Production of high quality Chinese rice wine largely depends on fermentation temperature. However, there is no report on the ethanol, sugars, and acids kinetics in the fermentation mash of Chinese rice wine treated at various temperatures. The effects of fermentation temperatures on Chinese rice wine quality were investigated. The compositions and concentrations of ethanol, sugars, glycerol, and organic acids in the mash of Chinese rice wine samples were determined by HPLC method. The highest ethanol concentration and the highest glycerol concentration both were attained at the fermentation mash treated at 23°C. The highest peak value of maltose (90 g/L) was obtained at 18°C. Lactic acid and acetic acid both achieved maximum values at 33°C. The experimental results indicated that temperature contributed significantly to the ethanol production, acid flavor contents, and sugar contents in the fermentation broth of the Chinese rice wines.
Chinese rice wine, a natural and nondistilled wine, is very popular in China and its market is speedily increasing [
Similar to sake and other rice wine varieties, the fermentation process of Chinese rice wine brewing can be divided into two stages: the main stage (also called primary fermentation) and the second stage (also called postfermentation). In the main stage, pre-steamed rice,
The main stage of the fermentation process is a typical simultaneous saccharification and fermentation (SSF) process as well as a semisolid state and semiliquor state fermentation (SSSLF) process. As the concentration of pre-steamed rice and wheat in mash is very high (can be as high as 45%), the SSF and SSSLF process may decrease yeast cell growth inhibition with high sugar concentration and facilitate ethanol production in Chinese rice wine brewing. The concentration of ethanol can thus be high and even more than 20% (v/v) in the final mash at the end of the main stage fermentation [
Temperature effects on wine fermentation have been widely investigated in beer [
The proportions of sugars, glycerol, ethanol, and organic acids are primarily responsible for the delicate taste and flavors of Chinese rice wine [
In the past, pursuit of high ethanol concentration is the main goal for wine fermentation. At present, volatile compounds in wine have become the new important parameters to evaluate the wine quality [
Due to the increasingly recognized importance of sugars and acids and their relationship to wine quality, it is important to investigate the effect of temperature on the yeast fermentation, organic acid, and glycerol compound during Chinese rice wine brewing. The experiment which simulated Chinese rice wine fermentation process was implemented at various temperatures (18°C, 23°C, 28°C, and 33°C) in a scale-down level. Based on previous research, 33°C is the highest temperature designed in plant fermentation process, 28°C is the desired temperature for this yeast cell growth [
The results of the study contributed significantly to the understanding of the role of temperature in ethanol, organic acids, glycerol, and sugars kinetics during Chinese rice wine brewing and also provided useful information to improve the quality of Chinese rice wine.
Yeast extract peptone dextrose medium (YPD) includes glucose 20 g/L, peptone 20 g/L, yeast extract 10 g/L, and agar 20 g/L.
Liquid yeast extract peptone dextrose medium (LYPD) includes glucose 20 g/L, peptone 20 g/L, and yeast extract 10 g/L.
The yeast strain was stored at 4°C on slants of yeast peptone dextrose agar medium (YPD). The yeast inoculum was transferred to a new slant of YPD and cultured for 24 h at 28°C. A sloop of yeast culture was added to 50 mL LYPD medium (250 mL flask) and cultured at 28°C for 18 h as yeast seed. The prepared yeast seed was diluted at a ratio of 1 : 10 with new LYPD (100 mL in 500 mL bottle) medium and cultured at 28°C for another 18 h.
Fermentation experiments were conducted in a 7 L tank fermenter (BioFlo IV, NBS Edison, NJ, USA). Sticky rice was steamed for 45 min and cooled at room temperature to 26°C. 1200 g steamed rice (dry weight), 204 g wheat
The fermentation process was maintained at constant temperature as designed for 4 days. 2 mL of samples from each experiment was taken out every 2 h and centrifuged at 10000 r/min for 3 min, filtered (0.22
Submicron-filtered HPLC-grade water was used. D-glucose, D-fructose, maltose, maltotriose, and sulfuric acid purchased from Sigma-Aldrich (St Louis, MO, USA) were used. HPLC-grade lactic acid, acetic acid, succinic acid, citric acid, malic acid, tartaric acid, propionic acid, ethanol, and acetonitrile purchased from Fisher (Pittsburgh, PA, USA) were used.
In order to compare the effect of various fermentation conditions on the Chinese rice wine brewing process, several enological parameters were determined offline right after the samples were taken out of the fermenter.
The concentrations of sugars, glycerol, ethanol, and organic acids were determined with HPLC (Agilent 1200 series). At the predetermined time, wine samples of 1 mL were taken for analyses. Durapore (PVDF, 0.45
Every 1000 mL mobile phase consisted of 1270
HPLC system (Agilent 1200 Series, Santa Clara, CA, USA) was used. Separate calibration standard curves were constructed.
The statistical analysis of the final lactic acids and ethanol concentrations was performed with SAS software (version 9.3; SAS Institute, Cary, NC).
Temperature is an important controlling parameter controlling the Chinese rice wine quality. However, it is still not clear why it affects the quality of Chinese rice wine. As ethanol is the main product of Chinese rice wine, exploring the effect of temperature on ethanol production is necessary for optimizing rice wine fermentation.
In this work, ethanol production under various temperatures (RT, 18, 23, 28, and 33°C) was investigated and presented in Table
Ethanol concentration (%, v/v ± SD) under various temperatures.
