Introduction

The trend towards the use of dairy by-products such as whey, skim milk, butter milk and permeate and some residues of agricultural crops such as broken chickpea seeds, wheat germ, wheat bran, rice bran and etc, as non-dairy by-products in the manufacture of functional dairy foods is increasing worldwide.

Whey is one of the most important dairy by-products. It is a by-product of cheese-making and casein manufacture in the dairy industry. Currently, whey and its components are recognized as value-added ingredients in many food products such as infant formulas, sports nutrition foods and other food products. Among whey components is the protein. Whey protein contains all 20 amino acids and all nine essential amino acids, and it is a rich and balanced source of the sulphur amino acids that serve a critical role as antioxidants.¹ Whey may also include low-molecular weight bioactive peptides formed by the enzymatic degradation of the caseins during the cheese making process.²

The broken chickpea seeds are one of the residues of the process of extracting chickpea seeds from their dry horns. Chickpea (Cicer arietinum L.) of legumes are rich in protein, vitamins, minerals and dietary fibers but low in fat^{3, 4} Besides promoting weight loss and preventing obesity and cardiovascular disease, Prebiotics are defined as “selectively fermented ingredients that result in specific changes, in the composition and/or activity of the gastrointestinal microbiota, thus conferring benefits upon host health.⁵ Prebiotics are also a substrate that is selectively utilized by host microorganisms conferring a health benefit.⁶

Both in vivo and in vitro studies have confirmed the positive influence of dietary fibers, as prebiotics, on survival of probiotic bacteria such as Bifidobacterium lactis Bb-12 and Lactobacillus acidophilius La-5.^{7, 8} showed that supplementing by chickpea flour enhanced the growth of Lactobacillus debrueckii ssp. bulgaricus in yoghurt. Yoghurt fortified with the watery extract of chickpea had higher Streptococcus thermophilus counts than that of the control without the extract.⁹ Thus, dietary fibers and protein contents of chickpea will be a useful ingredient to enhance the quality of any fermented dairy product.

In addition to the above, it should be noted that whey and chickpea are important sources of branched chain amino acids (BCAA_S).¹⁰ Branched chain amino acids (valine, leucine and isoleucine) have the effect of promoting protein synthesis and inhibiting protein collapse. Leucine is not only precursor for muscle protein synthesis, but also may play a role as a regulator of intracellular signaling pathways that are involved in the process of protein synthesis¹¹

Therefore, this study was conducted to investigate the effect of replacing chickpea components instead of milk components on the chemical, physical, nutritional, microbiological and sensory properties of like probiotic frozen yoghurt.

Materials and Methods

Materials            

Fresh skim milk (0.1% Fat and 9.43% solids not fat (S.N.F) and fresh cream (45% Fat and3.48 S.N.F) were obtained by separating raw buffalo’s milk using cream separator (Alfa Laval). Low heat skim milk powder made in USA (1.25% Fat and 94.75% S.N.F), cane sugar (sucrose) and raw vanilla were purchased from the local market. Lacta 9060 (combination of selected emulsifiers and stabilizers blend) was obtained from Misr Food Additives (MIFAD). Freeze dried ABT-2starter culture containing Lactobacillus acidophilus, Bifidobacterium bifidum and Streptococcus thermophilus was obtained from Chr. Hansen laboratories (Denmark). Broken chickpea seeds were obtained from Crops Research Institute, Agriculture Research Center, Ministry of Agriculture, Giza, Egypt. Broken chickpea was milled (using standard electric grinder, Moulinex^®, France) and sieved by stainless steel sieves (60 mesh) for obtaining chickpea’s powder. The powder product (5.85% fat, 89.04% S.N.F) was stored in glass bottles at^סC until use. Broken chickpea extract was prepared according to the following.

