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.     

酒酒球菌(Oenococcus oeni)胁迫适应性反应机制

苹果酸-乳酸发酵有利于提高葡萄酒品质,为了获得高活性的直投式酒酒球菌发酵制剂,从生理和分子生物学的角度理解该菌种胁迫耐受性增强的机制是必要的.本文就酒酒球菌利用苹果酸-乳酸发酵和膜结合的H -F0F1-ATP酶以维持细胞内环境的稳定和能量供给;胁迫适应过程中细胞膜组分的调整;小热休克蛋白Lo18等胁迫蛋白及其相应的基因的表达和调控等方面进行了综述.胁迫适应性反应机制的研究对发酵剂菌株的筛选、发酵剂的制备及其他工程菌株的构建具有重要意义.     

Cloning and expression of the malolactic gene of Pediococcus damnosus NCFB1832 in Saccharomyces cerevisiae

Wine production is characterized by a primary alcoholic fermentation, conducted by Saccharomyces cerevisiae, followed by a secondary malolactic fermentation (MLF). Although most lactic acid bacteria (LAB) have the ability to metabolize L-malate, only a few species survive the high ethanol and SO2 levels in wine. Wines produced in colder viticultural regions have a lower pH than wines produced in warmer regions. The decarboxylation of L-malate in these wines leads to an increase in pH, more organoleptic complexity and microbiological stability. MLF is, however, difficult to control and problems often occur during filtering of such wines. Pediococcus spp. are known to occur in high pH wines and have strong malolactic activity. However, some pediococci synthesize exocellular polysaccharides, which may lead to abnormal viscosity in wine. In this study, the malolactic gene from Pediococcus damnosus NCFB1832 (mleD) was cloned into S. cerevisiae and co-expressed with the malate permease gene (mae1) of Schizosaccharomyces pombe. Expression of the mleD gene was compared to the expression of two other malolactic genes, mleS from Lactococcus lactis MG1363 and mleA from Oenococcus oeni Lal1. The genetically modified strain of S. cerevisiae decreased the level of L-malate in grape must to less than 0.3 gl(-1) within 3 days. This is the first expression of a malolactic gene from Pediococcus in S. cerevisiae.     

Inhibition of bacterial growth and malolactic fermentation in wine by bacteriophage

Bacteriophage present in wine can attack bacterial starter cultures and inhibit the malolactic fermentation. The possibility of starter culture failure due to phage attack was studied in a commercial dry red wine of pH 3·23, inoculated with a multiple strain starter culture. During two stages of malolactic fermentation, bacterial growth and malate degradation in the wine were inhibited. A phage capable of lysing isolates of Leuconostoc oenos was isolated from the wine. The isolated phage had an icosahedral head of 42–45 nm diameter and a flexible, regularly cross-striated tail 197–207 nm long with a small baseplate. The results confirm that phage can attack bacterial starter cultures in wine at low pH.     

Inducing malolactic fermentation in wines

Malolactic fermentation (MLF) in wine can be accomplished by relying on the natural microflora or by inducing through inoculation of a specific strain(s) of malolactic bacteria, primarily strains of Leuconostoc oenos. Problems with inducing MLF include intrinsic factors of the grape must such as pH, presence of sulfur dioxide, and ethanol in addition to antagonism of malolactic bacteria by wine yeast. Current methods and new technology to improve the predictability of MLF are discussed.     

Bovine Bile Resistance Increases Leuconostoc oenos 44.40 Viability upon Lyophilization

A screening method based on the selection of strains of Leuconostoc oenos 44.40 resistant to bovine bile was developed to obtain strains of the organism more resistant to lyophilization damage. These strains could be used as starter cultures in the malolactic fermentation of wine. The strain resistant to bovine bile was 20% more viable after lyophilization than strains not resistant to bovine bile. This was confirmed in both laboratory-scale production (100 ml) and pilot-scale production (100 liters). Lyophilized cells of strains sensitive and resistant to bovine bile were inoculated into wine, and the malate metabolism by the organism was monitored in the wine. Resistance to bovine bile did not change the malate metabolism characteristic of the organism. A comparison was made of the fatty acid compositions of the two strains. There was a difference in the fatty acid distribution pattern for these two strains. The bovine bile-resistant strain contained more dodecanoic, hexadecanoic, and octadecanoic acid and less tetradecanoic and hexadecanoic acid than did the bovine bile-sensitive strain. Both strains contained high levels of C-19 cyclopropane fatty acid.     

