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Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process
Journal of Bioscience and Bioengineering VOL. xx No. xx, 1e6, 2016 www.elsevier.com/locate/jbiosc
Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process Xiao Lu Wei,1, 2, z Shuang Ping Liu,1, 2, 3, z Jian Shen Yu,4 Yong Jian Yu,5 Sheng Hu Zhu,5 Zhi Lei Zhou,1, 2, 3 Jian Hu,4 and Jian Mao1, 2, 3, * National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, Jiangsu 214122, China,1 School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China,2 National Engineering Research Center of Chinese Rice Wine, Shaoxing, Zhejiang 31200, China,3 Shanghai Jinfeng Wine Co., Ltd., Shanghai 200063, China,4 and Jiangsu Hengshun Vinegar Industry Co., Ltd., Zhenjiang 212043, PR China5 Received 21 August 2016; accepted 30 November 2016 Available online xxx
As a traditional fermented alcoholic beverage of China, Chinese rice wine (CRW) had a long history of more than 5000 years. Rice soaking process was the most crucial step during CRW brewing process, because rice soaking quality directly determined the quality of CRW. However, rice soaking water would cause the eutrophication of water bodies and waste of water. The longer time of rice soaking, the higher the content of biogenic amine, and it would have a huge impact on human health. An innovation brewing technology was carried out to exclude the rice soaking process and the Lactobacillus was added to make up for the total acid. Compared to the traditional brewing technology, the new technology saved water resources and reduced environmental pollution. The concentration of biogenic amine was also decreased by 27.16%, which improving the security of the CRW. The esters increased led to more soft-tasted CRW and less aging time; the quality of CRW would be improved with less alcohol. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: Chinese rice wine; Brewing; Rice soaking process; Lactobacillus; Biogenic amine]
As a special and traditional fermented alcoholic beverage of China, Chinese rice wine (CRW) has had a long history of more than 5000 years (1). Not only due to the abundant amino acids, sugar and other nutritional substances CRW hold, but its low alcohol (content) of 14e20% (v/v) (2), CRW has been popular in China. Recently, with a large amount of customers, production of CRW has been increasing gradually (3). CRW brewing process is mainly divided into two parts: the first step is the pretreatment of the raw materials, which is mainly the rice soaking. Rice soaking is the most crucial step of CRW brewing process, because rice soaking quality directly determines the quality of CRW (4), and its application in CRW brewing process has lasted for hundreds of years; the second step is the fermentation process, which refers to ferment steamed rice with wheat qu and the yeast (1,5). The rice soaking process always lasts quite a long time in the traditional CRW brewing. For example, traditional cooling of rice wine requires 16e20 days during soaking period. (The main changes in the rice soaking process are rice swelling after sucking in water (4), decomposition of main ingredients, the increase of water acidity, and so on.) Afterwards, rice can be steamed easily (6). High
* Corresponding author at: National Engineering Laboratory for Cereal Fermentation Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China. Tel./fax: þ86 510 85328273. E-mail address: [email protected] (J. Mao). z The first two authors contributed equally to this work.
concentration of lactic acid containing in rice soaking water (5e9 g/ L) can protect the metabolism of yeasts by inhibiting other bacteria growth. As a result, the acidity of rice soaking water is relatively high, usually between 6 and 15 g/L. With a plenty of nitrogen compounds, rice soaking water can cause the eutrophication of water bodies, which will result in serious environmental pollution (7). Waste soaking water treatment and water costs are the main burden for CRW winery (microorganisms in rice soaking water can use free amino acids to produce biogenic amines, which are also brought into the brewing process). The longer the rice soaks, the higher the biogenic amine of CRW contains, and it will have a huge impact on human health if the content is too high. In recent years, as increasing widespread attention towards food security, biogenic amine content in CRW appears to arise more public concerns than ever. From the perspective of country’s industrial adjustment and the long-term development of CRW industry, an eco-friendly and safety way must be found. Therefore, the solutions to economize water resources and decrease biogenic amines content have drawn much more attention than ever. The objective of the present work is to exclude rice soaking process, and economize all the water that used for rice soaking. Moreover, with the addition of Lactobacillus (8), it could make up for the insufficient of total acids and create low pH for CRW fermentation. A new CRW brewing process without rice soaking process is firstly developed in this paper, and will solve the problems such as rice soaking water’s pollution, waste of water resources and high content of biogenic amines.
1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
Please cite this article in press as: Wei, X. L., et al., Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
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Sample collection and processing Three fermentation samples (control of rice soaking, rice steaming with Lactobacillus and rice steaming without Lactobacillus) were taken. The fermentation mash is needed to be stirred evenly when sampled, then centrifuged to collect the supernatant and kept in ultra-low temperature freezer for later detection (including ethanol, total acids, pH, amino nitrogen (AN), reducing sugar, biogenic amine and volatile flavor). The proportions of sugars, ethanol, and organic acids were primarily responsible for the delicate taste and flavors of CRW (9). While detecting the total acids of the supernatant, the filter paper must be used to wipe the carbon dioxide out (because of the ethanol fermentation, which would increase the total acids to a great extent). The biogenic amines of CRW are in high concentration then, but low content of biogenic amines in CRW exactly meets the current market demand (10e12). Control of rice soaking technology and the moisture absorption of rice The control of rice soaking process was practiced by soaking the rice and water in proportion of 1:1 until the total acids of rice soaking water was 6e10 g/L. After draining the water, rice could be steamed then. The moisture absorption of rice was tested as follows, first and foremost, soaking 10 g of rice in water (28 C) and sampling some rice every 10 min; then drying the water of rice surface immediately with filter paper and weighing, besides, weighing the rice after 105 C drying to constant weight (4). The difference between these two was the moisture absorption of rice. Brewing with rice of different soaking time Rice was initially divided into five groups, each was soaking in water in proportion of 1:1, and soaking for 1e5 days, respectively. After soaking, rice with different soaking time was steamed and ready to ferment. The biogenic amine content of each group was detected and analyzed. Innovation of rice steaming technology The first step was spraying the water of 50 C about 150 mL/kg onto the rice, and the time was strictly controlled within 20 min. After spraying, the rice was steamed for the first time for approximately 7 min. Then sprayed for the second time about 150 mL/kg onto rice, the water temperature was controlled above 80 C. The second rice steaming time was 8 min and then spraying for the third time (the same to the second time). The final rice steaming time lasted for 10 min, after which cooling the steamed rice by air blast and fermenting with wheat qu, yeasts and other ingredients. The spraying water was recyclable, and there was fresh water supplement when it was water shortage. The Lactobacillus culturing and concentrated lyophilized cultures preparing The Lactobacillus was screened from fresh fermentation mash based on the next-generation sequencing technology (8), and then cultured through the three-level fermentation to get the Lactobacillus broths. The Lactobacillus in lyophilized preservation tube was inoculated to 150 mL MRS liquid medium and incubated at 37 C for 18 and 20 h anaerobically, which was as the seed liquid. Five percentage of quantity of the seed was inoculated to 400 mL MRS liquid medium, which was as the secondary seed liquid. Secondary seed with 5% quantity was inoculated into 6000 mL MRS liquid medium, and it was incubated at 37 C for 18e20 h, then to get triple Lactobacillus broths. The broths were centrifuged at 5000 r/min for 15 min to get Lactobacillus mud, which should be absterged twice with the sterile saline. The protective agent (90% skim milk, 10% trehalose) was used to dissolve Lactobacillus mud. Mixtures were freeze-dried for 48 h after precooling for 2 h, and then cultures were stored at 20 C for later use. Preparation and selection of Lactobacillus medium In order to facilitate the CRW winery from culturing Lactobacillus, malt medium, rice medium and glutinous rice medium were chosen as Lactobacillus medium. MRS liquid medium was impractical because of the high cost, and it also affected the flavor of CRW. Malt medium was made by mixing water and malt flour (v/v 4:1), then the mixtures were saccharified at 60 C for 4 h and sterilized at 115 C for 15 min. Rice (or glutinous rice) medium was made by mixing rice (or glutinous rice), malt flour and water (v/v/v 1:0.2:4), then saccharifying was done by adding enzymes and wheat qu at 60 C for 4 h. After that, sterilization was done at 115 C for 15 min. Lactobacillus lyophilized cultures were inoculated in MRS liquid medium for activation with a ratio of 5% based on the volume of (said MRS liquid medium), incubated at 37 C under anaerobic condition for 24 h, then 10% activated culture was transferred to new malt medium, rice medium and glutinous rice medium, respectively, and were incubated at 37 C for 60 h under anaerobic condition. The inoculation size of Lactobacillus must also be optimized. On the premise of not affecting rice wine fermentation, four kinds of inoculation size of Lactobacillus, namely, 2%, 4%, 8% and 12% were chosen for brewing experiment. CRW brewing process The rice steaming process was prepared as described in the Innovation of rice steaming technology section. After steaming, the 200 kg steamed rice, 175 kg water, 4.3 kg wheat qu, 21.5 kg rice wine starter (6& of yeast activated liquid was inoculated in rice medium, incubated at 28 C and 150 rpm for 24 h), 50 mL amylase (Suhong AA Plus), 50 mL glucoamylase (Suhong GA II) and Lactobacillus broth were added into the fermentation tank, the water temperature was controlled at 24e28 C. The primary fermentation was incubated at 28 C for 96 h with aeration once a day, and natural temperature with no aeration was suitable for post fermentation. The total fermentation hours were nearly 384 he432 h.