Preparation of broken chickpea extract

Figure 1: Flow chart of the preparation of broken chickpea extract.  Click here to View figure

Methods

Experimental procedure

Preparation of probiotic frozen yoghurt mixtures:

1. Two mixtures (A_control and B) were prepared from ingredients formed in calculated quantities as shown in Table (1) and standardized to contain 14% S.N.F, 8% fat, 13% sugar and 0.4% lacta 9060.¹²
2. Equal quantities of calculated skim milk and chickpea extract were heated to 90^○C and held for 10 min, then cooled down to the incubation temperature of 35^○C. These media inoculated with freeze dried ABT-2 starter culture (0.02%) and incubated at 35^○C until the pH
   value reached 5.5. These fermented media were chilled and stored at 4^○C.
3. The process used for manufacture of probiotic frozen yoghurt is shown in (Fig. 2).

Table 1: Formulation of different probiotic frozen yoghurt mixes

Figure 2: Flow chart of the process used for experimented frozen yoghurt making  Click here to View figure

Methods of Analysis

Microbiologically

The viable count of ABT cultures (Bifidobacterium bifidum, Lactobacillus acidophilus and Streptococcus thermophilus) was enumerated^{13, 14}

Chemically

Total solids, fat, total protein, ash, dietary fibers, minerals (Fe, Ca, P, Na, K, Mg and Zn) and vitamins contents were determined¹⁴ Amino acids content of the product was determined according to the method described in¹⁵ by using High Performance Amino Acid Analyzer. Branched chain amino acids BCAA_S (Leucine + Isoleucine + Valine) (g/100g protein) and Fisher ratio of the products were calculated by using the following equation as described by¹⁶

Physically

pH value was measured using a digital laboratory pH meter (HI931400 Hanna instruments) with glass electrode. Determination of dynamic viscosity (in Centi-poise, Cp) of aged probiotic frozen yoghurt mixes was carried out using a Brookfield viscometer (Brookfield DV-III, Brookfield Engineering Laboratories, Inc., USA). A Brookfield viscometer equipped with a spindle No.3 to run at 80 rpm in 250 ml cup. All samples were adjusted at 25±1°C before viscosity measurement. The overrun of probiotic frozen yoghurt samples were calculated by using the following equation as described by ¹⁷

The melting rate of probiotic frozen yoghurt samples was determined.¹⁸ The color attributes L (Brightness; 100: white, 0: black), a (+: red, – : green) and b (+: yellow, – : blue) of probiotic frozen yoghurt samples were evaluated using Hunter colorimeter (Hunter ultra-Scan. VIS).

Sensory Evaluation

All the resultant products were sensory rated for flavor (50), body & texture (40) and color & appearance (10) by 10 panelists of experienced staff members at Dairy Science Department, Cairo University and Agriculture Research Center, Ministry of Agriculture, Giza, Egypt according to the ice cream score card of.¹⁷ Total score of flavor, body & texture and color & appearance was defined as total acceptability (100 points).

Statistical Analysis

Statistical analysis was carried out by SPSS Vr. 20 program. Data were expressed as means ± S.D. and the statistical analysis were performed using one-way analysis of variance followed by Duncan’s tests. (SPSS, 20).¹⁹

Results and Discussion

Growth Behavior of ABT-2 Culture Strains in Chickpea Extracts

Frozen dairy products such as frozen yoghurt are an ideal carriers for the delivery of beneficial microorganisms to human intestines. In an attempt to increase its functional properties, whey and permeate as dairy by-products and broken chickpea seeds as non-dairy by-products were used in an initial experiment in the manufacture of frozen yoghurt. Therefore, the behavior of ABT-2 culture (Bifidobacterium bifidum, Lactobacillus acidophilus and Streptococcus thermophilus) in three extracts of grounded broken chickpea seeds with water, whey or permeate was investigated to determine the best extract for making probiotic frozen yoghurt. The mean value of viable counts of ABT-2 culture strains in the three extracts was estimated comparative to that in skim milk as shown in Fig (3).

Figure 3: Mean value of viable counts (log CFU/ml) of ABT culture in three chickpea extracts.  Click here to View figure

The obtained results showed that the three chickpea extracts were suitable environments for the growth and activity of probiotic bacteria. Whey/chickpea extract was the best extract as the number of viable cells of all ABT-2 culture strains reached a level approximately similar to that of the control skim milk. No microbial activity inhibition due to chickpea ingredients was detected. The dietary fibers and oligosaccharides of chickpea seeds as well as the whey lactoferrin may enhanced the growth and activity of ABT-2 culture strains^{8 ,20, 21} Therefore, this extract (6.5% fat and 9.34% S.N.F) was used for preparation of experimental probiotic frozen yoghurt mixes Figure 2.

