CN112625931A

CN112625931A - Method for improving tyrosol and tryptophol content of yellow wine by promoting yeast Ailixi approach

Info

Publication number: CN112625931A
Application number: CN202011295346.0A
Authority: CN
Inventors: 毛健; 刘双平; 周虞; 周佳冰
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2020-11-18
Filing date: 2020-11-18
Publication date: 2021-04-09
Anticipated expiration: 2040-11-18
Also published as: CN112625931B

Abstract

The invention discloses a method for improving the contents of tyrosol and tryptophol in yellow wine by promoting a yeast Ailixi approach, belonging to the technical field of yellow wine brewing. The invention verifies that aromatic alcohol is mainly synthesized by an Alixi pathway in the fermentation process of the yellow wine by saccharomyces cerevisiae, and the strength of the Alixi pathway is regulated by adding acid protease, so that the contents of beta-phenethyl alcohol, tyrosol and tryptophol reach 130.31 +/-2.13 mg/L, 111.38 +/-4.93 mg/L and 9.78 +/-0.12 mg/L when the addition amount of the acid protease is 20U/g, which are respectively 1.27, 1.56 and 2.52 of a control group. The method has simple process, the prepared yellow wine has stable quality, the content of aromatic alcohol and amino acid in the yellow wine is obviously improved, the content of aromatic alcohol in cooking wine and table vinegar prepared by taking the yellow wine as the raw material is also improved, and the flavor is more mellow.

Description

Method for improving tyrosol and tryptophol content of yellow wine by promoting yeast Ailixi approach

Technical Field

The invention relates to a method for improving the contents of tyrosol and tryptophol in yellow wine by promoting a yeast Ailixi way, belonging to the technical field of yellow wine brewing.

Background

The yellow wine as a traditional fermented wine in China has the characteristics of plump wine body, soft wine property and the like, and contains a large amount of functional components. The yellow wine brewing is a fermented alcoholic beverage with low alcohol content (14-20 vol%) brewed by taking grains as raw materials and jointly participating in brewing by multiple microorganisms (mould, yeast and bacteria), and the yellow wine brewing is mainly brewed by the processes of rice soaking, rice steaming, fermentation (pre-fermentation and post-fermentation), squeezing, clarification, filtration, wine decocting, ageing, blending and the like.

The higher alcohol is a generic term for alcohols containing more than 3 carbon atoms, and is a main byproduct in fermented wine, the aromatic alcohol is a higher alcohol with benzene ring, and the aromatic alcohol in the wine mainly comprises beta-phenethyl alcohol, tyrosol and tryptophol. Proper amount of aromatic alcohol can endow the fermented wine with unique flavor. Studies have shown that aromatic alcohols contribute positively to the organoleptic properties of wine, and they are also known as bioactive compounds and quorum sensing molecules. Aromatic alcohol is commonly present in yellow wine, is used as a main flavor substance in the yellow wine, and also has the effects of helping sleep, protecting heart and the like.

Besides drinking, yellow wine can also be used as a raw material of cooking wine and vinegar. Because the addition amount and the eating amount of the cooking wine and the vinegar are low, the content of aromatic alcohol of the cooking wine and the vinegar needs to be improved to achieve the effect of enhancing the flavor.

The content of aromatic alcohol in the existing yellow wine is uneven, the high content of aromatic alcohol in cooking wine and table vinegar is difficult to ensure, and in order to overcome the problems, a new solution needs to be explored.

Disclosure of Invention

The invention provides a method for promoting expression of saccharomyces cerevisiae ehrlichi pathway related genes and improving the content of yellow wine aromatic alcohol, so as to improve the contents of tyrosol and tryptophol in yellow wine, cooking wine and vinegar, and make the flavor of the yellow wine, cooking wine and vinegar better and mellow.

The first purpose of the invention is to provide a method for improving the yield of tyrosol and tryptophol of yellow wine yeast, which is realized by promoting the expression of Eichi pathway genes of saccharomyces cerevisiae; the promotion of the expression of the saccharomyces cerevisiae ehrlichia pathway gene comprises but is not limited to the enhancement of the expression amount of the gene by using a strong promoter or the increase of the content of nutrient substances in a fermentation environment.

In one embodiment, the ehrlichia pathway comprises transaminase-encoding gene BAT2^11-1Aromatic decarboxylase encoding gene ARO10^11-1Pyruvate decarboxylase gene PDC5^11-1The gene ADH1 encoding alcohol dehydrogenase^11-1、ADH4^11-1And a gene coding for tyrosinase-related protein TRP1^11-1、TRP2^11-1、TRP3^11-1、TRP4^11-1、TRP5^11-1At least one gene of (1).

In one embodiment, the ADH1^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 1; the ADH4^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 2; the ARO10^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 3; the BAT2^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 4; the GAP1^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 5; the PDC1^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 6; the PDC5^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 7; the PDC6^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 8; the TRP1^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 9; the TRP2^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 10; the TRP3^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 11; the TRP4^11-1The nucleotide sequence of (A) is shown as SEQ ID NO. 12; the TRP5^11-1The nucleotide sequence of (A) is shown in SEQ ID NO. 13.

In one embodiment, the yellow wine yeast is saccharomyces cerevisiae 11-1 or model bacterium BY 4743; the preservation number of the saccharomyces cerevisiae 11-1 is CCTCC NO: M2017488, and is disclosed in the patent application with the publication number of CN 107937295A.

In one embodiment, the method is performed by adding an acid protease to a yellow wine yeast fermentation environment; the fermentation loop contains protein or protein-containing substances.

The second purpose of the invention is to provide a method for improving the contents of tyrosol and tryptophol in yellow rice wine, wherein the method is to add acid protease in the blanking stage or the fermentation stage.

In one embodiment, the yellow wine production process comprises rice soaking, rice steaming, spreading for cooling, yeast adding, blanking and fermenting.

In one embodiment, the amount of the acidic protease added is 6 to 30U/g.

In one embodiment, 6-30U/g acid protease is added in the blanking stage.

In one embodiment, no acid protease is added in the blanking stage, and 6-30U/g protease is added in the fermentation stage.

In one embodiment, the method comprises the following steps

(1) Rice soaking: soaking glutinous rice serving as a raw material in water;

(2) and (3) steaming rice: cleaning the soaked glutinous rice in the step (1), and cooking the glutinous rice by a rice cooker until no white core exists;

(3) spreading for cooling: placing the cooked rice in the step (2) on a table, and cooling to room temperature;

(4) adding yeast: mixing the cooked rice spread in the step (3) with saccharifying enzyme, liquefying enzyme and raw wheat starter for saccharification and liquefaction, sterilizing and cooling, inoculating saccharomyces cerevisiae 11-1, and culturing to be mature for later use;

(5) blanking: transferring the rice added with the yeast in the step (4) into a fermentation tank, and adding water, wheat koji, the yeast and acid protease;

(6) fermentation: fermenting at 28 deg.C for five days. Harrowing is carried out after 8h of fermentation in the first day, and harrowing is carried out once every 24h in the last four days. Fermenting at 15 deg.C for fifteen days. Harrowing is carried out once every 48h during the post-fermentation period.

