres, vitamins and minerals
(Capurso et al., 2018; Jukanti et al., 2012; Milán-Carrillo et al., 2007).
Chickpea is rich in raffinose-family oligosaccharides (RFO), resistant
starches and fibres (Jukanti et al., 2012).
The functional properties of chickpea flour include its water reten-
tion capacity, emulsifying capacity, ability to form gels, and ability to
create a foam (Aguilar-Raymundo and Velez-Ruiz, 2013). Due to the
high nutritional and healthy benefits of Chickpea and a little informa-
tion about using of Chickpea in functional dairy products, this study
aimed to investigate the supplementation of stirred bio-yoghurt with
Chickpea flour as prebiotic, antioxidant and thickener agent.
2. Materials and methods
2.1. Materials
Raw cow's milk was obtained from the farm of Faculty of
Agriculture, Alexandria University. Its composition was 3% fat content,
3.1% total protein, 12.25% total solid (T.S) and total acidity % as lactic
acid was 0.172%. Skimmed milk powder (low heat) was obtained from
Arla company, United Kingdom. Whole chickpea (Garbanzo beans) was
obtained from the local market (Alex City, Egypt) and ground to a
particle size of 1.5 mm using mash.
2.1.1. Chemical and reagents
Methanol, chloroform, Ethanol, Amylalchol and sulfuric acid were
obtained from (Loba chemi, Indian); Lithium chloride was from (Merck,
German). Folin–Ciocalteu phenol reagent and 1,1-Diphenyl-2-pi-
crylhydrazyl (DPPH) were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Gas pack, Violet red bile lactose agar and Cysteine hydro-
chloride monohydrate were obtained from (Oxoid; England). Potato
dextrose agar was purchase from (Difco, Italy). De Man Rogosa and
Sharpe (MRS) was purchase from (LAB, England).
Yoghurt starter culture, LYOfast (Y 259 A) C 119368A, Freeze-
dried lactic cultures ferments lyophilizes was obtained from SACCO,
Italy. Probiotic culture (Bifidobacterium animalis subsp. lactis and
Lactobacillus acidophilus) was obtained from (SACOO Lyofast (AB1);
Italy. Also, Bifidobacterium bifidum (DSM 20082) was used as probiotic.
2.2. Methods
2.2.1. Chemical analysis of chickpea flour
Total solids and total nitrogen were determined according to AOAC
(2005). Flour fat was determined according to Folch et al. (1957). After
fat extraction, the fatty acid profile was carried out using ACME model
6100GC (Young LIN Instrument Co, Korea) fitted with a split injection
and FID detector. Nitrogen was used as carrier gas with a flow rate of
0.5 ml/min. The components were separated on 30 m SP-2380 fused-
silica capillary column with 0.25 mm and 0.2 μm film thickness (Su-
pelco, Bellefonte, PA) and the detector temperature was set at 260 °C.
The injector temperature was set at 220°C and in split mode (split ratio
1:80). The column was initially maintained at 140 °C for 5 min, and the
temperature was subsequently increased to 240 °C at the rat of 4°C /
min. The crude fibre was determined according to Joslyn (1970).
2.2.2. Determination of bioactive phytochemicals in chickpea
2.2.2.1. Preparation of chickpea extract. The chickpea flour was
expressed on a dry weight basis. The fine flour was extracted with
65% ethanol (1:20, w/v) at 80 °C for three hours (Mikulajová et al.,
2007). The extract was filtered for DPPH test and total phenolic content
determination.
2.2.2.2. Determination of total phenolic content in chickpea flour. Total
phenol contents were determined using a method developed by
(Singleton and Rossi, 1965). One and a half millilitres of
2
Annals of Agricultural Sciences xxx (xxxx) xxx–xxx
Folin–Ciocalteu's reagent (diluted 10 times) and 1.2 ml of Na2CO3
(7.5% w/v) solution were added to 300 μl of chickpea extract. Mixtures
were shaken and left to stand at room temperature for 30 min before
measuring absorbance at 765 nm using a spectrophotometer (Pg T80+,
England). The determination was done in triplicate. The total phenol
content (TPC) was expressed as gallic acid equivalent in mg/100 g of
sample.
2.2.2.3. Estimation of DPPH radical scavenging activity (1, 1-diphenyl-2
picrylhdydrazyl free radical scavenging activity). The scavenging activity
of the stable (DPPH) radical was determined according to a procedure
based on Miliauskas et al. (2004) and its modifications by Lim and
Quah (2007). Two millilitres of 0.15 mM DPPH was added to 1 ml of
extracts in different dilutions. A control was prepared by adding 2 ml of
DPPH to 1 ml of methanol. The contents of the tubes were mixed and
allowed to stand for 30 min, and absorbance was measured at 517 nm
using a spectrophotometer (Pg T80+, England). Triplicate tubes were
prepared for chickpea extract. The results were expressed as % radical
scavenging activity.
( A control − A sample )
Radical scavenging activity% = × 100
Acontrol
2.2.3. Stirred bio-yoghurt manufacture
2.2.3.1. Preparation of probiotic cultures. Probiotic culture (AB1) and
(DSM 20082) were inoculated in MRS media with 0.1 g Land 1% (v/v)
respectively then incubated at 37 °C for 48 h. The probiotics were
activated after three transfers for the preparation of cultures for yoghurt
making. The cultures were prepared by inoculating 100 mL of
reconstituted skim milk (RSM) supplemented with 1% (w/v) yeast
extract and 1 ml Cysteine hydrochloride monohydrate and then
incubated at 37 °C for 18 h, to obtain 2 × 108 CFU ml−1 (Crittenden
et al., 2005).
2.2.4. Stirred bio-yoghurt manufacture
Stirred bio-yoghurt was made using cow's milk by adding 8% sugar
and 3% skimmed milk powder (SMP). The adding skimmed milk
powder was replaced by chickpea flour (CPF) at 1% (1% chickpea flour
and 2% SMP), 2% (2% chickpea flour and 1% SMP), 3% (3% chickpea
flour) individually. The milk, after good mixing all the ingredients, was
heated separately at 90 °C for 5 min then cooled to 42 °C. Probiotic
starter culture was inoculated at 42 °C with level 8-log10 CFU ml-1 for
an hour before inoculation with the yoghurt culture (0.3 g L−1) and
fermented until pH 4.7. Then the bio-yoghurt was stirred and packaged
(Tamime and Robinson, 2007).
2.2.5. Physiochemical analysis of stirred bio-yoghurt
The pH was measured using a digital pH meter (MilwaukeeMi 150,
Italy). Total acidity, total solids, total nitrogen (TN) and fat contents
were determined in bio-stirred yoghurt according to the method of
AOAC (2005). The protein content was obtained by multiplying the
percentage of TN by 6.38.
2.2.6. Bioactive phytochemicals in stirred bio-yoghurt
2.2.6.1. Water extract of stirred bio-yoghurt. Water extraction of yoghurt
was carried out as described by (Shori and Baba, 2013). Bio-yoghurts
samples (10 g) were homogenized for 10 s with sterile distilled water
(2.5 ml) and adjusted the bio-yoghurt pH to 4.0 using HCl (0.1 M). The
volume was makeup to 50 ml by distilled water. Acidified yoghurts
were then incubated for 10 min at 45 °C followed by centrifugation
(5000 ×g, 4 °C/10 min). The pH of the resulting supernatant was then
adjusted to 7.0 using NaOH (0.1 M) followed by centrifugation
(5000 ×g, 4 °C/10 min). The clear supernatant obtained was stored
at −20 °C and used for analysis within 1–2 weeks of preparation.
H. Hussein, et al. Annals of Agricultural Sciences xxx (xxxx) xxx–xxx

