Whey/Broken Chickpea Extract for manufacture of Probiotic Frozen Yoghurt

Probiotic frozen yoghurt is one of the ideal probiotic dairy products for delivery of live probiotic micro-organisms to human diet and thus to human intestinal tract. Probiotic micro-organisms have beneficial effects on human health. These benefits include improvements to the intestinal microflora, anti tumoral activity, reduction of cholesterol in the blood, increased immune response and other health benefits. In this study five probiotic frozen yoghurt mixtures were prepared, Acontrol (mainly from fresh skim milk and skim milk powder), B (mainly from whey/broken chickpea extract and milled chickpea grains), AB25 (75% A + 25% B), AB50 (50% A + 50% B) and AB75 (25% A + 75% B). The resultant frozen yoghurts were sensory evaluated. Panelists arranged their preference as Acontrol> AB25> AB50> AB75> B. Physically, the melting resistance of frozen yoghurt increased with the increase of chickpea ingredients. Nutritionally, chickpea frozen yoghurt contained nutrients not found in Acontrol such as dietary fibers, iron, branched chain amino acids, zinc and vitamins B3, B9 and E. Microbiologically, the viable counts of ABT culture strains (Lactobacillus acidophilus + Bifidobacterium bifidum + Streptococcus thermophilus) in all frozen yoghurt samples remained within the limits recommended for the probiotic products. Microbiologically, large numbers (> 107 cfu/g) of probiotic microorganisms present in all stored samples indicated that the resulting frozen yogurt could represent a good source of Lactobacillus acidophilus and Bifidobacterium bifidum and commercially, this product was included in probiotic dairy products.


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 (BCAAS). 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 synthesis. 11 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 and 3.48 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 o C until use. Broken chickpea extract was prepared according to the following.

Preparation of Broken Chickpea Extract
Preparation of Broken Chickpea Extract steps is shown in Fig.1.

Experimental Procedure Preparation of Probiotic Frozen Yoghurt Mixtures
• Two mixtures (A control and B) were prepared from ingredients formed in calculated quantities as shown in The process used for manufacture of probiotic frozen yoghurt is shown in (Fig. 2).

Chemically
Total solids, fat, total protein, ash, dietary fibers, minerals (Fe, Ca, P, Na, K, Mg and Zn) and vitamins contents were determined 14 Amino acids content of the product was determined according to the method described in 15 by using High Performance Amino Acid Analyzer. Branched chain amino acids BCAAS (Leucine + Isoleucine + Valine) (g/100g protein) and Fisher ratio of the products were calculated by using the following equation as described by 16 Fisher ratio = 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 Overrun (%)= (Weight per gallon of frozen Youghurt mix -Weight per gallon of frozen Yoghurt / Weight per gallon of frozen Youghurt) × 100 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)

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).

Fig. 3: Flow chart of the process used for experimented frozen yoghurt making
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).

Sensory Evaluation of Experimented Frozen Yoghurt Samples
The resultant probiotic frozen yoghurt products (A control , AB 25 , AB 50 , AB 75 and B) were evaluated after 2 days of freezing for organoleptic properties ( Table 3).The panelists noted that B and AB 75 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 AB 75 and disappeared in AB 50 and AB 25 . They arranged their preference as A control >AB 25 >AB 50 >AB 75 >B. From the results of the sensory evaluation A, AB 25 and AB 50 treatments were selected for physical, chemical and microbiological examination.

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

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 and 25 for low fat ice cream containing Cactus pear pulp.
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 (AB 25 &AB 50 ). 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. Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05 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.

Nutritionally
The nutritive value of one serving (100 g) of frozen yoghurt samples (A control , AB 25 and AB 50 ) was estimated. The chemical composition, dietary fibers, minerals (Ca, P, K, Fe, Zn and Na) and vitamins (C, B 3 , B 2 , A, E and B 9 ) of the of the different samples were deter mined. Data obtained ( Figure 4) showed that AB 25

Nutritive Values of Frozen Yoghurts Protein
Essential and non-Essential amino acids (EAA) of frozen yoghurt samples (A control -,AB 25 and AB 50 ) 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 by 26   Values are means ± SD (n=3), mean number in the same column bearing different superscript letter are significantly different at p < 0.05

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 AB 25 -and AB 50 -samples but still within the limits recommended by 29 for the probiotic products (Table 8).   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.