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Influence of fermentation temperature and source of enzymes on enological characteristics of rice wine

2014; Wiley; Volume: 120; Issue: 3 Linguagem: Inglês

10.1002/jib.128

2050-0416

Dengfeng Liu, Hongtao Zhang, Baoguo Xu, Jinglu Tan,

Metabolomics and Mass Spectrometry Studies

Journal of the Institute of BrewingVolume 120, Issue 3 p. 231-237 Research articleFree Access Influence of fermentation temperature and source of enzymes on enological characteristics of rice wine Dengfeng Liu, Dengfeng Liu Key Laboratory of Industrial Advanced Process Control for Light Industry, Ministry of Education, Jiangnan University, Wuxi, 214122 China Department of Bioengineering, University of Missouri, Columbia, MO, 65211 USASearch for more papers by this authorHongtao Zhang, Hongtao Zhang Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122 ChinaSearch for more papers by this authorBaoguo Xu, Corresponding Author Baoguo Xu Key Laboratory of Industrial Advanced Process Control for Light Industry, Ministry of Education, Jiangnan University, Wuxi, 214122 ChinaCorrespondence to: B. Xu and J. Tan. E-mail: xbg@jiangnan.edu.cn; tanj@missouri.eduSearch for more papers by this authorJinglu Tan, Corresponding Author Jinglu Tan Department of Bioengineering, University of Missouri, Columbia, MO, 65211 USACorrespondence to: B. Xu and J. Tan. E-mail: xbg@jiangnan.edu.cn; tanj@missouri.eduSearch for more papers by this author Dengfeng Liu, Dengfeng Liu Key Laboratory of Industrial Advanced Process Control for Light Industry, Ministry of Education, Jiangnan University, Wuxi, 214122 China Department of Bioengineering, University of Missouri, Columbia, MO, 65211 USASearch for more papers by this authorHongtao Zhang, Hongtao Zhang Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122 ChinaSearch for more papers by this authorBaoguo Xu, Corresponding Author Baoguo Xu Key Laboratory of Industrial Advanced Process Control for Light Industry, Ministry of Education, Jiangnan University, Wuxi, 214122 ChinaCorrespondence to: B. Xu and J. Tan. E-mail: xbg@jiangnan.edu.cn; tanj@missouri.eduSearch for more papers by this authorJinglu Tan, Corresponding Author Jinglu Tan Department of Bioengineering, University of Missouri, Columbia, MO, 65211 USACorrespondence to: B. Xu and J. Tan. E-mail: xbg@jiangnan.edu.cn; tanj@missouri.eduSearch for more papers by this author First published: 14 April 2014 https://doi.org/10.1002/jib.128Citations: 6AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Abstract Rice wine samples were produced with four sources of enzymes, and with three fermentation temperatures. Key enological variables (including ethanol, the main sugars, glycerol and organic acids) were measured by HPLC at the end of primary fermentation (4 days) and at the end of the postfermentation period (40 days). The results showed that both source of enzymes and temperature had significant effects on the main flavour compounds. Ethanol varied initially, but its final concentration showed a weak dependence on the treatments. Faster starch hydrolysis led to higher initial sugar concentrations, but lower final sugar and higher final glycerol concentrations. Production of organic acids, lactic acid and acetic acid, in particular, depended on the fermentation temperature. The results provide insights into the rice wine fermentation process as affected by different enzymes and fermentation temperatures. Copyright © 2014 The Institute of Brewing & Distilling Introduction Rice wine is one of the oldest alcoholic beverages in the world. It is widely consumed in Asia and other parts of the world. It has unique aromas and subtle flavours 1. Research has shown that rice wine has numerous compounds that are beneficial to health 2-4. China alone has more than 700 