Chinese Rice Wine Fermentation Research Articles

Insights into the Biogenic Amine Metabolic Landscape during Industrial Semidry Chinese Rice Wine Fermentation

Inspired by concerns about food safety, the metabolic landscape of biogenic amines (BAs) was elucidated during industrial semidry Chinese rice wine fermentation. The main fermentation process represented the largest contribution to BA formation, which corresponded to 69.1% (54.3 mg/L). Principal component analysis revealed that total acid and ethanol were strongly correlated with BAs, indicating that BA formation favored acidic and stressful conditions. Other than putrescine (PUT), spermidine (SPD), and spermine (SPM), 5 BAs exhibited strong relationships with the precursor amino acids (R2 > 0.85). PUT was mainly decarboxylated from arginine (89.6%) whereas SPD (100%) and SPM (83.1%) were obtained from ornithine. Interestingly, some SPD could convert back to PUT (24.3%). All 8 BAs showed good relationships with lactic acid bacteria (LAB) (R2 around 0.75). Moreover, among the five main LAB genera, Lactobacillus had a positive correlation with BA formation.

The overexpression ofDUR1,2and deletion ofCAR1in an industrialSaccharomyces cerevisiaestrain 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

Correlation between the amino acid content in rice wine and protein content in glutinous rice

Chinese rice wine is a fermentation product of glutinous rice that contains high levels of protein and amino acids. The turnover and catabolism of amino acids by fermentative microorganisms plays an important role in wine quality. The fermentation of Chinese rice wine, using 34 different varieties of glutinous rice, and the analysis of the protein and amino acid content of the resultant rice wine using precolumn derivatization via high-performance liquid chromatography, are reported. A model of correlation and regression analysis of the protein content in the glutinous rice and the amino acids in rice wine was established. Results showed that the correlation coefficient between the total protein in glutinous rice and the total free amino acids in rice wine was 0.557, indicating a significant relevance. The population correlation coefficient between the total protein in the glutinous rice and the amino acids in the rice wine was high, i.e. R= 0.928. The correlation between the soluble protein content in the glutinous rice and the total free amino acids in the rice wine (or individual amino acids) was negligible. The total protein content in the rice variety was positively related to the sensory performance and free amino acid content of the resultant Chinese rice wine.

Comparative Transcriptomic Analysis of Saccharomyces cerevisiae under Limited Nitrogen Conditions during Fermentation of Chinese Rice Wine

Comparative Transcriptomic Analysis of Saccharomyces cerevisiae under Limited Nitrogen Conditions during Fermentation of Chinese Rice Wine

Effect of ‘wheat Qu’ addition on the formation of ethyl carbamate in Chinese rice wine with enzymatic extrusion liquefaction pretreatment

Ethyl carbamate (EC), which has been confirmed as probably carcinogenic to humans, can be formed during the alcoholic fermentation of Chinese rice wine. In this study, EC and two possible precursors (urea and arginine) were analysed in wines fermented from rice processed by enzymatic extrusion liquefaction pretreatment, with different wheat Qu content gradients from 6 to 18% (dry basis, d.b.). The EC concentration decreased 22.5% with the addition of wheat Qu reduced to 6% for the enzymatic extrusion-processed rice wine (EE). Moreover, although the arginine was generated 6.33–8.39 times higher in the enzymatic extruded rice, instead of the traditional treated rice at the same wheat Qu level, the corresponding level of urea formed from arginine was not high in the EE. In addition, rice treated by extrusion combined with enzymatic hydrolysis was found to be more utilizable and fermentable, and the lost quality of the final traditional rice wine with the low wheat Qu level (6–14%) could be improved using enzymatically extruded rice as the fermenting material. Therefore, it may be feasible to inhibit EC accumulation in Chinese rice wine by wheat Qu control, combined with enzymatic extrusion pretreatment for rice.

