Rice Wine Production Research Articles

Mechanism of Polygonum hydropiper reducing ethyl carbamate in Chinese rice wine (Huangjiu) brewing

Polygonum hydropiper (PH) is a rich source of active compounds and serves as a pivotal ingredient in Chinese rice wine (Huangjiu) production. This study investigates the impact of PH and Polygonum hydropiper extract (PHE) on ethyl carbamate (EC) production during Huangjiu fermentation. Our findings reveal that PH enhances the relative abundance of Bacillus subtilis in Huangjiu fermentation, thereby facilitating its interaction with Saccharomyces cerevisiae. Furthermore, PH modulates the urea metabolism of S. cerevisiae. In the PH-B. subtilis-S. cerevisiae fermentation system, the expression of DUR1,2 and DUR3 genes in S. cerevisiae is upregulated. This augmentation leads to increased urea uptake and metabolism by S. cerevisiae in the fermentation broth, subsequently reducing the urea concentration in the fermentation medium (The EC content in the CK group was approximately 355.55 % and 356.05 % higher than those in the PH and PHE groups, respectively). Consequently, PH demonstrates promise in reducing the EC concentration of Huangjiu, offering a novel approach to enhance the safety of Huangjiu consumption.

Advancements and Future Directions in Yellow Rice Wine Production Research

Yellow rice wine is a traditional fermented beverage in China. The microorganisms in the brewing process play a crucial role in shaping the composition and quality of the wine through their regulation of microbial growth, fermentation metabolites, metabolic balance, and ethanol production. Yellow rice wine not only has culinary value but also possesses potential medicinal value. This is attributed to the presence of polyphenolic compounds, antioxidants, and other natural products that can provide antioxidant and other probiotic effects. The fermentation process of yellow rice wine offers potential nutritional supplementation and improved digestion. While traditional brewing techniques have long been employed, modern biotechnology helps enhance the quality and stability of the wine by selecting suitable microbial strains, optimizing fermentation conditions, and precisely controlling the fermentation process. The collection of diverse fermentation microbes and the construction of specifically designed microbiota for yellow rice wine production could expedite the production of high-quality yellow rice wine. The ultimate goal is to enhance the nutritional value, conditioning function, and overall consumption experience of yellow rice wine. Future research will delve into exploring the relationship between microorganisms and active ingredients in yellow rice wine, improving both the quality and functionality of the wine.

Effect of ultrasound-assisted fermentation on physicochemical properties and volatile flavor compounds of Chinese rice wine

The traditional fermentation method for Chinese rice wine (RW) production has some limitations such as difficulty in controlling quality, long brewing time, and thin taste. In this study, ultrasonic technology was adopted by adding enzyme with the aim of improving the fermentation efficiency and volatile flavor quality of RW. Results showed that the ethanol yield was increased by 23.53 % and the fermentation time was reduced by nearly 2 days by ultrasonic treatment of (one-time, 28 kHz, 1 h, 35 W/L) on the first day. Flavor analysis (following the ultrasonic treatment) showed that the flavors, especially the esters and alcohols in RW, also enhanced most significantly. The contents of isobutyl acetate, ethyl butyrate, ethyl hexanoate, and phenethyl acetate increased by 58.03 %, 107.70 %, 31.84 %, and 18.71 %, respectively. The results showed that the brewing process could be simplified, brewing time could be reduced and volatile flavor of RW could be improved by ultrasound.

Application of multiple ultra-high-pressure homogenization to the pasteurization process of Japanese rice wine, sake

Hiire is a pasteurization process in the production of Japanese rice wine (sake), which stabilizes the quality of product; however, it also generates the carcinogen ethyl carbamate (EC). In this study, we investigated the application of ultra-high-pressure homogenization (UHPH) as an alternative sterilization method for sake production. Microbiological analysis revealed that multiple UHPH treatments sterilized hiochi lactobacilli (Lactobacillus fructivorans, L. homohiochii, L. casei, and L. hilgardii) and Saccharomyces cerevisiae. Enzyme activity assays revealed that α-amylase, glucoamylase, and acid-carboxypeptidase activities were reduced to less than 1% of the levels in non-pasteurized sake after four-time UHPH treatment. These results show that UHPH treatment meets the two requirements of the sake sterilization process sterilization and enzyme inactivation. The UHPH-processed sake did not show any significant changes in general properties but had reduced organic acid and aromatic component contents, with ethyl caproate content showing the most significant reduction of approximately 20%. Interestingly, EC was detected in pasteurized sake but not in UHPH-processed sake. These findings indicate that the UHPH technology could be used to inactivate microorganisms and enzymes in sake without generating EC.

