Refrigerated storage stability, probiotic survivability and techno-economic feasibility of dragon-fruit-enriched synbiotic buttermilk

Introduction

Fermented dairy beverages are widely used as carriers for beneficial microorganisms because the milk matrix supplies fermentable nutrients and can protect bacterial cells during manufacture, storage and passage through the gastrointestinal tract. A probiotic is a live microorganism that, when administered in an adequate amount, confers a health benefit on the host (Hill et al., 2014) [1] . A synbiotic combines live microorganisms with substrate(s) selectively utilized by host microorganisms to provide a health benefit (Swanson et al., 2020) [2] . Accordingly, the functional value of a synbiotic beverage depends not merely on adding a culture but on retaining an adequate viable population until consumption. Bifidobacterium species are valued in functional foods but are technologically demanding because their survival is affected by strain, oxygen exposure, acidity, storage temperature and the physicochemical characteristics of the food matrix. Refrigerated fermented products commonly undergo post-acidification, with declining pH and increasing titratable acidity, and these changes may progressively reduce probiotic survival (Mani-López et al., 2014; Shori, 2022) [3,4] . Reviews of dairy probiotic foods emphasize that formulation and matrix composition are decisive determinants of bifidobacterial stability (Sibanda et al., 2024) [5] . Red dragon fruit is a promising adjunct for cultured dairy beverages. Its carbohydrates and soluble fibre may support microbial metabolism, while its natural pigments and phytochemicals can improve product appeal and functional positioning. However, fruit addition also changes the acidity, nutrient environment and spoilage risk of a fermented beverage. A storage study is therefore essential before claims concerning probiotic delivery, shelf life or commercial feasibility can be made. Packaging may further influence stability through differences in oxygen, moisture and light barriers. Glass is chemically inert and offers a strong gas barrier, whereas PET is lighter and less breakable but can differ in permeability. Comparing product acidity and pH in the two packages during refrigeration provides a practical indication of whether either container accelerates post-acidification.

The present work focused on the two downstream objectives of the product-development study: (i) evaluating the viability of Bifidobacterium during refrigeration and (ii) assessing shelf life and techno-economic feasibility. The optimized beverage containing 10% dragon-fruit pulp was monitored for viable LAB and Bifidobacterium, hygiene indicators, packaging-related pH and acidity changes, and estimated production cost.

Materials and Methods

Product formulation and preparation

The optimized formulation (T 2 ) comprised 90% buttermilk and 10% red dragon-fruit pulp and contained 2% mixed lactic starter culture and 2% Bifidobacterium bifidum culture. Cow milk was heated at 90 °C for 5 min, cooled to 40-42 °C, inoculated with both cultures and incubated at 37 ± 1 °C for 6-8 h until a firm coagulum and a titratable acidity of approximately 0.7-0.9% lactic acid were obtained. The curd was churned, blended with pasteurized dragon-fruit pulp and permitted salt/spices, homogenized, filled into pre-sterilized bottles and held at 4 ± 1 °C.

Culture activation and maintenance

The freeze-dried B. bifidum and mixed lactic starter were activated through three successive transfers using 1% inoculum at 37 °C for 24 h. TPY broth supplemented with L-cysteine hydrochloride was used for B. bifidum; sterile skim milk or MRS broth was used for the mixed starter. Bifidobacterial cultures were incubated anaerobically at 37 °C for 24-48 h. Purity was checked by Gram staining and catalase reaction. Working cultures containing approximately 1 × 10 8 CFU/mL were stored at 4 ± 1 °C and subcultured at fortnightly intervals.

Refrigerated-storage design

The optimized beverage was examined on days 0, 2, 4, 6 and 8 of storage at 4 ± 1 °C. These actual sampling points were used for microbial quality, selective probiotic survivability and packaging comparisons. Glass and PET bottles were evaluated separately for pH and titratable acidity. All microbiological determinations were performed in triplicate.