2.2.7. Determination of total phenolic content in stirred bio-yoghurt Table 1

Total phenol contents were determined using a method developed Chemical composition of chickpea flour.
by Singleton and Rossi (1965) in bio-stirred yoghurt during cold storage Parameter Chemical composition of chickpea flour
for 21 days.
Moisture % 7.53
Protein% (TN*5.8) 20.1
2.2.8. Estimation of DPPH radical scavenging activity
Fat content% 5.7
The scavenging activity of the stable (DPPH) radical was de- Fatty acid %
termined according to a procedure of Miliauskas et al. (2004) and its Myristic acid 0.55
modifications by Lim and Quah (2007) in bio stirred yoghurt during Palmitic acid 22.45
cold storage for 21 days. Stearic acid 2.13
Behenic acid 0.35
Total saturated 25.48 ± 0.4
2.2.9. Ferric-reducing antioxidant power (FRAP) Linoleic acid 53.94
FRAP was done as described by Oyaizu (1986). This method based Oleic acid 15.5
on the presence of antioxidants in the sample to reduce the ferricyanide α –linolenic acid 4.57
Poullinic acid 0.35
complex to the ferrous form. One millilitre of extracts in different di-
Total unsaturated 74.36 ± 2.8
lutions was added to 2.5 ml phosphate buffer (0.1 M, pH 6.6) and Crude fibre% 3.8
2.5 ml potassium ferricyanide (1% w/v). The mixture was then in- Total solid% 92.47
cubated in a water bath at 50 °C for 20min, followed by 2.5 ml tri- TPC (mgGAE/g) 3.44
chloroacetic acid (10% w/v) solution. The contents of the tubes were DPPH inhibition activity % 80.64

mixed well, and 2.5 ml of solution was removed from each tube. To this,
2.5 ml solution, 2.5 ml water and 0.5 ml ferric chloride solution (0.1%
differences, the least significant difference (LSD) was calculated ac-
w/v) were added. The mixtures were allowed to stand for 30 min before
cording to Duncan (1995) for the comparison between means. The data
absorbance measurements were taken at 700 nm. Triplicate tubes were
presented, in the Tables, are the mean ( ± standard deviation) of three
prepared for each extract. The FRAP values, expressed in mg GAE/g,
experiments.
were derived from a standard curve.

