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Probiotics and Antimicrobial Proteins

, Volume 11, Issue 4, pp 1391–1402 | Cite as

Nisin Production by Enterococcus hirae DF105Mi Isolated from Brazilian Goat Milk

  • Danielle Nader Furtado
  • Lorenzo Favaro
  • Luis Augusto Nero
  • Bernadette Dora Gombossy de Melo Franco
  • Svetoslav Dimitrov TodorovEmail author
Original Research

Abstract

The purpose of this study was to select the promising biopreservation bacteriocin producer strain from goat milk and characterize the expressed bacteriocin, related to its physiological and biochemical properties and specificity of operon encoding production and expression of antimicrobial peptide. Brazilian goat milk was used as the source for the selection of bacteriocin-producing lactic acid bacteria. One strain (DF105Mi) stood out for its strong activity against several Listeria monocytogenes strains. Selected strain was identified based on the biochemical and physiological characteristics and 16s rRNA analysis. The bacteriocin production and inhibitory spectrum of strain DF105Mi were studied, together with the evaluation of the effect of temperature, pH, and chemicals on bacteriocin stability and production, activity, and adsorption to target cells as well as to the cell surface of bacteriocin producers. Physiological and bio-molecular analyses based on targeting of different genes, parts of nisin operon were performed in order to investigate the hypothesis that the studied strain can produce and express nisin. Based on biochemical, physiological, and 16s rRNA analysis, the strain DF105Mi was classified as Enterococcus hirae. The selected strain produces a bacteriocin which is stable in a wide range of pH (2.0–12.0), temperature (up to 120 °C), presence of selected chemicals and presents adsorption affinity to different test organisms, process influenced by environmental conditions. Higher bacteriocin production by Ent. hirae DF105Mi was recorded during stationary growth phase, but only when the strain was cultured at 37 °C. The strain’s genetic analysis indicated presence of the genes coding for the production of the bacteriocin nisin. This result was confirmed by cross-checking the sensitivity of the produced strain to commercial nisin A. The strong anti-Listeria activity, bacteriocin adsorption, and stability of produced bacteriocin indicate that Ent. hirae DF105Mi presents a differentiated potential application for biopreservation of fermented dairy products.

Keywords

Bacteriocins Enterococcus hirae Goat milk Biopreservation Nisin operon 

Notes

Funding

This work was supported by projects from USP Program for Visiting Professors (2016.1.920.93), FAPESP (process 2013/07914-8), FAPEMIG, and CNPq.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Research Involving Human Participants and/or Animals

All experiments performed in the present study do not involve human participants or animals.

Informed Consent

There is no need for informed consent because our study does not relate to human participants or animals.