Detection of main volatile flavor compounds in fermentation mash using headspace solid-phase micro-extraction and gas chromatographyemass spectrometry The headspace solid-phase micro-extraction (HS-SPME) method was referred to the report (5) and some adjustments. The alcohol content of CRW was diluted to 6% (v/v), then 6 mL diluted wine was taken to the head space bottle, 1.5 g NaCl and 15 mL internal standard (8800 mg/L 2-octanol) were added. A 50/30 mm divinylbenzene/carboxen/poly (dimethylsiloxane) (DVB/CAR/PDMS) coated fibers was used for the extractions of volatile compounds. The sample was equilibrated and extracted at 50 C for 40 min. After HS-SPME, the fiber was inserted into the injection port of gas chromatographyemass spectrometry (GCeMS) (250 C) for 7 min to desorb the analytes. The GCeMS conditions were according to the reports (13e15) and some adjustments. Chromatographic column: TG-WAXMS (30 m 0.25 mm 0.25 mm); injection port temperature: 250 C; temperature program: 40 C keep 3 min; 6 C/min up to 100 C; 10 C/min up to 230 C, keep 7 min; carrier gas: high purity helium (>99.999%), no shunt, flow rate of 1.0 mL/min; ionization method: EI; the emission current: 50 mA; electron energy: 70 eV; ion source temperature: 230; transmission line temperature: 250 C; scan range: 33e400 amu. The 2-octanol was used as the internal standard for semi-quantification of the volatiles according to the previous report (16). The detection of biogenic amine Samples (1 mL) were taken in different 10 mL plastic centrifuge tube, in each of the centrifuge tube mixed with 1 mL of the saturated NaHCO3 solution and 2 mL dansyl chloride solution (5 mg/mL in acetone), vortexed for 1 min, then mixtures were heated in water bath of 60 C for 30 min to derive. Mixtures were then placed at room temperature for a few minutes and 0.5 mL saturated NaCl solution was added (17). With absolute ether to be extraction agent, extraction was performed on a tabletop shaker for 2 min, the upper organic phase was taken, and the above extraction process was repeated twice. Finally, all the upper organic phase was merged with nitrogen gas blowing and 1 mL of acetonitrile dissolving after drying, then after the membrane filtration of 0.22 mm, it could (be directly into) the high performance liquid chromatography (HPLC) determination. A chromatographic column (250 mm 4.6 mm, XBridge C18, 5 mm) was used for all separations, with column temperature at 30 C and flow rate at 0.8 mL/min. In terms of mobile phase, ultrapure water was used as eluent A and acetonitrile was used as eluent B. For data analysis, the absorbance was read at 254 nm using a UV detector. Statistical analysis Volatile flavor and biogenic amine were also tested by using GCeMS and HPLC method separately, all analyses had three replicates.
RESULTS AND DISCUSSION Biogenic amine in the fermentation process of rice of different soaking time After rice soaking process, rice became loose for steaming, which was more beneficial to alcoholic fermentation (18). Rice could not be steamed ripe without soaking because of the gelatinization of rice starch, which needed to be at a certain temperature and moisture content conditions (6). Meanwhile, the traditional rice soaking process was last long and produced a large amount of rice soaking water that would pollute the environment. Fig. S1 shows that the longer the soaking time is, the more biogenic amine content generated in the fermentation process. Overall trends, biogenic amine content had been gradually increasing in the primary fermentation. On the fermentation time of 72 h, biogenic amine content reached the maximum, and then fell slightly. During the post fermentation, biogenic amine content had a downward trend. Based on this phenomenon, an innovative rice steaming technology was developed (described in the Innovation of rice steaming technology section) to replace the traditional rice soaking and steaming process to reduce the content of biogenic amines and waste water emissions. Innovation brewing of CRW In the developed rice steaming process, spraying water for 20 min onto rice induced quick absorption (Table 1). After it, the moisture content of rice was about 30% (Table 1) that achieved the same value in Fig. S2. After first steaming, the rice moisture content was increased by 2%e5%, and was up to 41%e44% after the final steaming. The figure of final rice moisture content was near that of the rice moisture content of control rice soaking (about 46%), which suggested that the rice of new steaming technology could achieve a certain degree of gelatinization and the new steaming technology could be applied to the CRW brewing process. However, with prolonging the soaking time, the components of rice reduced and transferred into water, like reducing sugar, total
Please cite this article in press as: Wei, X. L., et al., Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
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TABLE 1. Moisture content of rice at three different stages during the new steaming process. Sample
1 2 3 4
Rice moisture content after the first spraying (%)
Rice moisture content after first steaming (%)
Rice moisture content after final steaming (%)
30 29 31 29
33 32 33 34
42 44 43 41
The samples were collected from three different stages: after the first water spraying process, after the first rice steaming process, at the end of rice steaming process.
sugar, crude protein, crude fat and ash. Microorganism in soaking water used these compounds for growth, and increased the total acids in soaking water to 5e9 g/L (calculated in lactic acid). Some of these acids could be absorbed in rice and was subsequently carried into the fermentation process. Thus, the fermentation process had a low pH (about pH 3.5), which restrained bacteria from overgrowing. The new brewing process had no rice soaking, the fermentation process was not in low pH without accumulation of acids, then bacteria was likely to overgrow which might inhibit the growth of yeast and terminated the fermentation of alcohol. Although the rice soaking process was removed and the rice was steamed successfully by the innovative rice steaming technology, the acid in steamed rice was not enough to inhibit the growth of unexpected bacteria. On this basis, it was supposed that addition of Lactobacillus could create a low pH for fermentation and supply sufficient total acids. At the same time, there was no rice soaking water emissions that polluted the environment. Based on the fact, the Lactobacillus that produced no biogenic amines was screened and back-added into the fermentation mash. It also showed the Lactobacillus had high lactic acid production, and high ethanol tolerance which was preferable for CRW brewing (8). The growth of Lactobacillus in different culture medium In order to facilitate the CRW winery from producing, it was suitable to use activated fresh Lactobacillus broths instead of the concentrated lyophilized cultures. Therefore, the concentrated lyophilized cultures were inoculated into different culture medium to obtain activated fresh Lactobacillus broths. With the extension of culture time of Lactobacillus, the total acids of every medium increased. At the first 8 h, total acids increased slowly because of the adaptation to the environment; total acids increased rapidly between 8 and 24 h, showing that Lactobacillus metabolized actively; from 32 to 60 h, the total acids increased slowly. Malt medium had the highest total acids comparing with the other two mediums, and total acids of rice medium and glutinous rice medium were relatively similar to each other (Fig. 1A). However, the reducing sugar content in every medium descended gradually (Fig. 1B). From 0 to 4 h, the reducing sugar dropped most significantly; after 4 h, the overall decline was relatively flat. The reducing sugar content of malt medium, rice medium and glutinous rice medium were all reduced about 20 g/L. The reducing sugar in malt medium was significantly lower than the other two kinds of medium. In conclusion, malt medium was the best choice for culturing the Lactobacillus, which components in the above several kinds of the medium was the most close to MRS. However, for the actual production from the winery factory, rice medium as the culture medium of Lactobacillus was the right choice. The rice relative to glutinous rice was more economical and practical, and was consistent with the raw materials of CRW. It was also advantageous for the Lactobacillus to adapt to the fermentation environment quickly. Then it would not bring too much pressure from the outside on Lactobacillus. Outside the pressure, adding too much malt medium in the brewing process would affect the flavor of
FIG. 1. Changes of total acid (A) and reducing sugar (B) during culturing Lactobacillus in different mediums.