Lactobacillus plantarum F25‐4 assisted rice soaking reduces the cooking strength of japonica rice for Chinese rice wine brewing

SummaryThis study investigated the effect of Lactobacillus plantarum F25‐4 assisted rice soaking (LAS) on the cooking properties of japonica rice used for Chinese rice wine (CRW) production. The effects of LAS and natural soaking were compared based on the chemical composition, thermal characteristics, infrared spectroscopy and sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of rice flour. After LAS, the protein content of the rice was reduced by 17.63%, and the initial temperature (To), peak temperature (Tp) and conclusion temperature (Tc) were reduced by 36.86%, 17.89% and 1.61%, respectively, as revealed by differential scanning calorimetry analysis. The Zeta (ζ) potential value changed from −8.75 mV to −3.99 mV, and the minimum cooking time was reduced by 40% to only 15 min. The viscoelasticity of LAS rice increased after gelatinisation, as confirmed by confocal laser scanning microscope images. The glutelin precursor of the LAS rice disappeared, and the disorder of protein secondary structure increased, but there was no significant difference in the starch crystal area. LAS effectively improved the cooking properties of rice by changing the protein configuration on the surface of starch granules and reducing protein adsorption, leading to increased starch water absorption and decreased rice gelatinisation temperature. Our research suggests that LAS can reduce the cooking temperature of rice in CRW production, thus saving steam consumption during cooking.

Comparative analysis of microbial communities and volatile flavor components in the brewing of Hongqu rice wines fermented with different starters

As one of the quintessential representatives of Chinese rice wine, Hongqu rice wine is brewed with glutinous rice as the main raw material and Hongqu (Gutian Qu or Wuyi Qu) as the fermentation starter. The present study aimed to investigate the impact of Hongqu on the volatile compositions and the microbial communities in the traditional production of Gutian Hongqu rice wine (GT) and Wuyi Hongqu rice wine (WY). Through the OPLS-DA analysis, 3-methylbutan-1-ol, isobutanol, ethyl lactate, ethyl acetate, octanoic acid, diethyl succinate, phenylethyl alcohol, hexanoic acid and n-decanoic acid were identified as the characteristic volatile flavor components between GT and WY. Microbiome analysis revealed significant enrichments of Lactobacillus, Pediococcus, Aspergillus and Hyphopichia in WY brewing, whereas Monascus, Saccharomyces, Pantoea, and Burkholderia-Caballeronia-Paraburkholderia were significantly enriched in GT brewing. Additionally, correlation analysis showed that Saccharomyces, Lactobacillus, Weissella and Pediococcus were significantly positively correlated wih most characteristic volatile components. Conversely, Picha, Monascus, Franconibacter and Kosakonia showed significant negative correlations with most of the characteristic volatile components. Furthermore, bioinformatical analysis indicated that the gene abundances for enzymes including glucan 1,4-alpha-glucosidase, carboxylesterase, alcohol dehydrogenase, dihydroxy-acid dehydratase and branched-chain-amino-acid transaminase were significantly higher in WY compared to GT. This finding explains the higher content of higher alcohols and characteristic esters in WY relative to GT. Collectively, this study provides a theoretical basis for improving the flavor profile of Hongqu rice wine and establishing a solid scientific foundation for the sustainable development of Hongqu rice wine industry.

Environmental factors drive microbial succession and huangjiu flavor formation during raw wheat qu fermentation

As the core raw material of huangjiu (Chinese rice wine) production and brewing, changes of physichemical factors during the fermentation process significantly affect the succession of the microbial community of raw wheat qu (RWQ), also playing important role in huangjiu quality. In this report we analyzed the community structure and driving force RWQ microorganisms with different processes (manual qu, MAQ; mechanical qu, MEQ), and evaluating the flavor of huangjiu. The production procedure was divided into three stages. Dominant microorganisms were Aspergillus, Alternaria, Epicoccus, Bacillus, Pantoea, Saccharopolyspora and Staphylococcus. In early stage, microbial community of MAQ was significantly correlated with temperature and water content, oxygen in middle stage, while no significant driving force in the last stage. Water content and oxygen affected MEQ community significantly in early stage, while in the final stage, fungal community was affected by both temperature and water content, and bacterial community was significantly correlated with oxygen. Glucoamylase activity reached its maximum at 50 °C, while the α-amylase activity decreased with the rise of temperature. The highest amylase activity was obtained with 19% initial water addition. Amylase activity showed positive correlation with the thickness of RWQ. The total amount of organic acids was higher in mechanical huangjiu at the end of pre-fermentation. Contents of n-hexanol, ethyl isovalerate, ethyl hexanoate and guaiacol in manual huangjiu were significantly higher than those in mechanical one, while the content of 4-vinylguaiacol was lower.

