Screening, isolation, characterization, and optimization of BSH activity from potential probiotic isolates from various sources

Koushik Koujalagi; Alok Kumar Malaviya

doi:10.7324/JABB.2025.224557

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Abstract

Bile salt hydrolase (BSH)-producing probiotics can assimilate cholesterol from the body through de novo synthesis. The BSH enzyme was found in 23 of 513 isolates accessed from various sources. Five of the 23 BSH-positive strains have been selected for further study, based on their BSH activity, compared to two positive controls, Lactobacillus acidophilus and Enterococcus lactis. The Grams nature of the strains was determined and further examined for hemolytic activity, gelatinase, and catalase assay as per Indian Council for Medical Research—Department of Biotechnology recommendations. Two Enterococcus faecalis (CGz3 and CGz4) strains with γ-hemolytic, negative catalase, and gelatinase activity are selected for probiotic characterization, evaluating the organisms surface hydrophobicity, autoaggregation tests, tolerance to lysozyme, gastric acidity, bile salt and gastric juices (pepsin and pancreatin). The strain which withstands the harsh gastrointestinal conditions was considered for further experiments. To establish a standardized method to quantify the BSH activity of the potential probiotic isolate, substrate utilization was performed by screening sodium glycocholate (GCA) and taurocholic acid (TCA) at different concentrations. The optimal BSH activity was observed at the 16th hour and 0.1% (v/v) GCA. Based on the standardized protocol, factorial optimization of process parameters, such as pH, inoculum percentage, temperature, and revolutions per minute (RPM) was carried out for increased BSH activity. The optimal BSH activity was observed at pH 5.5 and 1% inoculum (v/v). The highest BSH activity was obtained at 40°C and 200 RPM. Among the other BSH-positive strains, E. faecalis CGz3 shows the best probiotic potential. The strain would be further studied for its ability to alleviate symptoms associated with non-alcoholic fatty liver disease (NAFLD), using a cell line-based study and associated gene regulation. In conclusion, E. faecalis CGz3 would have the potential to be used as a dietary supplement to treat metabolic disorders, such as hypercholesterolemia and NAFLD/metabolic-associated fatty liver disease.

Keyword: Bile salt hydrolase cholesterol assimilation probiotics process parameter optimization and thin-layer chromatography (TLC)

Citation:

Koujalagi K, Malaviya AK. Screening, isolation, characterization, and optimization of BSH activity from potential probiotic isolates from various sources. J Appl Biol Biotech. 2025;13(Suppl 1): 86–96. http://doi.org/10.7324/JABB.2025.224557

Copyright: Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license.

