In vitro effects of purified lacticin from whey isolated Lactococcus lactis culture on Staphylococcus aureus and MCF-7 breast cancer cells
,
Abstract
Background and Objectives: Bacteriocins are interested as antibacterial and anticancer agents due to their high specificity and low side effects. This study aimed to isolate bacteria which produce bacteriocins of the lacticin family from whey and to investigate their antibacterial and anticancer effects.
Materials and Methods: Lactic acid bacteria were isolated from different whey samples. The presence of the lacticin gene in the isolates was checked using PCR and then the inhibitory effects of their bacteriocin was investigated on Staphylococcus aureus utilizing well plate method. The protein content was separated by dialysis. The presence of lacticin was checked with the help of SDS-PAGE. The lacticin producing bacterium was identified through the sequencing of the 16S rRNA gene. Finally, the cytotoxicity of the obtained protein was studied on the MCF-7 breast cancer cells using MTT and scratching tests.
Results: The isolated lacticin-producing Lactococcus lactis was able to grow in acidic conditions (pH = 2.5 for 3 h) and in bile salts (0.3% for 24 h). The bacterium produced 4.2 μg/μl bacteriocin with a molecular weight of 3.1 KD. The lacticin showed antibacterial effect against S. aureus. The cancerous cells treated with lacticin had slower growth than the control in Scratch test. Based on the MTT results, more than 80% of cancerous cells were inhibited at a concentration of 7 μg/ml lacticin with IC50 = 5.2 μg/ml.
Conclusion: The bacteriocin produced in this study is a promising antibacterial and anticancer agent.
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12. Yoneyama F, Imura Y, Ohno K, Zendo T, Nakayama J, Matsuzaki K, et al. Peptide-lipid huge toroidal pore, a new antimicrobial mechanism mediated by a lactococcal bacteriocin, lacticin Q. Antimicrob Agents Chemother 2009; 53: 3211-3217.
13. Daba GM, Elkhateeb WA. Bacteriocins of lactic acid bacteria as biotechnological tools in food and pharmaceuticals: Current applications and future prospects. Biocatal Agric Biotechnol 2020; 28: 101750.
14. Song AA-L, In LLA, Lim SHE, Rahim RA. Erratum to: A review on Lactococcus lactis: from food to factory. Microb Cell Fact 2017; 16: 139.
15. Khemariya P, Singh S, Nath G, Gulati AK. Probiotic Lactococcus lactis: A review. Turk J Agric Food Sci Technol 2017; 5: 556-562.
16. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021; 71: 209-249.
17. Kushi LH, Doyle C, McCullough M, Rock CL, Demark-Wahnefried W, Bandera EV, et al. American cancer society guidelines on nutrition and physical activity for cancer prevention: reducing the risk of cancer with healthy food choices and physical activity. CA Cancer J Clin 2012; 62: 30-67.
18. Han KJ, Lee N-K, Park H, Paik H-D. Anticancer and anti-inflammatory activity of probiotic Lactococcus lactis NK34. J Microbiol Biotechnol 2015; 25: 1697-1701.
19. Rai N, Singh AK, Keshri PK, Barik S, Kamble SC, Singh SK, et al (2020). Probiotics for management of gastrointestinal cancers. In: Probiotic Research in Therapeutics. Publisher: Springer, Singapore. pp 191-209.
20. Mondragón-Parada ME, Nájera-Martínez M, Juárez-Ramírez C, Galíndez-Mayer J, Ruiz-Ordaz N, Cristiani-Urbina E. Lactic acid bacteria production from whey. Appl Biochem Biotechnol 2006; 134: 223-232.
21. Sequeiros C, Garcés ME, Vallejo M, Marguet ER, Olivera NL. Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae. Arch Microbiol 2015; 197: 449-458.
22. Balasingham K, Valli C, Radhakrishnan L, Balasuramanyam D. Probiotic characterization of lactic acid bacteria isolated from swine intestine. Vet World 2017; 10: 825-829.
23. Cui Y, Luo L, Wang X, Lu Y, Yi Y, Shan Y, et al. Mining, heterologous expression, purification, antibactericidal mechanism, and application of bacteriocins: A review. Compr Rev Food Sci Food Saf 2021; 20: 863-899.
