Lactobacillus plantarum : amino acid utilization

Thesis (MScAgric)--Stellenbosch University, 2018.ENGLISH ABSTRACT: Amino acid metabolism serves as a source of sulphur, carbon and nitrogen for bacteria growing in wine or grape juice. The metabolism of amino acids controls the rate of growth and malic acid degradation and also results in the formation of various aromatic compounds which may positively or negatively influence the aroma profile of wine. L. plantarum, a lactic acid bacterium (LAB), may be used as co-inoculant in high pH (≥ 3.5) grape juice for fast malic acid degradation and high aroma production. Since the research on L. plantarum nitrogen metabolism is scarce, the overall goal of this study was to better understand it. The first aim was to determine the amino acid requirements in L. plantarum for growth and malic acid degradation, through single amino acid omissions. This entailed inoculation of nitrogen starved L. plantarum strains into chemically defined media (in this case synthetic grape juice) in which one amino acid is removed at a time. The data suggests that amino acid trophic requirements in L. plantarum are highly strain dependent, although Leu, Ile, Val, Glu and Met were shown under our conditions to be essential amino acids and Gln, Gly, His, Lys and Trp were non-essential amino acids. In a subsequent experiment, 5 single amino acid omissions (Ala, Arg, Gln, Trp and Val) were selected to evaluate their effect on growth and malic acid uptake in synthetic grape juice. During malolactic fermentation (MLF) the removal of Ala and Val had completely repressed MLF induced by L. plantarum while the removal of Trp and Arg had somewhat repressed MLF. Only the removal Gln did not hinder MLF for at least one strain. The second aim was to determine the order of amino acid uptake by L. plantarum in synthetic grape juice using HPLC. Asp, Thr, Ser and Ala tends to be assimilated at a high rate within the first 72 h while the branched chain amino acids, aromatic amino acids (AAA) and Met are assimilated after 72 h. The third aim determined the amino acid uptake in Chardonnay grape juice. The assimilation pattern differed considerably between synthetic grape juice and Chardonnay grape juice. In contrast to synthetic grape juice Arg, Leu, Phe and Ala were preferred amino acid sources. It is thought that the differences could be attributed to mainly two factors: initial nitrogen concentration (40 mg N/L in SGJ vs 240 mg N/L in grape juice) and the pre-culture conditions. This study confirmed that higher nitrogen concentrations resulted in higher growth and quicker malic acid degradation. The high nitrogen requirement of certain amino acids combined with the harsh wine parameters experienced in sequential MLF might explain why L. plantarum struggles with MLF in this scenario. Further research should be directed towards identifying the preferred amino acids in dried and fresh L. plantarum starter cultures to assess if there is a difference. If nitrogen requirements continues to be investigated in L. plantarum successful tailored supplements can be created to aid the growth of L. plantarum in wine or grape juice.AFRIKAANSE OPSOMMING: Aminosuur metabolisme dien as ‘n bron van swawel, stikstof en koolstof vir bakterieë wat in wyn en druiwesap groei. Die metabolisme van aminosure beheer die tempo van groei, appelsuur afbraak asook die vorming van verskeie aromatiese verbindings wat die wyn aromaprofiel positief of negatief mag beïnvloed. L. plantarum, ‘n melksuurbakterium, mag gebruik word as ko-inokulant in hoë pH (≥ 3.5) druiwesap vir vinnige afbraak van appelsuur en hoë aroma produksie. Aangesien navorsing op die stikstof metabolisme van L. plantarum seldsaam is, was dit die algehele doelstelling van hierdie studie om dit beter te verstaan. Die eerste doelwit was om die aminosuur vereistes in L. plantarum te bepaal in terme van groei en appelsuur afbraak d. m. v. aminosuur weglatings. Dit behels die inokulasie van ‘n stikstof-uitgehongerde L. plantarum ras in chemiese gedefinieerde media (in hierdie geval sintetiese druiwesap) waarin een aminosuur op ‘n keer weggelaat is. Die data stel voor dat aminosuur trofiese vereistes in L. plantarum baie sterk afhanklik is van die ras wat gebruik word. Algeheel toon Leu, Ile, Val Glu en Met om essensiële aminosure te wees terwyl Gln, Gly, His, Lys en Trp toon om nie-essensiële aminosure te wees. In ‘n daaropvolgende eksperiment is 5 enkele aminosuur weglatings (Ala, Arg, Gln, Trp en Val) gekies om die effek op groei en appelsuur afbraak in sintetiese druiwesap te evalueer. Gedurende appelmelksuurgisting (AMG) het die weglating van Ala en Val die proses heeltemal onderdruk terwyl die weglating van Trp en Arg AMG slegs gedeeltelik onderdruk het. Slegs die weglating van Gln het glad nie AMG verhinder nie vir ten minste een ras. Die tweede doelwit het die volgorde van aminosuur opname deur L. plantarum in sintetiese druiwesap bepaal deur gebruik te maak van HPLC. Gevolglik, is bepaal dat Asp, Thr, Ser en Ala geneig is om eerste opgeneem te word teen ‘n hoë tempo binne die eerste 72 h van AMG terwyl Met, die vertakte ketting en aromatiese aminosure na 72 h geassimileer word. Die derde doelwit het die aminosuur opname in Chardonnay druiwesap bepaal. Die patroon van aminosuur assimilasie verskil heelwat tussen sintetiese druiwesap en Chardonnay druiwesap. In teenstelling met die sintetiese druiwesap, is Arg, Leu, Phe en Ala verkies as voorkeur bronne van aminosure in Chardonnay druiwesap. Die verskil tussen die resultate kan heelwaarskynlik toegeskryf word aan hoofsaaklik 2 faktore: die aanvanklike stikstof konsentrasie (40 mg N/L in sintetiese druiwesap en 240 mg N/L in druiwesap) en die vooraf kultiverings toestande. Hierdie studie bevestig dat hoër stikstof konsentrasies tot hoër groei en vinniger appelsuur afbraak lei. Die hoë stikstof vereistes tesame met die stresvolle wynkondisies wat verband hou met na alkoholiese fermentasie inokulasie mag verder verduidelik waarom L. plantarum sukkel onder hierdie toestande. Verdere navorsing behoort gerig te word om voorkeur aminosure in droë en vars aanvangskulture van L. plantarum te identifiseer, om te bepaal of daar ‘n verskil is. As stikstof vereistes in L. plantarum verder noukeurig ondersoek word kan stikstof aanvullings vervaardig word om L. plantarum te help met groei in wyn of druiwesap