(7) And (3) finished product: and after the fermentation is finished, filtering, squeezing and sterilizing to obtain the finished product yellow wine with high aromatic alcohol content.

In one embodiment, the proportion of rice soaked in step (1) is rice: water 1:1.2(w/w), and rice soaking time is 24 h.

In one embodiment, the temperature of the washing water in the step (2) is room temperature, and the steaming time is 20 min.

In one embodiment, the chilling temperature in step (3) is room temperature.

In one embodiment, the mass fractions of the saccharifying enzyme, the liquefying enzyme and the raw wheat starter in the step (4) are 1 per mill, 2 per mill and 13.4 percent, the temperature is 60 ℃, and the time is 4 hours; the inoculum size of the Saccharomyces cerevisiae was 5% (v/m).

In one embodiment, the sterilization conditions in step (5) are 115 ℃ for 20min, and the temperature is cooled to room temperature.

In one embodiment, the feed-water ratio in step (5) is 1: 1.35.

In one embodiment, the wheat koji in the step (5) is raw wheat koji and cooked wheat koji, and the raw wheat koji and the cooked wheat koji respectively account for 13.4% and 3.95% of the raw glutinous rice.

In one embodiment, the yeast in step (5) is added in an amount of 7.37% of the glutinous rice raw material, and the acid protease is derived from Aspergillus niger in an amount of 20U/g.

In one embodiment, the pre-fermentation temperature in step (6) is stabilized at 28 ℃ and the stationary culture is carried out for 5 days, and the post-fermentation temperature is stabilized at 16 ℃ and the stationary culture is carried out for 15 days.

In one embodiment, the press in step (6) is filtered twice with 8 layers of gauze.

In one embodiment, the sterilization temperature in step (6) is 85 ℃ and the sterilization time is 30 min.

The invention has the beneficial effects that:

1. according to the invention, through the research on the gene expression quantity of the related way of synthesizing aromatic alcohol by saccharomyces cerevisiae, the method is clear that the aromatic alcohol synthesized by saccharomyces cerevisiae in the yellow wine fermentation process mainly passes through the Alixi way, and the hydrolysis of protein in the raw materials is promoted by adding acid protease, so that the content of available amino acid of saccharomyces cerevisiae is increased, the expression of the related gene of the Alixi way of saccharomyces cerevisiae is promoted, and the effect of increasing the contents of tyrosol and tryptophol is achieved.

2. The method further improves the content of aromatic alcohol in the yellow wine by regulating and controlling the adding time of the acid protease, and can remarkably shorten the fermentation period, improve the content of aromatic alcohol in the yellow wine and enable the flavor of the yellow wine to be better and mellow while not remarkably influencing the physicochemical indexes of the yellow wine.

Drawings

FIG. 1 shows the content change of aromatic alcohol in the process of simulated fermentation of yellow wine;

FIG. 2 is the variation of amino acid content in the simulated fermentation process of yellow wine;

FIG. 3 shows the variation of the expression levels of Saccharomyces cerevisiae 11-1 and BY4743 genes during the simulated fermentation of yellow wine;

FIG. 4 shows promoter strengths of aromatic alcohol related genes in Saccharomyces cerevisiae 11-1 and BY4743 during simulated fermentation of yellow wine;

FIG. 5 is the influence of different addition strategies of acid protease on the physicochemical indexes of yellow wine in examples 2-6;

FIG. 6 shows the effect of different addition strategies of acid protease on the physicochemical indexes of yellow wine in examples 7 to 11;

FIG. 7 is the effect of different 2-6 acid protease addition strategies on amino acids in yellow wine in the examples;

FIG. 8 is the effect of different 7-11 acid protease addition strategies on amino acids in yellow wine according to the examples;

FIG. 9 is the effect of different 2-6 acid protease addition strategies on yellow wine aromatic alcohol in the examples;

FIG. 10 is a graph showing the effect of different 7-11 acid protease addition strategies on yellow wine aromatic alcohol in examples;

FIG. 11 is the effect of example 4 on the Ehrlich pathway of Saccharomyces cerevisiae by addition of an acidic protease.

Detailed Description

Introduction of raw materials in the examples:

glutinous rice is purchased from Wuxi rice.

Raw wheat koji and cooked wheat koji from guyuelongshan shaoxing wine, gmbh.

The acid protease with the enzyme activity of 5 ten thousand U/g is purchased from Beijing Soilebao science and technology Limited.

Beta-phenylethyl alcohol, tyrosol and tryptophol were all of chromatographic grade and purchased from Sigma.

The YPD preparation method comprises the following steps: 1% yeast extract, 2% peptone, 2% glucose.

TB Green Fast qPCR Mix was purchased from Bao bioengineering (Dalian) Co., Ltd.

Trizol reagent, DEPC water, was purchased from Biotechnology engineering (Shanghai) Inc.

Example 1

(1) Preparing yellow wine simulation liquid, wherein the proportion of the yellow wine simulation liquid is as follows: 1.8kg of steamed rice, 1.4kg of clean water, 17.2g of inoculated raw wheat starter, 2.6mL of liquefying enzyme with the enzyme activity of 86.54U/mL and 1.28mL of saccharifying enzyme with the enzyme activity of 175.78U/mL. Saccharifying at 60 deg.C for 6 hr, filtering with 8 layers of gauze, and sterilizing.

(2) Inoculating with 5% inoculum concentration of 10⁸And (3) carrying out yellow wine simulated fermentation on the CFU/mL saccharomyces cerevisiae 11-1 or the model bacterium BY 4743.

(3) The dynamic changes of aromatic alcohol and free amino acid in the fermentation process are measured by high performance liquid chromatography.

FIGS. 1(a), (b) and (c) show the changes of beta-phenylethyl alcohol, tyrosol and tryptophol, respectively, in a simulated fermentation process. As can be seen from FIG. 1, aromatic alcohol in both groups of samples is produced in large quantity during the primary fermentation (0 h-120 h), and rises with the increase of fermentation time, and has no obvious change during the secondary fermentation, mainly because the secondary fermentation temperature is not favorable for the growth and metabolism of yeast, so that the aromatic alcohol cannot be normally synthesized.

FIGS. 2(a), (b) and (c) show the changes in phenylalanine, tyrosine and tryptophan, respectively, during simulated fermentation. As can be seen from FIGS. 2(a) and (b), phenylalanine and tyrosine are both consumed in the fermentation system of Saccharomyces cerevisiae 11-1 and model bacterium BY4743 for 72h before fermentation, and the ehrlichia pathway is the main synthesis pathway of beta-phenylethyl alcohol and tyrosol in the process. During the subsequent fermentation, the phenylalanine and tyrosine contents do not greatly float, and the contents of beta-phenethyl alcohol and tyrosol are still increased, which indicates that the saccharomyces cerevisiae mainly synthesizes the beta-phenethyl alcohol and tyrosol through the shikimic acid pathway at the time. As can be seen from FIG. 2(c), the tryptophan consumption is mainly concentrated in the 24 th fermentation period, and the content thereof is substantially maintained during the subsequent fermentation period, which indicates that the Saccharomyces cerevisiae synthesizes tryptophol mainly through the Elishi pathway 24h before fermentation.

