Impact of the protease-secreting yeast Metschnikowia pulcherrima IWBT Y1123 on wine properties and response of protease production to nitrogen sources

Thesis (MScAgric)--Stellenbosch University, 2019.ENGLISH ABSTRACT: Yeast of oenological origin belong to various genera and harbour unique metabolic properties that may significantly impact aspects of wine processing and composition. Extracellular aspartic proteases, and their secretion by wine yeasts, have received much attention due to their protein degradative ability as a possible solution to the quality problem of wine turbidity. This fault is generally addressed by wine-makers with the use of bentonite, but this fining agent is associated with various technological and organoleptic issues. A key contributing factor to haze formation in wine is the presence of heat unstable grape proteins, and their removal by proteases therefore presents an attractive alternative to the use of bentonite. The yeast Metschnikowia pulcherrima IWBT Y1123 has been isolated from grape juice and secretes an extracellular aspartic protease named MpAPr1. This enzyme demonstrated activity against grape proteins and reduced wine haze-forming potential under winemaking conditions after 48 h incubation with a purified exogenous MpAPr1 preparation. However, inoculation of M. pulcherrima IWBT Y1123 as a co-starter culture to wine fermentation for the secretion of MpAPr1 directly into the matrix presents a possible time- and cost-effective alternative that would eliminate the need for enzyme purification steps. Nevertheless, understanding protease regulation by environmental conditions and relating protease secretion and activity to its impact on wine properties could prove useful when considering inoculation strategies for this yeast. This study sought to establish the potential for developing co-inoculation strategies of M. pulcherrima IWBT Y1123 with the efficient fermenter Saccharomyces cerevisiae, by assessing the impact of nitrogen sources and protein availability on protease production and activity by M. pulcherrima IWBT Y1123, as well as the impact of protease production and activity in grape juice inoculated with M. pulcherrima IWBT Y1123 on grape protein content, haze formation potential and wine volatile aroma profile. Protease production was shown to be subject to nitrogen catabolite repression in the presence of preferred sources of nitrogen, as well as induction by proteins. Upon inoculation into grape juice, an up-regulation of MpAPr1 gene expression could be observed, as well as protease production and activity. With regard to the impact of M. pulcherrima IWBT Y1123 co-inoculation with S. cerevisiae on wine properties at the end of fermentation, total protein content and haze forming potential were lower compared to controls and the volatile profile was altered. Future work should focus on enhancing protease production by M. pulcherrima IWBT Y1123 to improve its viability as a commercial protease-producing yeast strain. Nevertheless, the results generated in this study contribute to knowledge in both fundamental and biotechnological aspects of protease secretion by M. pulcherrima IWBT Y1123.AFRIKAANSE OPSOMMING: Gis van oenologiese oorsprong behoort aan verskeie genera en bevat unieke metaboliese eienskappe wat die aspekte van wynverwerking en samestelling aansienlik kan beïnvloed. Ekstrasellulêre aspartiensuur protease, en hul afskeiding deur wyngiste, het baie aandag gekry vanweë hul proteïenafbrekende vermoë as 'n moontlike oplossing vir die kwaliteitsprobleem van wyn wasigheid. Die probleem word oor die algemeen deur wynmakers aangespreek deur die gebruik van bentoniet, maar hierdie klei word geassosieer met verskeie tegnologiese en organoleptiese probleme. 'n Belangrike bydraende faktor vir die vorming van wasigheid in wyn is die teenwoordigheid van hitte-onstabiele druiweproteïene, en hul verwydering deur protease bied dus 'n aantreklike alternatief tot bentoniet. Die gis Metschnikowia pulcherrima IWBT Y1123 is van druiwesap geïsoleer, en skei 'n ekstrasellulêre aspartiensuur protease genaamd MpAPr1 af. Hierdie ensiem het voorheen aktiwiteit teen druiweproteïene gedemonstreer, en na 48 uur inkubasie onder wynmaak omstandighede het ‘n gesuiwerde MpAPr1 voorbereiding die potensiaal vir die vorming van wasigheid verminder. Die inenting van M. pulcherrima IWBT Y1123 as 'n mede-kultuur vir wynfermentasie vir die afskeiding van MpAPr1 direk in die matriks, bied egter 'n moontlike tyd- en koste-effektiewe alternatief wat die behoefte aan ensiem-suiweringstappe sal elimineer. Nietemin kan die kennis van protease regulasie deur omgewingsomstandighede, en proteasekresie en aktiwiteit met betrekking tot die impak daarvan op wyn eienskappe, nuttig wees as inentingstrategieë vir hierdie gis oorweeg word. Hierdie studie het gepoog om die potensiaal vir die ontwikkeling van gesamentlike inentingstrategieë van M. pulcherrima IWBT Y1123 met die effektiewe fermentor Saccharomyces cerevisiae, te bepaal deur die impak van stikstofbronne en proteïenbeskikbaarheid op protease produksie en aktiwiteit deur M. pulcherrima IWBT Y1123 te evalueer, sowel as die impak van protease produksie en aktiwiteit in druiwesap met M. pulcherrima IWBT Y1123 ingeënt op druiweproteïeninhoud, wasigheids-vormingspotensiaal en aroma-profiel. Protease produksie het getoon dat dit onderhewig is aan stikstof kataboliet-onderdrukking in die teenwoordigheid van voorkeurbronne van stikstof, sowel as induksie deur proteïene. Met inenting in druiwesap kan 'n opwaartse regulering van MpAPr1-gene-uitdrukking waargeneem word, sowel as proteaseproduksie en -aktiwiteit. Met betrekking tot die impak van M. pulcherrima IWBT Y1123 mede-inenting met S. cerevisiae op wyn eienskappe aan die einde van fermentasie, was die totale proteïeninhoud en wasigheidsvormende potensiaal laer in vergelyking met kontrole, en die vlugtige profiel is verander. Toekomstige werk moet fokus op die verbetering van protease produksie deur M. pulcherrima IWBT Y1123 om die lewensvatbaarheid daarvan as kommersiële protease-produserende gissoort te verbeter. Nietemin dra die resultate wat in hierdie studie gegenereer word, by tot kennis in beide fundamentele en biotegnologiese aspekte van proteasekresie deur M. pulcherrima IWBT Y1123