Physico-chemical and cytotoxic analysis of a novel large molecular weight bacteriocin produced by Lactobacillus casei TA0021
,
Abstract
Background and Objectives: Antimicrobial peptides produced by lactic acid bacteria have gained enormous attention owing to their health benefits. This study aimed to isolate, purify and characterize the antibacterial protein produced by autochthonous Lactobacillus casei TA0021 strain.
Materials and Methods: The antagonistic activity of L. casei TA0021 against a number of pathogenic bacteria was tested by agar well diffusion assay. The antimicrobial agent in the neutralized supernatant fluids was subjected to the action of proteolytic enzymes, catalase, lipase and lysozyme, and their tolerance to variable pH and temperature was estimated. The proteinaceous antagonistic compound was precipitated by 60% w/v ammonium sulphate, desalted and subjected to cation exchange and gel filtration chromatography. Approximate molecular weight of Lactocin was determined by SDS-PAGE and non-denaturing gel electrophoresis. Hemoglobin release assay and cytotoxicity effect of Lactocin TA0021 was determined. The results were statistically analyzed.
Results: The antagonistic agent active against Salmonella Typhimurium and Shigella flexneri appeared resistant to catalase and lipase treatments, while sensitive to the tested proteolytic enzymes. Lactocin TA0021 resisted acidic pH values of 3.0, while alkaline pH values of ˃9 completely destroyed the activity. The antibacterial peptide was approximately 68 KDa and heat labile as lost its activity at 100°C after 5 minutes. The bacteriocin was non-toxic to MRC-5 cell lines and non-hemolytic. Purification method lead to increase in antibacterial activity while, subsequent decrease in recovery and yield was observed with increasing purification fold.
Conclusion: The purified antimicrobial protein from L. casei TA0021 might be used for application in medicinal and food products.
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12. Karimi S, Azizi F, Nayeb-Aghaee M, Mahmoodnia L. The antimicrobial activity of probiotic bacteria Escherichia coli isolated from different natural sourcesagainst hemorrhagic E. coli O157:H7. Electron Physician 2018; 10:6548-6553.
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14. Vuyst L, Callewaert R, Crabbe K. Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiol 1996; 143:817-827.
15. Elayaraja S, Annamalai N, Mayavu P, Balasubramanian T. Production, purification and characterization of bacteriocin from Lactobacillus murinus AU06 and its broad antibacterial spectrum. Asian Pac J Trop Biomed 2014; 4(Suppl 1):S305-311.
16. Vijay Simha B, Sood SK, Kumariya R, Garsa AK. Simple and rapid purification of pediocin PA-1 from Pediococcus pentosaceous NCDC 273 suitable for industrial application. Microbiol Res 2012; 167:544-549.
17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
18. Schagger H. Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Anal Biochem 1987;166: 368-379.
19. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol 2015; 111:A3.B.1-3.
20. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55-63.
21. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986; 89: 271-277.
22. Orlando A, Linsalata M, Russo F. Antiproliferative effects on colon adenocarcinoma cells induced by co-administration of vitamin K1 and Lactobacillus rhamnosus GG. Int J Oncol 2016; 48: 2629-2638.
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24. Sharma SK, Joshi VK, Sharma S. (2012) Probiotics: Concepts and Applications in Food. in: Food biotechnology: principles and practices. Eds, Joshi VK and Singh RS. IK International Publishing House Pvt Ltd, 1st ed. New Dehli, India . pp.781-798.
25. Lopetuso LR, Giorgio ME, Saviano A, Scaldaferri F, Gasbarrini A, Cammarota G. Bacteriocins and bacteriophages: Therapeutic weapons for gastrointestinal diseases? Int J Mol Sci 2019; 20:E183.
26. Toba T, Yoshioka E, Itoh T. Potential of Lactobacillus gasseri isolated from infant faeces to produce bacteriocin. Lett Appl Microbiol 2008; 12:228-231.
27. Toba T, Yoshioka E, Itoh T. Acidophilucin A, a new heat labile bacteriocin produced by Lactobacillus acidophilus LAPT 1060. Let Appl Microbiol 1991;12: 106-108.
28. Toba T, Yoshioka E, Itoh T. Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp lactis. Let Appl Microbiol 1991;12: 43-45.
29. Gaspar C, Donders GG, Palmeira de Oliveira R, Queiroz JA, Tomaz C, Martinez de Oliveira J, et al. Bacteriocin production of the probiotic Lactobacillus acidophilus KS400. AMB Express 2018; 8:153.
30. Milioni C, Martínez B, Degl’Innocenti S, Turchi B, Fratini F, Cerri D, et al. A Novel bacteriocin produced by Lactobacillus plantarum LpU4 as a valuable candidate for biopreservation in artisanal raw milk cheese. Dairy Sci Technol 2015; 95:479-494.