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.     

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.     

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.     

Degradation of free and sulfur-dioxide-bound acetaldehyde by malolactic lactic acid bacteria in white wine

AIMS: Acetaldehyde is the major carbonyl compound formed during winemaking and has implications for sensory and colour qualities of wines as well as for the use of the wine preservative SO(2). The current work investigated the degradation of acetaldehyde and SO(2)-bound acetaldehyde by two commercial Oenococcus oeni starters in white wine. METHODS AND RESULTS: Wines were produced by alcoholic fermentation with commercial yeast and adjusted to pH 3.3 and 3.6. While acetaldehyde was degraded rapidly and concurrently with malic acid at both pH values, SO(2)-bound acetaldehyde caused sluggish bacterial growth. Strain differences were small. CONCLUSIONS: Efficient degradation of acetaldehyde can be achieved by commercial starters of O. oeni. According to the results, the degradation of acetaldehyde could not be separated from malolactic conversion by oenococci. While this may be desirable in white winemaking, it may be necessary to delay malolactic fermentation (MLF) in order to allow for colour development in red wines. SO(2)-bound acetaldehyde itself maybe responsible for the sluggish or stuck MLF, and thus bound SO(2) should be considered next to free SO(2) in order to evaluate malolactic fermentability. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study provides new results regarding the metabolism of acetaldehyde and SO(2)-bound acetaldehyde during the MLF in white wine. The information is of significance to the wine industry and may contribute to reducing the concentration of wine preservative SO(2).     

A multiplex PCR method for simultaneous species identification and strain typification of Oenococcus oeni

Selected starter cultures of Oenococcus oeni are widely used to initiate malolactic fermentation (MLF) in wine. Nevertheless, the inoculated culture does not always develop as expected and undesired strains can grow causing wine spoilage. Therefore, methods that can reliably differentiate O. oeni strains are essential to monitor the population dynamics of MLF. This work presents a new multiplex PCR method that allows the simultaneous species identification and strain typification of O. oeni, based on the combined use of species-specific PCR primers and a Random Polymorphic DNA (RAPD)-PCR primer. This method represents an useful tool for the control of wine MLF.     

Impact of winemaking practices on arginine and citrulline metabolism during and after malolactic fermentation

AIMS: To study arginine degradation and carcinogenic ethyl carbamate precursor citrulline formation during and after malolactic fermentation (MLF). METHODS AND RESULTS: MLF was induced in white wine with two commercial Oenococcus oeni strains under different winemaking conditions regarding the type of alcoholic fermentation (spontaneous, induced) and the lees management (racked, on lees). Arginine degradation and citrulline formation did not occur during malic acid degradation in any treatment. In five of the six treatments in which arginine degradation took place, it occurred 3 weeks after malic acid depletion and significant amounts of citrulline were formed. Presence of yeast lees in wines led to increased citrulline formation. Conclusions: This study suggests that arginine metabolism is inhibited in oenococci at low pH values (< 3.5) and that in the postalcoholic fermentation phase, citrulline formation from arginine degradation can be avoided if MLF is induced by pure cultures of O. oeni with inhibition of the bacterial biomass after malic acid depletion. Residual yeast lees in the wine have been identified as a significant risk factor for increased citrulline formation. SIGNIFICANCE AND IMPACT OF THE STUDY: Conclusions drawn from this study allow reducing the risk of carcinogenic ethyl carbamate formation from citrulline excretion by wine lactic acid bacteria.     

