Academic literature on the topic 'Malolactic bacteria'

Abstract The research investigated the accumulation of malic acid and diacetyl in the white wines during the alcoholic fermentation of must with a Saccharomyces cerevisiae wine yeasts isolated by the author and Oenococcus oeni malolactic bacteria in a concentration of 106 CFU/mL. The two aroma compounds were detected and quantified in the resulting wines, making a comparison between the two technological systems, i.e.: co-inoculation of malolactic bacteria and sequential inoculation. Based on our determinations, it was ascertained that the Oenococcus oeni malolactic bacteria co-inoculation system in fermentative processes leads to a substantial reduction of malolactic bacteria and diacetyl concentrations. Thus, harmonious, balanced white wines are obtained, as specific to the area.

Malolactic enzyme of lactic acid bacteria catalyzes the decarboxylation of L-malate to L-lactate. The appropriate enzyme of Lactobacillus casei, Leuconostoc oenos, and Leuconostoc mesenteroides, as well as the malic enzyme of Lactobacillus casei, were purified to electrophoretic homogeneity by salmine sulphate precipitation, ion-exchange chromatography, hydrophobic chromatography, and gel filtration. The malolactic enzymes investigated were similar and showed only minor variations in the isoelectric point and the temperature optimum. The molecular weight of the subunit of all malolactic enzymes was about 65 000. Aggregates were formed, depending on the pH. The optimum activity of malolactic enzyme was observed at pH 5.8–6.0, and at this pH the dimer was stable. In addition to Mn2+ and NAD, the malolactic enzyme required K+, which was replaceable by NH4+, for maximum activity. The Km values for L-malate were 10.9 mM (Leuconostoc mesenteroides B116) and 3 mM (Leuconostoc oenos). The Km values for Mn2+ were 0.1 mM (Leuconostoc mesenteroides B116) and 0.017 mM (Leoconostoc oenos). Malic enzyme oxidatively decarboxylates L-malate to pyruvate. This enzyme consists of a 37 000 subunit that forms dimers and tetramers. The NAD-dependent malic enzyme of Lactobacillus casei decarboxylates oxalacetate and is therefore regarded as a L-malate:NAD+ oxidoreductase (oxalacetate decarboxylating), EC 1.1.1.38. Key words: malolactic enzyme, malic enzyme, Lactobacillus, Leuconostoc.

ABSTRACT Oenococcus oeni is often employed to perform the malolactic fermentation in wine production, while nonoenococcal lactic acid bacteria often contribute to wine spoilage. Two real-time PCR assays were developed to enumerate the total, and nonoenococcal, lactic acid bacterial populations in wine. Used together, these assays can assess the spoilage risk of juice or wine from lactic acid bacteria.

ABSTRACT The lack of malolactic activity in H+-ATPase-deficient mutants of Oenococcus oeni selected previously was analyzed at the molecular level. Western blot experiments revealed a spot at 60 kDa corresponding to the malolactic enzyme only in the parental strain. Moreover, the mleA transcript encoding the malolactic enzyme was not detected by reverse transcription (RT)-PCR analysis of mutants. These results suggest that the malolactic operon was not transcribed in ATPase-deficient mutants. The mleR gene encoding a LysR-type regulatory protein which should be involved in expression of the malolactic genes was described previously for O. oeni. Results obtained in this study show that the mleR transcript was not detected in the mutants by RT-PCR. No mutation in the nucleotide sequences of the mleR gene and the malolactic operon was found. The effect of a reduction in H+-ATPase activity on l-malate metabolism was then investigated by using other malolactic bacteria. Spontaneous H+-ATPase-deficient mutant strains of Lactococcus lactis and Leuconostoc mesenteroides were isolated by using neomycin resistance. Two mutants were selected. These mutants exhibited ATPase activities that were reduced to 54 and 70% of the activities obtained for the L. lactis and L. mesenteroides parental strains, respectively. These mutants were also acid sensitive. However, in contrast to the ATPase-deficient mutants of O. oeni, activation of l-malate metabolism was observed with the L. lactis and L. mesenteroides mutants under optimal or acidic growth conditions. These data support the suggestion that expression of the genes encoding malolactic enzymes in O. oeni is regulated by the mleR product, as it is in L. lactis. Nevertheless, our results strongly suggest that there is a difference between the regulation of expression of the malolactic locus in O. oeni and the regulation of expression of this locus in less acidophilic lactic acid bacteria.

<p style="text-align: justify;">A method is described, by wich the malolactic fermentation is induced through direct inoculation with an industrial preparation of <em>L. plantarum</em> (CHL2). When inoculated into must, this homofermentative lactobacillus metabolizes very little sugar, and is unable to produce acetic acid. The inoculated population of bacteria has a high malolactic activity even at low pH values, which is closely related to cell viability. The decarboxylation of malic acid starts immediately after inoculation and proceeds at a high rate during the first days of alcoholic fermentation. As the alcohol concentration rises, the lactobacilli are gradually eliminated, so inoculation with the malolactic culture has to be done very early in the vinification process, in order to achieve a complete malolactic fermentation. At the end of alcoholic fermentation, the malolactic fermentation is finished, and the bacterial inoculum has been completely killed of.</p><p style="text-align: justify;">The efficiency of this new process is demonstrated in laboratory experiments and field trials with different types of must and different vinification procedures. The advantages of the method, which the winemakers would particularly appreciate, include the direct inoculation mode and considerable gain in terms of production time.</p>

The effects of different malolactic bacteria fermentation techniques on the bioconversion of aromatic compounds in cool-climate grape wines were examined. During three wine seasons, red and white grape wines were produced using various malolactic fermentation induction techniques: Coinoculation, sequential inoculation, and spontaneous process. Volatile compounds (diacetyl and the products of its metabolism, and selected ethyl fatty acid esters) were extracted by solid phase microextraction. Compounds were identified with a multidimensional gas chromatograph—GC × GC-ToFMS with ZOEX cryogenic (N2) modulator. Sensory evaluation of the wines was also performed. It was found that the fermentation-derived metabolites studied were affected by the malolactic bacteria inoculation regime. Quantitatively, ethyl lactate, diethyl succinate, and ethyl acetate dominated as esters with the largest increase in content. The total concentration of ethyl esters was highest for the coinoculation technique, while the highest concentration of diacetyl was noted for the spontaneous technique. Controlled malolactic fermentation, especially using the coinoculation technique, can be proposed as a safe and efficient enological practice for producing quality cool-climate grape wines enriched with fruity, fresh, and floral aromas.

