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.     

Effect of citrulline metabolism in Saccharomyces cerevisiae on the formation of ethyl carbamate during Chinese rice wine fermentation

Urea, as the main precursor of ethyl carbamate (EC), has received extensive attention. Here, we have metabolically engineered an industrial yeast strain – Saccharomyces cerevisiae N85 – to investigate the contribution of the EC precursor citrulline to the concentration of EC in Chinese rice wine. The results showed that the citrulline biosynthetic pathway of the modified strain N85‐arg3 was completely suppressed by deletion of ARG3, encoding ornithine carbamoyltransferase. However, there were no significant differences in the levels of citrulline or EC between N85‐arg3 and the parental strain N85 during fermentation. In addition, we over‐expressed ARG1 (encoding argininosuccinate synthase) and ARG4 (encoding argininosuccinate lyase) to construct the engineered strains N85^ARG1,4 and N85^ARG1,4‐arg3. The citrulline contents in Chinese rice wine fermented with N85^ARG1,4 and N85^ARG1,4‐arg3 were respectively 24.1 and 20.4% less than that of N85. However, the contents of EC were 23.8 and 28.5% more than that of N85. These results suggested that reducing the formation of EC during Chinese rice wine fermentation by genetically engineering citrulline metabolism in S. cerevisiae was not a viable proposition. 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.     

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     

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.     

Enhancement of urea uptake in Chinese rice wine yeast strain N85 by the constitutive expression of DUR3 for ethyl carbamate elimination

Most of the fermented alcoholic beverages, particularly Chinese rice wine, contain the potentially human carcinogenic compound ethyl carbamate (EC). As a major EC precursor in Chinese rice wine, urea in fermentations can be transported into the yeast cell by urea permease and finally metabolized by urea carboxylase and allophanate hydrolase in vivo. To eliminate EC in Chinese rice wines, the present study constructed high urea uptake yeast strains N1‐D, N2‐D and N‐D, by introducing a strong promoter (PGK1p) into the urea permease gene (DUR3) of the industrial Chinese rice wine yeast N85, and by the restoration of the URA3 gene at the same time. With these self‐cloned, high urea uptake strains, the urea and EC in the terminal Chinese rice wine samples were reduced to different extents. With two copies of overexpressed DUR3, the N‐D strain could reduce the urea and the EC by 53.4 and 26.1%, respectively. No difference in fermentation characteristics was found between the engineered strains and the parental industrial yeast strain N85. These results could help to optimize the genetic manipulation strategy for EC elimination in Chinese rice wine production. Copyright © 2015 The Institute of Brewing & Distilling     

黄酒酵母的改良及对产物中尿素和氨基甲酸乙酯含量的影响

氨基甲酸乙酯（EC)是一种潜在致癌物,在黄酒发酵过程中尿素是它的前体物质。该研究通过紫外诱变和基因过表达筛选获得改良黄酒酵母菌种,并对黄酒产品的理化指标进行检测可知,与出发菌株相比,改造菌株的发酵性能和黄酒的出酒率、酒精度、总糖、总酸、氨基酸态氮和β-苯乙醇没有明显的差异,而诱变菌株JF501-A62发酵产物尿素含量降低了67%,EC含量降低了59%;基因过表达菌株JF501-B5发酵产物尿素含量降低了88%,EC含量降低了63%。两者均有很好的发酵性能,并取得了较好地降低产品中尿素含量、进而降低氨基甲酸乙酯含量的效果。与紫外诱变相比,基因过表达的改良方法获得了尿素含量更低的菌株,并贮存6个月之后产品中的EC含量更低。     

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.     

脲基酰胺酶基因在黄酒酵母中的整合型表达

氨基甲酸乙酯是一种人类的潜在致癌物,在许多发酵酒中均有存在,主要来源于发酵过程中酵母代谢产生的尿素。以pYX212为载体,将脲基酰胺酶基因DUR1,2克隆到TPI强启动子和终止子之间的位点,再通过同源重组的方式将受强启动子调控的目的基因整合到黄酒酵母的基因组中,最终获得一株低产尿素的胞内脲基酰胺酶基因组成型高表达的黄酒酵母85#DUR1,2。在实验室规模的黄酒酿造实验中,85#DUR1,2产尿素量为8.34mg/L,比出发菌株降低了69.9%,贮存一段时间后的酒液中氨基甲酸乙酯含量比出发菌株降低了40.5%,而发酵性能、酒精度、总酸及氨基态氮与出发菌株无显著差异。     