CRW. Therefore, after the activation of Lactobacillus lyophilized cultures in MRS liquid medium, the activated culture could be transplanted to the malt medium for culturing. Finally, the culture could be transplanted to rice medium for actual production. Thus, it could ensure not only the vitality of Lactobacillus but also the flavor of CRW. Influence of different inoculation size of Lactobacillus on CRW The purpose of Lactobacillus back-added was to provide a low pH for fermentation and improve the total acids (up to 5 g/L) of fermentation mash on the premise of not affecting rice wine fermentation. Lactobacillus overgrew and increased the total acids which would lead to the acidification of the wine. The detection results of fermentation mash with different inoculation size of Lactobacillus are shown in Table 2. With the inoculation size increasing, after the fermentation was finished, the total acids were higher. Because superabundant acids affected the metabolism of yeasts that making use of reducing sugar to produce alcohol, the fermentation process became more difficult. Alcohol rose faster under this craft, alcohol content was almost around 7% (v/v) at 24 h (Table 2). At the end of the primary fermentation, alcohol content reached to 15% (v/v), then rose 2e3% (v/v) or so during the post fermentation, so adding the Lactobacillus
Please cite this article in press as: Wei, X. L., et al., Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
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TABLE 2. The detection results of fermentation mash with different inoculation size of Lactobacillus. Inoculation amount Fermentation of Lactobacillus (%) time (h)
2
Physical and chemical indicators Total acid (g/L)
pH
2.71 3.42 3.60 3.94 2.88 3.92 4.25 5.05 3.16 4.53 5.42 5.95 3.34 5.22 5.95 6.52
3.89 3.68 3.55 3.51 3.76 3.50 3.84 3.89 3.37 3.46 3.73 3.67 3.33 3.41 3.62 3.50
24 96 192 384 24 96 192 384 24 96 192 384 24 96 192 384
4
8
12
Alcohol Reducing content (%) sugar (g/L) 10.70 14.40 15.80 17.20 7.20 15.60 16.50 18.24 7.10 14.90 15.90 16.13 6.60 13.70 14.40 15.62
53.56 8.07 4.21 2.01 67.00 6.80 3.33 2.30 65.80 7.03 4.06 2.70 64.40 10.51 6.18 3.75
AN 0.08 0.14 0.25 0.32 0.03 0.07 0.27 0.29 0.02 0.07 0.26 0.28 0.02 0.07 0.27 0.27
to improve total acids played a vital role in the primary fermentation. The post fermentation with higher alcohol content inhibited the activity of Lactobacillus, and acids’ producing ability of it was extremely low. The optimum inoculation size of Lactobacillus was 4%. When inoculation size was 2%, the total acids could not meet the winery requirements; when inoculation sizes were 8% and 12%, the total acid contents were both beyond the scope of the winery standard, which made the rice wine taste poorly. Therefore, the most appropriate inoculation size was 4%. The detection results of different fermentation process With rice medium culturing Lactobacillus and inoculation size of 4% to brew, fermentation mash of rice steaming with Lactobacillus, rice steaming without Lactobacillus and control of rice soaking were tested and compared. The results are shown in Table 3. The total acids of culture medium (about 1 L) were 11.34 g/L, while the total acids of fermentation system (500 kg) were only 0.023 g/L, so the added culture medium could hardly affect the acids of the fermentation system. However, the alcohol content was around 7e9% (v/v) at the end of 24 h of fermentation, and the total acids of rice steaming with Lactobacillus were 4e5 g/L. At the end of the fermentation (384 h), the total acids content of rice steaming without Lactobacillus (sample L) and control of rice soaking (sample J) were between 3.5 and 4 g/L. The acidity was insufficient, could not
TABLE 3. The detection results of fermentation mash with different crafts. Sample
D
L
J
Fermentation time (h)
24 96 192 288 384 24 96 192 288 384 24 96 192 288 384
Physical and chemical indicators Total acid (g/L)
pH
Alcohol content (%)
Reducing sugar (g/L)
AN
4.56 4.78 4.90 4.95 5.01 3.52 2.79 3.31 3.59 3.45 2.97 3.33 3.88 4.31 4.06
3.73 3.71 3.97 3.99 3.99 3.69 4.79 4.75 4.76 4.44 3.78 4.19 4.39 4.39 4.40
8.15 15.10 14.60 15.90 17.60 9.20 13.90 14.80 14.60 15.20 7.75 16.90 17.00 17.60 18.00
13.70 3.40 2.10 3.11 1.56 20.40 7.60 6.07 5.01 4.81 35.10 3.40 2.59 1.90 1.14
0.03 0.19 0.25 0.53 0.82 0.02 0.69 0.72 0.78 0.79 0.02 0.41 0.55 0.64 0.68
FIG. 2. Changes of biogenic amine (A), putrescine (B) and tyramine (C) in different fermentation process.
D: rice steaming with Lactobacillus of 4%; L: rice steaming without Lactobacillus; J: control of rice soaking.
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reach the winery requirements. However, the total acids of rice steaming with Lactobacillus (sample D) were 5.01 g/L, which improved about 1.5 g/L compared to sample L and met the needs. The total acids of the sample L during 24 he96 h were decreasing and this may be due to the fast increase of AN content from 0.02 to 0.69. Some free acid radical ions may turn into amino acids, which may cause the decrease of total acids. It may also have certain effects on the metabolism of the Lactobacillus. After 96 h, the concentration of AN had little change and the total acids increased slightly because of the metabolism of the Lactobacillus. At the same time, alcohol content, reducing sugar, amino nitrogen (AN) and pH were also conformed to the enterprise standard. As a result, rice steaming with Lactobacillus (sample D) was quite a good new CRW brewing technology, which reduced waste water emissions and the content of biogenic amines. Biogenic amine in fermentation mash Changes of biogenic amines in different fermentation mash are shown in Fig. 2A. The diagram shows that control of rice soaking of biogenic amine content was higher than the other two crafts and at a high level, about 30 mg/L. Rice steaming without Lactobacillus of biogenic amine was mainly increased during the primary fermentation, and had a slight increase during the post fermentation. At the end of the fermentation, the total content of biogenic amines in rice steaming with Lactobacillus was around 21 mg/L, compared to control of rice soaking and rice steaming without Lactobacillus, which were reduced by 27.16% and 20.59%, respectively. On account of Lactobacillus cultures of low pH, it reduced the fermentation pH and largely inhibited the growth of the other bacteria metabolism of generating biogenic amines. Therefore,
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with Lactobacillus culture of 4% to brew, it could effectively reduce the biogenic amine amount of CRW, and at the same time increase the total acids, which making the rice wine more secure and having a better taste. Biogenic amines in CRW were mainly putrescine, tyramine and so on (19,20). Putrescine of control of rice soaking was the maximum one, around 9 mg/L (Fig. 2B). At the end of the fermentation of rice steaming with Lactobacillus, the putrescine was only about 3.5 mg/L, which was one third of control of rice soaking, so reducing the content of putrescine also largely reduced the total content of biogenic amines. Fig. 2C shows the tyramine changes of different fermentation processes. Tyramine content of control of rice soaking was the highest, while rice steaming with Lactobacillus was the lowest. In addition to the increase of 0.5 mg/L during the primary fermentation, there was almost no changes in the post fermentation. To certain extent, tyramine could be detrimental to the human bodies, which was the most toxic biogenic amine compared with others (21,22). Malolactic fermentation was confirmed to be the main origin of tyramine (23). Thus, reducing tyramine content by adding Lactobacillus could improve the safety of CRW. Main volatile flavor compounds in fermentation mash Volatile flavor compounds played an important role in the typical taste of CRW (13,24). From the different fermentation mash, it mainly determined the 15 kinds of ester substances, 10 kinds of alcohols, 5 kinds of aldehyde and 4 kinds of phenol. Fig. 3A shows the changes of total volatile esters in different fermentation processes. With the prolonging of fermentation time, the total volatile esters content showed an increasing trend on the
FIG. 3. Changes of total volatile ester (A), alcohol (B), aldehyde (C) and phenol (D) in different fermentation process.