(4) Collecting fermentation liquid in different fermentation periods, extracting total RNA of yeast by adopting a Trizol method, synthesizing cDNA by utilizing a reverse transcription kit, and determining the relative expression quantity of genes by adopting a qPCR method. By the above method, the expression amounts of 31 genes in total, i.e., yeast ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, SFA1, ARO10, PDC1, PDC5, PDC6, THI3, ARO3, ARO4, AR O1, ARO2, ARO7, PHA2, ARO8, ARO9, BAT1, BAT2, AGP1, GAP1, BAP2, TRP1, TRP2, TRP3, TRP4, TRP5, and TYR1, were determined.

The expression quantity of the beta-phenethyl alcohol related gene synthesized BY Saccharomyces cerevisiae 11-1 and model bacterium BY4743 is shown in FIG. 3, the Ehrlich pathway starts from amino acid transamination, and analysis of the expression quantity of the related gene of two bacterial transaminases from the figure shows that the BAT2 has large variation difference and the BAT2 has the first 72h^11-1The expression level in Saccharomyces cerevisiae 11-1 is high, and research shows that BAT2 as isozyme in cytoplasm can express a large amount in stationary phase, so that it may be one key gene in the synthetic pathway of beta-phenethyl alcohol in 11-1. The first 72h on the ehrlichia pathway were then analyzed one by one with BAT2^11-1The genes with similar expression quantity changes can be preliminarily obtained into GAP1 in 72h of saccharomyces cerevisiae 11-1 before fermentation^11-1、BAT2^11-1、PDC1^11-1、PDC5^11-1、PDC6^11-1、 ADH1^11-1With ADH6^11-1Is a potential key gene for synthesizing beta-phenethyl alcohol, because only Aro10p and Pdc5p can catalyze the decarboxylation of phenylpyruvic acid which is an aromatic substrate, and at least one PDC gene is required to exist independently of the activity of the phenylpyruvic acid decarboxylase of ARO10, the key decarboxylase gene for synthesizing the beta-phenethyl alcohol in the fermentation process of yellow wine is PDC5^11-1GAP1 in Saccharomyces cerevisiae 11-1 72h before fermentation^11-1、BAT2^11-1、PDC5^11-1、ADH1^11-1With ADH6^11-1Is a key gene for potential beta-phenethyl alcohol synthesis. Different from the saccharomyces cerevisiae 11-1, the content of beta-phenethyl alcohol synthesized BY BY4743 is lower and more stable, and the change of the expression quantity of the related gene of BY4743 is analyzed to find ARO8^BYThe expression level is higher in the first 72h, the expression is smoother, and the affinity of the enzyme coded by ARO8 and phenylalanine is 20 times that of tryptophan, so ARO8 is suspected^BYMay be a key gene in the BY4743 phenethyl alcohol synthesis pathway, and then the first 72h ehrlichia pathway and ARO8 are analyzed one BY one^BYThe genes with similar expression quantity changes can be found 72h before fermentation，AGP1^BY、ARO8^BY、ARO10^BY、ADH5^BYWith ADH6^BYIs a potential beta-phenethyl alcohol synthesis key gene of model bacterium BY 4743.

When the fermentation lasts for 96h to 120h, the phenylalanine content in the fermentation liquor is basically unchanged and is at a lower level, the beta-phenylethyl alcohol is still continuously increased, and the ehrlichia way can generate the beta-phenylethyl alcohol by using the phenylalanine, so that the two strains in the time period can synthesize the beta-phenylethyl alcohol by the shikimic acid way. At this stage, the expression levels of the genes of the saccharomyces cerevisiae 11-1 and the model bacterium BY4743 are not high, and the changes are stable. The enzymes coded by ARO3 and ARO4 are respectively feedback inhibited by phenylalanine and tyrosine as isoenzymes, the content of phenylalanine is very low at 72 h-120 h of fermentation, therefore ARO3 starts to express to synthesize part of DAHP, and further analysis shows that ARO3^11-1、ARO1^11-1、ARO2^11-1、ARO7^11-1、PHA2^11-1、ARO10^11-1、PDC5^11-1、 ADH1^11-1、ADH2^11-1Probably the key gene for synthesizing beta-phenethyl alcohol in the time period 11-1, ARO3 in BY4743^BY、 ARO1^BY、ARO2^BY、ARO7^BY、PHA2^BY、PDC5^BY、ADH6^BYMay be the key gene for synthesizing beta-phenethyl alcohol in the time period.

As can be seen from FIG. 3, Saccharomyces cerevisiae 11-1 shows genes involved in the synthesis of tryptophol and tyrosol at 24h and 48h of fermentation (TRP 1)^11-1、 TRP2^11-1、TRP3^11-1、TRP4^11-1、TRP5^11-1、TYR1^11-1) All have higher expression level, but the previous research shows that the saccharomyces cerevisiae 11-1 consumes a large amount of tyrosine and tryptophan in the 48h before fermentation, which indicates that 11-1 plays an important role in synthesizing tyrosol and tryptophol through the Aliskiric pathway in the period. Transaminase Bat2p in the tyrosol pathway^11-1Aromatic decarboxylase Aro10p^11-1And Pdc5p^11-1And alcohol dehydrogenase Adh1p^11-1，Adh4p^11-1All have higher expression level, so the key pathway for synthesizing tyrosol in the pre-fermentation process of 11-1 is formed by the combined use of an ehrlichia pathway and a shikimic acid pathwayTo accomplish this, the key gene may be BAT2^11-1、TYR1^11-1、ARO10^11-1、PDC5^11-1、ADH1^11-1And ADH4^11-1. Analysis of tryptophol pathway related genes to find ARO8 in the ehrlichia pathway^11-1、ARO9^11-1Is not highly expressed and the shikimic acid pathway comprises TRP1^11-1The five genes have longer paths and lower efficiency, which is also one of the reasons for the lower synthesis amount of the tryptophol of the saccharomyces cerevisiae. Like tyrosol, the key gene in the tryptophol pathway may be BAT2^11-1、TRP1^11-1、TRP2^11-1、TRP3^11-1、TRP4¹¹ ^-1、 TRP5^11-1、ARO10^11-1、PDC5^11-1、ADH1^11-1And ADH4^11-1. Since tyrosine and tryptophan are both consumed in large quantities in the early stage of fermentation, the synthesis of tyrosol and tryptophol by yeast is mainly based on the Aliskir pathway.