2.3. Microbiology analysis of stirred bio-yoghurt 3. Results and discussion

Appropriate serial dilutions of bio-yoghurt samples were prepared
for microbial enumeration, by using 2% sodium citrate. The enumera-
tion of yeasts and moulds was carried out as recommended by (Okoye
and Animalu, 2009), using potato dextrose agar (Difico, Italy) acidified
with 10% tartaric acid and incubated at 25 °C for 5 days. Violet red bile
lactose agar (Oxide, England) was used to coliform count according to
the Marth, 1978. The plates were incubated at 37 °C for 24h.
The enumeration of probiotic starters Bifidobacterium bifidium (DSM
20082) and (Bifidobacterium animalis subsp. lactis and Lactobacillus
acidophilus) Lyofast AB1 was done as described in Technical (2005)
using MRS agar (LABM, England) supplemented with 0.05 g L−1 Cy-
steine hydrochloride monohydrate (Oxide, England) and one g L−1
Lithium chloride (Merck). The plates were incubated at 37 °C for 72 h
under anaerobic conditions using a gas pack (Oxide, England).
2.4. Viscosity
Stirred bio-yoghurt viscosity was measured during cold storage for
21 days using a digital rotary viscometer (Model NDJe9S, U.S.A) with a
spindle No. 4at 12 rpm rotation speed for 50 s. The samples were
measured at 10°C. The test was replicated three times.
2.5. Sensory evaluation
The sensorial properties of stirred bio-yoghurt samples (appearance,
aroma, texture, flavour/taste and overall preference) were assessed by a
consumer-oriented panel of 20 panelists using a 9-point hedonic scale.
Panelists are comprising of staff members and assistants at Agriculture
Faculty, Alexandria University during storage for 21 days (Clark et al.,
2009).
2.6. Statistical analysis
All statistical analyses for the result of microbial and physico-
chemical descriptive were performed using SAS version 9.1 (SAS
Institute Inc.), analysis of variance with three factorials (treatments and
storage period) were conducted by the procedure of General Linear
Model. The significance levels of p < .05 were used for statistical
3.1. Physicochemical properties of chickpea flour and stirred bio-yoghurt
supplemented with chickpea flour
The data in Table 1 clearly showed the chemical composition of
chickpea. Chickpea flour contain 20.1% protein, 5.7% fat, 3.8% crude
fibre. High protein content in chickpea flour (20.5%) was also reported
by Wallace et al., 2016. The fatty acids of chickpea flour counted of
25.48% saturated fatty acids and 74.36% unsaturated fatty acids, the
dominant saturated fatty acid is palmitic acid and the dominant un-
saturated fatty acid is Linoleic acid.
Data in Table 2 showed the effect of different concentrations 1%,
2% and 3% of chickpea flour on the physicochemical properties of
stirred bio-yoghurt. The pH values in control and all treatments with
chickpea flour were in the range of 4.63 to 3.91. The lowest pH value
was found in the sample made with 3% chickpea flour at the 21-days
using probiotic culture AB1. The pH value of control samples was sig-
nificantly higher when compared with treatments made with chickpea
flour. There was a decline in pH value during storage up to 21-days at
4 °C in all treatments. The overall titratable acidity (TA) of all samples
generally increased during storage up to 21-days. The TA of the bio-
yoghurt was significantly higher in bio-yoghurts containing chickpea
flour than control bio-yoghurt, and the highest (TA) was in bio-yoghurt
supplemented with 3% chickpea flour. The acidity of yoghurt is in-
creased due to the fermentation of lactose by lactic acid bacteria in
yoghurt during the storage time. Although the pH of yoghurt should be
4.7 or lower FDA (2015), the pH of the final product mainly depends on
the preferences of customers. Customers may accept a yoghurt with a
pH of 4.2, even yoghurt with the pH of 3.7–3.8, such as Greek-style
yoghurt that contains a high total solid and fat (Robinson and
Itsaranuwat, 2008). According to Zare et al. (2012), the acidity value of
yoghurt with probiotic bacteria increased with adding some pulse in-
gredients, such as chickpea flour and pea protein.
The result showed that the control had lower fat (3.14%) comparing
with that in chickpea yoghurt 3%, 2% and 1% (3.3%, 3.26% and 3.2%,
respectively). This is due to the high-fat content in chickpeas flour
(5.7%) when compared with the fat in skimmed milk powder (SMP) as
shown in Table 1. Despite the high-fat content in chickpeas, the highest
percentage of fatty acids is unsaturated fatty acids such as Linoleic acid

3
H. Hussein, et al. Annals of Agricultural Sciences xxx (xxxx) xxx–xxx

Table 2
Physicochemical properties of stirred bio-yoghurt supplemented with chickpea flour.
Samples Storage period (days) pH Titratable acidity% Fat content % Protein% Total Solid %