Temperature | RT | 18°C | 23°C | 28°C | 33°C |
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Ethanol (%, v/v) |
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Detailed evaluation of the effect of various temperatures on ethanol production is an efficient way to analyze the kinetics of ethanol fermentation during Chinese rice wine production which are shown in Figure
The profiles of ethanol concentration under various temperatures.
For the first 20 hours, the ethanol concentration increased with temperature. The highest concentration was achieved at 33°C and the lowest concentration was detected at 18°C. From 20 h to 70 h, the profiles of ethanol concentration under 23°C, 28°C, and RT were all similar. After 20 h, ethanol concentration under 33°C only has just a little fluctuation. At 18°C the ethanol concentration kept growing and was much higher than that of 33°C, but slightly lower than that at other temperatures at the 70 h. However, from 70 h to 100 h, the profile of ethanol concentration at RT and 23°C increased quickly, which only slightly increased at 28°C. From 100 h to 140 h, the ethanol concentration at 18°C increased quickly. However, under the other temperature, there were only slight variations of the ethanol concentrations. At 33°C, the ethanol concentration only slightly increased from 20 h to 140 h. All these data indicate that temperatures have different effect on ethanol production kinetics at different fermentation stages.
Ethanol production kinetics at various stages of Chinese rice wine brewing under designed temperatures was further analyzed (Table
Effect of various temperatures on the ethanol concentration (%, v/v ± SD) at different stages.
Temperature | Ethanol production (%, v/v ) | |||||||
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0–14 h | 14–24 h | 24–37 h | 37–47 h | 47–57 h | 57–74 h | 74–96 h | 96–140 h | |
RT |
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18°C |
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23°C |
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28°C |
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33°C |
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Sugars in the fermentation mash of Chinese rice wine not only are important nutrient components for rice wine production but also contribute to its taste and flavor. Chinese rice wine (commonly known as Shaoxing huangjiu) is divided into four types according to its total sugar contents as shown in the Introduction. Among it, semidry rice wine is the most popular [
The production of sugars and glycerol in the fermentation mash of Chinese rice wine fermentation with designed experiments, including glucose, maltose, maltotriose, and glycerol, was analyzed. The fermentation profiles of sugars and glycerol are shown in Figure
Time course of changes in productive concentration of sugars and glycerol under various temperatures: (a) glycerol, (b) fructose, (c) maltotriose, (d) maltose, and (e) glucose.
However, the concentrations for maltotriose at various temperatures are different from that for glycerol and fructose. The maximum concentrations of maltotriose at various temperatures were in the descending order of 33°C 18°C, 28°C, RT, and 23°C (Figure
The fermentation kinetics of maltotriose and maltose are quite similar. This phenomenon can be explained below. Under low and high temperature, the fermentation rate is low. As maltose and maltotriose were utilized slowly by the microbial cells, both the observed residual maltose and maltotriose were higher. However, the low fermentation rate is different between low and high temperatures. The cellular metabolic activity at low temperature is generally low which conceivably caused retarded ethanol biosynthesis. On the contrary, at higher temperature, cellular aging process was accelerated which reduced ethanol formation at most of the fermentation processes. In addition,
A certain amount of acids played an important role in the flavor of rice wine and gradually converted into aromatic esters during storage. Therefore, the total acid content in the fermentation mash of Chinese rice wine is a key parameter to evaluate and control the fermentation process during industrial Chinese rice wine brewing [
The fermentation profiles of organic acids are shown in Figure
Effect of changing temperature on lactic acid concentration (g/L ± SD) at different stages.
Temperature | Lactic acid concentration (g/L) | |||||||
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0–14 h | 14–24 h | 24–37 h | 37–47 h | 47–57 h | 57–74 h | 74–96 h | 96–140 h | |
RT |
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18°C |
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23°C |
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28°C |
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33°C |
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Time course of changes in the concentration of organic acids under various temperatures: (a) succinic acid, (b) lactic acid, (c) acetic acid, (d) pyruvate acid, (e) malic acid, and (f) tartaric acid.
Only the concentrations of lactic acid and acetic acid at 33°C are statistically significantly higher than that at other temperatures which agrees with that of Mao [
Results from this study have shown that different fermentation temperatures affect the levels of sugars, glycerol concentration, and organic acid in the fermentation medium for Chinese rice wine production. The highest concentration of ethanol was achieved at 23°C. The lowest concentration of ethanol was at 33°C.
Higher temperatures can enhance organic acid production through stimulation of the growth of
Although it is proven that temperatures can affect the production of ethanol, glycerol, and organic acid, their optimal level in Chinese rice wine fermentation and how to accurately control their ratio through controlling temperatures are still not clear. Consequently, developing a kinetic model to describe the effect of temperatures on ethanol, glycerol, and organic acid production during Chinese rice wine fermentation is needed and is currently in progress.
The authors declare that there is no conflict of interests regarding the publication of this paper.
The authors thank Drs. Feng Ding, Ya Guo, Ru Dai, and Ti Zhang and Ms. Connie Liu and Mr. Lakdas Fernando for their technical assistance. Shaoxing Nverhong Rice Wine Company is acknowledged for the material support. This work was supported in part by research grants from the National Science Foundation of China (21276111, and 21206053), Zhejiang Science and Technology Project (2011C12033), China Scholarship Council (no. 2010679023), and Jiangnan University P.h.D. Research Fund (no. 20110149).