References

  1. 1.
    De Vuyst L, Leroy F (2007) Bacteriocins from lactic acid bacteria: production, purification, and food applications. J Mol Microbiol Biotechnol 13:194–199PubMedGoogle Scholar
  2. 2.
    Favaro L, Penna ALB, Todorov SD (2015) Bacteriocinogenic LAB from cheeses–application in biopreservation? Trends Food Sci Technol 41:37–48Google Scholar
  3. 3.
    Varsha KK, Nampoothiri KM (2016) Appraisal of lactic acid bacteria as protective cultures. Food Control 69:61–64Google Scholar
  4. 4.
    Todorov SD, Franco BDGM, Tagg JR (2019) Bacteriocins of Gram positive bacteria having activity spectra extending beyond closely-related species. Benef Microb 10:315–328Google Scholar
  5. 5.
    Leroy F, De Vuyst L (2004) Lactic acid bacteria as functional starter cultures for the food fermentation industry. Trends Food Sci Technol 15:67–78Google Scholar
  6. 6.
    Ross RP, Morgan S, Hill C (2002) Preservation and fermentation: past, present and future. Int J Food Microbiol 79:3–16PubMedGoogle Scholar
  7. 7.
    Gillor O, Etzion A, Riley M (2008) The dual role of bacteriocins as anti-and probiotics. Appl Microbiol Biotechnol 81:591–606PubMedPubMedCentralGoogle Scholar
  8. 8.
    Favaro L, Todorov SD (2017) Bacteriocinogenic LAB strains for fermented meat preservation: perspectives, challenges, and limitations. Prob Antimicrob Prot 9:444–458Google Scholar
  9. 9.
    O’Bryan C, Crandall P, Ricke S, Ndahetuye J (2015) Lactic acid bacteria (LAB) as antimicrobials in food products: types and mechanisms of action. Handbook Nat Antimicrob Food Saf Qual 6:117–129Google Scholar
  10. 10.
    Cintas L, Casaus P, Fernandez M, Hernández P (1998) Comparative antimicrobial activity of enterocin L50, pediocin PA-1, nisin A and lactocin S against spoilage and foodborne pathogenic bacteria. Food Microbiol 15:289–298Google Scholar
  11. 11.
    Dimov S, Peykov S, Raykova D, Ivanova P, Kirilov N, Dalgalarrondo M, Chobert J, Haertlé T, Ivanova I (2009) A newly discovered bacteriocin produced by Enterococcus faecalis 3915. Benef Microb 1:43–51Google Scholar
  12. 12.
    Eguchi T, Kaminaka K, Shima J, Kawamoto S, Mori K, Choi S-H, Doi K, Ohmomo S, Ogata S (2001) Isolation and characterization of enterocin SE-K4 produced by thermophilic enterococci, Enterococcus faecalis K-4. Biosci Biotechnol Biochem 65:247–253PubMedGoogle Scholar
  13. 13.
    Foulquié Moreno M, Callewaert R, Devreese B, Van Beeumen J, De Vuyst L (2003) Isolation and biochemical characterisation of enterocins produced by enterococci from different sources. J Appl Microbiol 94:214–229PubMedGoogle Scholar
  14. 14.
    El-Ghaish S, Hadji-Sfaxi I, Ahmadova A, Choiset Y, Rabesona H, Sitohy M, Haertlé T, Chobert J (2011) Characterization of two safe Enterococcus strains producing enterocins isolated from Egyptian dairy products. Benef Microb 2:15–27Google Scholar
  15. 15.
    Todorov SD, Favaro L, Gibbs P, Vaz-Velho M (2012) Enterococcus faecium isolated from Lombo, a Portuguese traditional meat product: characterisation of antibacterial compounds and factors affecting bacteriocin production. Benef Microb 3:319–330Google Scholar
  16. 16.
    Graham CE, Cruz MR, Garsin DA, Lorenz MC (2017) Enterococcus faecalis bacteriocin EntV inhibits hyphal morphogenesis, biofilm formation, and virulence of Candida albicans. Proc Nat Acad Sci USA 114:4507–4512Google Scholar
  17. 17.
    Giraffa G (2002) Enterococci from foods. FEMS Microbiol Rev 26:163–171PubMedGoogle Scholar
  18. 18.
    Khan H, Flint S, Yu P-L (2010) Enterocins in food preservation. Int J Food Microbiol 141:1–10PubMedGoogle Scholar
  19. 19.
    Moreno MF, Sarantinopoulos P, Tsakalidou E, De Vuyst L (2006) The role and application of enterococci in food and health. Int J Food Microbiol 106:1–24Google Scholar
  20. 20.
    Lôbo RB, Facó O, Lôbo ABO, Villela LV (2010) Brazilian goat breeding programs. Small Rum Res 89:149–154Google Scholar
  21. 21.
    Facó O, Lôbo RB, Gouveia AG, de Paiva Guimarães MM, Fonseca JF, dos Santos TNM, da Silva MAA, Villela LV (2011) Breeding plan for commercial dairy goat production systems in southern Brazil. Small Rumin Res 98:164–169Google Scholar
  22. 22.