d
Control 1 1 4.63 0.74 ± 0.002 3.12 ± 0.017 4.36 ± 0.07 22.06 ± 0.01c
7 4.4 0.903 ± 0.002c 3.12 ± 0.017 ND 22.17 ± 0.089 b
14 4.29 0.945 ± 0.002b 3.15 ± 0.028 ND 22.21 ± 0.021b
21 4.22 1.02 ± 0.003a 3.17 ± 0.017 4.4 ± 0.06 22.37 ± 0.023 a
Mean 3.137 ± 0.01D 4.38 ± 0.042A 221. 7 ± 0.029A
Control 2 1 4.57 0.774 ± 0.004d 3.1 ± 0.00 4.33 ± 0.03 21.91 ± 0.023c
7 4.37 0.888 ± 0.00c 3.1 ± 0.00 ND 22.11 ± 0.044b
14 4.25 0.948 ± 0.002b 3.17 ± 0.01 ND 22.019 ± 0.023a
21 4.18 1.071 ± 0.001a 3.2 ± 0.00 4.37 ± 0.035 22.22 ± 0.027a
Mean 3.14 ± 0.013D 4.35 ± 0.021A 22.11 ± 0.04A
1 1 4.51 0.825 ± 0.001d 3.17 ± 0.017 4.27 ± 0.02 22.12 ± 0.06 b
7 4.27 0.945 ± 0.009c 3.23 ± 0.017 ND 22.21 ± 0.035 a
14 4.18 1.038 ± 0.001b 3.22 ± 0.017 ND 22.24 ± 0.041a
21 3.99 1.158 ± 0.007a 3.23 ± 0.017 4.31 ± 0.01 22.27 ± 0.02 a
Mean 3.2 ± 0.01C 4.28 ± 0.01B 22.221 ± 0.025A
2 1 4.47 0.885 ± 0.004d 3.22 ± 0.017 4.20 ± 0.02 22.15 ± 0.076 c
7 4.2 1.029 ± 0.003c 3.23 ± 0.016 ND 22.22 ± 0.023 bc
14 4.09 1.101 ± 0.007b 3.25 ± 0.00 ND 22.25 ± 0.025 ab
21 3.97 1.17 ± 0.001a 3.28 ± 0.016 4.23 ± 0.018 22.30 ± 0.015 a
Mean 3.24 ± 0.01B 4.21 ± 0.015B 22.23 ± 0.024A
3 1 4.45 0.888 ± 0.003d 3.25 ± 0.00 4.00 ± 0.024 22.187 ± 0.023c
7 4.16 1.083 ± 0.003c 3.2 ± 0.017 ND 22.24 ± 0.02 bc
14 4.06 1.119 ± 0.001b 3.28 ± 0.017 ND 22.28 ± 0.036 ab
21 3.94 1.206 ± 0.003a 3.33 ± 0.017 4.05 ± 0.023 22.31 ± 0.023 a
Mean 3.28 ± 0.011A 4.0 ± 0.016C 22.25 ± 0.18A
4 1 4.47 0.885 ± 0.0032d 3.15 ± 0.00 4.24 ± 0.02 21.99 ± 0.035c
7 4.25 0.948 ± 0.00c 3.17 ± 0.017 ND 22.14 ± 0.024b
14 4.15 1.089 ± 0.001b 3.2 ± 0.00 ND 22.21 ± 0.014 ab
21 3.94 1.185 ± 0.002a 3.25 ± 0.00 4.27 ± 0.01 22.27 ± 0.015a
Mean 3.2 ± 0.12C 4.26 ± 0.01B 22.15 ± 0.033A
5 1 4.43 0.9 ± 0.001d 3.25 ± 0.00 4.18 ± 0.01 22.07 ± 0.036 c
7 4.18 1.071 ± 0.001c 3.25 ± 0.00 ND 22.19 ± 0.023 b
14 4.08 1.134 ± 0.001b 3.27 ± 0.17 ND 22.27 ± 0.018 a
21 3.95 1.188 ± 0.001a 3.28 ± 0.017 4.21 ± 0.012 22.31 ± 0.023 a
Mean 3.26 ± 0.01B 4.2 ± 0.01B 22.21 ± 0.031A
6 1 4.41 0.903 ± 0.001d 3.27 ± 0.00 3.98 ± 0.026 22.1 ± 0.035 c
7 4.14 1.095 ± 0.001c 3.28 ± 0.17 ND 22.17 ± 0.037 b
14 4.04 1.143 ± 0.002b 3.32 ± 0.17 ND 22.27 ± 0.021 a
21 3.91 1.221 ± 0.001a 3.35 ± 0.00 4.02 ± 0.031 22.33 ± 0.014 a
Mean 3.3 ± 0.011A 4.0 ± 0.019C 22.22 ± 0.03A

Means (n = 3 ± SD) with the same letters are not significantly different.
Control1: stirred bio-yoghurt (3%S.M.P, probiotic culture B. bifidum); Control 2: stirred bio-yoghurt (3%S.M.P, probiotic culture B.animalis sub sp. lactis and
L.acidophilus)SACCO lyofast AB1. Sample 1: stirred bio-yoghurt supplemented with 1% chickpea flour,2% S.M.P) by using probiotic culture B. bifidum; Sample 2:
stirred bio-yoghurt supplemented with 2% chickpea flour,1% S.M.P) by using probiotic culture B. bifidum; Sample 3: stirred bio-yoghurt supplemented with 3%
chickpea flour) by using probiotic culture B. bifidum; Sample 4: stirred bio –yoghurt supplemented with 1% chickpea flour,2% S.M.P) by using probiotic culture
AB1;Sample 5: stirred bio-yoghurt supplemented with 2% chickpea flour,1% S.M.P) by using probiotic culture AB1;
Sample 6: stirred bio-yoghurt supplemented with 3% chickpea flour) by using probiotic culture AB1.

53.49%. The fat content in all treatments, including control was not
significantly changed during storage for up to 21 days. These results are
in agreement with that reported by Salem and Massoud (2003).
Protein content was at the level of 4.35%, 4.00%, 4.2% and 4.26%
in control, 3% chickpea flour, 2% chickpea flour and 1% chickpea flour,
respectively. The low protein level in treatments made with chickpea
flour when compared with control is due to the lower protein content of
chickpea flour (20.1%) than that in skimmed milk powder (34%) as
shown in Table 1. There was no significant difference in total solids
among all treatments, this shows that the replacement of skimmed milk
powder (SMP) with chickpea flour has no effect on the total solids of
stirred bio-yoghurt.
These results show a slightly increasing of total solids in all treat-
ments during storage period at 4 °C. This could be related to slow loss of
moisture during storage by evaporation, a similar observation was re-
ported by (Farag et al., 2007; Roushdy and El-Saadaney, 2007).
The results showed that no significant difference on physicochem-
ical properties of stirred bio-yoghurt supplemented with chickpea flour
when made using probiotic cultures mix of (B.animalis subsp. lactis and
L.acidophilus) or single culture of (B.bifidum).
3.2. Microbiological analysis of stirred bio-yoghurt supplemented with
different dosages of Chickpea flour
The health benefit of probiotic food products depends on the
number of active cells at the consumption Korbekandi et al. (2011).
Therefore, it is essential to have a high survival rate of the probiotic
cultures over the shelf life of the final products Cruz et al. (2010).
The results of Fig. 1 (A and B) showed the survival of probiotic
cultures (B.bifidum) and (B.animalis subsp. lactis and L.acidophilus) cul-
ture in stirred bio-yoghurt supplemented with chickpea flour during
storage period for 21 days at 4 °C. The viable probiotic culture was
exceeded recommended level (1 million cells/g) as all treatments and
control have viable probiotic culture above (106 CFU g−1) during sto-
rage period up to 21 days. The count of probiotics in stirred bio-yoghurt
with 3% chickpea (log 8.63 CFU g−1) was the highest at 1 day of sto-
rage, whereas the count was at lowest rate in control stirred bio-yoghurt
(8.3 CFU g−1). There was a decline in the count of probiotics during the
storage period, but bio-yoghurt supplemented with chickpea still had a
positive effect on survival rate when compared with control.
The viable count of probiotic culture B.bifidum has the same trend,
while the viable count of probiotic cultures (B.animalis subsp. lactis and