Earlier studies found that the model bacterium BY4743 consumed a certain amount of tyrosine and tryptophan in the early stage of fermentation, and as can be seen from FIG. 3, BY4743 consumed a key gene (TRP 1) for the synthesis of tryptol and tyrosol during the early fermentation (0 h-120 h)^BY、TRP2^BY、 TRP3^BY、TRP4^BY、TRP5^BY、TYR1^BY) Relative expression is low, and ARO8 is simultaneously^BY，ARO9^BYThe expression level of (4) is higher, which indicates that tyrosol and tryptophol are mainly synthesized through an ehrlichia pathway in the BY4743 pre-fermentation process. Analysis of genes related to the Ehrlich pathway revealed ARO10^BY、ADH4^BYWith ADH5^BYThe expression level is higher in 72 hours before fermentation, so that the key gene for synthesizing tyrosol and tryptophol in 72 hours before fermentation of the model bacterium BY4743 is ARO8^BY、ARO9^BY、ARO10^BY、ADH4^BYWith ADH5^BY。

In conclusion, the key pathways for synthesizing aromatic alcohol in different fermentation periods of the saccharomyces cerevisiae 11-1 and the model bacterium BY4743 are the same, the early fermentation stage mainly comprises the Aliskiving pathway, the late fermentation stage mainly comprises the shikimic acid pathway, but the involved key genes are different, and the strain difference exists between the two.

(5) And designing a corresponding enzyme cutting site by taking pY26TEF-GPD as a framework, and constructing an expression vector by utilizing an enzyme cutting method to construct a corresponding expression vector.

(6) The expression vector was introduced into Saccharomyces cerevisiae BY4743 using lithium acetate transformation.

(7) The article clones and expresses promoters of genes related to aromatic alcohol synthesis in yellow wine yeast 11-1 and model bacterium BY4743, and then measures the relative strength of the promoters BY using a flow cytometer.

The yellow wine yeast 11-1 and BY4743 genomes are respectively used as templates, promoters of yellow wine yeast 11-1 and BY4743 aromatic alcohol related genes are obtained through PCR amplification, the strength of the obtained promoters is measured and shown in figure 4, wherein the amplification primer sequences of the related promoters are shown in table 1. From the figure, it is clear that 25 pairs of promoters obtained by cloning have different strengths and certain diversity. Wherein ARO4 in Saccharomyces cerevisiae 11-1^11-1、TRP1^11-1、TRP5^11-1Promoter strength on the three shikimic acid pathways was not significantly different compared to model bacteria (P > 0.05), even ARO1^11-1、ARO7^11-1And TRP4^11-1There is a certain reduction in the starting strength of (c). Analysis of the intensity of the relevant promoter in the Ehrlichia pathway revealed that BAT1 was found in Saccharomyces cerevisiae 11-1^11-1With BAT2^11-1Compared with the model bacteria, the promoter strength of the transaminase related gene is respectively increased by 66.67 percent and 43.40 percent, the promoter strength is obviously improved (P is less than 0.05), and the amino acid transfer gene GAP1^11-1And AGP1^11-1The promoter strength is also obviously improved (P is less than 0.05). Analysis of genes related to pyruvate decarboxylase and alcohol dehydrogenase shows that ARO10 in saccharomyces cerevisiae 11-1^11-1、PDC1^11-1、PDC5^11-1、ADH5^11-1With ADH6^11-1Compared with BY4743, the promoter strength is respectively increased BY 47.44%, 40.51%, 54.11%, 143.90% and 13.31%, and is also significantly improved (P is less than 0.05). And amino acid transaminase gene ARO9 from Saccharomyces cerevisiae 11-1^11-1The strength of the promoter is obviously lower than BY4743(P < 0.05), and the reduction amplitude is higher than that of other genes. In conclusion, the promoter strength of the related genes in the aromatic alcohol pathway of the saccharomyces cerevisiae has certain diversity. The base mutation of the promoter ensures that the saccharomyces cerevisiae 11-1 mugwortTransaminase in the rihig pathway (BAT 1)^11-1And BAT2^11-1) With amino acid transferases (GAP 1)^11-1And AGP1^11-1) The promoter strength of the related gene is obviously improved, and meanwhile, the promoter strength of the shikimic acid pathway related gene is not greatly improved compared with BY 4743. The enhancement of the promoter strength of the related gene in the ehrlichia pathway enables the expression level of the related gene of the saccharomyces cerevisiae 11-1 to be higher than that of the model bacterium BY 4743. The early research result shows that the Aliskiria pathway is the main pathway for synthesizing aromatic alcohol BY the saccharomyces cerevisiae 96h before fermentation, so that the related genes in the Aliskiria pathway of saccharomyces cerevisiae 11-1 have higher expression level compared with the model bacterium BY4743, which also enables the amino acid in the fermented mash to be more effectively utilized, and more aromatic alcohol is synthesized accordingly.

TABLE 1 primers for amplification of reporter gene eGFP and aromatic alcohol promoter

Example 2

(1) Rice soaking: soaking glutinous rice in the raw material of rice in proportion: water 1:1.2(w/w), and rice soaking time is 24 h. (ii) a

(2) And (3) steaming rice: cleaning the soaked glutinous rice with room temperature water, and steaming for 20min with a rice steamer until no white core exists;

(3) spreading for cooling: placing the cooked rice on a table, and cooling to room temperature;

(4) and (3) yeast: mixing the cooled cooked rice with saccharifying enzyme (1 ‰), liquefying enzyme (2 ‰), and raw wheat starter (13.4%), saccharifying and liquefying at 60 deg.C for 4h, sterilizing at 115 deg.C for 20min, cooling, inoculating 5% Saccharomyces cerevisiae 11-1, culturing, and standing;

(5) blanking: transferring the cooled rice to a fermentation tank, adding water (135%), wheat starter (13.4%), cooked wheat starter (3.95%), yeast (7.37%), and acid protease (6U/g);

(6) fermentation: fermenting at 28 deg.C for five days. Harrowing is carried out after 8h of fermentation in the first day, and harrowing is carried out once every 24h in the last four days. Fermenting at 15 deg.C, standing, and fermenting for fifteen days. Harrowing is carried out once every 48h during the post-fermentation period.

(7) And (3) finished product: filtering with 8 layers of gauze after fermentation, and sterilizing at 85 deg.C for 30min to obtain final product yellow wine.

(8) Determining amino acids, beta-phenylethyl alcohol, tyrosol and tryptophol in the fermentation process by high performance liquid chromatography; the determination of the alcoholic strength, the acidity, the content of reducing sugar and amino acid nitrogen refers to the yellow wine national standard GB/T13662-2018.

Example 3

(5) blanking: transferring the cooled rice to a fermentation tank, adding water (135%), wheat starter (13.4%), cooked wheat starter (3.95%), yeast (7.37%), and acid protease (10U/g);

Example 4

(5) blanking: transferring the cooled rice to a fermentation tank, adding water (135%), wheat starter (13.4%), cooked wheat starter (3.95%), yeast (7.37%), and acidic protease 15U/g;

Example 5

(5) blanking: transferring the cooled rice to a fermentation tank, adding water (135%), wheat starter (13.4%), cooked wheat starter (3.95%), yeast (7.37%), and acidic protease (20U/g);

Example 6

(5) blanking: transferring the cooled rice to a fermentation tank, adding water (135%), wheat starter (13.4%), cooked wheat starter (3.95%), yeast (7.37%), and acidic protease (30U/g);

