Potential formation of ethyl carbamate in simulated wine inoculated with Oenococcus oeni and Lactobacillus plantarum

The production of ethyl carbamate (EC) and its precursor citrulline by two strains of Oenococcus oeni and one of Lactobacillus plantarum during malolactic fermentation (MLF) was studied in different conditions of pH, temperature, and ethanol and l -malic acid concentrations. The potential EC, defined as the concentration of EC after wine is heated at 80 °C for 48 h, was also investigated. The O. oeni strains were able to degrade arginine in the conditions studied and to excrete some citrulline. In these strains, the conditions that led to a slight increase in EC were a high ethanol concentration, low pH and a high l -malic acid concentration. However, the potential EC increased with higher pH. In the case of L. plantarum , arginine was not degraded and citrulline was not produced, although the potential EC was comparable with that of the O. oeni strains studied.     

Production of biogenic amines by lactic acid bacteria: screening by PCR, thin-layer chromatography, and high-performance liquid chromatography of strains isolated from wine and must

Biogenic amines are frequently found in wine and other fermented food. We investigated the ability of 133 strains of lactic acid bacteria isolated from musts and wines of different origins to produce histamine, tyramine, and putrescine. We detected the genes responsible for encoding the corresponding amino acid decarboxylases through PCR assays using two primer sets for every gene: histidine decarboxylase (hdc), tyrosine decarboxylase (tdc), and ornithine decarboxylase (odc); these primers were taken from the literature or designed by us. Only one strain of Lactobacillus hilgardii was shown to possess the hdc gene, whereas four strains of Lactobacillus brevis had the tdc gene. None of the Oenococcus oeni strains, the main agents of malolactic fermentation, was a biogenic amine producer. All PCR amplicon band-positive results were confirmed by thin-layer chromatography and high-performance liquid chromatography analyses.     

Arginine metabolism by wine Lactobacilli isolated from wine

Lactobacillus hilgardii is a very common heterofermentative bacterium found in wine, associated mainly with several kinds of negative alterations. It is also known as a spoilage organism in soft drinks and other fermented beverages. It is able to break down arginine, one of the most abundant amino acids in wine, through the arginine deiminase (ADI) pathway. The first step of this metabolism may lead to the excretion of citrulline. This feature has an important enological implication since citrulline can react spontaneously with ethanol to form ethyl carbamate. Carcinogenic effects of this compound have been observed when administrated at high concentrations to laboratory animals. To complete the understanding of this catabolic pathway in L. hilgardii, arginine and citrulline utilization were investigated under different conditions. Moreover, ATP production from these amino acids was also monitored. From these data, arginine degradation via the ADI pathway can be considered to be a mechanism of energy production and pH regulation. However, if arginine degradation is beneficial for the bacteria, improving its growing ability and its adaptability, this increases the risk of degradation of the organoleptic and hygienic properties of wine.     

Arginine dihydrolase pathway in Lactobacillus plantarum from orange.

Lactobacillus plantarum N8 and N4 strains isolated from orange degraded L-arginine to citrulline, ornithine and ammonia. Citrulline and ornithine were consumed. Lactobacillus plantarum N4 utilized arginine and ornithine to a higher extent than Lactobacillus plantarum N8. Urea was not detected during arginine degradation, indicating that the amino acid degradation was carried out only by the arginine dihydrolase pathway. Citrulline increased the growth of the two strains, arginine only increased the growth of Lactobacillus plantarum N4. Ornithine did not modify the growth of the strains studied. With different behavior, Lactobacillus plantarum N8 and N4 strains were able to derive energy and ammonia from arginine or citrulline catabolism. This is interesting for microorganisms developing in a stressful environment.     

