Development of a Probiotic-Based Health Supplement: Screening, Characterization, and Stability Assessment

Abstract

Probiotic-based health supplements have gained considerable attention due to their potential benefits in improving gut health, immunity, and overall well-being. This study focuses on the screening, characterization, and formulation of a probiotic health supplement, emphasizing stability assessment to ensure long-term efficacy. Lactic acid bacteria (LAB) were isolated from dairy samples collected from various sources and characterized using biochemical and molecular techniques. Their ability to survive under different pH conditions, bile salt concentrations, NaCl levels, and cholesterol assimilation was evaluated. Acid tolerance and adhesion assays were performed to determine their probiotic potential. A probiotic formulation for skin health was developed incorporating a probiotic blend, hyaluronic acid, grape seed extract, vitamins, and inulin. The formulation was subjected to stability testing at controlled environmental conditions to assess microbial viability and physicochemical properties. This research provides a comprehensive approach to developing a stable probiotic-based health supplement with enhanced functional benefits.

Keywords

Probiotic supplement, Lactic acid bacteria, Stability assessment, Gut health, Formulation.

Introduction

Table 1 Morphological, cultural and biochemical characteristics of isolated Lactobacillus spp. from curd samples

 

Legend:

Identification of Lactic Acid Bacteria

Table 2 Sugar fermentation chart of isolated Lactobacillus spp. from curd sample

 

Legend:

The ability of probiotic bacteria to tolerate salt stress is an important characteristic for their survival in food products and the human body. The study evaluated NaCl tolerance at concentrations ranging from 1% to 6%, measuring bacterial growth through absorbance at 600 nm. Many isolates displayed strong growth at 4% NaCl, indicating good salt tolerance. However, at 6% NaCl, only a few isolates, such as Isolate 12, continued to thrive, demonstrating robust resistance to salt stress. This suggests that certain isolates could be used in probiotic formulations where salt tolerance is required.

The optimal growth of an organism at different pH levels by measuring absorbance at 600 nm. The pH values tested include 2.00, 3.00, and 3.50, with growth measurements recorded under different conditions represented by I? to I??. At pH 2.00, the absorbance values range from 0.09 to 0.29, indicating moderate growth. At pH 3.00, the values increase slightly, ranging from 0.11 to 0.32, suggesting improved growth. The highest growth is observed at pH 3.50, with absorbance values between 0.09 and 0.33, indicating that this pH is the most favorable for the organism. Overall, the trend shows that growth improves as pH increases, suggesting that the organism thrives better in slightly less acidic conditions.

The acid tolerance test evaluated the survival of bacterial isolates under acidic conditions, mimicking the stomach environment. Bacterial counts were taken at 0 hours and 3 hours, and results showed that some isolates maintained better survival over time. Isolate 10 exhibited the highest survival rate, reducing from 102 × 10¹ CFU to 61 × 10¹ CFU, followed by Isolate 12, which reduced from 119 × 10¹ CFU to 55 × 10¹ CFU. These findings indicate that these isolates have strong acid resistance, which is essential for probiotics to survive in the digestive system.

Cholesterol assimilation was assessed by testing bacterial growth in a medium containing 0.3% oxgal and 100 mcg/mL cholesterol, measuring absorbance at 550 nm. The results showed that isolates had varying abilities to assimilate cholesterol. Isolate 12 recorded the highest absorbance (0.49), followed by Isolate 10 (0.472) and Isolate 13 (0.467). These results suggest that certain isolates have potential cholesterol-lowering properties, making them suitable for functional food applications aimed at improving heart health.