FIGS. 5(a), (b), (c) and (d) show the effect of adding acid protease on reducing sugar, alcohol content, acidity and amino acid nitrogen in yellow wine during blanking. As can be seen from fig. 5(a), the addition of acid protease during the blanking accelerates the utilization of reducing sugar by yeast, and the yeast in the sample of example 6 has the highest utilization efficiency of reducing sugar; the alcoholic strength changes are shown in fig. 5(b), the addition of acid protease with different concentrations can accelerate the synthesis of ethanol during the primary fermentation of yellow wine, the fermentation period can be shortened to a certain extent, the alcoholic strength at the end of the fermentation in the embodiment 5 is obviously higher than that of a control group, about 1% vol is increased compared with that of the control group, and the fermentation efficiency is the highest compared with that of other sample groups; as can be seen from FIG. 5(c), the acidity is not greatly affected by the addition of acidic protease during blanking, the acidity of the sample group is not significantly different from that of the control group (P > 0.05), and the acidity of example 5 reaches 3.88 + -0.12 g/L; as can be seen from fig. 5(d), the content of amino acid nitrogen in different samples is not greatly different in 72h before fermentation, and the rising rate of amino acid nitrogen in yellow wine to which acid protease with different concentrations is added from 96h after fermentation is obviously higher than that of the control group, wherein the content of amino acid nitrogen in the yellow wine of example 6 is significantly higher than that of the control group at the end of fermentation, reaching 1.27 ± 0.01g/L, which is 1.73 times of that of the control group, so that the addition of acid protease during blanking can improve the alcoholic strength of yellow wine to a certain extent and shorten the fermentation period, but the alcoholic strength does not continuously increase with the addition of acid protease, and the result shows that the alcoholic strength is highest when the addition of acid protease reaches 20U/g, and the content of amino acid nitrogen is also suitable at this time.

Example 7

(5) blanking: transferring the cooled rice to a fermentation tank, adding water (135%), raw wheat starter (13.4%), cooked wheat starter (3.95%), yeast (7.37%), and no acid protease;

(6) fermentation: fermenting at 28 deg.C for five days. Harrowing is carried out after 8h of fermentation in the first day, and harrowing is carried out once every 24h in the last four days. Post-fermentation is carried out at 15 ℃, 6U/g acid protease is added when the post-fermentation is started, and then the mixture is kept still for fifteen days. Harrowing is carried out once every 48h during the post-fermentation period.

Example 8

(6) fermentation: fermenting at 28 deg.C for five days. Harrowing is carried out after 8h of fermentation in the first day, and harrowing is carried out once every 24h in the last four days. Post-fermentation is carried out at 15 ℃, 10U/g acid protease is added when the post-fermentation is started, and then the mixture is left for fermentation for fifteen days. Harrowing is carried out once every 48h during the post-fermentation period.

Example 9

(6) fermentation: fermenting at 28 deg.C for five days. Harrowing is carried out after 8h of fermentation in the first day, and harrowing is carried out once every 24h in the last four days. Post-fermentation is carried out at 15 ℃, 15U/g acid protease is added when the post-fermentation is started, and then the mixture is left for fermentation for fifteen days. Harrowing is carried out once every 48h during the post-fermentation period.

Example 10

(6) fermentation: fermenting at 28 deg.C for five days. Harrowing is carried out after 8h of fermentation in the first day, and harrowing is carried out once every 24h in the last four days. Post-fermentation is carried out at 15 ℃, 20U/g acid protease is added when the post-fermentation is started, and then the mixture is left for fermentation for fifteen days. Harrowing is carried out once every 48h during the post-fermentation period.

Example 11

(6) fermentation: fermenting at 28 deg.C for five days. Harrowing is carried out after 8h of fermentation in the first day, and harrowing is carried out once every 24h in the last four days. Post-fermentation is carried out at 15 ℃, 30U/g acid protease is added when the post-fermentation is started, and then the mixture is left for fermentation for fifteen days. Harrowing is carried out once every 48h during the post-fermentation period.

Comparative example 1

(6) fermentation: fermenting at 28 deg.C for five days. Harrowing is carried out after 8h of fermentation in the first day, and harrowing is carried out once every 24h in the last four days. Post-fermenting at 15 deg.C without adding acid protease, and standing for fifteen days. Harrowing is carried out once every 48h during the post-fermentation period.

The physical and chemical index changes of the yellow wine prepared in the examples 2, 4 and 6 and the yellow wine prepared in the comparative example 1 are shown in fig. 5.

FIGS. 6(a) and (b) show the effect of adding acid protease on the alcoholic strength and acidity of yellow wine during post-fermentation, respectively. As can be seen from fig. 6(a), the addition of the acid protease during the post-fermentation period has little influence on the alcoholic strength of the yellow wine, and no significant difference (P > 0.05) exists among the groups of samples, because the reducing sugar is consumed at 120h of the pre-fermentation period, while the yeast mainly uses glucose as raw material to ferment to form ethanol, and because the reducing sugar is maintained at a very low level all the time during the post-fermentation period, the alcoholic strength of the yellow wine is not increased along with the addition of the acid protease; the change of acidity is shown in FIG. 6(b), the acidity is not greatly affected by adding different concentrations of acidic protease during the post-fermentation, only the samples of example 9 and example 10 have significant difference (P < 0.05) from the control group at the end of the fermentation, and the acidity increases are only 0.39g/L and 0.55 g/L; as can be seen from FIG. 6(b), unlike the addition of the acid protease during the blanking, the content of amino acid nitrogen in each sample group to which the acid protease is added during the post-fermentation period is not so much different from that in the control group (P < 0.05), but the increase of amino acid nitrogen is lower than that in the sample to which the protease is added during the blanking, which is probably because the optimum temperature of the acid protease is 28 ℃ to 45 ℃ and the fermentation temperature (16 ℃) is far lower than the optimum temperature thereof, the activity of the acid protease is inhibited, so that the addition of the acid protease does not significantly improve the content of the amino acid nitrogen. In conclusion, the influence of the raw materials and the fermentation temperature in the fermented mash during the post-fermentation period is small, and the influence of the addition of the acid protease on the alcoholic strength, acidity and amino acid nitrogen of the yellow wine during the post-fermentation period is not large.

FIGS. 7(a), (b), (c) and (d) show the effect of adding acid protease on total free amino acids, phenylalanine, tyrosine and tryptophan of yellow wine during the material dropping process, respectively. As can be seen from FIG. 7(a), the content of free amino acids in the sample of example 2 was not significantly different from that in the control group (P > 0.05) at the end of the primary fermentation, but the content of free amino acids in the sample was significantly higher than that in the control group (P < 0.05) with increasing addition amount, and the content of free amino acids in the sample of example 6 was 2994.96. + -. 6.29mg/L, which is 2.35 times that in the control group. According to the analysis of the change of free amino acid in the post-fermentation period, the content of the free amino acid in each group of samples increases along with the increase of the post-fermentation time, the content of the free amino acid in the samples of the examples 5 and 6 at the end of the fermentation is obviously higher than that in the control group and other sample groups (P < 0.05), the content of the free amino acid reaches 5739.30 +/-73.57 mg/L and 6460.31 +/-57.64 mg/L respectively, and is 1.97 and 2.22 times of that in the control group, and therefore, the release of the amino acid in the yellow wine can be obviously improved by adding the acid protease during the blanking process. Further analysis revealed that the free amino acid content in each sample group was still increasing continuously, and the amino acid in the starting material was not completely released in example 6.

