Development of probiotic enriched chakka whey drink

Introduction

Whey, a by-product obtained during the manufacture of cheese, chhana, paneer, and Chakka, has gained considerable attention due to its high nutritional value. But whey is not utilise efficiently and discarded in environment although often underutilized, whey contains lactose, high-quality proteins, minerals, and water-soluble vitamins, retaining nearly half of the total solids of milk. Globally, whey production is estimated at 180-190 million tonnes annually, yet less than 50% is effectively utilized, highlighting the need for its value addition (Borkar, 2020; Madavi et al., 2025; Mirzakulova et al., 2025) [9,13,16] . Probiotics and prebiotics have emerged as key components in the development of functional foods. Probiotics such as Lactobacillus and Bifidobacterium contribute to gastrointestinal health, while prebiotics act as substrates that promote their growth and activity (Al-Habsi et al., 2024) [2] . The combination of both, known as synbiotics, enhances microbial viability, improves gut health, and provides additional metabolic and immunological benefits. Techniques such as encapsulation further improve probiotic stability and survival under adverse conditions, increasing their effectiveness in food applications (Sarita et al., 2025; Pal and Bhowal, 2023; Al-Habsi et al., 2024) [22,21,2] . In this context, the development of a probiotic-enriched chakka whey drink represents a sustainable and functional approach to utilize dairy by-products while addressing the growing demand for health-promoting beverages. Incorporation of probiotics into whey enhances its nutritional and therapeutic value, contributing to improved gut health, immunity, and overall well-being. Furthermore, synbiotic formulations offer superior benefits by modulating intestinal microbiota, enhancing nutrient absorption, and preventing various gastrointestinal disorders. Therefore, the present study focuses on the development and evaluation of a probioticenriched chakka whey drink as an innovative functional beverage.

Materials and Methods

The present investigation was undertaken to develop a functional dairy beverage, namely a probiotic-enriched chakka whey drink, and to evaluate the growth pattern of probiotic microorganisms in the developed product. The study included preparation of whey drink, incorporation of probiotic and prebiotic components, and subsequent evaluation through physicochemical, microbiological, and sensory analyses. The experiment was designed in two parts. The first experiment aimed to optimize the process for the development of probiotic chakka whey drink using three treatments, namely (T 1 ) control (without probiotic and prebiotic), (T 2 ) probiotic-added, and (T 3 ) synbiotic (probiotic with prebiotic inulin), each replicated six times. The whey drink was prepared following the method described by Drgalic et al., (2005) [11] with suitable modifications. Fresh buffalo milk procured from the cattle breeding farm was filtered and standardized to 4% fat and 8.5% solids-not-fat using Pearson's square method. The standardized milk was heated, cooled to incubation temperature (37±1 °C), and inoculated with Streptococcus lactis culture at 1% (v/v), followed by incubation for 14-16 hours to obtain curd. The curd was transferred into muslin cloth and allowed to drain under gravity to obtain whey. The extracted whey was incorporated with prebiotic inulin at 1% (w/v) as per treatment requirements and subjected to heat treatment at 63 °C for 30 minutes. After cooling to 37±1 °C, the whey was inoculated with Lactobacillus acidophilus NCDC 14 at 2% (v/v) and incubated at 37±1 °C for 24 hours under anaerobic conditions. The fermented whey was subsequently cooled to 4±1 °C to arrest microbial activity. Sugar was added at 8% (w/v), and the final product was filled into sterilized glass bottles, capped, and stored under refrigerated conditions (4±1 °C) until further analysis. The flowchart for the preparation of chakka whey drink is as follows: In the second experiment, the developed whey drink samples were analysed to study the growth pattern of probiotic microorganisms along with quality attributes. Proximate composition, including fat, solids-not-fat, total solids, and protein, was determined using standard methods of AOAC, 2016 [3] and AOAC, 2019 [4] . Physico-chemical properties such as pH and titratable acidity were evaluated following AOAC, (2016) [3] procedures. Microbiological quality, including, psychrophilic count, yeast and mould count, and Lactobacillus acidophilus count, was determined as per standard methods described by APHA (1984) [5] , and results were expressed as log₁₀ CFU/mL. Sensory evaluation of the product was carried out by a semi-trained panel using a 9-point hedonic scale as described by Nelson and Trout (1964) [19] to assess appearance, flavour, taste, and overall acceptability. The experimental data were statistically analysed using Completely Randomized Design (CRD) as described by Snedecor and Cochran (1989) [25] .

