Biochemical characterization and evaluation of the oenological attributes of Lachancea species

Thesis (MScAgric)--Stellenbosch University, 2017.ENGLISH SUMMARY: Previously thought to be the catalyst for spoilt wine, several non-Saccharomyces yeasts are now commercially available for winemaking. Increasing investigations into this group of microorganisms has emphasized their oenological potential in terms of their contribution to the organoleptic profile of the resulting wines. However, their sub-optimal fermentation performances, force their combined inoculation with the main wine yeast Saccharomyces cerevisiae. Among the non-Saccharomyces yeasts, Lachancea thermotolerans has been reported to bring about an overall improvement in wine acidity through the production of L-lactic acid and reduction of acetic acid as well as increased glycerol and 2-phenylethanol levels, which can impart a sweet taste and flowery aromas to wine, respectively. Its secretion of hydrolytic enzymes of oenological relevance has also been reported. Other species of the Lachancea genus (L. fermentati and L. lanzarotensis) have also been isolated from the fermentation environment, but have not received the same degree of focus as L. thermotolerans. The aim of this study was to investigate the oenological potential of these species, regarding their expression of oenologically relevant enzymes, their fermentation attributes and the expression and location of β-glucosidase during fermentation of synthetic and real grape must (Muscat d’Alexandrie). All the Lachancea spp. illustrated β-glucosidase activity in initial plate screenings, while none exhibited protease or pectinase activities. L. thermotolerans and L. fermentati also displayed higher tolerance to ethanol and SO2 when exposed to varying concentrations of these inhibitory compounds. Higher fermentation performance was demonstrated by L. thermotolerans strains and L. fermentati, which performed much better in Muscat grape juice, and was the only Lachancea spp. to complete the fermentation in monoculture. During these fermentations, all three Lachancea spp. illustrated β-glucosidase activity, where the highest levels were expressed by a L. thermotolerans strain, Y940. Most of the enzyme activity originated from the cell walls of the yeasts, while lower levels were isolated in the intracellular region and none determined to be released into the medium. The mixed culture fermentations resulted in wine products with significantly different chemical compositions compared to the S. cerevisiae fermentation. L. thermotolerans and L. lanzarotensis mixed culture fermentations resulted in similar wines with increased production of common compounds including isoamyl alcohol, butanol and ethyl phenylacetate. L. fermentati seemingly produced wines dissimilar to the other Lachancea spp., producing levels of acetic acid perhaps flagging this species as a potential spoilage microorganism. This study provided novel information on relatively uninvestigated Lachancea spp., where their oenological potential was highlighted by the ability to express β-glucosidase enzymes during fermentations and producing wines significantly different from S.AFRIKAANS OPSOMMING: ‘n Aantal nie-Saccharomyces giste, voorheen aan gedink as die kataliste vir bederfde wyn, is nou kommersieël beskikbaar vir wynbereiding. In diepte ondersoeke na hierdie groep mikro-organisms het hul wynkundige potensiaal, in terme van hul bydra tot die organoleptiese profiel van die gevolglike wyn, beklemtoon. Alhoewel, hul sub-optimale fermentasie gedrag forseer hul gekombineerde inokulasie saam met die hoof wyn gis Saccharomyces cerevisiae. Onder die nie-Saccharomyces giste, word berig dat Lachancea thermotolerans ‘n algehele verbetering in wyn suurheid te weeg bring deur die produksie van L-melksuur en ‘n verlaging in asynsuur asook ‘n verhoging in gliserol en 2-fenieletanol vlakke, wat onderskeidelik ‘n soet smaak en blommerige aroma aan die wyn kan verleen. Die afskeiding van hidrolitiese ensieme van wynkundige belang is ook al berig. Ander spesies van die Lachancea genus (L. fermentati en L. lanzarotensis) is ook al uit fermentasie omgewings ge-isoleer, maar hul is nie so in deeglik soos L. thermotolerans bestudeer nie. Die doel van hierdie studie was om die wynkundige potensiaal van hierdie spesies te ondersoek, met betrekking tot die uitdrukking van wynkundige relevante ensieme, hul fermentasie eienskappe en die setel van β-glukosidase gedurende fermentasie in sintetiese en egte druiwe sap (Muscat d’Alexandrie). Al die Lachancea spp. het β-glukosidase aktiwiteit getoon in die plaat siftings, maar het geen protease of pektinase aktiwiteit getoon nie. L. thermotolerans en L. fermentati het ‘n hoë toleransie vir etanol en SO2 vertoon in ‘n inhibiese sifting met verkillende konsentrasies van hierdie inhibitoriese komponente. L. thermotolerans rasse en L. fermentati het ook hoër fermentasie prestasie getoon; hierdie spesies was die enigste Lachancea spp. wat as monokulture die fermentasie voltooi het en goeie prestasie in Muscat druiwe sap getoon het. Gedurende hierdie fermentasies het al drie Lachancea spp. β-glukosidase aktiwiteit getoon, met die hoogste vlakke wat deur L. thermotolerans ras Y940 uitgedruk is. Meeste van die ensiem aktiwiteit het in die selwand voorgekom, terwyl laer vlakke uit die intrasellulêre area ge-isoleer is en geen ensiem aktiwiteit in die medium as uitgeskeide ensiem voorgekom het nie. Die gemengde kultuur fermentasies het gelei tot wynprodukte met beduidend anderse chemiese samestelling in vergelyking met S. cerevisiae fermentasie. L. thermotolerans en L. lanzarotensis gemengde kultuur fermentasies het soortgelyke wyne met hoër produksie van algemene verbindings, insluitend isoamiel alkohol, butanol, en etiel fenielasetaat, opgelewer. L. fermentati het oënskynlik wyne geproduseer wat heelwat anders is as die ander Lachancea spp., met vlakke van asynsuur wat moontlik ‘n aanduiding is dat die spesie ‘n potensiele bederf mikroorganisme is. Hierdie studie het nuwe inligting oor die relatief onbestudeerde Lachancea spp. getoon, waar hul wynkundige potensiaal uitgelig is deur die vermoë om β-glukosidase ensieme uit te druk gedurende fermentasies en om wyne te produseer wat beduidend anders is as die van S. cerevisiae