rice wineries 5. Chinese rice wine, one of the rice wine categories, is made by fermenting glutinous rice with yeast (Saccharomyces cerevisiae) and Chinese wheat Qu, which has been used to produce alcoholic beverages since ancient times. Similar to sake and other rice wine varieties, the Chinese rice wine-making process has two features. First, it uses a simultaneous saccharification and fermentation (SSF) process; and second, Chinese wheat Qu, instead of purified enzymes, is used for liquefaction and saccharification. Wheat Qu contains microorganisms, but additional yeast is often used for fermentation. Figure 1 shows a flowchart of the Chinese rice wine production process. The SSF process reduces yeast cell exposure to high sugar concentrations during fermentation and thus ethanol concentrations over 20% (v/v) can be achieved 6. Figure 1Open in figure viewerPowerPoint Flowchart of the Chinese rice wine production process. Chinese wheat Qu is mainly a culture of Aspergillus spp. and is similar to the Koji used for sake production. To make wheat Qu, wheat grain is milled, mixed with natural water, and pressed in a mould. Then, it is incubated at 28 − 30°C for 48 h and dried at 45°C until the moisture is lower than 12% (w/w) 7. The naturally fermented wheat Qu contains complex enzymes and various microorganisms, including bacteria, yeasts and fungi 8. Different kinds of wheat Qu are in use by the rice wine industry. One, named Shengqu, is prepared by the traditional method described as above. Another, called Shuqu, is made through the same process but with two fungal strains to produce enzymes of higher saccharifying activity 6, 9. When purified enzymes instead of wheat Qu are used for rice wine-making, ethanol production can be enhanced, but flavour loss has been observed 10. This suggests that the complex microorganisms and enzyme systems in wheat Qu are responsible for producing certain flavours and aroma compounds in rice wines. In the rice wine-making process, many flavour compounds are produced. Research shows that Chinese rice wines contain 21 varieties of amino acids, nine varieties of organic acids, over 10 varieties of esters, and various vitamins 11. Ethanol concentration is usually between 15 and 20% (v/v). Rice wines have sweet, sour, spicy, bitter and other flavours. The proportions of sugars, glycerol, ethanol and organic acids are primarily responsible for the delicate taste and flavours of Chinese rice wines 12. Sugars and glycerol contribute to sweetness and smoothness, ethanol results in a spicy taste and organic acids give rise to acidic flavours. Malic acid and acetic acid are very sharp and strong, and thus the concentrations of these two acids should be kept low 12. Lactic acid gives smooth dryness and is desirable. A wine yields subtle flavours and full body by having complementary proportions of these components. Chinese rice wine-making is a two-stage fermentation process. In the first stage, which lasts approximately 4 days, the main product is ethanol and the by-products are organic acids, glycerol and other components. In the second stage, fermentation continues for an additional 40 days. During this time, the ethanol content is further increased, and organic acids and other flavours change gradually. There has been research about pH and temperature changes during the fermentation process 13. Others have studied the origins of colour, aroma and flavour compounds 12, 14-17. Li and Feng compared the main gustatory substances in different rice wines of different ages 18. Purified enzymes have been used to enhance saccharification and ethanol production in rice wine-making 10. There has been little published work about the dependence of flavour development on fermentation temperature and source of enzymes. In this research, the effects of