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Reference

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2. Kim DH, Park MH, Choi YJ, Chung KW, Park CH, Jang EJ, et al. Molecular study of dietary heptadecane for the anti-inflammatory modulation of NF-kB in the aged kidney. PLoS One 2013;8(3):e59316. https://doi.org/10.1371/journal.pone.0059316
3. Riaz Rajoka MS, Mehwish HM, Siddiq M, Haobin Z, Zhu J, Yan L, et al. Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk. Lwt 2017; 84:271-80. https://doi.org/10.1016/j.lwt.2017.05.055
4. Havenaar R, Huis In't Veld JHJ. Probiotics: a general view. Lact Acid Bact 1992;1:151-70. https://doi.org/10.1007/978-1-4615-3522-5_6
5. Kimoto H, Ohmomo S, Okamoto T. Cholesterol removal from media by lactococci. J Dairy Sci 2002;85(12):3182-8. https://doi.org/10.3168/jds.S0022-0302(02)74406-8
6. Lechner K, Mckenzie AL, Kränkel N, Von Schacky C, Worm N, Nixdorff U, et al. High-risk atherosclerosis and metabolic phenotype: the roles of ectopic adiposity, atherogenic dyslipidemia, and inflammation. Metab Syndr Relat Disord 2020;18(4):176-85. https://doi.org/10.1089/met.2019.0115
7. De Rodas BZ, Gilliland SE, Maxwell CV. Hypocholesterolemic action of Lactobacillus acidophilus ATCC 43121 and calcium in swine with hypercholesterolemia induced by diet. J Dairy Sci 1996 Dec;79(12):2121-8. https://doi.org/10.3168/jds.S0022-0302(96)76586-4
8. Kneifel W, Jaros D, Erhard F. Microflora and acidification properties of yogurt and yogurt-related products fermented with commercially available starter cultures. Int J Food Microbiol 1993;18(3):179-89. https://doi.org/10.1016/0168-1605(93)90043-G
9. Mombelli B, Gismondo MR. The use of probiotics in medical practice. Int J Antimicrob Agents 2000;16(4):531-6. https://doi.org/10.1016/S0924-8579(00)00322-8
10. Krasaekoopt W, Bhandari B, Deeth H. Evaluation of encapsulation techniques of probiotics for yoghurt. Int Dairy J [Internet] 2003;13(1):3-13. Available via https://www.sciencedirect.com/ science/article/pii/S0958694602001553 https://doi.org/10.1016/S0958-6946(02)00155-3
11. Power DA, Burton J, Chilcott C, Dawes P, Tagg J. Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Streptococcus salivarius K12. Eur J Clin Microbiol Infect Dis 2008;27:1261-3. https://doi.org/10.1007/s10096-008-0569-4
12. Vinderola CG, Reinheimer JA. Lactic acid starter and probiotic bacteria: a comparative "in vitro" study of probiotic characteristics and biological barrier resistance. Food Res Int [Internet] 2003;36:895-904. Available via https://api.semanticscholar.org/ CorpusID:83480721 https://doi.org/10.1016/S0963-9969(03)00098-X
13. Chikai T, Nakao H, Uchida K. Deconjugation of bile acids by human intestinal bacteria implanted in germ-free rats. Lipids [Internet] 1987;22(9):669-71. doi: 10.1007/BF02533948 https://doi.org/10.1007/BF02533948
14. Roy A, Panja AS, Maitra M, Bandopyadhyay B. Isolation and characterizations of probiotics from bovine (Cow) milk. Res Rev Biotechnol Biosci 2019;6(2):24-32.
15. Acurcio LB, Souza MR, Nunes AC, Oliveira DLS, Sandes SHC, Alvim LB. Isolation, enumeration, molecular identification and probiotic potential evaluation of lactic acid bacteria isolated from sheep milk. Arq Bras Med Vet Zootec 2014;66(3):940-8. https://doi.org/10.1590/1678-41625796
16. Shafakatullah N, Chandra M. Screening of raw Buffalo's milk from Karnataka for potential probiotic strains. Res J Recent Sci Res J Recent Sci 2014;3(9):73-8.
17. Kimprasit T, Sukontasing S, Amavisit P. In vitro selection of potential lactic acid bacteria isolated from ducks and geese in Thailand. Kasetsart J Nat Sci 2013;47(2):261-70.
18. Knarreborg A, Engberg RM, Jensen SK, Jensen BB. Quantitative determination of bile salt hydrolase activity in bacteria isolated from the small intestine of chickens. Appl Environ Microbiol. 2002;68(12):6425-8. https://doi.org/10.1128/AEM.68.12.6425-6428.2002
19. Idoui T. Probiotic properties of Lactobacillus strains isolated from gizzard of local poultry. Iran J Microbiol 2014;6(2):120-6.
20. Pramono_Screening and characterization of bacteria like producing lactic acid bacteria from mud cr.pdf.
21. Buntin N, Chanthachum S, Hongpattarakere T. Screening of lactic acid bacteria from gastrointestinal tracts of marine fish for their potential use as probiotics. Songklanakarin J Sci Technol 2008;30(SUPPL. 1):141-8.