Microbiological quality

For general aerobic enumeration, a 10-mL beverage sample was homogenized with 90 mL sterile 0.1% peptone water and serially diluted. One-millilitre aliquots were pour-plated with 15-20 mL molten plate-count agar at 45 ± 1 °C and incubated at 37 ± 1 °C for 48 ± 2 h. Yeasts and moulds were enumerated by spreading 0.1 mL of appropriate dilutions on potato dextrose agar acidified to pH 3.5 ± 0.1, followed by incubation at 25 ± 1 °C for 5 days. Coliforms were assessed by the violet-red bile agar pour-plate procedure with an overlay and incubation at 37 ± 1 °C for 24 ± 2 h. Results were expressed as CFU/mL; absence of colonies at the tested dilution was recorded as not detected (ND). The procedures were adapted from ISO 4833-1:2013 [6] , ISO 21527-1:2008 [7] and ISO 4832:2006 [8] , respectively.

LAB and Bifidobacterium survivability

For selective LAB enumeration, serial dilutions were pour-plated on de Man-Rogosa-Sharpe agar and incubated anaerobically at 37 ± 1 °C for 48-72 h. B. bifidum was enumerated on MRS agar supplemented with 0.05% L-cysteine hydrochloride and incubated anaerobically at 37 ± 1 °C for 72 h. Plates containing 30-300 colonies were counted, and viable populations were calculated from colony number, dilution factor and plated volume and expressed as CFU/mL. MRS medium was based on de Man et al. (1960) [9] .

Packaging response

The pH of beverage samples stored in glass and PET bottles was measured on each sampling day using a calibrated digital pH meter. Titratable acidity was determined by neutralization with standard alkali and expressed as percentage lactic acid. Standard error (SE) and critical difference (CD) at the 5% level were available for each storage-day comparison.

Techno-economic assessment

Ingredient, culture, fruit, packaging and labelling costs were calculated using the prevailing purchase prices recorded during the experiment. Processing cost was taken as 20% of raw-material cost. Total batch cost, cost per litre and cost per 200-mL serving were then calculated. The estimate excludes profit margin, distribution, marketing, depreciation and taxes and therefore represents production cost rather than a final retail price. CFU/mL (13.5%). Despite this reduction, the final count remained high. Refrigerated storage slows bacterial metabolism but does not completely prevent injury caused by acidity, nutrient depletion or accumulated fermentation products. A gradual loss of viable cells is consequently expected in cultured dairy products. Coliforms were not detected at any sampling point, supporting satisfactory hygienic preparation and handling under the conditions used. Yeast and mould were absent through day 6 but reached 1.12 × 10 4 CFU/mL on day 8. This late appearance is the clearest shelf-life constraint in the dataset. Although the probiotic population remained abundant, a functional count alone cannot establish acceptable shelf life when spoilage organisms emerge. Therefore, the evidence supports a conservative refrigerated shelf life of up to 6 days for the present process unless day-8 acceptability is demonstrated against an applicable microbiological specification and confirmed in replicate production batches. CFU/mL, within the range frequently cited as desirable for probiotic dairy carriers (Shori, 2022) [4] . This comparison describes numerical retention; statistical significance cannot be inferred because dispersion estimates for the viability table were not reported. The retention may reflect the nutrient-rich milk matrix and the contribution of fermentable carbohydrates or soluble fibre from dragon fruit. Conversely, the downward trend is consistent with the sensitivity of bifidobacteria to increasing acidity and oxygen. Shori (2022) [4] emphasized that food matrix, strain, active ingredients and storage conditions jointly govern bifidobacterial survival, while Sibanda et al. (2024) [5] identified shelf-life viability as a continuing challenge in dairy foods. The present decline was smaller than the substantial reductions that can occur in some commercial fermented products, although direct comparisons require identical strains, media and storage durations.

Results and Discussion

Microbial quality during refrigerated storage

Survival of LAB and Bifidobacterium

The general LAB dataset in Table 1 and the selective survivability dataset in Table 2 were generated for different analytical purposes and should not be treated as interchangeable. Their different numerical levels likely reflect the enumeration media and operational definitions used. Future studies should specify the starter organisms recovered by each medium and present counts as log 10 CFU/mL with standard deviations to improve comparability. [3] similarly reported lower pH and higher titratable acidity during storage of yogurts and fermented milks.