Sequencing-based screening of functional microorganism to decrease the formation of biogenic amines in Chinese rice wine

As a traditional alcoholic beverage, Chinese rice wine with a unique flavor and high nutritional value has been popular in China for thousands of years. The biogenic amines in Chinese rice wine and other fermented food are attracting much more attention than ever. There are numerous microorganisms accompanying the fermentation of Chinese rice wine. Based on the next-generation sequencing technology, an in vivo screening process was carried out to find the strains without biogenic amines formation, and then these strains were screened and back-added into the fermentation mash. It was found that the Lactobacillus might not produce biogenic amines by the correlation analysis of bacteria community succession and biogenic amines change. After that, three Lactobacillus strains without biogenic amines formation were screened and identified as Lactobacillus plantarum and L. hilgardii. The L. plantarum JN01 showed high lactic acid production, low acetic acid production, and high ethanol tolerance which was preferable for Chinese rice wine brewing. When 0.001 gDCW/t of L. plantarum JN01 was used to inoculate the fermentation mash, the biogenic amines concentration was significantly decreased 24%. Although the formation of biogenic amines was further inhibited by increasing the dose of L. plantarum JN01, the total acid and the residue sugar would increase resulting an incomplete fermentation. In this paper, we firstly developed an effective method to decrease the formation of biogenic amines in traditional fermentation foods based on the next-generation sequencing technology. This research provided new strategy to decrease biogenic amines in fermentation food.

Constitutive expression of the DUR1,2 gene in an industrial yeast strain to minimize ethyl carbamate production during Chinese rice wine fermentation.

Urea and ethanol are the main precursors of ethyl carbamate (EC) in Chinese rice wine. During fermentation, urea is generated from arginine by arginase in Saccharomyces cerevisiae, and subsequently cleaved by urea amidolyase or directly transported out of the cell into the fermentation liquor, where it reacts with ethanol to form EC. To reduce the amount of EC in Chinese rice wine, we metabolically engineered two yeast strains, N85(DUR1,2) and N85(DUR1,2)-c, from the wild-type Chinese rice wine yeast strain N85. Both new strains were capable of constitutively expressing DUR1,2 (encodes urea amidolyase) and thus enhancing urea degradation. The use of N85(DUR1,2) and N85(DUR1,2)-c reduced the concentration of EC in Chinese rice wine fermented on a small-scale by 49.1% and 55.3%, respectively, relative to fermentation with the parental strain. All of the engineered strains showed good genetic stability and minimized the production of urea during fermentation, with no exogenous genes introduced during genetic manipulation, and were therefore suitable for commercialization to increase the safety of Chinese rice wine.

Bacterial succession and the dynamics of volatile compounds during the fermentation of Chinese rice wine from Shaoxing region.

Shaoxing rice wine is one of the most typical representatives of Chinese rice wine. It is brewed under non-sterile condition with various microorganism growing at the same time and forms a special flavor. The aims of this study was to monitor the bacterial succession by MiSeq pyrosequencing and the volatile compound dynamics by HS-SPME/GC–MS during brewing process. Moreover, the volatile compounds and bacterial community were analyzed by partial least squares regression to evaluate the effect of bacteria on volatile compounds formation. The results showed that there were ten dominating genera during Shaoxing rice wine fermentation process. Ten genera, Bacillus, Leuconostoc, Lactococcus, Weissella, Thermoactinomyces, Pseudomonas, Saccharopolyspora, Staphylococcus, Enterobacter and Lactobacillus, were identified as the main bacteria. The Bacillus and Lactobacillus dominated the Chinese rice wine ecosystems. In addition, a total of 64 volatile compounds were identified, mainly esters, alcohols, carbonyl compound and phenols. Pseudomonas were involved in synthesis of a wide variety of volatile compounds. Thermoactinomyces, Bacillus and Lactococcus also played critical roles in the formation of volatile compounds.

Characterization of Volatile Flavor Compounds in Chinese Rice Wine Fermented from Enzymatic Extruded Rice.