24. Shin GW, Palaksha KJ, Kim YR, Nho SW, Cho JH, Heo NE, et al. Immunoproteomic analysis of capsulate and non-capsulate strains of Lactococcus garvieae. Vet Microbiol 2007; 119: 205-212.
25. Rabilloud T, Vuillard L, Gilly C, Lawrence JJ. Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol (Noisy-le-grand) 1994; 40: 57-75.
26. Ashrafian B, Hosseini-Abari A. Investigation of bioactivity of unsaturated oligo galacturonic acids produced from apple waste by Alcaligenes faecalis AGS3 and Paenibacillus polymyxa S4 pectinases. Sci Rep 2022; 12: 15830.
27. Bergkessel M, Guthrie C. Colony PCR. Methods Enzymol 2013; 529: 299-309.
28. Woodman ME, Savage CR, Arnold WK, Stevenson B. Direct PCR of intact bacteria (colony PCR). Curr Protoc Microbiol 2016; 42: A.3D.1-A.3D.7.
29. Chaudhry G, Zeenia, Safdar N, Begum S, Akim AM, Sung YY, et al. Cytotoxicity assays for cancer drug screening: methodological insights and considerations for reliable assessment in drug discovery. Braz J Biol 2024; 84: e284409.
30. Liang C-C, Park AY, Guan J-L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2: 329-333.
31. Afshari A, Hashemi M, Tavassoli M, Eraghi V, Noori SMA. Probiotic bacteria from 10 different traditional Iranian cheeses: Isolation, characterization, and investigation of probiotic potential. Food Sci Nutr 2022; 10: 2009-2020.
32. Otaghvar HA, Hosseini M, Tizmaghz A, Shabestanipour G, Noori H. A review on metastatic breast cancer in Iran. Asian Pac J Trop Biomed 2015; 5: 429-433.
33. Aghamiri S, Zandsalimi F, Raee P, Abdollahifar M-A, Tan SC, Low TY, et al. Antimicrobial peptides as potential therapeutics for breast cancer. Pharmacol Res 2021; 171: 105777.
34. Jin G, Weinberg A. Human antimicrobial peptides and cancer. Semin Cell Dev Biol 2019; 88: 156-162.
35. Li S, Wang Y, Xue Z, Jia Y, Li R, He C, et al. The structure-mechanism relationship and mode of actions of antimicrobial peptides: A review. Trends Food Sci Technol 2021; 109: 103-115.
36. Browne K, Chakraborty S, Chen R, Willcox MD, Black DS, Walsh WR, et al. A new era of antibiotics: the clinical potential of antimicrobial peptides. Int J Mol Sci 2020; 21: 7047.
37. He J-F, Jin D-X, Luo X-G, Zhang T-C. LHH1, a novel antimicrobial peptide with anti-cancer cell activity identified from Lactobacillus casei HZ1. AMB Express 2020; 10: 204.
38. Fornitano ALP, Amêndola I, Santos SSF, Silva CRG, Leão MVP. Lactobacillus rhamnosus versus Staphylococcus aureus: influence on growth and expression of virulence factors. J Dent Maxillofacial Res 2019; 2: 29-33.
39. Gao X, Wang Z, Li X, Zhang X, Du S, Jia M, et al. A new Lactobacillus gasseri strain HMV18 inhibits the growth of pathogenic bacteria. Food Sci Hum Wellness 2022; 11: 247-254.
40. Layus BI, Gerez CL, Rodriguez AV. Antibacterial activity of Lactobacillus plantarum CRL 759 against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Arab J Sci Eng 2020; 45: 4503-4510.
41. Teuber M. Lactococcus. Bergey's manual of systematics of archaea and bacteria, Springer, 2015.
42. Kondrotiene K, Zavistanaviciute P, Aksomaitiene J, Novoslavskij A, Malakauskas M. Lactococcus lactis in Dairy Fermentation—Health-Promoting and probiotic properties. Fermentation 2024; 10: 16.
43. Mataragas M. Investigation of genomic characteristics and carbohydrates’ metabolic activity of Lactococcus lactis subsp. lactis during ripening of a Swiss-type cheese. Food Microbiol 2020; 87: 103392.