22. Sedlá?ková P, Horá?ková Š, Shi T, Kosová M, Plocková M. Two different methods for screening of bile salt hydrolase activity in Lactobacillus strains. Czech J Food Sci 2015;2015(1):13-8. https://doi.org/10.17221/299/2014-CJFS
23. Coico R. Gram staining basic protocol commonly used techniques. Curr Protoc Microbiol 2005;3-4. https://doi.org/10.1002/9780471729259.mca03cs00
24. Shuhadha MFF, Panagoda GJ, Madhujith T, Jayawardana NWIA. Evaluation of probiotic attributes of Lactobacillus sp. isolated from cow and buffalo curd samples collected from Kandy. Ceylon Med J 2017;62(3):159. https://doi.org/10.4038/cmj.v62i3.8519
25. Perin LM, Miranda RO, Todorov SD, de Franco BDGM, Nero LA. Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk. Int J Food Microbiol [Internet] 2014;185:121-6; doi: 10.1016/j. ijfoodmicro.2014.06.001 https://doi.org/10.1016/j.ijfoodmicro.2014.06.001
26. Ruiz-Moyano S, Martín A, Benito MJ, Casquete R, Serradilla MJ, de Córdoba MG. Safety and functional aspects of pre-selected lactobacilli for probiotic use in Iberian dry-fermented sausages. Meat Sci [Internet] 2009;83(3):460-7; doi: 10.1016/j.meatsci.2009.06.027 https://doi.org/10.1016/j.meatsci.2009.06.027
27. Nueno-Palop C, Narbad A. Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut. Int J Food Microbiol 2011;145(2-3):390-4. https://doi.org/10.1016/j.ijfoodmicro.2010.12.029
28. Liu Z, Jiang Z, Zhou K, Li P, Liu G, Zhang B. Screening of bifidobacteria with acquired tolerance to human gastrointestinal tract. Anaerobe 2007;13(5-6):215-9. https://doi.org/10.1016/j.anaerobe.2007.05.002
29. Balcázar JL, Vendrell D, de Blas I, Ruiz-Zarzuela I, Muzquiz JL, Girones O. Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish. Aquaculture 2008;278(1-4):188-91. https://doi.org/10.1016/j.aquaculture.2008.03.014
30. Vizoso Pinto MG, Franz CMAP, Schillinger U, Holzapfel WH. Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products. Int J Food Microbiol 2006;109(3):205-14. https://doi.org/10.1016/j.ijfoodmicro.2006.01.029
31. Osmanagaoglu O, Kiran F, Ataoglu H. Evaluation of in vitro probiotic potential of Pediococcus pentosaceus OZF isolated from human breast milk. Probiotics Antimicrob Proteins 2010;2(3):162-74. https://doi.org/10.1007/s12602-010-9050-7
32. Gregory R, Pascoe TA. The quantitative determination of bile acids by means of a new color reaction and monochromatic light. J Biol Chem [Internet] 1929;83(1):35-42; doi: 10.1016/S0021-9258(20)70835-5 https://doi.org/10.1016/S0021-9258(20)70835-5
33. Ahmadova A, Todorov SD, Choiset Y, Rabesona H, Mirhadi Zadi T, Kuliyev A, et al. Evaluation of antimicrobial activity, probiotic properties and safety of wild strain Enterococcus faecium AQ71 isolated from Azerbaijani Motal cheese. Food Control [Internet] 2013;30(2):631-41; doi: 10.1016/j.foodcont.2012.08.009 https://doi.org/10.1016/j.foodcont.2012.08.009
34. Qin X, Singh KV, Weinstock GM, Murray BE. Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence. Infect Immun 2000;68(5):2579-86. https://doi.org/10.1128/IAI.68.5.2579-2586.2000
35. Liong MT, Shah NP. Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains. Int Dairy J [Internet] 2005;15(4):391-8. Available via https://www. sciencedirect.com/science/article/pii/S0958694604002109 https://doi.org/10.1016/j.idairyj.2004.08.007
36. Begley M, Hill C, Gahan CGM. Bile salt hydrolase activity in probiotics. Appl Environ Microbiol 2006;72(3):1729-38. https://doi.org/10.1128/AEM.72.3.1729-1738.2006
37. Malaviya A, Malviya S, Agarwal A, Mishra M, Dalmida V. Saccharomyces-eukaryotic probiotic for human applications. In: High value fermentation products, John Wiley & Sons, Hoboken, NJ, pp 211-29, 2019. https://doi.org/10.1002/9781119555384.ch11
38. Zhu Y, Li T, Din AU, Hassan A, Wang Y, Wang G. Beneficial effects of Enterococcus faecalis in hypercholesterolemic mice on cholesterol transportation and gut microbiota. Appl Microbiol Biotechnol 2019;103(7):3181-91. https://doi.org/10.1007/s00253-019-09681-7
39. Asiva Noor Rachmayani. No Covariance structure analysis of health-related indicators in elderly people living at home, focusing on subjective health Title. 2015;6.

