Ferulic Acid Production in the Brewing of Rice Wine (Sake)

The traditional Japanese alcoholic beverage sake is produced by fermentation of rice by Saccharomyces cerevisiae and Aspergillus oryzae. A. oryzae releases ferulic acid, an antioxidant, from steamed rice during the fermentation process. The concentration of ferulic acid increased with time during fermentation and the production rate peaked 9–12 days post inoculation. Analysis of the fermentation cultures of Aspergillus oryzae, by high‐performance liquid chromatography (HPLC), revealed that p‐coumaric acid induced an 18.9‐fold increase in the level of ferulic acid. Furthermore, SDS‐PAGE analysis revealed an increase or decrease in the level of specific proteins after the addition of p‐coumaric acid to fermentation cultures of Aspergillus oryzae. Ferulate esterase (FAE) activity was observed in the fermented sake ten days following the start of the fermentation process. These results suggest that the level of ferulic acid is regulated by the enzymes synthesized by A. oryzae during the sake brewing process.     

Changes in Volatile Compounds of Chinese Rice Wine Wheat Qu During Fermentation and Storage

In order to understand the optimum time of using wheat Qu for Chinese rice wine brewing on the basis of the contents of volatile compounds, changes in volatile compounds of Chinese rice wine wheat Qu during fermentation and storage were studied by headspace solid phase microextraction (HS‐SPME) combined with gas chromatography‐mass spectrum (GC‐MS). It was shown that the concentrations of most groups of volatiles, including alcohols, aldehydes, aromatic compounds, phenols, sulphides, furans, and nitrogen‐containing compounds, increased progressively from the first day to the fourth day of fermentation, and then decreased gradually. A minority group of volatile compounds such as acids and ketones peaked after one day of fermentation, esters increased slowly from the first day to the eighth day of fermentation. Lactones and esters rose slowly from the first day to the fifteenth day of fermentation respectively, and decreased afterwards, whereas the evolution of terpenes was somewhat erratic. These results suggested that changes in the concentrations of most groups of volatile compounds during fermentation and storage were uniform. This made it possible to determine the optimum level of fermentation and storage for Chinese rice wine wheat Qu on the basis of the contents of volatile compounds.     

采用GC/MS和HPLC-FLD2种方法测定黄酒中的EC含量

针对黄酒特性,采用固相萃取-气相色谱/质谱联用法(SPE-GC/MS)和高效液相色谱-荧光法(HPLC-FLD)分别测定黄酒中氨基甲酸乙酯(EC)含量,并进行了系统优化和比对。结果表明,SPE-GC/MS和HPLC-FLD法均能准确检测黄酒中的EC含量,测定结果可靠。其中,SPE-GC/MS法灵敏度较高,已被国际权威机构AOAC、OIV和欧盟纳入标准检测方法,宜选作我国黄酒EC含量检测方法标准。而HPLC-FLD法测定黄酒中EC更为简单快速,成本较低,易于掌握,适用于生产企业过程监控。     

孝感米酒中乳酸菌的分离及其对黄酒品质的影响

目的:探明孝感米酒中乳酸菌的种类及其对黄酒品质的影响。方法:采用传统可培养方法使用MRS培养基、厌氧工作站,对孝感米酒中乳酸菌进行分离,并接入黄酒对黄酒发酵过程进行干预,进而通过分析黄酒有机酸与滋味等理化特征来评价乳酸菌的发酵特性。结果:从孝感米酒中分离到9株乳酸菌,基于16S rDNA基因对乳酸菌分离株进行了系统发育分析,结果显示它们属于魏斯氏菌属(Weissella)、片球菌属(Pediococcus),种水平上分为戊糖片球菌(Pediococcus pentosaceus)、融合魏斯氏菌(Weissella confusa)、食窦魏斯氏菌(Weissell acibaria)。将这9株菌应用于黄酒发酵制作,在菌株融合魏斯氏菌MJ3-1与MJ4-1、戊糖片球菌MJ6-1、食窦魏斯氏菌MJ8-1干预下,黄酒中乳酸含量显著增加(p<0.05),苹果酸含量显著减少(p<0.05);同时黄酒的滋味中咸味和丰度有显著增加(p<0.05)。总体上来说,乳酸菌对黄酒滋味的影响由酸味、鲜味与丰度引起。结论:融合魏斯氏菌MJ4-1能增加黄酒乳酸,并降低苹果酸含量作用最强,适用于黄酒复合型发酵剂的研发。     