<p style="text-align: justify;">The red wine« Appellation d'Origine Naoussa de qualité supérieure » stemming from the « Xynomavro » vine cultivated in this wine-growing region of western Macedonia (Greece) needs the advantages coming of malolatic fermentation. This wine, in spite of its incontestable qualities has a strong astringent and a high level of acidity, the malic acid contributing essentially to these two characters.</p><p style="text-align: justify;">Because of the interest malolactic fermentation presents for this region, we have collaborated with the wine-producters to insure, at first, the generally admitted conditions which favour the evolution of malolactic fermentation, then to study it.</p><p style="text-align: justify;">The latter includes : a) the control of the conversion of malic acid into lactic acid by chromatography paper and b) the enumeration, isolation and identification of lactic acid bacteria.</p><p style="text-align: justify;">During this two-year-long research, 187 samples have been taken from 50 different tanks during 12 samplings. The isolation of lactic acid bacteria was done by plating dilutions of wine samples and their identification with the API system and other physico-chemical and enzymatic methods.</p><p style="text-align: justify;">Data processing has been done by computer and given a strain group distribution according to their similarity.</p><p style="text-align: justify;">The results show that the homofermentative lactic acid bacteria - mainly <em>Lactobacillus plantarum</em> - are more numerous than the heterofermentative ones before the malolactic fermentation. They decrease during the fermentative process and disappear with its completion.</p><p style="text-align: justify;">The heterofermentative lactic aeid baeteria, whose main specie is <em>Leuconostoc oenos</em> evolves inversely.</p><p style="text-align: justify;">A low increase of <em>Pediococcus</em> has been observed too after the beginning of malolactic fermentation. A statistical analysis confirms the main points of this research.</p><p style="text-align: justify;">The application of malolactic fermentation becomes more efficient the second year; it is favoured in press-wines and for temperatures kept at 20-25°C; the bacteria population is also increased.</p><p style="text-align: justify;">The persued reduction of total acidity in wines, which underwent malolactic fermentation, was considerable.</p>

Malolactic fermentation (MLF) is an important process in the wine production. MLF results from the metabolism of certain lactic acid bacteria (LAB) and consists in the conversion of malate to lactate and CO2. Except deacidification, MLF can improve the quality and microbiological stability of the wines. The aim of this project was to investigate the preservation and utilization of the LAB with particular reference to the MLF. In order to measure the effect of various preservation methods and their productivity, an assay of cell vitality was developed. This measured the capacity to overcome and recover from freezing and freeze-drying. It was shown that this method was easily used and reliable. The effect of cultural conditions on the cryotolerance and vitality of the LAB was investigated, including: (1) the growth phase, (2) the growth temperature, (3) the medium pH, (4) composition of the medium, and (5) preincubation conditions. The optimal cultural conditions to obtain higher vitality after freezing varied with the species of LAB. When the pH of culture medium was controlled at pH 5 the LAB attained the highest vitality after freezing. When L. plantarum was preincubated in 5g/l yeast extract solution at 25°C for 1 hour, the survival rate of L. plantarum greatly increased, from 5.2% to 46.5%. The conditions of freeze-drying of the LAB were investigated. It was found that 4% yeast extract was the most effective protectant for L. plantarum and L. brevis and 5% glutamate were the best protectant for O. oeni. When the LAB was frozen quickly at -65°C, the vitality obtained was higher than those frozen slowly at -20°C after freeze-drying. Another factor to be considered important was ethanol tolerance when the freeze-dried malolactic bacteria were used in wines. Among the suspension media tested, 5% glutamate and 10% sucrose were the best for freeze-dried L. brevis and O. oeni respectively to obtain high vitality in high ethanol solutions. These studies showed that there were no consistent underlying processes that could be easily identified and that preservation was a species specific, multifactorial process. The MLF was then investigated further by studying the effect of wine components on the batch MLF of L. brevis and O. oeni using a defined synthetic wine. This uniquely allowed a systematic study of the MLF in high alcohol environments. Alcohol tolerance was dependent on temperature and important fermentation intermediates such as citrate, pyruvate and malate. Malolactic fermentations were inhibited when glucose concentration was 2 g/1 to 6 g/1. The inhibition to MLF of O. oeni caused by glucose was relieved when fructose was present. Nutritional status was also an important factor that affected the MLF, when the synthetic wine did not contain added yeast extract, malic acid degradation of L brevis and O. oeni was low (6.1% and 54.3% respectively). Rapid and continuous malolactic fermentation was achieved in the membrane bioreactor (MBR) with high cell density of O. oeni (greater than 108CFU/ml). More than 95% degradation of malic acid in the synthetic wine was obtained at 0.48 1/h of flow rate and 10.4 h residence time. High ethanol concentration of wine was main factor that caused the loss in malic acid degrading activity of O. oeni in the MBR. The poor nutritional condition of wine was not the main factor causing loss in the stability of malic acid degrading activity of O. oeni. The shear stress had little influence on the malic acid degradation of O. oeni under the conditions investigated. Ethanol stress adaptation could improve the stability of malic acid degrading activity of O. oeni in the MBR.

Mestrado em Biotecnologia
O Oenococcus oeni é uma espécie de bactéria de elevado interesse, nomeadamente enológico, porque é capaz de realizar a fermentação maloláctica (desacidificação do vinho) e reúne as condições essenciais para poder sobreviver às condições adversas do vinho. Por outro lado ao alta-pressão é uma tecnologia com grande potencial para explorar novas e promissoras aplicações na biotecnologia. Neste trabalho pretendeu-se avaliar o efeito de um tratamento de alta-pressão no metabolismo desta bactéria, nomeadamente na descarboxilação do ácido L-málico, no metabolismo de açúcares e no crescimento bacteriano. O tratamento de 50 MPa durante 8 h e 100 MPa durante 0.5 h não resultaram em alterações significativas no metabolismo das bactérias. O tratamento de 100 MPa, durante 8 e 60 h resultaram numa redução da quantidade de ácido L-láctico produzido, propondo-se que também foi produzido ácido D-láctico a partir do ácido L-málico. O tratamento de 300 MPa durante 0.5 h resultou na completa inactivação das bactérias. Assim conclui-se que alta-pressão é uma tecnologia que permite a alteração do metabolismo, nomeadamente a modificação da especificidade da enzima maloláctica, e a inactivação de Oenococcus oeni.
The Oenococcus oeni is a bacterial species with high interest, especially winemaking, because it is able to carry out malolactic fermentation (wine desacidification) and has the essentials conditions to be able to survive in the wine harsh conditions. On the other hand the high-pressure is a technology with great potential to explore new and promising applications in biotechnology. In this work the aim was to evaluate the effect of high pressure treatments in the metabolism of the bacteria, especially in the decarboxylation of L-malic acid, in the sugars metabolism and bacterial growth. The treatment of 50 MPa during 8 hours and 100 MPa during 0.5 h did not result in significant alterations in the bacteria metabolism. The treatment at 100 MPa during 8 and 60 h resulted in a reduction of the amount of L-lactic acid produced. It was proposed that was also produced D-lactic acid from L-malic acid. The treatment of 0.5 MPa during 300 h resulted in complete inactivation of bacteria. It is concluded that high pressure is a technology that allows the alteration of the metabolism, particularly change of malolactic enzyme specificity, and inactivation of Oenococcus oeni.