FIGS. 8(a), (b), (c) and (d) show the effect of adding acid protease on total free amino acids, phenylalanine, tyrosine and tryptophan of yellow wine during post-fermentation, respectively. As can be seen from FIG. 8(a), the content of free amino acids in the samples of three groups of examples 7,8 and 9 was not significantly different from that in the control group (P > 0.05) during 144h fermentation, mainly because the post-fermentation temperature (16 ℃) inhibited the activity of the acid protease, and a small amount of the acid protease could not rapidly release the free amino acids in the fermented mash. The free amino acid content of the yellow wine samples of examples 10 and 11 reached 5136.66 + -88.81 mg/L and 5485.01 + -80.48 mg/L at the end of fermentation, respectively, which were 1.76 and 1.88 times higher than the control, but were lower than the samples of examples 5 and 6 (5739.30 + -73.57 mg/L and 6460.31 + -57.64 mg/L), and this difference was probably due to the fact that the acid protease had a higher enzyme activity during the pre-fermentation (28 ℃), the pre-fermentation released more free amino acid in the beer, and a portion of the free amino acid was absorbed by the yeast and autolyzed and released into the beer during the post-fermentation, compared to the post-fermentation. In conclusion, the release of amino acids in yellow wine is better than the addition of the acid protease in the post-fermentation period by adding the acid protease during the blanking.

FIGS. 9(a), (b) and (c) show the effect of adding acid protease on yellow wine beta-phenylethyl alcohol, tyrosol and tryptophol during blanking. As can be seen from FIG. 9, with the increase of the addition amount of the acid protease, the synthesis rates of the three aromatic alcohols are greatly increased, and the aromatic alcohol content in the sample group is significantly higher than that in the control group (P < 0.05) at the end of the fermentation. However, the higher the amount of the acid protease is, the better, calculation shows that there is no significant difference (P > 0.05) in the aromatic alcohol content between the samples of example 5 and example 6 at the end of fermentation, and the aromatic alcohol content in example 5 has reached a peak value, at which the content of beta-phenylethyl alcohol, tyrosol and tryptophol is 130.31 + -2.13 mg/L, 111.38 + -4.93 mg/L and 9.78 + -0.12 mg/L, which are 1.27, 1.56 and 2.52 times of the control group, respectively, so 20U/g of the acid protease is more suitable. Although the content of aromatic alcohol in the yellow wine can be obviously improved by adding the acid protease during the blanking period, the content of aromatic alcohol does not continuously increase along with the addition of the acid protease, which is probably because the content of available free amino acid in the fermented mash is improved by adding the acid protease, the content of available reducing sugar in the fermented mash still keeps unchanged, when the content of the free amino acid reaches the critical value, the growth metabolism of yeast reaches the peak value due to the limitation of the reducing sugar, and the yield of aromatic alcohol also reaches the peak value at the moment and cannot be continuously improved along with the addition of the acid protease.

FIGS. 10(a), (b) and (c) show the effect of adding acid protease on yellow wine beta-phenylethyl alcohol, tyrosol and tryptophol during post-fermentation, respectively. As can be seen from fig. 10, there is no significant difference (P > 0.05) between the sample groups and the control group at 144h of the fermentation, and there is no significant difference (P > 0.05) between the tyrosol and the tryptophol content of the sample groups of examples 7,8 and 9 and the control group at the end of the fermentation, while the content of aromatic alcohol in the sample group of example 11 is significantly higher than the control group, but the content of β -phenylethanol, tyrosol and tryptophol is only increased by 11.50%, 4.16% and 4.18% compared with the control group, and is lower than the increase of aromatic alcohol in the sample group of example 6 (24.80%, 44.97% and 145.00%). The analysis reason is probably that the post-fermentation temperature is lower and is not suitable for the normal metabolism of the yeast, and the previous research shows that the addition of the acid protease during the post-fermentation period does not obviously improve the content of free amino acid in the fermented mash, and the nitrogen source which can be utilized by the yeast is not much different from a control group; and a large amount of yeast die during the post-fermentation period, the cell viability is far inferior to that during the pre-fermentation period, and finally the content of aromatic alcohol in the yellow wine cannot be effectively improved due to the addition of the acid protease during the post-fermentation period.

Example 5 changes in relative expression levels of the 24h and 72h genes in the fermentation are shown in fig. 11(a) and (b), and it can be seen from fig. 11(a) that the expression levels of shikimate pathway-related genes such as ARO3, ARO4, PHA2, ARO1, ARO2 and the like do not increase with the addition of acid protease at the 24h of the fermentation, but the expression levels of ARO3, ARO4 and ARO7 decrease slightly at the 24h of the fermentation, which may be due to the increase of the free amino acid content at the early stage of the fermentation by acid protease, and it was found that the expression of ARO3 is feedback-inhibited by phenylalanine, while the expression of ARO4 and ARO7 is feedback-inhibited by tyrosine. Further analysis shows that the expression levels of the genes related to the Aliskiria pathways, such as ARO8, ARO9, BAT1, ARO10, PDC5, ADH1, ADH6, GAP1 and BAP2, are remarkably improved compared with the genes related to ARO3, probably because the Aro8p has higher affinity with tyrosine and phenylalanine, and the ARO10 can improve the expression level under the induction of tryptophan and phenylalanine. Since Gap1p and Bap2p are main permeases for transporting aromatic amino acids and branched-chain amino acids into cells, respectively, the addition of acid proteases can significantly increase their expression levels even in the early stage of fermentation.

The relative expression level of the 72h gene in fermentation was changed as shown in FIG. 11(b), and the shikimic acid pathway-related genes were not changed much. Different from 24h, the expression levels of BAT2, ADH2 and ADH3 were all significantly increased at 72h of fermentation, and conversely, the expression level of BAT1 began to decrease. This is probably due to the fact that BAT1 is inhibited in stationary phase, whereas BAT2 is mainly expressed in large amounts in stationary phase, and the Saccharomyces cerevisiae has reached stationary phase already at 72h fermentation, while the addition of acid protease further promotes its expression. The research shows that the ADH2 and the ADH3 have the capacity of decomposing ethanol, the expression of the ADH2 and the ADH3 is inhibited by glucose, the speed of glucose utilization by yeast is accelerated by adding the acid protease, the content of reducing sugar in fermented mash is lower than that of a control group when the fermentation is carried out for 72 hours, and the content of alcohol substances is higher than that of the control group, so that the expression levels of the ADH2 and the ADH3 are obviously improved when the fermentation is carried out for 72 hours.