麦芽在黄酒生产中的应用

通过在黄酒发酵过程中以麦芽部分代替麦曲,探索麦芽对黄酒生产的影响。结果表明,麦芽、麦曲最佳质量配比为1∶1,其总量为原料米的10%(w/w),酿出的成品黄酒色泽橙黄、麦香浓郁、口味淡爽、风格独特。试验发现珍珠岩和硅藻土共助滤可有效改善黄酒的沉淀问题,其总用量可控制在2～3 g/L黄酒。结果表明,黄酒发酵过程中,添加麦芽可减少麦曲用量,改善黄酒的品质及风味。     

Determination of β‐Phenylethanol in Chinese Rice Wine by Reverse Phase High‐Performance Liquid Chromatography

The objective of this study was to develop a reverse phase HPLC method for determining the content of β‐phenylethanol in Chinese rice wine. The β‐phenylethanol was separated using a Synergi Hydro‐RP C18 (4.6 mm × 250 mm, 4 μm) column, CH₃OH:H₂O (50:50, v/v) as the mobile phase, and a diode array detector set at 210 nm. The results showed that when 5.00?30.00 mg/L of β‐phenylethanol was fortified, the average recoveries were between 99.5% and 99.8%, the RSD was 0.6% (n = 6), and the limit of determination was 0.05 mg/L. The calibration curve was linear over a range of 0.5 mg/L to 50 mg/L (R = 0.9999). The results of this method were almost the same as that of the gas chromatography method. The developed method is suitable to determine the β‐phenylethanol content in Chinese rice wine.     

米糠中阿魏酸的提取及荧光分光光度法检测

确定了新鲜米糠中阿魏酸的最佳提取工艺。采用高效液相色谱法对提取产品的定性检测,荧光分光光度法对提取产品的定量检测。阿魏酸在0.2～2.2μg/ml浓度范围内呈良好的线性关系,相关系数为0.99957,平均回收率为99.0%。此法具有准确、简便、快速、灵敏等优点。     

The Influence of Yeast Strains on the Volatile Flavour Compounds of Chinese Rice Wine

In order to assess the influence of yeast strains on volatile flavour profiles of Chinese rice wine, small‐scale Chinese rice wine brewing was carried out with eight yeast strains from three different typical Chinese rice wine brewing regions. The volatile flavour compounds were extracted by headspace solid phase microextraction (HS‐SPME) and analyzed by gas chromatography‐mass spectrometry (GC‐MS). The volatile flavour profiles of the different Chinese rice wines showed statistically significant differences depending on the yeast strains used. Yeast strains from the Shaoxing region showed a higher capacity to produce the esters 2‐phenylethanol and 3‐methylthiopropanol, while yeast strains from the Shanghai region stood out for their production of branched‐chain higher alcohols. Chinese rice wine fermented with a yeast strain from the Jiangsu region had the highest levels of organic acids. Using principal component analysis of the Chinese rice wine volatile flavour compounds, the eight yeast strains could be classified into three groups according to their origins. This is the first report about the volatile flavour characteristics of Chinese rice wine yeast. The data obtained in this work shows that the yeast strains contributed significantly to the flavour differences of the Chinese rice wines from the different regions.     