Dissertation (PhD Agric)--University of Stellenbosch, 2004.
ENGLISH ABSTRACT: Malolactic fermentation (MLF) is conducted by lactic acid bacteria (LAB) and entails the decarboxylation of L-malate to L-Iactate through a reaction catalysed by the malolactic enzyme (MLE). The consequence of this conversion is a decrease in total acidity. MLF plays a part in microbial stabilisation and due to the metabolic activity of the bacteria the organoleptic profile of the wine is modified. In some wines MLF is considered as spoilage, especially in warm viticultural regions with grapes containing less malic acid. In addition to undesirable organoleptic changes, MLF can alter wine colour, and biogenic amines may be produced. To induce MLF we provided s. cerevisiae with the enzymatic activities required for MLF, which is then conducted by the yeast during alcoholic fermentation. The malolactic enzyme-encoding gene (mieD) was cloned from Pediococcus damnosus NCFB 1832, characterised and expressed in S. cerevisiae. The activity of this enzyme was compared to two other malolactic genes, mieS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni La11, expressed in the same yeast strain. All three recombinant strains of S. cerevisiae converted L-malate to L-Iactate in synthetic grape must, reaching L-malate concentrations of below 0.3 gIL within 3 days. However, a lower conversion rate and a significant lower final L-Iactate level were observed with the yeast expressing mieD. In order to inhibit MLF, we show that the growth of O. oeni, the main organism responsible for MLF, could be safely repressed with a ribosomaly synthesised antimicrobial peptide, pediocin PD-1, produced by P. damnosus NCFB 1832, without effecting yeast growth. Pediocin PD-1 is stable in wine at 4°C-100°C, and ethanol or S02 does not affect its activity. The peptide was purified to homogeneity and sequence analysis suggests that the peptide is a member of the lantibiotic family of bacteriocins. The molecular mass was estimated by mass spectroscopy to be 2866.7 ± 0.4 Da. Pediocin PD-1 forms pores in sensitive cells, as indicated by the efflux of K+ from O. oeni, combined with inhibition of cell wall biosynthesis, leading to cell lysis. Loss of cell K+was reduced at low temperatures, presumably as a result of the increased ordering of the lipid hydrocarbon chains in the cytoplasmic membrane. Although pediocin PD-1 is active over a broad pH range, optimal activity was recorded at pH 5.0. The petide is, however, more stable between pH 2.0 and 5.0, with the best stability observed between pH 3.0 and 4.0. Pediocin PD-1 provides a safer biological alternative than chemical preservatives such as S02.
AFRIKAANSE OPSOMMING: Appelmelksuurgisting (AMG) word deur sekere melksuurbakterieë (MSB) uitgevoer en verwys na die dekarboksilering van L-malaat na L-Iaktaat, 'n reaksie gekataliseer deur die appelmelksuurensiem (AME). AMG verlaag die suurvlakke in wyn, speel 'n rol in mikrobiologiese stabiliteit, en verander die organoleptiese profiel van die wyn. In sommige wyne word AMG beskou as bederf, veral in warm wynbou streke met minder malaat in druiwe. AMG kan ongewenste organoleptiese veranderinge teweeg bring, die wyn se kleur beinvloed, en tot die produksie van biogene amiene lei. Vir die bevordering van AMG het ons S. eerevisiae met die ensiematiese aktiwiteit benodig vir AMG voorsien wat dan veilig deur die gis tydens alkoholiese fermentasie uitgevoer word. 'n AME-koderende geen (mIeD) is uit Pedioeoeeus damnosus NCFB 1832 gekloneer, gekarakteriseer en in S. Cerevisiae uitgedruk. Die aktiwiteit van die ensiem is vervolgens vergelyk met die aktiwitet van twee ander AME gene, mIeS van Laetoeoeeus laetis MG1363 en mleA van Oenoeoeeus oeni Lal1, uitgedruk in dieselfde gisras. AI drie rekombinante gisrasse het L-malaat binne die bestek van drie dae na L-Iaktaat omgeskakel en die finale L-malaat vlakke was minder as 0.3 gIL. Die tempo van omkakeling was egter laer in die gis wat die mIeD geen uitdruk en die finale L-Iaktaat vlakke was veel laer. Om AMG te inhibeer is die groei van O. oeni, die organisme hoofsaaklik verantwoordelik vir AMG, onderdruk deur die byvoeging van 'n ribosomaal gesintetiseerde antimikrobiese peptied, pediocin PD-1, geproduseer deur P. damnosus NCFB 1832. Gisgroei is nie geaffekteer nie. Pediocin PD-1 is stabiel in wyn by temperature wat wissel tussen 4°C en 100°C, en die aktiwiteit van die peptied word nie geaffekteer deur ethanol of S02 nie. Die peptied is gesuiwer volgens In eenvoudige metode wat amoniumsulfaat-presipitasie en katioon uitruilings-ehromatografie insluit. Aminosuur volgorde bepaling van gesuiwerde peptied dui daarop dat pediocin PD-1 tot die lantibiotiese familie van bakteriosiene behoort. Die molekulêre massa van die peptied, soos bepaal deur massa spektroskopie, is 2866.7 ± 0.4 Da. Pediocin PD-1 vorm porieë in selmembrane van sensitiewe selle soos aangedui deur die uitvloei van K+vanuit O. oeni selle. Die peptied kombineer hierdie aksie met die inhibisie van selwand biosintese wat lei tot sel lise. Verlies van sellulêre K+verminder by laer temperature, waarskynlik as gevolg van verandering in die lipied- en protein inhoud van die sitoplasmiese membraan. Alhoewel die peptied aktief is oor 'n breë pH grens, is die antimikrobiese aksie optimaal by pH 5.0. Die peptied is meer stabiel tussen pH 2.0 en 5.0 en toon die beste stabiliteit tussen pH 3.0 en 4.0. Peiocin PD-1 is 'n veilige biologiese alternatief vir chemiese preserveermiddels soos S02.