The adhesion study was conducted to evaluate the ability of Isolate 10, Isolate 12, Isolate 5, Isolate 6, and Isolate 4 to attach to HT-29 human colon carcinoma cell lines. Adhesion ability is crucial for probiotic strains as it determines their ability to colonize the intestinal lining, provide health benefits, and competitively exclude pathogens. The adhesion ability of isolates was evaluated based on the number of bacteria adhering per microscopic field, with classification as strongly adhesive (>100 bacteria per field), moderately adhesive (41–100 bacteria per field), and weakly adhesive (≤40 bacteria per field). The experimental setup included HT-29 cell seeding at 3 × 10? cells/well with a 48-hour incubation period, while probiotic strains were seeded at 6 × 10? – 41 × 10? cells/well, with an adhesion time of 2 hours. These criteria allowed for the accurate assessment of probiotic adhesion potential.

Table 3: Results of Adhesion Study

 

The adhesion study on HT-29 cell lines revealed that Isolate 5 (IL-5) and Isolate 10 (I-10) were the strongest adhesive isolates, with adhesion scores of 56 × 10? CFU/well and 41 × 10? CFU/well, respectively. Their high adhesion is likely due to surface proteins, extracellular polysaccharides (EPS), and bacterial aggregation factors, enhancing their colonization efficiency and ability to exclude pathogens. Moderately adhesive isolates, Isolate 4 (IL-4) and Isolate 6 (IL-6), demonstrated adhesion scores of 49.1 × 10? CFU/well and 39.4 × 10? CFU/well, respectively, indicating decent binding ability but lower colonization potential compared to the strongest isolates. The least adhesive isolate, Isolate 12 (I-12), showed the lowest adhesion at 13.6 × 10? CFU/well, suggesting limited gut persistence despite potential probiotic benefits. Isolate 5 (IL-5) and Isolate 10 (I-10) demonstrated the strongest adhesion, making them ideal probiotic candidates for gut health applications. Isolate 4 (IL-4) and Isolate 6 (IL-6) showed moderate adhesion, indicating some probiotic potential but lower colonization efficiency than the strongest isolates. In contrast, Isolate 12 (I-12) exhibited the weakest adhesion, making it less suitable for gut colonization. These results suggest that Isolate 5 and Isolate 10 could be effectively incorporated into probiotic formulations, while Isolate 12 may require further optimization to enhance its adhesion properties.

A probiotic-based oral health supplement was formulated using Isolate 10 and Isolate 5. These isolates were selected due to their strong probiotic characteristics, including acid and bile tolerance, cholesterol assimilation, and adhesion ability. The supplement was developed to prevent acne and improve gut health, aligning with the study’s objectives.

 

A 6-month accelerated stability study was conducted on the probiotic health supplement at 38°C and 90% relative humidity. The supplement remained stable in terms of color, odor, pH, and microbial viability over six months. However, a slight decrease in probiotic count was observed over time, with the total bacterial count reducing from 5.3 × 10? CFU to 3.77 × 10? CFU by the sixth month. Despite this reduction, the supplement maintained an acceptable shelf-life stability.

