ISOLATION AND CHARACTERIZATION OF POTENTIAL PROBIOTICS FROM FERMENTED RAGI (ELEUSINE CORACANA)
Objective: The aim of this study was to isolate, characterize and identify the potential lactic acid bacteria from fermented ragi millet (Eleusine coracana).
Methods: A total of 177 isolates were isolated from the fermented ragi porridge. All the isolates were subjected to preliminary screening (Gram staining and catalase test). The physiological features of the probiotic isolates such as tolerance to pH (2.0, 4.0), phenol (0.3%, 0.5%) and bile salt (0.3%) were carried out. Antibiotic susceptibility test was performed using disc diffusion method. Free radical scavenging assay was conducted by 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. The isolates were further characterized for their cholesterol-lowering property. Based on their survival ability and biochemical tests, the selected potent isolates were identified by 16S rRNA gene sequence by PCR and phylogenetic analysis. The 16S rRNA gene sequencing confirmed that isolates were Lactococcus lactis RS01, Lactobacillus plantarum RS09, Lactobacillus plantarum RS16 and Lactobacillus plantarum RS23.
Results: Twenty five isolates were screened from the 177 total isolates based on their morphological and biochemical characteristics (Gram staining and catalase test). All the isolates showed tolerance against acid, phenol and bile salts. However, RS09 and RS23 exhibited maximum viable count and demonstrated good tolerance at the end of 3 h. Highest antioxidant activity was recorded in RS09 ranging from 32-85%. Among the tested strains, the degradation rate of supernatants, RS09 (61.9%), RS23 (60%) and RS01 (58.97%) and RS16 (56.2%) showed the highest cholesterol assimilation rate. In addition, they also found to be resistant and sensitive to few antibiotics.
Conclusion: Based on the obtained results, these isolates are ideal in vitro probiotic strains and can be used further for in vivo evaluation.
2. Ahmend AA. In vitro screening of Lactobacillus species from homemade yogurt for antagonistic effects against common bacterial pathogens. Jordan J Biol Sci 2013;6:211-6.
3. Jood S, Khetarpaul N, Goyal R. Effect of germination and probiotic fermentation on pH, titratable acidity, dietary fibre, Î²-Glucan and vitamin content of sorghum-based food mixtures. J Nutr Food Sci 2012;2:164.
4. Chen YS, Liou MS, Ji SH, Yu CR, Pan SF, Yanagida F. Isolation and characterization of lactic acid bacteria from Yantsai-shin (Fermented broccoli stems), a traditional fermented food in Taiwan. J Appl Microbiol 2013;115:125-32.
5. Abdulla AA, Abed TA, Saeed AM. Adhesion, autoaggregation and hydrophobicity of six Lactobacillus strains. Br Microbiol Res J 2014;4:381-91.
6. Suskovic J, Bla`enka Kos, Jasna Begano V, Andreja Lebo Pavunc, Ksenija Habjani, Srecko Mato. Antimicrobial activityâ€“the most important property of probiotic and starter lactic acid bacteria. Biotechnology 2010;48:296-307.
7. Deshmukh PV, Thorat PR. Isolation and extraction of bacteriocins produced by lactic acid bacteria isolated from raw milk samples. Indian Streams Res J 2013;3:1-7.
8. Fuller R. Probiotics in man and animals. J Appl Bacteriol 1989;66:365-78.
9. Catanzaro J, Green L. Microbial ecology and probiotics in human medicine (Part II). Altern Med Rev 1997;2:296-305.
10. O'Riordan K, Andrews D, Buckle K, Conway P. Evaluation of microencapsulation of a Bifidobacterium strain with starch as an approach to prolonging viability during storage. J Appl Microbiol 2001;91:1059-66.
11. Gilliland SE, Staley TE, Bush LJ. Importance in bile tolerance of Lactobacillus acidophilus used as a diatery adjunct. J Dairy Sci 1984;67:3045-51.
12. Suneeti Gore, Anjali Paul, Yashada Bhagwat. Comparative evaluation of commercially available probiotics products. Int J Curr Pharm Res 2017;9:26-30.
13. EFSA. Technical guidance prepared by the panel on additives and products or substances used in animal feed (FEEDAP) on the update of the criteria used in the assessment of bacterial resistance to antibiotics of human or veterinary importance. EFSA J 2008;732:1-15.
14. Danielson AD, Peo ER, Shahani KM, Lewis AJ, Whalen PJ, Amer MA. Anticholesterolemic property of Lactobacillus acidophilus yogurt fed to mature boars. J Anim Sci 1989;67:966-74.
15. Teply M Cistemlekarskekultury. Phara SNTL Nakladatelstvi, Technicke Litertury. In: Starters for fermented milks. Kurmann JA. Ed. IDF Bulletin 1984;227:41-55.
16. Rahman MK. Moazzem Hossain, Mahbubur Rahman SM. Isolation, characterization, and properties study of probiotic lactic acid bacteria of selected yoghurt from Bangladesh. Afr J Microbiol Res 2015;10:23-31.
17. Kuskoski EM, Asuero AG, Troncoso AM, Mancini-Filho J, Fett R. Application of several chemical methods to determine antioxidant activity in fruit pulps. Cienciay TecnologÃa de Alimentos. Campinas 2005;25:726-32.
18. Holt JG, Krieg NR, Sneath PHA, Staley JT, Williams ST. Bergey's Manual of Determinative Bacteriology. 9th ed. Baltimore: Williams and Wilkins; 1994.
19. James Owusu Kwarteng, Kwaku Tano Debrah, Fortune Akabanda, Lene Jespersen. Technological properties and probiotic potential of Lactobacillus fermentum strain isolated from West African fermented millet dough. BMC Microbiol 2015;15:261.
