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Li X.,Jiangnan University | Shen C.,Jiangnan University | Wu D.,Jiangnan University | Lu J.,Jiangnan University | And 3 more authors.
Journal of the Institute of Brewing

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. © 2015 The Institute of Brewing & Distilling. Source

Wu D.,Jiangnan University | Li X.,Jiangnan University | Shen C.,Jiangnan University | Lu J.,Jiangnan University | And 3 more authors.
International Journal of Food Microbiology

Saccharomyces cerevisiae metabolizes arginine to ornithine and urea during wine fermentations. In the fermentation of Chinese rice wine, yeast strains of S. cerevisiae do not fully metabolize urea, which will be secreted into the spirits and spontaneously reacts with ethanol to form ethyl carbamate, a potential carcinogenic agent for humans. To block the pathway of urea production, we genetically engineered two haploid strains to reduce the arginase (encoded by CAR1) activity, which were isolated from a diploid industrial Chinese rice wine strain. Finally the engineered haploids with opposite mating type were mated back to diploid strains, obtaining a heterozygous deletion strain (CAR1/. car1) and a homozygous defect strain (car1/. car1). These strains were compared to the parental industrial yeast strain in Chinese rice wine fermentations and spirit production. The strain with the homozygous CAR1 deletion showed significant reductions of urea and EC in the final spirits in comparison to the parental strain, with the concentration reductions by 86.9% and 50.5% respectively. In addition, EC accumulation was in a much lower tempo during rice wine storage. Moreover, the growth behavior and fermentation characteristics of the engineered diploid strain were similar to the parental strain. © 2014 Elsevier B.V. Source

Wang P.,Jiangnan University | Sun J.,Jiangnan University | Li X.,Jiangnan University | Wu D.,Jiangnan University | And 5 more authors.
Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment

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. © 2014 Taylor & Francis. Source

Yellow And Co and CHINA SHAOXING RICE WINE GROUP Corporation | Date: 1999-07-06

wine, fermented spirits and liqueurs.

Wu D.,Jiangnan University | Li X.,Jiangnan University | Shen C.,Jiangnan University | Lu J.,Jiangnan University | And 3 more authors.
Journal of the Institute of Brewing

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 G418r 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. © 2013 The Institute of Brewing & Distilling. Source

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