Effect of packaging on post-acidification

At days 0, 2, 4 and 8, the absolute glass-PET pH difference was 0.10 unit and exceeded the reported CD for the corresponding day (0.083-0.094); there was no difference on day 6. For acidity, the packages were identical on days 0 and 4, and their 0.01-percentage-point differences on days 2, 6 and 8 exceeded the reported CD only on day 2 (0.008). Thus, statistically detectable point differences occurred, but neither package showed consistently superior control of both variables. The two materials produced broadly comparable acidification profiles over the short storage period.

Techno-economic feasibility

Conclusion

Dragon-fruit-enriched synbiotic buttermilk retained high viable populations during 8 days at 4 ± 1 °C. Selective LAB and Bifidobacterium counts decreased by only 3.23% and 3.36%, respectively, and Bifidobacterium remained above 8.6 × 10 7 CFU/mL. Glass and PET bottles both supported comparable short-term storage, although continued post-acidification occurred. Coliforms were absent throughout; however, yeast and mould appeared on day 8. The safest conclusion from the available evidence is therefore a provisional refrigerated shelf life of 6 days rather than 8 days. The calculated production cost of ₹14.22 per 200-mL serving suggests affordability at the experimental scale, with packaging as the largest cost component. Further work should validate shelf life across independent batches, include sensory data at every storage point, report replicate variability and determine an applicable microbiological acceptance limit before commercial release.

Study Limitations

The storage experiment covered only 8 days and did not report replicate dispersion for viable counts. Sensory acceptance during storage and package-specific probiotic counts were not available. The cost analysis used prevailing experimental prices and omitted several commercial overheads. In addition, the original study used the term 'synbiotic' operationally for the combined culture-and-fruit formulation; definitive classification under the current ISAPP definition would require evidence that the fruit-derived substrate is selectively utilized and that the combined product confers a health benefit (Swanson et al., 2020) [2] . These limitations restrict generalization but do not alter the observed direction of change.

Acknowledgement

AcknowledgementsThe authors acknowledge the Department of Food Microbiology and Safety, College of Food Technology, Vasantrao Naik Marathwada Krishi Vidyapeeth, Parbhani, Maharashtra, India, for providing facilities for the investigation.

References

1. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nature Reviews Gastroenterology & Hepatology. 2014;11:506-514.
2. Swanson KS, Gibson GR, Hutkins R, Reimer RA, Reid G, Verbeke K, Scott KP, Holscher HD, Azad MB, Delzenne NM, Sanders ME. The International Scientific Association for Probiotics and Prebiotics consensus statement on the definition and scope of synbiotics. Nature Reviews Gastroenterology & Hepatology. 2020;17:687-701.
3. Mani-López E, Palou E, López-Malo A. Probiotic viability and storage stability of yogurts and fermented milks prepared with several mixtures of lactic acid bacteria. Journal of Dairy Science. 2014;97(5):2578-2590.
4. Shori AB. Application of Bifidobacterium spp. in beverages and dairy food products: An overview of survival during refrigerated storage. Food Science and Technology. 2022;42:e41520.
5. Sibanda T, Marole TA, Thomashoff UL, Thantsha MS, Buys EM. Bifidobacterium species viability in dairy-based probiotic foods: Challenges and innovative approaches for accurate viability determination and monitoring of probiotic functionality. Frontiers in Microbiology. 2024;15:1327010.
6. International Organization for Standardization. ISO 4833-1:2013: Microbiology of the food chain-Horizontal method for the enumeration of microorganisms-Part 1: Colony count at 30 °C by the pour plate technique. ISO; 2013.
7. International Organization for Standardization. ISO 215271:2008: Microbiology of food and animal feeding stuffsHorizontal method for the enumeration of yeasts and mouldsPart 1: Colony count technique in products with water activity greater than 0.95. ISO; 2008.
8. International Organization for Standardization. ISO 4832:2006: Microbiology of food and animal feeding stuffs-Horizontal method for the enumeration of coliforms-Colony-count technique. ISO; 2006.
9. de Man JC, Rogosa M, Sharpe ME. A medium for the cultivation of lactobacilli. Journal of Applied Bacteriology. 1960;23(1):130135.