Thesis (MScAgric)--Stellenbosch University, 2014.
ENGLISH ABSTRACT: Wine is a product of the fermentation of grape juice. Alcoholic fermentation is mainly conducted by the yeast Saccharomyces cerevisiae which metabolises grape sugars to mainly ethanol, CO2 and glycerol. Aside from these primary fermentation compounds, the yeast also produces many secondary metabolic by-products that are important to wine quality and style. Malolactic fermentation (MLF) is a secondary fermentation that normally occurs after alcoholic fermentation. Lactic acid bacteria (LAB) are responsible for the conversion of malic acid to lactic acid and CO2 during MLF, which is important for wine deacidification and also contributes to microbial stability. Malolactic fermentation and LAB strains can also influence the aroma profile of wines. The main genera associated with this process are Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Oenococcus oeni is the main species associated with MLF because it is able to survive the harsh physiochemical environment of winemaking. Recently L. plantarum has also been introduced as a commercial MLF starter culture. Research has started to focus on the potential of wine yeast and LAB interactions or combinations to alter the wine aroma profile via the production and/or degradation of aroma compounds. The overriding goal of this study is to unravel the interactions between wine yeast and different LAB strains and their impact on wine aroma and flavour. The first aim was to assess LAB growth during co- and sequential inoculation strategies, the ability to complete MLF and the impact on the production of aroma compounds in combination with two different yeast strains in a medium containing full complement of nitrogen supplementation. Malolactic fermentation was successful in the different inoculation strategies and the bacterial combination (L. plantarum and O. oeni) completed MLF in the shortest time. The impact of the bacterial strains on the modification of aroma compounds was bigger in co- than sequential inoculation. A general increase in total esters (contributing to the fruity character of wines) especially ethyl lactate and ethyl acetate was observed. The production of esters, volatile fatty acids and higher alcohols proved to be dependent on either the yeast strain used and/or the LAB strains used. The second aim of the research was to assess the effect of NH4Cl (ammonium) and amino acids supplementation on yeast and LAB strains (both in co- and sequential inoculation strategies) and the impact on the aroma profile of the fermented must. Fermentations supplemented with ammonia as sole nitrogen source showed the highest total bacterial growth in terms of cell numbers. Malolactic fermentation was completed in the shortest time with O. oeni and the bacterial combination inoculums. The co-inoculated strategies in combination with amino acids supplementation showed the biggest impact on the aroma compound profiles of the different fermentation strategies and bacterial treatments. A general increase in total esters was observed for NH4Cl additions with ethyl lactate and ethyl acetate showing the highest concentrations. The concentration of esters, volatile fatty acids and higher alcohols were strongly influenced by the yeast and the single LAB strains used. The results generated from this study showed that the chemical composition of the fermentation medium and the selection of yeast and LAB strains are important because these factors have an influence on the aroma and flavour profiles of wines.
AFRIKAANSE OPSOMMING: Wyn is die produk van gefermenteerde druiwe. Die gis, Saccharomyces cerevisiae is verantwoordelik vir alkoholiese fermentasies waar druiwe suikers na hoofsaaklik etanol, CO2 en gliserol gemetaboliseer word. Die gis produseer ook sekondêre metaboliete wat ‘n belangrike bydrae lewer tot wynstyl en kwaliteit. Appelmelksuurgisting (AMG) is ‘n sekondêre fermentasie wat gewoonlik na alkoholiese fermentasie plaasvind. Melksuurbakterieë (MSB) speel ‘n sleutel rol in die omskakeling van appelsuur na melksuur en CO2 gedurende AMG. Hierdie fermentasie lei tot ‘n afname in die suurheidsgraad en verbeter die mikrobiese stabiliteit van die wyn. Appelmelksuurgisting en MSB rasse kan die aroma- en geurprofiel van wyne beïnvloed. Die belangrikste genera wat met AMG geassosieer word is Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Oenococcus oeni is die mees algemene ras wat vir AMG gebruik word omdat dit in uiterste wyn toestande kan oorleef. Mees onlangs is Lactobacillus plantarum as kommersiële aanvangskultuur vir AMG geïdentifiseer. Navorsing het onlangs meer begin fokus op gis en MSB interaksie of kombinasies as ‘n strategie om die aroma profiele van wyne te verander. Die hoofdoel van die studie is om die interaksie tussen wyngiste en verskillende MSB rasse en die effek op die aroma profile van wyne te bestudeer. Die eerste doelwit was om die impak van die twee giste op die groei en AMG vermoeë van MSB gedurende ko- en sekwensiële inokulasie praktyke en die impak op die produksie van aroma komponente, in ‘n medium wat die volledige stikstof aanvullings bevat, te bestudeer. Appelmelksuurgisting was suksesvol in die verskillende inokulasie praktyke en die bakteriese kombinasie (L. plantarum en O. oeni) het AMG in die kortste tyd voltooi. Die impak van die bakteriese rasse op die modifikasie van die aroma komponente was groter met ko- as sekwensiële inokulasies. Daar was ‘n toename in die totale esterkonsentrasies veral in etiellaktaat en etielasetaat. Die produksie van esters, vlugtige vetsure en hoër alkohole word beïnvloed deur die gisras en MSB rasse wat gebruik word. Die tweede doelwit was om die impak van NH4Cl (ammonium) en aminosure aanvullings op die gis- en MSB rasse gedurende ko- en sekwensiële inokulasie strategieë te bepaal. Melksuurbakterieë se groei was beter met die ammonium aanvulling. Appelmelksuurgisting was in die kortste tyd voltooi met O. oeni en die bakteriese kombinasie. Die ko-inokulasie praktyke in kombinasie met die kompleks aminosure aanvulling het die grootste impak op die produksie van aroma komponente gehad. Daar was weereens ‘n toename in die totale esterkonsentrasies vir die NH4Cl aanvulling, veral in etiellaktaat en etielasetaat. Die gis en MSB rasse speel ‘n rol by die produksie en konsentrasies van esters, vlugtige vetsure en hoër alkohole. Die resultate van hierdie studie bewys dat die chemiese samestelling van die fermentasie medium, die seleksie van gis- en MSB rasse is belangrik omdat hierdie faktore die aroma en geur profiele van wyne beïnvloed.

Thesis (PhD)--University of Stellenbosch, 2011.