不同酒类酒球菌苹果酸—乳酸发酵对葡萄酒中氨基酸的影响

不同酒类酒球菌茵株完成MLF后,葡萄酒中氨基酸均发生显著变化:种类增多,诸多氨基酸含量提高。SD-2a发酵酒样中的丙氨酸和谷氨酸、SD-1b发酵酒样中甘氨酸、丝氨酸和脯氨酸、SD-2h酒样中谷氨酸、甘氨酸、丝氨酸、脯氨酸含量明显增加;含量呈极显著增加的氨基酸为:在SD-2h发酵酒样中的丙氨酸、在SD-1b发酵酒样中的丙氨酸、缬氨酸和谷氨酸;对照菌株31DH发酵酒样中各种氨基酸的增减没有达到显著水平。葡萄酒学院分离筛选的3个菌株发酵酒样中的精氨酸含量增加,而对照菌株31DH则有所降低。因此,3个菌株代谢特性良好,在葡萄酒中不会导致致癌物质——氨基甲酸乙酯的前体物脲、瓜氨酸等的过多合成。鉴于各种氨基酸独特的生化和生理特性,3株酒类酒球菌菌株完成MLF后,氨基酸种类、含量的增加可以提高葡萄酒的营养价值和保健功能。     

Effect of phenolic aldehydes and flavonoids on growth and inactivation of Oenococcus oeni and Lactobacillus hilgardii

The aim of this work was to investigate the effect of wine phenolic aldehydes, flavonoids and tannins on growth and viability of strains of Oenococcus oeni and Lactobacillus hilgardii. Cultures were grown in ethanol-containing MRS/TJ medium supplemented with different concentrations of phenolic aldehydes or flavonoids and monitored spectrophotometrically. The effect of tannins was evaluated by monitoring the progressive inactivation of cells in ethanol-containing phosphate buffer supplemented with grape seed extracts with different molecular weight tannins. Of the phenolic aldehydes tested, sinapaldehyde, coniferaldehyde, p-hydroxybenzaldehyde, 3,4-dihydroxybenzaldehyde and 3,4,5-trihydroxybenzaldehyde significantly inhibited the growth of O. oeni VF, while vanillin and syringaldehyde had no effect at the concentrations tested. Lact. hilgardii 5 was only inhibited by sinapaldehyde and coniferaldehyde. Among the flavonoids, quercetin and kaempferol exerted an inhibitory effect especially on O. oeni VF. Myricetin and the flavan-3-ols studied (catechin and epicatechin) did not affect considerably the growth of both strains. Condensed tannins (particularly tetramers and pentamers) were found to strongly affect cell viability, especially in the case of O. oeni VF. In general, this strain was found to be more sensitive than Lact. hilgardii 5 to the phenolic compounds studied. This work contributes to the knowledge of the effect of different phenolic compounds on the activity of wine lactic acid bacteria, which, especially in the case of aldehydes and of different molecular weight fractions of tannins, is very scarce.     

Both arginine and fructose stimulate pH-independent resistance in the wine bacteria Oenococcus oeni

The wine bacteria Oenococcus oeni has to cope with harsh environmental conditions including an acidic pH, a high alcoholic content, and growth inhibitory compounds such as fatty acids, phenolic acids and tannins. So how can O. oeni bacteria naturally present on the surface of grape berries acquire a natural resistance that will alleviate the effect of wine stresses? One mechanism displayed by O. oeni and many other bacteria against the damaging effects of acid environments is arginine consumption through the arginine deiminase pathway. Various studies have shown that the bacterial protection conferred by arginine depends on the rise in pH associated with ammonia production. However, many experimental results disagree with this point of view. The aim of this study was to clarify the protective effect of arginine on O. oeni stress adaptation. Is it only by increasing the pH through ammonia production that this effect is triggered, or does stimulation of appropriate cellular responses play an additional role? This study shows that: (a) arginine in combination with fructose triggers the expression of a subset of genes which are also stress-responsive; (b) cultivation of O. oeni in a fructose- and arginine-supplemented medium prior to wine exposure protects bacteria against subsequent wine shock, and (c) this acquired stress resistance is independent of pH.     

