Identification of enterocins and in vitro characterization of their antimicrobial and anticancer activity in Enterococcus strains isolated from traditional fermented products

Subject Areas : Food Hygiene

Faezeh Salek ¹ (Department of Food Hygiene, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran.)
Hamid Mirzaei ² (Department of Food Hygiene, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran)
Jalil Khandaghi ³ (Department of Food Science and Technology, Sarab Branch, Islamic Azad University, Sarab, Iran)
Afshin Javadi ⁴ (Department of Food Hygiene, Faculty of Veterinary Medicine, Tabriz Medical Sciences, Islamic Azad University, Tabriz, Iran)
Yosef Nami ⁵ (Department of Food Biotechnology, Branch for Northwest & West region, Agricultural Biotechnology Research, Institute of Iran, Agricultural Research, Education and Extension Organization (AREEO), Tabriz, Iran)

Received: 2023-05-16 Accepted : 2023-08-13 Published : 2023-06-22

Keywords: Enterococcus, bacteriocin, Antimicrobial, Antitumor,

Abstract :

Replacing naturally occurring bioactive substances, such as microbial metabolites, with synthetic preservative compounds, and getting access to their beneficial properties, is expanding every day. Meanwhile, the focus of many researchers' attention has been on bacteriocins, which have multiple physiological functions, including antibacterial and anticancer properties. In this study, the enterocin production genes of Enterococcus isolates from two fermented dairy products (Motal cheese and Tarkhineh) were tracked using a molecular method. The kinetics of bacteriocin-like substance production, as well as its stability (at high temperatures, different pH levels, and under different enzyme influences), were investigated. The antagonistic (well diffusion method) and anticancer (with MTT colorimetric technique) characteristics of enterocins generated by these isolates were also investigated in two human cancer cell lines. The results of the PCR test showed that isolates of Enterococcus faecium AUT-7KB and Enterococcus faecalis KUMS-T48 contained all three studied enterocin production genes, and the bacteriocin-like metabolites of these strains had strong antagonistic effects on Listeria monocytogenes and Bacillus cereus. These metabolites had high stability at different pH and became ineffective under the influence of protease enzymes (trypsin and pepsin), which indicated their protein nature. Additionally, metabolites of the KUMS-T48 isolate showed strong cytotoxic effects on two cancer cell lines, AGS and HT-29. According to the findings of the current research, enterocins of the E. faecalis KUMS-T48 strain are suggested as a suitable candidate for further anti-tumor studies, such as in vivo studies, after complete investigations of safety aspects.

References:

• Aguilar, C., Vanegas, C. and Klotz, B. (2011). Antagonistic effect of Lactobacillus strains against Escherichia coli and Listeria monocytogenes in milk. Journal of Dairy Research, 78(2): 136-143.


• Ahmadova, A., Todorov, S.D., Choiset, Y., Rabesona, H., Zadi, T.M., Kuliyev, A., et al. (2013). Evaluation of antimicrobial activity, probiotic properties and safety of wild strain Enterococcus faecium AQ71 isolated from Azerbaijani Motal cheese. Food Control, 30(2): 631-641.


• An, B.C., Hong, S., Park, H.J., Kim, B.K., Ahn, J.Y., Ryu, Y., et al. (2019). Anti-colorectal cancer effects of probiotic-derived p8 protein. Genes, 10(8): 624.


• Aran, H., Biscola, V., El-Ghaish, S., Jaffrès, E., Dousset, X., Pillot, G., et al. (2015). Bacteriocin-producing Enterococcus faecalis KT2W2G isolated from mangrove forests in southern Thailand: purification, characterization and safety evaluation. Food Control, 54: 126-134.


• Araújo, T.F. and Ferreira, C.L. (2013). The genus Enterococcus as probiotic: safety concerns. Brazilian Archives of Biology and Technology, 56: 457-466.


