Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In the current study, two isolates showing killer activity against several wine yeast species in a
previous study were identified to strain level and found to belong to the yeast species Candida
pyralidae. The identified yeast strains and a Kluyveromyces wickerhamii yeast strain used as a
control exhibited killer activity against B. bruxellensis known for its spoilage characteristics in
red wine, and against several strains of the genus Brettanomyces on white and red grape juice
medium. The killer yeasts inhibited neither the growth of S. cerevisiae nor that of the lactic acid
bacteria Oenococcus oeni and Lactobacillus plantarum strains. Yeasts are reported to secrete
killer toxins, which can play a role in yeast microbial interactions under winemaking conditions.
The C. pyralidae strains were found to secrete two novel killer toxins, designated CpKT1 and
CpKT2. These killer toxins were stable and active under winemaking conditions, pH 3.5 - 4.5
and temperature ranges between 15 and 25°C. Ethanol and sugar concentrations found during
winemaking did not affect the activity and stability of these killer toxins. Although, the killer
toxins differed with regards to their biochemical and environmental stability and activity, they
were found to have a similar mode of action. The killer toxins induced a fungistatic effect on
B. bruxellensis sensitive cells in addition to binding to the cell wall of the sensitive cells, inducing
cell surface and plasma membrane damage as did the Kwkt killer toxin secreted by
K. wickerhamii. According to the author’s knowledge this is the first report on the identification of
novel killer toxins secreted by C. pyralidae strains isolated from a wine environment as well as
the identification of the mode of action of killer toxins on B. bruxellensis cells. This indeed
provides great research scope in this field.
The exoproteomes consisting of the killer toxins Kwkt, CpKT1 and CpKT2 revealed the
presence of exo-glucanases and glucosidases, respectively. The enzymes KwExg1 (exoglucanase)
and KwSun4 (glucosidase) retrieved from K. wickerhamii’s exoproteome were
identified as the potential toxins, but their killer activity could not be confirmed. These findings
suggest that hydrolytic enzymes possess killer activity, as previously reported in literature.
However, further investigation is needed to identify the killer toxins characterized in this study. / AFRIKAANSE OPSOMMING: In die huidige studie is twee isolate wat in ’n vorige studie “killer” aktiwiteit teenoor verskeie
wyngisspesies vertoon het, tot op rasvlak geïdentifiseer en daar is gevind dat hulle aan die
gisspesie Candida pyralidae behoort. Die geïdentifiseerde gisrasse en ’n Kluyveromyces
wickerhamii gisras wat as kontrole gebruik is, het “killer” aktiwiteit getoon teenoor B.
bruxellensis, wat bekend is vir sy bederfkarakter in rooi wyn, en ook teenoor verskeie rasse van
die genus Brettanomyces in wit en rooi druiwesapmedium. Die “killer” giste het nie die groei van
óf S. cerevisiae óf van die melksuurbakteria Oenococcus oeni en Lactobacillus plantarum-rasse
geïnhibeer nie. Giste word berig om “killer” gifstowwe uit te skei, wat ’n rol kan speel in gis
mikrobiese interaksies onder wynbereidingstoestande.
Die C. pyralidae-rasse is gevind om twee nuwe “killer” gifstowwe af te skei, wat CpKT1 en
CpKT2 genoem is. Hierdie “killer” gifstowwe was stabiel en aktief onder
wynbereidingstoestande, pH 3.5 - 4.5 en temperatuur tussen 15 en 25°C. Die etanol- en
suikerkonsentrasies wat onder wynbereiding voorkom, het nie die aktiwiteit en stabiliteit van
hierdie “killer” gifstowwe beïnvloed nie. Hoewel die “killer” gifstowwe met betrekking tot hulle
biochemiese en omgewingstabiliteit en aktiwiteit verskil het, is daar gevind dat hulle ’n eenderse
modus van aksie het. Die “killer” gifstowwe het ’n fungistatiese effek op B. bruxellensis
sensitiewe selle geïnduseer, buiten dat dit aan die selwand van die sensitiewe selle gebind het,
en het seloppervlak- en plasma-membraanskade geïnduseer, net soos die Kwkt “killer” gifstof
wat deur K. wickerhamii afgeskei is. So ver die skrywer weet, is hierdie die eerste verslag van
die identifisering van nuwe “killer” gifstowwe wat deur C. pyralidae rasse afgeskei word wat uit
’n wynomgewing geïsoleer is, asook van die identifikasie van die modus van aksie van “killer”
gifstof op B. bruxellensis selle. Dit verbreed dus beslis die navorsingsomvang van hierdie
gebied.
Die eksoproteome, bestaande uit die “killer” gifstowwe Kwkt, CpKT1 en CpKT2, het die
teenwoordigheid van ekso-glukanases en glukosidases onderskeidelik onthul. Die ensieme
KwExg1 (eksoglukanase) en KwSun4 (glukosidase) wat vanuit K. wickerhamii se eksoproteoom
herwin is, is as die potensiële gifstowwe geïdentifiseer, maar hulle “killer” aktiwiteit kon nie
bevestig word nie. Hierdie bevindings suggereer dat hidrolitiese ensieme “killer” aktiwiteit besit,
soos voorheen in die literatuur berig is. Verdere ondersoeke word egter benodig om die “killer”
gifstowwe wat in hierdie studie gekarakteriseer is, te identifiseer.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/97035 |
| Date | 04 1900 |
| Creators | Mehlomakulu, Ngwekazi Nwabisa |
| Contributors | Divol, Benoit T., Setati, Evodia, 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 | Unknown |
| Type | Thesis |
| Format | 161 pages : illustrations |
| Rights | Stellenbosch University |
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