Identification of lactic acid bacteria isolated from South African brandy base wines

In brandy base wines, no sulphur dioxide is used and it therefore is ideal for the proliferation of lactic acid bacteria. As part of an extensive taxonomic survey within the ecological framework of South African vineyards and wineries, and the influence of naturally occurring lactic acid bacteria on the quality of wine and brandy, a total of 54 strains were isolated from grape juice and at different stages of brandy base wine production. The strains were identified using numerical analysis of total soluble cell protein patterns, 16S rRNA sequence analyses and polymerase chain reaction (PCR) using species-specific primers. The predominant species was Oenococcus oeni (22 strains), but Lactobacillus brevis (8 strains), Lactobacillus paracasei (8 strains) and Lactobacillus plantarum (6 strains) were also isolated frequently. Many of the O. oeni strains were isolated from brandy base wines after completion of spontaneous malolactic fermentation (MLF). The Lactobacillus spp. were isolated from all the different stages of brandy base wine production. Lb. plantarum was the dominant species in the juice, but disappeared during the later stages of production. However, Lactobacillus hilgardii, Lb. brevis and Lb. paracasei were also isolated from base wine after spontaneous MLF. Strains identified as Lactobacillus vermiforme were isolated during the alcoholic fermentation and after MLF have been completed. Total soluble cell protein patterns grouped O. oeni strains into two phenotypic groups. Two phenotypic clusters have also been identified for the Lb. brevis isolates. The Lb. paracasei isolates all grouped in one cluster. This is the first report of the presence of Lb. paracasei and Lb. vermiforme in brandy base wines. The presence of the Lactobacillus spp. could be correlated to the decrease in quality of the base wine and distillate, while O. oeni strains were found to have a more favourable influence on the quality of base wine and distillates. These results shed some light on the ecology and oenological influence of lactic acid bacteria (LAB) on the quality of South African brandy.     

Wine composition plays an important role in the control of carcinogenic precursor formation by Lactobacillus hilgardii X1B

BACKGROUND: Lactobacillus hilgardii, a wine lactic acid bacterium, is able to use arginine, through the arginine deiminase pathway with the formation of citrulline, a precursor of the carcinogen ethyl carbamate. The influence of different Argentine wine varieties (Merlot, Cabernet Sauvignon and Malbec), on bacterial growth and arginine metabolism was examined. Furthermore, the effect of different components normally present in wines on the enzyme activities of the arginine deiminase system was determined. RESULTS: Malbec wine under all conditions assayed (33, 50 and 100% supplemented wine:basal media) showed higher arginine consumption and citrulline production than the other wines, as well as the highest bacterial growth and survival of Lactobacillus hilgardii X₁B. Glucose and L ‐malic inhibited both arginine deiminase enzymes while fructose and citric acid only inhibited arginine deiminase. The red wines assayed in this study had different composition, and this is an explanation for the different behavior of the bacterium. CONCLUSION: The highest citrulline production in Malbec wine could be correlated with its lower concentrations of glucose, fructose, citric and phenolic acid than the other wines. Therefore, a wine with lower concentration of these sugars and acids could be dangerous due to the formation of ethyl carbamate precursors. Copyright © 2012 Society of Chemical Industry     

Environmental pH determines citrulline and ornithine release through the arginine deiminase pathway in Lactobacillus fermentum IMDO 130101

Sourdough lactic acid bacteria (LAB) need to be adapted to a highly acidic and, therefore, challenging environment. Different mechanisms are employed to enhance competitiveness, among which conversion of arginine into ornithine through the arginine deiminase (ADI) pathway is an important one. A combined molecular and kinetic approach of the ADI pathway in Lactobacillus fermentum IMDO 130101, a highly competitive sourdough LAB strain, identified mechanisms with advantageous technological effects and quantified the impact of these effects. First, molecular analysis of the arcBCAD operon of 4.8 kb revealed the genes encoding the enzymes ornithine transcarbamoylase, carbamate kinase, arginine deiminase, and an arginine/ornithine (A/O) antiporter, respectively, with an additional A/O antiporter 702.5 kb downstream of the ADI operon. The latter could play a role in citrulline transport. Second, pH-controlled batch fermentations were carried out, generating data for the development of a mathematical model to describe the temporal evolution of the three amino acids involved in the ADI pathway (arginine, citrulline, and ornithine) as a result of the activity of these enzymes and transporter(s). Free arginine in the medium was converted completely into a mixture of citrulline and ornithine under all conditions tested. However, the ratio between these end-products and the pattern of their formation showed variation as a function of environmental pH. Under optimal pH conditions for growth, citrulline release and some further conversion into ornithine was observed. When growing under sub-optimal pH conditions, ornithine was the main product of the ADI pathway. These kinetic data suggest a role in adaptation of L. fermentum IMDO 130101 to growth under sub-optimal conditions.     

