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     

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     

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     

Effects of overexpression of the alcohol acetyltransferase‐encoding gene ATF1 and disruption of the esterase‐encoding gene IAH1 on the flavour profiles of Chinese yellow rice wine

Alcohol acetyltransferase (AATase), which is mainly encoded by ATF1, is one of the most important enzymes for acetate ester synthesis. On the other hand, isoamyl acetate is degraded into a higher alcohol under the catalysis of IAH1‐encoded esterase. In this study, Chinese Saccharomyces cerevisiae was used as the parent strain to construct an ATF1 overexpression and IAH1 disruption mutant. The results show that after 5 days of pre‐fermentation, the concentrations of ethyl acetate, isoamyl acetate and isobutyl acetate in the yellow rice wines fermented with EY1 (pUC‐PIAK) increased to 468.94 mg L^?1 (which is approximately 22‐fold higher than that of the parent cell RY1), 99.86 and 7.69 mg L^?1 respectively. Meanwhile, isoamyl alcohol production was reduced to 56.37 mg L^?1 (which is approximately 50% of that produced by the parent strain RY1). Therefore, ATF1 overexpression and IAH1 disruption can significantly increase acetate esters contents and reduce isoamyl alcohol content in Chinese yellow rice wine, thereby paving the way for breeding an excellent yeast strain for high‐quality Chinese yellow rice wine production.     

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     

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.     

Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation

The complex metabolic processes of yeast influence wine fermentation and therefore the quality of wine. Wine yeasts, owing to their being typically prototrophic and often polyploid, have been restricted in terms of exploiting classical recombinant genetic techniques to improve their characteristics. To overcome this problem, haploids have been isolated from a commercial Chinese rice wine strain N85, by disruption of the HO gene. In this study, the Cre–loxP system and a removable G418^r marker were used to construct an HO disruption cassette. Most of the heterologous sequences of constructed disruption cassette were successfully excised from the genome of the haploids by loop‐out of the KanMX gene, through induced expression of the Cre recombinase. The removal of the resistant marker ensures the biological safety of the strains. As expected, no difference in fermentation capacity between the parental and the haploid strains was seen. The present work reports the construction of an HO disruption cassette by touchdown polymerase chain reaction and its application with a Chinese rice wine yeast for haploid isolation and to broaden physiological investigations and industrial applications. Copyright © 2013 The Institute of Brewing & Distilling     

Chemical and volatile composition of lychee wines fermented with four commercial Saccharomyces cerevisiae yeast strains

This study evaluated the chemical and volatile composition of lychee wines fermented with four commercial yeast strains of Saccharomyces cerevisiae: EC‐1118, R2, 71B and MERIT.ferm. Yeast cell population, pH, malic acid, ammonia and some amino acids had significant differences between strain 71B and other strains. There were strain variations with regard to degradation and retention of some juice volatiles as well as formation of new volatile compounds such as most esters and certain terpene compounds. Ethyl octanoate had the highest odour activity value (OAV of 500–1100), followed by ethyl hexanoate (about 50–85), among the common odorants in all lychee wines. Ethyl octanoate reached its highest OAV (1077) in the lychee wine fermented with strain EC1118, whereas ethyl hexanoate had the highest OAV (85) in the wine fermented with strain MERIT.ferm. Cis‐rose oxide, the character‐impact volatile in lychee juice with highest OAV (161), was dramatically reduced to trace levels after fermentation.     

Growth and fermentation kinetics of a mixed culture of Saccharomyces cerevisiae var. bayanus and Williopsis saturnus var. saturnus at different ratios in longan juice (Dimocarpus longan Lour.)

Cofermentation of longan juice by mixed cultures of Saccharomyces cerevisiae var. bayanus EC‐1118 and Williopsis saturnus var. saturnus CBS254 at two inoculation ratios (EC‐1118:CBS254 = 1:100 and 1:1000 cfu mL^?1) was performed to ascertain their impact on longan wine aroma compound formation. The results showed improved aroma compound profiles in the longan wine fermented with mixed yeasts in comparison with the longan wines fermented with single yeasts in terms of increased production of acetate esters, fatty acid ethyl esters, alcohols and carboxylic acids. The impact of cofermentation on longan wine aroma formation was affected by the ratio of S. cerevisiae EC‐1118 to W. saturnus CBS254 with 1:100 cfu mL^?1 being more effective. This research suggests that the inoculation ratio of mixed yeasts may be used as an effective means of manipulating longan wine aroma.     

Determination of ethyl carbamate in Chinese yellow rice wine using high‐performance liquid chromatography with fluorescence detection

Ethyl carbamate (EC) is a naturally occurring component in the fermented products, especially the Chinese yellow rice wine. EC has been found showing a potential for carcinogenity and causing food safety problems. This study is to investigate the application of the existing HPLC‐FLD method to the analysis of EC in Chinese yellow rice wine, and to validate it for EC analysis with high through‐out and applicability in Chinese yellow rice wine sample. The parameters examined in this study were fully evaluated. Results indicated that good linearity was obtained with a correlation coefficient exceeding 0.9990, the limit of detection and quantification was 73.2 and 243.9 μg L^?1, respectively. Recoveries ranged between 98.30% and 101.30%, and the precision of this method was lower than 5% (RSD). The method conducted in this work was successfully applied to determine EC concentration in Chinese yellow rice wine samples from different origins. The negative correlation between EC formation and urea level in yellow rice wine samples is evaluated with the developed HPLC‐FLD method.     