source of enzymes and fermentation temperature were analysed in terms of the main sugars, glycerol, ethanol and organic acids at two stages of the rice wine production process. These groups of compounds are known to be the main enological variables responsible for the flavours of rice wines 12. The results provide further insights into flavour development in the process. Materials and Method Source of enzymes Two kinds of Chinese wheat Qu were supplied by the Nverhong Wine Company (Shaoxing, Zhejiang, China). One was Shengqu, which is naturally fermented and is a source of many enzymes such as amylase, maltase, amyloglucosidase and protease (hereafter referred to as Qu 1); the other was Shuqu, which is fermented with two fungal strains to give additional saccharifying activity (hereafter referred to as Qu 2). Yeast strain Lalvin EC-1118 (S. cerevisiae; Lallemand Australia Pty Ltd, Underdale, SA, Australia) was used in this project. Purified α-amylase from Aspergillus oryzae (powder, ~30 U/mg) and amyloglucosidase from Aspergillus niger (≥300 U/mL, aqueous solution) were purchased from Sigma-Aldrich (St Louis, MO, USA). Sample preparation To study the effects of source of enzymes and fermentation temperature, the following treatments were applied in a randomized experiment with two replications: source of enzymes – purified, Qu 1, Qu 2 and Qu 1 + 2; primary fermentation temperature – 22, 26 and 30°C. The Qu 1 + 2 treatment consisted of equal proportions of Qu 1 and Qu 2. Based on previous research, 26°C is the desired temperature for cell growth, and 30°C is favourable for ethanol production 19, 20. As a result, these two temperatures were chosen. A low temperature of 22°C was added for comparison purposes. Rice wine samples were prepared by following the procedures used in the industry (Fig. 1). Glutinous rice was steeped in tap water (pH 4.6) for 2 days. In this process, lactic acid was produced naturally 7, which retards the growth of other microbes 21. The steeped rice was steamed for 45 min in a steam cooker and then cooled to room temperature. In the meantime, 1 g of EC1118 yeast was activated in 10 mL sterile water at 42°C for 20 min. The activated yeast was added to 300 mL YPD medium and cultured at 24°C for 24 h. A 10 mL aliquot of yeast cells was mixed with 100 g of steamed glutinous rice, 20 g of wheat Qu or purified enzymes (0.05 g α-amylase with 100 μL amyloglucosidase) and 300 mL of tap water in a 500 mL flask. The concentrations used are typical proportions used in the rice wine industry 22. The single-cell plating technique was used to determine the concentration of living yeast cells, which was 8 × 106 cells/mL. Since materials are not sterile in rice wine production, elevated yeast cell concentrations are commonly used to ensure dominance of the inoculated yeast strain 8, 11, 23. The mash was incubated at 22, 26 or 30°C under stable conditions for the 4-day phase of primary fermentation. This is a typical duration used in the rice wine industry 22. After primary fermentation, samples were taken and filtered for analysis. Postfermentation was carried out at 8°C for 40 days. After postfermentation, the mash was centrifuged and the supernatant (rice wine) was analysed. Chemicals HPLC-grade acetonitrile, citric acid, tartaric acid, malic acid, succinic acid, lactic acid, acetic acid, propionic acid, and ethanol were purchased from Fisher (Pittsburgh, PA, USA). Maltotriose, maltose, d-glucose, d-fructose and sulphuric acid were purchased from Sigma-Aldrich (St Louis, MO, USA). Submicron-filtered HPLC-grade water was used. Measurement Organic acids, sugars, glycerol and ethanol were measured by HPLC. One-millilitre wine samples were filtered through 0.45 µm pore Durapore (PVDF) membrane filters (Fisher, Pittsburgh, PA, USA). An Aminex HPX-87H column (300 × 7.8 mm; Bio-Rad Laboratories, Richmond, CA, USA) was used to measure organic acids, sugars, glycerol and ethanol. The column was kept at 55°C with a Bio-Rad HPLC column heater and protected with a Bio-Rad 125–0131 guard cartridge (Bio-Rad Laboratories; Richmond, CA, USA). The eluted compounds (organic acids, sugars, glycerol and ethanol) were simultaneously detected with an LC-90 UV detector (Perkin-Elmer, Waltham, MA, USA) and an RID-6A detector (Shimadzu, Kyoto, Japan). The solvent delivery system was driven with a Series-410 quaternary pump (Perkin-Elmer, Waltham, MA, USA). The HPLC conditions used were as follows: flow rate was 0.5 mL/min; the mobile phase consisted of 60 mL acetonitrile and 1270 μL sulphuric acid for every 1000 mL solvent; run time for every sample was 30 min; injection volume was 20 μL per fixed loop; and two injections were conducted for each sample. A Varian model-410 autosampler was used for injection, and the Varian Galaxie chromatography data system (Palo Alto, CA, USA) was used to acquire and process data. Data analysis Pure sugars, glycerol, organic acids and ethanol were used as measurement standards. Measured peaks were identified based on retention time and quantified by using the spiking technique. A standard curve was developed for every compound to quantify the sample concentrations 7. SAS (version 9.3; SAS Institute, Cary, NC, USA) was used for the statistical analysis of sugars, glycerol, organic acids and ethanol. Analysis of variance was performed and Tukey's test was used to find significant differences among different treatments at p = 0.05 level. Results and discussion The effects of source of enzymes and fermentation temperature were analysed in terms of the measured enological variables after primary fermentation and after postfermentation. The measurements gave a profile of the product at two stages of the fermentation process and comparison of the two snapshots provided some insights to the progression of the process. Enological characteristics after primary fermentation As sugars, glycerol and organic acids constitute the key flavours of Chinese rice wine and are mainly produced during primary fermentation 6, these enological variables after 4 days of primary fermentation were examined. The measured concentrations are summarized in Table 1. Table 1. Enological variables after primary fermentation Measured enological variables Treatment Purified Qu 1 Qu 2 Qu 1 + 2 22°C 26°C 30°C 22°C 26°C 30°C 22°C 26°C 30°C 22°C 26°C 30°C Ethanol 5.27F ** Means with the same letter are not significantly different at p = 0.05. 4.65F 8.88C 8.87C|D 8.22D 6.33E 9.98A 9.72A 10.19A 9.59A|B 9.01B|C 8.94B|C Glycerol 5.15A 3.57B 5.30A 3.57B|C|D 2.73E|F 2.46F 4.09B 3.53B|C|D 4.08B|C|D 3.77B|C|D 3.37C|D|E 3.13D|E|F Glucose 45.18A 46.86A 28.54B 0.32C 0.78C 24.01C 3.62C 1.9C 2.02C 3.89C 3.41C 2.79C Maltose 1.02G|F 13.72B 0G 4.99C|D 5.85C 24.01A 4.46C|D|E 2.58E|F 0.45G 3.68D|E 2.54E|F 1.57G|F Maltotriose 0F 7.41C 0F 8.68B 11.68A 11.66A 3.71E 2.9E 0.90F 5.60D 4.81D 2.88E Fructose 0C 0C 0C 0.03C 0C 0C 0.33A 0C 0C 0.17B 0C 0C Lactic acid 7.39B|C|D 2.2E 1.84E 5.89C|D 5.53C|D 10.89A 6.43B|C|D 4.56D|E 7.21B|C|D 5.99C|D 9.47A|B 8.43A|B|C Acetic Acid 1.24B|C 0.99C|D 0.71D|E 0.59E 0.59E 1.63A 1.36A|B|C 1.16B|C 1.32A|B|C 1.19B|C 1.11B|C 1.40A|B Malic acid 0.06C|D 0.54A 0.11B|C 0.03D 0.14B|C 0.17B 0.07C|D 0.14B 0.14B 0.02D 0.11B 0.13B Tartaric Acid 0H 0.3C|D 0.30C|D 0.13F 0.33A|B 0.35A 0.09G 0.28D 0.24E 0.1G 0.32B|C 0.29C|D Succinic Acid 0.34B|C|D 1.07A|B|C 0.99A|B|C|D 0.12D 1.27A 0.38A|B|C|D 0.41A|B|C|D 1.21A|B 0.78A|B|C|D 0.25C|D 0.72A|B|C|D 0.75A|B|C|D Citric acid 0.01F 0.25B 0.11C|D|E|F 0.05E|F 0.15B|C|D|E 0.39A 0.11D|E|F 0.24B|C 0.21B|C|D 0.1D|E|F 0.17B|C|D|E 0.14B|C|D|E|F Propionic Acid 0F 0F 0F 0F 0.01E|F 0.04E 0.09D 0.12C|D 0.21B 0.13C 0.13C 