Saccharification Efficiency Improvement by Eurotium cristatum and its Mechanism Study during the Glutinous Rice Wine Fermentation

AbstractTo verify whether Eurotium cristatum promotes the saccharification efficiency of glutinous rice wine is the aim of this study. Thus, Anhua black tea (containing substantial E. cristatum) is used for co‐fermentation with liqueur koji in glutinous rice fermentation. The glutinous rice starch is analyzed to infer the effect of E. cristatum on saccharification efficiency, and the quality of co‐fermented glutinous rice wine is evaluated. The results show that E. cristatum can contribute to the saccharification efficiency of glutinous rice wine. Saccharification efficiency reaches a maximum at 40 g 100 g−1 when 1.0 g liqueur koji and 0.6 g Anhua black tea are added in 100 g glutinous rice during the fermentation process at 32 °C for 3 days. In the glutinous rice starch, co‐fermentation by E. cristatum results in increased amylose content, decreased molecular weight, smaller starch particles, and obvious crystal structure. These phenomena in the starch molecules express the mechanism of saccharification efficiency promotion by E. cristatum. What's more, the glutinous rice wine co‐fermented by Anhua black tea has potential health functions for the human body and presents better sensory evaluation than the traditional one. Therefore, it is feasible and significant for the co‐fermentation with E. cristatum during glutinous rice wine production.

Power ultrasound to enhance the fermentation process of traditional Taiwanese rice wine

AbstractRice wine is a fermented product that can be distilled to produce rice spirit, which is an ingredient in several Asian dishes. The industry usually uses traditional practices for rice wine production. This study aims to evaluate the possibility of enhancing Taiwanese rice wine fermentation through the application of ultrasound as an emerging technology. In this regard, in an academic‐industrial cooperation, a commercial process was modified by incorporating a 550‐W power horn ultrasound treatment after the saccharification step. Independent parameters were sonication times of 0, 3, 6, 9, and 12 min (referred to as control, US3, US6, US9, and US12, respectively) and fermentation time (0, 2, 4, 6, qnd 8 days), and dependent parameters were total phenolic content (TPC), total acidity, alcohol content, CIE color values, pH, total acidity, electrical conductivity (EC), and refractive index (RI). The results showed that US3 provided the highest alcohol content while the physical characteristics, including pH, total acidity, EC, and RI, were not significantly different from those of the control sample. At the same time, sonication alters color values (L*, a*, b*, color intensity, and overall color changes) and TPC. Besides, US12 treatment yielded a rice wine with the highest TPC, that is, 306.77 ± 1.90 mg GAE/L sample. Therefore, sonication conditions can be optimized to achieve the desired properties such as the highest alcohol content or highest TPC. This study revealed that a relatively mild sonication process can enhance the fermentation process to achieve higher alcohol content which could be related to the stress‐response of yeast (Saccharomyces cerevisiae) and modification of fermentation medium. This can be considered an effort to contribute to sustainable development goals (SDGs) and sustainability by increasing resource efficiency. Besides, a more intense sonication resulted in a fermented product with higher amounts of bioactive compounds through the improved extraction process. According to the observations, power ultrasound is a promising pretreatment to boost the fermentation process and rice wine quality through positive effects on both brewer's yeast and fermentation substrates.Practical ApplicationsThe wine processing industry is seeking innovative approaches to enhance the fermentation yield, reduce fermentation time, and enhance the production capacity. In this sense, the results of this industrial‐academic collaborative research showed that power ultrasound has the potential to improve rice wine fermentation to meet the above‐mentioned industrial demands. This emerging technology at appropriate processing conditions enhanced the percentage of alcohol in the final product which could be considered as an approach to enhance the profit of wineries through enhancing the resource efficiency, that is, more amount of product from the same amount of raw materials. At the same time, this sonication‐based technique did not negatively affect the physicochemical properties of the rice wine. The production platform explored in this study is scalable and can be implemented at industrial levels in wineries.