Microbial characterization of Iranian traditional Lighvan cheese over manufacturing and ripening via culturing and PCR-DGGE analysis: identification and typing of dominant lactobacilli

This paper reports on the diversity and dynamics of the dominant microbial populations during manufacturing and ripening of Lighvan, a traditional, starter-free Iranian cheese made from raw ewe and goat’s milk as determined by culturing and PCR-DGGE. Similar dominant populations, composed of Lactococcus lactis and Lactobacillus spp. strains, were found by both techniques. However, discrepancies regarding the identity of the Lactobacillus species were encountered. Lactobacillus curvatus and Lactobacillus sakei proved to be dominant by PCR-DGGE; in contrast, Lactobacillus paraplantarum, Lactobacillus paracasei, Lactobacillus brevis and Lactobacillus plantarum were the majority cultivable organisms. RAPD typing of lactobacilli isolates showed wide genetic diversity among the species. Moreover, strain compositions change over time; L. brevis and L. paraplantarum were dominant in milk and were replaced by L. plantarum and L. paracasei strains as ripening progressed.     

The overexpression of DUR1,2 and deletion of CAR1 in an industrial Saccharomyces cerevisiae strain and effects on nitrogen catabolite repression in Chinese rice wine production

Urea is acknowledged as the predominant precursor of ethyl carbamate (EC) in Chinese rice wine. During Chinese rice wine fermentation, urea accumulates owing to the nitrogen catabolite repression effect when preferred nitrogen sources are available. In previous research, two metabolically engineered strains were constructed with overexpression of DUR1,2 and deletion of CAR1 from an industrial Chinese rice wine yeast N85. The decreasing effect of urea and EC was demonstrated during small‐scale Chinese rice wine fermentations. The present study compared the urea utilization rate of the parental and metabolically engineered yeast strains, using a preferred and non‐preferred nitrogen source culture media, leading to alleviated urea accumulation and thus EC formation. The qRT‐PCR results showed that, in all of the culture media, DUR1,2 was overexpressed with the inserted strong promoter PGK1p. During pilot scale fermentations, the urea and EC content decreased with the engineered strains. These results confirmed that the engineered strains could resist the nitrogen catabolite repression effect. Copyright © 2016 The Institute of Brewing & Distilling     

Beer enemy number one: genetic diversity,physiology and biofilm formation of Lactobacillus brevis

Lactobacillus brevis is the most significant beer spoilage bacteria worldwide. It is found as a contaminant at all stages of brewing, including during primary and secondary fermentation, storage, filtration and the packaging process. In production with flash pasteurisation and subsequent hygienic filling, avoiding and tracing secondary contaminations is the key to a microbiologically stable product. However, L. brevis strains vary in their spoilage potential and can grow in many different beer types. This study presents a physiological test scheme for growth potential and biofilm formation in various media. It was determined that a large number of L. brevis strains can form biofilms as a first coloniser. The identification of the species alone is therefore not enough to be sure of the spoilage risk, which shows the need for a more in depth differentiation. DNA fingerprint techniques are crucial to differentiate isolates of this species at strain level. The rep‐PCR fingerprint system (GTG)₅ was used to differentiate a selected collection of 20 isolates, which were characterised in growth and biofilm formation in various media. The data showed a high variation within the selected isolates. As second step, generated fingerprint clusters of L. brevis were traced back to contamination sources in a German brewery, revealing a high number of isolates with potentially varying growth, spoilage and biofilm potential. L. brevis being the demonstrator species, the PCR system used is a powerful and compatible tracing and troubleshooting tool for all kinds of spoilage bacteria in the brewing industry. © 2019 The Institute of Brewing & Distilling     

黄酒中不产生物胺乳酸菌的筛选及应用

为筛选不产生物胺的乳酸菌菌株,对黄酒发酵醪液及米浆水中的微生物进行培养及与分离纯化,发现3株菌不具有氨基酸脱羧酶活性,但具有一定的生物胺降解能力。使用黄酒发酵液培养该3株菌,发现在2.50%Vol的黄酒培养液中,3株菌能正常生长;在8.80%Vol黄酒培养液中,菌株5-4和8-3生长能力明显弱于14-2-1;在15.70%Vol黄酒培养液中,3株菌均受到严重抑制。菌株14-2-1生长速率最快,繁殖最旺,经鉴定为植物乳杆菌,同时菌株产酸能力较强,产酸速率也较快,表现出一定的优越性,应用于黄酒酿造中,能有效降低黄酒中生物胺的含量。     