0.25A Total sugar 46.20B 67.99A 28.54D 14.01E|F 18.31E 38.34C 12.11F|G 7.38H|G 3.36H 13.34E|F 10.76F|G 7.23H|G Total acids 9.05B|C|D 5.34E|F 4.05F 6.79D|E|F 7.98C|D|E 13.83A 8.55B|C|D|E 7.70D|E 10.09B|C|D 7.78D|E 12.05A|B 11.37A|B|C * Means with the same letter are not significantly different at p = 0.05. Ethanol is a major component of rice wine. Under different fermentation temperatures, the concentration of ethanol treated with different sources of enzymes (purified enzymes and Qu) were different (see Table 1) at 4 days. Table 2 illustrates the Tukey's groupings of ethanol more visually. There were six different groups, but only five were distinct. Tukey's groupings revealed that ethanol concentrations for the batches treated with Qu 2 (the Qu 2 and the Qu 1 + 2 treatments) were significantly higher, and Qu 1 and purified enzymes yielded relatively lower ethanal concentrations. Table 2. Tukey's grouping for ethanol after primary fermentation Tukey grouping Mean N Treatment A** Means with the same letter are not significantly different at p = 0.05. 10.19 4 30°C, Qu 2 A 9.98 4 22°C, Qu 2 A 9.72 4 26°C, Qu 2 B A 9.59 4 22°C, Qu 1 + 2 B C 9.01 4 26°C, Qu 1 + 2 B C 8.94 4 30°C, Qu 1 + 2 C 8.88 4 30°C, Purified D C 8.87 4 22°C, Qu 1 D 8.22 4 26°C, Qu 1 E 6.33 4 30°C, Qu 1 F 5.27 4 22°C, Purified F 4.65 4 26°C, Purified * Means with the same letter are not significantly different at p = 0.05. Temperature effects on ethanol were largely insignificant. The only exception was 30°C, which resulted in higher ethanol concentration in the purified treatment, but lower production in the Qu 1 experiment. The results showed that the source of enzymes significantly affected ethanol production during primary fermentation, and temperature had little effect except at the high value (30°C). This agreed with previous research 6, 20. At the end of primary fermentation, glycerol concentrations were significantly different. Compared with ethanol, there were stronger interactions between source of enzymes and temperature, but in general, the Tukey's groupings differed more among different sources of enzymes than they did among different temperatures (Table 1). In particular, glycerol in the purified treatments was significantly higher than that in the Qu treatments, and temperature made no significant difference in batches treated with Qu 2 (Qu 2 or Qu 1 + 2). The results revealed that the source of enzymes had a stronger influence than temperature on the glycerol concentration during primary fermentation. Maltose and maltotriose levels for Qu 1 were significantly higher than those for the other treatments (Table 1). Fructose was detected in the Qu treatments, but was not found in the purified treatments at the lowest temperature (22°C) and was not present in the higher temperature (26 or 30°C) treatments. Total sugars are one of the key factors that affect the flavouring of rice wines. The total sugars in the batches treated with purified enzymes were higher than those treated with Qu. There were eight different levels of total sugars in the Tukey's groupings. Both temperature and enzyme source made significant differences in the total sugars after primary fermentation. However, for the same temperature, there were no significant differences in total sugars between Qu 2 and Qu 1 + 2. This shows that Qu 2 had stronger hydrolysing power than Qu 1 9 and dominated sugar production during primary fermentation. Malic acid was significantly different among the different temperatures, but not among the different sources of enzymes, except for the purified treatment at 26°C. This indicates that temperature influenced malic acid production more than the source of enzymes. Lactic acid was not different among the enzyme treatments at 22°C, it was significantly higher in the Qu 1 treatments at 