EFFECT OF WHEY PROTEIN CONCENTRATE ON MANUFACTURING OF NONFAT FERMENTED MILK

-Consumer perception of food gave a golden opportunity to synbiotic fermented dairy products, since that have been playing a major role in the part of the diet of people throughout the world for their taste, nutritive value and therapeutic properties. “Synbiotic food” contains probiot ic microorganisms and prebiotic substances. Thus, it is essential to determine the composition, titratable acidity and textural properties to provide the best synbiotic fermented product. Different combination of Lactobacillus acidophilus and Lactobacillus casei were studied to cull the best combination on the basis of acid production for preparation of nonfat fermented milk. Keeping in view of the above said observations of the best inoculums percentage (1%) of Lactobacillus acidophilus and Lactobacillus casei at 1:1 ratio was selected and further planned and conducted to examine the effect of various level of whey protein concentrate in fatless fermented synbiotic fermented milk for optimizing the best combination ratio of total solids which would give the maximum sensory grade. The titratable acidity of nonfat fermented milk was measured at 2h time interval during the incubation period. After reached to the desire acidity level the different combination of products was subjected to sensory evaluation. Synbiotic Nonfat fermented milk added with 3% whey protein concentrate level prepared with 1% inoculum of Lactobacillus acidophilus and Lactobacillus casei at 1:1 ratio was observed the best and showed highest overall grade for acceptance of the product.


Introduction
The synbiotic fermented dairy foods namely symbiotic dahi are inherent part of human diet.The potential benefits of synbiotic-dahi are increased safety, economic value of the product, nutritional value, digestibility, making the food more suitable for subsequent processing, acceptability, enhancing the flavour and palatability, ease of storage and transportation, shelf life with decreased toxicity, cooking time and processing time.During recent years, an increasing interest has developed in foods that contribute to a positive effect on health beyond their nutritional value.Probiotic foods contain microorganisms or components of microbial cells that have a beneficial effect on the health and well being of the consumer host [1].Sometimes the probiotic organisms fail to grow properly and to fight against pathogenic organisms due to lack of their foods.This has led to the coining of the term synbiotic food, which includes probiotic microorganisms and their food materials known as prebiotic substances in the system.Yadav, et al., (2007) found that Lactococcus lactis ssp diacetylactis, Lactobacillus acidophilus and Lactobacilus casei had the ability to produce oligosaccharides due to transgalactosidal and lactose hydrolysis activities of beta-galactosidase [2].Champagne, et al., (1996) suggested that whey protein concentrate could be successfully used to prepare starter cultures because it gave higher population of bacteria than when milk was used as the medium [3].Dave & Shah, (1998) found that whey protein concentrate contains amino nitrogen, which was used as a catalator for the growth of microorganisms.They found that supplementation of 2% whey protein concentrate with milk to prepare yoghurt by inoculating Lactobacillus acidophilus, Biofidobacterium & Strptococcus thermophilus (ABT) culture decreased fermentation time by 4 h to reach pH 4.5 as compare to yogurt preparation having same culture but without whey protein concentrate [4].Martin-Diana, et al., (2003) suggested that supplementation of 3% whey protein concentrate (35) with goat milk to prepare fermented goat's milk by inoculating ABT culture, decreased fermentation time by 2 h [5].Martin-Diana, et al. (2003) suggested that 3% whey protein concentrate supplementation into goat milk to prepare fermented goat milk increased the Bifidobacterial growth and viability of Lactobacilli [5].Janer, et al., (2004) reported that addition of 2% whey protein concentrate into skimmed milk to prepare fatless fermented milk by inoculating Bifidobacterium lactis, increased the counts of Bifidobacterium lactis by 3.5 times at 6 th hour of incubation as compare to plain fatless fermented milk [6].Needs, et al., (2000) observed that denaturation of β-lactoglobulin found in whey protein concentrate and its interaction with casein micelles had been shown to highly influence gel fermented milk properties [7).Shanker, et al., (2000) reported that denaturation of whey protein during heat treatment highly influenced viscosity due to an increase of protein binding capacity that resulted in a higher gel viscosity during coagulation [8].Martin-Diana, et al., (2003) investigated that increase in the content of solid not fat was required in fat less fermented milk to obtain a satisfactory curd tension [5].Whey protein concentrates might be used as a cheaper and readily available additive to increase viscosity, reduce whey synergism and give impart desirable texture characteristics in fermented milk.This study was planned to evaluate different nonfat fermented synbiotic milk Available online at http://www.bioinfopublication.org/jouarchive.php?opt=&jouid=BPJ0000234