Evaluation of six commercial koji on the formation of biogenic amines and higher alcohols in rice wine

Commercial koji are increasingly used as the microbial inoculum in the production of rice wine. This study reports on the use of six commercial koji on rice wine brewing and shows that the microbial composition varied significantly among the different koji. Of the koji, AQ (An Qi) and YB (Yi Bin) contained fewer microflora but exhibited higher starch degradation and wine production efficiency. The Two-way Orthogonal PLS model and Pearson correlation analysis suggests that the presence of biogenic amines in rice wine was related to contaminating microorganisms in the koji including Lactobacillus, Kosakonia, Pantoea, Ralstonia, Kosakonia and Pantoea. The formation of higher alcohols is related to Saccharomyces species. Screening of beneficial yeasts with low production of higher alcohols and avoiding contamination by unwanted bacteria are suggested for improving the quality of rice wine. © 2022 The Institute of Brewing & Distilling.

Identification of Yeast and Mould Isolated from murcha in Nepal for Rice Wine Production

HIGHLIGHTS Two novel yeast strains were identified by sequencing for rice wine production. Rice wine (low alcohol by volume) was prepared from local raw materials of Nepal. Yeast and mould isolated from Parbat murcha showed potential fermentative characters.

Isolation and characterization of yeast for the production of rice wine with low fusel alcohol content.

Fusel alcohols (FAs) are a type of flavor compound found in rice wine. An overly high FA content not only leads to spicy, bitter, and astringent taste but also has side effects. Therefore, screening for yeast that produce low FA contents has attracted much attention. Thirty-two yeast strains were isolated from fermenting material during Luzhou-flavor liquor production in this study. Strain YB-12 was selected as a suitable candidate for rice wine production. The strain was identified as a member of the genus Meyerozyma based on phylogenetic analysis using 26S rDNA gene sequences. The ability of strain YB-12 to produce ethanol was similar to that of Saccharomyces cerevisiae NRRL Y-567, while isobutanol and isoamyl alcohol production was only 53.96% and 50.23%, respectively, of that of NRRL Y-567. The FA yield of rice wine produced with strain YB-12 was reduced to 51.85% in a 20 L fermenter. These results demonstrate that strain YB-12 presents promising characteristics for use in the production of rice wine with a potentially low content of FAs.

Study on the evaluation standard of extruded glutinous rice starch with thermostable α‐ amylase for making Chinese rice wine

SummaryTo promote the industrial production of Chinese rice wine made by enzymatic extrudates, the first is to ensure the consistency of the raw material. That is, an evaluation standard of enzymatic extrudate should be set up. It was studied for the first time. By studying the changes of seven indexes of samples at different sites of extruder barrel and the die along the extrusion direction and making the correlation analysis to select the representative indexes. The results showed that the change trends of dextrose equivalent (DE), molecular weight distribution (MWD) and oligosaccharide composition (OC) were the same, and these indexes were more affected by mechanical force than by temperature. The ability of degradation of different zones was: conveying area < kneading area << exit of the die. There was little difference in extent of gelatinisation (GE) at the temperature where the enzyme activity was relatively high. Different from DE value, water solubility index (WSI) rose gradually. Surprisingly, DE was negatively correlated with GE, and positively correlated with oligosaccharides except DP1 and DP6, and had no significant correlation with water absorption index (WAI) and WSI. In conclusion, DE, WSI, DPD (especially DP6) could be used as a comprehensive evaluation index of enzymatic extrudates.

Novel Angiotensin-I Converting Enzyme Inhibitory Peptides Isolated From Rice Wine Lees: Purification, Characterization, and Structure-Activity Relationship.

The bioactive peptides that can inhibit angiotensin-I converting enzyme (ACE, EC. 3. 4.15.1) are considered as possible cures of hypertension. Food-derived angiotensin-I converting enzyme inhibitory (ACEi) peptides have gained more attention because of their reduced side effects. In this study, we reported the method for purifying ACEi peptides from the lees of traditional Chinese rice wine and evaluated the product's biochemical properties. After three steps of reversed-phase high-performance liquid chromatography (RP-HPLC), for the first time, we isolated, purified, and identified two novel peptides: LIIPQH and LIIPEH, both of which showed strong ACEi activity (IC50-values of 120.10 ± 9.31 and 60.49±5.78 μg/ml, respectively). They were further categorized as mixed-type ACE inhibitors and were stable against both ACE and gastrointestinal enzymes during in vitro digestion. Together, these results suggest that the rice wine lees that produced as a by-product during rice wine production can be utilized in various fields related to functional foods and antihypertensive medicine.

