Thesis (MScAgric)--University of Stellenbosch, 2002. / 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53113 |
| Date | 03 1900 |
| Creators | Du Toit, Corina |
| Contributors | Du Toit, M., Pretorius, I. S., Van Rensburg, P., Stellenbosch University. Faculty of AgriSciences. Dept. of Viticulture and Oenology. Institute for Wine Biotechnology. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 98 p. : ill. |
| Rights | Stellenbosch University |
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