Effect of ethanol and low pH on citrulline and ornithine excretion and arc gene expression by strains of Lactobacillus brevis and Pediococcus pentosaceus

The accumulation of citrulline and ornithine in wine or beer as a result of the arginine catabolism of some lactic acid bacteria (LAB) species increases the risk of ethyl carbamate and putrescine formation, respectively. Several LAB species, which are found as spoilage bacteria in alcoholic beverages, have been reported to be arginine degrading. This study evaluates the effect of ethanol content and low pH on the excretion of citrulline and ornithine by two strains belonging to the potential contaminant species Lactobacillus brevis and Pediococcus pentosaceus. In the conditions that most affected cell viability, arginine consumption per cell increased noticeably, indicating that arginine utilization may be a stress responsive mechanism. L. brevis showed a higher accumulation of ornithine in the media than P. pentosaceus. In the presence of ethanol, a higher expression of the arcC gene was found in P. pentosaceus, which resulted in a lower excretion of citrulline and ornithine than in L. brevis. This suggests that L. brevis is more likely to produce these amino acids, which are precursors of ethyl carbamate and putrescine.     

客家娘酒中氨基甲酸乙酯回归分析

以广东客家娘酒中氨基甲酸乙酯为对象,研究了总糖、总酸、酒精度、多酚、低聚糖、尿素、有机酸和氨基酸与氨基甲酸乙酯之间的相互关系.通过相关性分析表明,尿素、瓜氨酸和精氨酸与氨基甲酸乙酯呈现显著的负相关,鸟氨酸与氨基甲酸乙酯呈现显著的正相关,其他物质与氨基甲酸乙酯没有显著的相关性.对氨基甲酸乙酯、尿素、瓜氨酸、精氨酸和鸟氨酸进行多重回归分析,建立了氨基甲酸乙酯与其之间的定量关系,得到的回归方程.     

Comparative study of the inhibitory effects of wine polyphenols on the growth of enological lactic acid bacteria

This paper reports a comparative study of the inhibitory potential of 18 phenolic compounds, including hydroxybenzoic acids and their derivatives, hydroxycinnamic acids, phenolic alcohols and other related compounds, stilbenes, flavan-3-ols and flavonols, on different lactic acid bacteria (LAB) strains of the species Oenococcus oeni, Lactobacillus hilgardii and Pediococcus pentosaceus isolated from wine. In general, flavonols and stilbenes showed the greatest inhibitory effects (lowest IC?? values) on the growth of the strains tested (0.160-0.854 for flavonols and 0.307-0.855 g/L for stilbenes). Hydroxycinnamic acids (IC?? > 0.470 g/L) and hydroxybenzoic acids and esters (IC?? >1 g/L) exhibited medium inhibitory effect, and phenolic alcohols (IC?? > 2 g/L) and flavanol-3-ols (negligible effect) showed the lowest effect on the growth of the LAB strains studied. In comparison to the antimicrobial additives used in winemaking, IC?? values of most phenolic compounds were higher than those of potassium metabisulphite for O. oeni strains (e.g., around 4-fold higher for quercetin than for potassium metabisulphite), but lower for L. hilgardii and P. pentosaceus strains (e.g., around 2-fold lower for quercetin). Lysozyme IC?? values were negligible for L. hilgardii and P. pentosaceus, and were higher than those corresponding to most of the phenolic compounds tested for O. oeni strains, indicating that lysozyme was less toxic for LAB than the phenolic compounds in wine. Scanning electron microscopy confirmed damage of the cell membrane integrity as a consequence of the incubation with antimicrobial agents. These results contribute to the understanding of the inhibitory action of wine phenolics on the progress of malolactic fermentation, and also to the development of new alternatives to the use of sulphites in enology.     

