Evaluation of citrate metabolism in Oenococcus oeni and Lactobacillus plantarum

Pretorius, Natasha (2016-12)

Thesis (MSc)--Stellenbosch University, 2016.

Thesis

ENGLISH ABSTRACT: Citrate positive (cit+) Oenococcus oeni and Lactobacillus plantarum are able to degrade citrate during fermentation. These bacteria possess the citrate permease and citrate lyase enzymes which are responsible for citrate transport and degradation, respectively. Citrate negative (cit-) strains lack either one of these genes rendering them incapable of citrate degradation. Citrate metabolism results in the production of compounds such as D-lactate, acetate, diacetyl, acetoin and 2,3-butanediol, which can influence the aroma profile and quality of wine. Of these compounds, diacetyl is the most important compound produced via citrate metabolism that can give rise to a buttery aroma important to create certain wine styles. The aim of this study was to determine the effect of glucose, fructose and pH on citrate metabolism by a cit+ and a cit- strain from both species of O. oeni and Lb. plantarum. The effects of these factors were determined by focussing specifically on the consumption of citrate, the relative expression (RE) of the beta subunit (citE) of the citrate lyase gene and the production of D-lactate, acetate, diacetyl and acetoin. Synthetic wine was used to determine the influence of different glucose and fructose concentrations (115 g/L, 50 g/L and 2.5 g/L) and different pH levels (3.0, 3.5, 4.0 and 5.0) as these represent the differences either between warm and cool climate or co-inoculation and sequential malolactic fermentation. The results obtained for all the treatments showed that glucose had the smallest impact on citrate metabolism. In the glucose treatments, the cit+ O. oeni strain completely consumed citrate and the cit- O. oeni did not consume citrate but both strains produced trace amounts of diacetyl and acetoin. The cit- strain produced less D-lactate and acetate than the cit+ O. oeni strain in most of the glucose treatments. The lowest RE of citE in the cit+ O. oeni strain was seen in the 115 g/L glucose treatment where malate and citrate were consumed the fastest. Both the Lb. plantarum strains partially consumed citrate. The cit+ Lb. plantarum strain produced more diacetyl and acetoin than the cit- Lb. plantarum strain in all the glucose treatments. In the 2.5 g/L glucose treatment, the D-lactate and acetate concentrations were lower than in the other two treatments, which might be due to the lower initial glucose concentration in this treatment. However, the cit+ Lb. plantarum strain produced more diacetyl and acetoin in the 2.5 g/L glucose treatment compared to the other two treatments. The Lb. plantarum strains produced more D-lactate, diacetyl and acetoin than the O. oeni strains. In the fructose treatments, the cit- O. oeni strain did not consume any citrate and the cit- Lb. plantarum strain partially consumed citrate. The cit+ Lb. plantarum strain was able to consume more citrate in the fructose treatments and therefore produced more diacetyl and acetoin in these treatments. The highest RE in the cit+ O. oeni strain was seen in the 115 g/L fructose treatment where the malate and citrate were slightly delayed. The cit+ strains produced similar amounts of D-lactate and acetate than the cit- strains. The Lb. plantarum strains produced more D-lactate, diacetyl and acetoin than the O. oeni strains, as was seen in the glucose treatments. In all the pH conditions tested, the cit- O. oeni strain did not consume citrate and produced less D-lactate, diacetyl and acetoin than the cit+ O. oeni strain. The cit+ O. oeni strain consumed more citrate and produced more diacetyl and acetoin in the pH 3.0 and 3.5 treatments than in the pH 4.0 and 5.0 treatments. The Lb. plantarum strains consumed more citrate and therefore produced more D-lactate, acetate, diacetyl and acetoin in the pH 3.5, 4.0 and 5.0 treatments than in the pH 3.0 treatment. The highest RE in the cit+ O. oeni strain was seen in the pH 3.0 treatment where the malate and citrate consumption were slightly delayed as was seen in the fructose treatments. The Lb. plantarum strains produced more D-lactate and acetoin than the O. oeni strains in most of the treatments. The cit+ Lb. plantarum strain produced the highest diacetyl in the pH 4.0 and 5.0 treatments, but the cit+ O. oeni strain produced the highest diacetyl in the pH 3.0 and 3.5 treatments. The measurement of 2,3-butanediol could not be performed in this study to determine whether acetoin was converted and if this is the reason why diacetyl concentrations were relatively low taking into consideration the amount of citrate consumed in some of the treatments. In conclusion, this study showed that different sugar concentrations, pH levels and the lactic acid bacteria strains used to induce malolactic fermentation can influence citrate metabolism, which can ultimately influence the final diacetyl and acetoin concentrations and therefore the wine style. The results obtained in this study can be used by winemakers to obtain or avoid a buttery, creamy aroma in their wines.