44. Abbdul-Kaliq MT, Rasool KH, Essa RH. Effect of crude bacteriocin isolated from locally Lactococcus lactis on cancer cell lines. Indian J Forensic Med Toxicol 2020; 14: 281-288.
45. Hamdan AM, Alyami A, DorghamMM, Awad H, Hamdy R. Cytotoxicity and antimicrobial activities of the novel lactic acid bacterium, Lactobacillus brevis A13, isolated from the gut of the polychaete, Perinereis nuntia. Egypt J Aquat Biol Fish 2022; 26: 533-547.
46. Avand A, Akbari V, Shafizadegan S. In vitro cytotoxic activity of a Lactococcus lactis antimicrobial peptide against breast cancer cells. Iran J Biotechnol 2018; 16(3): e1867.
47. Mohammedsaeed W (2015). Characterisation of the potential of probiotics or their extracts as therapy for skin. The University of Manchester. https://research.manchester.ac.uk/en/studentTheses/characterisation-of-the-potential-of-probiotics-or-their-extracts
2. Didari T, Solki S, Mozaffari S, Nikfar S, Abdollahi M. A systematic review of the safety of probiotics. Expert Opin Drug Saf 2014; 13: 227-239.
3. Ayivi RD, Gyawali R, Krastanov A, Aljaloud SO, Worku M, Tahergorabi R, et al. Lactic acid Bacteria: food safety and human health applications. Dairy 2020; 1: 202-232.
4. Tsilingiri K, Barbosa T, Penna G, Caprioli F, Sonzogni A, Viale G, et al. Probiotic and postbiotic activity in health and disease: comparison on a novel polarised ex-vivo organ culture model. Gut 2012; 61: 1007-1015.
5. Soltani S, Hammami R, Cotter PD, Rebuffat S, Said LB, Gaudreau H, et al. Bacteriocins as a new generation of antimicrobials: toxicity aspects and regulations. FEMS Microbiol Rev 2021; 45: fuaa039.
6. Trejo-González L, Gutiérrez-Carrillo A-E, Rodríguez-Hernández A-I, Del Rocío López-Cuellar M, Chavarría-Hernández N. Bacteriocins produced by LAB isolated from cheeses within the period 2009–2021: a review. Probiotics Antimicrob Proteins 2022; 14: 238-251.
7. O’Connor PM, Kuniyoshi TM, Oliveira RP, Hill C, Ross RP, Cotter PD. Antimicrobials for food and feed; a bacteriocin perspective. Curr Opin Biotechnol 2020; 61: 160-167.
8. Van Staden Adu P, Heunis T, Smith C, Deane S, Dicks LM. Efficacy of lantibiotic treatment of Staphylococcus aureus-induced skin infections, monitored by in vivo bioluminescent imaging. Antimicrob Agents Chemother 2016; 60: 3948-3955.
9. Baindara P, Chaudhry V, Mittal G, Liao LM, Matos CO, Khatri N, et al. Characterization of the antimicrobial peptide penisin, a class Ia novel lantibiotic from Paenibacillus sp. strain A3. Antimicrob Agents Chemother 2015; 60: 580-591.
10. Piper C, Casey PG, Hill C, Cotter PD, Ross RP. The lantibiotic lacticin 3147 prevents systemic spread of Staphylococcus aureus in a murine infection model. Int J Microbiol 2012; 2012: 806230.
11. Fujita K, Ichimasa S, Zendo T, Koga S, Yoneyama F, Nakayama J, et al. Structural analysis and characterization of lacticin Q, a novel bacteriocin belonging to a new family of unmodified bacteriocins of gram-positive bacteria. Appl Environ Microbiol 2007; 73: 2871-2877.
12. Yoneyama F, Imura Y, Ohno K, Zendo T, Nakayama J, Matsuzaki K, et al. Peptide-lipid huge toroidal pore, a new antimicrobial mechanism mediated by a lactococcal bacteriocin, lacticin Q. Antimicrob Agents Chemother 2009; 53: 3211-3217.