黄酒前酵中生物胺生成规律的研究

本研究对黄酒前酵工序的生物胺生成规律及影响因素进行了分析探讨。采用反相高效液相色谱技术,改进了生物胺定量检测法,该检测体系准确可靠,样品峰型对称分离度好且缩短了检测时间。分析了前酵工序中主要微生物,氨基酸,发酵醪酸度、糖度、酒精度、p H对生物胺生成的影响。发现前酵工序对成品酒生物胺的影响度为77.67%,第一次开耙阶段生物胺总量增幅最大,达到7.63 mg/L。研究发现主要酿造微生物中乳酸菌总数与生物胺总量成正相关,乳酸菌在搭窝期间生长速率最大,为7.13×106CFU/(m L·h)。在前酵工序中生物胺总量与发酵醪酸度、酒精度、p H呈显著正相关,与糖度呈负相关;前酵过程中主要生物胺与前体氨基酸也呈明显正相关。本研究分析了黄酒前酵中生物胺生成规律,有助于建立降低黄酒生物胺含量的更安全、科学的工艺。     

快速浸米酿制黄酒新工艺研究

研究快速淋米酿造和传统浸米工艺酿造黄酒生产过程,监控发酵过程酸度和酒精度的变化规律,对比分析发酵酒理化指标和风物成分。结果表明:米最佳吸水时间为1 h,快速淋米工艺发酵酒的酸度、氨氮、pH等理化指标升高明显,而β-苯乙醇含量降低;快速淋米工艺发酵酒的醛类、酮类、酯类、酸类等挥发性风味物质含量较传统浸米工艺发酵酒有所提高,醇类物质含量减少。     

黄酒中氨基甲酸乙酯的分析与控制

氨基甲酸乙酯是一种存在于黄酒中的致癌物质。本文采用气质联用法检测了广东市场上42种黄酒样品中氨基甲酸乙酯含量,28个样品中EC含量超过30μg/kg唱,占66．67％,其中7个样品中EC含量超过了100μg/kg（日本清酒中EC的限量标准）,最高的高达1210μg/kg。研究了脲酶对黄酒处理的效果,结果发现,一定条件下添加脲酶能够去除酒里的尿素,破坏产生氨基甲酸乙酯的主要途径,从而控制氨基甲酸乙酯含量不再剧增。并在单因素试验结果的基础上,进行正交实验,得出脲酶最佳的处理条件为酶浓度150mg／L、处理温度为25℃,pH值为3．5,处理时间10d。该条件下进行处理,可使酒中氨基甲酸乙酯含量降低45．83％。     

Identification of Low Molecular Weight Peptides in Chinese Rice Wine (Huang Jiu) by UPLC‐ESI‐MS/MS

The aim of this work was to increase the general knowledge of peptide identification and to examine the low molecular weight peptides in Chinese rice wine (Huang Jiu) as there are very few publications in the literature on this topic. Chinese rice wine (Huang Jiu) has a high content of peptides and more than 500 peptides have been tentatively identified by ultra‐performance liquid chromatography‐quadrupole time‐of‐flight tandem mass spectrometry (UPLC‐ESI‐MS/MS), including 43 potential bio‐active peptides and 3 sensory‐active peptides, based on published references to date. These peptides have been tentatively identified according to the MS/MS spectra which produced sufficient information of product ions (complementary y ions and b ions). Results from this work indicated that it is feasible to produce rice wine with rich bioactive peptides and that there is potential to examine for rice wine authenticity and traceability based on the origin of the peptides.     

Simultaneous Determination of 15 Phenolic Constituents of Chinese Black Rice Wine by HPLC‐MS/MS with SPE

This study established a new method for quantitative and qualitative determination of certain components in black rice wine, a traditional Chinese brewed wine. Specifically, we combined solid‐phase extraction and high‐performance liquid chromatography (HPLC) with triple quadrupole mass spectrometry (MS/MS) to determine 8 phenolic acids, 3 flavonols, and 4 anthocyanins in black rice wine. First, we clean samples with OASIS HLB cartridges and optimized extraction parameters. Next, we performed separation on a SHIM‐PACK XR‐ODS column (I.D. 3.0 mm × 75 mm, 2.2 μm particle size) with a gradient elution of 50% aqueous acetonitrile (V/V) and water, both containing 0.2% formic acid. We used multiple‐reaction monitoring scanning for quantification, with switching electrospray ion source polarity between positive and negative modes in a single chromatographic run. We detected 15 phenolic compounds properly within 38 min under optimized conditions. Limits of detection ranged from 0.008 to 0.030 mg/L, and average recoveries ranged from 60.8 to 103.1% with relative standard deviation ≤8.6%. We validated the method and found it to be sensitive and reliable for quantifying phenolic compounds in rice wine matrices.     