Enzymatic extrusion, instead of traditional steam cooking, to treat rice is an efficient and alternative pretreatment for Chinese rice wine fermentation. In order to determine the formation of volatiles in enzymatic extrusion-processed rice wine (EE), and to confirm its characteristic flavor compounds, headspace solid-phase micro-extraction followed by GC-MS was used. A total of 66 volatile compounds were identified in EE. During fermentation, most volatiles generated from enzymatic extruded rice had the similar trends with those from steam-cooked rice, but the differences in the concentration of volatiles indicated a changed balance of flavors release caused by enzymatic extrusion. Besides, the concentrations and sorts of volatiles in EEs fermented from different rice particle sizes, were not dramatically different. By principal component analysis, EE could be distinctly separated from other traditional Chinese rice wines according to its characteristic volatiles, namely, 2-heptanol, 1-octen-3-ol, ethyl 4-hydroxybenzoate, methylpentyl 2-propenoate, γ-hexalactone, and 4-vinylguaiacol. Enzymatic extrusion liquefaction has been a popular thermal treatment for cereals, and gradually being applied in fermentation and liquor-making industry all over the world. The characterization of volatile flavor compounds in Chinese rice wine processed by enzymatic extrusion liquefaction pretreatment, might be made use not only for a better understanding of this new-type rice wine, but for the further utilization of enzymatic extrusion in other wine or alcohol production as well.

Decreased production of higher alcohols by Saccharomyces cerevisiae for Chinese rice wine fermentation by deletion of Bat aminotransferases.

An appropriate level of higher alcohols produced by yeast during the fermentation is one of the most important factors influencing Chinese rice wine quality. In this study, BAT1 and BAT2 single- and double-gene-deletion mutant strains were constructed from an industrial yeast strain RY1 to decrease higher alcohols during Chinese rice wine fermentation. The results showed that the BAT2 single-gene-deletion mutant strain produced best improvement in the production of higher alcohols while remaining showed normal growth and fermentation characteristics. Furthermore, a BAT2 single-gene-deletion diploid engineered strain RY1-Δbat2 was constructed and produced low levels of isobutanol and isoamylol (isoamyl alcohol and active amyl alcohol) in simulated fermentation of Chinese rice wine, 92.40 and 303.31mg/L, respectively, which were 33.00 and 14.20% lower than those of the parental strain RY1. The differences in fermentation performance between RY1-Δbat2 and RY1 were minor. Therefore, construction of this yeast strain is important in future development in Chinese wine industry and provides insights on generating yeast strains for other fermented alcoholic beverages.

Measurement of fermentation parameters of Chinese rice wine using Raman spectroscopy combined with linear and non-linear regression methods

Effective fermentation monitoring is a growing need during the manufacture of wine due to the rapid pace of change in the wine industry. Ethanol and reducing sugar are two most important process variables indicating the status of Chinese rice wine (CRW) fermentation process. In this study, the potentials of Raman spectroscopy (RS) as a rapid process analytical technique to monitor the evolution of these two chemical parameters involved in CRW fermentation process and to group samples according to different fermentation stages were investigated. The results demonstrated that compared with the PLS model using all wavelengths of Raman spectra, the prediction precision of model based on the spectral variables selected by competitive adaptive reweighted sampling (Cars) was significantly improved. In addition, nonlinear models outperformed linear models in predicting fermentation parameters. After systemically comparison and discussion, it was found that for both ethanol and glucose, Cars-support vector machine (Cars-SVM) models gave the best results with the highest prediction precisions. Moreover, the results obtained from discriminant partial least squares analysis (DPLS) showed that good performances were obtained with an average correct classification rate of 94.9% for different fermentation stages. The overall results indicated that RS combined with efficient variable selection algorithm and nonlinear regression tool could be utilized as a rapid method to monitor CRW fermentation process.

Reduction of biogenic amines production by eliminating the PEP4 gene in Saccharomyces cerevisiae during fermentation of Chinese rice wine

Biogenic amines in Chinese rice wine have a potential threat of toxicity to human health. In this study, PEP4 gene in Saccharomyces cerevisiae was knocked out in order to evaluate its effect on biogenic amines production; the enzyme encodes proteinase A (PrA), an enzyme that is responsible for the production of free amino acids. It was found that compared to the wild type strain, the PrA activity and amino acid concentration decreased significantly, and the production of biogenic amines in this knockout strain decreased by 25.5%, from 180.1mg/L to 134.2mg/L. Especially, tyramine, cadaverine and histamine concentrations were also decreased by 57.5%, 24.6% and 54.3%, respectively. The main reason for the decrease of biogenic amines may be due to the low concentration of free amino acids. Our results provide a new strategy to minimize the biogenic amine production during fermentation of Chinese rice wine.