30°C, lower in the purified treatment at 26°C, and higher in the Qu 1 and Qu 1 + 2 treatments at 30°C. Acetic acid was significantly higher in the Qu 1 treatment at 30°C and lower in the purified enzyme treatment at 30°C. There were strong interaction effects between temperature and source of enzyme for acids. The high temperature (30°C) generally produced more acids in the Qu treatments, but led to more variations. This agreed with previous research 24. Enological characteristics after postfermentation The concentrations of sugars, glycerol, organic acids, and ethanol concentration, as well as pH after postfermentation, are shown in Table 3. After postfermentation, ethanol content became more uniform than after primary fermentation. Although there were still five Tukey groups (Table 4), the groups overlapped a great deal except for Qu 1 at 30°C. There were some interactions and differences, but the final ethanol concentration was only a weak function of the treatments. Table 3. Enological variables after postfermentation Measured enological variables Treatment Purified Qu 1 Qu 2 Qu 1 + 2 22°C 26°C 30°C 22°C 26°C 30°C 22°C 26°C 30°C 22°C 26°C 30°C Ethanol 11.40C|D* 11.44C|D 11.60B|C|D 11.70A|C|C|D 11.11D 9.50E 12.38A|B 12.50A 11.94A|B|C 12.06A|B|C 12.11A|B|C 11.00D Glycerol 6.61A 6.41A|B 6.32A|B 3.23D|E 2.90E 3.45D 5.96B 6.07B 5.97B 5.05C 5.15C 5.04C Glucose 0E 0E 0E 1.56B 1.57B 3.84A 0.09D|E 0.09D|E 0.03D|E 0.23C|D|E 0.36C|D 0.55C Maltose 0B 0A 0B 0.13B 0B 0.52A 0B 0B 0B 0B 0B 0B Maltotriose 0B 0B 0B 0.18B 0.23B 4.42A 0B 0B 0B 0B 0B 0B Fructose 0D 0D 0D 0.28B 0.24B 0.13C 0.51A 0.48A 0.4525A 0.49A 0.48A 0.4575A Lactic acid 2.03E 2.04E 1.67E 6.64D 6.23D 10.01B 6.54D 6.12D 7.64C|D 6.52D 14.37A 9.10B|C Acetic Acid 1.33A|B 1.41A|B 0.81B|C|D 0.58D 0.61C|D 1.70A 1.46A 1.52A 1.62A 1.33A|B 1.58A 1.20A|B|C Malic acid 0.06A 0.1A 0.12A 0.03A 0.17A 0.04A 0.04A 0.06A 0.08A 0.16A 0.02A 0.04A Tartaric Acid 0.03C|D 0D 0D 0.04B|C|D 0.09B 0.27A 0.03B|C|D 0.03B|C|D 0.08B|C 0.03B|C|D 0.01D 0.02C|D Succinic Acid 1.89A 0.81A 0.93A 1.13A 1.00A 1.60A 1.51A 1.01A 0.67A 0.38A 0.50A 0.60A Citric acid 0.06B|C 0.08B|C 0.10A|B|C 0.10A|B|C 0.14A 0.10A|B|C 0.09A|B|C 0.09A|B|C 0.07B|C 0.12A|B 0.05C 0.10A|B|C Propionic Acid 0.01D 0.32A|B|C|D 0.01D 0.11C|D 0.22A|B|C|D 0.12B|C|D 0.29A|B|C|D 0.58A|B|C|D 0.43A|B|C|D 0.47A|B|C 0.32A 0.55A|B Total sugar 0.00E 0.00E 0.00E 2.14B 2.03B 8.91A 0.60D 0.56D 0.49D 0.73C|D 0.84C|D 1.01C Total acids 5.38E 4.75E 3.64E 8.61D 8.46D 13.83B 9.95C|D 9.15C|D 10.58C|D 8.99C|D 17.10A 11.60B|C pH 3.97C 3.93C 4.23A 3.89C|D 3.79D 4.20A 4.01B|C 4.13A|B 3.90C|D 3.95C 4.18A 4.02B|C * Means with the same letter are not significantly different at p = 0.05. Table 4. Tukey's grouping for ethanol after postfermentation Tukey Grouping Mean N Treatment A** Means with the same letter are not significantly different at p = 0.05. 12.50 4 26°C, Qu 2 B A 12.38 4 22°C, Qu 2 B A C 12.18 4 26°C, Qu 1 + 2 B A C 12.06 4 22°C, Qu 1 + 2 B A C 11.94 4 30°C, Qu 2 B D A C 11.70 4 22°C, Qu 1 B D C 11.60 4 30°C, Purified D C 11.44 4 26°C, Purified D C 11.40 4 22°C, Purified D 11.11 4 26°C, Qu 1 D 11.00 4 30°C, Qu 1 + 2 E 9.50 4 30°C, Qu 1 * Means with the same letter are not significantly different at p = 0.05. Glycerol in the purified treatments was significantly higher than that in the other treatments, and the batches treated with Qu 1 yielded lower glycerol than the other treatments in the final product. There were no significant differences among the three temperatures treated with the same source of enzymes. This indicates that the source of the enzymes was more influential than temperature on the final glycerol concentration. The glycerol content in the Qu 1 treatments was closer to the desirable levels suggested in previous research 25. Maltotriose and maltose were detected in the batches treated