Effect of Simultaneous Inoculation with Yeast and Bacteria on Fermentation Kinetics and Key Wine Parameters of Cool-Climate Chardonnay

Inoculating grape musts with wine yeast and lactic acid bacteria (LAB) concurrently in order to induce simultaneous alcoholic fermentation (AF) and malolactic fermentation (MLF) can be an efficient alternative to overcome potential inhibition of LAB in wines because of high ethanol concentrations and reduced nutrient content. In this study, the simultaneous inoculation of yeast and LAB into must was compared with a traditional vinification protocol, where MLF was induced after completion of AF. For this, two suitable commercial yeast-bacterium combinations were tested in cool-climate Chardonnay must. The time courses of glucose and fructose, acetaldehyde, several organic acids, and nitrogenous compounds were measured along with the final values of other key wine parameters. Sensory evaluation was done after 12 months of storage. The current study could not confirm a negative impact of simultaneous AF/MLF on fermentation success and kinetics or on final wine parameters. While acetic acid concentrations were slightly increased in wines after simultaneous AF/MLF, the differences were of neither practical nor legal significance. No statistically significant differences were found with regard to the final values of pH or total acidity and the concentrations of ethanol, acetaldehyde, glycerol, citric and lactic acids, and the nitrogen compounds arginine, ammonia, urea, citrulline, and ornithine. Sensory evaluation by a semiexpert panel confirmed the similarity of the wines. However, simultaneous inoculation led to considerable reductions in overall fermentation durations. Furthermore, differences of physiological and microbiological relevance were found. Specifically, we report the vinification of “super-dry” wines devoid of glucose and fructose after simultaneous inoculation of yeast and bacteria.     

Effect of simultaneous inoculation with yeast and bacteria on fermentation kinetics and key wine parameters of cool-climate chardonnay

Inoculating grape musts with wine yeast and lactic acid bacteria (LAB) concurrently in order to induce simultaneous alcoholic fermentation (AF) and malolactic fermentation (MLF) can be an efficient alternative to overcome potential inhibition of LAB in wines because of high ethanol concentrations and reduced nutrient content. In this study, the simultaneous inoculation of yeast and LAB into must was compared with a traditional vinification protocol, where MLF was induced after completion of AF. For this, two suitable commercial yeast-bacterium combinations were tested in cool-climate Chardonnay must. The time courses of glucose and fructose, acetaldehyde, several organic acids, and nitrogenous compounds were measured along with the final values of other key wine parameters. Sensory evaluation was done after 12 months of storage. The current study could not confirm a negative impact of simultaneous AF/MLF on fermentation success and kinetics or on final wine parameters. While acetic acid concentrations were slightly increased in wines after simultaneous AF/MLF, the differences were of neither practical nor legal significance. No statistically significant differences were found with regard to the final values of pH or total acidity and the concentrations of ethanol, acetaldehyde, glycerol, citric and lactic acids, and the nitrogen compounds arginine, ammonia, urea, citrulline, and ornithine. Sensory evaluation by a semiexpert panel confirmed the similarity of the wines. However, simultaneous inoculation led to considerable reductions in overall fermentation durations. Furthermore, differences of physiological and microbiological relevance were found. Specifically, we report the vinification of "super-dry" wines devoid of glucose and fructose after simultaneous inoculation of yeast and bacteria.     

Methionine catabolism and production of volatile sulphur compounds by OEnococcus oeni