ENGLISH ABSTRACT: During winemaking, a number of biochemical changes occur as a result of the metabolic activity of wine lactic acid bacteria (LAB) associated with malolactic fermentation (MLF). The latter process, which occurs mostly after alcoholic fermentation by wine yeasts, involves the conversion of L-malate to L-lactate and CO2, thus resulting to wine acidity reduction, microbiological stabilization and alterations of wine organoleptic quality. Although Oenococcus oeni is predominantly the most preferred species suitable for carrying out MLF in wine owing to its desirable oenological properties, Lactobacillus plantarum has also been considered as a potential candidate for MLF induction. Other species in the genera of Lactobacillus and Pediococcus are often associated with wine spoilage. These microorganisms induce wine spoilage by producing off-flavours derived from their metabolic activity. It is therefore of paramount importance to understand the mechanism by which wine microbiota cause spoilage. The purpose of this study was to investigate the presence of genes encoding enzymes of oenological relevance in wine-associated LAB strains. In order to achieve this, different sets of specific primers were designed and employed for a wide-scale genetic screening of wine LAB isolates for the presence of genes encoding enzymes involved in various metabolic pathways, such as citrate metabolism, amino acid metabolism, hydrolysis of glycosides, degradation of phenolic acids as well as proteolysis and peptidolysis. PCR detection results showed that the majority of the tested strains possessed most of the genes tested for. It was also noted that, among the O. oeni strains tested for the presence of the pad gene encoding a phenolic acid decarboxylase, only two strains possessed this gene. None of the O. oeni strains has previously been shown to possess the pad gene, and this study was the first to report on the presence of this gene in O. oeni strains. In an attempt to genetically characterize this putative gene, DNA fragments from the two positive O. oeni strains were sequenced. The newly determined sequences were compared to other closely related species. Surprisingly, no match was found when these sequences were compared to the published genomes of three O. oeni strains (PSU-1, ATCC BAA-1163 and AWRI B429). This reinforced a speculation that the pad gene in these two strains might have been acquired via the horizontal gene transfer. In addition, it remains to be further determined if the presence of this gene translates to volatile phenol production in wine. In this study, a novel strain isolated from South African grape and wine samples was also identified and characterized. The identification of this strain was performed through the 16S rDNA sequence analysis, which indicated that this strain belongs to Lactobacillus florum (99.9% sequence identity). A novel PCR assay using a species-specific primer for the rapid detection and identification of Lb. florum strains was also established. For further characterization, this strain was also investigated for the presence of genes encoding enzymes of oenological relevance. PCR detection results indicated that the Lb. florum strain also possess some of the genes tested for. In addition to genetic screening of wine LAB isolates for the presence of different genes, this study was also aimed at evaluating the regulation of the mleA gene encoding malate decarboxylase in three oenological strains of O. oeni. The regulation of this gene was tested in a synthetic wine medium under various conditions of pH and ethanol. From the expression analysis, it was observed that the mleA gene expression was negatively affected by high ethanol content in the medium. On the other hand, low pH of the medium seemed to favour the expression of this gene as the mleA gene expression was more pronounced at pH 3.2 than at pH 3.8. The findings from this study have shed more light on the distribution of a wide array of enzyme-encoding genes in LAB strains associated with winemaking. However, it remains unknown if the enzymes encoded by these genes are functional under oenological conditions, given that wine is such a hostile environment encompassing a multitude of unfavourable conditions for the enzymes to work on. Evaluating the expression of these genes will also help give more insights on the regulation of the genes under winemaking conditions.
AFRIKAANSE OPSOMMING: Gedurende wynmaak, sal 'n aantal biochemiese veranderinge plaasvind as gevolg van die metaboliese aktiwiteit van wyn melksuurbakterieë (MSB) wat betrokke is by appelmelksuurgisting (AMG). Die laasgenoemde proses, wat meestal na alkoholiese fermentasie deur wyngiste plaasvind, behels die omskepping van L-malaat na L-laktaat en CO2, om sodoende die wyn se suur te verminder, mikrobiologiese stabiliteit en verandering van wyn organoleptiese kwaliteit. Alhoewel Oenococcus oeni hoofsaaklik die mees gewenste spesies is wat geskik is vir die uitvoering van AMG in wyn weens sy geskikte wynkundige eienskappe, Lactobacillus plantarum word ook beskou as 'n potensiële kandidaat vir AMG induksie. Ander spesies in die genera Lactobacillus en Pediococcus word dikwels geassosieer met wynbederf. Hierdie mikro-organismes veroorsaak wynbederf deur die produksie van wangeure as gevolg van hul metaboliese aktiwiteite. Dit is dus van kardinale belang dat die meganisme van die wynbederf verstaan word. Die doel van hierdie studie was om die teenwoordigheid van koderend ensieme gene van wynkundige belang in wynverwante MSB stamme te ondersoek. Ten einde dit te bereik, was verskillende stelle van spesifieke peilers ontwerp en toegepas vir 'n groot skaal se genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van ensiemkoderende gene betrokke by verskeie metaboliese paaie, soos sitraat metabolisme, aminosuur metabolisme, hidrolise van glikosiede, agteruitgang van fenoliese sure sowel as proteolise en peptidolise. PKR opsporings resultate het getoon dat die meerderheid van die stamme getoets, die meeste van die gene getoets voor besit. Dit is ook opgemerk dat, onder die O. oeni stamme getoets vir die teenwoordigheid van die pad geen, slegs twee stamme hierdie geen besit. Geen O. oeni stamme het voorheen gewys dat hul die pad geen besit, en hierdie studie was die eerste bewys oor die teenwoordigheid van hierdie geen in O. oeni stamme. In 'n poging om die geen geneties te karakteriseer, is DNA-fragmente van die twee positiewe O. oeni stamme se sekwens volgorde bepaal. Die DNA volgorde is vergelyk met ander nouverwante spesies. Verrassend, was geen passende DNA volgorde gevind met die gepubliseerde genome van drie O. oeni stamme (PSU-1, ATCC BAA-1163 en AWRI B429) nie. Dit versterk die spekulasie dat die pad geen in hierdie twee stamme via die horisontale geen-oordrag verkry is. Verder moet dit nog bepaal word of die teenwoordigheid van hierdie geen lei na vlugtige fenol produksie in wyn. In hierdie studie, is ongeïdentifiseerde stam geïsoleerd van Suid-Afrikaanse druiwe en wyn monsters ook geïdentifiseer en karakteriseer. Die identifisering van hierdie stam is uitgevoer deur middel van die 16S rDNA volgorde analise, wat aangedui het dat hierdie stam behoort aan Lactobacillus florum (99.9% volgorde identiteit). PKR toetse met behulp van die spesie-spesifieke peiler vir die vinnige opsporing en identifikasie van Lb. florum stamme is ook ontwikkel. Vir verdere karakterisering, was hierdie stam ook ondersoek vir die teenwoordigheid van koderende ensiem gene van wynkundige belang. PKR opsporings resultate het aangedui dat die Lb. florum stam ook oor 'n paar van die gene getoets voor besit. Bykomend tot genetiese toetsing van wyn MSB isolate vir die teenwoordigheid van verskillende gene, het die studie ook die evaluering van die regulering van die mleA geen, kodering malaatdekarboksilase in drie wyn stamme van O. oeni. Die regulering van hierdie geen was getoets in die sintetiese wynmedium onder verskillende pH en etanol kondisies. Van die uitdrukkingsresultate, is daar waargeneem dat die mleA geenuitdrukking is negatief geraak deur hoë etanol-inhoud in die medium. Aan die ander kant, in die lae pH medium was die uitdrukking van hierdie geen bevoordeel by pH 3.2 as by pH 3.8. Die bevindinge van hierdie studie het meer lig gewerp op die verspreiding van die wye verskeidenheid van ensiem-koderende gene in MSB stamme wat verband hou met wynmaak. Dit bly egter steeds onbekend of die ensieme gekodeer deur hierdie gene funksioneel is onder wynkondisies, gegewe dat wyn so 'n vyandige omgewing is menigte ongunstige toestande vir die werking van ensieme. Evaluering van die uitdrukking van hierdie gene sal ook help om meer insigte gee oor die regulering van die gene onder wynmaak toestande.