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whole. The total volatile easers of rice steaming with Lactobacillus were more than others, the increase of the esters might be generated by metabolic reaction of Lactobacillus. Esters were the largest part of volatile compounds in CRW, and were mainly produced by the esterification of alcohol and acid (25). There were two ways to form esters: one was from the biosynthesis of microorganism such as ester-producing yeasts in fermentation mash, the other was generated by organic chemical reactions. The latter under the normal condition of the reaction was especially slow, often needed a long time to make the esterification reaction equalized. When under the normal fermentation, esters of fermentation mash mainly stemmed from the metabolism of ester yeasts (5). Esters could impact the taste of CRW for the low threshold concentrations and desirable fruity and flowery flavors. Furthermore, wine would become mellower with the longer aging time due to increasing ester substances (26). Therefore, adding the Lactobacillus may reduce the aging time of rice wine and improve the taste of CRW. The higher alcohols were one of the most important volatile compounds in CRW, which were mainly generated by the metabolism of yeasts that used sugar and amino acid (27). In the process of amino acid metabolism, amino acid could generate higher alcohols by deamination or decarboxylation reaction of losing a carbon atom (28). The higher alcohols in CRW were the main cause of being unwell after drinking CRW. Thus, it was appropriate to reduce higher alcohols content in CRW, which was beneficial to improve the quality and the comfort level of drinking CRW. Fig. 3B shows the changes of alcohols under different fermentation process, rice steaming with Lactobacillus of higher alcohol content was lower than rice steaming without Lactobacillus and control of rice soaking. During the post fermentation, higher alcohols mainly came from amino acid metabolism of yeasts, so the reduction of higher alcohols in rice steaming with Lactobacillus was due to the coordinated growth of Lactobacillus and yeast, which suppressed the yeast metabolism of amino acids into the higher alcohols. Changes of aldehyde under different fermentation process are shown in Fig. 3C, the aldehyde content of rice steaming with Lactobacillus was lower than others, especially was only about 5 mg/L, control of rice soaking was significantly higher than the other groups, aldehydes in CRW was a dynamic change process under the influence of multiple factors. There was no significant distinctions between the different fermentation process of the phenols content, and the inoculation of Lactobacillus had no obvious influence on phenols (Fig. 3D). Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2016.11.014. ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (2016YFD0400504), the ‘863’ Program (2013AA102203-06), the National Natural Science Foundation of China (31271839 and 31571823), the Fundamental Research Funds for the Central Universities of China (JUSRP11509) and achievements transformation of industry-university-research cooperation in Shanghai Jinshan district (2016-CXY-01). References 1. Yu, L. J., Ding, F., and Ye, H.: Analysis of characteristic flavour compounds in Chinese rice wines and representative fungi in wheat Qu samples from different regions, J. Inst. Brew., 118, 114e119 (2012). 2. Que, F., Mao, L. C., Zhu, C. G., and Xie, G. F.: Antioxidant properties of Chinese yellow wine, its concentrate and volatiles, Lwt-Food Sci. Technol., 39, 111e117 (2006). 3. Chen, S., Wang, D., and Xu, Y.: Characterization of odor-active compounds in sweet-type Chinese rice wine by aroma extract dilution analysis with special emphasis on sotolon, J. Agric. Food Chem., 61, 9712e9718 (2013).
4. Tomochika, M., Yoshifumi, K., and Yoshinori, W.: Water absorption characteristics and structural properties of rice for sake brewing, J. Biosci. Bioeng., 106, 258e262 (2008). 5. Luo, T., Fan, W., and Xu, Y.: Characterization of volatile and semivolatile compounds in Chinese rice wines by headspace solid phase microextraction followed by gas chromatography-mass spectrometry, J. Inst. Brew., 114, 172e179 (2008). 6. Tomochika, M., Akiko, T., Atsushi, K., Yoshifumi, K., and Yoshinori, W.: Water-absorption rate equation of rice for brewing sake, J. Biosci. Bioeng., 103, 60e65 (2007). 7. Xu, E. B., Wu, Z. Z., Wang, F., Li, H. Y., Xu, X. M., Jin, Z. Y., and Jiao, A. Q.: Impact of high-shear extrusion combined with enzymatic hydrolysis on rice properties and Chinese rice wine fermentation, Food Bioprocess Technol., 8, 589e604 (2015). 8. Liu, S. P., Yu, J. X., Wei, X. L., Ji, Z. W., Zhou, Z. L., Meng, X. Y., and Mao, J.: Sequencing-based screening of functional microorganism to decrease the formation of biogenic amines in Chinese rice wine, Food Control, 64, 98e104 (2016). 9. Cao, Y., Xie, G. F., Wu, C., and Lu, J.: A study on characteristic flavor compounds in traditional Chinese rice wine-Guyue Longshan rice wine, J. Inst. Brew., 116, 182e189 (2010). 10. Alvarez, M. A. and Victoria Moreno-Arribas, M.: The problem of biogenic amines in fermented foods and the use of potential biogenic amine-degrading microorganisms as a solution, Trends Food Sci. Technol., 39, 146e155 (2014). 11. Pineda, A., Carrasco, J., Pena-Farfal, C., Henriquez-Aedo, K., and Aranda, M.: Preliminary evaluation of biogenic amines content in Chilean young varietal wines by HPLC, Food Control, 23, 251e257 (2012). 12. Yongmei, L., Xin, L., Xiaohong, C., Mei, J., Chao, L., and Mingsheng, D.: A survey of biogenic amines in Chinese rice wines, Food Chem., 100, 1424e1428 (2007). 13. Chen, S. and Xu, Y.: The influence of yeast strains on the volatile flavour compounds of Chinese rice wine, J. Inst. Brew., 116, 190e196 (2010). 14. Dong, L., Piao, Y., Zhang, X., Zhao, C., Hou, Y., and Shi, Z.: Analysis of volatile compounds from a malting process using headspace solid-phase microextraction and GC-MS, Food Res. Int., 51, 783e789 (2013). 15. Xu, E. B., Long, J., Wu, Z. Z., Li, H. Y., Wang, F., Xu, X. M., Jin, Z. Y., and Jiao, A. Q.: Characterization of volatile flavor compounds in Chinese rice wine fermented from enzymatic extruded rice, J. Food Sci., 80, 1476e1489 (2015). 16. Wang, P., Mao, J., Meng, X., Li, X., Liu, Y., and Feng, H.: Changes in flavour characteristics and bacterial diversity during the traditional fermentation of Chinese rice wines from Shaoxing region, Food Control, 44, 58e63 (2014). 17. Romano, A., Klebanowski, H., La Guerche, S., Beneduce, L., Spano, G., Murat, M.-L., and Lucas, P.: Determination of biogenic amines in wine by thinlayer chromatography/densitometry, Food Chem., 135, 1392e1396 (2012). 18. Li, H., Jiao, A., Xu, X., Wu, C., Wei, B., Hu, X., Jin, Z., and Tian, Y.: Simultaneous saccharification and fermentation of broken rice: an enzymatic extrusion liquefaction pretreatment for Chinese rice wine production, Bioprocess Biosyst. Eng., 36, 1141e1148 (2013). 