Effect of chickpea ingredients on experimented frozen yoghurt color

Of course, a clean white color is often demanded color of a naturally frozen yoghurt. A_control– frozen yoghurt (without chickpea ingredients) exhibited more whiteness as indicated by a high L* value, and low a* and b* values. B-frozen yoghurt (100% chickpea) showed greater yellowish color value (b*) and lesser greenish color value (-a*) than the others (Table 2). Therefore addition of chickpea ingredients decreased the whiteness of the product and this decrease was proportional to replacement ratio of skim milk and skim milk powder with whey/chickpea extract and milled chickpea seeds in experimented frozen yoghurts (Table 1).

Table 2: Color values of experimented frozen yoghurt

(L*):The lightness, (b*):The yellowness and (a*): The redness

Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05

Sensory evaluation of experimented frozen yoghurt samples

The resultant probiotic frozen yoghurt products (A_control, AB₂₅, AB₅₀, AB₇₅ and B) were evaluated after 2 days of freezing for organoleptic properties (Table 3). The panelists noted that Band AB₇₅ frozen yoghurt melted slowly in the mouth than the others. In other words panelists agreed on the sense of coarseness in sample B, which decreased in AB₇₅ and disappeared in AB₅₀ and AB₂₅. They arranged their preference as A_control >AB₂₅>AB₅₀>AB₇₅> B. From the results of the sensory evaluation A, AB₂₅ and AB₅₀ treatments were selected for physical, chemical and microbiological examination.

Table 3: Sensory evaluation of experimented frozen yoghurt

Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05

Physical properties of the selected probiotic frozen yoghurt mixes

Replacement of non-fat milk solids with chickpea ingredients caused a significant decrease in specific gravity (Sp.gr) and weight per gallon and increased viscosity of treated frozen yoghurt mixes as shown in Table (4).

The high viscosity of treated frozen yoghurt mixes is due to their high content of starch and fibers. Gelatinization of starch is the most prominent change taking place during boiling of chickpea seeds through preparation of its extract. Gelatinized starch has high water holding capacity and it can influence the rheological properties of frozen yoghurt mix.^{22, 23}

Table 4: Specific gravity (Sp.gr), weight per gallon (Wt/gal) and viscosity of the selected probiotic frozen yoghurt mixes^*

weight per gallon (Ibs) = Sp.gr×8.345 (cp) = centipoise.

Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05

Physical properties of the resultant probiotic frozen yoghurt

With an increase in the level of replacing of non fat milk solids with chickpea ingredients there was a corresponding significant increase in specific gravity and weight per gallon and significant decrease in the overrun of treated frozen yoghurt (Table 5). This lowering in the overrun is due to the high viscosity of mixes which caused a less air incorporation (Table 4). These results are in agreement with the results reported by ²⁴ for grape wine lee ice cream, ²⁵ for functional ice cream fortified with date pulp and²⁵ for low fat ice cream containing Cactus pear pulp.

Table 5: Specific gravity (Sp.gr), weight per gallon (Wt/gal) and overrun (%) of the resultant probiotic frozen yoghurt.^*

Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05

Melting resistant of frozen yoghurt samples expressed as a percentage of melted frozen yoghurt during 60 minutes at intervals of 15 minutes is recorded in Table (6).The recorded data indicated that frozen yoghurt samples without chickpea (A_control) lost their shape and melted more quickly than those with chickpea (AB₂₅&AB₅₀). In other words the melting resistance increased significantly as the percentage of whey/ broken chickpea extract increased. A probable cause for this behavior was the impact of fibers and starch of chickpea seeds on the viscosity of the treated frozen yoghurt mixes. These results were in agreement with those reported by. ²³ They found that ice creams with the high level of gelatinized malted ragi flour had the highest melting resistance. They attributed that to fibers and starch in ragi flour which may act as stabilizers and thus caused increase in melting resistance of resultant ice cream.