Determination of the relationship between oxalate degradation and exopolysaccharide production by different Lactobacillus probiotic strains

The aim of this study was to evaluate the protective effects of exopolysaccharide (EPS) production on resistance to gastrointestinal digestive conditions and oxalate by probiotic strains of the species Lactobacillus rhamnosus, Lactobacillus fermentum and Lactobacillus brevis. The oxalate‐degrading ability of the strains was determined using an enzymatic assay. The correlation between oxalate degradation rate and EPS production was not significant (P > 0.05); however, the high‐EPS‐producing L. fermentumIP5 and L. brevisYG7 strains showed high oxalate‐degrading activity, whereas the low‐EPS‐producing strain L. fermentumBP5 demonstrated low oxalate‐degrading activity. The present findings suggest that dietary supplementation with the probiotic strain L. fermentumIP5 could be a promising strategy for the prevention of oxalate stone disease.     

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.     

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.     

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.     

Identification and molecular characterisation of lactobacilli isolated from traditional Koopeh cheese

This study was undertaken in order to phenotype and genotype wild‐type lactic acid bacteria isolated from Koopeh cheese of West Azerbaijan. Lactic acid bacteria (LAB) isolates were identified by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) and confirmed by phylogenetic analysis. Genotyping based on phylogenetic analysis of 16s rDNA gene revealed that LAB isolated from Koopeh cheese were mostly Lactobacillus plantarum as well as other species including Lactobacillus brevis, Entrococcus faecium and Enterococcus durans. It was concluded that a combined phenotypic and genotypic method could be used as a reliable technique for the identification and differentiation of LAB from dairy products.     

Rapid determination of ethyl carbamate in Chinese rice wine using headspace solid‐phase microextraction and gas chromatography–mass spectrometry

Ethyl carbamate (EC) is a naturally occurring toxic contaminant that may imply a risk to human health and is usually found in alcoholic beverages such as Chinese rice wine. An automated procedure for the rapid determination of EC in Chinese rice wine was developed by headspace solid‐phase microextraction (HS‐SPME) coupled to gas chromatography–mass spectrometry (GC‐MS). Using propyl carbamate as internal standard, the optimized HS‐SPME sampling with a polyacrylic fibre was 45 min at 70°C after applying 38.8% NaCl to saturate the sample. This method showed good linearity over a range of 25–600 µg L^?1 (R² = 0.997). The recovery, relative standard deviation and limit of detection were 90.21–97.35%, lower than 2.89% and 1.19 µg L^?1, respectively. Additionally, the ethanol concentration had no effect on the analysis of EC. The total analysis time of 57 min per sample in continuous determination was twice as fast as the widely used solid‐phase extraction–GC‐MS method. This solvent‐free HS‐SPME‐GC‐MS procedure is suitable for the rapid, automated, and therefore convenient, determination of EC in Chinese rice wine. Copyright © 2012 The Institute of Brewing & Distilling     

Determination of fumonisins B1 and B2 in Chinese rice wine by HPLC using AQC precolumn derivatisation

BACKGROUND: Fumonisins are a group of naturally occurring mycotoxins produced by various Fusarium species that commonly infect maize and other cereals, including sorghum and rice. In this study a sensitive and selective method was developed for the determination of fumonisins B₁ and B₂ (FB₁ and FB₂) in Chinese rice wine. The method is based on high‐performance liquid chromatography and fluorescence detection following precolumn derivatisation with 6‐aminoquinolyl‐N‐hydroxysuccinimidyl carbamate (AQC). RESULTS: FB₁ and FB₂ in Chinese rice wine were extracted and purified using strong anion exchange cartridges and derivatised with AQC at room temperature. The AQC derivatives were stable for 5 days. Optimal fluorescence was obtained at an excitation wavelength of 246 nm and an emission wavelength of 390 nm. Chromatography was performed using a C₁₈ column and gradient elution at 1 mL min^?1 with methanol and 0.05 mol L^?1 phosphate buffer at pH 4. The limit of detection was 6 µg L^?1 for both FB₁ and FB₂. The method was successfully applied to the determination of FB₁ and FB₂ in Chinese rice wine, with recoveries of 87.5–94.5% being obtained. CONCLUSION: The established method was stable and sensitive for the determination of FB₁ and FB₂ in Chinese rice wine. © 2012 Society of Chemical Industry