    Furtado DN, Todorov SD, Landgraf M, Destro MT, Franco BDGM (2014) Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: characterization of the bacteriocin. Braz J Microbiol 45:1541–1550PubMedGoogle Scholar
  23. 23.
    Favaro L, Basaglia M, Casella S, Hue I, Dousset X, Franco BDGM, Todorov SD (2014) Bacteriocinogenic potential and safety evaluation of non-starter Enterococcus faecium strains isolated from home made white brine cheese. Food Microbiol 38:228–239PubMedGoogle Scholar
  24. 24.
    Schirru S, Favaro L, Mangia NP, Basaglia M, Casella S, Comunian R, Fancello F, Franco BDGM, Oliveira RPS, Todorov SD (2014) Comparison of bacteriocins production from Enterococcus faecium strains in cheese whey and optimised commercial MRS medium. Ann Microbiol 64:321–331Google Scholar
  25. 25.
    Furtado DN, Todorov SD, Landgraf M, Destro MT, Franco BDGM (2015) Bacteriocinogenic Lactococcus lactis subsp lactis DF04Mi isolated from goat milk: application in the control of Listeria monocytogenes in fresh Minas-type goat cheese. Braz J Microbiol 46:201–206PubMedPubMedCentralGoogle Scholar
  26. 26.
    Perin LM, Miranda RO, Todorov SD, Franco BDGM, Nero LA (2014) Virulence, antibiotic resistance and biogenic amines of bacteriocinogenic lactococci and enterococci isolated from goat milk. Int J Food Microbiol 185:121–126PubMedPubMedCentralGoogle Scholar
  27. 27.
    Todorov SD, Dicks LMT (2005a) Optimization of bacteriocin ST311LD production by Enterococcus faecium ST311LD, isolated from spoiled black olives. J Microbiol 43:370–374PubMedGoogle Scholar
  28. 28.
    Schillinger U, Lücke F-K (1987) Identification of lactobacilli from meat and meat products. Food Microbiol 4:199–208Google Scholar
  29. 29.
    Stiles ME, Holzapfel WH (1997) Lactic acid bacteria of foods and their current taxonomy. Int J Food Microbiol 36:1–29PubMedGoogle Scholar
  30. 30.
    Felske A, Rheims H, Wolterink A, Stackebrandt E, Akkermans AD (1997) Ribosome analysis reveals prominent activity of an uncultured member of the class Actinobacteria in grassland soils. Microbiol 143:2983–2989Google Scholar
  31. 31.
    Costa Y, Galimand M, Leclercq R, Duval J, Courvalin P (1993) Characterization of the chromosomal aac (6′)-Ii gene specific for Enterococcus faecium. Antimicrob Agents Chem 37:1896–1903Google Scholar
  32. 32.
    Robredo B, Singh KV, Baquero F, Murray BE, Torres C (1999) From vanA Enterococcus hirae to vanA Enterococcus faecium: a study of feed supplementation with avoparcin and tylosin in young chickens. Antimicrob Agents Chem 43:1137–1143Google Scholar
  33. 33.
    De Kwaadsteniet M, Fraser T, Van Reenen C, Dicks L (2006) Bacteriocin T8, a novel class IIa sec-dependent bacteriocin produced by Enterococcus faecium T8, isolated from vaginal secretions of children infected with human immunodeficiency virus. Appl Environ Microbiol 72:4761–4766PubMedPubMedCentralGoogle Scholar
  34. 34.
    Veljovic K, Terzic-Vidojevic A, Vukasinovic M, Strahinic I, Begovic J, Lozo J, Ostojic M, Topisirovic L (2007) Preliminary characterization of lactic acid bacteria isolated from Zlatar cheese. J Appl Microbiol 103:2142–2152PubMedGoogle Scholar
  35. 35.
    Olasupo NA, Schillinger U, Narbad A, Dodd H, Holzapfel WH (1999) Occurrence of nisin Z production in Lactococcus lactis BFE 1500 isolated from wara, a traditional Nigerian cheese product. Int J Food Microbiol 53:141–152PubMedGoogle Scholar
  36. 36.
    Li H, O'Sullivan DJ (2002) Heterologous expression of the Lactococcus lactis bacteriocin, nisin, in a dairy Enterococcus strain. Appl Environ Microbiol 68:3392–3400PubMedPubMedCentralGoogle Scholar
  37. 37.
    AlKhatib Z, Lagedroste M, Fey I, Kleinschrodt D, Abts A, Smits SH (2014) Lantibiotic immunity: inhibition of nisin mediated pore formation by NisI. PLoS One 9:e102246.  https://doi.org/10.1371/journal.pone.0102246 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Pisano MB, Fadda ME, Melis R, Ciusa ML, Viale S, Deplano M, Cosentino S (2015) Molecular identification of bacteriocins produced by Lactococcus lactis dairy strains and their technological and genotypic characterization. Food Control 51:1–8Google Scholar
  39. 39.