AFRIKAANS OPSOMMING: Sitraat positiewe (sit+) Oenococcus oeni en Lactobacillus plantarum is in staat om sitraat af te breek gedurende die fermentasie. Hierdie bakterieë besit die sitraat permease en sitraat liase ensieme wat verantwoordelik is vir die vervoer en afbraak van sitraat, onderskeidelik. Sitraat negatiewe (sit-) bakterieë is nie in staat om sitraat af te breek nie, omdat hul nie die gene besit wat kodeer vir hierdie ensieme nie. Verskeie metaboliete naamlik D-laktaat, asetaat, diasetiel, asetoïen en 2,3-butaandiol word geproduseer tydens sitraat metabolisme wat die aroma en kwaliteit van die wyn kan beïnvloed. Van hierdie komponente is diasetiel die belangrikste metaboliet wat geproduseer word vanaf sitraat en gee `n botteragtige karakter wat belangrik is vir spesifieke wynstyle. Die doel van hierdie studie was om te bepaal watter effek glukose, fruktose en pH sal hê op sitraat metabolisme van sit+ en sit- O. oeni en Lb. plantarum isolate. Die effek van hierdie faktore was bepaal deur spesifiek te fokus op die sitraat verbruik, relatiewe ekspressie (RE) van die beta subeenheid (citE) van sitraat liase geen en die produksie van D-laktaat, asetaat, diasetiel, asetoïen en 2,3-butaandiol. Sintetiese wyn was gebruik om die invloed van verskillende glukose en fruktose konsentrasies (115 g/L, 50 g/L en 2.5 g/L) en verskillende pH vlakke (3.0, 3.5, 4.0 en 5.0) te evalueer, aangesien hierdie faktore die verskil tussen ko- en sekwensiële appelmelksuurgisting asook tussen warm en koel klimate verteenwoordig. Die resultate verkry tydens hierdie studie het aangedui dat glukose die kleinste impak gehad het op sitraat metabolisme. In al die glukose behandelinge het die sit+ O. oeni alle sitraat verbruik en die sit- O. oeni geen sitraat afgebreek nie, maar beide het min diasetiel en asetoïen geproduseer. Die sit- het ook minder D-laktaat en asetaat geproduseer as die sit+ O. oeni in meeste van die glukose behandelinge. Die laagste RE van citE in die sit+ O. oeni was gesien in die 115 g/L glukose behandeling waar die malaat en sitraat die vinnigste afgebreek was. Beide die Lb. plantarum isolate het net `n gedeelte van die sitraat verbruik. Die sit+ Lb. plantarum het egter meer diasetiel en asetoïen geproduseer as die sit- Lb. plantarum in al die glukose behandelinge. In die 2.5 g/L glukose behandeling was die D-laktaat en asetaat heelwat minder as in die ander twee behandelinge as gevolg van die laer aanvanklike glukose konsentrasie. Die sit- Lb. plantarum het egter meer diasetiel en asetoïen in die 2.5 g/L glukose behandeling geproduseer in vergelyking met die ander twee behandelinge. Die Lb. plantarum isolate het ook meer D-laktaat, diasetiel en asetoïen as die O. oeni isolate geproduseer. In die fruktose behandelinge het die sit- O. oeni weereens nie die sitraat afgebreek nie en die sit- Lb. plantarum net sitraat gedeeltelik af te breek. Die sit+ Lb. plantarum isolaat was instaat om meer sitraat te gebruik in die fruktose behandelinge en het dus meer diasetiel en asetoïen geproduseer in die hierdie behandelinge. Die hoogste RE van citE in die sit+ O. oeni was gesien in die 115 g/L fruktose behandeling waar die malaat en sitraat verbruik effens vertraag was. Die sit+ isolate het egter min of meer dieselfde hoeveelheid D-laktaat en asetaat geproduseer as die sit-isolate. Die Lb. plantarum isolate het meer D-laktaat, diasetiel en asetoïen geproduseer as die O. oeni isolate soos wat gesien was in die glukose behandelinge. In al die pH behandelinge het die sit- O. oeni nie die sitraat afgebreek nie en dus minder D-laktaat, diasetiel en asetoïen as die sit+ O. oeni. Die sit+ O. oeni het meer sitraat afgebreek en dus meer diasetiel en asetoïen in die pH 3.0 en 3.5 behandelinge as in die pH 4.0 en 5.0 behandelinge. Die Lb. plantarum isolate het meer sitraat afgebreek en dus meer D-laktaat, asetaat, diasetiel en asetoïen in die pH 3.5, 4.0 en 5.0 behandelinge as in die pH 3.0 behandeling. Die hoogste RE van citE in die sit+ O. oeni was gesien in die pH 3.0 behandeling waar die malaat en sitraat verbruik effens vertraag was soos wat gesien was in die fruktose behandelinge. Die Lb. plantarum isolate het meer D-laktaat en asetoïen geproduseer as die O. oeni isolate in meeste van die behandelinge. Die sit+ Lb. plantarum isolate het meer diasetiel as die ander isolate in die pH 4.0 en 5.0 behandelinge geproduseer, maar die sit+ O. oeni het meer diasetiel in die pH 3.0 en 3.5 behandelinge geproduseer. Die een beperking van die studie was dat 2,3-butaandiol nie gemeet kon word nie en daarom kon daar nie vasgestel word of die asetoïen omgeskakel was nie en of dit die rede is hoekom die diasetiel konsentrasies so laag was al was sitraat heeltemal gemetaboliseer in sommige behandelinge. Ten slotte, hierdie studie het aangedui dat verskillende suiker konsentrasies, pH vlakke en tipe melksuurbakterieë wat gebruik word om appelmelksuurgisting te induseer `n invloed het op sitraat metabolisme wat uiteindelik die finale diasetiel en asetoïen konsentrasies kan beïnvloed en dus die wyn styl. Die resultate verkry deur die studie kan moontlik gebruik word deur wynmakers om `n botteragtige aroma te verkry of te vermy in hul wyne.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/100214
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