Fermentation optimization of pediocin PD-1 production and a comparative study of the effect of pediocin PD-1, plantaricin 423 and nisin on biofilms of Oenococcus oeni

Nel, Hannes Augustinus (2001-12)

Thesis (MSc)--Stellenbosch University, 2001.

Thesis

ENGLISH ABSTRACT: Lactic acid bacteria are present in many foods and beverages and are used as starter cultures in the production of a variety of fermented products. Many of these bacteria produce ribosomally synthesized antimicrobial peptides (bacteriocins), which inhibit the growth of bacteria genetically closely related to the producer cell. Since many of these target bacteria include foodbome pathogens such as Bacillus spp., Clostridium spp., Listeria spp., and Staphylococcus spp., the practical importance of these peptides as food preservatives has been well documented and, in the case of nisin and pediocin PA-I, commercially explored. The increased demand from health conscious consumers for foods with no chemical preservatives is putting renewed pressure on the producer to supply a "clean and green" product, but with the same or even an extended shelf life. Various research groups are screening lactic acid bacteria for production of novel broad-spectrum antimicrobial peptides or are exploring the possibilities of altering known bacteriocins to inhibit Gram-negative bacteria, yeasts and molds. Pediocin PD-I, produced by Pediococcus damnosus NCFB 1832, belongs to the class Ila bacteriocins, i.e. heat-stable Listeria-active peptides, containing the YGNGV -consensus sequence in the N-terminal region. Little is known about the production and mode of activity of pediocin PD-I. In this study, production of pediocin PD-I was significantly increased by optimizing the growth medium, De Man Rogosa and Sharpe (MRS) broth. Addition of bacteriological peptone (1.7%, w/v), manganous sulphate (0.014%, w/v) and Tween 80 (3%, v/v), and lowering of the pH during fermentation stimulated pediocin PD-I production and the level of organic acids produced. Maximum levels of bacteriocin activity were recorded at an initial pH of 6.7 in the latter medium. Under these conditions the specific bacteriocin activity increased by a factor of approximately six after 55 h of fermentation. The effect of pediocin PD-I, plantaricin 423, produced by Lactobacillus plantarum 423, and commercial grade nisin (Aplin and Barrett Ltd., Trowbrige, Wilts, England) was tested against planktonic cells of Oenococcus oeni and a biofilm of the cells established on stainless steel surfaces identical to those used in wineries. After 5 h of treatment with 3000 AU (arbitrary units )/ml of each bacteriocin, all planktonic cells of 0. oeni in a modified Chardonnay must medium were killed. All viable cells in the biofilm were killed after only 1 h in the presence of 3000 AU/ml of anyone of the bacteriocins. In addition, pediocin PD-I, plantaricin 423 and nisin removed the biofilms from the surfaces and reduced the biomass either completely, as in the case of pediocin PD-I, or by 58% and 50% as in the case of plantaricin 423 and nisin, respectively. These same results were recorded after 5 h of treatment with 3000 AU/ml in a modified Chardonnay must medium. To our knowledge this is the first report of controlling biofilm formation of malolactic bacteria on stainless steel surfaces with natural antimicrobial peptides. This implies that, apart from being very effective in controlling the cell numbers of free-living cells of 0. oeni, the three bacteriocins, especially pediocin PD-I, could also be used as natural sanitizers. The fact that the production and activity levels ofpediocin PD-I could be increased without genetically modifying the producer strain is an added advantage.