4
H. Hussein, et al.
Fig. 1. Count of probiotic culture (a) B. bifidum, (b) B.animalis ssp. lactis and L.acidophilus in stirred bio-yoghurt supplemented with chickpea flour.
A: using (B. bifidum) as probiotic culture
B: using AB1 (B.animalis ssp. lactis and L.acidophilus) as probiotic culture.
L. acidophilus) was significantly higher compared with the viable count
of probiotic culture (B.bifidum) with same treatments. Based on the
previous result, there was a positive relation between chickpea con-
centration and survival of probiotic bacteria (B.animalis subsp. lactis and
L.acidophilus) in stirred bio-yoghurt during storage. Chickpea is a rich in
total oligosaccharides, about 144.9 mg/g, among pulses which mainly
composed of raffinose, ciceritol, and staehyose (Han and Baik, 2006).
Raffinose and stachyose that present in chickpeas are considered a good
source of prebiotics. The addition of raffinose family oligosaccharides
had a positive influence on the survival of Bafidobacterium lactis Bb-12
and L. acidophilius La-5(Martinez-Villaluenga et al., 2006). Hernandez-
Hernandez et al. (2012) suggested that galacto oligosaccharides were
an excellent supplement for stimulating the growth and improved the
survival of probiotic Lactobacillus strains.
Moreover, chickpea flour increased the viability of probiotic bac-
teria during the storage period. The control sample, without chickpea
flour, has the lowest viable counts of probiotic culture (B.animalis
subsp. lactis and L. acidophilus) during the 21 days of storage. The
highest survival of probiotic bacteria (B.animalis subsp. lactis and
5
Annals of Agricultural Sciences xxx (xxxx) xxx–xxx
L.acidophilus) was observed in the bio-yoghurts containing 3% chickpea
flour on the first day (P < .05). Although the viable count of probiotic
culture (B.animalis subsp. lactis and L.acidophilus)and (B.bifidum) de-
creased gradually during the storage period, yoghurt samples held
probiotic values of log 7 CFUg−1 at day 21. In contrast, the viable count
of the control sample (probiotic culture: B.bifidum) was at the level of
log 6 CFUg−1at 21 day. According to the definition based on FDA
(2015), the minimum level of live and active culture should be 7 log
CFU ml−1 after production and ideally contained an expectation 6 log
CFU ml−1 through the shelf life Hill et al. (2014). As a result, chickpea
flour at a concentration of 3% had the strongest stimulatory effect on
the viable counts of probiotic culture (B.animalis subsp. lactis and
L.acidophilus) and (B.bifidum). Natural compounds in chickpea could act
as a supplementary energy source or exert antioxidant effects. The
observed increase in viability of probiotic culture (B.animalis subsp.
lactis and L.acidophilus) and (B.bifdum) has been attributed to dietary
fibre oligosaccharids, some ion especially iron and phenolic compounds
as the main constituents of chickpea Segev et al. (2011). These results
agree with Agil et al. (2013), who used lentil a type of legumes as
H. Hussein, et al.
prebiotic to enhance survival of probiotic growth in yoghurt. Results
confirmed that the antioxidant capacity of lentil polysaccharides in
enhancing the growth of probiotic bacteria in yoghurt during manu-
facturing as well as surviving the probiotic bacteria during storage
period over 28 days.
All samples of stirred bio-yoghurt were free of coliform, yeast and
mould after storage up to 21 days due to the good hygienic conditions
of manufacture.
3.3. Bioactive compounds of stirred bio-yoghurt supplemented with chickpea
flour
The result in Table 1 showed that chickpea has high total phenolic
content TPC (3.44 mg GAE/g) and antioxidant activity AA% (80.64%).
These results were in agreement with Han and Baik, 2008. So, the
presence of chickpea flour at different concentrations in stirred bio-
yoghurt influence the level of TPC and AA% in stirred bio-yoghurt
Fig. 2 (A, B and C). The TPC values were 5.75, 5.62, 5.5 and 4.1 mg
6
Annals of Agricultural Sciences xxx (xxxx) xxx–xxx
Fig. 2. Effect of chickpea on (a) total phenolic, (b)
DPPH inhibition (%), and (C) FRAP value, of stirred
bio-yoghurt during 21 day of storage at 4 °C.
A: TPC (mgGAE/g) of stirred bio-yoghurt supple-
mented with chickpea flour during storage period.
B: DPPH Inhibition activity % of stirred bio-yoghurt
supplemented with chickpea flour during storage
period.
C: FRAP (mgGAE/g) of stirred bio-yoghurt supple-
mented with chickpea flour during storage period.
Control (2): stirred bio-yoghurt (without prebiotics,
probiotic culture B. animalis subsp. lactis and
L.acidophilus); Sample 4: stirred bio –yoghurt (1%
chickpea flour with probiotic culture culture B. ani-
malis subsp. lactis and L.acidophilus).; Sample 5:
stirred bio –yoghurt (2% chickpea flour with pro-
biotic culture B. animalis subsp. lactis and
L.acidophilus); Sample 6: stirred bio – yoghurt (3%
chickpea flour with probiotic culture B. animalis
subsp. lactis and L.acidophil.
GAE/g, in 3%, 2%, 1% and control sample, respectively at the 1st day of
manufacturing. Adding various levels of chickpea flour to bio-yoghurt
caused an increase in TPC of the stirred bio-yoghurts, the previous
study reported that the chickpea flour has a higher content of phenolic
compounds during fermentation process Fernandez-Orozco et al.
(2009).
A significant decline in TPC in all samples was noticed during the
21 days of storage at 4 °C compared to their initial values at 1st day of