Effects of sterilization temperature on the concentration of ethyl carbamate and other quality traits in Chinese rice wine

Ethyl carbamate (EC), which is present in Chinese rice wine, has a large potential for carcinogenicity and genotoxicity. EC is produced during the process of rice wine fermentation and storage. High concentrations of precursors, as well as high temperatures, will significantly accelerate the formation of EC. The present work aims to reduce EC formation by optimizing the production process, especially the boiling procedure. With various boiling sterilization temperatures, EC accumulated to different concentrations but the lower the temperature, the less EC was formed. To preserve the quality traits of Chinese rice wine, including biological and non‐biological stability, as well as the sugar component, an 80°C boiling temperature is suggested. The present study provides direction for process optimization, which combined with improved production technology and metabolic engineered yeast strains, can reduce the content of EC in Chinese rice wine. Copyright © 2014 The Institute of Brewing & Distilling     

Cloning,sequencing and disruption of the ARG8 gene encoding acetylornithine aminotransferase in the petite-negative yeast Kluyveromyces lactis

A recombinant plasmid was isolated from a Kluyveromyces lactis genomic DNA library which complements a Saccharomyces cerevisiae arg8 mutant defective in the gene encoding acetylornithine aminotransferase. The complementation activity was found to reside within a 2.0 kb DNA fragment. Nucleotide sequence analysis revealed an open reading frame able to encode a 423-residue protein sharing 68·1% and 35·0% sequence identities with the products of the ARG8 and argD genes of S. cerevisiae and Escherichia coli. That the cloned gene, KlARG8, is the functional equivalent of S. cerevisiae ARG8 was supported by a gene disruption experiment which showed that K. lactis strains carrying a deleted chromosomal copy of KlARG8 are auxotrophic for arginine. The nucleotide sequence of KlARG8 has been submitted to GenBank under Accession Number U93209.     

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。     

YDL080C和LEU2基因敲除对工业黄酒酵母异戊醇生成量的影响

通过敲除生成途径中关键酶的编码基因,对异戊醇在工业黄酒酵母N85中的生成进行了研究。采用融合PCR技术,分别构建类丙酮酸脱羧酶基因（YDL080C）缺失组件\     

低产尿素黄酒酵母工程菌的酿造特性

为考察酵母工程菌在黄酒酿造过程中的发酵性能及其降低发酵液中尿素和氨基甲酸乙酯(ethyl carba-mate,EC)的能力,以前期构建的降低黄酒中尿素和EC效果最好的酵母工程菌N85^DUR1,2-c为研究对象,利用单因素试验考察黄酒发酵工艺对其降低发酵液中尿素和EC能力的影响,并对其在生产试验过程中的发酵性能进行研究。结果表明,酵母接种量、发酵温度以及麦曲添加量等工艺参数对工程菌N85^DUR1,2-c低产尿素和EC的性能没有明显的影响,且含量低于亲本菌株。50 kL生产试验表明,工程菌N85^DUR1,2-c所酿黄酒中理化指标含量正常,符合黄酒国标的要求。而N85^DUR1,2-c发酵液中尿素和EC的含量分别为(2.4±0.2)mg/L和(14.9±0.6)μg/L,较亲本菌株分别降低了90.7%和54.6%,且贮存过程中EC含量增加缓慢。说明酵母工程菌N85^DUR1,2-c在不改变黄酒优良品质的前提下,能够显著地降低发酵液中尿素的含量,可以从根源上减少黄酒中EC的积累,提高饮用...     

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     

Flavour characteristics of rice wine fermented with mixed starter by moulds and yeast strains

Mixed starter of pure mould and yeast was designed to circument the problem of flavour characteristics in rice wine. The result underlined that the mixed starter (Q7) made by simultaneous inoculation of Rhizopus oryzae, Saccharomyces cerevisiae, Wickerhamomyces anomalus and Candida glabrata had the highest contents of α-amylase (8.02 ± 0.96 U/g) and sensory scoring (92.05 ± 2.24), presenting relatively strong flower-aroma, wine-aroma, Qu-aroma and rice-aroma. Meanwhile, the total concentration of flavour compounds in rice wine (RQ, 125.828 mg L⁻¹) obtained from rice fermentation using Q7 were better than the commercially available rice wine (SS, 125.828 mg L⁻¹). The E-nose analysis showed that the flavour components in RQ differed dramatically from SS. The E-tongue results indicated that the sourness, sweetness and umami aftertaste were greatly higher in RQ, and the bitterness and astringency are lower compared with SS. This study provided a method to improve the flavour and quality of rice wine.     

Optimization of fermentation conditions for Chinese bayberry wine by response surface methodology and its qualities

Colour and flavour are both important challenges for bayberry wine production because of a shortage of selected strains. The yeast Saccharomyces cerevisiae strain YF152, separated from a natural Chinese bayberry fermentation mash [alcohol tolerance capacity (18.0%), SO₂ (200 mg/L), sugar content (40%) and pH (2.50)] was employed to brew Chinese bayberry wine. Response surface methodology was used to simultaneously analyse the effects of the fermentation conditions on Chinese bayberry wine. The optimum conditions were found to be a temperature of 26.5 °C, an initial sugar content of 22.0°Brix, an inoculum size of 4.0% and an initial pH of 2.90. Under these conditions, the final alcohol content of the bayberry wine was 13.4%, the anthocyanin content was 77 mg/L and the residual sugar content was 1 g/L. These numbers agreed well with the predicted values. The major characteristic flavour components of the wine were 3‐methyl‐1‐butanol, 2‐phenylethanol, ethyl acetate, 2‐methylbutyl acetate and acetic acid. Copyright © 2016 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。