19. Huang, K. J., Wei, C. Y., Liu, W. L., Xie, W. Z., Zhang, J. F., and Wang, W.: Ultrasound-assisted dispersive liquid-liquid microextraction combined with high-performance liquid chromatography-fluorescence detection for sensitive determination of biogenic amines in rice wine samples, J. Chromatogr. A, 1216, 6636e6641 (2009). 20. Xia, X. L., Zhang, Q. W., Zhang, B., Zhang, W. J., and Wang, W.: Insights into the biogenic amine metabolic landscape during industrial semidry Chinese rice wine fermentation, J. Agric. Food Chem., 64, 7385e7393 (2016). 21. Chu, C. H. and Bejldanes, L. F.: Effect of diamines, polyamines and tuna fish extracts on the binding of histamine to mucin in vitro, J. Food Sci., 47, 79e80 (1982). 22. Bjeldanes, L. F., Schutz, D. E., and Morris, M. M.: On the aetiology of scombroid poisoning: cadaverin potentiation of histamine toxicity in the Guinea pig, Food Cosmet. Toxicol., 16, 157e159 (1978). 23. Herbert, Paulo, Cabrita, Maria J., Ratola, Nuno, Laureano, Olga, and Alves, Arminda: Free amino acids and biogenic amines in wines and musts from the Alentejo region. Evolution of amines during alcoholic fermentation and relationship with variety, sub-region and vintage, J. Food Eng., 66, 315e322 (2005). 24. Molina, A. M., Guadalupe, V., Varela, C., Swiegers, J. H., Pretorius, I. S., and Agosin, E.: Differential synthesis of fermentative aroma compounds of two related commercial wine yeast strains, Food Chem., 117, 189e195 (2009). 25. Mo, X., Fan, W., and Xu, Y.: Changes in volatile compounds of Chinese rice wine wheat Qu during fermentation and storage, J. Inst. Brew., 115, 300e307 (2009). 26. Fan, W. L. and Qian, M. C.: Headspace solid phase microextraction and gas chromatographyolfactometry dilution analysis of young and aged Chinese “Yanghe Daqu” liquors, J. Agric. Food Chem., 53, 7931e7938 (2005). 27. Wondra, M. and Berovic, M.: Analyses of aroma components of chardonnay wine fermented by different yeast strains, Food Technol. Biotechnol., 39, 141e148 (2001). 28. Hazelwood, L. A., Daran, J.-M., van Maris, A. J. A., Pronk, J. T., and Dickinson, J. R.: The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism, Appl. Environ. Microbiol., 74, 2259e2266 (2008).
Please cite this article in press as: Wei, X. L., et al., Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process Xiao Lu Wei,1, 2, z Shuang Ping Liu,1, 2, 3, z Jian Shen Yu,4 Yong Jian Yu,5 Sheng Hu Zhu,5 Zhi Lei Zhou,1, 2, 3 Jian Hu,4 and Jian Mao1, 2, 3, * National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, Jiangsu 214122, China,1 School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China,2 National Engineering Research Center of Chinese Rice Wine, Shaoxing, Zhejiang 31200, China,3 Shanghai Jinfeng Wine Co., Ltd., Shanghai 200063, China,4 and Jiangsu Hengshun Vinegar Industry Co., Ltd., Zhenjiang 212043, PR China5 Received 21 August 2016; accepted 30 November 2016 Available online xxx
As a traditional fermented alcoholic beverage of China, Chinese rice wine (CRW) had a long history of more than 5000 years. Rice soaking process was the most crucial step during CRW brewing process, because rice soaking quality directly determined the quality of CRW. However, rice soaking water would cause the eutrophication of water bodies and waste of water. The longer time of rice soaking, the higher the content of biogenic amine, and it would have a huge impact on human health. An innovation brewing technology was carried out to exclude the rice soaking process and the Lactobacillus was added to make up for the total acid. Compared to the traditional brewing technology, the new technology saved water resources and reduced environmental pollution. The concentration of biogenic amine was also decreased by 27.16%, which improving the security of the CRW. The esters increased led to more soft-tasted CRW and less aging time; the quality of CRW would be improved with less alcohol. Ó 2016, The Society for Biotechnology, Japan. All rights reserved. [Key words: Chinese rice wine; Brewing; Rice soaking process; Lactobacillus; Biogenic amine]
As a special and traditional fermented alcoholic beverage of China, Chinese rice wine (CRW) has had a long history of more than 5000 years (1). Not only due to the abundant amino acids, sugar and other nutritional substances CRW hold, but its low alcohol (content) of 14e20% (v/v) (2), CRW has been popular in China. Recently, with a large amount of customers, production of CRW has been increasing gradually (3). CRW brewing process is mainly divided into two parts: the first step is the pretreatment of the raw materials, which is mainly the rice soaking. Rice soaking is the most crucial step of CRW brewing process, because rice soaking quality directly determines the quality of CRW (4), and its application in CRW brewing process has lasted for hundreds of years; the second step is the fermentation process, which refers to ferment steamed rice with wheat qu and the yeast (1,5). The rice soaking process always lasts quite a long time in the traditional CRW brewing. For example, traditional cooling of rice wine requires 16e20 days during soaking period. (The main changes in the rice soaking process are rice swelling after sucking in water (4), decomposition of main ingredients, the increase of water acidity, and so on.) Afterwards, rice can be steamed easily (6). High
* Corresponding author at: National Engineering Laboratory for Cereal Fermentation Technology, School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China. Tel./fax: þ86 510 85328273. E-mail address: [email protected] (J. Mao). z The first two authors contributed equally to this work.
concentration of lactic acid containing in rice soaking water (5e9 g/ L) can protect the metabolism of yeasts by inhibiting other bacteria growth. As a result, the acidity of rice soaking water is relatively high, usually between 6 and 15 g/L. With a plenty of nitrogen compounds, rice soaking water can cause the eutrophication of water bodies, which will result in serious environmental pollution (7). Waste soaking water treatment and water costs are the main burden for CRW winery (microorganisms in rice soaking water can use free amino acids to produce biogenic amines, which are also brought into the brewing process). The longer the rice soaks, the higher the biogenic amine of CRW contains, and it will have a huge impact on human health if the content is too high. In recent years, as increasing widespread attention towards food security, biogenic amine content in CRW appears to arise more public concerns than ever. From the perspective of country’s industrial adjustment and the long-term development of CRW industry, an eco-friendly and safety way must be found. Therefore, the solutions to economize water resources and decrease biogenic amines content have drawn much more attention than ever. The objective of the present work is to exclude rice soaking process, and economize all the water that used for rice soaking. Moreover, with the addition of Lactobacillus (8), it could make up for the insufficient of total acids and create low pH for CRW fermentation. A new CRW brewing process without rice soaking process is firstly developed in this paper, and will solve the problems such as rice soaking water’s pollution, waste of water resources and high content of biogenic amines.