Results

Formulation of Chakka Whey Drink

Three formulations of chakka whey drink were developed under different treatment combinations, namely control (T 1 ), probiotic (T 2 ), and synbiotic (T 3 ). The control sample (T 1 ) consisted of chakka whey with sugar, while T 2 included Lactobacillus acidophilus and sugar. The synbiotic treatment (T 3 ) comprised Lactobacillus acidophilus, inulin and sugar. The detailed formulation is presented in Table 1. Chakka whey + Sugar @ 8%w/v T2 Chakka whey + L. acidophilus @ 2% v/v +Sugar @ 8%w/v T3 Chakka whey + L. acidophilus @ 2% v/v+ Inulin @ 1%w/v +Sugar @ 8%w/v

Proximate Composition Fat

Fat content of chakka whey drink decreased significantly (p<0.01) during refrigerated storage (Table 2). The control treatment (T 1 ) showed a decline from 0.22 to 0.19%, while T 2 and T 3 decreased from 0.21 to 0.14% and 0.19 to 0.14%, respectively. Among treatments, T 1 consistently exhibited higher fat content compared to T 2 and T 3 .

Solids-Not-Fat (SNF)

SNF content increased significantly (p<0.01) during storage (Table 2). Values increased from 5.77 to 5.97% in T 1 , 6.02 to 6.28% in T 2 , and 6.08 to 6.31% in T 3 . The highest SNF content was recorded in T 3 , followed by T 2 and T 1 .

Total Solids

Total solids showed a significant increase (p<0.05) during storage (Table 2). T 1 increased from 6.03 to 6.15%, while T 2 and T 3 increased from 6.24 to 6.40% and 6.27 to 6.40%, respectively. Synbiotic treatment (T 3 ) consistently showed higher total solids.

Protein

Protein content decreased significantly (p<0.01) in all treatments during storage (Table 2). T 1 decreased from 0.73 to 0.59%, T 2 from 0.72 to 0.60%, and T 3 from 0.82 to 0.74%. Among treatments, T 3 maintained the highest protein content throughout storage.

Physico-Chemical Properties pH

The pH of chakka whey drink decreased significantly (p<0.01) during storage (Table 3). The values declined from 4.84 to 4.69 in T 1 , 4.78 to 4.68 in T 2 , and 4.78 to 4.66 in T 3 .

The lowest pH values were observed in T 3 .

Titratable Acidity

Titratable acidity increased significantly (p<0.01) during storage (Table 4). Acidity values increased from 0.78 to 0.89% in T 1 , 0.81 to 0.96% in T 2 , and 0.84 to 0.99% in T 3 .

The highest acidity was recorded in T 3 , followed by T 2 and T 1 .

Microbial Analysis Psychrophilic Count

Psychrophilic count was not detected up to day 6 but showed a significant increase (p<0.01) by day 8 (Table 5).

Counts ranged from 1.13 to 1.47 log₁₀ CFU/mL, with higher values in T 2 and T 3 compared to T 1 . Yeast and Mould Count: Yeast and mould were not detected in any treatment throughout the storage period.