DISCUSSION

The study aimed to identify and evaluate Lactobacillus spp. from curd samples, assess their probiotic properties, and formulate a stable probiotic-based oral health supplement. The results provide insights into their potential applications in gut health and disease prevention. Bile tolerance is a key determinant of probiotic efficacy, as bacteria must withstand bile concentrations in the gastrointestinal tract to colonize and exert health benefits ²⁵. The average bile salt concentration in the human intestine ranges from 0.3% to 2.0% ²⁶. The isolates demonstrated significant survival at bile concentrations of 0.5%–2.5%, with Isolate 12 showing the highest tolerance at 2.5%. This suggests that the selected Lactobacillus strains have enhanced bile resistance, a crucial factor for gut persistence²⁷. These results align with previous studies indicating that Lactobacillus spp. can adapt to bile stress by modifying their cell membrane composition ²⁸. Salt tolerance is essential for probiotic strains used in food and pharmaceutical formulations. The study evaluated bacterial growth at NaCl concentrations of 1%–6%, with strong growth observed at 4%. The human intestine generally has an osmolarity equivalent to 0.9% NaCl ²⁹. The ability of some isolates to thrive at 6% NaCl suggests potential for their application in fermented foods and high-salt environments³⁰. Isolate 12 exhibited the highest tolerance, making it suitable for food-based probiotic formulations. The stomach’s acidic environment (pH 1.5–3.5) poses a challenge for probiotics ³¹. In this study, bacterial growth improved as pH increased from 2.0 to 3.5. The highest growth was observed at pH 3.5, indicating the isolates' ability to survive gastric conditions. Isolate 10 showed the highest survival rate under acidic conditions, making it a strong candidate for probiotic applications ³². These findings align with research demonstrating that Lactobacillus spp. enhance acid resistance through the production of protective proteins and biofilm formation ³³. Probiotic strains can lower serum cholesterol by assimilating dietary cholesterol and modifying bile salt metabolism ³⁴. The study demonstrated varying cholesterol assimilation abilities, with Isolate 12 showing the highest absorption (0.49 absorbance), followed by Isolates 10 and 13. This suggests that these strains may contribute to cholesterol reduction, aligning with previous research showing that Lactobacillus strains can enzymatically deconjugate bile acids, reducing cholesterol reabsorption ³⁵. Probiotic adhesion to intestinal cells is crucial for colonization and pathogen exclusion³⁶. The adhesion study categorized isolates into strong, moderate, and weak adhesive groups. Isolates 5 and 10 were the most adhesive, suggesting their ability to persist in the gut and confer probiotic benefits. Strong adhesion is often linked to surface proteins and extracellular polysaccharides, enhancing probiotic efficacy ³⁷. These results are consistent with studies demonstrating that Lactobacillus adhesion improves gut microbiota stability and immune modulation ³⁸. A 6-month accelerated stability study assessed the probiotic supplement’s viability under storage conditions. The total bacterial count decreased from 5.3 × 10? CFU to 3.77 × 10? CFU, indicating a decline in viability over time. However, the formulation-maintained stability in terms of sensory characteristics, pH, and microbial safety. Previous studies suggest that probiotic viability can be improved through encapsulation techniques and optimized storage conditions ³⁹. Despite the decline, the probiotic count remained within acceptable limits, confirming the supplement’s effectiveness ⁴⁰.

CONCLUSION

The study successfully identified probiotic Lactobacillus strains with strong bile and acid resistance, cholesterol assimilation, and adhesion properties. The formulated supplement  demonstrated stability, making it a potential candidate for gut health applications. Future research should explore methods to enhance probiotic viability and optimize formulation strategies for commercial applications.

ACKNOWLEDGMENT

We sincerely thank Vital Nutraceuticals Pvt. Ltd., Ambernath, Maharashtra, for their support in developing the probiotic-based health supplement. Our gratitude also goes to NBHC Lab for conducting the stability tests. Their contributions were invaluable to this project.