Thesis (MScAgric (Viticulture and Oenology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: The quality of wine is influenced and determined by various factors, one of which includes the process of malolactic fermentation (MLF). MLF plays an integral role in the flavour and sensory profile of most red wines as well as some white wines like Chardonnay. This process is conducted by lactic acid bacteria (LAB), specifically of the genera Oenococcus, Lactobacillus, Pediococcus and Leuconostoc. Of these, Oenococcus oeni is best adapted to survive in the harsh wine environment. MLF is defined as the conversion of L-malic acid to L-lactic acid and carbon dioxide. The conversion of the dicarboxylic malic acid to the monocarboxylic lactic acid results in a decrease in acidity and an increase in pH, to give a softer mouthfeel and more favourable flavour profile. A further reason for conducting MLF in wine includes the improvement of microbial stability due to the removal of malic acid as a possible substrate for microorganisms. Recently, research focus has shifted to the ability of MLF and LAB to alter the aroma profile of wine via the production and/or modification of certain aroma compounds. In order for wine LAB to conduct MLF, they need to be able to survive the harsh and challenging wine environment. Conditions in South African wines are particularly challenging due to the long, hot ripening seasons resulting in high sugar concentrations which give high ethanol concentrations. Some LAB also struggle to adapt to an environment with high pH and low malic acid concentrations. These factors, combined with the use of sulphur dioxide, cause LAB to struggle in conducting and completing successful MLF. Many of the commercial starter cultures that are currently available contain LAB that have not been isolated from South African wine and are therefore not optimal for use under these challenging wine conditions. Oenococcus oeni is also the single LAB culture present in all commercially available starter cultures. The overriding goal of this study was to create a MLF starter culture containing a mixture of LAB cultures, namely O. oeni and Lactobacillus plantarum, which can successfully convert malic acid to lactic acid, ensure microbial stability, but also make a positive contribution to the wine aroma profile. Lactobacillus plantarum has previously been considered for possible use in a commercial starter culture. The LAB isolates used in this study were selected from the Institute for Wine Biotechnology culture collection as well as isolated from spontaneous MLF. The first objective was to characterise these LAB strains for important traits and for possible use as a MLF starter culture. A total of 23 strains were identified as O. oeni and 19 strains as Lb. plantarum. The identified strains were screened in a synthetic wine medium for their ability to convert malic acid to lactic acid. Based on the LAB strain performance in the synthetic wine medium, seven strains of both O. oeni and Lb. plantarum were selected. These 14 strains were screened for the presence of genes encoding for enzymes responsible for biogenic amine production and were found to contain none of the genes associated with the formation of histamine, tyramine or putrescine. The LAB strains were genetically screened for enzymes associated with aroma modification by LAB during MLF. The enzymes of interest that were screened for included β-glucosidase, esterase, protease and phenolic acid decarboxylase (PAD). The Lb. plantarum strains were found to possess more diverse enzymatic profiles related to aroma than O. oeni. The biggest differences were observed for the presence of β-glucosidase and PAD. The second objective was to perform small-scale fermentations with the individual LAB isolates. The individual isolates were evaluated in Pinotage and based on these results; three strains of each O. oeni and Lb. plantarum were selected for evaluation in mixed culture fermentations. The mixed cultures were evaluated in Pinotage, Shiraz and Cabernet Sauvignon in the 2008 vintage. As a third objective, the wines were also analytically and sensorially evaluated to investigate the changes in the aroma profile that could be attributed to the presence of the mixed LAB isolates. Based on the fermentation data as well as data pertaining to the aroma modification, three mixed cultures were selected for evaluation in the 2009 vintage in Pinotage, Cabernet Sauvignon and Chardonnay. The mixed cultures were able to successfully complete MLF in fermentation periods comparable to that of a commercial culture used as control. The different LAB cultures had distinct and diverse effects on the wine aroma profile. The O. oeni strain played a larger role in the ester concentration present after MLF, while the Lb. plantarum strain had a larger effect on the higher alcohol and volatile fatty acid concentration upon completion of MLF. The results generated by this novel study clearly indicate the potential of a mixed LAB starter culture for conducting MLF. The mixed cultures successfully completed MLF and made a positive contribution to the wine aroma profile.
AFRIKAANSE OPSOMMING: Die kwaliteit van wyn word beïnvloed en bepaal deur verskeie faktore en wynbereidings prosesse, wat die proses van appelmelksuurgisting (AMG) insluit. AMG speel ’n integrale rol in die sensoriese profiel van meeste rooiwyne, sowel as sommige witwyne soos Chardonnay. AMG word gedefinieër as die omskakeling van L-appelsuur na L-melksuur en koolstofdioksied. Hierdie omskakeling kan toegeskryf word aan die teenwoordigheid van melksuurbakterieë (MSB), spesifiek spesies van die genera Oenococcus, Lactobacillus, Pediococcus en Leuconostoc. Vanuit hierdie wyn MSB, is Oenococcus oeni die spesies wat die beste aanpas en oorleef onder stresvolle wyn kondisies. Die omskakeling van appelsuur, ’n dikarboksielsuur, na melksuur, ’n monokarboksielsuur, lei tot ‘n vermindering in suurheid en ’n verhoging in pH. Hierdie vermindering in suurheid gee ’n sagter en meer geronde mondgevoel aan die wyn en dra by tot ‘n meer aangename geurprofiel. ’n Verdere rede vir AMG in wyn is om mikrobiese stabiliteit te verseker deurdat appelsuur verwyder word as ’n moontlike koolstof substraat vir mikroörganismes. Onlangs het navorsing begin fokus op AMG en die vermoë van MSB om die aroma profiel van wyn te beïnvloed deur die produksie/modifisering van sekere aroma komponente. Vir MSB om AMG te kan deurvoer, moet hulle kan oorleef in die stresvolle wynomgewing. Wyntoestande in Suid-Afrika is veral uitdagend vir die oorlewing van mikroörganismes as gevolg van lang, warm somers wat lei tot ’n matriks met ’n hoë suikerkonsentrasie en wyn met ’n hoë etanolkonsentrasie. ‘n Omgewing met ‘n hoë pH en lae appelsuur konsentrasie, kan ook bydrae tot stresvolle kondisies vir MSB. Hierdie parameters, tesame met die gebruik van swaweldioksied, maak dit moeilik vir MSB om AMG te inisieer en te voltooi. Sommige van die kommersiële aanvangskulture wat tans beskikbaar is, bevat nie MSB wat onder Suid-Afrikaanse wyntoestande geïsoleer is nie en daarom is dit nie altyd optimaal vir gebruik nie. Oenococcus oeni is ook die enkele MSB kultuur wat in alle kommersiële kulture gebruik word. Die hoofdoelwit van hierdie studie was om ’n potensiële kommersiële aanvangskultuur te ontwikkel wat ‘n mengsel van MSB bevat. Hierdie aanvangskultuur moet AMG suksesvol kan voltooi, mikrobiologiese stabiliteit bevorder en steeds die wynaroma positief kan beïnvloed. Bakterierasse van O. oeni en Lb. plantarum is geselekteer vir gebruik in hierdie studie. Lactobacillus plantarum het reeds in vorige studies potensiaal getoon as ‘n moontlike aanvangskultuur. Die MSB isolate vir hierdie studie is geselekteer uit die Instituut vir Wynbiotegnologie se kultuurversameling en geïsoleer uit spontane AMG fermentasies. Die eerste doelwit was om hierdie MSB isolate te karakteriseer vir belangrike eienskappe en die moontlike gebruik as ’n kommersiële AMG aanvangskultuur. ‘n Totaal van 23 O. oeni en 19 Lb. plantarum isolate is geïdentifiseer. Hierdie isolate is in ’n sintetiese wynmedium geëvalueer vir hul vermoë om appelsuur na melksuur om te skakel. Op grond van hul reaksie in die sintetiese wynmedium, is sewe isolate van elk van die O. oeni en Lb. plantarum geselekteer. Hierdie 14 isolate is ondersoek vir die teenwoordigheid van die gene wat kodeer vir biogeenamien produksie en daar is gevind dat geen van die isolate enige van die biogeenamien gene wat ondersoek is, naamlik histamien, tiramien en putresien besit nie. Die MSB isolate is geneties ondersoek vir die teenwoordigheid van dié gene wat kodeer vir ensieme wat die aromaprofiel tydens AMG beïnvloed. Dié ensieme sluit β-glukosidase, esterase, protease, fenoliese suurdekarboksilase en sitraatliase in. Daar is gevind dat die Lb. plantarum isolate meer diverse ensiemprofiele as O. oeni besit. Die grootste verskille in die ensiemprofiele kan toegeskryf word aan die teenwoordigheid van β-glukosidase en fenoliese suurdekarboksilase. Die tweede doelwit was om kleinskaalse AMG fermentasies met die individuele MSB isolate uit te voer. Die individuele isolate is in Pinotage geëvalueer. Volgens hierdie resultate is drie isolate van elk van die O. oeni en Lb. plantarum geselekteer om in gemengde kulture getoets te word. Die gemengde kulture is in Pinotage, Shiraz en Cabernet Sauvignon in 2008 geëvalueer. As ’n derde doelwit is hierdie wyne ook analities en sensories geëvalueer om die veranderinge in die aromaprofiele as gevolg van die teenwoordigheid van die MSB te ondersoek. Op grond van die fermentasiedata, sowel as die data oor die aromaveranderinge, is drie gemengde kulture geselekteer vir evaluering in Pinotage, Cabernet Sauvignon en Chardonnay in 2009. Die gemengde kulture kon AMG suksesvol voltooi met fermentasietempo’s wat vergelykbaar was met dié van ‘n kommersiële AMG kultuur wat as kontrole gebruik is. Die verskillende MSB kulture het spesifieke en uiteenlopende uitwerkings op die wynaroma gehad. Die O. oeni isolaat in die gemengde kultuur blyk ‘n belangriker rol te speel in die esterkonsentrasie na AMG, terwyl die Lb. plantarum isolaat ’n groter effek het op die hoër alkohol en vlugtige vetsuurinhoud na AMG. Die resultate wat deur hierdie unieke studie gegenereer is, gee ’n aanduiding van die potensiaal van ’n gemengde MSB aanvangskultuur vir AMG. Die gemengde kulture kon AMG suksesvol voltooi en ‘n positiewe bydrae tot die aromaprofiel van die wyn lewer.

