The evaluation of bacteriocins and enzymes for biopreservation of wine

Du Toit, Corina (2002-03)

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

Thesis

ENGLISH ABSTRACT: The winemaking process involves a number of microorganisms, each with its own role. Yeasts are responsible for the alcoholic fermentation, the lactic acid bacteria (LAB) are Gram-positive bacteria associated with must and wine and perform the malolactic fermentation (MLF), while the acetic acid bacteria (AAB) are Gram-negative bacteria converting ethanol to acetic acid. These microorganisms are present in the cellar and fermentation tanks and can be seen either as beneficial or as wine spoilage microorganisms because, under certain circumstances, they affect the wine quality if they should grow in the wine or must. Strict measures need to be implemented in the cellar during the winemaking process to ensure microbiological stability. This can be achieved through good microbiological practices and, additionally, chemical preservatives. Sulphur dioxide (S02) is widely used as the primary preservative in winemaking. However, consumer resistance has been building up against the use of chemical preservatives, due to the possible health risks and a decrease in nutritional value and sensorial quality of certain foods and beverages. Biopreservation as an alternative to the traditionally-used chemical preservation is a new approach and has been attracting much attention. This implies the use of the natural microflora and/or their antibacterial products, such as bacteriocins and bacteriolytic enzymes (e.g. lysozyme). Bacteriocins from LAB are proteins or protein complexes, produced by Gram-positive bacteria, with antibacterial activity against closely-related Gram-positive species. Lysozyme occurs in substances such as hen egg white and has lytic activity against Gram-positive bacteria. ' The bacteriocins nisin, of the class I lantibiotics, and pediocin PA-1 and leucocin BTA 11a, of the class lIa Listeria-active bacteriocins, have been investigated for the biopreservation of wine. Nisin, however, is the only bacteriocin that has been approved for use as a preservative, while pediocin is likely to follow in approval. Lysozyme has been approved for use in winemaking by the Office International de la Vigne et du Vin (OIV). The main objectives of this study were to determine whether these substances showed any antimicrobial action against wine-associated microorganisms, namely LAB, AAB and yeasts. The stability and suitability of the bacteriocins and lysozyme as antimicrobials in wine was researched, especially when used in combination. Possible synergistic or antagonistic interactions between the bacteriocins were also investigated by means of the microtitre broth dilution method and scanning electron microscopy, as well as at what concentration and combinations the bacteriocins were most effective against increasing LAB concentrations. It was found that nisin, pediocin and leucocin were effective to varying degrees against a test panel of LAB type and reference strains, as well as wine isolates. Nisin repeatedly had the highest level of inhibition against all the LAB tested, followed by pediocin and leucocin. There was no inhibition of the wine-associated AAB and yeasts tested. Pediocin stability was evaluated in simulated wine must and proved to be stable for at least 20 days, without being affected by the sulphur or alcohol content. A low pH, however, led to a more rapid decrease in activity. The same was found for nisin and leucocin in other studies. Combinations of bacteriocins at increasing concentrations were evaluated against increasing concentrations of a LAB wine isolate. When used in pairs (namely, nisinleucocin, nisin-pediocin and pediocin-Ieucocin), the combinations were most effective against lower concentrations of bacteria, namely 102 and 104 cfu/ml. At lower concentrations, the pairs of bacteriocins were much less effective against the higher bacterial concentrations of 106 and 108 cfu/ml. Leucocin-pediocin combinations were the least effective, while nisin-Ieucocin combinations were marginally more effective than the nisin-pediocin combinations. The most pronounced effect was observed when all three the bacteriocins were used together. Combinations of bacteriocins had no inhibitory effect against AAB. Pediocin and lysozyme was used in combination against the same wine isolate, but no conclusive conclusions could be drawn in this experiment. __ Scanning electron microscopy was used to investigate any disturbances in cell morphology when bacteriocins were added to LAB. The above-mentioned LAB was subjected to bacteriocins used singularly and also in combinations of equal amounts of bacteriocins. The action of the bacteriocins led to major disturbances in cell morphology. Once again, the combination of leucocin-pediocin was the least effective, even less so than when the single bacteriocins were used. The nisin-pediocin and nisin-Ieucocin combinations seemed to be more effective in causing cell disturbances and perturbations. The microtitre broth dilution methodwas used to further characterise the nature of the interaction of the pairs of bacteriocins. This test showed clearly that the bacteriocins had definite interactions. By adding one bacteriocin to varying concentrations of another bacteriocin, the inhibitory action of the second bacteriocin was affected, either increasing or decreasing its effectiveness. The most important factor to consider seems to be the ratio at which the bacteriocins should be used together, leading either to synergism or antagonism, and this also implies a very complex interaction. This project indicated that it is indeed possible to use both bacteriocins and lysozyme in "Vine preservation, both being stable in wine environments and effective against LAB without affecting the yeast fermentation. Bacteriocins could also be used in combination, to broaden the inhibition spectrum, as well as possibly increasing the inhibitory potential of the individual antimicrobials. The underlying interactions in such combinations should be carefully researched, however, when considering using combinations of antimicrobials in food and beverage products. Further attention can also be given to finding biopreservatives against the Gram-negative AAB, as well as to research the interaction of the pairs of bacteriocins over time. Another point to consider would be the engineering of yeasts or bacteria to produce these antibacterial substances in situ as part of their metabolism.

