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, citrulline and ornithine metabolism by lactic acid bacteria from wine

The catabolism of arginine, an amino acid found in grape juice and wine, citrulline and ornithine was investigated in four lactic acid bacteria. Only Lactobacillus hilgardii X1B catabolized arginine and excreted citrulline into the medium. The recovery of arginine as ornithine was lower than the expected theoretical value. The arginase-urease pathway was not detected indicating that the amino acid degradation was carried out only by the arginine dihydrolase pathway. Oenococcus oeni m, a strain not able to utilize arginine, degraded citrulline that was completely recovered as ornithine, ammonia and CO2. Lactobacillus hilgardii X1B catabolized citrulline but it was only 44% recovered as ornithine. The citrulline utilization by Oenococcus oeni m may be important for two reasons: it can gain extra energy for growth from citrulline metabolism, and the amino-acid diminution could avoid the possibility of ethyl carbamate formation from the citrulline naturally present in wine.     

Citrulline production by lactic acid bacteria in Chinese rice wine

Ethyl carbamate (EC) is a carcinogenic compound derived from the spontaneous reaction of ethanol with urea or citrulline in Chinese rice wine. Polymerase chain reaction–denaturing gradient gel electrophoresis showed that five species, Lactobacillus fermentum, Lactobacillus plantarum, Lactobacillus brevis, Lactococcus lactis and Lactobacillus coryniformis were the most abundant bacteria in the Chinese rice wine production process. Five strains belonging to these species can degrade arginine primarily in the exponential growth phase and accumulate citrulline in MRS‐Arg medium. In addition, an L. brevis strain was shown to be capable of assimilating citrulline, indicating the potential of this strain suggesting a potential route to reduce citrulline content and ethyl carbamate formation in Chinese rice wine fermentation. Copyright © 2018 The Institute of Brewing & Distilling     

Contribution of citrulline to the formation of ethyl carbamate during Chinese rice wine production

Ethyl carbamate is a well-known carcinogen and widely occurs in Chinese rice wine. To provide more clues to minimise ethyl carbamate accumulation, the levels of possible precursors of ethyl carbamate in Chinese rice wine were investigated by HPLC. Studies of the possible precursors of ethyl carbamate in Chinese raw rice wine with various additives and treatments indicated that significant amounts of urea can account for ethyl carbamate formation. It was also recognised that citrulline is another important precursor that significantly affects ethyl carbamate production during the boiling procedure used in the Chinese rice wine manufacturing process. Besides urea and citrulline, arginine was also found to be an indirect ethyl carbamate precursor due to its ability to form urea and citrulline by microorganism metabolism.     

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.     

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.     

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.     

Assessing the Potential Content of Ethyl Carbamate in White,Red, and Rosé Wines as a Key Factor for Pursuing Urea Degradation by Purified Acid Urease

The ethyl carbamate (EC) content of a wine after a given temperature‐time storage was theoretically predicted from the potential concentration of ethyl carbamate (PEC), as determined via an accelerated EC formation test. Such information was used to decide whether an enzymatic treatment was needed to reduce the wine urea level before bottling/aging. To this end, 6 white, red, and rosé wines, manufactured in Italy as such or enriched with urea, were tested for their PEC content either before or after enzymatic treatment using a purified acid urease preparation derived from Lactobacillus fermentum. The treatment was severely affected by the total phenolic content (TP) of the wine, the estimated pseudo‐first‐order kinetic rate constant for NH₃ formation reducing by a factor of approximately 2000 as the TP increased from 0 to 1.64 g L^‐1. Such a sensitivity to TP was by far greater than that pertaining to a killed cell‐based enzyme preparation used previously. Urea hydrolysis was successful at reducing EC concentration in wines with low levels of TP and other EC precursors.     

Exposure assessment and risk characterisation of ethyl carbamate from Korean traditional fermented rice wine,Takju and Yakju

Ethyl carbamate is one of the most hazardous chemicals naturally occurring in food, and is present in alcoholic beverages. Korean traditional rice wine, Takju and Yakju, is frequently consumed in Korea, but there have been no studies characterising the risks of ethyl carbamate in these products. In order to assess and characterise the exposure risk of ethyl carbamate in Korean traditional rice wines, ethyl carbamate was investigated by means of GC-MS. The analytical methods were optimised and validated through determining linearity, detection limit, quantification limit, recovery and precision. A total of 283 traditional Korean rice wines, including 175 Takju and 108 Yakju samples, were analysed. Exposure assessment was performed by factoring in ethyl carbamate content, daily consumption and body weight. Daily exposures of ethyl carbamate were estimated for adults in four age groups, and risks of ethyl carbamate were characterised by the margin of exposure, which is more than 10 000. Based on this study, the risks of ethyl carbamate in Korean traditional rice wine were shown to be of low concern.     