黄酒发酵液中产生物胺乳酸菌的分离鉴定与评价

从黄酒发酵液中分离得到6株乳酸菌,16S rDNA序列分析和生理生化鉴定结果表明:菌株R1、R4、R5、R8、R20为Lactobacillus rossiae,菌株R2为Lactobacillus casei。采用平板检测法对6株乳酸菌产生物胺能力进行了评价,结果显示只有乳酸菌R1具有产生物胺的能力。利用HPLC检测对平板检测结果进行了验证,结果表明乳酸菌R1能产598.61 mg/L的腐胺,其他菌株不具有产生物胺的能力。平板检测与HPLC检测结果相一致,表明平板检测可作为一种有效、操作简单、费用低的定性评价乳酸菌产生物胺能力的手段。     

Enhancing the levels of 4‐vinylguaiacol and 4‐vinylphenol in pilot‐scale top‐fermented wheat beers by response surface methodology

Top‐fermented wheat beers are desired by consumers because of their distinctive clove‐like and phenolic aroma, in which two characteristic flavour substances, 4‐vinylguaiacol (4VG) and 4‐vinylphenol (4VP), are of vital importance. In this paper, the levels of 4VG and 4VP were the subject of an intensive and detailed investigation. The influence of wheat malt proportion, mashing‐in temperature, boiling time and fermentation temperature on 4VG and 4VP was studied by pilot‐scale brewing experiments using a 10 hL mashing vessel and 20 hL fermentation tanks. Response surface methodology (RSM) was used to explore the possibilities for enhancing the concentration of 4VG and 4VP in top‐fermented wheat beers. Statistical analysis of the results showed that for the ranges of wheat malt proportion studied, mashing‐in temperature, boiling time and fermentation temperature all had a significant influence (p <0.05) on 4VG and 4VP production. Based on the response surface plots, the optimal brewing parameters that maximized 4VG and 4VP levels were as follows: (a) wheat malt proportion 40%; (b) mashing‐in temperature 44°C; (c) boiling time 88 min; and (d) fermentation temperature 19.5°C, resulting in significantly increased levels of 4VG and 4VP (2.418 mg/L and 1.402 mg/L, respectively). These brewing and fermentation parameters were concluded to be the optimal conditions to highlight the typical flavour and aroma of top‐fermented wheat beers. Copyright © 2015 The Institute of Brewing & Distilling     

Effect of ‘wheat Qu’ on the fermentation processes and volatile flavour‐active compounds of Chinese rice wine (Huangjiu)

Wheat Qu is a unique saccharifying agent used for Chinese rice wine brewing. In recent years, the impact of wheat Qu on Chinese rice wine flavour has attracted great interest. In this study, Chinese rice wine fermentations were conducted with different saccharifying agents (wheat Qu and mixed commercial enzymes) to study the effect of wheat Qu on the fermentation process and aroma characteristics of Chinese rice wine. Our results showed that wheat Qu not only acted as a saccharifying agent, but also increased yeast activity and the formation of aroma compounds. A higher yeast growth and fermentation rate, in addition to a lower yeast death rate, were observed when wheat Qu was used as a saccharifying agent. Global analysis of the aromatic profiles revealed that the use of wheat Qu greatly promoted the formation of the aroma compounds during a Chinese rice wine fermentation. The concentrations of the volatile phenolic compounds and higher alcohols were higher in the Chinese rice wine samples fermented with wheat Qu. It was concluded that the role of wheat Qu cannot simply be replaced by commercial enzymes and that the Chinese rice wine aromatic profiles can be modulated depending on the amount of wheat Qu used in the fermentation. Copyright © 2013 The Institute of Brewing & Distilling     