In conclusion, the addition of the acid protease can increase the content of free amino acid and aromatic alcohol in the yellow wine to a certain extent. The method is characterized in that the acid protease with different concentrations is added during blanking, so that the content of total free amino acids and aromatic amino acids at the end of yellow wine fermentation can be remarkably increased, and when the addition amount of the acid protease is 30U/g, the content of the total free amino acids in a sample is remarkably higher than that of other sample groups (P is less than 0.05) and reaches 6460.31 +/-57.64 mg/L, although the content of the free amino acids in the yellow wine can be increased by adding the acid protease during post-fermentation, the improvement effect is obvious when no blanking occurs. The content of three aromatic alcohols in the yellow wine is obviously increased along with the increase of the addition amount of the acid protease during blanking, and when the addition amount reaches 20U/g, the content of the aromatic alcohol reaches a peak value, and the content of beta-phenethyl alcohol, tyrosol and tryptophol is 130.31 +/-2.13 mg/L, 111.38 +/-4.93 mg/L and 9.78 +/-0.12 mg/L respectively, which is far higher than that of a sample group added with the acid protease during post-fermentation. Further, analysis on the change of the expression quantity of the genes related to the saccharomyces cerevisiae in the sample with the addition of the acid protease of 20U/g shows that the addition of the acid protease promotes the expression of the genes related to the ehrlichia pathway during the pre-fermentation period of the saccharomyces cerevisiae 11-1, the utilization capacity of the saccharomyces cerevisiae on amino acid is improved, and the saccharomyces cerevisiae has the highest fermentation efficiency when the addition of the acid protease is 20U/g. Therefore, the addition of 20U/g acid protease during blanking can effectively improve the utilization capacity of the amino acid of the saccharomyces cerevisiae and obviously improve the content of aromatic alcohol in the yellow wine. The yellow wine is applied to the production of cooking wine and vinegar, can increase the content of aromatic alcohol in the cooking wine and the vinegar, has mellow flavor, and is more easily accepted by consumers.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

SEQUENCE LISTING

<110> university of south of the Yangtze river

<120> method for promoting yeast Ailixi approach to improve contents of tyrosol and tryptophol in yellow wine

<130> BAA200763A

<160> 13

<170> PatentIn version 3.3

<210> 1

<211> 1047

<212> DNA

<213> Saccharomyces cerevisiae

<400> 1

atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taagttggaa 60

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tctggtgtct gtcacactga cttgcacgct tggcatggtg actggccatt gccaactaag 180

ttaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240

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ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600

attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660

gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720

cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttctac cagatacgtt 780

agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840

gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct 900

gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt 960

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aagatggcta tcattgacaa caacgtcact ccagctgttg ctgtcaacga tccatctacc 540

atgtttggtt tgccacctgc tttgactgct gctactggtc tagatgcttt gactcactgt 600

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attgatttga tcaatgaaag cttagtcgct gcatacaaag acggtaaaga caagaaggcc 720

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atcaaggctt tgcacgtttt aaacagaacc atgaacattc caagaaactt gaaagaatta 1020

ggtgttaaaa ccgaagattt tgaaattttg gctgaacacg ccatgcatga tgcctgccat 1080

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<210> 3

<211> 1908

<212> DNA

<213> Saccharomyces cerevisiae

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aaatcagttt tcggtgttcc tggtgacttc aacttatctc tattagaata tctctattca 180

cctagtgttg aatcagctgg cctaagatgg gtcggcacgt gtaatgaact gaacgccgct 240

tatgcggccg acggatattc ccgttactct aataagattg gctgtttaat aaccacgtat 300

ggcgttggtg aattaagcgc cttgaacggt atagccggtt cgttcgctga aaatgtcaaa 360

gttttgcaca ttgttggtgt ggccaagtcc atagattcgc gttcaagtaa ctttagtgat 420

cggaacctac atcatttggt cccacagcta catgattcaa attttaaagg gccaaatcat 480

aaagtatatc atgatatggt aaaagataga gtcgcttgct cggtagccta cttggaggat 540

attgaaactg catgtgacca agtcgataat gttatccgcg atatttacaa gtattctaaa 600

cctggttata tttttgttcc tgcagatttt gcggatatgt ctgttacatg tgataatttg 660

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actgggattt ggaatttttc cactgttatg ggaaaatctg taattgatga gtcaaaccca 900

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aaagaactat acaaacgcat tgacgtttct aaactttctt tgcaatatga ttcaaatgta 1200

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aaaagaagcg ggatagaact tttagaagtc aaattaggcg aattggattt ccccgaacag 1860

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<213> Saccharomyces cerevisiae

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ctaattggga aactgatcca gcaagataag tacttagttc ctgaaggaaa aggttactct 420

ttatatatca ggcctacatt aatcggcact acggccggtt taggggtttc cacgcctgat 480

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<212> DNA

<213> Saccharomyces cerevisiae

<400> 5

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gtttctattg acgaaacagg ttcagggtcc aaatggcaag actttaaaga ttctttcaaa 180

agggtaaaac ctattgaagt tgatcctaat ctttcagaag ctgaaaaagt ggctatcatc 240

actgcccaaa ctccattgaa gcaccacttg aagaatagac atttgcaaat gattgccatc 300

ggtggtgcca tcggtactgg tctgctggtt gggtcaggta ctgcactaag aacaggtggt 360

cccgcttcgc tactgattgg atgggggtct acaggtacca tgatttacgc tatggttatg 420

gctctgggtg agttggctgt tatcttccct atttcgggtg ggttcaccac gtacgctacc 480

agatttattg atgagtcctt tggttacgct aataatttca attatatgtt acaatggttg 540

gttgtgctac cattggaaat tgtctctgca tctattactg taaatttctg gggtacagat 600

ccaaagtata gagatgggtt tgttgcgttg ttttggcttg caattgttat catcaatatg 660

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gttgttgggt tcatcatctt aggtatcatt ctaaactgtg gtggtggtcc aacaggtggt 780

tacattgggg gcaagtactg gcatgatcct ggtgcctttg ctggtgacac tccaggtgct 840

aaattcaaag gtgtttgttc tgtcttcgtc accgctgcct tttcttttgc cggttcagaa 900

ttggttggtc ttgctgccag tgaatccgta gagcctagaa agtccgttcc taaggctgct 960

aaacaagttt tctggagaat caccctattt tatattctgt cgctattaat gattggtctt 1020

ttagtcccat acaacgataa aagtttgatc ggtgcctcct ctgtggatgc tgctgcttca 1080

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gttatcttga ttgccgtgtt atctgtcggt aactctgcca tttatgcatg ttccagaaca 1200