with Qu 1, but not in the other treatments (Table 3). Fructose was detected in all of the Qu treatments, but not in the purified treatments. In general, Qu 1 yielded the highest sugar concentrations, while purified enzymes gave the lowest sugar concentrations in the final product. Comparison with the profile after primary fermentation indicates that slower hydrolysis during primary fermentation led to higher final sugar and lower glycerol concentrations, or vice versa. Malic acid and succinic acid were not significantly different among the three temperatures, although they were different after primary fermentation. Lactic acid was lower in the purified enzyme treatments than in the Qu treatments. Desirable lactic acid concentrations 25 resulted from lower temperatures. Acetic acid was lower in the Qu 1 treatments than in the others at 22 and 26°C, and lower in the purified enzyme treatments at 30°C. High temperatures appeared to lead to an overproduction of acetic acid in the Qu treatments because of the complex microorganism and enzyme systems in Qu. Qu 1 or Qu 1 + 2 at low temperature gave the most desirable acid levels according to existing research 25. The pH level is also one of the key variables in Chinese rice wine fermentation. Low pH may cause rancidity of the broth. Suitable pH values are 3.0–4.5 13 and can inhibit the growth of bacteria, which can produce waste by-products in rice wine production. Lower pH occurred in batches with Qu 1 at the lower temperatures. Summary In summary, the results of this research showed the following: (a) fermentation temperature and source of enzymes can significantly affect the concentrations of the main sugars, glycerol and main organic acids at different stages of the fermentation process; (b) ethanol concentration initially (after primary fermentation) varied with source of enzyme, but eventually became largely independent of enzyme source or fermentation temperature; (c) glycerol initially depended on both enzyme source and temperature with interactions, but the temperature effects essentially disappeared after postfermentation; (d) faster hydrolysis resulted in higher initial but lower final sugar concentrations accompanied by higher final glycerol content, or vice versa; (e) initial malic acid concentration depended more on temperature than enzyme source but the reverse was true after postfermentation, and higher acetic and lactic acid concentrations resulted from higher temperatures; and (f) Qu 1, by itself or in combination with Qu 2, at low temperature (22°C) resulted in desirable levels of glycerol and the main organic acids according to the existing literature. Acknowledgements The authors thank Dr Feng Ding, Dr Ya Guo, Ms Connie Liu and Mr Lakdas Fernando for their technical assistance. The Shaoxing Nverhong Rice Wine Company is acknowledged for the material support. This work was supported in part by NSFC (21276111, 21206053), Zhejiang Science and Technology Project (2011C12033), China Scholarship Council (no. 2010679023), 111 Project (B12018), and Jiangnan University PhD Research Fund (JUDCF10062, no. 20110149). References 1 Zhang, Z. and Wu, G. (1999) The brewing technology development of yellow wine, Liquor Making 2, 45– 47. CASPubMedWeb of Science®Google Scholar 2 Que, F., Mao, L., Zhu, C., and Xie, G. (2006) Antioxidant properties of Chinese yellow wine, its concentrate and volatiles, LWT-Food Sci. Technol. 