    Yildirim Z, Avsar YK, Yildirm M (2002) Factors affecting the adsorption of buchnericin LB, a bacteriocin produced by Lactocobacillus buchneri. Microbiol Res 157:103–107PubMedGoogle Scholar
  40. 40.
    Todorov SD (2008) Bacteriocin production by Lactobacillus plantarum AMA-K isolated from Amasi, a Zimbabwean fermented milk product and study of adsorption of bacteriocin AMA-K to Listeria spp. Braz J Microbiol 38:178–187Google Scholar
  41. 41.
    Yang R, Johnson MC, Ray B (1992) Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl Environ Microbiol 58:3355–3359PubMedPubMedCentralGoogle Scholar
  42. 42.
    Siragusa GR (1992) Production of bacteriocin inhibitory to Listeria species by Enterococcus hirae. Appl Environ Microbiol 58:3508–3513PubMedPubMedCentralGoogle Scholar
  43. 43.
    Gupta A, Tiwari SK (2015) Probiotic potential of bacteriocin-producing Enterococcus hirae strain LD3 isolated from dosa batter. Ann Microbiol 65:2333–2342Google Scholar
  44. 44.
    De Vuyst L, Vandamme EJ (1994) Antimicrobial potential of lactic acid bacteria, Bacteriocins of lactic acid bacteria. Springer, Berlin, pp 91–142Google Scholar
  45. 45.
    Todorov SD, Wachsman MB, Knoetze H, Meincken M, Dicks LMT (2005) An antibacterial and antiviral peptide produced by Enterococcus mundtii ST4V isolated from soya beans. Int J Antimicrob Agents 25:508–513PubMedGoogle Scholar
  46. 46.
    Schirru S, Todorov SD, Favaro L, Mangia NP, Basaglia M, Casella S, Comunian R, Franco BDGM, Deiana P (2012) Sardinian goat’s milk as source of bacteriocinogenic potential protective cultures. Food Control 25:309–320Google Scholar
  47. 47.
    Aspri M, O'Connor PM, Field D, Cotter PD, Ross P, Hill C, Papademas P (2017) Application of bacteriocin-producing Enterococcus faecium isolated from donkey milk, in the bio-control of Listeria monocytogenes in fresh whey cheese. Int Dairy J 73:1–9Google Scholar
  48. 48.
    Xi Q, Wang J, Du R, Zhao F, Han Y, Zhou Z (2018) Purification and characterization of bacteriocin produced by a strain of Enterococcus faecalis TG2. Appl Biochem Biotechnol 184:1106–1119PubMedGoogle Scholar
  49. 49.
    Ko S-H, Ahn C (2000) Bacteriocin production by Lactococcus lactis KCA2386 isolated from white kimchi. Food Sci Biotechnol 9:263–269Google Scholar
  50. 50.
    Tomé E, Todorov SD, Gibbs PA, Teixeira PC (2009) Partial characterization of nine bacteriocins produced by lactic acid bacteria isolated from cold-smoked salmon with activity against Listeria monocytogenes. Food Biotechnol 23:50–73Google Scholar
  51. 51.
    Cavicchioli V, Camargo A, Todorov SD, Nero L (2017) Novel bacteriocinogenic Enterococcus hirae and Pediococcus pentosaceus strains with antilisterial activity isolated from Brazilian artisanal cheese. J Dairy Sci 100:2526–2535PubMedGoogle Scholar
  52. 52.
    Huycke MM (2002) Physiology of enterococci, The Enterococci. In: American Society of Microbiology, pp 133–175Google Scholar
  53. 53.
    Aymerich T, Artigas M, Garriga M, Monfort J, Hugas M (2000) Effect of sausage ingredients and additives on the production of enterocin A and B by Enterococcus faecium CTC492. Optimization of in vitro production and anti-listerial effect in dry fermented sausages. J Appl Microbiol 88:686–694PubMedGoogle Scholar
  54. 54.
    Sarantinopoulos P, Leroy F, Leontopoulou E, Georgalaki MD, Kalantzopoulos G, Tsakalidou E, De Vuyst L (2002) Bacteriocin production by Enterococcus faecium FAIR-E 198 in view of its application as adjunct starter in Greek Feta cheese making. Int J Food Microbiol 72:125–136PubMedGoogle Scholar
  55. 55.
    Yang E, Fan L, Yan J, Jiang Y, Doucette C, Fillmore S, Walker B (2018) Influence of culture media, pH and temperature on growth and bacteriocin production of bacteriocinogenic lactic acid bacteria. AMB Express 8:10PubMedPubMedCentralGoogle Scholar
  56. 56.
    Poeta P, Costa D, Rojo-Bezares B, Zarazaga M, Klibi N, Rodrigues J, Torres C (2007) Detection of antimicrobial activities and bacteriocin structural genes in faecal enterococci of wild animals. Microbiol Res 162:257–263PubMedGoogle Scholar
  57. 57.