13. Daba GM, Elkhateeb WA. Bacteriocins of lactic acid bacteria as biotechnological tools in food and pharmaceuticals: Current applications and future prospects. Biocatal Agric Biotechnol 2020; 28: 101750.
14. Song AA-L, In LLA, Lim SHE, Rahim RA. Erratum to: A review on Lactococcus lactis: from food to factory. Microb Cell Fact 2017; 16: 139.
15. Khemariya P, Singh S, Nath G, Gulati AK. Probiotic Lactococcus lactis: A review. Turk J Agric Food Sci Technol 2017; 5: 556-562.
16. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021; 71: 209-249.
17. Kushi LH, Doyle C, McCullough M, Rock CL, Demark-Wahnefried W, Bandera EV, et al. American cancer society guidelines on nutrition and physical activity for cancer prevention: reducing the risk of cancer with healthy food choices and physical activity. CA Cancer J Clin 2012; 62: 30-67.
18. Han KJ, Lee N-K, Park H, Paik H-D. Anticancer and anti-inflammatory activity of probiotic Lactococcus lactis NK34. J Microbiol Biotechnol 2015; 25: 1697-1701.
19. Rai N, Singh AK, Keshri PK, Barik S, Kamble SC, Singh SK, et al (2020). Probiotics for management of gastrointestinal cancers. In: Probiotic Research in Therapeutics. Publisher: Springer, Singapore. pp 191-209.
20. Mondragón-Parada ME, Nájera-Martínez M, Juárez-Ramírez C, Galíndez-Mayer J, Ruiz-Ordaz N, Cristiani-Urbina E. Lactic acid bacteria production from whey. Appl Biochem Biotechnol 2006; 134: 223-232.
21. Sequeiros C, Garcés ME, Vallejo M, Marguet ER, Olivera NL. Potential aquaculture probiont Lactococcus lactis TW34 produces nisin Z and inhibits the fish pathogen Lactococcus garvieae. Arch Microbiol 2015; 197: 449-458.
22. Balasingham K, Valli C, Radhakrishnan L, Balasuramanyam D. Probiotic characterization of lactic acid bacteria isolated from swine intestine. Vet World 2017; 10: 825-829.
23. Cui Y, Luo L, Wang X, Lu Y, Yi Y, Shan Y, et al. Mining, heterologous expression, purification, antibactericidal mechanism, and application of bacteriocins: A review. Compr Rev Food Sci Food Saf 2021; 20: 863-899.
24. Shin GW, Palaksha KJ, Kim YR, Nho SW, Cho JH, Heo NE, et al. Immunoproteomic analysis of capsulate and non-capsulate strains of Lactococcus garvieae. Vet Microbiol 2007; 119: 205-212.
25. Rabilloud T, Vuillard L, Gilly C, Lawrence JJ. Silver-staining of proteins in polyacrylamide gels: a general overview. Cell Mol Biol (Noisy-le-grand) 1994; 40: 57-75.
26. Ashrafian B, Hosseini-Abari A. Investigation of bioactivity of unsaturated oligo galacturonic acids produced from apple waste by Alcaligenes faecalis AGS3 and Paenibacillus polymyxa S4 pectinases. Sci Rep 2022; 12: 15830.
27. Bergkessel M, Guthrie C. Colony PCR. Methods Enzymol 2013; 529: 299-309.
28. Woodman ME, Savage CR, Arnold WK, Stevenson B. Direct PCR of intact bacteria (colony PCR). Curr Protoc Microbiol 2016; 42: A.3D.1-A.3D.7.
29. Chaudhry G, Zeenia, Safdar N, Begum S, Akim AM, Sung YY, et al. Cytotoxicity assays for cancer drug screening: methodological insights and considerations for reliable assessment in drug discovery. Braz J Biol 2024; 84: e284409.
30. Liang C-C, Park AY, Guan J-L. In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc 2007; 2: 329-333.
31. Afshari A, Hashemi M, Tavassoli M, Eraghi V, Noori SMA. Probiotic bacteria from 10 different traditional Iranian cheeses: Isolation, characterization, and investigation of probiotic potential. Food Sci Nutr 2022; 10: 2009-2020.