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1. Collado MC, Meriluoto J, Salminen S. Adhesion and aggregation properties of probiotic and pathogen strains [Internet]. Eur Food Res Technol 2008; 226:1065-73. Available via https://api. semanticscholar.org/CorpusID:85423542 https://doi.org/10.1007/s00217-007-0632-x

2. Kim DH, Park MH, Choi YJ, Chung KW, Park CH, Jang EJ, et al. Molecular study of dietary heptadecane for the anti-inflammatory modulation of NF-kB in the aged kidney. PLoS One 2013;8(3):e59316. https://doi.org/10.1371/journal.pone.0059316

3. Riaz Rajoka MS, Mehwish HM, Siddiq M, Haobin Z, Zhu J, Yan L, et al. Identification, characterization, and probiotic potential of Lactobacillus rhamnosus isolated from human milk. Lwt 2017; 84:271-80. https://doi.org/10.1016/j.lwt.2017.05.055

4. Havenaar R, Huis In't Veld JHJ. Probiotics: a general view. Lact Acid Bact 1992;1:151-70. https://doi.org/10.1007/978-1-4615-3522-5_6

5. Kimoto H, Ohmomo S, Okamoto T. Cholesterol removal from media by lactococci. J Dairy Sci 2002;85(12):3182-8. https://doi.org/10.3168/jds.S0022-0302(02)74406-8

6. Lechner K, Mckenzie AL, Kränkel N, Von Schacky C, Worm N, Nixdorff U, et al. High-risk atherosclerosis and metabolic phenotype: the roles of ectopic adiposity, atherogenic dyslipidemia, and inflammation. Metab Syndr Relat Disord 2020;18(4):176-85. https://doi.org/10.1089/met.2019.0115

7. De Rodas BZ, Gilliland SE, Maxwell CV. Hypocholesterolemic action of Lactobacillus acidophilus ATCC 43121 and calcium in swine with hypercholesterolemia induced by diet. J Dairy Sci 1996 Dec;79(12):2121-8. https://doi.org/10.3168/jds.S0022-0302(96)76586-4

8. Kneifel W, Jaros D, Erhard F. Microflora and acidification properties of yogurt and yogurt-related products fermented with commercially available starter cultures. Int J Food Microbiol 1993;18(3):179-89. https://doi.org/10.1016/0168-1605(93)90043-G

9. Mombelli B, Gismondo MR. The use of probiotics in medical practice. Int J Antimicrob Agents 2000;16(4):531-6. https://doi.org/10.1016/S0924-8579(00)00322-8

10. Krasaekoopt W, Bhandari B, Deeth H. Evaluation of encapsulation techniques of probiotics for yoghurt. Int Dairy J [Internet] 2003;13(1):3-13. Available via https://www.sciencedirect.com/ science/article/pii/S0958694602001553 https://doi.org/10.1016/S0958-6946(02)00155-3

11. Power DA, Burton J, Chilcott C, Dawes P, Tagg J. Preliminary investigations of the colonisation of upper respiratory tract tissues of infants using a paediatric formulation of the oral probiotic Streptococcus salivarius K12. Eur J Clin Microbiol Infect Dis 2008;27:1261-3. https://doi.org/10.1007/s10096-008-0569-4