1389-1723/$ e see front matter Ó 2016, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
Please cite this article in press as: Wei, X. L., et al., Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
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Sample collection and processing Three fermentation samples (control of rice soaking, rice steaming with Lactobacillus and rice steaming without Lactobacillus) were taken. The fermentation mash is needed to be stirred evenly when sampled, then centrifuged to collect the supernatant and kept in ultra-low temperature freezer for later detection (including ethanol, total acids, pH, amino nitrogen (AN), reducing sugar, biogenic amine and volatile flavor). The proportions of sugars, ethanol, and organic acids were primarily responsible for the delicate taste and flavors of CRW (9). While detecting the total acids of the supernatant, the filter paper must be used to wipe the carbon dioxide out (because of the ethanol fermentation, which would increase the total acids to a great extent). The biogenic amines of CRW are in high concentration then, but low content of biogenic amines in CRW exactly meets the current market demand (10e12). Control of rice soaking technology and the moisture absorption of rice The control of rice soaking process was practiced by soaking the rice and water in proportion of 1:1 until the total acids of rice soaking water was 6e10 g/L. After draining the water, rice could be steamed then. The moisture absorption of rice was tested as follows, first and foremost, soaking 10 g of rice in water (28 C) and sampling some rice every 10 min; then drying the water of rice surface immediately with filter paper and weighing, besides, weighing the rice after 105 C drying to constant weight (4). The difference between these two was the moisture absorption of rice. Brewing with rice of different soaking time Rice was initially divided into five groups, each was soaking in water in proportion of 1:1, and soaking for 1e5 days, respectively. After soaking, rice with different soaking time was steamed and ready to ferment. The biogenic amine content of each group was detected and analyzed. Innovation of rice steaming technology The first step was spraying the water of 50 C about 150 mL/kg onto the rice, and the time was strictly controlled within 20 min. After spraying, the rice was steamed for the first time for approximately 7 min. Then sprayed for the second time about 150 mL/kg onto rice, the water temperature was controlled above 80 C. The second rice steaming time was 8 min and then spraying for the third time (the same to the second time). The final rice steaming time lasted for 10 min, after which cooling the steamed rice by air blast and fermenting with wheat qu, yeasts and other ingredients. The spraying water was recyclable, and there was fresh water supplement when it was water shortage. The Lactobacillus culturing and concentrated lyophilized cultures preparing The Lactobacillus was screened from fresh fermentation mash based on the next-generation sequencing technology (8), and then cultured through the three-level fermentation to get the Lactobacillus broths. The Lactobacillus in lyophilized preservation tube was inoculated to 150 mL MRS liquid medium and incubated at 37 C for 18 and 20 h anaerobically, which was as the seed liquid. Five percentage of quantity of the seed was inoculated to 400 mL MRS liquid medium, which was as the secondary seed liquid. Secondary seed with 5% quantity was inoculated into 6000 mL MRS liquid medium, and it was incubated at 37 C for 18e20 h, then to get triple Lactobacillus broths. The broths were centrifuged at 5000 r/min for 15 min to get Lactobacillus mud, which should be absterged twice with the sterile saline. The protective agent (90% skim milk, 10% trehalose) was used to dissolve Lactobacillus mud. Mixtures were freeze-dried for 48 h after precooling for 2 h, and then cultures were stored at 20 C for later use. Preparation and selection of Lactobacillus medium In order to facilitate the CRW winery from culturing Lactobacillus, malt medium, rice medium and glutinous rice medium were chosen as Lactobacillus medium. MRS liquid medium was impractical because of the high cost, and it also affected the flavor of CRW. Malt medium was made by mixing water and malt flour (v/v 4:1), then the mixtures were saccharified at 60 C for 4 h and sterilized at 115 C for 15 min. Rice (or glutinous rice) medium was made by mixing rice (or glutinous rice), malt flour and water (v/v/v 1:0.2:4), then saccharifying was done by adding enzymes and wheat qu at 60 C for 4 h. After that, sterilization was done at 115 C for 15 min. Lactobacillus lyophilized cultures were inoculated in MRS liquid medium for activation with a ratio of 5% based on the volume of (said MRS liquid medium), incubated at 37 C under anaerobic condition for 24 h, then 10% activated culture was transferred to new malt medium, rice medium and glutinous rice medium, respectively, and were incubated at 37 C for 60 h under anaerobic condition. The inoculation size of Lactobacillus must also be optimized. On the premise of not affecting rice wine fermentation, four kinds of inoculation size of Lactobacillus, namely, 2%, 4%, 8% and 12% were chosen for brewing experiment. CRW brewing process The rice steaming process was prepared as described in the Innovation of rice steaming technology section. After steaming, the 200 kg steamed rice, 175 kg water, 4.3 kg wheat qu, 21.5 kg rice wine starter (6& of yeast activated liquid was inoculated in rice medium, incubated at 28 C and 150 rpm for 24 h), 50 mL amylase (Suhong AA Plus), 50 mL glucoamylase (Suhong GA II) and Lactobacillus broth were added into the fermentation tank, the water temperature was controlled at 24e28 C. The primary fermentation was incubated at 28 C for 96 h with aeration once a day, and natural temperature with no aeration was suitable for post fermentation. The total fermentation hours were nearly 384 he432 h.
Detection of main volatile flavor compounds in fermentation mash using headspace solid-phase micro-extraction and gas chromatographyemass spectrometry The headspace solid-phase micro-extraction (HS-SPME) method was referred to the report (5) and some adjustments. The alcohol content of CRW was diluted to 6% (v/v), then 6 mL diluted wine was taken to the head space bottle, 1.5 g NaCl and 15 mL internal standard (8800 mg/L 2-octanol) were added. A 50/30 mm divinylbenzene/carboxen/poly (dimethylsiloxane) (DVB/CAR/PDMS) coated fibers was used for the extractions of volatile compounds. The sample was equilibrated and extracted at 50 C for 40 min. After HS-SPME, the fiber was inserted into the injection port of gas chromatographyemass spectrometry (GCeMS) (250 C) for 7 min to desorb the analytes. The GCeMS conditions were according to the reports (13e15) and some adjustments. Chromatographic column: TG-WAXMS (30 m 0.25 mm 0.25 mm); injection port temperature: 250 C; temperature program: 40 C keep 3 min; 6 C/min up to 100 C; 10 C/min up to 230 C, keep 7 min; carrier gas: high purity helium (>99.999%), no shunt, flow rate of 1.0 mL/min; ionization method: EI; the emission current: 50 mA; electron energy: 70 eV; ion source temperature: 230; transmission line temperature: 250 C; scan range: 33e400 amu. The 2-octanol was used as the internal standard for semi-quantification of the volatiles according to the previous report (16). The detection of biogenic amine Samples (1 mL) were taken in different 10 mL plastic centrifuge tube, in each of the centrifuge tube mixed with 1 mL of the saturated NaHCO3 solution and 2 mL dansyl chloride solution (5 mg/mL in acetone), vortexed for 1 min, then mixtures were heated in water bath of 60 C for 30 min to derive. Mixtures were then placed at room temperature for a few minutes and 0.5 mL saturated NaCl solution was added (17). With absolute ether to be extraction agent, extraction was performed on a tabletop shaker for 2 min, the upper organic phase was taken, and the above extraction process was repeated twice. Finally, all the upper organic phase was merged with nitrogen gas blowing and 1 mL of acetonitrile dissolving after drying, then after the membrane filtration of 0.22 mm, it could (be directly into) the high performance liquid chromatography (HPLC) determination. A chromatographic column (250 mm 4.6 mm, XBridge C18, 5 mm) was used for all separations, with column temperature at 30 C and flow rate at 0.8 mL/min. In terms of mobile phase, ultrapure water was used as eluent A and acetonitrile was used as eluent B. For data analysis, the absorbance was read at 254 nm using a UV detector. Statistical analysis Volatile flavor and biogenic amine were also tested by using GCeMS and HPLC method separately, all analyses had three replicates.