International Journal of Microbiology Research
Abstract-Consumer perception of food gave a golden opportunity to synbiotic fermented dairy products, since that have been playing a major role in the part of the diet of people throughout the world for their taste, nutritive value and therapeutic properties."Synbiotic food" contains probiotic microorganisms and prebiotic substances.Thus, it is essential to determine the composition, titratable acidity and textural properties to provide the best synbiotic fermented product.Different combination of Lactobacillus acidophilus and Lactobacillus casei were studied to cull the best combination on the basis of acid production for preparation of nonfat fermented milk.Keeping in view of the above said observations of the best inoculums percentage (1%) of Lactobacillus acidophilus and Lactobacillus casei at 1:1 ratio was selected and further planned and conducted to examine the effect of various level of whey protein concentrate in fatless fermented synbiotic fermented milk for optimizing the best combination ratio of total solids which would give the maximum sensory grade.The titratable acidity of nonfat fermented milk was measured at 2h time interval during the incubation period.After reached to the desire acidity level the different combination of products was subjected to sensory evaluation.Synbiotic Nonfat fermented milk added with 3% whey protein concentrate level prepared with 1% inoculum of Lactobacillus acidophilus and Lactobacillus casei at 1:1 ratio was observed the best and showed highest overall grade for acceptance of the product. .

Materials and Methods
Two strains of lactic acid bacteria in freeze dried ampules form namely Lactobacillus acidophilus and Lactobacillus casei were obtained from national collection of dairy culture centre, NDRI, Karnal, India.Both the cultures were maintained in sterile litmus milk media and activated by three successive transfers at 24 h interval before use.Skim milk (10% TS) was prepared by dissolving 10.5 gm of skimmed milk powder in 100 ml of distilled water.Whey protein concentrate about 3.17 gm was added into 100 ml Skim milk (10% TS) to maintain 13% TS.Then fatless milk was heated up to 115 0 C for 15 min and cooled down to 38 -40 0 C. 1% Mixed culture of Lactobacillus acidophilus and Lactobacillus casei having 1:1 ratio was inoculated into the fatless milk and incubated for 7 h at 37 0 C. The acidity was measured at each 2 h interval during the incubation period of milk as per provision of ISI handbook of food analysis, part XI Dairy products, 1981 [9].Lactic acid bacteria count at the 7 th hour in the incubation period of the samples was carried out by using deMann Rogosa and Sharpe agar (MRS).(As per provision of Manual in Dairy Bacteriology, ICAR-Sub committee on Dairy Education, 1972) [9].Fatless fermented milk prepared by different combination of whey protein concentrate and skimmed milk powder were judged independently by a selected panel of six experienced members.The products were evaluated in respect to flavour, body and texture, colour and appearance and overall acceptability using a 9-point Hedonic scale evaluation card specially prepared for this purpose [10].

Result & Discussion Acid producing ability of different lactic cultures
Two types of single lactic cultures viz.Lactobacillus acidophilus and Lactobacillus casei, their combinations with different ratios viz.Lactobacillus acidophilus + Lactobacillus casei ( 1 : 1, 3 : 1 & 1 : 3), 1% rate of inoculums were studied with a view to select a suitable starter culture or combination for the preparation of fatless fermented milk.The respective data are presented in The acid producing ability of the cultures was measured in term of percentage of titratable acidity (TA %).The results pertaining to the acid producing abilities of above mentioned lactic cultures and combination thereof are presented and discussed as below.It might be observed that the variation in TA values among the type of cultures, the period of incubation and their interaction were significant (P<0.05).(Average of three replications)
The rate & increase in titratable acidity increased significantly (P<0.05) in case of culture type C1 + C2 (1 : 1) than that of single cultures or other combinations because of more synergism effect of this type of culture combination as compare to others, at the same time lactose was more used by C1 + C2 culture combination.Hence, 1% culture of C1 + C2 combination with ratio 1:1 was taken as inoculums to prepare fatless fermented milk.

Effect of whey protein concentrate on titratable acidity with different incubation period
The selected combination of culture [C1 + C2 (1: 1)] @ 1% inoculum was inoculated to prepare fatless fermented milk.The different percentages of WPC were used like 1%, 2%, 3%, and 4%.The SMP percentage was taken for making the fatless fermented milk in such a way that total solids in the fatless fermented milk remained 13%, were studied with a view to select a suitable WPC combination.These data are presented in  and in [Fig- 2].The acid production was measured in term of percentage of titratable acidity.It may be observed that changes in TA% with respect to period of incubation, combination of WPC and their interaction were significant (P<0.05).Titratable acidity was increased with increase of percentage of WPC and incubation period.Fatless fermented milk prepared with the addition of 4% WPC showed the highest percentage of TA i.e., 2.265%.It may be due to the most favorable situation of growth to lactobacilli as a result of addition of WPC.Martin-Diana, et al. (2003) studied the development of a fermented goat's milk containing probiotic bacteria and found that supplementation of 3% whey protein concentrate increased viability of Lactobacilli.While Janer, et al., (2004) reported that addition of 2% whey protein concentrate into skimmed milk to prepare fat less fermented milk increased the lactic count.Culture used @ 1% with 1:1 of C1 + C2 (Average of three replications)