AFRIKAANSE OPSOMMING: Daar is 'n verskeidenheid mikroorganismes in die wynrnaakproses betrokke, elkeen met sy eie rol. Giste is vir die alkoholiese fermentasie verantwoordelik, die Gram-positiewe melksuurbakterieë (MSB) wat in mos en wyn voorkom, is vir die appelmelksuurgisting (AMG) verantwoordelik, terwyl die Gram-negatiewe asynsuurbakterieë (ASB) etanol in asynsuur omskakel. Hierdie mikroorganismes is in die wynkelder en fermentasietenke teenwoordig en kan as óf gunstig óf ongunstig beskou word, afhangende van die toestande waaronder hulle groei en hoe die wyn daardeur beïnvloed word. Om mikrobiologiese stabiliteit in wyn te verseker, moet daar streng higiëniese maatreëls in die kelder toegepas word en word daar ook van addisionele chemiese preserveermiddels gebruik gemaak. Swaweidioksied (S02) word tans algemeen as pnmere preserveermiddel in die wynbedryf gebruik. Weens die moontlike gesondheidsrisiko's wat S02 mag inhou en die moontlike verlaging van die voedingswaarde en sensoriese gehalte waarmee dit in sommige voedsel- en drankprodukte geassosieer word, bou daar tans verbruikersweerstand teen die gebruik daarvan as chemiese preserveermiddelop. Biopreservering is 'n alternatief tot hedendaagse chemiese preservering en het reeds baie belangstelling ontlok. Hierdie metode impliseer die gebruik van die natuurlike mikroflora en/of die antimikrobiese produkte van hierdie rnikroërqanisrnes, soos bakteriosiene en bakteriolitiese ensieme (bv. lisosiem). Bakteriosiene van MSB is proteïene of proteïenkomplekse met antimikrobiese aktiwiteit teen naby-verwante Grampositiewe spesies. Lisosiem kom in produkte soos hoendereierwit voor en het litiese aktiwiteit teen Gram-positiewe bakterieë. Die bakteriosiene nisien, wat tot die klas I lantibiotiese bakteriosiene behoort, en pediosien PA-1 en leukosien B-TA11a, wat tot die klas lIa Listeria-aktiewe bakteriosiene behoort, is as moontlike biopreserveringsagense in wyn ondersoek. Nisien is egter tot op hede die enigste bakteriosien wat amptelik vir gebruik as 'n preserveermiddel in voedsel goedgekeur is, terwyl pediosien moontlik sal volg. Lisosiem is vir gebruik in wynmaak deur die Office International de la Vigne et du Vin (OIV) goedgekeur. Die hoofdoelwitte van hierdie studie was om te bepaal of die bogenoemde stowwe antimikrobiese werking teen wyngeassosieerde mikroorganismes het, soos die ongewenste MSB, ASB en giste. Die stabiliteit en geskiktheid van dié bakteriosiene en lisosiem as antimikrobiese middels in wyn is ook ondersoek, veral wanneer hulle in kombinasie vir preservering gebruik is. 'n Mikrotiterverdunningsboeljon-metode en skanderingselektronmikroskopie is gebruik om moontlike sinergistiese en antagonistiese interaksies tussen bogenoemde bakteriosienpare te ondersoek. Terselfdertyd is die effektiefste konsentrasies en kombinasies van bakteriosiene teen stygende MSB-getalle bepaal. Daar is bevind dat nisien, pediosien en leukosien in verskillende mates teen 'n toetspaneel van MSB tipe- en verwysingsrasse, asook MSB-wynisolate, effektief is. Nisien was herhaaldelik die effektiefste teen dié MSB, gevolg deur pediosien en dan leukosien. Die bakteriosiene was nie teen die wyngeassosieerde ASB of giste wat getoets is, effektief nie. Daar is ook bewys dat pediosien vir tot 20 dae stabiel in 'n gesimuleerde wynomgewing was, sonder dat die alkohol- of die swaweldioksiedkonsentrasie 'n invloed op die aktiwiteit gehad het nie. 