Table 6: Melting resistance of the resultant probiotic frozen yoghurt.^*

Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05

The functional evaluation

Chemical composition of the resultant probiotic frozen yoghurt

Total solids and fat contents of all frozen yoghurt were within the limited levels of the standards given by¹² as shown in Table (7).With increased levels of replacement of fresh skim milk and skim milk powder with chickpea ingredients the protein and ash contents of the resulting frozen yoghurt significantly decreased, while the total carbohydrates content significantly increased as compared to A_control frozen yoghurt which may be attributed to the high lactose content of whey/chickpea extract.

Table 7: Chemical composition of the selected probiotic frozen yoghurt^*

^* Total protein of A_control = TN х 6.38, AB₂₅ = TN х 6.35, AB₅₀ = TN х 6.32

Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05

Nutritionally

The nutritive value of one serving (100 g) of frozen yoghurt samples (A_control, AB₂₅ and AB₅₀) was estimated. The chemical composition, dietary fibers, minerals (Ca, P, K, Fe, Zn and Na) and vitamins (C, B₃, B₂, A, E and B₉ ) of the of the different samples were determined. Data obtained (Figure 4) showed that AB₂₅– and AB₅₀– frozen yoghurt samples had chemical composition approximately similar to that of A_control, but contained nutrients not found in A_control such as dietary fibers and iron and contained higher content of zinc (0.94 and 1.00 versus 0.88 mg) and low content of sodium (102.58 and 85.92 versus 119.21 mg ), vitamin B₃ (0.20 and 0.22 versus 0.19 mg), vitamin B₉ (19.65 and 28.62 versus10.66 mg) and vitamin E (0.05 and 0.09 versus 0.04 mg).

Figure 4: Nutritive value of one serving (100 g) of frozen yoghurt  Click here to View figure

Nutritive values of frozen yoghurts protein

Essential and non-Essential amino acids (EAA) of frozen yoghurt samples (A_control-,AB₂₅ and AB₅₀) were also determined Figure 5, 6. The percentage of all amino acids slightly decreased as the percentage of whey/chickpea extract increased in the mixture, but remained within the established limits approved by²⁶ for yoghurt.The branched chain amino acids BCAA_S (isoleucine, leucine and valine) percentage for all amino acids was 46.63 for A_control ,46.70 for AB₂₅– and 46.85% for AB₅₀-. BCAA_S are important for muscle protein synthesis and have a positive effect on mental state ¹⁰.Also, a high fisher ratio was noticed in all frozen yoghurt samples (A_control = 2.26, AB₂₅ = 2.36 and AB₅₀ = 2.39). Fisher ratio is important for maintenance of hepatic function reserve and prevention of progression to liver failure²⁸

Figure 5: Essential amino acids composition and nutritional values of frozen yoghurt samples protein  Click here to View figure

Figure 6: Non-Essential amino acids composition of frozen yoghurt samples protein.  Click here to View figure

Microbiologically

There was no significant difference between the viable counts of ABT culture strains in all fresh frozen yoghurt samples but they were reduced in A_control sample at the end of the storage period compared to AB₂₅– and AB₅₀– samples but still within the limits recommended by ²⁹for the probiotic products (Table 8).

Table 8: Changes in the number of viable count of ABT culture strains during the storage period at -23 ° C for 21 days

^* See Table 1

Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05

Conclusion

From the above, it could be concluded that using mixture of whey as dairy by product and broken chickpea as non-dairy by product in the manufacture of probiotic frozen yoghurt was succeed. The resulting frozen yoghurt can be successfully used commercially to deliver probiotic bacteria such as Lactobacillus acidophilus and Bifidobacterium bifidum to consumers from different age groups.

Acknowledgment

A lot of thanks to my young colleague Dr. Fouad M F Elshaghabee lecturer in Dairy Science Department, Faculty of Agriculture, Cairo University, Egypt for his kindly help through preparation the microbiological experiment, providing us with the ABT culture and enumeration the probiotic microorganisms.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

Conflict of Interest

The authors do not have any conflict of interest.

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