Assessment of antibiotic susceptibility within lactic acid bacteria strains isolated from wine

Susceptibility to 12 antibiotics was tested in 75 unrelated lactic acid bacteria strains of wine origin of the following species: 38 Lactobacillus plantarum, 3 Lactobacillus hilgardii, 2 Lactobacillus paracasei, 1 Lactobacillus sp, 21 Oenococcus oeni, 4 Pediococcus pentosaceus, 2 Pediococcus parvulus, 1 Pediococcus acidilactici, and 3 Leuconostoc mesenteroides. The Minimal Inhibitory Concentrations of the different antibiotics that inhibited 50% of the strains of the Lactobacillus, Leuconostoc and Pediococcus genera were, respectively, the following ones: penicillin (2, < or =0.5, and < or =0.5 microg/ml), erythromycin (< or =0.5 microg/ml), chloramphenicol (4 microg/ml), ciprofloxacin (64, 8, and 128 microg/ml), vancomycin (> or =128 microg/ml), tetracycline (8, 2, and 8 microg/ml), streptomycin (256, 32, and 512 microg/ml), gentamicin (64, 4, and 128 microg/ml), kanamycin (256, 64, and 512 microg/ml), sulfamethoxazole (> or =1024 microg/ml), and trimethoprim (16 microg/ml). All 21 O. oeni showed susceptibility to erythromycin, tetracycline, rifampicin and chloramphenicol, and exhibited resistance to aminoglycosides, vancomycin, sulfamethoxazole and trimethoprim, that could represent intrinsic resistance. Differences were observed among the O. oeni strains with respect to penicillin or ciprofloxacin susceptibility. Antibiotic resistance genes were studied by PCR and sequencing, and the following genes were detected: erm(B) (one P. acidilactici), tet(M) (one L. plantarum), tet(L) (one P. parvulus), aac(6')-aph(2") (four L. plantarum, one P. parvulus, one P. pentosaceus and two O. oeni), ant(6) (one L. plantarum, and two P. parvulus), and aph(3')-IIIa (one L. plantarum and one O. oeni). This is the first time, to our knowledge, that ant(6), aph(3')-IIIa and tet(L) genes are found in Lactobacillus and Pediococcus strains and antimicrobial resistance genes are reported in O. oeni strains.     

Antimicrobial activity of nisin against Oenococcus oeni and other wine bacteria

Nisin is a bacteriocin used against food spoilage bacteria. Sulphur dioxide is a potent antioxidant as well as an antimicrobial agent widely used in the wine industry. In this study we describe the effect of these important antibacterial agents on the growth of a collection of 64 lactic acid bacteria (23 Oenococcus, 29 Lactobacillus, 3 Leuconostoc and 9 Pediococcus), 23 acetic acid bacteria and 20 yeast isolates, most of them recovered from wine. Minimal inhibitory concentrations (MIC) and minimal bactericide concentrations of nisin, potassium metabisulphite and ethanol were determined. Nisin MIC(50) values for the tested isolates were as follows: 0.024, 12.5, 200 and > or micro for oenococci, lactobacilli-pediococci-leuconostoc, acetic acid bacteria and yeasts, respectively. Synergistic effects on bacterial growth inhibition were observed, and potassium metabisulphite MIC(50) values decreased from one to three orders of dilution when it was combined with subinhibitory concentrations of nisin in the growth media. This effect was observed in all lactic acid bacteria species of our study. Significant differences in nisin sensitivity were observed between Gram-positive and Gram-negative bacteria, and between Oenococcus oeni and other species of lactic acid bacteria. It is concluded that appropriate combinations of nisin and metabisulphite could control the growth of spoilage bacteria in wine and therefore allow a decrease in the levels of sulphur dioxide currently used by the wine industry.     