manufacturing Fig. (2-A). Such a phenomenon was also observed by
(Trigueros et al., 2014; Varela-Santos et al., 2012). In fact, the gel
matrix of yoghurt might protect the phenolics during storage to some
extent; however, the declination is thought to be triggered because of
the degradation of phenolic compounds, mainly flavonoids, which is
influenced by the contact with oxygen (Mang et al., 2015). This result
was consistent with the results of a previous study showing that TPC
values of yoghurt supplemented with grape and callus extracts were
decreased when the storage period was longer (Karaaslan et al., 2011).
Although the TPC decreased during the 21 days of storage, a
H. Hussein, et al.
considerable amount remained in the fortified yoghurt with chickpea
flour. This could be attributed to flavonoids, which are the most stable
among all phenolics in yoghurt matrix (Trigueros et al., 2014).
Total antioxidant activity potential of stirred bio-yoghurt with
chickpea flour was determined by two methods, namely, DPPH radical
scavenging activity and Ferric Reducing Antioxidant Power (FRAP)
methods. Several tests were used because the antioxidant capacities of
samples might be influenced by several factors and could not be fully
described by one single method. As well as, most natural antioxidants
components are multifunctional. Therefore, different methods for an-
tioxidant activity assessments should be carried out to understand the
mechanisms of antioxidant action (Zhang et al., 2009).
The stable radical DPPH was used widely for the measurement of
primary antioxidant activity. DPPH radical-scavenging was used to
determine the antioxidant activity of yoghurt supplemented with
chickpea flour with different concentrations and control (stirred bio-
yoghurt) at different days of storage Fig. 2-B. Results show that the
presence of chickpea flour during stirred bio-yoghurt fermentation in-
creased (p < .05) the antioxidant capacity in all stirred bio-yoghurts
supplemented with chickpea flour at 1st day, and then there was a
reduction in antioxidant capacity during storage, but the samples made
with chickpea flour still have higher level of antioxidant capacity when
compared to control. The degradation of milk proteins to release phe-
nolic amino acids and non-phenolic compounds may influence the total
phenolic, where the phenolic content is the most factor affecting the
antioxidant activity. This result agreed with Shori (2013), who reported
7
Annals of Agricultural Sciences xxx (xxxx) xxx–xxx
Fig. 3. Viscosity of stirred bio-yoghurt
supplemented with chickpea flour.
A: Viscosity of stirred bio –yoghurt made by
probiotic culture B. bifidum (DSM 20082);
B: Viscosity of stirred bio –yoghurt made by
probiotic culture B. animalis subsp. lactis
and L.acidophilus (AB1)
Control (1): stirred bio-yoghurt (without
prebiotics, probiotic culture B. bifidum);
Control (2): stirred bio-yoghurt (without
prebiotics, probiotic culture B. animalis
subsp. lactis and L.acidophilus); sample1:
stirred bio –yoghurt (1% chickpea flour
with probiotic culture B. bifidum); Sample
2: stirred bio –yoghurt (2% chickpea flour
with probiotic culture B. bifidum). Sample
3: stirred bio – yoghurt (3% chickpea flour
with probiotic culture B. bifidum); Sample
4: stirred bio –yoghurt (1% chickpea flour
with probiotic culture culture B. animalis
subsp. lactis and L. acidophilus).; Sample 5:
stirred bio –yoghurt (2% chickpea flour
with probiotic culture B. animalis subsp.
lactis and L.acidophilus); Sample 6: stirred
bio – yoghurt (3% chickpea flour with pro-
biotic culture B. animalis subsp. lactis and L.
acidophilus.
that the soybean has antioxidant properties associated with isoflavones
and polyphenolic compounds (Slavin et al., 2009). Additionally, some
organic acids are produced during fermentation (Correia et al., 2004) as
a result of microbial metabolic activity could be other sources of anti-
oxidant activities. In the current study, there was an increasing in TPC
and antioxidant activity of yoghurt supplemented with chickpea, this
due to the fermentation improves antioxidative activity by increasing
the release of flavonoids (chickpea its rich) from plant-based foods, it is
a useful method for increasing the supply of natural antioxidants.
During the fermentation, cereal breakdown and some bioactive com-
pounds are synthesized. Hur et al. (2014) reported that the total phe-
nols were increased during the fermentation of plant parts by lactic acid
bacteria and increasing in antioxidative activity due to the increase of
total phenolic compounds. Phenolic compounds can act as hydrogen
donors, reducing agents, and singlet oxygen quenchers, thus reducing
oxidation of the product.
FRAP reflected total antioxidant power and was determined for both
stirred bio-yoghurt supplemented with chickpea flour and control. The
ability of antioxidant compounds in stirred bio-yoghurt with different
concentrations of chickpea flour to reduce ferric Fe3+ to the ferrous
form Fe2+ is showing in (Fig. 2-C). The results revealed that reducing
power values of all chickpea bio-yoghurt and control were decreased
throughout the overall storage period compared to their initial values
(2.08, 1.90, 1.74 and 0.3 mg GAE/g to 1.61, 1.53, 1.42 and 0.15 mg
GAE/g, respectively). This result was in agreement with the result of
Trigueros et al. (2014), who reported that the longer storage period
H. Hussein, et al. Annals of Agricultural Sciences xxx (xxxx) xxx–xxx