Effect of baking technique for rice wine production and the characteristics of baked rice wine.

Driven by the requirement to reduce the emission of wastewater in the brewing process, it is necessary to improve and innovate the fermentation technology of Chinese rice wine. In this study, the baking technique used to brew rice wine was explored. Rice wine was brewed based on baked glutinous rice undergoing different high-temperature stages, designated as baked rice wine (BRW1, BRW2 and BRW3). X-ray diffraction and Fourier transform infrared analysis revealed that baking treatment under 110 °C 0.5 h + 170 °C 30 min relatively changed the crystal properties and short-range molecular order of starch. Compared with the traditional rice wine (RW) from steamed rice, the alcohol content in BRWs was nearly twice that of RW, especially in BRW3. The contents of protein, ascorbic acid and total phenols in BRWs were significantly higher than that in RW. Besides, BRWs presented more abundance in the contents of volatile compounds, free amino acids and certain organic acids, including volatile esters and alcohols and 17 amino acids, which would give rice wine a pleasant aroma and a more comprehensive taste. Furthermore, analysis of the antioxidant capacity indicated a functional difference between RW and BRWs. It was feasible to produce rice wine using baked rice. The baking method allowed for several advantages, including the improvement of alcohol yield, fermentation efficiency, new typicality and stronger antioxidant capacity. This work is expected to provide a foundation for related research. © 2021 Society of Chemical Industry.

Fermentation profiling of rice wine produced by Aspergillus oryzae KSS2 and Rhizopus oryzae KJJ39 newly isolated from Korean fermentation starter

The objective of this study was to determine the fermentation characteristics of rice wine produced by koji inoculated with Aspergillus oryzae KSS2 and Rhizopus oryzae KJJ39 on moisturized wheat-bran and rice grain. We also compared rice wine samples produced in this study and three commercial Makgeolli. The alcohol content was about 12% higher in the rice wine samples fermented by wheat-bran koji than in the other samples. In all of the samples, the range of pH value was 3.8–4.6 and the total acid was below 0.5. The soluble solid content was highest in the rice wine sample prepared by the wheat-bran R. oryzae KJJ39 koji (15.5°Brix) and overall relatively higher in the samples with wheat-bran koji than rice koji. The content of reducing sugar was twofold higher in rice wine prepared by koji inoculated with R. oryzae KJJ39 than A. oryzae KSS2. The volatile acid content was higher in rice wine produced by the wheat-bran A. oryzae KSS2 koji than the others. In the analyses of five organic acids, A. oryzae KSS2 was found to produce more malic acid and fumaric acid while R. oryzae KJJ39 to do more citric acid, lactic acid and acetic acid. The rice wine sample prepared with the wheat-bran A. oryzae KSS2 koji contained much higher concentration of sucrose, maltose and amino acids. Comprehensively, the results of fermentation profiling suggest that both A. oryzae KSS2 and R. oryzae KJJ39 can be applied to the production of rice wine as a valuable fungal isolate for fermentation start.

Effects of simultaneous and sequential cofermentation of Wickerhamomyces anomalus and Saccharomyces cerevisiae on physicochemical and flavor properties of rice wine.

Microorganism species and inoculation fermentation methods have great influence on physicochemical and flavor properties of rice wine. Thus, this work investigated microbial interactions and physicochemical and aroma changes of rice wine through different inoculation strategies of Wickerhamomyces anomalus (W. anomalus) and Saccharomyces cerevisiae (S. cerevisiae). The results underlined that inoculation strategies and non‐Saccharomyces yeasts all affected the volatile acidity, total acidity, and alcohol content of rice wine. The sequential cofermentation consumed relatively more sugar and resulted in the higher ethanol content, causing reduced thiols and increased alcohols, esters, phenylethyls, and terpenes, which was more conducive to improve rice wine flavor than simultaneous cofermentation. Moreover, simultaneous cofermentation increased fatty aroma of rice wine, while sequential cofermentation increased mellow and cereal‐like flavor. These results confirmed that sequential cofermentation of S. cerevisiae and W. anomalus was a choice for the future production of rice wine with good flavor and quality.

Profiling of Fungal Diversity and Fermentative Yeasts in Traditional Chinese Xiaoqu.