Ethyl carbamate formation regulated by ornithine transcarbamylase and urea metabolism in the processing of Chinese yellow rice wine

Ethyl carbamate (EC) is a potential carcinogenic compound present in most of the fermented foods. In this work, EC was inhibited through different strategies during vinification of Chinese yellow rice wine. EC can be inhibited by the use of ornithine in contrast to the control at peak point. However, the utilisation of urease resulted in little inhibitive effect on EC. The comparative data of intracellular ornithine transcarbamylase (OTCase) and arginine deiminase (ADI) among four experiments showed that EC was positively regulated by intracellular OTCase, but ADI was not determined. Extracellular urea and citrulline content was significantly increased by adding ornithine (P < 0.05), whereas ethanol played a minor role in EC formation. The correlation analysis between EC and OTCase or urea revealed a linear association (correlation coefficients above 0.8). These findings suggested that OTCase may be a required factor regulating EC formation during the brewing of Chinese yellow rice wine.     

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 acids on glucose and organic acid metabolism by lactic acid bacteria from wine

The influence of phenolic (p-coumaric, caffeic, ferulic, gallic and protocatechuic) acids on glucose and organic acid metabolism by two strains of wine lactic acid bacteria (Oenococcus oeni VF and Lactobacillus hilgardii 5) was investigated. Cultures were grown in modified MRS medium supplemented with different phenolic acids. Cellular growth was monitored and metabolite concentrations were determined by HPLC-RI. Despite the strong inhibitory effect of most tested phenolic acids on the growth of O. oeni VF, the malolactic activity of this strain was not considerably affected by these compounds. While less affected in its growth, the capacity of L. hilgardii 5 to degrade malic acid was clearly diminished. Except for gallic acid, the addition of phenolic acids delayed the metabolism of glucose and citric acid in both strains tested. It was also found that the presence of hydroxycinnamic acids (p-coumaric, caffeic and ferulic) increased the yield of lactic and acetic acid production from glucose by O. oeni VF and not by L. hilgardii 5. The results show that important oenological characteristics of wine lactic acid bacteria, such as the malolactic activity and the production of volatile organic acids, may be differently affected by the presence of phenolic acids, depending on the bacterial species or strain.     

Effects of storage time and temperature on the concentration of ethyl carbamate and its precursors in wine

A 3-year study was carried out on the effects of time and temperature on the concentration of ethyl carbamate in wine. The study monitored the changing concentration of ethyl carbamate and of urea and citrulline, which are two major precursors of ethyl carbamate in wine. In addition to the formation of ethyl carbamate, both urea and citrulline decayed in other reactions. Kinetic analysis was carried out to model the formation of ethyl carbamate and its dependence on the concentrations of ethanol, urea and citrulline. This led to the development of an equation that can be used to predict the concentration of ethyl carbamate in wine at the point of consumption, resulting from any given storage time and temperature profile. The results were in good agreement with data obtained from similar studies.     

Temperature and Light Effects on Ethyl Carbamate Formation in Wine During Storage

The formation and/or increase of ethyl carbamate during storage of wine was monitored at 43°C, 32°C and 22°C in the dark, at 22°C under fluorescent light and in a greenhouse with naturally fluctuating temperature and light conditions simulating commercial display of wines. The ethyl carbamate concentration of fifteen selected red and white table and dessert wines was determined initially, and after 3, 6, and 12 months storage. In most wines ethyl carbamate increased as a function of time and temperature. After 12 months in the dark at 43°C, the ethyl carbamate in two red wines increased 20 to 30 times. It increased three to nine-fold in nine of the wines, but remained unchanged in four. Light did not significantly influence ethyl carbamate formation, whether in clear or green bottles.     