31. Souzaa E, Oliveira P de Souza de Azevedoa, Domínguezb J, Convertic A, Pinheiro P de Souza Oliveiraa. Influence of temperature and pH on the production of biosurfactant, bacteriocin and lactic acid by Lactococcus lactis CECT-4434. CYTA-J Food 2017; 15:525-530.
32. Klaenhammer TR, Fremaux C, Ahn C, Milton K. (1993) Molecular biology of bacteriocins produced by Lactobacillus. In: Bacteriocins of Lactic Acid Bacteria. eds, Hoover D. G. and Steenson L. R. New York: Academic press Inc. pp.151-180.
33. Mokoena MP. Lactic acid bacteria and their bacteriocins: classification, biosynthesis and applications against uropathogens: A mini-review. Molecules 2017; 22(8): E1255.
34. Rammelsberg M, Radler F. Antibacterial polypeptides of Lactobacillus species. J Appl Microbiol 1990; 69: 177-184.
35. Bemena1 LD, Mohamed LA, Fernandes AM, Lee1 BH. Applications of bacteriocins in food, livestock health and medicine. Int J Curr Microbiol App Sci 2014;3: 924-949.
36. Piard JC, Desmazeaud M. Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antibacterial substances. Dairy Sci Technol 1992;72: 113-142.
2. Zhao S, Han J, Bie X, Lu Z, Zhang C, Lv F. Purification and characterization of plantaricin JLA-9: a novel bacteriocin against Bacillus spp. produced by Lactobacillus plantarum JLA-9 from Suan-Tsai, a traditional Chinese fermented cabbage. J Agric Food Chem 2016; 64:2754-2764.
3. Alvarez-Sieiro P, Montalbán-López M. Bacteriocins of lactic acid bacteria: extending the family. Appl Microbiol Biotechnol 2016; 100: 2939-2951.
4. Zhang J, Liu G, Shang N, Cheng W, Chen S, Li P. Purification and partial amino acid sequence of pentocin 31-1, an anti-Listeria bacteriocin produced by Lactobacillus pentosus 31-1. J Food Prot 2009; 72:2524-2529.
5. Aslim B, Yuksekdag ZN, Sarikaya E, Beyatli Y. Determination of the bacteriocin-like substances produced by some lactic acid bacteria isolated from Turkish dairy products. LWT Food Sci Tech 2005; 38:691-694.
6. Amortegui J, Rodríguez-López A, Rodríguez D, Carrascal A, Alméciga-Díaz C. Characterization of a new bacteriocin from Lactobacillus plantarum LE5 and LE27 isolated from ensiled corn. Appl Biochem Biotechnol 2014;172:3374-3389.
7. Hathout AS, Aly SE. Role of lactic acid bacteria as a biopreservative agent of Talbina. J Am Sci 2010; 6:889-898.
8. Iranmanesh M, Ezzatpanah H, Mojgani N, Karimi Torshizi MA, Aminafshar M, Maohamadi M. Isolation of lactic acid bacteria from ewe milk, traditional yoghurt and sour buttermilk in Iran. Eur J Food Res Rev 2012;2:79-92.
9. Mojgani N, Ameli M, Vaseji N, Hejazi MA, Torshizi MAK, Amirinia C. Growth control of Listeria monocytogenes in experimental cheese samples by Lactobacillus casei RN 78 and its bacteriocin. Afr J Microbiol Res 2010; 4:1044-1050.
10. Gharsallaoui A, Joly C, Oulahal N, Pascal Degraeve. Nisin as a food preservative: Part 2: Antimicrobial polymer materials containing Nisin. Crit Rev Food Sci Nutr 2016; 56:1275-1289.
11. Jamaluddin N, Stuckey D, Arbakariya B, Wong F. Novel approaches to purifying bacteriocin: A review. Crit Rev Food Sci Nutr 2018;58:2453-2465.
12. Karimi S, Azizi F, Nayeb-Aghaee M, Mahmoodnia L. The antimicrobial activity of probiotic bacteria Escherichia coli isolated from different natural sourcesagainst hemorrhagic E. coli O157:H7. Electron Physician 2018; 10:6548-6553.
13. Hockett KL, Baltrus DA. Use of the soft-agar overlay technique to screen for bacterially produced inhibitory compounds. J Vis Exp 2017; 119: 10.3791/55064.
14. Vuyst L, Callewaert R, Crabbe K. Primary metabolite kinetics of bacteriocin biosynthesis by Lactobacillus amylovorus and evidence for stimulation of bacteriocin production under unfavourable growth conditions. Microbiol 1996; 143:817-827.
15. Elayaraja S, Annamalai N, Mayavu P, Balasubramanian T. Production, purification and characterization of bacteriocin from Lactobacillus murinus AU06 and its broad antibacterial spectrum. Asian Pac J Trop Biomed 2014; 4(Suppl 1):S305-311.