AIMS: During malolactic fermentation (MLF), the secondary metabolisms of lactic acid bacteria (LAB) contribute to the organoleptic modification of wine. To understand the contribution of MLF, we evaluated the capacity of various wine LAB to metabolize methionine. METHODS AND RESULTS: Using gas chromatography (GC) coupled either with mass spectrometry (MS) or a flame photometry detector in sulphur mode (FPD), we studied this metabolism in laboratory media and wine. In laboratory media, several LAB isolated from wine were able to metabolize methionine. They formed methanethiol, dimethyl disulphide, 3-(methylsulphanyl)propan-1-ol and 3-(methylsulphanyl)propionic acid. These are known to have powerful characteristic odours and play a role in the aromatic complexity of wine. In various red wines, after MLF only the 3-(methylsulphanyl)propionic acid concentration increased significantly, as verified with several commercial starter cultures. This compound, which is characterized by chocolate and roasted odours, could contribute to the aromatic complexity produced by MLF. CONCLUSIONS: This study shows that LAB isolated from wine, especially OEnococcus oeni strains, the major species in MLF, are able to metabolize methionine to form volatile sulphur compounds. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the capacity of wine LAB to metabolize methionine.     

Development of a New Method for Detection and Identification of Oenococcus oeni Bacteriophages Based on Endolysin Gene Sequence and Randomly Amplified Polymorphic DNA

Malolactic fermentation (MLF) is a biochemical transformation conducted by lactic acid bacteria (LAB) that occurs in wine at the end of alcoholic fermentation. Oenococcus oeni is the main species responsible for MLF in most wines. As in other fermented foods, where bacteriophages represent a potential risk for the fermentative process, O. oeni bacteriophages have been reported to be a possible cause of unsuccessful MLF in wine. Thus, preparation of commercial starters that take into account the different sensitivities of O. oeni strains to different phages would be advisable. However, currently, no methods have been described to identify phages infecting O. oeni. In this study, two factors are addressed: detection and typing of bacteriophages. First, a simple PCR method was devised targeting a conserved region of the endolysin (lys) gene to detect temperate O. oeni bacteriophages. For this purpose, 37 O. oeni strains isolated from Italian wines during different phases of the vinification process were analyzed by PCR for the presence of the lys gene, and 25 strains gave a band of the expected size (1,160 bp). This is the first method to be developed that allows identification of lysogenic O. oeni strains without the need for time-consuming phage bacterial-lysis induction methods. Moreover, a phylogenetic analysis was conducted to type bacteriophages. After the treatment of bacteria with UV light, lysis was obtained for 15 strains, and the 15 phage DNAs isolated were subjected to two randomly amplified polymorphic DNA (RAPD)-PCRs. By combining the RAPD profiles and lys sequences, 12 different O. oeni phages were clearly distinguished.     

Impact of different malolactic fermentation inoculation scenarios on Riesling wine aroma

During malolactic fermentation (MLF), lactic acid bacteria influence wine aroma and flavour by the production of volatile metabolites and the modification of aroma compounds derived from grapes and yeasts. The present study investigated the impact of different MLF inoculation strategies with two different Oenococcus oeni strains on cool climate Riesling wines and the volatile wine aroma profile. Four different timings were chosen for inoculation with bacteria to conduct MLF in a Riesling must/wine with a high acidity (pH 2.9–3.1). Treatments with simultaneous inoculation showed a reduced total fermentation time (alcoholic and malolactic) compared to the sequential inoculations. No negative impact of simultaneous alcoholic and malolactic fermentation on fermentation success and on the final wine volatile aroma composition was observed. Compared to sequential inoculation, wines with co-inoculation tended to have higher concentrations of ethyl and acetate esters, including acetic acid phenylethylester, acetic acid 3-methylbutylester, butyric acid ethylester, lactic acid ethylester and succinic acid diethylester. Results of this study provide some alternatives to diversify the number of wine styles by safely conducting MLF in low-pH, cool-climate white musts with potential high alcohol content.     