Lactobacillus acidophilus Count: Probiotic counts decreased significantly (p<0.01) during storage (Table 6). In T 2 , counts decreased from 8.41 to 7.62 log₁₀ CFU/mL, while in T 3 they decreased from 8.78 to 7.94 log₁₀ CFU/mL. Higher counts were maintained in T 3 compared to T 2 . Mean ± SE with different superscripts in a row and subscripts in a column differ significantly at 1% (p≤0.01) and 5% (p≤ 0.05) levels of significance.

Sensory Evaluation Appearance

Appearance scores differed significantly among treatments (p<0.01) (Table 7). T 3 recorded the highest mean score (8.70), followed by T 2 (8.20) and T 1 . Storage had no significant effect.

Flavour

Flavour scores were significantly influenced by treatments (p<0.01). T 3 showed the highest mean score (8.80), followed by T 2 (8.12) and T 1 (7.88).

Taste

Taste scores differed significantly among treatments (p<0.01), with T 3 recording the highest score (8.80), followed by T 2 (8.27) and T 1 (7.68). Storage effect was nonsignificant.

Overall Acceptability

Overall acceptability was significantly affected by both treatment and storage (p<0.01). T 3 recorded the highest score (8.98), followed by T 2 (8.23) and T 1 (7.77). Scores decreased slightly during storage but remained within acceptable limits. Mean ± SE with different superscripts in a row and subscripts in a column differ significantly at 1 % (p≤0.01) and 5% (p≤0.05) levels of significance.

Cost Structure

The cost of production per litre of chakka whey drink was Rs. 142.61, Rs. 152.61, and Rs. 172.51 for T 1 , T 2 , and T 3 , respectively (Table 8). The synbiotic treatment (T 3 ) showed the highest production cost.

Discussion

The present investigation on the development of probioticenriched chakka whey drink revealed significant changes in proximate composition, physico-chemical properties, microbiological quality, and sensory attributes during refrigerated storage. The observed variations among treatments and storage periods indicate the influence of probiotic and prebiotic incorporation on product quality and stability. A significant decrease in fat content was observed during storage in all treatments, with a more pronounced reduction in probiotic (T 2 ) and synbiotic (T 3 ) samples. This decline may be attributed to physicochemical instability and possible oxidative changes in fat fractions during storage. Similar findings have been reported by Madhavi (2009) [14] and Naik et al., (2023) [17] , who observed a gradual reduction in fat content in whey-based beverages during refrigerated storage. The comparatively lower fat content in T 2 and T 3 suggests the influence of microbial activity on fat destabilization.

In contrast, solids-not-fat (SNF) and total solids content increased significantly during storage, with the highest values observed in synbiotic treatment (T 3 ). The increase in SNF and total solids may be due to slight moisture loss and improved solubilization of whey constituents over time.

These results are in agreement with studies conducted by Ahmed et al., (2023) [1] and Nedanovska et al., (2022) [18] , who reported similar increasing trends in whey-based functional beverages. The higher values recorded in T 3 may be attributed to the addition of inulin, which contributes additional solids to the formulation. Protein content showed a significant decline during storage; however, synbiotic treatment (T 3 ) exhibited better protein retention compared to other treatments. The decrease in protein content may be due to denaturation and aggregation of whey proteins during storage. Similar observations have been reported by Zheng (2023) [26] and Singh et al., (2022) [23] . The improved protein stability in T 3 may be attributed to the protective effect of prebiotics, which help in maintaining protein integrity by stabilizing the matrix system.

The pH of all samples decreased significantly, while titratable acidity increased during storage, indicating progressive acidification of the whey drink. This trend is primarily due to the metabolic activity of probiotic microorganisms producing organic acids. Similar results have been reported by Madhavi (2009) [14] and Ahmed et al., (2023) [1] in fermented whey beverages. The greater reduction in pH and higher acidity observed in synbiotic treatment (T 3 ) confirms the synergistic effect of probiotics and prebiotics in enhancing fermentation activity.