RESULTS AND DISCUSSION Biogenic amine in the fermentation process of rice of different soaking time After rice soaking process, rice became loose for steaming, which was more beneficial to alcoholic fermentation (18). Rice could not be steamed ripe without soaking because of the gelatinization of rice starch, which needed to be at a certain temperature and moisture content conditions (6). Meanwhile, the traditional rice soaking process was last long and produced a large amount of rice soaking water that would pollute the environment. Fig. S1 shows that the longer the soaking time is, the more biogenic amine content generated in the fermentation process. Overall trends, biogenic amine content had been gradually increasing in the primary fermentation. On the fermentation time of 72 h, biogenic amine content reached the maximum, and then fell slightly. During the post fermentation, biogenic amine content had a downward trend. Based on this phenomenon, an innovative rice steaming technology was developed (described in the Innovation of rice steaming technology section) to replace the traditional rice soaking and steaming process to reduce the content of biogenic amines and waste water emissions. Innovation brewing of CRW In the developed rice steaming process, spraying water for 20 min onto rice induced quick absorption (Table 1). After it, the moisture content of rice was about 30% (Table 1) that achieved the same value in Fig. S2. After first steaming, the rice moisture content was increased by 2%e5%, and was up to 41%e44% after the final steaming. The figure of final rice moisture content was near that of the rice moisture content of control rice soaking (about 46%), which suggested that the rice of new steaming technology could achieve a certain degree of gelatinization and the new steaming technology could be applied to the CRW brewing process. However, with prolonging the soaking time, the components of rice reduced and transferred into water, like reducing sugar, total
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TABLE 1. Moisture content of rice at three different stages during the new steaming process. Sample
1 2 3 4
Rice moisture content after the first spraying (%)
Rice moisture content after first steaming (%)
Rice moisture content after final steaming (%)
30 29 31 29
33 32 33 34
42 44 43 41
The samples were collected from three different stages: after the first water spraying process, after the first rice steaming process, at the end of rice steaming process.
sugar, crude protein, crude fat and ash. Microorganism in soaking water used these compounds for growth, and increased the total acids in soaking water to 5e9 g/L (calculated in lactic acid). Some of these acids could be absorbed in rice and was subsequently carried into the fermentation process. Thus, the fermentation process had a low pH (about pH 3.5), which restrained bacteria from overgrowing. The new brewing process had no rice soaking, the fermentation process was not in low pH without accumulation of acids, then bacteria was likely to overgrow which might inhibit the growth of yeast and terminated the fermentation of alcohol. Although the rice soaking process was removed and the rice was steamed successfully by the innovative rice steaming technology, the acid in steamed rice was not enough to inhibit the growth of unexpected bacteria. On this basis, it was supposed that addition of Lactobacillus could create a low pH for fermentation and supply sufficient total acids. At the same time, there was no rice soaking water emissions that polluted the environment. Based on the fact, the Lactobacillus that produced no biogenic amines was screened and back-added into the fermentation mash. It also showed the Lactobacillus had high lactic acid production, and high ethanol tolerance which was preferable for CRW brewing (8). The growth of Lactobacillus in different culture medium In order to facilitate the CRW winery from producing, it was suitable to use activated fresh Lactobacillus broths instead of the concentrated lyophilized cultures. Therefore, the concentrated lyophilized cultures were inoculated into different culture medium to obtain activated fresh Lactobacillus broths. With the extension of culture time of Lactobacillus, the total acids of every medium increased. At the first 8 h, total acids increased slowly because of the adaptation to the environment; total acids increased rapidly between 8 and 24 h, showing that Lactobacillus metabolized actively; from 32 to 60 h, the total acids increased slowly. Malt medium had the highest total acids comparing with the other two mediums, and total acids of rice medium and glutinous rice medium were relatively similar to each other (Fig. 1A). However, the reducing sugar content in every medium descended gradually (Fig. 1B). From 0 to 4 h, the reducing sugar dropped most significantly; after 4 h, the overall decline was relatively flat. The reducing sugar content of malt medium, rice medium and glutinous rice medium were all reduced about 20 g/L. The reducing sugar in malt medium was significantly lower than the other two kinds of medium. In conclusion, malt medium was the best choice for culturing the Lactobacillus, which components in the above several kinds of the medium was the most close to MRS. However, for the actual production from the winery factory, rice medium as the culture medium of Lactobacillus was the right choice. The rice relative to glutinous rice was more economical and practical, and was consistent with the raw materials of CRW. It was also advantageous for the Lactobacillus to adapt to the fermentation environment quickly. Then it would not bring too much pressure from the outside on Lactobacillus. Outside the pressure, adding too much malt medium in the brewing process would affect the flavor of
FIG. 1. Changes of total acid (A) and reducing sugar (B) during culturing Lactobacillus in different mediums.
CRW. Therefore, after the activation of Lactobacillus lyophilized cultures in MRS liquid medium, the activated culture could be transplanted to the malt medium for culturing. Finally, the culture could be transplanted to rice medium for actual production. Thus, it could ensure not only the vitality of Lactobacillus but also the flavor of CRW. Influence of different inoculation size of Lactobacillus on CRW The purpose of Lactobacillus back-added was to provide a low pH for fermentation and improve the total acids (up to 5 g/L) of fermentation mash on the premise of not affecting rice wine fermentation. Lactobacillus overgrew and increased the total acids which would lead to the acidification of the wine. The detection results of fermentation mash with different inoculation size of Lactobacillus are shown in Table 2. With the inoculation size increasing, after the fermentation was finished, the total acids were higher. Because superabundant acids affected the metabolism of yeasts that making use of reducing sugar to produce alcohol, the fermentation process became more difficult. Alcohol rose faster under this craft, alcohol content was almost around 7% (v/v) at 24 h (Table 2). At the end of the primary fermentation, alcohol content reached to 15% (v/v), then rose 2e3% (v/v) or so during the post fermentation, so adding the Lactobacillus
Please cite this article in press as: Wei, X. L., et al., Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
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TABLE 2. The detection results of fermentation mash with different inoculation size of Lactobacillus. Inoculation amount Fermentation of Lactobacillus (%) time (h)
2
Physical and chemical indicators Total acid (g/L)
pH
2.71 3.42 3.60 3.94 2.88 3.92 4.25 5.05 3.16 4.53 5.42 5.95 3.34 5.22 5.95 6.52
3.89 3.68 3.55 3.51 3.76 3.50 3.84 3.89 3.37 3.46 3.73 3.67 3.33 3.41 3.62 3.50
24 96 192 384 24 96 192 384 24 96 192 384 24 96 192 384
4
8
12
Alcohol Reducing content (%) sugar (g/L) 10.70 14.40 15.80 17.20 7.20 15.60 16.50 18.24 7.10 14.90 15.90 16.13 6.60 13.70 14.40 15.62
53.56 8.07 4.21 2.01 67.00 6.80 3.33 2.30 65.80 7.03 4.06 2.70 64.40 10.51 6.18 3.75
AN 0.08 0.14 0.25 0.32 0.03 0.07 0.27 0.29 0.02 0.07 0.26 0.28 0.02 0.07 0.27 0.27
to improve total acids played a vital role in the primary fermentation. The post fermentation with higher alcohol content inhibited the activity of Lactobacillus, and acids’ producing ability of it was extremely low. The optimum inoculation size of Lactobacillus was 4%. When inoculation size was 2%, the total acids could not meet the winery requirements; when inoculation sizes were 8% and 12%, the total acid contents were both beyond the scope of the winery standard, which made the rice wine taste poorly. Therefore, the most appropriate inoculation size was 4%. The detection results of different fermentation process With rice medium culturing Lactobacillus and inoculation size of 4% to brew, fermentation mash of rice steaming with Lactobacillus, rice steaming without Lactobacillus and control of rice soaking were tested and compared. The results are shown in Table 3. The total acids of culture medium (about 1 L) were 11.34 g/L, while the total acids of fermentation system (500 kg) were only 0.023 g/L, so the added culture medium could hardly affect the acids of the fermentation system. However, the alcohol content was around 7e9% (v/v) at the end of 24 h of fermentation, and the total acids of rice steaming with Lactobacillus were 4e5 g/L. At the end of the fermentation (384 h), the total acids content of rice steaming without Lactobacillus (sample L) and control of rice soaking (sample J) were between 3.5 and 4 g/L. The acidity was insufficient, could not
TABLE 3. The detection results of fermentation mash with different crafts. Sample
D
L
J
Fermentation time (h)
24 96 192 288 384 24 96 192 288 384 24 96 192 288 384
Physical and chemical indicators Total acid (g/L)
pH
Alcohol content (%)
Reducing sugar (g/L)
AN
4.56 4.78 4.90 4.95 5.01 3.52 2.79 3.31 3.59 3.45 2.97 3.33 3.88 4.31 4.06
3.73 3.71 3.97 3.99 3.99 3.69 4.79 4.75 4.76 4.44 3.78 4.19 4.39 4.39 4.40
8.15 15.10 14.60 15.90 17.60 9.20 13.90 14.80 14.60 15.20 7.75 16.90 17.00 17.60 18.00
13.70 3.40 2.10 3.11 1.56 20.40 7.60 6.07 5.01 4.81 35.10 3.40 2.59 1.90 1.14
0.03 0.19 0.25 0.53 0.82 0.02 0.69 0.72 0.78 0.79 0.02 0.41 0.55 0.64 0.68
FIG. 2. Changes of biogenic amine (A), putrescine (B) and tyramine (C) in different fermentation process.