20. Shehata MG, El Sohaimy SA, Malak A, El-Sahn Youssef MM. Screening of isolated potential probiotic lactic acid bacteria for cholesterol lowering property and bile salt hydrolase activity. Ann Agric Sci 2016;61:65-75.
21. Krishnamoorthy M, Arjun P. Probiotic and antimicrobial activity of bacteria from fermented toddy of Cocus nucifera. J Acad Indus Res 2012;1:127-31.
22. Hyronimus B, Le Marrec C, Hadj Sassi A, Deschamps A. Acid and bile tolerance of spore-forming lactic acid bacteria. Int J Food Microbiol 2002;61:193-7.
23. Faye T, Tamburello A, Vegarud GE, Skeie S. Survival of lactic acid bacteria from fermented milks in an in vitro digestion model exploiting sequential incubation in human gastric and duodenum juice. J Dairy Sci 2012;95:558-66.
24. Garcia Ruiz A, de Llano DG, Esteban Fernandez A, Requena T, Bartolome B, Moreno Arriba SMV. Assessment of probiotic properties in lactic acid bacteria isolated from wine. Food Microbiol 2014;44:220-5.
25. Turchi B, Mancni S, Fratini F, Pedonese F, Nuvoloni R, Bertelloni F, et al. Preliminary evaluation of probiotic potential of Lactobacillus plantarum strains isolated from Italian food products. World J Microbiol Biotechnol 2013;29:1913-22.
26. Ruby Yadav, Anil K, Puniyaand Pratyoosh Shukla. Probiotic properties of Lactobacillus plantarum RYPR1 from an indigenous fermented beverage raabadi. Front Microbiol 2016;7:1683.
27. Zavaglia AG, Kociubinski G, Perez P, Disalvo E, de Antoni G. Effect of bile on the lipid composition and surface properties of bifidobacteria. J Appl Microbiol 2002;93:794-9.
28. Jicheng Wang, Xiao Dong, Yuyu Shao, Huiling Guo, Lin Pan, Wenyan Hui, et al. Genome adaptive evolution of Lactobacillus casei under long-term antibiotic selection pressures. BMC Genomics 2017;18:320.
29. Danielsen M, Wind A. Susceptibility of Lactobacillus spp. to antimicrobial agents. Int J Food Microbiol 2003;82:1-11.
30. Jena, PK, Trivedi D, Thakore K, Chaudhary H, Giri SS, Seshadri S. Isolation and characterization of probiotic properties of Lactobacilli isolated from rat fecal microbiota. Microbiol Immunol 2013;57:407-41.
31. Guo Zhuang, Xiao-Ming Liu, Qiu Xiang Zhang, Feng Wei Tian, Hao Zhang, He-Ping Zhang, et al. Research advances with regards to clinical outcome and potential mechanisms of the cholesterol-lowering effects of probiotics. Clin Lipidol 2012;7:501-7.
32. Liong MT, Shah NP. Acid and bile tolerance and the cholesterol removal ability of Lactobacilli strains. J Dairy Sci 2005;88:55-66.
33. Lee BJ, Kim JS, Kang YM, Lim JH, Kim YM, Lee MS, et al. Antioxidant activity and Î³-aminobutyric acid (GABA) content in sea tangle fermented by Lactobacillus brevis BJ20 isolated from traditional fermented foods. Food Chem 2010;122:271-6.
34. Yeong Ji Oh, Jung DS. Evaluation of probiotic properties of Lactobacillus and Pediococcus strains isolated from Omegisool, a traditionally fermented millet alcoholic beverage in Korea. LWT Food Sci Technol 2015;63:437-44.
35. Sun YP, Chou CC, Yu RC. Antioxidant activity of Lactic acid bacteria fermented cheese cabbage. Food Chem 2009;115:912-7.
36. Cheng H, Jiang N. Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol Lett 2006;28:55-9.
37. Sumi Das Purkhayastha, Bhattacharya MK, Prasad HK, Upadhyaya H, Pal K, Sharma Gd. Antimicrobial activity of Lactobacillus fermentum, a volvo vaginal isolate. Asian J Pharm Clin Res 2015;8:371-2.
38. Drancourt M, Bollet C, Carlioz A, Martelin R, Grayral JP, Raoult D. 16S Ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates. J Clin Microbiol 2000;38:3623-30.
39. Sghir A, Gramet G, Suau A, Rochet V, Pochart P, Dore J. Quantification of bacterial groups within human fecal microbiota by oligonucleotide probe hybridization. Appl Environ Microbiol 2000;66:2263-6.
40. Greetham HL, Giffard C, Hutson RA, Collins MD, Gibson GR. Bacteriology of the Labrador dog gut: a cultural and genotypic approach. J Appl Microbiol 2002;93:640-6.
41. Heilig HGHJ, Zoetenda EG, Vaughan EE, Marteau P, Akkermans ADL, De Vos WM. Molecular diversity of Lactobacillus ssp. and other lactic acid bacteria in the human intestine as determined by specific amplification of 16S ribosomal DNA. Appl Environ Microbiol 2002;68:114-23.
42. Pandey P, Kang SC, Maheswari DK. Isolation of endophytic plant growth-promoting Burkholderia spp. MSSP from root nodules of Mimosa pudica. Curr Sci 2005;89:177-80.
43. Alschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol 1990;215:403-10.
44. Thompson JD, Gibsom TJ, Plewnaik F, Jeanmougin F, Higgins DG. The clustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 1997;24:4876-82.
45. Hemal Sadrani, Jayantilal Dave, Bharatkumar Rajiv Manuel Vyas. Screening of potentially probiotic Lactobacillus strains isolated from fermented foods, fruits and of human origin. Asian J Pharm Clin Res 2014;7 Suppl 2:216-25.