Microbiological analysis revealed a significant increase in total plate count during storage, with higher counts observed in probiotic and synbiotic treatments. This indicates active microbial growth and survival in the whey matrix. Similar findings were reported by Dharani et al., (2021) [10] and Atallah and Gemiel (2020) [7] . Psychrophilic counts remained low and appeared only during later stages of storage, indicating good initial microbiological quality and effectiveness of refrigeration. The absence of yeast and mould throughout the storage period further confirms the microbiological safety of the product. The viability of Lactobacillus acidophilus decreased during storage but remained within the recommended therapeutic level, particularly in synbiotic treatment (T 3 ). The higher survival rate in T 3 may be attributed to the presence of inulin, which acts as a protective and nutritive substrate for probiotic bacteria. These findings are consistent with reports by Drgalic et al., (2005) [11] and Skryplonek and Jasińska (2015) [24] , who observed enhanced probiotic survival in synbiotic formulations. Sensory evaluation indicated that synbiotic treatment (T 3 ) received significantly higher scores for appearance, flavour, taste, and overall acceptability compared to other treatments. The improved sensory attributes may be attributed to the presence of inulin, which enhances mouthfeel and flavour profile. Similar improvements in sensory quality of synbiotic dairy products have been reported by Aryana et al., (2007) [6] and Minj and Vij (2017) [15] . Although sensory scores decreased slightly during storage, all treatments remained within acceptable limits throughout the study period.

The cost analysis indicated that synbiotic treatment (T 3 ) had the highest production cost, followed by probiotic (T 2 ) and control (T 1 ). The increased cost is mainly due to the addition of probiotic cultures and prebiotic inulin. However, the enhanced nutritional, functional, and sensory qualities of the synbiotic product justify the higher cost of production.

Overall, the findings of the present study demonstrate that incorporation of probiotics and prebiotics significantly improves the functional, microbiological, and sensory quality of chakka whey drink. The synbiotic formulation (T 3 ) showed superior performance in terms of nutritional value, probiotic viability, and consumer acceptability, making it a promising functional dairy beverage.

Conclusion

The present study demonstrates that chakka whey, a nutrient-rich dairy by-product, can be effectively utilized for the development of a probiotic and synbiotic functional beverage with desirable quality attributes. Incorporation of Lactobacillus acidophilus and inulin significantly influenced physicochemical properties, microbial viability, and sensory characteristics during refrigerated storage. The synbiotic formulation exhibited improved probiotic survivability, acceptable microbial quality, and superior sensory scores compared to the control and probiotic-only treatments.

Among the treatments evaluated, the synbiotic chakka whey drink (T 3 ) showed the most favourable results in terms of probiotic viability, product stability, and overall acceptability, indicating its potential as a functional dairy beverage. These findings support the utilization of chakka whey as a sustainable raw material for the development of value-added, health-promoting drinks with good shelf life and consumer acceptability.