Table 3
Sensory evaluation of stirred bio-yoghurt supplemented with chickpea flour.
Treatments Storage period (day) Appearance (9) Aroma (9) Texture (9) Flavour/taste (9) Overall preference (9)

Control (1) 1 8.6 ± 0.24 8.6 ± 0.24 8.6 ± 0.24 8 ± 0.0 8.6 ± 0.24
7 8.8 ± 0.2 8.6 ± 0.24 8.8 ± 0.2 8.6 ± 0.24 8.6 ± 0.24
14 8.4 ± 0.24 8.4 ± 0.24 8.2 ± 0.2 8.4 ± 0.24 8.6 ± 0.24
21 7.6 ± 0.24 8.2 ± 0.2 8.2 ± 0.2 8 ± 0.316 8 ± 0.316
Mean 8.35 ± 0.15A 8.45 ± 0.11A 8.40 ± 0.011A 8.40 ± 0.011A
Control (2) 1 8.6 ± 0.24 8.6 ± 0.24 8.6 ± 0.24 8.6 ± 0.24 8.6 ± 0.24
7 8.6 ± 0.24 8.6 ± 0.24 8.6 ± 0.24 8.6 ± 0.24 8.6 ± 0.24
14 8.6 ± 0.24 8.6 ± 0.24 8.4 ± 0.24 8.6 ± 0.24 8.4 ± 0.24
21 8.2 ± 0.2 8.2 ± 0.2 8.4 ± 0.24 8.2 ± 0.2 8.2 ± 0.2
Mean 8.50 ± 0.11A 8.50 ± 0.11A 8.50 ± 0.11A 8.50 ± 0.11A
Sample 1 1 9 9 9 9 9
7 9 9 9 9 9
14 9 9 9 9 9
21 8.8 ± 0.2 8.8 ± 0.2 8.4 ± 0.24 8.4 ± 0.24 8.4 ± 0.24
Mean 8.95 ± 0.05A 8.95 ± 0.05A 8.85 ± 0.08A 8.85 ± 0.08A
Sample 2 1 8.6 ± 0.24 8.8 ± 0.2 8.8 ± 0.2 8.8 ± 0.2 9
7 8.6 ± 0.24 8.8 ± 0.2 8.8 ± 0.2 8.8 ± 0.2 8.8 ± 0.2
14 8.6 ± 0.24 8.8 ± 0.2 8.6 ± 0.24 8.4 ± 0.24 8.6 ± 0.24
21 8.4 ± 0.24 8.6 ± 0.24 8 ± 0.31 8.2 ± 0.2 8.2 ± 0.2
Mean 8.65 ± 0.13A 8.9 ± .006A 8.55 ± 0.13A 8.55 ± 0.13A
Sample 3 1 7.8 ± 0.2 8 ± 0.316 7.8 ± 0.2 7 ± 0.316 7.2 ± 0.2
7 7.8 ± 0.2 8 ± 0.316 7.8 ± 0.2 7 ± 0.316 7.2 ± 0.2
14 7.8 ± 0.2 7.8 ± 0.37 7.8 ± 0.2 7 ± 0.31 7 ± 0.0
21 7.2 ± 0.2 7.2 ± 0.2 7.4 ± 0.24 6.4 ± 0.24 6.4 ± 0.24
Mean 7.65 ± 0.109B 7.75 ± 0.16B 7.70 ± 0.10B 6.85 ± 0.15B
Sample 4 1 9 9 9 9 9
7 9 9 9 9 9
14 9 9 9 9 9
21 8.8 ± 0.2 8.8 ± 0.2 8.6 ± 0.24 8.6 ± 0.24 8.4 ± 0.24
Mean 8.95 ± 0.05A 8.95 ± 0.05A 8.90 ± 0.06A 8.9 ± 0.068A
Sample 5 1 8.6 ± 0.24 8.8 ± 0.2 8.6 ± 0.24 8.8 ± 0.2 8.6 ± 0.24
7 8.6 ± 0.24 8.8 ± 0.2 8.6 ± 0.24 8.8 ± 0.2 8.4 ± 0.24
14 8.6 ± 0.24 8.8 ± 0.2 8.2 ± 0.37 8.6 ± 0.24 8.4 ± 0.24
21 8.4 ± 0.24 8.6 ± 0.24 8.2 ± 0.37 8.2 ± 0.37 8 ± 0.316
Mean 8.65 ± 0.13A 8.75 ± 0.099A 8.4 ± 0.15A 8.6 ± 0.133A
Sample 6 1 7.6 ± 0.24 8.2 ± 0.2 8. ± 0.316 7 ± 0.316 7.4 ± 0.24
7 7.6 ± 0.24 8.2 ± 0.2 8. ± 0.316 7 ± 0.316 7.4 ± 0.24
14 7.6 ± 0.24 8.2 ± 0.2 7.8 ± 0.2 6.8 ± 0.2 7 ± 0.316
21 7 ± 0.316 7.4 ± 0.24 7.4 ± 0.24 6.6 ± 0.24 6.6 ± 0.24
Mean 7.4 ± 0.135B 8.0 ± 0.125B 7.8 ± 0.13B 6.85 ± 0.15B

