Characterization of thoeniicin 447 produced by Propionibacterium thoenii

Van der Merwe, Iansha (Iansha Rosalia), 1975- (2002-12)

Thesis (MSc)--University of Stellenbosch, 2002.

Thesis

ENGLISH ABSTRACT: Antimicrobial peptides continue to be one of the most important classes of food additives. The food industry is especially interested in the application of naturally occuring and biologically derived preservatives. Among the metabolites of industrial importance produced by propionibacteria are peptides called bacteriocins. Bacteriocins are ribosomally synthesized peptides with antagonistic activity against closely related microorganisms. Many microorganisms associated with food produce bacteriocins, which have stimulated interest in the use of these peptides as natural food preservatives. Numerous bacteriocins are produced by lactic acid bacteria, but only a few have been reported for propionibacteria. Since propionic acid bacteria have GRAS (generally regarded as safe) status, their metabolic compounds should be safe for human consumption. Propionibacterium thoenii 447, isolated from Emmentaler cheese, produces a bacteriocin-like peptide, named thoeniicin 447, with a narrow spectrum of activity. The peptide displays a bactericidal mode of action against Lactobacillus delbrueckii subsp. bulgaricus and a bacteriostatic action against Propionibacterium acnes. Optimal bacteriocin production was detected during the early stationary growth phase. The peptide is resistant to heat treatments of 60°C and 80°C for 15 and 30 min and to 100°C for 15 min, but loses 80% of its activity after autoclaving (10 min at 121°C). Thoeniicin 447 remains active after incubation in buffers with pH values ranging from 1-10. The peptide is inactivated by pepsin, pronase, a-chymotrypsin, trypsin and Proteinase K. Thoeniicin 447 was partially purified by ammonium sulfate precipitation, followed by SP-Sepharose cation exchange chromatography. The estimated size of thoeniicin 447, according to tricine-SDSPAGE, is approximately 6 kDa. Based on DNA sequencing, the mature peptide is 7130 Da in size and homologous to propionicin Tl produced by P. thoenii strain 419. Thoeniicin 447 is a relatively small, cationic and heat-stable peptide and can therefor be classified as a member of class II bacteriocins. These features are very similar to those of bacteriocins produced by lactic acid bacteria. However, no unique classification system has been proposed for bacteriocins of propionibacteria. As a member of the genus Propionibacterium, P. thoenii 447 is generally regarded as safe. This, together with the narrow spectrum of activity, particularly the action against P. acnes, heat tolerance of thoeniicin 447 and its activity over a wide pH range renders the peptide suitable for possible pharmaceutical applications.

AFRIKAANSE OPSOMMING: Antimikrobiese middels sal deurgaans beskou word as een van die belangrikste klasse van voedsel bymiddels. Die voedselindustrie is veral geïnteresseerd in die toepassing van preserveermiddels van 'n meer natuurlike en biologiese oorsprong. Onder die metaboliese produkte van industriële belang wat deur propionibakterieë geproduseer word is antimikrobiese peptiede (bakteriosiene). Bakteriosiene is ribosomaal-gesintetiseerde peptiede met 'n antagonistiese aktiwiteit teenoor naverwante bakterieë. Verskeie bakteriosiene word deur melksuurbakterieë geproduseer, terwyl slegs enkele vir propionibakterieë beskryf is. Baie van hierdie propionibakterieë word in die algemeen as veilig beskou en het GRAS status. Die metaboliete wat hulle produseer behoort dus veilig vir menslike gebruik te wees. Propionibacterium thoenii 447 is uit Emmentaler kaas geisoleer en produseer 'n bakteriosien-agtige peptied, naamlik thoeniicin 447 met 'n beperkte spektrum van aktiwiteit. Die peptied het 'n bakteriosidiese werking teenoor Lactobacillus delbrueckii subsp. bulgaricus en 'n bakteriostatiese werking teenoor Propionibacterium acnes. Optimum bakteriosien produksie is verkry tydens die vroeë stationêre groeifase. Die peptied is bestand teen hittebehandelings van 60°C en 80°C vir 15 en 30 min, asook 100°C vir 15 min, maar verloor 80% van sy aktiwiteit na outoklavering (lOmin by 121°C). Die peptied blyaktief na inkubasie in buffers van pH 1-10. Die peptied word deur pepsien, pronase, uchymotripsien, tripsien en Proteinase K geïnaktiveer. Thoeniicin 447 is met behulp van ammoniumsulfaat-presipitasie, gevolg deur SPSepharose katioon-uitruilchromatografie gedeeltelik gesuiwer. Skeiding op "n trisien-SDS poliakrielarnied-jel het 'n aktiewe band van ongeveer 6 kDa getoon. Volgens die DNA volgorde bepaling is thoeniicin 447, 7130 Da in grootte en homoloog aan Propionicin Tl, geisoleer vanaf P. thoenii stam 419. Thoeniicin 447 is 'n relatiewe klein, kationiese en hitte-bestande peptied en kan op grond hiervan as 'n lid van die klas II bakteriosiene geklassifiseer word. Hierdie eienskappe is soortgelyk aan die eienskappe van bakteriosiene geproduseer deur melksuurbakterieë. Tot op hede is geen klassifikasiesisteem vir die bakteriosiene van propionibakterieë voorgestel nie. As 'n lid van die genus Propionibacterium, word P. thoenii 447 in die algemeen as veilig beskou. Dit, tesame met die nou spektrum van aktiwiteit, veral teenoor P. acnes, die hittetoleransie van thoeniicin 447, asook die aktiwiteit oor 'n wye pH-grens, maak die peptied geskik vir moontlike farmaseutiese toepassings.

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