乌衣红曲黄酒生产工艺研究

通过对乌衣红曲黄酒传统发酵工艺进行分析和研究,获得乌衣红曲黄酒酿造的最佳工艺参数为:浸曲温度控制在24℃左右、持续时间为2d、酵母添加量为0.25‰;发酵温度25℃左右、乌衣红曲的添加量为20%、发酵时间为20d,此参数为乌衣红曲黄酒的工业生产提供了理论依据。     

Determination of γ‐aminobutyric acid in Chinese rice wines and its evolution during fermentation

γ‐Aminobutyric acid (GABA) is a functional amino acid that is widely present in Chinese rice wine. In this study, high‐performance liquid chromatography coupled with ultraviolet detection (HPLC‐UV) was established for the determination of γ‐aminobutyric acid in 22 Chinese rice wines collected from the Shaoxing region of China. Furthermore, the evolution of GABA was studied in Chinese rice wine during primary and post‐fermentation process. Results showed that the HPLC method was reliable with good linearity, accuracy, precision and stability. Additionally, the GABA content varied significantly in the 22 Chinese rice wines, and the content was much higher in wine samples with long aging periods. Regarding the evolution of GABA in Chinese rice wine during the brewing process, the level slowly increased during primary fermentation. A decrease in GABA was observed in the wine at the early stage of the post‐fermentation process. However, a marked increase on the GABA content occurred in wine at the late stage of post‐fermentation. The findings from this study are that HPLC can be successfully applied to determine GABA in Shaoxing brewed rice wines, and further provide useful information on quality control of such wines. Copyright © 2017 The Institute of Brewing & Distilling     

A comparative study on the fungal communities of wheat Qu for Qingshuang‐type Chinese rice wine

A traditional culture‐dependent method and ribosomal intergenic spacer analysis (RISA) were employed to investigate the fungal communities in two representative samples of wheat Qu (S1, S2). A total of seven species were isolated on media of potato dextrose agar, Czapek and wheat Qu extraction. Owing to the difference in the culture media, a high variability of predominant species was observed. RISA profiles of total DNA exhibited distinguishable bands corresponding to nine fungal species. Similar RISA profiles of two starter cultures were obtained. Sequencing of the internal transcribed spacer fragments from fungal isolates and RISA fingerprint profiles revealed the presence of nine species of fungi in wheat Qu S1: Lichtheimia ramosa, Absidia corymbifera, Aspergillus sydowii, Aspergillus oryzae, Rhizomucor pusillus, Eurotium amstelodami, Issatchenkia orientalis, Emericella nidulans and Cladosporium cladosporioides. In comparison with S1, S2 had a richer fungal diversity. The fungal species derived from S1 were all present in S2 coupled with another two species, namely, Aspergillus niger and Clavispora lusitaniae. Among all species isolated, Aspergillus sydowii was the dominant species in Qingshuang‐type Chinese rice wine wheat Qu. Copyright © 2012 The Institute of Brewing & Distilling     

黄酒发酵醪中特有乳酸杆菌检测和培养基的研究及初步鉴定

通过可溶性淀粉的添加量、培养温度、"X"物质的添加量三因素三水平对黄酒发酵醪中乳酸杆菌培养基配方和培养方法的正交试验研究,以及性能的测定和初步鉴定,表明:黄酒发酵醪中乳酸杆菌培养、分离的培养基配方和培养方法为:加饭酒稀释至酒精度14.5%voL、加"X"物质的量1.3%、加可溶性淀粉0.5%或不加、培养温度18℃或30℃、压盖密封培养;黄酒后发酵醪中的优势菌,在上述培养基中18℃生长良好,25℃及以上生长不良或生长极慢,初步鉴定为乳酸杆菌的新种,命名为黄酒乳酸杆菌Ⅱ-1(Lactobacillus chinese-rice-wineⅡ-1)。黄酒前发酵醪中的优势菌,在上述培养基中30℃生长良好,20℃及以下生长缓慢,初步鉴定为乳酸杆菌的新种,命名为黄酒乳酸杆菌Ⅱ-2(Lactobacillus chinese-rice-wineⅡ-2)。