39, 111– 117. CrossrefCASWeb of Science®Google Scholar 3 Xie, G. (2009) Current research on the health functions of Japanese sake and its inspiration to Chinese rice wine, China Brew. 7, 10– 11. PubMedWeb of Science®Google Scholar 4 Xie, G., Dai, J., Zhao, G., Shuai, G., and Li, L. (2005) γ-Aminobutyric acid in the rice wine and its healthy function, China Brew. 3, 49– 50. Web of Science®Google Scholar 5 Shao, J., Huang, C., Yu, L., and Zhan, Y. (2009) Investigation and development research of Zhejiang rice wine manufacturing, Zhejiang Statistics Chinese 8, 12– 14. Google Scholar 6 Zhou, J. (1996) Chinese Rice Wine Brewing Process, 2nd edn, China Light Industry Press, Beijing. Google Scholar 7 Mo, X., Fan, W., and Xu, Y. (2012) Changes in volatile compounds of Chinese rice wine wheat Qu during fermentation and storage, J. Inst. Brew. 115, 300– 307. Wiley Online LibraryGoogle Scholar 8 Luo, T., Fan, W., and Xu, Y. (2012) Characterization of volatile and semi-volatile compounds in Chinese rice wines by headspace solid phase microextraction followed by gas chromatography–mass spectrometry, J. Inst. Brew. 114, 172– 179. Wiley Online LibraryWeb of Science®Google Scholar 9 Shou, Q., Yang, G., Chen, X., and Zhao, G. (2008) Properties comparison between uncooked wheat starter and cooked wheat starter for yellow rice wine, Liquor Making Sci. Technol. 4, 92– 95. Google Scholar 10 He, J. and Huang, H. (2007) Application of enzyme engineering in wine industry, Liquor Making Sci. Technol. 3, 57– 60. Web of Science®Google Scholar 11 Luo, T., Fan, W., and Xu, Y. (2007) The review of volatile and nonvolatile compounds in Chinese rice wine, Liquor Making 34, 44– 48. CASGoogle Scholar 12 Wang, J. (2004) Analysis of composition and source of color, aroma, taste, type in rice wine, China Brew. 4, 6–10, 18. CASGoogle Scholar 13 Wei, T. (2005) Investigation on pH change during the fermentation of yellow rice wine, Liquor Making Sci. Technol. 3, 78– 79. Google Scholar 14 Zhao, M., Leng, Y., and Li, P. (2009) Process analysis of yellow rice wine fermentation and its key points control, Jiangsu Condiment Subsidiary Food 29, 30– 34. Google Scholar 15 Shou, H., Ling, Z., Yang, X., and Xie, G. (2007) Relationship between metabolites of wheat Qu microorganisms and flavor of rice wine, China Brew. 8, 55– 57. Google Scholar 16 Chen, N., Chen, N., Wang, Q., and Yang, H. (2012) The HPLC analysis of rice wine, Anhui Agri. Sci. Bull. 18, 177– 179. Google Scholar 17 Zhao, M., Leng, Y., Ye, H., Zhao, Y., and Wang, F. (2010) Analysis of yellow wine big pots fermentation and sugar metabolism in fermentation process, Liquor Making 2, 58– 61. Web of Science®Google Scholar 18 Li, H., and Feng, T. (2012) Determination of the main gustatory substances in rice wine with different ages and types, China Brew. 31, 172– 175. Web of Science®Google Scholar 19 Russell, I. (2003) Understanding yeast fundamentals, in The Alcohol Textbook, 4th edn ( K. A. Jacques, Lyons, T. P., and Kelsall, D. R., Eds), pp. 85– 119, Nottingham University Press, Nottingham. Google Scholar 20 Torija, M. J., Rozès, N., Poblet, M., Guillamón, J. M., and Mas, A. (2003) Effects of fermentation temperature on the strain population of Saccharomyces cerevisiae, Int. J. Food Microbiol. 80, 47– 53. CrossrefCASPubMedWeb of Science®Google Scholar 21 Mao, Q. (2001) Positive and negative effect of Lactobacillus lactis in the fermentation of yellow wine, Liquor Making 28, 72– 75. CASGoogle Scholar 22 Xu, Y., Wang, D., Fan, W., Mu, X., and J. Chen (2010) Traditional Chinese biotechnology, in Biotechnology in China II: Chemicals, Energy and Environment ( G. T. Tsao, P. Ouyang, and J. Chen, Eds), pp. 189– 233, Springer, Berlin. Google Scholar 23 Chen, S., and Xu, Y. (2012) The influence of yeast strains on the volatile flavour compounds of Chinese rice wine, J. Inst. Brew. 116, 190– 196. Wiley Online LibraryWeb of Science®Google Scholar 24 Mao, Q. (2004) Discussion on composition changes during rice wine fermentation, China Brew. 141, 1– 5. Google Scholar 25 Hu, Z., and Xie, G. (2008) Rice Wine, Zhejiang Science and Technology Press, Hangzhou. Google Scholar Citing Literature Volume120, Issue32014Pages 231-237 FiguresReferencesRelatedInformation