Technological properties of Oenococcus oeni strains isolated from typical southern Italian wines

Aims: To isolate indigenous Oenococcus oeni strains suitable as starters for malolactic fermentation (MLF), using a reliable polyphasic approach. Methods and Results: Oenococcus oeni strains were isolated from Nero di Troia wines undergoing spontaneous MLF. Samples were taken at the end of alcoholic fermentation and during MLF. Wine samples were diluted in a sterile physiological solution and plated on MRS and on modified FT80. Identification of O. oeni strains was performed by a polymerase chain reaction (PCR) experiment using strain‐specific primers. Strains were further grouped using a multiplex RAPD‐PCR analysis. Then, six strains were inoculated in two wine‐like media with two different ethanol concentrations (11 and 13% vol/vol) with a view to evaluate their capacity to grow and to perform MLF. In addition, a quantitative PCR (qRT‐PCR) approach was adapted to monitor the physiological state of the strains selected. Conclusion: A positive correlation between the malolactic activity performance and the ability to develop and tolerate stress conditions was observed for two selected O. oeni strains. Significance and Impact of the Study: The results reported are useful for the selection of indigenous MLF starter cultures with desired oenological traits from typical regional wines. It should be the base for the improvement in organoleptic quality of typical red wine.     

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.     

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.     

Implications of new research and technologies for malolactic fermentation in wine

The initial conversion of grape must to wine is an alcoholic fermentation (AF) largely carried out by one or more strains of yeast, typically Saccharomyces cerevisiae. After the AF, a secondary or malolactic fermentation (MLF) which is carried out by lactic acid bacteria (LAB) is often undertaken. The MLF involves the bioconversion of malic acid to lactic acid and carbon dioxide. The ability to metabolise l-malic acid is strain specific, and both individual Oenococcus oeni strains and other LAB strains vary in their ability to efficiently carry out MLF. Aside from impacts on acidity, LAB can also metabolise other precursors present in wine during fermentation and, therefore, alter the chemical composition of the wine resulting in an increased complexity of wine aroma and flavour. Recent research has focused on three main areas: enzymatic changes during MLF, safety of the final product and mechanisms of stress resistance. This review summarises the latest research and technological advances in the rapidly evolving study of MLF and investigates the directions that future research may take.     

苹果酸-乳酸发酵能量产生机理

苹果酸乳酸发酵(MLF)是现代葡萄酒酿造工艺中重要的生物降酸手段。MLF是L苹果酸在乳酸菌的苹果酸乳酸酶催化下转变成L乳酸的酶促反应过程,该过程没有底物水平的磷酸化,但在MLF过程中苹果酸确实能够刺激细菌的比生长速率,增加细菌生物量。进一步的研究表明,代谢能的产生并非由于苹果酸的脱羧反应,而主要源于跨膜的L苹果酸摄入和(或)L乳酸的流出,从而产生跨膜质子电动势(pmf)(一小部分能量可能由L苹果酸的代谢中间产物丙酮酸产生)。按照化学渗透理论,pmf驱动F0F1ATPase合成ATP用于细菌生长。本文还对苹果酸的运输机制进行了综述。     

Performance and diacetyl production of commercial strains of malolactic bacteria in wine

This study compares 11 commercial cultures of Leuconostoc oenos and Lactobacillus plantarum in Cabernet Sauvignon, Pinot Noir and Chardonnay wines. Performance of the cultures was found to be greatly influenced by wine type. Better survival of the bacteria was observed in Cabernet Sauvignon and Pinot Noir wines. The time necessary to complete malolactic fermentation (MLF) was 65 ± 14 d for Chardonnay, 71 ± 3 d for Cabernet Sauvignon, and 25 ± 8 d for Pinot Noir. The maximal rate of malate utilization was 0·4 g d^-1 for Pinot Noir, and 0·2 g d^-1 for the two other wine types. Final diacetyl concentration was lower in Chardonnay wines (highest 0·58 mg l^-1) compared to the other wines (highest 5·8 mg l^-1). Malic and citric acid were co-metabolized by all strains. None of the strains metabolized glycerol. Significant differences in final diacetyl concentration of wine vinified with the different strains were found. Panelists could reliably differentiate MLF wines from non-MLF wines, irrespective of their diacetyl content, indicating that diacetyl is not the only important MLF flavour.     

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.