Influence of different yeasts on the growth of lactic acid bacteria in wine

The influence of various yeasts on the growth of lactic acid bacteria in wine was tested by inoculating Lactobacillus hilgardii, L. brevis and two strains of Leuconostoc mesenteroides into experimental wines made with twelve different yeasts of the genus Saccharomyces. Wines made from juice which had been infected with several spoilage yeasts and then fermented with a wine yeast were also tested in this way. It was found that the yeasts differed considerably in their effects on bacterial growth. In some of the experimental wines bacterial growth was delayed or failed altogether. Generally, the unfavourable influence of any yeast on bacterial growth was much reduced if the wines were left in contact with the yeast cells for some weeks after the fermentation. The significance of these results in relation to the occurrence of malo-lactic fermentation in commercial wineries is discussed.     

The kinetics of the arginine deiminase pathway in the meat starter culture Lactobacillus sakei CTC 494 are pH-dependent

Lactobacillus sakei is frequently present as the dominant lactic acid bacterium in spontaneously fermented meat products, demonstrating its competitiveness in and adaptation to the meat environment. Since meat is generally low in carbohydrate content, the ability to utilize other energy sources to generate ATP, such as arginine via the arginine deiminase (ADI) pathway, represents a competitive benefit. In this study, the kinetics of growth and arginine conversion capabilities of Lb. sakei CTC 494 were analyzed, and a model was set up to describe the influence of pH on growth and arginine conversion. A series of in vitro batch fermentations using reconstituted MRS medium at different constant pH values (pH 4.50-pH 7.75) was performed. Arginine conversion through the ADI pathway, which was activated from the stationary growth phase on, resulted in the production of both citrulline and ornithine for all pH conditions tested. However, the pattern and the ratio of the end-products of the ADI pathway were influenced by pH. For certain pH values (between pH 5.0 and 6.5), a further conversion of citrulline into ornithine was found when all arginine was depleted. Characterization of responses of the ADI pathway in Lb. sakei CTC 494 to environmental conditions will allow a better understanding and control of this important starter culture in meat fermentations.     

葡萄酒中氨基甲酸乙酯的含量测定及酿造工艺对其含量的影响

调查了我国干红葡萄酒(77款)和干白葡萄酒(17款)中氨基甲酸乙酯(EC)含量情况,并探讨了酿酒工艺(果胶酶、酵母、乳酸菌和发酵温度等)对葡萄酒中氨基甲酸乙酯(EC)含量的影响。研究结果显示:干红葡萄酒中EC含量范围在7.00～26.80μg/L,平均浓度为17.90μg/L;干白葡萄酒中EC含量范围在6.40～21.58μg/L,平均浓度为11.35μg/L。葡萄酒酿造过程中EC的生成主要受酵母菌和乳酸菌种类的影响,酒精发酵温度的降低和果胶酶的使用有助于减少葡萄酒中EC的含量。     

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.     

东山老米酒酿造过程中酵母菌的分离鉴定及其品质分析

目的 本文对传统东山老米酒的发酵特性进行了比较全面研究。方法 首先利用26S rDNA序列分析法对酒曲中的酵母菌进行了分离鉴定,借助高效液相色谱法测定酒中的氨基甲酸乙酯,利用气相色谱-质谱联用法分析了老米酒中的风味物质,同时还用氨基酸自动分析仪测定了游离氨基酸种类。结果 分离得到的菌株有3株为酿酒酵母,1株为白色隐球菌;老米酒中氨基甲酸乙酯含量处于较低水平,尤其是陈酿中仅含0.6 mg/L;风味物质以醇、酯类为主,如苯乙醇、琥珀酸二乙酯,酯类随酒的陈化而逐渐增多;老米酒中组氨酸和精氨酸含量较高,导致酒中苦味氨基酸含量高于60%,同时氨基酸含量在陈化过程中逐渐下降。结论 东山老米酒的发酵以酵母发酵为主,发酵产生的氨基甲酸乙酯的含量处于较低水平,且随着老米酒的陈化而降低。     

Application of an Acid Urease to Wine: Determination of Trace Urea in Wine

An improved method of urea detection having a sensitivity to 0.05 ppm, applicable to white wines, was developed by modifying Jansen's procedure. The urea concentration in many commercial bottled wines was found to be around 2 ppm. The method was utilized to the urea degradation by an acid urease which degraded naturally occurring urea of 0.5–2 ppm at the same reaction rate as that for the spiked urea of 30 ppm in the same white wine. Wine treatment with the acid urease also reduced ethyl carbamate formed by heating.