'n Lae pH het geblyk om die grootste invloed op die afname in aktiwiteit te hê. Hierdie bevindinge ten opsigte van pediosien het die resultate van nisien en leukosien in ander, soortgelyke ondersoeke, bevestig. Die werking van toenemende konsentrasies van bakteriosienkombinasies (as pare van nisien-Ieukosien, nisien-pediosien, leukosien-pediosien, en al drie saam as nisienpediosien- Ieukosien) teen toenemende getalle van In wyngeïsoleerde MSB is geëvalueer. Wanneer die bakteriosiene in pare gebruik is, was die kombinasies die effektiefste teen laer MSB selgetalle (102 en 104 kfe/ml), terwyl dit baie minder effektief teen hoër selgetalle (106 en 108 kfe/ml) was, veral wanneer lae bakteriosienkonsentrasies gebruik is. Die nisien-Ieukosien kombinasiewas tot 'n geringe mate meer effektief as die nisien-pediosien kombinasie. Die leukosien-pediosien kombinasie het die laagste effektiwiteit van.al die pare bakteriosiene wat gebruik is, getoon. Die sterkste werking is waargeneem toe al drie die bakteriosiene saam teen bogenoemde MSB gebruik is. Die bakteriosien kombinasies het geen effek teen ASB gehad nie. Pediosien en lisosiem is ook in kombinasie teen dieselfde wynisolaat gebruik, maar geen oortuigende afleidings kon van hierdie eksperiment gemaak word nie. Skanderingselektronmikroskopie is gebruik om enige morfologiese verandering in die MSB-wynisolaat waar te neem wanneer bakteriosiene daarby gevoeg is. Dieselfde wynisolaat is weer gebruik en bakteriosiene is by die bakterieë gevoeg, enkelvoudig asook in kombinasies (soos voorheen gebruik) teen gelyke hoeveelhede. Die werking van die bakteriosien het gelei na merkbare veranderinge in selmorfologie, en die kombinasie van pediosien-Ieukosien was weereens die minste effektief. Die mikrotiterverdunningsboeljon-metode is gebruik om die aard van die bakteriosieninteraksies verder te karakteriseer. Die toetse het duidelik aangedui dat die bakteriosiene op mekaar reageer. Deur een bakteriosien tot variërende konsentrasies van 'n ander bakteriosien te voeg, is die inhibitoriese werking van die tweede bakteriosien geaffekteer deurdat die effektiwiteit daarvan toegeneem of afgeneem het. Dit het ook geblyk dat die belangrikste faktor wat hier in ag geneem moet word die verhouding is waarteen die bakteriosiene met mekaar gebruik word, aangesien dit tot óf sinergisme óf antagonisme kan lei. Dft dui op 'n baie komplekse interaksie. Die resultate van hierdie projek het dus daarop gedui dat dit inderdaad moontlik is om beide bakteriosien en lisosiem in wynpreservering te gebruik, aangesien beide nie net stabiel in 'n wynomgewing is nie, maar ook effektief is teen MSB sonder dat die gisfermentasies geaffekteer word. Bakteriosiene kan ook in kombinasie gebruik word om die inhibisie spektrum te verbreed, en om ook moontlik die inhibisiepotensiaal van die individuele peptiede te verhoog. Onderliggende interaksies by sulke kombinasies moet egter sorgvuldig ondersoek word wanneer daar oorweeg word om kombinasies van hierdie antimikrobiese middels in voedsel- en drankprodukte te gebruik. Verder moet daar ook aandag geskenk word om biopreserveermiddels te vind wat ook teen die Gram-negatiewe ASB effektief is, asook aan die aard van die verloop van interaksies van pare van bakteriosiene oor tyd. Nog 'n punt om te oorweeg is die manipulasie van giste of bakterieë omdie antimikrobiese peptiede in situ, as deel van hulle metabolisme, te produseer.

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