16. Vijay Simha B, Sood SK, Kumariya R, Garsa AK. Simple and rapid purification of pediocin PA-1 from Pediococcus pentosaceous NCDC 273 suitable for industrial application. Microbiol Res 2012; 167:544-549.
17. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem 1951; 193: 265-275.
18. Schagger H. Tricine-sodium dodecylsulfate polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 KDa. Anal Biochem 1987;166: 368-379.
19. Strober W. Trypan blue exclusion test of cell viability. Curr Protoc Immunol 2015; 111:A3.B.1-3.
20. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65:55-63.
21. Denizot F, Lang R. Rapid colorimetric assay for cell growth and survival. Modifications to the tetrazolium dye procedure giving improved sensitivity and reliability. J Immunol Methods 1986; 89: 271-277.
22. Orlando A, Linsalata M, Russo F. Antiproliferative effects on colon adenocarcinoma cells induced by co-administration of vitamin K1 and Lactobacillus rhamnosus GG. Int J Oncol 2016; 48: 2629-2638.
23. Paiva C E, Paulo I, Lima S N, Sakamoto I, Paiva R. Articles with short titles describing the results are cited more often. Clinics (Sao Paulo) 2012;67:509-513.
24. Sharma SK, Joshi VK, Sharma S. (2012) Probiotics: Concepts and Applications in Food. in: Food biotechnology: principles and practices. Eds, Joshi VK and Singh RS. IK International Publishing House Pvt Ltd, 1st ed. New Dehli, India . pp.781-798.
25. Lopetuso LR, Giorgio ME, Saviano A, Scaldaferri F, Gasbarrini A, Cammarota G. Bacteriocins and bacteriophages: Therapeutic weapons for gastrointestinal diseases? Int J Mol Sci 2019; 20:E183.
26. Toba T, Yoshioka E, Itoh T. Potential of Lactobacillus gasseri isolated from infant faeces to produce bacteriocin. Lett Appl Microbiol 2008; 12:228-231.
27. Toba T, Yoshioka E, Itoh T. Acidophilucin A, a new heat labile bacteriocin produced by Lactobacillus acidophilus LAPT 1060. Let Appl Microbiol 1991;12: 106-108.
28. Toba T, Yoshioka E, Itoh T. Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp lactis. Let Appl Microbiol 1991;12: 43-45.
29. Gaspar C, Donders GG, Palmeira de Oliveira R, Queiroz JA, Tomaz C, Martinez de Oliveira J, et al. Bacteriocin production of the probiotic Lactobacillus acidophilus KS400. AMB Express 2018; 8:153.
30. Milioni C, Martínez B, Degl’Innocenti S, Turchi B, Fratini F, Cerri D, et al. A Novel bacteriocin produced by Lactobacillus plantarum LpU4 as a valuable candidate for biopreservation in artisanal raw milk cheese. Dairy Sci Technol 2015; 95:479-494.
31. Souzaa E, Oliveira P de Souza de Azevedoa, Domínguezb J, Convertic A, Pinheiro P de Souza Oliveiraa. Influence of temperature and pH on the production of biosurfactant, bacteriocin and lactic acid by Lactococcus lactis CECT-4434. CYTA-J Food 2017; 15:525-530.
32. Klaenhammer TR, Fremaux C, Ahn C, Milton K. (1993) Molecular biology of bacteriocins produced by Lactobacillus. In: Bacteriocins of Lactic Acid Bacteria. eds, Hoover D. G. and Steenson L. R. New York: Academic press Inc. pp.151-180.
33. Mokoena MP. Lactic acid bacteria and their bacteriocins: classification, biosynthesis and applications against uropathogens: A mini-review. Molecules 2017; 22(8): E1255.
34. Rammelsberg M, Radler F. Antibacterial polypeptides of Lactobacillus species. J Appl Microbiol 1990; 69: 177-184.
35. Bemena1 LD, Mohamed LA, Fernandes AM, Lee1 BH. Applications of bacteriocins in food, livestock health and medicine. Int J Curr Microbiol App Sci 2014;3: 924-949.
36. Piard JC, Desmazeaud M. Inhibiting factors produced by lactic acid bacteria. 2. Bacteriocins and other antibacterial substances. Dairy Sci Technol 1992;72: 113-142.
| Files | ||
| Issue | Vol 11 No 5 (2019) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v11i5.1958 | |
| Keywords | ||
| Bacteriocin; Antimicrobial; Lactobacillus casei; Cytotoxicity; Heat labile | ||
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How to Cite
1.
Noroozi E, Mojgani N, Motevaseli E, Modarressi MH, Tebianian M. Physico-chemical and cytotoxic analysis of a novel large molecular weight bacteriocin produced by Lactobacillus casei TA0021. Iran J Microbiol. 2019;11(5):397-405.