• Azad, A., Naghavy, N. and Karbasizade, V. (2021). Antibacterial activity of Bacteriocin produced by Lactococcus lactis isolates from dairy products against Foodborne Pathogens. International Journal of Molecular and Clinical Microbiology, 11(1): 1416-1422.


• Balla, E., Dicks, L., Du Toit, M., Van Der Merwe, M. and Holzapfel, W. (2000). Characterization and cloning of the genes encoding enterocin 1071A and enterocin 1071B, two antimicrobial peptides produced by Enterococcus faecalis BFE 1071. Applied and Environmental Microbiology, 66(4): 1298-1304.


• Ben Braiek, O. and Smaoui, S. (2019). Enterococci: between emerging pathogens and potential probiotics. BioMed Research International, 2019: 1-13


• Brown, J.M. and Attardi, L.D. (2005). The role of apoptosis in cancer development and treatment response. Nature Reviews Cancer, 5(2): 231.


• Cesa-Luna, C., Alatorre-Cruz, J. M., Carreno-Lopez, R., Quintero-Hernandez, V. and Baez, A. (2021). Emerging applications of bacteriocins as antimicrobials, anticancer drugs, and modulators of the gastrointestinal microbiota. Polish Journal of Microbiology, 70(2): 143-159.


• Chopra, L., Singh, G., Kumar Jena, K. and Sahoo, D.K. (2015). Sonorensin: a new bacteriocin with potential of an anti-biofilm agent and a food biopreservative. Scientific Reports, 5(1): 1-13.


• De arauz, L.J., Jozala, A.F., Mazzola, P.G. and Penna, T.C.V. (2009). Nisin biotechnological production and application: a review. Trends in Food Science & Technology, 20(3-4): 146-154.


• Divyashri, G., Krishna, G. and Prapulla, S. (2015). Probiotic attributes, antioxidant, anti-inflammatory and neuromodulatory effects of Enterococcus faecium CFR 3003: in vitro and in vivo evidence. Journal of Medical Microbiology, 64(12): 1527-1540.


• Giraffa, G. (2003). Functionality of enterococci in dairy products. International Journal of Food Microbiology, 88(2-3): 215-222.


• Gorbach, S.L. (2000). Probiotics and gastrointestinal health. The American Journal of Gastroenterology, 95(1): S2-S4.


• Haghshenas, B., Nami, Y., Abdullah, N., Radiah, D., Rosli, R. and Khosroushahi, A.Y. (2014). Anti-proliferative effects of Enterococcus strains isolated from fermented dairy products on different cancer cell lines. Journal of Functional Foods, 11: 363-374.


• Hammami, I., Rhouma, A., Jaouadi, B., Rebai, A. and Nesme, X. (2009). Optimization and biochemical characterization of a bacteriocin from a newly isolated Bacillus subtilis strain 14B for biocontrol of Agrobacterium spp. strains. Letters in Applied Microbiology, 48(2): 253-260.


• Hammami, R., Zouhir, A., Hamida, J.B. and Fliss, I. (2007). BACTIBASE: a new web-accessible database for bacteriocin characterization. BMC Microbiology, 7(1): 1-6.


• Hanchi, H., Mottawea, W., Sebei, K. and Hammami, R. (2018). The genus Enterococcus: between probiotic potential and safety concerns—an update. Frontiers in Microbiology, 9: 1791.


• Hassan, M., Diep, D.B., Javadzadeh, Y., Dastmalchi, S., Nes, I.F., Sharifi, Y., et al. (2012). Prevalence of bacteriocin activities and bacteriocin-encoding genes in enterococcal clinical isolates in Iran. Canadian Journal of Microbiology, 58(4): 359-368.


• Hirayama, K. and Rafter, J. (2000). The role of probiotic bacteria in cancer prevention. Microbes and Infection, 2(6): 681-686.