Oenococcus oeni, l’espècie de bacteris làctics (BL) principal responsable de la fermentació malolàctica (FML), s'ha aïllat repetidament dels vins, però quasi mai des del raïm. En aquesta tesi, la biodiversitat de BL de l'ecosistema vinya-vi va ser establerta i també es va realitzar un ampli estudi sobre els BL autòctons de la regió vitivinícola catalana del Priorat. Es van identificar i tipificar un total de 1.904 aïllats de BL, de raïm de Garnatxa i de Carinyena, així com de vins de diferents cellers. Al voltant del 70% dels aïllats van ser O. oeni, principalment de vi, però sorprenentment, 53 d'ells es van aïllar de raïm. Es van identificar i tipificar també altres espècies no-Oenococcus, éssent Lactobacillus plantarum la predominant en raïm. La presència d'Oenococcus i Lactobacillus en raïm també es va confirmar per seqüenciació massiva. Es va estudiar la possibilitat d'utilitzar algunes d'aquestes soques autòctones; s'en van seleccionar i caracteritzar 45 BL en base a la degradació de l'àcid L-màlic, la resistència al pH baix i a les altes concentracions d'etanol, i l'absència de gens d'amines biògenes. Les tres soques amb les característiques més desitjables es van inocular en vins reals, dels quals la FML es va dur a terme amb èxit. Les característiques dels vins obtinguts van suggerir el possible ús d'una de les soques com a bona candidata per a un potencial cultiu iniciador. Per tant, les soques autòctones podrien ser utilitzades, després de ser seleccionades, com inòcul de vins reals, ja que estan ben adaptades a les condicions d'aquesta àrea específica i poden mantenir les característiques del terroir.
Oenococcus oeni, la especie de bacterias lácticas (BL) principal responsable de la fermentación maloláctica (FML), se ha aislado repetidamente de los vinos, pero casi nunca desde uva. En esta tesis, se estableció la biodiversidad de BL del ecosistema viñedo-vino y también se realizó un amplio estudio sobre las BL autóctonas de la región vitivinícola catalana del Priorat. Se identificaron y tipificaron un total de 1.904 aislados de BL, de uva de Garnacha y de Cariñena, así como de vinos de diferentes bodegas. Alrededor del 70% de los aislados fueron O. oeni, principalmente de vino, pero sorprendentemente, 53 de ellos se aislaron de uva. Se identificaron y tipificarn también otras especies no- Oenococcus, siendo Lactobacillus plantarum la predominante en uva. La presencia de Oenococcus y Lactobacillus en uva también se confirmó por secuenciación masiva. Se estudió la posibilidad de utilizar algunas de estas cepas autóctonas; se seleccionaron y caracterizaron 45 BL en base a la degradación del ácido L-málico, la resistencia al pH bajo y a las altas concentraciones de etanol, y la ausencia de genes de amines biógenas. Las tres cepas con las características más deseables se inocularon en vinos reales, de los cuales la FML se llevó a cabo con éxito. Las características de los vinos obtenidos sugirieron el posible uso de una de las cepas como buena candidata para un potencial cultivo iniciador. Por lo tanto, las cepas autóctonas podrían ser utilizadas como inóculo de vinos reales, ya que están bien adaptadas a las condiciones de esta área específica y pueden mantener las características del terroir.
Oenococcus oeni, the lactic acid bacterium (LAB) mainly responsible for malolactic fermentation (MLF), has been repeatedly isolated from wines, but hardly ever from grapes. In this work, the LAB biodiversity of the vine-wine ecosystem was established, and a large survey of autochthonous LAB from the Catalan wine region of Priorat was achieved, too. A total of 1,904 LAB isolates, from Grenache and Carignan grape berries and from wines of different cellars, were identified and typed. Around 70% of isolates were O. oeni, mostly from wines, but remarkably, 53 of them were isolated from grapes. Other non-Oenococcus species were also identified and typed, being Lactobacillus plantarum the predominant one in grapes. The presence of Oenococcus and Lactobacillus in grapes was also confirmed by high-throughput sequencing. The possibility of using some of these autochthonous strains was studied; 45 LAB were selected and characterized in base of their degradation of L-malic acid, the resistance to low pH and high ethanol, and the absence of biogenic amine genes. The three strains with the most desirable characteristics were inoculated in real wines and MLF was carried out successfully. The characteristics of the obtained wines suggested the possible use of one of the strains as good candidate for starter culture. Thereby, autochthonous strains have the potential to be used, after being selected, as inoculum of real wines, they are well adapted to the conditions of this specific area and can keep the terroir characteristics.