References

1. Ahmed T, Sabuz AA, Mohaldar A, Fardows HS, Inbaraj BS, Sharma M, et al. Development of novel whey-mango based mixed beverage: effect of storage on physicochemical, microbiological, and sensory analysis. Foods. 2023;12(2):237-237.
2. Al-Habsi N, Al-Khalili M, Haque SA, Elias M, Olqi NA, Al Uraimi T. Health benefits of prebiotics, probiotics, synbiotics, and postbiotics. Nutrients. 2024;16(22):3955-3955.
3. AOAC. Official methods of analysis. Method No. 2003.06. 20th ed. Washington (DC): Association of Official Analytical Chemists; 2016.
4. AOAC. Official methods of analysis of AOAC International. 21st ed. Gaithersburg (MD): AOAC International; 2019.
5. APHA. Compendium of methods for the microbiological examination of food. Speck ML, editor. Washington (DC): American Public Health Association; 1984.
6. Aryana KJ, Plauche S, Rao RM, McGrew P, Shah NP. Fat-free plain yogurt manufactured with inulins of various chain lengths and Lactobacillus acidophilus. J Food Sci. 2007;72(3):M79-M84.
7. Atallah AA, Gemiel DG. Preparation of new carbonated beverages based on hydrolyzed whey by fruit and some herbs. Am J Food Sci Technol. 2020;8(2):49-55.
8. BAHS. Basic Animal Husbandry Statistics. New Delhi: Dept. of Animal Husbandry & Dairying, Ministry of Fisheries, Animal Husbandry & Dairying, Govt. of India; 2025.
9. Borkar MA. Preparation of chakka whey beverage blended with watermelon (Citrullus lanatus) juice [dissertation]. Akola: Dr. Panjabrao Deshmukh Krishi Vidyapeeth; 2020.
10. Drgalic I, Tratnik L, Bozanic R. Growth and survival of probiotic bacteria in reconstituted whey. Le Lait. 2005;85(3):171-179.
11. González-Herrera SM, Bermúdez-Quiñones G, OchoaMartínez LA, Rutiaga-Quiñones OM, Gallegos-Infante JA. Synbiotics: a technological approach in food applications. J Food Sci Technol. 2021;58(3):811-824.
12. Madavi VS, Patil BD, Kudale SM, Jodave RR, Narale SB, Kamble DK. Process standardization for preparation of whey beverage added with jamun (Syzygium cumini) juice. Plant Arch. 2025;25(1):32343240.
13. Madhavi TV. Development of synbiotic whey drink [thesis]. Anand: Anand Agricultural University; 2009.
14. Minj J, Vij S. Effect of prebiotic inulin on the fermentation and growth kinetics pattern of probiotic yoghurt bacteria. Int J Curr Microbiol Appl Sci. 2017;6(12):1755-1768.
15. Mirzakulova A, Sarsembaeva T, Suleimenova Z, Kowalski Ł, Gajdzik B, Wolniak R, et al. Whey: composition, processing, application, and prospects in functional and nutritional beverages-a review. Foods. 2025;14(18):3245-3245.
16. Naik B, Kohli D, Walter N, Gupta AK, Mishra S, Khan JM, et al. Whey-carrot based functional beverage: development and storage study. J King Saud Univ Sci. 2023;35(6):102775-102775.
17. Nedanovska E, Jakopović KL, Daniloski D, Vaskoska R, Vasiljevic T, Barukčić I. Effect of storage time on the microbial, physicochemical and sensory characteristics of ovine whey-based fruit beverages. Int J Food Sci Technol. 2022;57(8):5388-5398.
18. Nelson JA, Trout GM. Judging dairy products. 4th ed. Olsen Publishing Co.; 1964. p. 357.
19. OECD, FAO. OECD-FAO agricultural outlook 20252034: dairy and dairy products. Paris: OECD Publishing; 2025.
20. Pal AD, Bhowal S. Analysis of physiochemical and functional properties of oat-based synbiotic beverage enriched with phytonutrients. J Sci Res. 2023;15(3):769-781.
21. Sarita B, Samadhan D, Hassan MZ, Kovaleva EG. A comprehensive review of probiotics and human health: current perspectives and applications. Front Microbiol. 2025;15:1487641-1487641.
22. Singh R, Rathod G, Meletharayil GH, Kapoor R, Sankarlal VM, Amamcharla JK. Invited review: shelfstable dairy protein beverages-scientific and technological aspects. J Dairy Sci. 2022;105(12):93279346.
23. Skryplonek K, Jasińska M. Fermented probiotic beverages based on acid whey. Acta Sci Pol Technol Aliment. 2015;14(4):397-405.
24. Snedecor GW, Cochran WG. Statistical methods. Ames (IA): Iowa State University Press; 1989.
25. Zheng YY. Changes in the physicochemical, flavor and stability of whey protein solutions during UHT processing and storage: a review. Food Sci Anim Prod. 2023;1:9240006-9240006.