The occurrence of malolactic fermentation in brandy base wine and its influence on brandy quality

AIMS: In this study we determined the extent to which lactic acid bacteria (LAB) occurred in brandy base wines, their ability to catalyse the malolactic fermentation (MLF) and the effect of MLF on the quality of the base wine and the brandy distillate. METHODS AND RESULTS: Lactic acid bacteria were isolated and enumerated from grape juice, experimental and commercially produced brandy base wines. Spontaneous MLF occurred in approximately 50% of the commercial base wines. The occurrence of MLF had an influence on the quality of the base wines and the resulting distillates. In samples where MLF occurred there was a loss of fruitiness and in the intensity of aroma. Volatile compounds like iso-amyl acetate, ethyl acetate, ethyl caproate, 2-phenethyl acetate and hexyl acetate decreased in samples having undergone MLF, while ethyl lactate, acetic acid and diethyl succinate increased in the same samples. CONCLUSIONS: Spontaneous malolactic fermentation does occur in commercial brandy base wines and it has an influence on base wine and brandy quality. SIGNIFICANCE AND IMPACT OF THE STUDY: This study showed that MLF influences the quality of the base wine and the resulting distillate and with this in mind commercial base wine producers should be able to produce brandy of higher quality.     

Growth and Arginine Metabolism of the Wine Lactic Acid Bacteria Lactobacillus buchneri and Oenococcus oeni at Different pH Values and Arginine Concentrations

During malolactic fermentation (MLF) in grape must and wine, heterofermentative lactic acid bacteria may degrade arginine, leading to the formation of ammonia and citrulline, among other substances. This is of concern because ammonia increases the pH and thus the risk of growth by spoilage bacteria, and citrulline is a precursor to the formation of carcinogenic ethyl carbamate (EC). Arginine metabolism and growth of Lactobacillus buchneri CUC-3 and Oenococcus oeni strains MCW and Lo111 in wine were investigated. In contrast to L. buchneri CUC-3, both oenococci required a higher minimum pH for arginine degradation, and arginine utilization was delayed relative to the degradation of malic acid, the main aim of MLF. This allows the control of pH increase and citrulline formation from arginine metabolism by carrying out MLF with pure oenococcal cultures and inhibiting cell metabolism after malic acid depletion. MLF by arginine-degrading lactobacilli should be discouraged because arginine degradation may lead to the enhanced formation of acids from sugar degradation. A linear relationship was found between arginine degradation and citrulline excretion rates. From this data, strain-specific arginine-to-citrulline conversion ratios were calculated that ranged between 2.2 and 3.9% (wt/wt), and these ratios can be used to estimate the contribution of citrulline to the EC precursor pool from a given amount of initial arginine. Increasing arginine concentrations led to higher rates of growth of L. buchneri CUC-3 but did not increase the growth yield of either oenococcus. These results suggest the use of non-arginine-degrading oenococci for inducing MLF.     

Genomic variations of <Emphasis Type="Italic">Oenococcus oeni</Emphasis> strains and the potential to impact on malolactic fermentation and aroma compounds in wine

Malolactic fermentation (MLF) is the bacterially driven decarboxylation of l-malic acid to l-lactic acid and carbon dioxide, and brings about deacidification, flavour modification and microbial stability of wine. The main objective of MLF is to decrease wine sourness by a small increase in wine pH via the metabolism of l-malic acid. Oenococcus oeni is the main lactic acid bacterium to conduct MLF in virtually all red wine and an increasing number of white and sparkling wine bases. Over the last decade, it is becoming increasingly recognized that O. oeni exhibits a diverse array of secondary metabolic activities during MLF which can modify the sensory properties of wine. These secondary activities include the metabolism of organic acids, carbohydrates, polysaccharides and amino acids, and numerous enzymes such as glycosidases, esterases and proteases, which generate volatile compounds well above their odour detection threshold. Phenotypic variation between O. oeni strains is central for producing different wine styles. Recent studies using array-based comparative genome hybridization and genome sequencing of three O. oeni strains have revealed the large genomic diversity within this species. This review will explore the links between O. oeni metabolism, genomic diversity and wine sensory attributes.     