Mestrado Vinifera Euromaster - Viticulture and Enology - Instituto Superior de Agronomia
This research work addresses the under explored interaction between malolactic bacteria and non-Saccharomyces yeast, specifically commercial strains of Metschnikowia Pulcherrima and Oenococcus oeni. Chemically defined media and two chardonnay juices were used to evaluate sequential and co-inoculated malolactic fermentation strategies, investigating the effect of M. Pulcherrima on O. oeni viability and malolactic fermentation kinetics. L-malic acid consumption, and bacteria viability were tracked during fermentation, along with sampling for organic acid analysis. Our findings showed the addition of M. pulcherrima significantly influenced malolactic fermentation kinetics and increased O. oeni bacteria viability in chemically defined media, and select Chardonnay treatments. Further work is investigating the source of commensalism between M. pulcherrima and O. oeni. Exploring alternative strain pairs and understanding non- Saccharomyces and bacteria interactions will help increase winemakers control when implementing co-inoculation strategies for malolactic fermentation. This has a promising application in the industrial production of sparkling base wine, increasing malolactic fermentation efficiency and closing the gap for potential spoilage associated with conducting malolactic after alcoholic fermentation
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Thesis (MSc (Institute for Wine Biotechnology))--Stellenbosch University, 2010.
ENGLISH ABSTRACT: The process of winemaking involves a number of microorganisms, contributing both positively and negatively to the final product. Lactic acid bacteria (LAB) are present at all stages of vinification and therefore play a major role in the production of wine, especially red wine. LAB are responsible for malolactic fermentation (MLF), which can be desirable or unwanted depending on the style of wine. LAB can also be responsible for spoilage, and production of off flavours resulting in a decrease in the quality of the finished wine. Spoilage occurs if the wrong species are present at the wrong time and can also occur as a result of spontaneous MLF. It is therefore necessary to control the population of indigenous LAB present in the wine. Plantaricins are bacteriocins produced by Lactobacillus plantarum strains and have the potential to inhibit closely related strains that occupy the same ecological niche. This makes them promising for the control of LAB during the winemaking process. Inhibition of the indigenous LAB microflora could help to prevent the formation of undesirable off-flavours, as well as allowing for control over MLF. The use of plantaricin-producing starter cultures could also lead to a reduction in the amount of sulphur dioxide used in wine. The purpose of this study was to investigate the potential of L. plantarum strains isolated from wine to produce plantaricins under winemaking conditions. This potential was evaluated by investigating the expression of plantaricin genes under winemaking conditions. The first objective was to screen nineteen strains of L. plantarum isolated from South African red wines, as well as a commercial strain; for various genes responsible for the production of plantaricins, including structural, transport and regulatory genes. Results showed that the twenty strains contained at least 16 of the 24 genes (previously reported to be associated with the plantaricin locus for various L. plantarum strains) screened for. Only orfZ123 and orf345 genes yielded no positive results in any of the strains. The second objective was to sequence selected plantaricin genes (plnE, plnF, plnN, plnG and plnB) to determine the variation in nucleotide and amino acid sequences of these genes among the different wine L. plantarum isolates. High homology was found between the nucleotide sequences of the strains and none of the amino acid substitutions in the protein sequences occurred in conserved regions. The nucleotide sequence of plnN was identical in all but one of the strains and similarity of the plnB sequence ranged from 96% to 100%. Similarity of the plnG nucleotide sequence ranged from 99% to 100%. The plnE nucleotide sequence was identical in all but two strains and there were only two groups in terms of nucleotide sequence for plnF, with only two changes between the groups. The third objective was the evaluation of plantaricin production using plate assays mimicking certain wine parameters (pH and ethanol concentration). All twenty strains showed inhibitory activity to varying degrees against a panel of nine indicator microorganisms, including Enterococcus faecalis, Listeria monocytogenes and potential wine spoilage organisms, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides. Addition of 10% ethanol and a low pH of 3.5 decreased both the bacteriocin production as well as the spectrum of activity. Seven of the twenty strains, however, showed good bacteriocin activity under all conditions. The fourth objective was to investigate the expression of two plantaricin structural genes (plnEF and plnJK) and the transporter gene (plnG) under winemaking conditions. Two strains (R1122 and 113.1) were chosen, based on the results from the previous objectives, as starter cultures for MLF in synthetic wine media and Riesling wine. Low wine pH (3.2) and high wine pH (3.8) levels were investigated in conjunction with ethanol concentrations of 0%, 12% and 15%. All three of the genes were expressed to varying degrees depending on the fermentation condition. High ethanol and low pH generally decreased expression of the structural plantaricin genes. The influence on expression of the transporter gene was different, with low pH and presence of ethanol resulting in an increase in gene expression. The genes were also expressed in wine, although at a lower level relative to expression in the synthetic wine media. The presence of sensitive bacteria in the wine seemed to increase expression of the structural genes. Furthermore, expression of the mle gene responsible for MLF was investigated under the same winemaking conditions. Expression was shown to be inducible by malic acid, and negatively affected by the presence of ethanol but positively influenced by a lowering in pH from 3.8 to 3.2. This study confirms that plantaricin genes are expressed under winemaking conditions, which in turn indicates that the plantaricins could be produced under winemaking conditions. This confirms the potential use of these plantaricin-producing strains as starter cultures for MLF with the ability to inhibit indigenous LAB, however, presence of the plantaricin protein in wine still needs to be confirmed. It will also need to be established whether the protein is biologically active and not inhibited by wine-related factors.
AFRIKAANSE OPSOMMING: Die proses van wynmaak bevat 'n verskeidenheid mikroorganismes, wat postiewe en negatiewe bydrae kan lewer tot die finale produk. Melksuurbakterieë is teenwoordig by alle stadiums van wynmaak en speel 'n belangrike rol in die produksie van wyn. Melksuurbakterieë is verantwoordelik vir appelmelksuur gisting (AMG), wat gewens of ongewens kan wees, afhangende van die styl van die wyn. Melksuurbakterieë kan ook verantwoordelik wees vir bederf van wyn, asook die produksie van ongewenste geure wat bydrae tot ʼn toename in die kwaliteit van die wyn. Bederf van wyn kan gebeur as die verkeerde spesies voorkom op die verkeerde tyd en kan ook gebeur as ʼn gevolg van spontane AMG. Dit is dus nodig om die populasie van natuurlike melksuurbakterieë in wyn te beheer. Plantarisiene, geproduseer deur Lactobacillus plantarum wyn-isolate, het die potensiaal om naby verwante stamme se groei te inhibeer wat in dieselfde nis voorkom. Hierdie eienskap maak hul belowend vir die beheer van melksuurbakterieë se groei gedurende die wynmaakproses. Inhibering van die natuurlike mikroflora kan help om die vorming van ongewenste geure te verhoed, sowel as om AMG te beheer. Die gebruik van aanvangskulture, wat plantarisiene kan produseer, kan lei tot ’n vermindering in die gebruik van swaweldioksied in die wynindustrie. Die doel van hierdie studie was om die potensiaal van L. plantarum stamme, geïsoleer vanuit wyn, te ondersoek vir hul vermoë om plantaricins te produseer in toestande wat die wynmaakproses naboots. Die potensiaal was ondersoek deur te kyk na die uitdrukking van plantarisien-produserende gene onder wynmaak toestande. Die eerste objektief was om die 19 L. plantarum stamme, geïsoleer vanuit Suid-Afrikaanse rooi wyne, asook n kommersiele stam, te ondersoek vir die teenwoordigheid van verskeie gene wat verantwoordelik is vir die produksie van plantarisiene, sowel as strukturele, transporter en regulerende gene. Al twintig van hierdie stamme het ten minste 16 uit die 24 gene bevat waarvoor ondersoek was. OrfZ123 en orf345 het egter geen positiewe resultate opgelewer in enige van die stamme nie. Die tweede objektief was om die DNA-volgorde te bepaal van spesifieke gene (plnE, plnF, plnN, plnG, sowel as plnB) en sodoende die variasie in nukleotied en aminosuur volgorde van hierdie gene in die verskillende L. plantarum wyn-isolate te bepaal. Hoë vlakke van homologie was gevind en geen van die aminosuur veranderings het in behoue gebiede plaasgevind nie. Die nukleotied volgorde van plnN was identies in al die stamme, behalwe vir een, en die ooreenkomste tussen die plnB volgorde het varieër van 96% tot 100%. Die ooreenkomste tussen die plnG nukleotied volgorde het varieër van 99% to 100%. Die plnE nukleotied volgorde was identies in al die stamme, behalwe vir twee, en daar was net twee groepe in terme van nukleotied volgorde vir plnF, met net twee veranderinge tussen die groepe. Die derde objektief was om die vermoë van die stamme om plantaricins the produseer, deur gebruik te maak van plaat assays, onder verskillende wyntoestande te ondersoek. Die twinting stamme het verskillende vlakke van inhibering teenoor die nege toets-organismes getoon, wat Enterococcus faecalis, Listeria monocytogenes sowel as potensiele wyn bederf organismes, Lactobacillus spp, Pediococcus spp and Leuconostoc mesenteroides insluit. Die byvoeging van 10% etanol en ’n lae pH van 3.5, het beide bakteriosien produksie inhibeer, sowel as die spektrum van aktiwiteit verminder. Sewe van die stamme het egter steeds goeie aktiwiteit getoon onder al die kondisies wat getoets was. Die vierde objektief was om die uitdrukking van twee plantaricin strukturele gene (plnEF en plnJK), sowel as die transporter geen (plnG) onder wynmaak omstandighede te ondersoek. Twee stamme (R1122 en 113.1) was gekies as aanvangskulture vir AMG in sintesiese wyn media, sowel as Riesling wyn. Hierdie twee stamme was gekies op grond van die resultate wat van die vorige objektiewe verkry was. Lae wyn pH (3.2) en hoë wyn pH (3.8) was ondersoek in samewerking met verskillende etanol konsentrasies wat 0%, 12% en 15% etanol insluit. Al drie hierdie gene was uitgedruk teen verskillende vlakke, afhangende van die verskeie fermentasie kondisies. Hoë etanol en lae pH lei oor die algemeen tot ʼn toename in uitdrukking van die strukturele plantarisien gene. Die invloed op uitdrukking van die transporter geen was verskillend, want lae pH en die teenwoordigheid van etanol het gelei tot ʼn verhoging in geen uitdrukking. Die gene was uitegdruk in wyn, maar was teen laer vlakke relatief tot uitdrukking in die sintetiese wyn media. Dit blyk dat die teenwoordigheid van sensitiewe bakterieë in die wyn tot ‘n hoër uitdrukking van die strukturele gene lei. Die uitdrukking van die mle geen, verantwoordelik vir AMG, was ook onder dieselfde wynmaak kondisies ondersoek. Die uitdrukking was geïnduseer deur appelsuur, negatief beïnvloed deur die teenwoordigheid van etanol, maar positief beïnvloed deur ’n verlaging in pH van 3.8 tot 3.2. Hierdie studie toon dat plantaricin gene uitegedruk word onder wynmaak toestande en dat plantaricins moontlik onder hierdie toestande geproduseer kan word. Die potensiaal van hierdie stamme word getoon om as aanvangskulture gebruik te word vir AMG, om sodoende die groei van natuurlike melksuur bakterieë te inhibeer. Die teenwoordigheid van die plantarisien peptied in die wyn moet egter nog bewys word. Daar sal ook vasgestel moet word of die peptied biologies aktief is en nie deur wynverwante faktore geïnhibeer word nie.