To increase the safety and quality of baijiu and rice wine in China, controlling the use of traditional Xiaoqu by studying the beneficial yeasts present has recently been considered. The fungal diversity of six Chinese Xiaoqu including five traditional and one commercial samples was investigated to screen fermentative yeasts with low yields of higher alcohols. A high throughput sequencing approach detected fifteen fungal species with relative abundance higher than 1%, and displayed dissimilarities of fungal diversity among Xiaoqu samples. The 15 fungal species were composed of 11 filamentous fungi with Rhizopus arrhizus as the most common specie and four yeast species, containing Hyphopichia burtonii, Saccharomyces cerevisiae, Saccharomycopsis fibuligera, and Saccharomycopsis malanga. Classic culture-dependent approaches, including 5.8S-ITS-RFLP analysis and D1/D2 sequencing of the 26S rRNA gene, identified nine yeast species in the five traditional Chinese Xiaoqu. In addition to the four yeast species also detected by high throughput sequencing approach, the other five yeast species isolated were Clavispora lusitaniae, Cryptococcus neoformans, Komagataella pastoris, Trichosporon asahii, and Wickerhamomyces anomalus. Further micro-fermentations of rice wine were performed using 19 single yeast isolates, and after the fermentation of rice wine, higher alcohols and ethanol were analyzed by gas chromatography. Two yeast strains, Saccharomyces cerevisiae FBKL2.8022 and Wickerhamomyces anomalus FBKL2.8023, were found to have low yields of higher alcohols and could produce 11.70%vol and 7.10%vol ethanol separately. This study for the first time, to the best of our knowledge, explored the fungal resources in traditional Xiaoqu from different regions of Guizhou, China. The screened S. cerevisiae and W. anomalus strains could be used to establish specific starters to promote the standardization of the production of baijiu and rice wine.

Influence of vacuum soaking on the brewing properties of japonica rice and the quality of Chinese rice wine

Soaking is an important process in Chinese rice wine brewing. In this study, the influence of vacuum soaking on Chinese rice wine production was investigated. Rice subjected to a 1-hvacuum soaking process or a traditional 2-days soaking process was steamed and fermented. Our results showed that vacuum soaking led to similar absorbed water but less leached solids compared with traditional soaking and showed limited influence on the physiochemical characteristics of steamed rice. Monitoring of the fermentation process suggested that the content of amino acid nitrogen in the vacuum-soaked group was significantly higher than that of the traditional-soaked group, while the other indexes were similar. The detection of flavor substances in the rice wine indicated that the contents of organic acids and free amino acids were higher in the vacuum-soaked group, and the main kinds of volatile flavor compounds from the two groups were similar. Additionally, sensory evaluation reflected that the rice wine brewed with rice subjected to either of the two different soaking treatments had similar sensory performances. Our research indicated that vacuum soaking could effectively shorten the soaking time of rice in Chinese rice wine production, thus shortening the brewing cycle without sacrificing the quality of the rice wine.

Metabolic engineering of Saccharomyces cerevisiae using the CRISPR/Cas9 system to minimize ethyl carbamate accumulation during Chinese rice wine fermentation.

Ethyl carbamate (EC) is a potential carcinogen to humans that is mainly produced through the spontaneous reaction between urea and ethanol during Chinese rice wine brewing. We metabolically engineered a strain by over-expressing the DUR3 gene in a previously modified strain using an improved CRISPR/Cas9 system to further decrease the EC level. Homologous recombination of the DUR3 over-expression cassette was performed at the HO locus by individual transformation of the constructed plasmid CRISPR-DUR3-gBlock-HO, generating the engineered strain N85DUR1,2/DUR3-c. Consequently, the DUR3 expression level was significantly enhanced in the modified strain, resulting in increased utilization of urea. The brewing test showed that N85DUR1,2/DUR3-c reduced urea and EC concentrations by 92.0% and 58.5%, respectively, compared with those of the original N85 strain. Moreover, the engineered strain showed good genetic stability in reducing urea content during the repeated brewing experiments. Importantly, the genetic manipulation had a negligible effect on the growth and fermentation characteristics of the yeast strain. Therefore, the constructed strain is potentially suitable for application to reduce urea and EC contents during production of Chinese rice wine. KEY POINTS: • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation. • An efficient CRISPR vector was constructed and applied for DUR3 over-expression. • Multi-modification of urea cycle had synergistic effect on reducing EC level. • Fermentation performance of engineered strain was similar with the parental strain. • No residual heterologous genes were left in the genome after genetic manipulation.