• Jafari-Nasab, T., Khaleghi, M., Farsinejad, A. and Khorrami, S. (2021). Probiotic potential and anticancer properties of Pediococcus sp. isolated from traditional dairy products. Biotechnology Reports, 29: e00593.


• Kemnitzer, W., Drewe, J., Jiang, S., Zhang, H., Crogan-Grundy, C., Labreque, D., et al. (2008). Discovery of 4-aryl-4 H-chromenes as a new series of apoptosis inducers using a cell-and caspase-based high throughput screening assay. 4. Structure–activity relationships of N-alkyl substituted pyrrole fused at the 7, 8-positions. Journal of Medicinal Chemistry, 51(3): 417-423.


• Khodaii, M. and Soltani Nezhad, S. (2017). Isolation and screening of bacteriocin-producing bacteria from native dairy products of Kerman province and study of antibacterial activity of produced bacteriocin. Journal of Food Microbiology, 4(3): 69-79. [In persian]


• Kiani, A., Nami, Y., Hedayati, S., Jaymand, M., Samadian, H. and Haghshenas, B. (2021 a). Tarkhineh as a new microencapsulation matrix improves the quality and sensory characteristics of probiotic Lactococcus lactis KUMS-T18 enriched potato chips. Scientific Reports, 11(1): 12599.


• Kiani, A., Nami, Y., Hedayati, S., Komi, D.E.A., Goudarzi, F. and Haghshenas, B. (2021 b). Application of Tarkhineh fermented product to produce potato chips with strong probiotic properties, high shelf-life, and desirable sensory characteristics. Frontiers in Microbiology, 12: 1-13.


• Kouhi, F., Mirzaei, H., Nami, Y., Khandaghi, J. and Javadi, A. (2021). Potential probiotic and safety characterisation of Enterococcus bacteria isolated from indigenous fermented Motal cheese. International Dairy Journal, 105247.


• Lee, J.W., Shin, J.G., Kim, E.H., Kang, H.E., Yim, I.B., Kim, J.Y., et al. (2004). Immunomodulatory and antitumor effects in vivo by the cytoplasmic fraction of Lactobacillus casei and Bifidobacterium longum. Journal of Veterinary Science, 5(1): 41-48.


• Martin-Visscher, L.A., Yoganathan, S., Sit, C.S., Lohans, C.T. and Vederas, J.C. (2011). The activity of bacteriocins from Carnobacterium maltaromaticum UAL307 against Gram-negative bacteria in combination with EDTA treatment. FEMS Microbiology Letters, 317(2): 152-159.


• Mirdamadi, S. and Tangestani, M. (2011). Screening and characterization of bacteriocins produced by some Strains of Lactobacillus spp isolated from Iranian Dairy Products. Journal of Food Hygiene, 1(3): 55-69. [In persian]


• Mirzaei, M., Mirdamadi, S., Ehsani, M.R., Aminlari, M. and Hosseini, E. (2015). Purification and identification of antioxidant and ACE-inhibitory peptide from Saccharomyces cerevisiae protein hydrolysate. Journal of Functional Foods, 19: 259-268.


• Mojsova, S., Krstevski, K., Dzadzovski, I., Popova, Z. and Sekulovski, P. (2015). Phenotypic and genotypic characteristics of enterocin producing enterococci against pathogenic bacteria. Macedonian Veterinary Review, 38(2): 209-216.


• Molham, F., Khairalla, A.S., Azmy, A.F., El-Gebaly, E., El-Gendy, A.O. and AbdelGhani, S. (2021). Anti-Proliferative and Anti-Biofilm Potentials of Bacteriocins Produced by Non-Pathogenic Enterococcus sp. Probiotics and Antimicrobial Proteins, 13(2): 571-585.


• Moslehishad, M., Ehsani, M.R., Salami, M., Mirdamadi, S., Ezzatpanah, H., Naslaji, A.N. and Moosavi-Movahedi, A.A. (2013). The comparative assessment of ACE-inhibitory and antioxidant activities of peptide fractions obtained from fermented camel and bovine milk by Lactobacillus rhamnosus PTCC 1637. International Dairy Journal, 29(2): 82-87.