32. Otaghvar HA, Hosseini M, Tizmaghz A, Shabestanipour G, Noori H. A review on metastatic breast cancer in Iran. Asian Pac J Trop Biomed 2015; 5: 429-433.
33. Aghamiri S, Zandsalimi F, Raee P, Abdollahifar M-A, Tan SC, Low TY, et al. Antimicrobial peptides as potential therapeutics for breast cancer. Pharmacol Res 2021; 171: 105777.
34. Jin G, Weinberg A. Human antimicrobial peptides and cancer. Semin Cell Dev Biol 2019; 88: 156-162.
35. Li S, Wang Y, Xue Z, Jia Y, Li R, He C, et al. The structure-mechanism relationship and mode of actions of antimicrobial peptides: A review. Trends Food Sci Technol 2021; 109: 103-115.
36. Browne K, Chakraborty S, Chen R, Willcox MD, Black DS, Walsh WR, et al. A new era of antibiotics: the clinical potential of antimicrobial peptides. Int J Mol Sci 2020; 21: 7047.
37. He J-F, Jin D-X, Luo X-G, Zhang T-C. LHH1, a novel antimicrobial peptide with anti-cancer cell activity identified from Lactobacillus casei HZ1. AMB Express 2020; 10: 204.
38. Fornitano ALP, Amêndola I, Santos SSF, Silva CRG, Leão MVP. Lactobacillus rhamnosus versus Staphylococcus aureus: influence on growth and expression of virulence factors. J Dent Maxillofacial Res 2019; 2: 29-33.
39. Gao X, Wang Z, Li X, Zhang X, Du S, Jia M, et al. A new Lactobacillus gasseri strain HMV18 inhibits the growth of pathogenic bacteria. Food Sci Hum Wellness 2022; 11: 247-254.
40. Layus BI, Gerez CL, Rodriguez AV. Antibacterial activity of Lactobacillus plantarum CRL 759 against methicillin-resistant Staphylococcus aureus and Pseudomonas aeruginosa. Arab J Sci Eng 2020; 45: 4503-4510.
41. Teuber M. Lactococcus. Bergey's manual of systematics of archaea and bacteria, Springer, 2015.
42. Kondrotiene K, Zavistanaviciute P, Aksomaitiene J, Novoslavskij A, Malakauskas M. Lactococcus lactis in Dairy Fermentation—Health-Promoting and probiotic properties. Fermentation 2024; 10: 16.
43. Mataragas M. Investigation of genomic characteristics and carbohydrates’ metabolic activity of Lactococcus lactis subsp. lactis during ripening of a Swiss-type cheese. Food Microbiol 2020; 87: 103392.
44. Abbdul-Kaliq MT, Rasool KH, Essa RH. Effect of crude bacteriocin isolated from locally Lactococcus lactis on cancer cell lines. Indian J Forensic Med Toxicol 2020; 14: 281-288.
45. Hamdan AM, Alyami A, DorghamMM, Awad H, Hamdy R. Cytotoxicity and antimicrobial activities of the novel lactic acid bacterium, Lactobacillus brevis A13, isolated from the gut of the polychaete, Perinereis nuntia. Egypt J Aquat Biol Fish 2022; 26: 533-547.
46. Avand A, Akbari V, Shafizadegan S. In vitro cytotoxic activity of a Lactococcus lactis antimicrobial peptide against breast cancer cells. Iran J Biotechnol 2018; 16(3): e1867.
47. Mohammedsaeed W (2015). Characterisation of the potential of probiotics or their extracts as therapy for skin. The University of Manchester. https://research.manchester.ac.uk/en/studentTheses/characterisation-of-the-potential-of-probiotics-or-their-extracts
| Files | ||
| Issue | Vol 17 No 3 (2025) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v17i3.18828 | |
| Keywords | ||
| Probiotic Lacticin Lactococcus lactis Anticancer Antibacterial | ||
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How to Cite
1.
HosseinzadehM, Doudi M, Ahadi AM. In vitro effects of purified lacticin from whey isolated Lactococcus lactis culture on Staphylococcus aureus and MCF-7 breast cancer cells. Iran J Microbiol. 2025;17(3):451-459.