12. Vinderola CG, Reinheimer JA. Lactic acid starter and probiotic bacteria: a comparative "in vitro" study of probiotic characteristics and biological barrier resistance. Food Res Int [Internet] 2003;36:895-904. Available via https://api.semanticscholar.org/ CorpusID:83480721 https://doi.org/10.1016/S0963-9969(03)00098-X

13. Chikai T, Nakao H, Uchida K. Deconjugation of bile acids by human intestinal bacteria implanted in germ-free rats. Lipids [Internet] 1987;22(9):669-71. doi: 10.1007/BF02533948 https://doi.org/10.1007/BF02533948

14. Roy A, Panja AS, Maitra M, Bandopyadhyay B. Isolation and characterizations of probiotics from bovine (Cow) milk. Res Rev Biotechnol Biosci 2019;6(2):24-32.

15. Acurcio LB, Souza MR, Nunes AC, Oliveira DLS, Sandes SHC, Alvim LB. Isolation, enumeration, molecular identification and probiotic potential evaluation of lactic acid bacteria isolated from sheep milk. Arq Bras Med Vet Zootec 2014;66(3):940-8. https://doi.org/10.1590/1678-41625796

16. Shafakatullah N, Chandra M. Screening of raw Buffalo's milk from Karnataka for potential probiotic strains. Res J Recent Sci Res J Recent Sci 2014;3(9):73-8.

17. Kimprasit T, Sukontasing S, Amavisit P. In vitro selection of potential lactic acid bacteria isolated from ducks and geese in Thailand. Kasetsart J Nat Sci 2013;47(2):261-70.

18. Knarreborg A, Engberg RM, Jensen SK, Jensen BB. Quantitative determination of bile salt hydrolase activity in bacteria isolated from the small intestine of chickens. Appl Environ Microbiol. 2002;68(12):6425-8. https://doi.org/10.1128/AEM.68.12.6425-6428.2002

19. Idoui T. Probiotic properties of Lactobacillus strains isolated from gizzard of local poultry. Iran J Microbiol 2014;6(2):120-6.

20. Pramono_Screening and characterization of bacteria like producing lactic acid bacteria from mud cr.pdf.

21. Buntin N, Chanthachum S, Hongpattarakere T. Screening of lactic acid bacteria from gastrointestinal tracts of marine fish for their potential use as probiotics. Songklanakarin J Sci Technol 2008;30(SUPPL. 1):141-8.

22. Sedlá?ková P, Horá?ková Š, Shi T, Kosová M, Plocková M. Two different methods for screening of bile salt hydrolase activity in Lactobacillus strains. Czech J Food Sci 2015;2015(1):13-8. https://doi.org/10.17221/299/2014-CJFS

23. Coico R. Gram staining basic protocol commonly used techniques. Curr Protoc Microbiol 2005;3-4. https://doi.org/10.1002/9780471729259.mca03cs00

24. Shuhadha MFF, Panagoda GJ, Madhujith T, Jayawardana NWIA. Evaluation of probiotic attributes of Lactobacillus sp. isolated from cow and buffalo curd samples collected from Kandy. Ceylon Med J 2017;62(3):159. https://doi.org/10.4038/cmj.v62i3.8519

25. Perin LM, Miranda RO, Todorov SD, de Franco BDGM, Nero LA. Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk. Int J Food Microbiol [Internet] 2014;185:121-6; doi: 10.1016/j. ijfoodmicro.2014.06.001 https://doi.org/10.1016/j.ijfoodmicro.2014.06.001

26. Ruiz-Moyano S, Martín A, Benito MJ, Casquete R, Serradilla MJ, de Córdoba MG. Safety and functional aspects of pre-selected lactobacilli for probiotic use in Iberian dry-fermented sausages. Meat Sci [Internet] 2009;83(3):460-7; doi: 10.1016/j.meatsci.2009.06.027 https://doi.org/10.1016/j.meatsci.2009.06.027