• Motahari, P., Amini-Bayat, Z. and Mirdamadi, S. (2017). Bacteriocins: New generation of antimicrobial peptides. The Journal of Qazvin University of Medical Sciences, 21(2): 79-94. [In persian]


• Nascimento, M.D., Moreno, I. and Kuaye, A.Y. (2010). Antimicrobial activity of Enterococcus faecium FAIR-E 198 against gram-positive pathogens. Brazilian Journal of Microbiology, 41: 74-81.


• Nunez, M., Rodriguez, J., Garcia, E., Gaya, P. and Medina, M. (1997). Inhibition of Listeria monocytogenes by enterocin 4 during the manufacture andripening of Manchego cheese. Journal of Applied Microbiology, 83(6): 671-677.


• Rajaram, G., Manivasagan, P., Thilagavathi, B. and Saravanakumar, A. (2010). Purification and characterization of a bacteriocin produced by Lactobacillus lactis isolated from marine environment. Advance Journal of Food Science and Technology, 2(2): 138-144.


• Sami, A., Abbasgholizadeh, N. and Khandaghi, J. (2022). Evaluation of Safety Aspects and Antagonistic Activity of Enterococcus Strains Isolated from Traditional Pot Cheese. Journal of Health, 13(1), 7-16. [In persian]


• Śliżewska, K., Markowiak-Kopeć, P. and Śliżewska, W. (2020). The role of probiotics in cancer prevention. Cancers, 13(1): 1-22.


• Sobrino-López, A. and Martín-Belloso, O. (2008). Use of nisin and other bacteriocins for preservation of dairy products. International Dairy Journal, 18(4), 329-343.


• Sonsa-Ard, N., Rodtong, S., Chikindas, M.L. and Yongsawatdigul, J. (2015). Characterization of bacteriocin produced by Enterococcus faecium CN-25 isolated from traditionally Thai fermented fish roe. Food Control, 54: 308-316.


• Strompfová, V., Lauková, A., Simonová, M. and Marciňáková, M. (2008). Occurrence of the structural enterocin A, P, B, L50B genes in enterococci of different origin. Veterinary Microbiology, 132(3-4): 293-301.


• Todorov, S. and Dicks, L. (2006). Screening for bacteriocin-producing lactic acid bacteria from boza, a traditional cereal beverage from Bulgaria: Comparison of the bacteriocins. Process Biochemistry, 41(1): 11-19.


• Tsai, Y.T., Cheng, P.C., Fan, C.K. and Pan, T.M. (2008). Time-dependent persistence of enhanced immune response by a potential probiotic strain Lactobacillus paracasei subsp. paracasei NTU 101. International Journal of Food Microbiology, 128(2): 219-225.


• Wang, C.Z., Calway, T. and Yuan, C.S. (2012). Herbal medicines as adjuvants for cancer therapeutics. The American Journal of Chinese Medicine, 40(4): 657-669.


• Wong, S., McLaughlin, J., Cheng, D. and Witte, O.N. (2003). Cell context–specific effects of the BCR-ABL oncogene monitored in hematopoietic progenitors. Blood, the Journal of the American Society of Hematology, 101(10): 4088-4097.


• Xi, Q., Wang, J., Du, R., Zhao, F., Han, Y. and Zhou, Z. (2018). Purification and characterization of bacteriocin produced by a strain of Enterococcus faecalis TG2. Applied Biochemistry and Biotechnology, 184: 1106-1119.


• Yerlikaya, O. and Akbulut, N. (2020). In vitro characterisation of probiotic properties of Enterococcus faecium and Enterococcus durans strains isolated from raw milk and traditional dairy products. International Journal of Dairy Technology, 73(1): 98-107.