A mathematical model of the link between growth and L-malic acid consumption for five strains of Oenococcus oeni

In winemaking, after the alcoholic fermentation of red wines and some white wines, L-malic acid must be converted into L-lactic acid to reduce the acidity. This malolactic fermentation (MLF) is usually carried out by the lactic acid bacteria Oenococcus oeni. Depending on the level of process control, selected O. oeni is inoculated or the natural microbiota of the cellar is used. This study considers the link between growth and MLF for five strains of O. oeni species. The kinetics of growth and L-malic acid consumption were followed in modified MRS medium (20 °C, pH 3.5, and 10 % ethanol) in anaerobic conditions. A large variability was found among the strains for both their growth and their consumption of L-malic acid. There was no direct link between biomass productivities and consumption of L-malic acid among strains but there was a link of proportionality between the specific growth of a strain and its specific consumption of L-malic acid. Experiments with and without malic acid clearly demonstrated that malic acid consumption improved the growth of strains. This link was quantified by a mathematical model comparing the intrinsic malic acid consumption capacity of the strains.     

Characterization and technological properties of Oenococcus oeni strains from wine spontaneous malolactic fermentations: a framework for selection of new starter cultures

Aims:  To characterize the genetic and phenotypic diversity of 135 lactic acid bacteria (LAB) strains isolated from Italian wines that undergone spontaneous malolactic fermentation (MLF) and propose a multiphasic selection of new Oenococcus oeni malolactic starters.
Methods and Results:  One hundred and thirty-five LAB strains were isolated from 12 different wines. On the basis of 16S amplified ribosomal DNA restriction analysis (ARDRA) with three restriction enzymes and 16S rRNA gene sequencing, 120 O. oeni strains were identified. M13-based RAPD analysis was employed to investigate the molecular diversity of O. oeni population. Technological properties of different O. oeni genotypes were evaluated in synthetic medium at increasing selective pressure, such as low pH (3·5, 3·2 and 3·0) and high ethanol values (10, 11 and 13% v/v). Finally, the malolactic activity of one selected strain was assessed in wine by malolactic trial in winery.
Conclusions:  The research explores the genomic diversity of wine bacteria in Italian wines and characterizes their malolactic metabolism, providing an efficient strategy to select O. oeni strains with desirable malolactic performances and able to survive in conditions simulating the harsh wine environment.
Significance and Impact of the Study:  This article contributes to a better understanding of microbial diversity of O. oeni population in Italian wines and reports a framework to select new potentially O. oeni starters from Italian wines during MLF.     

Multi-Loci Sequence Typing (MLST) for Two Lacto-Acid Bacteria (LAB) Species: <Emphasis Type="Italic">Pediococcus parvulus</Emphasis> and <Emphasis Type="Italic">P. damnosus</Emphasis>

The control of wine microbial population during and beyond fermentation is of huge importance for wine quality. Lactic acid bacteria (LAB) in wine are responsible for malolactic fermentation (MLF) which can be desired in some cases and undesirable in others. Some LAB do not perform MLF and their uncontrolled growth could contribute to severe wine spoilage such as undesired flavours. Their identification and detection is considered crucial for numerous biotechnological applications in food fermentations, where, through acidification and secretion of bacteriocins, they contribute to reduce food spoilage and growth of pathogenic microorganisms. LAB have traditionally been classified using morphological or biochemical features. Primary isolation, biochemical identification and phenotypic analysis are laborious, time consuming and inaccurate and often lead to misidentification within some genera such as Pediococcus. Molecular identification based on suitable marker genes could be an attractive alternative to conventional morphological and biochemical methods. We assessed here the applicability of four housekeeping genes recA, rplB, pyrG and leuS in combination with the mle gene in multi-loci sequence typing (MLST) of Pediococcus parvulus and Pediococcus damnosus. Sequencing and comparative analysis of sequence data were performed on 19 strains collected during wine fermentation. A combination of these five marker genes allowed for a clear differentiation of the strains analysed, indicating their applicability in molecular typing. Analysis of the observed nucleotide polymorphisms allowed designing highly discriminative primers for a multi-loci sequence typing (MLST) method that proved successful in detecting a particular isolate or sequence type of P. parvulus when using either conventional PCR or Real Time PCR.     

Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra

Aims: This study was designed to isolate and characterize the lactic acid microbiota of the musts and wines of a young denomination of origin area, Ribeira Sacra in north‐west Spain. Methods and Results: Over three consecutive years (2007, 2008 and 2009), we examined musts and wines from four cellars in different zones of the region. Through biochemical and genetic tests, 459 isolates of lactic acid bacteria (LAB) were identified as the following species: Lactobacillus alvei (0·7%), Lactobacillus brevis (1·7%), Lactobacillus frumenti (0·9%), Lactobacillus kunkeei (12%), Lactobacillus plantarum (6·5%), Lactobacillus pentosus (0·9%), Lactococcus lactis ssp. lactis (3%), Leuconostoc citreum (0·7%), Leuconostoc fructosum (synon. Lactobacillus fructosum) (3·7%), Leuconostoc mesenteroides ssp. mesenteroides (2·8%), Leuconostoc pseudomesenteroides (0·2%), Oenococcus oeni (59%), Pediococcus parvulus (7%) and Weisella paramesenteroides (synon. Leuconostoc paramesenteroides) (0·9%). Of these species, O. oeni was the main one responsible for malolactic fermentation (MLF) in all cellars and years with the exception of Lact. plantarum, predominant in 2007, in one cellar, and Lact. brevis, Lact. frumenti and Ped. parvulus coexisting with O. oeni in one cellar in 2009. Different strains (84) of LAB species (14) were identified by biochemical techniques (API strips, the presence of plasmids, enzyme activities and MLF performance) and molecular techniques (PCR). All assays were carried out with every one of the 459 isolates. To select candidates for use as culture starters, we assessed malolactic, β‐glucosidase and tannase activities, the presence of genes involved in biogenic amine production and plasmid content. Conclusions: A high diversity of LAB is present in the grape musts of Ribeira Sacra but few species are responsible for MLF; however, different strains of such species are involved in the process. As far as we are aware, this is the first report of Lact. frumenti thriving in wine. Significance and Impact of the Study: Information on LAB populations in must and wine is presented. A large collection of well‐characterized strains of LAB are available as starter cultures to winemakers.     

Preliminary characterization of wine lactobacilli able to degrade arginine

Lactobacillus strains able to degrade arginine were isolated and characterized from a typical red wine. All the strains were gram-positive, catalase-negative and produced both D- and L-lactate from glucose. Strains L2, L3, L4, and L6 were able to produce CO₂ from glucose; however, production of CO₂ from glucose was not observed in strains L1 and L5, suggesting that they belong to the homofermentative wine lactic acid bacteria (LAB) group. All of the lactobacilli were tested for their ability to ferment 49 carbohydrates. The sugar fermentation profile of strain L1 was unique, suggesting that this strain belonged to Lactococcus lactis ssp. cremoris, a non-typical wine LAB. Furthermore, a preliminary typing was performed by using a random amplified polymorphic DNA analysis (RAPD-PCR analysis).     

Starter cultures as biocontrol strategy to prevent Brettanomyces bruxellensis proliferation in wine

Brettanomyces bruxellensis is a common and significant wine spoilage microorganism. B. bruxellensis strains generally detain the molecular basis to produce compounds that are detrimental for the organoleptic quality of the wine, including some classes of volatile phenols that derive from the sequential bioconversion of specific hydroxycinnamic acids such as ferulate and p-coumarate. Although B. bruxellensis can be detected at any stage of the winemaking process, it is typically isolated at the end of the alcoholic fermentation (AF), before the staring of the spontaneous malolactic fermentation (MLF) or during barrel aging. For this reason, the endemic diffusion of B. bruxellensis leads to consistent economic losses in the wine industry. Considering the interest in reducing sulfur dioxide use during winemaking, in recent years, biological alternatives, such as the use of tailored selected yeast and bacterial strains inoculated to promote AF and MLF, are actively sought as biocontrol agents to avoid the “Bretta” character in wines. Here, we review the importance of dedicated characterization and selection of starter cultures for AF and MLF in wine, in order to reduce or prevent both growth of B. bruxellensis and its production of volatile phenols in the matrix.