Application of FT-NIR spectroscopy and FT-IR spectroscopy to Chinese rice wine for rapid determination of fermentation process parameters

NIR and MIR combined with chemometric tools were used to monitor time-related changes during Chinese rice wine fermentation.

Rapid Determination of Process Variables of Chinese Rice Wine Using FT-NIR Spectroscopy and Efficient Wavelengths Selection Methods

There is a growing need for the effective fermentation monitoring during the manufacture of wine due to the rapid pace of change in the wine industry. In this study, Fourier transform near-infrared (FT-NIR) spectroscopy combined with chemometrics was applied to monitor time-related changes during Chinese rice wine (CRW) fermentation. Various wavelength selection methods and support vector machine (SVM) algorithm were used to improve the performances of partial least squares (PLS) models. In total, ten different calibration models were established. It was observed that the performances of models based on wavelength variables selected by variable selection methods were much better than those based on the full spectrum. In addition, nonlinear models outperformed linear models in prediction of fermentation parameters. After systemically comparing and discussing, it was found that for both ethanol and total acid, genetic algorithm-support vector machine (GA-SVM) models obtained the best result with excellent prediction accuracy. The correlation coefficients (R2 (pre)), root mean square error of prediction (RMSEP), and the residual predictive deviation (RPD) for the prediction set were 0.94, 3.02 g/L, and 8.7 for ethanol and 0.97, 0.10 g/L, and 6.1 for total acid, respectively. The results of this study demonstrated that FT-NIR could monitor and control CRW fermentation process rapidly and efficiently with efficient variable selection algorithms and nonlinear regression tool.

Impact of High-Shear Extrusion Combined With Enzymatic Hydrolysis on Rice Properties and Chinese Rice Wine Fermentation

To better understand how high-shear extrusion combined with enzymatic hydrolysis affects the rice properties and subsequent fermentation, a twin screw extruder incorporated with rice treatment with thermostable α-amylase was used for simultaneous saccharification and fermentation (SSF) of Chinese rice wine. Seven pretreatment methods for rice were compared, and their impacts on rice properties and quality indices of final wine were studied. Enzymatic extruded rice had higher water solubility index (WSI), extent of gelatinization (GE), but lower water absorption index (WAI) and viscosity than those of other treated rice due to a strong synergy of shear action and enzymatic hydrolysis on the degradation and gelatinization of starch. The modification of enzymatic autoclaved rice was smaller than that of enzymatic extruded rice mainly for the lack of mechanical shearing force. Although extrusion with smaller particle size tended to have higher susceptibility to enzyme, the effect was not dramatic. This implied that manipulating particle size had limited effect on the modification of rice properties. Correlation analysis showed that the ethanol yield (EY), fermentation efficiency (FE), amino acid (AA), and soluble solid content (SSC) of finished rice wine increased as rice properties improved by enzymatic extrusion. Overall, simultaneous high-shear extrusion and enzymatic hydrolysis can be an attractive alternative to obtain modified rice with desirable properties for manufacturing Chinese rice wine or other fermentation products.

Monitoring of fermentation process parameters of Chinese rice wine using attenuated total reflectance mid-infrared spectroscopy

There is a growing need for the effective fermentation monitoring during the manufacture of wine due to the rapid pace of change in the industry. In this study, the potential of attenuated total reflectance mid-infrared (ATR-MIR) spectroscopy to monitor time-related changes during Chinese rice wine (CRW) fermentation was investigated. Interval partial least-squares (i-PLS) and support vector machine (SVM) were used to improve the performances of partial least-squares (PLS) models. In total, four different calibration models, namely PLS, i-PLS, SVM and interval support vector machine (i-SVM), were established. It was observed that the performances of models based on the efficient spectra intervals selected by i-PLS were much better than those based on the full spectrum. In addition, nonlinear models outperformed linear models in predicting fermentation parameters. After systemically comparison and discussion, it was found that i-SVM model gave the best result with excellent prediction accuracy. The correlation coefficients (R2 (pre)), root mean square error (RMSEP (%)) and the residual predictive deviation (RPD) for the prediction set were 0.96, 6.92 and 14.34 for total sugar, 0.97, 3.32 and 12.64 for ethanol, 0.93, 3.24 and 9.3 for total acid and 0.95, 6.33 and 8.46 for amino nitrogen, respectively. The results demonstrated that ATR-MIR combined with efficient variable selection algorithm and nonlinear regression tool as a rapid method to monitor and control CRW fermentation process was feasible.