AFRIKAANSE OPSOMMING: Melksuurbakterieë is teenwoordig in verskeie soorte voedsel- en drankprodukte en word as suurselkulture in die produksie van 'n verskeidenheid gefermenteerde produkte gebruik. Baie van hierdie bakterieë produseer ribosomaal-vervaardigde antimikrobiese peptiede (bakteriosiene) wat die groei van ander bakterieë, geneties naverwant aan die produserende organisme, inhibeer. Omdat baie van hierdie bakterieë voedselpatogene soos Bacillus spp., Clostridium spp., Listeria spp. en Staphylococcus spp. insluit, is die praktiese belang van hierdie peptiede reeds deeglik ondersoek en word, soos in die geval van nisien en pediosien PA-I, kommersieel gebruik. Die toenemende behoefte van die verbruiker na voedselprodukte met geen chemiese preserveermiddels plaas nuwe druk op die vervaardiger om veilige voedselprodukte te produseer, maar met dieselfde of selfs langer rakleeftyd. Verskeie navorsingsgroepe bestudeer melksuurbakterieë vir die produksie van unieke antimikrobiese peptiede met 'n wye spektrum van inhibisie en ondersoek ook die moontlikhede om hierdie bakteriosiene geneties te manipuleer ten einde Gram-negatiewe bakterieë, giste en swamme te inhibeer. Pediosien PD-l, geproduseer deur Pediococcus damnosus NCFB 1832, word as 'n klass na bakteriosien geklassifiseer. Hierdie groep sluit in die hitte-stabiele Listeria-aktiewe peptiede, met 'n YGNGV-konsensus volgorde in die N-terminale deel van die peptied. Min is egter bekend oor die meganisme van werking van hierdie bakteriosiene. In hierdie studie is die produksie van pediosien PD-l betekenisvol verhoog met die optimalisering van die vloeibare groeimedium De Man Rogosa en Sharpe (MRS). Die toevoeging van bakteriologiese peptone (1.7%, miv), mangaan sulfaat (0.014%, miv) en Tween 80 (3.0%, v/v) en 'n afname in die pH gedurende groei het pediosien PD-l-poduksie gestimuleer en sodoende ook die vlak van organiese sure wat geproduseer is. Maksimum vlakke van bakteriosien-aktiwiteit is in hierdie medium met 'n aanvangs-pH van 6.7 waargeneem. Onder hierdie omstandighede, en na 55 uur van fermentasie, het die spesifieke aktiwiteit van die bakteriosien met 'n faktor van ongeveer ses verhoog. Die effek van pediosien PD-l, plantarisien 423, geproduseer deur Lactobacillus plantarum 423, en 'n kommersiële graad nisien (Aplin and Barrett Ltd., Trowbride, Wilts, Engeland) is teen die planktoniese selle van Oenococcus oeni en 'n biofilm van hierdie selle, gevestig op 'n vlekvrye staaloppervlak identies aan wat in wynkelders gebruik word, getoets. Na 5 ure van behandeling met 3000 AB (arbitrêre eenhede)/ml van elke bakteriosien, is al die planktoniese selle van O. oeni in 'n gemodifiseerde Chardonnay mos-medium vernietig. Alle lewensvatbare selle in die biofilm is ook na slegs 1 uur in die teenwoordigheid van 3000 AE/ml van enige een van hierdie bakteriosiene vernietig. Verdermeer het pediosien PD-I, plantarisien 423 en nisien ook die biofilm op die vlekvrye staal-oppervlak verwyder. In die geval van pediosien PD-I is 'n totale afname van die biomassa-oppervlak waargeneem, terwyl plantarisien 423 en nisien 58% en 50% van die totale biomassa verwyder het. Hierdie resultate is na 5 ure van behandeling (3000 AE/ml) in 'n gemodifiseerde Chardonnay mos-medium waargeneem. Sover ons kennis strek is hierdie die eerste verslag rakende die gebruik van natuurlike antimikrobiese peptiede om biofilm-vorming deur appel-melksuurbakterieë op vlekvrye staal oppervlaktes te beheer. Dit impliseer dat bakteriosiene, spesifiek pediosien PD-I, benewens die beheer van planktoniese selle van appel-melksuurbakterieë, ook as natuurlike oppervlak-reinigers gebruik kan word. Die feit dat die produksie en aktiwiteitsvlakke van pediosien PD-I verhoog kon word sonder om die organisme geneties te modifiseer is 'n verdere voordeel.

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