REFERENCES

1. 1. 1. 1. Turnbaugh, P.J., Ley, R.E., Hamady, M., Fraser-Liggett, C.M., Knight, R., & Gordon, J.I. (2021). Human gut microbiome and health: A review. Nature Reviews Microbiology, 19(3), 171-181.
         2. Cho, I., & Blaser, M.J. (2020). The microbiome in health and disease. Cell Host & Microbe, 17(1), 92-103.
         3. LeBlanc, J.G., Milani, C., de Giori, G.S., Sesma, F., van Sinderen, D., & Ventura, M. (2019). Role of the gut microbiome in vitamin synthesis. Trends in Microbiology, 27(10), 853-867.
         4. Kamada, N., Seo, S.U., Chen, G.Y., & Núñez, G. (2018). Gut microbiota and pathogen defense. Frontiers in Immunology, 9, 1335. https://doi.org/10.3389/fimmu.2018.01335
         5. Petersen, C., & Round, J.L. (2019). Dysbiosis and its clinical implications. Gut, 68(3), 526-535.
         6. O’Neill, C.A., Monteleone, G., McLaughlin, J.T., & Paus, R. (2020). Microbiome and skin disorders: An emerging connection. Journal of Dermatological Science, 98(2), 123-128.
         7. FAO/WHO (2002). Guidelines on probiotics in food. Food and Agriculture Organization/World Health Organization. https://www.fao.org/3/a0512e/a0512e.pdf
         8. Hill, C., Guarner, F., Reid, G., Gibson, G.R., Merenstein, D.J., Pot, B., ... & Sanders, M.E. (2021). Probiotics and human health: Systematic review. The Lancet, 398(10295), 437-446.
         9. Liu, Y., Alookaran, J.J., & Rhoads, J.M. (2019). Lactic acid bacteria: Mechanisms and benefits. Current Opinion in Microbiology, 49, 64-70.
         10. Marco, M.L., Pavan, S., & Kleerebezem, M. (2018). Probiotic fermentation and immune modulation. Applied and Environmental Microbiology, 84(4), e00989-18.
         11. Gopal, P.K., Prasad, J., Gill, H.S., & Shu, Q. (2020). Lactobacillus adhesion and gut colonization. Journal of Applied Microbiology, 128(3), 734-749.
         12. Jones, M.L., Martoni, C.J., Parent, M., & Prakash, S. (2019). Bifidobacterium and cholesterol reduction. Journal of Nutritional Biochemistry, 64, 74-82.
         13. Shinde, T., Perera, A.P., Vemuri, R., Gondalia, S.V., Beale, D.J., Karpe, A.V., & Stanley, R. (2020). Stability challenges in probiotic formulations. Trends in Food Science & Technology, 104, 152-161.
         14. Fang, Y., Li, X., Xue, H., & Liu, X. (2021). Effect of storage conditions on probiotic viability. Food Microbiology, 98, 103758. https://doi.org/10.1016/j.fm.2020.103758
         15. Zuo, F., Yu, R., Khaskheli, G.B., Chen, S., & Menghe, B. (2018). Probiotic delivery and gastrointestinal survival. Critical Reviews in Food Science and Nutrition, 58(14), 2588-2602.
         16. Chidre Prabhurajeshwar, Revanasiddappa Kelmani Chandrakanth.(2017). Probiotic potential of Lactobacilli with antagonistic activity against pathogenic strains: An in vitro validation for the production of inhibitory substances. Biomedical journal. 40 (27), November,270-283.
         17. Graciela FVD, Maria PT. (2001). Food microbiology protocols probiotic properties of Lactobacilli. Totowa: Spencer Humana Press Inc.
         18. Quwehand AC, Vesterlund S. (2004). Antimicrobial components from lactic acid bacteria. In: Salminen Seppo, von Wright Atte, Ouwehand Arthur, editors. Lactic acid bacteria, microbiological and functional aspects; 375-396.
         19. M.G. Shehata, S.A. El Sohaimy, Malak A. El-Sahn, M.M. Youssef. (2016). Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Annals of Agricultural Science.61(1), 65–75.
         20. Ghosh, A. R. (2011). Probiotic Potentials Among Lactic Acid Bacteria Isolated from Available online through. 2 (2). 602-609
         21. Raj Kumar Duary, Yudhishthir Singh Rajput, Virender Kumar Batish, and Sunita Grover. (2011). Assessing the adhesion of putative indigenous probiotic lactobacilli to human colonic epithelial cells. Indian J Med Res., Nov; 134(5): 664–671.