Adaptive evolution of Saccharomyces cerevisiae with enhanced ethanol tolerance for Chinese rice wine fermentation.

High tolerance towards ethanol is a desirable property for the Saccharomyces cerevisiae strains used in the alcoholic beverage industry. To improve the ethanol tolerance of an industrial Chinese rice wine yeast, a sequential batch fermentation strategy was used to adaptively evolve a chemically mutagenized Chinese rice wine G85 strain. The high level of ethanol produced under Chinese rice wine-like fermentation conditions was used as the selective pressure. After adaptive evolution of approximately 200 generations, mutant G85X-8 was isolated and shown to have markedly increased ethanol tolerance. The evolved strain also showed higher osmotic and temperature tolerances than the parental strain. Laboratory Chinese rice wine fermentation showed that the evolved G85X-8 strain was able to catabolize sugars more completely than the parental G85 strain. A higher level of yeast cell activity was found in the fermentation mash produced by the evolved strain, but the aroma profiles were similar between the evolved and parental strains. The improved ethanol tolerance in the evolved strain might be ascribed to the altered fatty acids composition of the cell membrane and higher intracellular trehalose concentrations. These results suggest that adaptive evolution is an efficient approach for the non-recombinant modification of industrial yeast strains.

Decreased ethyl carbamate generation during Chinese rice wine fermentation by disruption of CAR1 in an industrial yeast strain

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.

Changes in flavour characteristics and bacterial diversity during the traditional fermentation of Chinese rice wines from Shaoxing region

As a traditional alcoholic beverage, Chinese rice wine is quite popular for its unique flavor and high nutritional value. In this study, we investigated the changes of flavor compounds in varying stages of fermentation by HS-SPME-GC-MS and HPLC. In addition, 16s rDNA-PCR was utilized to analyze the changes of bacterial communities during fermentation of Chinese rice wine from the Shaoxing region. It was noted that the content of flavor compounds including esters, higher alcohols, amino acids and organic acids was different in varying stages of fermentation. In general, the flavor compounds of Chinese rice wine were mainly produced after pre-fermentation. The results also showed that the bacterial community structures and diversity of Chinese rice wine varied significantly during different fermentation stages, and more than 10 genera of bacteria was detected in Chinese rice wine fermentation broth. Among these specific bacteria identified in the study, Lactobacillus (the predominant genus) and Bacillus might take an active part in flavor development in Chinese rice wine. This implied that these bacteria might play significant contributions on the flavor of Chinese rice wine. 2014 Published by Elsevier Ltd.

Molecular identification of lactic acid bacteria in Chinese rice wine using species-specific multiplex PCR

Chinese rice wine (CRW), a national unique and traditional food, has more than 4,000 years of history and is very popular in China. Lactic acid bacteria (LAB) play important roles in the fermentation of CRW because of its strong lactic acid production and high acid tolerance. It would be of practical interest to isolate the LAB species from CRW and to develop a simple and effective method for its accurate identification. In this study, 11 CRW-related LAB strains, frequently occurring in the fermentation of CRW, were isolated or collected and analyzed with the nested specifically amplified polymorphic DNA (nSAPD)-PCR technique. Eleven species-specific sequence characterized amplified region (SCAR) markers were generated from 11 polymorphic nSAPD bands. Three multiplex PCR assays were further established for the joint use of the 11 species-specific SCAR markers. Thus, a simple rapid molecular method was developed based on the application of nSAPD, SCAR, and multiplex PCR techniques, which allowed a rapid and simultaneous detection of 11 CRW-related LAB species. The simple rapid PCR-based molecular method can be used as a useful tool for the identification of LAB species in the traditionally fermented foods, especially in the rapid developing CRW industry in China.