    Ahmadova A, Todorov SD, Choiset Y, Rabesona H, Zadi TM, Kuliyev A, Franco BDGM, Chobert J-M, Haertlé T (2013) Evaluation of antimicrobial activity, probiotic properties and safety of wild strain Enterococcus faecium AQ71 isolated from Azerbaijani Motal cheese. Food Control 30:631–641Google Scholar
  58. 58.
    Mogoşanu GD, Grumezescu AM, Bejenaru C, Bejenaru LE (2017) Natural products used for food preservation, food preservation. Elsevier, Amsterdam, pp 365–411Google Scholar
  59. 59.
    Chikindas ML, Weeks R, Drider D, Chistyakov VA, Dicks LM (2018) Functions and emerging applications of bacteriocins. Curr Opin Biotechnol 49:23–28PubMedPubMedCentralGoogle Scholar
  60. 60.
    McAuliffe O, Ross RP, Hill C (2001) Lantibiotics: structure, biosynthesis and mode of action. FEMS Microbiol Rev 25:285–308PubMedGoogle Scholar
  61. 61.
    Ra R, Beerthuyzen MM, de Vos WM, Saris PE, Kuipers OP (1999) Effects of gene disruptions in the nisin gene cluster of Lactococcus lactis on nisin production and producer immunity. Microbiol 145:1227–1233Google Scholar
  62. 62.
    Broadbent JR, Kondo JK (1991) Genetic construction of nisin-producing Lactococcuslactis subsp. cremoris and analysis of a rapid method for conjugation. Appl Environ Microbiol 57:517–524PubMedPubMedCentralGoogle Scholar
  63. 63.
    Paiva AD, Breukink E (2013) Antimicrobial peptides produced by microorganisms. In: Antimicrobial peptides and innate immunity. Springer, pp., pp 53–95Google Scholar
  64. 64.
    Gobbetti M, De Angelis M, Di Cagno R, Minervini F, Limitone A (2007) Cell–cell communication in food related bacteria. Int J Food Microbiol 120:34–45PubMedGoogle Scholar
  65. 65.
    Kleerebezem M (2004) Quorum sensing control of lantibiotic production; nisin and subtilin autoregulate their own biosynthesis. Peptides 25:1405–1414PubMedGoogle Scholar
  66. 66.
    Draper LA, Ross RP, Hill C, Cotter PD (2008) Lantibiotic immunity. Curr Protein Peptide Sci 9:39–49Google Scholar
  67. 67.
    Rodali VP, Lingala VK, Karlapudi AP, Indira M, Venkateswarulu TC, John Babu D (2013) Biosynthesis and potential application of bacteriocins. J Pure Appl Microbiol 7:2933–2945Google Scholar
  68. 68.
    Kjos M, Oppegard C, Diep DB, Nes IF, Veening J-W, Nissen-Meyer J, Kristensen T (2014) Sensitivity of two-peptide bacteriocin lactocin G in dependent on UppP, an enzyme involved in cell-wall synthesis. Mol Microbiol 92:1177–1187PubMedGoogle Scholar
  69. 69.
    Pingitore EV, Todorov SD, Sesma F, Franco BDGM (2012) Application of bacteriocinogenic Enterococcus mundtii CRL35 and Enterococcus faecium ST88Ch in the control of Listeria monocytogenes in fresh Minas cheese. Food Microbiol 32:38–47Google Scholar
  70. 70.
    Todorov S, Onno B, Sorokine O, Chobert J-M, Ivanova I, Dousset X (1999) Detection and characterization of a novel antibacterial substance produced by Lactobacillus plantarum ST31 isolated from sourdough. Int J Food Microbiol 48:167–177PubMedGoogle Scholar
  71. 71.
    Todorov SD, Dicks LMT (2005b) Pediocin ST18, an anti-listerial bacteriocin produced by Pediococcus pentosaceus ST18 isolated from boza, a traditional cereal beverage from Bulgaria. Process Biochem 40:365–370Google Scholar
  72. 72.
    Ivanova I, Kabadjova P, Pantev A, Danova S, Dousset X (2000) Detection, purification and partial characterization of a novel bacteriocin substance produced by Lactococcus lactis subsp. lactis B14 isolated from boza-Bulgarian traditional cereal beverage. Biocatal 41:47–53Google Scholar
  73. 73.
    Atrih A, Rekhif N, Moir A, Lebrihi A, Lefebvre G (2001) Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19. Int J Food Microbiol 68:93–104PubMedGoogle Scholar

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Authors and Affiliations

  1. 1.Food Research Center (FoRC), Faculdade de Ciências Farmacêuticas, Departamento de Alimentos e Nutrição Experimental, Laboratório de Microbiologia de AlimentosUniversidade de São PauloSão PauloBrazil
  2. 2.Department of Agronomy Food Natural resources Animals and Environment (DAFNAE)University of Padova, AgripolisLegnaroItaly
  3. 3.Departamento de VeterináriaUniversidade Federal de Viçosa, Campus UFVViçosaBrazil

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