Means (n = 3 ± SD) with the same letters are not significantly different.
Control1: stirred bio-yoghurt (3%S.M.P, probiotic culture B. bifidum); Control 2: stirred bio-yoghurt (3%S.M.P, probiotic culture B. animalis sub sp. lactis and
L.acidophilus) SACCO lyofast AB1. Sample 1: stirred bio-yoghurt supplemented with 1% chickpea flour,2% S.M.P) by using probiotic culture B. bifidum; Sample 2:
stirred bio-yoghurt supplemented with 2% chickpea flour,1% S.M.P) by using probiotic culture B. bifidum; Sample 3: stirred bio-yoghurt supplemented with 3%
chickpea flour) by using probiotic culture B. bifidum; Sample 4: stirred bio-yoghurt supplemented with 1% chickpea flour,2% S.M.P) by using probiotic culture
AB1;Sample 5: stirred bio-yoghurt supplemented with 2% chickpea flour,1% S.M.P) by using probiotic culture AB1;Sample 6: stirred bio-yoghurt supplemented
with 3% chickpea flour) by using probiotic culture AB1.

decreased the reducing power of pomegranate yoghurt.
The reduction in antioxidant activities (DPPH scavenging activity
and FRAP) of chickpea bio-yoghurt likely occurred due to oxidative
degradation and polymerization of phenolic compounds during storage
of chickpea bio-yoghurts (Dufresne and Farnworth, 2001). This de-
creasing is related to binding effect between polyphenol and dairy
protein. For instance, the addition of milk to tea reduced the anti-
oxidant capacity of the antioxidants present in tea. The binding proteins
and polyphenols reduce antioxidant activity by lowering the number of
free hydroxyls (Dubeau et al., 2010). Because of the polyphenol-protein
complexes, antioxidants bioavailability might be decreased. This phe-
nomenon might also occur in other products containing both poly-
phenols and proteins. Also, Anabeh et al. (2017) reported that the de-
cline in the antioxidant activity of quinoa yoghurt during storage is
related to the extensive proteolysis that produces smaller and less
bioactive peptides.
3.4. Viscosity of stirred bio-yoghurt supplemented with chickpea flour
bio-yoghurt. Chickpea flour has some functional properties including its
water retention capacity, emulsifying capacity, ability to form gels, and
ability to form a foam, which effects on yoghurt viscosity (Aguilar-
Raymundo and Velez-Ruiz, 2013). The results showed that there was no
significant difference in viscosity values between samples made by two
probiotic cultures (B.animalis subsp.lactis and L.acidophilus) and (B.bi-
fidum). Nevertheless, there is a significant increase (p < .05) of visc-
osity during storage of all treatments (Fig. 3).
These results accord to the earlier studies (Cruz et al., 2013), who
reported a higher apparent viscosity of yoghurt supplemented with
oligofructose at the 1st day of storage and a greater viscosity of yoghurt
supplemented with oligofructose at the 28 day of storage. The main
effect observed on a stirred bio-yoghurt is an increase of viscosity due to
the bacterial activity, which decreases the pH during aging and increase
the strength of the protein network (Ebrahimi et al., 2015).
3.5. Sensory evaluation of stirred bio-yoghurt supplemented with chickpea
flour

The estimated value of viscosity Fig. 3 (A-B) indicated that the The sensory characteristic is the main factor affecting the con-
addition of chickpea flour increased the viscosity (p < .05) of stirred sumption of the products and its acceptability. Sensory results of stirred

8
H. Hussein, et al.
bio-yoghurt supplemented with chickpea flour were presented in
Table 3. It was clear that the addition of chickpea flour at 1% and 2%
improved the sensory characteristics of fresh products (1 day). There is
no significant different (p < 0.05) between 1%, 2% and control bio-
yoghurt samples, but sample contains 3% Chickpea flour received a low
score of sensory properties. This is related to reduction the properties of
body and texture by high dosage of Chickpea flour, as well as changing
the colour to yellowish, but the taste was accepted. After storage, the
acidity increased gradually in stirred bio-yoghurt supplemented with
chickpea flour but not significantly affect the flavour. All samples had
8% of sugar, which improve the acceptability of all samples. There was
no significant difference between samples made with two probiotic
cultures (B.animalis subsp. lactis and L.acidophilus) and (B.bifidium).
Consumers are interested to define the defects of food products, the
accepted and no accepted qualities of flavours and aromas, and the
important points in the selection of high-quality and high nutrition
dairy products would enable the consumer to purchase wisely and
economically dairy products. The dairy industry knows that the in-
creased sales of dairy products depend on that pleasant, good flavour
sensation and the cost. The total cost of ingredients used to produce 1
ton of stirred bio-yoghurt supplemented with chickpea to replace the
SMP in the base blend (control) partially or totally is calculated. The
total cost for control was 9100 L.E; it decreased to 8900, 8700 and
8500 L.E in stirred bio-yoghurt supplemented with chickpea flour at the
level of 1, 2 and 3%, respectively.
4. Conclusion
This study intended to develop novel bio-yoghurt and to evaluate
the effects of segmented bio-yoghurt with various levels of chickpea
flour on the viability of probiotic starters and the quality of the final
product during storage. Results confirmed that chickpea flour enhanced
the population of probiotic bacteria in bio-yoghurt during storage
period over 21-days. Based on overall preference, bio-yoghurt with 1
and 2% chickpea flour have good sensorial property. Generally,
chickpea is promised new and valuable component in fermented dairy
products by enhancing the growth of probiotic bacteria as well as in-
crease the antioxidant capacity. Supplemented bio-yoghurt could con-
sume as a functional food with therapeutic properties and adjunct nu-
tritional benefits. Taste and colour of bio-yoghurt supplemented with
chickpea may also attract new consumers. Future studies with animal
models and human clinical trials to determine the health-related
properties of yoghurt and other fermented dairy products are necessary.
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