D: rice steaming with Lactobacillus of 4%; L: rice steaming without Lactobacillus; J: control of rice soaking.
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INNOVATION CHINESE RICE WINE BREWING TECHNOLOGY
reach the winery requirements. However, the total acids of rice steaming with Lactobacillus (sample D) were 5.01 g/L, which improved about 1.5 g/L compared to sample L and met the needs. The total acids of the sample L during 24 he96 h were decreasing and this may be due to the fast increase of AN content from 0.02 to 0.69. Some free acid radical ions may turn into amino acids, which may cause the decrease of total acids. It may also have certain effects on the metabolism of the Lactobacillus. After 96 h, the concentration of AN had little change and the total acids increased slightly because of the metabolism of the Lactobacillus. At the same time, alcohol content, reducing sugar, amino nitrogen (AN) and pH were also conformed to the enterprise standard. As a result, rice steaming with Lactobacillus (sample D) was quite a good new CRW brewing technology, which reduced waste water emissions and the content of biogenic amines. Biogenic amine in fermentation mash Changes of biogenic amines in different fermentation mash are shown in Fig. 2A. The diagram shows that control of rice soaking of biogenic amine content was higher than the other two crafts and at a high level, about 30 mg/L. Rice steaming without Lactobacillus of biogenic amine was mainly increased during the primary fermentation, and had a slight increase during the post fermentation. At the end of the fermentation, the total content of biogenic amines in rice steaming with Lactobacillus was around 21 mg/L, compared to control of rice soaking and rice steaming without Lactobacillus, which were reduced by 27.16% and 20.59%, respectively. On account of Lactobacillus cultures of low pH, it reduced the fermentation pH and largely inhibited the growth of the other bacteria metabolism of generating biogenic amines. Therefore,
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with Lactobacillus culture of 4% to brew, it could effectively reduce the biogenic amine amount of CRW, and at the same time increase the total acids, which making the rice wine more secure and having a better taste. Biogenic amines in CRW were mainly putrescine, tyramine and so on (19,20). Putrescine of control of rice soaking was the maximum one, around 9 mg/L (Fig. 2B). At the end of the fermentation of rice steaming with Lactobacillus, the putrescine was only about 3.5 mg/L, which was one third of control of rice soaking, so reducing the content of putrescine also largely reduced the total content of biogenic amines. Fig. 2C shows the tyramine changes of different fermentation processes. Tyramine content of control of rice soaking was the highest, while rice steaming with Lactobacillus was the lowest. In addition to the increase of 0.5 mg/L during the primary fermentation, there was almost no changes in the post fermentation. To certain extent, tyramine could be detrimental to the human bodies, which was the most toxic biogenic amine compared with others (21,22). Malolactic fermentation was confirmed to be the main origin of tyramine (23). Thus, reducing tyramine content by adding Lactobacillus could improve the safety of CRW. Main volatile flavor compounds in fermentation mash Volatile flavor compounds played an important role in the typical taste of CRW (13,24). From the different fermentation mash, it mainly determined the 15 kinds of ester substances, 10 kinds of alcohols, 5 kinds of aldehyde and 4 kinds of phenol. Fig. 3A shows the changes of total volatile esters in different fermentation processes. With the prolonging of fermentation time, the total volatile esters content showed an increasing trend on the
FIG. 3. Changes of total volatile ester (A), alcohol (B), aldehyde (C) and phenol (D) in different fermentation process.
Please cite this article in press as: Wei, X. L., et al., Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.014
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whole. The total volatile easers of rice steaming with Lactobacillus were more than others, the increase of the esters might be generated by metabolic reaction of Lactobacillus. Esters were the largest part of volatile compounds in CRW, and were mainly produced by the esterification of alcohol and acid (25). There were two ways to form esters: one was from the biosynthesis of microorganism such as ester-producing yeasts in fermentation mash, the other was generated by organic chemical reactions. The latter under the normal condition of the reaction was especially slow, often needed a long time to make the esterification reaction equalized. When under the normal fermentation, esters of fermentation mash mainly stemmed from the metabolism of ester yeasts (5). Esters could impact the taste of CRW for the low threshold concentrations and desirable fruity and flowery flavors. Furthermore, wine would become mellower with the longer aging time due to increasing ester substances (26). Therefore, adding the Lactobacillus may reduce the aging time of rice wine and improve the taste of CRW. The higher alcohols were one of the most important volatile compounds in CRW, which were mainly generated by the metabolism of yeasts that used sugar and amino acid (27). In the process of amino acid metabolism, amino acid could generate higher alcohols by deamination or decarboxylation reaction of losing a carbon atom (28). The higher alcohols in CRW were the main cause of being unwell after drinking CRW. Thus, it was appropriate to reduce higher alcohols content in CRW, which was beneficial to improve the quality and the comfort level of drinking CRW. Fig. 3B shows the changes of alcohols under different fermentation process, rice steaming with Lactobacillus of higher alcohol content was lower than rice steaming without Lactobacillus and control of rice soaking. During the post fermentation, higher alcohols mainly came from amino acid metabolism of yeasts, so the reduction of higher alcohols in rice steaming with Lactobacillus was due to the coordinated growth of Lactobacillus and yeast, which suppressed the yeast metabolism of amino acids into the higher alcohols. Changes of aldehyde under different fermentation process are shown in Fig. 3C, the aldehyde content of rice steaming with Lactobacillus was lower than others, especially was only about 5 mg/L, control of rice soaking was significantly higher than the other groups, aldehydes in CRW was a dynamic change process under the influence of multiple factors. There was no significant distinctions between the different fermentation process of the phenols content, and the inoculation of Lactobacillus had no obvious influence on phenols (Fig. 3D). Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2016.11.014. ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (2016YFD0400504), the ‘863’ Program (2013AA102203-06), the National Natural Science Foundation of China (31271839 and 31571823), the Fundamental Research Funds for the Central Universities of China (JUSRP11509) and achievements transformation of industry-university-research cooperation in Shanghai Jinshan district (2016-CXY-01). References 1. Yu, L. J., Ding, F., and Ye, H.: Analysis of characteristic flavour compounds in Chinese rice wines and representative fungi in wheat Qu samples from different regions, J. Inst. Brew., 118, 114e119 (2012). 2. Que, F., Mao, L. C., Zhu, C. G., and Xie, G. F.: Antioxidant properties of Chinese yellow wine, its concentrate and volatiles, Lwt-Food Sci. Technol., 39, 111e117 (2006). 3. Chen, S., Wang, D., and Xu, Y.: Characterization of odor-active compounds in sweet-type Chinese rice wine by aroma extract dilution analysis with special emphasis on sotolon, J. Agric. Food Chem., 61, 9712e9718 (2013).
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Please cite this article in press as: Wei, X. L., et al., Innovation Chinese rice wine brewing technology by bi-acidification to exclude rice soaking process, J. Biosci. Bioeng., (2016), http://dx.doi.org/10.1016/j.jbiosc.2016.11.014