酒酒球菌（Oenococcus oeni）相关生物组学研究进展

酒酒球菌（Oenococcus oeni）是触发葡萄酒苹果酸-乳酸发酵的主要微生物,而苹果酸-乳酸发酵有利于提高葡萄酒品质,为了进一步提高酒酒球菌在葡萄酒酿造工业中的应用,通过各种生物组学方法来探究酒酒球菌的生物调节和代谢体系是必要的。本文对不同生物组学的研究手段进行汇总,并对其在酒酒球菌相关研究中的应用进行归纳和展望。     

Use of a polymerase chain reaction for specific detection of the malolactic fermentation bacterium Oenococcus oeni (formerly Leuconostoc oenos) in grape juice and wine samples

A PCR method has been developed that enables rapid and direct identification of the malolactic bacterium Oenococcus oeni from grape must or wine samples. Two primers, based on unique, highly conserved regions within the 16S rRNA gene of O. oeni , were used to amplify a 995 bp fragment which is specific for O. oeni . Other species of bacteria from Lactobacillus, Pediococcus and Acetobacter which may be found in grape must or wine were not detected using this technique. This diagnostic test is able to specifically detect in the order of 10³colony forming units per mL of O. oeni in a wine sample, and can be used for monitoring bacterial growth during malolactic fermentation.     

Molecular cloning, heterologous expression, and characterization of Ornithine decarboxylase from Oenococcus oeni

Ornithine decarboxylase (ODC) is responsible for the production of putrescine, the major biogenic amine found in wine. Oenococcus oeni is the most important lactic acid bacterium in the winemaking process and is involved in malolactic fermentation. We report here the characterization of ODC from an O. oeni strain isolated from wine. Screening of 263 strains isolated from wine and cider from all over the world revealed that the presence of the odc gene appears to be strain specific in O. oeni. After cloning, heterologous expression in Escherichia coli, and characterization, the enzyme was found to have a molecular mass of 85 kDa and a pI of 6.2 and revealed maximal activity at pH 5.5 and an optimum temperature of 35°C. Kinetic studies showed that O. oeni ODC is specific for L-ornithine with a K(m) value of 1 mM and a V(max) of 0.57 U·mg(-1). The hypothesis that cadaverine, which results from lysine decarboxylation, may be linked to putrescine production is not valid since O. oeni ODC cannot decarboxylate L-lysine. As no lysine decarboxylase was detected in any of the O. oeni genomes sequenced, cadaverine synthesis may result from another metabolic pathway. This work is the first characterization of an ODC from a lactic acid bacterium isolated from a fermented product.     

酒酒球菌中生物胺相关基因的检测

酒酒球菌在葡萄酒苹果酸-乳酸发酵过程中可通过脱羧基作用将氨基酸转化为生物胺.该研究采用PCR技术对27株酒酒球菌中与生物胺产生相关的基因-组氨酸脱羧酶基因、鸟氨酸脱羧酶基因、酪氨酸脱羧酶基因进行了检测,研究了这些菌株产生生物胺的特性.结果显示,所有菌株均不含有组氨酸脱羧酶基因、鸟氨酸脱羧酶基因、酪氨酸脱羧酶基因,不具有产生组胺、腐胺和酪胺的能力,因而具有较高的生物胺代谢安全性.     

葡萄酒中酒类酒球菌的分离——抑制剂对酵母菌生长的抑制效应

从葡萄酒中分离酒类酒球菌时 ,酵母菌是最主要的干扰菌。作者通过在ATB和改良MRS琼脂培养基中添加放线菌酮、山梨酸和制霉菌素 ,研究了不同抑制剂对酵母生长的抑制效应。结果表明 ,采用ATB + 5 0mg/L放线菌酮、改良MRS + 5 0mg/L放线菌酮、改良MRS + 10 0mg/L山梨酸、改良MRS + 5 0mg/L制霉菌素选择性培养基 ,能够完全抑制酿酒酵母的生长。