Effect of whey protein concentrates on population of Lactic acid bacteria and Titratable acidity at 7 th hour of incubation
The selected combination of culture [C1 + C2 (1:1)] @ 1% inoculum was inoculated to prepare fatless fermented milk.The different percentage of WPC was used like 1%, 2%, 3% and 4%.The SMP percentage was taken for the making the fatless fermented milk in such a way that total solids in the fatless fermented milk remained 13%.The data related to population of Lactic acid bacteria and TA% at 7 th hour of incubation is presented in .Titratable acidity, population of Lactic acid bacteria were increased with increase of percentage of WPC at 7 th hour of incubation.The fatless fermented milk prepared with the addition of 4% WPC showed the highest percentage of TA i.e.,1.053% and also in case of LAB population i.e., 155 × 10 7 cfu/gm.It may be due to more quantities of nutrition available resulting to more number of Lactose utilizable lactobacilli and more utilization of lactose by microorganisms.Martin-Diana, et al., (2003) suggested that 3% WPC supplementation into goat milk to prepare fermented goat's milk increased the bifidobacterial growth and viability of Lactobacilli.Janer, et al., (2004) reported that addition of 2% whey protein concentrate into skimmed milk to prepare fatless fermented milk by inoculating Bifidobacterium Lactis, increased the counts of Bifidobacterium lactis by 3.5 times at 6 th hour of incubation as compare to plain fatless fermented milk.The effect of addition of whey protein concentrate on the organoleptic characteristics of fermented milk was therefore, studied with a view to assess to their efficiency to improve the acceptability.All five sample of fermented milk exhibited distinct separation of whey on top and the cream coloured was observed on top of samples.Flavour was almost similar in second, third and fourth samples but in fifth one distinct whey flavour was evaluated so obtained less overall score among all samples.Combination-4 (i.e., 3% WPC) secured overall acceptability more among all might be due to good texture, less whey separation and absence of whey flavour, whereas combination-1 (i.e., 0% WPC) obtained least score of overall acceptability due to more separation of whey.Martin-Diana, et al., (2003) reported that addition of 3% whey protein concentrate into fatless fermented milk reduced whey synersis and gave impart desirable texture characteristics in fermented milk.The finding of this investigation is therefore, in accordance with the above reports.It is observed that average score of fatless fermented milk prepared with 3% WPC in overall acceptance characteristics was maximum i.e., 9.0.Although the maximum acidity (1.053% TA) was produced by fat less fermented milk prepared with 4% WPC but it failed to be accepted organoleptically.Now the fatless fermented milk prepared with 3% WPC was selected on the basis of overall acceptability by the technical exports or judges.

Conclusion
Two cultures named Lactobacillus acidophilus (C1) and Lactobacillus casei (C2) alone and their combination 1:1, 3:1 and 1:3 were inoculated @ 1% for preparation of fatless fermented milk having 13% TS.C1 + C2 with 1:1 ratio culture combination was used to prepare fatless fermented milk by adding whey protein concentrate.Whey protein concentrate 1%, 2%, 3%, and 4% were added into skim milk in such a way that the total solid remained 13% in fat less fermented milk.By increasing the rate of WPC from 0 to 4% the titratable acidity and population of lactic acid bacteria were increased in fatless fermented milk.4% WPC added fatless fermented milk gave higher TA% and LAB population than 3% WPC added fatless fermented milk.Five samples of fatless fermented milk, varying with different combinations of WPC like 0%, 1%, 2%, 3%, and 4% were judged organoleptically.3% WPC added fatless fermented milk was scored highest overall grade for acceptance of the product.Therefore, addition of 3% whey protein concentrate level with 1% inoculum of Lactobacillus acidophilus and Lactobacillus casei at 1:1 ratio was observed the best and showed highest overall grade for acceptance of the product and this can be recommended to the dairy Industry to introduce in the market.

Table - 3
Lactic acid bacteria count at 7 th hour of incubation of fat less fermented milk having different combination of WPC by remaining the T.S constant as 13%

Sensory evaluation of whey protein concentrate added fatless fermented milk
A sensory panel comprising of six experienced members evaluated the Flavour, Body & Texture, Colour & Appearance and Overall Acceptability of the product by using 9-point Hedonic scale.Sensory evaluation was done for five samples of fatless fermented milk.First one was prepared from only skim milk having 13% T.S. Second one was prepared by adding 12% T.S. from SMP and 1% T.S. from WPC, third one was prepared by adding 11% T.S. from SMP & 2% T.S. from WPC, fourth one was prepared by adding 10% T.S. from SMP & 3% T.S. from WPC and fifth one was prepared by adding 9% T.S. from SMP & 4% T.S. from WPC.The respective observations are depicted in.