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 carbamate, 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N85DUR1,2-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N85DUR1,2-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N85DUR1,2-c所酿黄酒中理化指标含量正常,符合黄酒国标的要求。而N85DUR1,2-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N85DUR1,2-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,提高饮用安全性。
The purpose of this study was to investigate the fermentation performance of the engineered Saccharomyces cerevisiae strain and its capacity to decrease urea and ethyl carbamate (EC) in Huangjiu brewing. Single factor experiments were conducted to evaluate the effect of fermentation parameters on the brewing performance of the constructed S. cerevisiae strain N85DUR1,2-c, which performed best in reducing the concentrations of urea and EC. Meanwhile, an industrial production experiment was carried out in a Huangjiu brewery to study its fermentation characteristics. The results showed that the parameters including inoculation concentration, fermentation temperature and the amount of wheat Qu had no significant influences on urea and EC elimination property of N85DUR1,2-c. At industrial fermentation in 50 kiloliters tank, the contents of ethanol, total sugar, amino acid nitrogen, total acid and main flavour compounds in the fermentation liquor of N85DUR1,2-c were basically in accordance with the China national standard of Huangjiu. In addition, the concentrations of urea and EC were (2.4±0.2) mg/L and (14.9±0.6) μg/L in the liquor fermented by N85DUR1,2-c, which were 90.7% and 54.6% respectively which was less than in parental strain. Moreover, EC content in the Huangjiu sample fermented with N85DUR1,2-c increased at a much lower rate during storage compared with control. The concentration of urea can be significantly reduced in the Huangjiu sample fermented by constructed strain N85DUR1,2-c. Therefore, the EC level in rice wine could be fundamentally reduced, which is beneficial to its safety.
[1] 张顺荣, 范文来, 徐岩. 不同香型白酒中氨基甲酸乙酯的研究与风险评估 [J]. 食品与发酵工业, 2016, 42(5): 198-202.
[2] WU P G, PAN X D, WANG L Y, et al. A survey of ethyl carbamate in fermented foods and beverages from Zhejiang, China [J]. Food Control, 2012, 23(1): 286-288.
[3] NBREGA I C, PEREIRA J A, PAIVA J E, et al. Ethyl carbamate in cachaça (Brazilian sugarcane spirit): Extended survey confirms simple mitigation approaches in pot still distillation [J]. Food Chemistry, 2011, 127(3): 1 243-1 247.
[4] UTHURRY C A, VARELA F, COLOMO B, et al. Ethyl carbamate concentrations of typical Spanish red wines [J]. Food Chemistry, 2004, 88(3): 329-336.
[5] LIU Y P, DONG B, QIN Z S, et al. Ethyl carbamate levels in wine and spirits from markets in Hebei Province, China [J]. Food Additives and Contaminants: Part B, 2011, 4(1): 1-5.
[6] 华颖, 茅佩卿, 刘柱. GC-MS法测定黄酒和食用酒精中的氨基甲酸乙酯 [J]. 食品与发酵工业, 2019, 45(14): 196-202.
[7] KIM Y G, LYU J, KIM M K, et al. Effect of citrulline, urea, ethanol, and urease on the formation of ethyl carbamate in soybean paste model system [J]. Food Chemistry, 2015, 189:74-79.
[8] TANG A, CHUNG S, KWONG K, et al. Ethyl carbamate in fermented foods and beverages: Dietary exposure of the Hong Kong population in 2007-2008 [J]. Food Additives and Contaminants: Part B, 2011, 4(3): 195-204.
[9] FU M L, LIU J, CHEN Q H, et al. Determination of ethyl carbamate in Chinese yellow rice wine using high-performance liquid chromatography with fluorescence detection [J]. International Journal of Food Science and Technology, 2010, 45(6): 1 297-1 302.
[10] WANG P H, SUN J Y, LI X M, et al. Contribution of citrulline to the formation of ethyl carbamate during Chinese rice wine production [J]. Food Additives and Contaminants: Part A, 2014, 31(4): 587-592.
[11] WU D H, LI X M, SHEN C, et al. Decreased ethyl carbamate generation during Chinese rice wine fermentation by disruption of CAR1 in an industrial yeast strain [J]. International Journal of Food Microbiology, 2014, 180:19-23.
[12] WU D H, LI X M, LU J, et al. Constitutive expression of the DUR1, 2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation [J]. FEMS Microbiology Letters, 2016, 363(1): 1-6.
[13] WU D H, LI X M, SHEN C, et al. Isolation of a haploid from an industrial Chinese rice wine yeast for metabolic engineering manipulation [J]. Journal of the Institute of Brewing, 2013, 119(4): 288-293.
[14] 中国国家标准化管理委员会. GB/T 13662—2018 黄酒 [S]. 北京: 中国标准出版社, 2018.
[15] 邢江涛, 钟其顶, 熊正河, 等. 高效液相色谱-荧光检测器法测定黄酒中尿素含量 [J]. 酿酒科技, 2011(3): 104-106.
[16] 任江伟. 黄酒中氨基甲酸乙酯生成动力学的研究 [D]. 无锡: 江南大学, 2007.
[17] ZHAO X R, ZOU H J, FU J W, et al. Nitrogen regulation involved in the accumulation of urea in Saccharomyces cerevisiae [J]. Yeast, 2013, 30(11): 437-447.
[18] ZHAO X R, ZOU H J, DU G C, et al. Effects of nitrogen catabolite repression-related amino acids on the flavour of rice wine [J]. Journal of the Institute of Brewing, 2015, 121(4): 581-588.
[19] 戈瑚瑚, 倪莉. 黄酒饮后产生上头上火的原因探究 [J]. 食品与发酵工业, 2010, 36(8): 136-139.
[20] WU P G, CAI C G, SHEN X H, et al. Formation of ethyl carbamate and changes during fermentation and storage of yellow rice wine [J]. Food Chemistry, 2014, 152: 108-112.
[21] WU H M, CHEN L, PAN G S, et al. Study on the changing concentration of ethyl carbamate in yellow rice wine during production and storage by gas chromatography/mass spectrometry [J]. European Food Research and Technology, 2012, 235(5): 779-782.