         22. Lalla, J. K., Nandedkar, S. Y., Paranjape, M. H., & Talreja, N. B. (2001). Clinical trials of ayurvedic formulations in the treatment of acne vulgaris. Journal of Ethnopharmacology, 78, 99–102.
         23. ANNEX V ASEAN GUIDELINES ON STABILITY STUDY AND SHELF-LIFE OF. (n.d.). 0–21.
         24. Benkerroum, N., Daoudi, A., Hamraoui, T., Ghalfi, H., Thiry, C., Duroy, M., Roblain, D. (2005). Lyophilized preparations of bacteriocinogenic Lactobacillus curvatus and Lactococcus lactis subsp . lactis as potential protective adjuncts to control Listeria monocytogenes in dry-fermented sausages. 56–63.
         25. 1. Ouwehand, A. C., Salminen, S., & Isolauri, E. (2002). Probiotic survival in the gastrointestinal tract. International Dairy Journal, 12(2-3), 173-182.
         26. 2. Begley, M., Hill, C., & Gahan, C. G. M. (2005). The interaction between bacteria and bile. FEMS Microbiology Reviews, 29(4), 625-651.
         27. 3. Gilliland, S. E., Staley, T. E., & Bush, L. J. (1984). Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. Journal of Dairy Science, 67(12), 3045-3051.
         28. 4. Saarela, M., Mogensen, G., Fondén, R., Mättö, J., & Mattila-Sandholm, T. (2000). Probiotic bacteria: Safety, functional and technological properties. Journal of Biotechnology, 84(3), 197-215.
         29. 5. FAO/WHO. (2002). Guidelines for the evaluation of probiotics in food. Food and Agriculture Organization of the United Nations/World Health Organization.
         30. 6. Kailasapathy, K., & Chin, J. (2000). Survival and therapeutic potential of probiotic organisms with reference to Lactobacillus acidophilus and Bifidobacterium spp. Immunology and Cell Biology, 78(1), 80-88.
         31. 7. Marteau, P., Minekus, M., Havenaar, R., & Huis in’t Veld, J. H. (1997). Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: Validation and the effects of bile. Journal of Dairy Science, 80(6), 1031-1037.
         32. 8. Charteris, W. P., Kelly, P. M., Morelli, L., & Collins, J. K. (1998). Development and application of an in vitro methodology to determine the transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species in the upper human gastrointestinal tract. Journal of Applied Microbiology, 84(5), 759-768.
         33. 9. Corcoran, B. M., Stanton, C., Fitzgerald, G. F., & Ross, R. P. (2005). Survival of probiotic lactobacilli in acidic environments is enhanced in the presence of metabolizable sugars. Applied and Environmental Microbiology, 71(6), 3060-3067.
         34. 10. Pereira, D. I., & Gibson, G. R. (2002). Cholesterol assimilation by lactic acid bacteria and bifidobacteria isolated from the human gut. Applied and Environmental Microbiology, 68(9), 4689-4693.
         35. 11. Liong, M. T., & Shah, N. P. (2005). Acid and bile tolerance and cholesterol removal ability of lactobacilli strains. Journal of Dairy Science, 88(1), 55-66.
         36. 12. Ouwehand, A. C., Kirjavainen, P. V., Shortt, C., & Salminen, S. (1999). Probiotic and other functional microbes: From markets to mechanisms. Current Opinion in Biotechnology, 10(5), 483-487.
         37. 13. Adlerberth, I., Ahrné, S., Johansson, M. L., Molin, G., Hanson, L. A., & Wold, A. E. (1996). A mannose-specific adherence mechanism in Lactobacillus plantarum conferring binding to the human colonic cell line HT-29. Applied and Environmental Microbiology, 62(7), 2244-2251.
         38. 14. Vinderola, G., & Reinheimer, J. (2003). Lactic acid starter and probiotic bacteria: A comparative “in vitro” study of probiotic characteristics and biological barrier resistance. Food Research International, 36(9-10), 895-904.
         39. 15. Shah, N. P. (2000). Probiotic bacteria: Selective enumeration and survival in dairy foods. Journal of Dairy Science, 83(4), 894-907.
         40. 16. Mattila-Sandholm, T., Myllärinen, P., Crittenden, R., Mogensen, G., Fonden, R., & Saarela, M. (2002). Technological challenges for future probiotic foods. International Dairy Journal, 12(2-3), 173-182.