27. Nueno-Palop C, Narbad A. Probiotic assessment of Enterococcus faecalis CP58 isolated from human gut. Int J Food Microbiol 2011;145(2-3):390-4. https://doi.org/10.1016/j.ijfoodmicro.2010.12.029

28. Liu Z, Jiang Z, Zhou K, Li P, Liu G, Zhang B. Screening of bifidobacteria with acquired tolerance to human gastrointestinal tract. Anaerobe 2007;13(5-6):215-9. https://doi.org/10.1016/j.anaerobe.2007.05.002

29. Balcázar JL, Vendrell D, de Blas I, Ruiz-Zarzuela I, Muzquiz JL, Girones O. Characterization of probiotic properties of lactic acid bacteria isolated from intestinal microbiota of fish. Aquaculture 2008;278(1-4):188-91. https://doi.org/10.1016/j.aquaculture.2008.03.014

30. Vizoso Pinto MG, Franz CMAP, Schillinger U, Holzapfel WH. Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products. Int J Food Microbiol 2006;109(3):205-14. https://doi.org/10.1016/j.ijfoodmicro.2006.01.029

31. Osmanagaoglu O, Kiran F, Ataoglu H. Evaluation of in vitro probiotic potential of Pediococcus pentosaceus OZF isolated from human breast milk. Probiotics Antimicrob Proteins 2010;2(3):162-74. https://doi.org/10.1007/s12602-010-9050-7

32. Gregory R, Pascoe TA. The quantitative determination of bile acids by means of a new color reaction and monochromatic light. J Biol Chem [Internet] 1929;83(1):35-42; doi: 10.1016/S0021-9258(20)70835-5 https://doi.org/10.1016/S0021-9258(20)70835-5

33. Ahmadova A, Todorov SD, Choiset Y, Rabesona H, Mirhadi Zadi T, Kuliyev A, et al. Evaluation of antimicrobial activity, probiotic properties and safety of wild strain Enterococcus faecium AQ71 isolated from Azerbaijani Motal cheese. Food Control [Internet] 2013;30(2):631-41; doi: 10.1016/j.foodcont.2012.08.009 https://doi.org/10.1016/j.foodcont.2012.08.009

34. Qin X, Singh KV, Weinstock GM, Murray BE. Effects of Enterococcus faecalis fsr genes on production of gelatinase and a serine protease and virulence. Infect Immun 2000;68(5):2579-86. https://doi.org/10.1128/IAI.68.5.2579-2586.2000

35. Liong MT, Shah NP. Bile salt deconjugation ability, bile salt hydrolase activity and cholesterol co-precipitation ability of lactobacilli strains. Int Dairy J [Internet] 2005;15(4):391-8. Available via https://www. sciencedirect.com/science/article/pii/S0958694604002109 https://doi.org/10.1016/j.idairyj.2004.08.007

36. Begley M, Hill C, Gahan CGM. Bile salt hydrolase activity in probiotics. Appl Environ Microbiol 2006;72(3):1729-38. https://doi.org/10.1128/AEM.72.3.1729-1738.2006

37. Malaviya A, Malviya S, Agarwal A, Mishra M, Dalmida V. Saccharomyces-eukaryotic probiotic for human applications. In: High value fermentation products, John Wiley & Sons, Hoboken, NJ, pp 211-29, 2019. https://doi.org/10.1002/9781119555384.ch11

38. Zhu Y, Li T, Din AU, Hassan A, Wang Y, Wang G. Beneficial effects of Enterococcus faecalis in hypercholesterolemic mice on cholesterol transportation and gut microbiota. Appl Microbiol Biotechnol 2019;103(7):3181-91. https://doi.org/10.1007/s00253-019-09681-7

39. Asiva Noor Rachmayani. No Covariance structure analysis of health-related indicators in elderly people living at home, focusing on subjective health Title. 2015;6.