     

ADI pathway and histidine decarboxylation are reciprocally regulated in Lactobacillus hilgardii ISE 5211: proteomic evidence

Amine production by amino acid decarboxylation is a common feature that is used by lactic acid bacteria (LAB) to complement lactic fermentation, since it is coupled with a proton-extruding antiport system which leads to both metabolic energy production and the attenuation of intracellular acidity. Analogous roles are played in LAB by both malolactic fermentation (MLF) and the arginine deiminase (ADI) pathway. The present investigation was aimed at establishing reciprocal interactions between amino acid decarboxylation and the two above mentioned routes. The analyses were carried out on a Lactobacillus hilgardii strain (ISE 5211) that is able to decarboxylate histidine to histamine, which had previously been isolated from wine and whose complete genome is still unknown. The 2DE proteomic approach, followed by MALDI TOF–TOF and De Novo Sequencing, was used to study the protein expression levels. The experimental evidence has indicated that malate does not influence histidine decarboxylase (HDC) biosynthesis and that histidine does not affect the malolactic enzyme level. However, the expression of the ADI route enzymes, arginine deiminase and ornithine transcarbamylase, is down-regulated by histidine: this biosynthetic repression is more important (4-fold) in cultures that are not supplemented with arginine, but is also significant (2-fold) in an arginine supplemented medium that normally induces the ADI pathway. On the other hand, arginine partially represses HDC expression, but only when histidine and arginine are both present in the culture medium. This proteomic study has also pointed out a down-regulation exerted by histidine over sugar metabolism enzymes and a GroEL stress protein. These data, together with the reciprocal antagonism between arginine deimination and histidine decarboxylation, offer clue keys to the understanding of the accumulation of lactate, amine, ammonia and ethylcarbamate in wine, with consequent implications on different health risk controls.     

Predominance of malolactic fermentation overd-glucose utilization inLactobacillus plantarum andLactobacillus curvatus wine strains

Summary Two selected wine strains of the genusLactobacillus (L. plantarum 197 andL. curvatus 783) were tested for their ability to complete malolactic fermentation (MLF) in a synthetic medium (PBM-broth) supplemented withL-malic acid (7.5–74.6 mM) andD-glucose (5.5–55 mM). The 24 directed fermentation assays, 12 for each bacterial strain, were carried out at 20°C and pH 3.5. MLF was completed (residualL-malic acid 0.2 mM) in eight days in 19 of the 24 fermentation assays, even in the presence of 74.6 mML-malic acid or 55.5 mMD-Glucose utilization was generally simultaneous to MLF but was completed (residual concentrations 0.2 mM) only in 6 of the 24 fermentation assays. These results support the use of these strains in directed MLF assays at the very differentL-malic acid andD-glucose concentrations tested.     

Development of a sequence-characterized amplified region marker-targeted quantitative PCR assay for strain-specific detection of Oenococcus oeni during wine malolactic fermentation

Control over malolactic fermentation (MLF) is a difficult goal in winemaking and needs rapid methods to monitor Oenococcus oeni malolactic starters (MLS) in a stressful environment such as wine. In this study, we describe a novel quantitative PCR (QPCR) assay enabling the detection of an O. oeni strain during MLF without culturing. O. oeni strain LB221 was used as a model to develop a strain-specific sequence-characterized amplified region (SCAR) marker derived from a discriminatory OPA20-based randomly amplified polymorphic DNA (RAPD) band. The 5' and 3' flanking regions and the copy number of the SCAR marker were characterized using inverse PCR and Southern blotting, respectively. Primer pairs targeting the SCAR sequence enabled strain-specific detection without cross amplification of other O. oeni strains or wine species of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and yeasts. The SCAR-QPCR assay was linear over a range of cell concentrations (7 log units) and detected as few as 2.2 × 10(2) CFU per ml of red wine with good quantification effectiveness, as shown by the correlation of QPCR and plate counting results. Therefore, the cultivation-independent monitoring of a single O. oeni strain in wine based on a SCAR marker represents a rapid and effective strain-specific approach. This strategy can be adopted to develop easy and rapid detection techniques for monitoring the implantation of inoculated O. oeni MLS on the indigenous LAB population, reducing the risk of unsuccessful MLF.     

Saccharomyces cerevisiae and Oenococcus oeni immobilized in different layers of a cellulose/starch gel composite for simultaneous alcoholic and malolactic wine fermentations

The production of a two-layer composite biocatalyst for immobilization of two different microorganisms for simultaneous alcoholic and malolactic fermentation (MLF) of wine in the same bioreactor is reported. The biocatalyst consisted of a tubular delignified cellulosic material (DCM) with entrapped Oenococcus oeni cells, covered with starch gel containing the alcohol resistant and cryotolerant strain Saccharomyces cerevisiae AXAZ-1. The biocatalyst was found effective for simultaneous low temperature alcoholic fermentation resulting to conversion of malic acid to lactic acid in 5 days at 10 °C. Improvement of wine quality compared with wine fermented with S. cerevisiae AXAZ-1 immobilized on DCM was attributed to MLF as well as to increased ester formation and lower higher alcohols produced at low fermentation temperatures (10 °C) as shown by GC and headspace SPME GC/MS analysis. Scanning electron microscopy showed that the preparation of a three-layer composite biocatalyst is also possible. The significance of such composite biocatalysts is the feasibility of two or three bioprocesses in the same bioreactor, thus reducing production cost in the food industry     

Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine

The main objectives of this study were the search for enzymatic activities responsible for biogenic amine (BA) degradation in lactic acid bacteria (LAB) strains isolated from wine, their identification, and the evaluation of their applicability for reducing BAs in wine. Fifty-three percent of the 76 LAB cell extracts showed activity against a mixture of histamine, tyramine, and putrescine when analyzed in-gel. The quantification of the degrading ability for each individual amine was tested in a synthetic medium and wine. Most of the bacteria analyzed were able to degrade the three amines in both conditions. The highest percentages of degradation in wine were those of putrescine: up to 41 % diminution in 1 week. Enzymes responsible for amine degradation were isolated and purified from Lactobacillus plantarum J16 and Pediococcus acidilactici CECT 5930 strains and were identified as multicopper oxidases. This is the first report of an efficient BA reduction in wine by LAB. Furthermore, the identity of the enzymes involved has been revealed.     

Comparative metabolic profiling to investigate the contribution of <Emphasis Type="Italic">O. oeni</Emphasis> MLF starter cultures to red wine composition

In this research work we investigated changes in volatile aroma composition associated with four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures in South African Shiraz and Pinotage red wines. A control wine in which MLF was suppressed was included. The MLF progress was monitored by use of infrared spectroscopy. Gas chromatographic analysis and capillary electrophoresis were used to evaluate the volatile aroma composition and organic acid profiles, respectively. Significant strain-specific variations were observed in the degradation of citric acid and production of lactic acid during MLF. Subsequently, compounds directly and indirectly resulting from citric acid metabolism, namely diacetyl, acetic acid, acetoin, and ethyl lactate, were also affected depending on the bacterial strain used for MLF. Bacterial metabolic activity increased concentrations of the higher alcohols, fatty acids, and total esters, with a larger increase in ethyl esters than in acetate esters. Ethyl lactate, diethyl succinate, ethyl octanoate, ethyl 2-methylpropanoate, and ethyl propionate concentrations were increased by MLF. In contrast, levels of hexyl acetate, isoamyl acetate, 2-phenylethyl acetate, and ethyl acetate were reduced or remained unchanged, depending on the strain and cultivar evaluated. Formation of ethyl butyrate, ethyl propionate, ethyl 2-methylbutryate, and ethyl isovalerate was related to specific bacterial strains used, indicating possible differences in esterase activity. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, although further investigation is needed in this regard. This study provided insight into metabolism in O. oeni starter cultures during MLF in red wine.