attgttgccc tagctgaaca gagatttatg ccagaaatct tttcctacgt tgaccgtaag 1260

ggtagaccat tggtgggaat tgctgtcaca tgtgcattcg gtcttattgc gtttgttgcc 1320

gcctccaaaa aggaaggtga agttttcaac tggttactag ccttgtctgg gttgtcatct 1380

ctattcacat ggggtggtat ctgtatttgt cacattcgtt tcagaaaggc attggccgcc 1440

caaggaagag gcttggatga attgtctttc aagtctccta ccggtgtttg gggttcctac 1500

tgggggttat ttatggttac tattatgttc attgcccaat tctacgttgc tctattcccc 1560

gtgggagatt ctccaagtgc ggaaggtttc ttcgaagctt atctatcctt cccacttgtt 1620

atggttatgt acatcggaca caagatctat aagaggaatt ggaagctttt catcccagca 1680

gaaaagatgg acattgatac gggtagaaga gaagtcgatt tagatttgtt gaaacaagaa 1740

attgcagaag aaaaggcaat tatggccaca aagccaagat ggtatagaat ctggaatttc 1800

tggtgttaa 1809

<210> 6

<211> 1692

<212> DNA

<213> Saccharomyces cerevisiae

<400> 6

atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60

accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120

gaaggtatga gatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt 180

tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240

gctttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300

gtcccatcca tctctgctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360

gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420

gacattgcta ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480

agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540

ttgcaaactc caattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600

attgacacca tcttggcttt ggtcaaggat gctaagaacc cagttatctt ggctgatgct 660

tgttgttcca gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720

ccagctttcg tcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt 780

ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840

ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900

tacaagacca agaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960

ttcccaggtg tccaaatgaa attcgttttg caaaagttgt tgactactat tgctgacgcc 1020

gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080

gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140

ggtgatattg tcattgctga aaccggtacc tccgctttcg gtatcaacca aaccactttc 1200

ccaaacaaca cctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260

gctaccttgg gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320

ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380

ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440

cacggtccaa aggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500

actttcggtg ctaaggacta cgaaacccac agagtcgcta ccaccggtga atgggacaag 1560

ttgacccaag acaagtcttt caacgacaac tctaagatca gaatgattga aatcatgttg 1620

ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680

gctaagcaat aa 1692

<210> 7

<211> 1692

<212> DNA

<213> Saccharomyces cerevisiae

<400> 7

atgtctgaaa taaccttagg taaatattta tttgaaagat tgagccaagt caactgtaac 60

accgtcttcg gtttgccagg tgactttaac ttgtctcttt tggataagct ttatgaagtc 120

aaaggtatga gatgggctgg taacgctaac gaattgaacg ctgcctatgc tgctgatggt 180

tacgctcgta tcaagggtat gtcctgtatt attaccacct tcggtgtcgg tgaattgtct 240

gctttgaatg gtattgccgg ttcttacgct gaacatgtcg gtgttttgca cgttgttggt 300

gttccatcca tctcttctca agctaagcaa ttgttgttgc atcatacctt gggtaacggt 360

gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc catgatcact 420

gatattgcta acgctccagc tgaaattgac agatgtatca gaaccaccta cactacccaa 480

agaccagtct acttgggttt gccagctaac ttggttgact tgaacgtccc agccaagtta 540

ttggaaactc caattgactt gtctttgaag ccaaacgacg ctgaagctga agctgaagtt 600

gttagaactg ttgttgaatt gatcaaggat gctaagaacc cagttatctt ggctgatgct 660

tgtgcttcta gacatgatgt caaggctgaa actaagaagt tgatggactt gactcaattc 720

ccagtttacg tcaccccaat gggtaagggt gctattgacg aacaacaccc aagatacggt 780

ggtgtttacg ttggtacctt gtctagacca gaagttaaga aggctgtaga atctgctgat 840

ttgatattgt ctatcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactcc 900

tacaagacca aaaatatcgt cgaattccac tctgaccaca tcaagatcag aaacgccacc 960

ttcccaggtg ttcaaatgaa atttgccttg caaaaattgt tggatgctat tccagaagtc 1020

gtcaaggact acaaacctgt tgctgtccca gctagagttc caattaccaa gtctactcca 1080

gctaacactc caatgaagca agaatggatg tggaaccaat tgggtaactt cttgagagaa 1140

ggtgatattg ttattgctga aaccggtact tccgccttcg gtattaacca aactactttc 1200

ccaacagatg tatacgctat cgtccaagtc ttgtggggtt ccattggttt cacagtcggt 1260

gctctattgg gtgctactat ggccgctgaa gaacttgatc caaagaagag agttatttta 1320

ttcattggtg acggttctct acaattgact gttcaagaaa tctctaccat gattagatgg 1380

ggtttgaagc catacatttt tgtcttgaat aacaacggtt acaccattga aaaattgatt 1440

cacggtcctc atgccgaata taatgaaatt caaggttggg accacttggc cttattgcca 1500

acttttggtg ctagaaacta cgaaacccac agagttgcta ccactggtga atgggaaaag 1560

ttgactcaag acaaggactt ccaagacaac tctaagatta gaatgattga agttatgttg 1620

ccagtctttg atgctccaca aaacttggtt aaacaagctc aattgactgc cgctactaac 1680

gctaaacaat aa 1692

<210> 8

<211> 1692

<212> DNA

<213> Saccharomyces cerevisiae

<400> 8

ttattgtttg gcatttgtag cggcagtcaa ttgcgcttgt ttgatcaaac tttccggagc 60

atcaaagacg ggcagtttca gttcaattag tctgatcacc gagtttttct ggaactctga 120

atcagtggtt aaggcgtccc actcgcccgt agtggcgatc ttgtgatttt cgtacttttt 180

cgcaccaaat gcgggcaaca gggcgaggtg atcccaggtc tggatttcgt tgtactctgc 240

gtgaggccca tgaatcagct tttcgatagt gtagccgtcg ttgttaagga caaaaagata 300

cggctttaac ccccatctga tcatggtgga gatttcttgg acggttaact gcaaagaccc 360

gtcacctatg aataagatga ctctcttgtt ggggtcaatc tcctcagcgg caaaggcagc 420

acctaaagtt gctcctgttg taaaaccgat ggacccccac aacacctgcg agataccgta 480

ggcgtcctta ggaaagatag tttgattgat accgaaggca gacgtgccgg tctcggaaat 540

gataacatca ccttcttgca agaatttgga caattcgttc cacaaccact cttgtttcaa 600

gggcgtgcta gcaggtacac ctttgtttgc gggagttttg gttggtacgg gaacgctctt 660

gtagccctta acaacatcgg gaataacctt cagtaagttt tgtagtgcaa atttcatttg 720

tacaccggag aacgtagcgt tcttcacctt tacgtaatcg gaatgaaact ccactacatt 780

tttagtcttg taggagtagg aaaacgaacc tgtgttaaaa tcagagagca aagcaccgac 840

cgaaaggatc aaatcagccg actcaacggc ctgtttcacg tctggtttgg acagcgttcc 900

cacataaaca ccgccatatc tgggatgctg ttcatctatt gaccctttac ctaaaggtgt 960

cacaaaagct gggaattgcg tcaaatcaat taacttctgg gtttcctttt taacgttgtg 1020

cctagaagca caggcatccg atagtatgac agggtttttc gaattctgga tcaattctag 1080

tacagtatca ataacttcct tttcagcttc aggatcgtta ggttttaatg atagatcaat 1140

cggtttttcc aaaagagaac caggaacctt tagatctacc aaattcgctg gcaaccccaa 1200

gtagctaggc ctttgtgtta taaatgttgt cctgatcaac ctatcgattt ctgaaggggc 1260

tgtagcaatg tctgtaatca ttgatgtagt ttctgagata ttggcggaca tcctgtgaaa 1320

aacagtaaaa tcaccgttac ccaaggtatg atgcaacaac aattgcttag cctgagcgga 1380

gatagagggg acaccaacaa catgcagtac accgacgtgt tctgcatacg atcctgcaat 1440

accattcaag gcagataatt cacccacgcc aaaagtagtt accagcacag ataaaccctt 1500

gatgcgtgcg taaccatcgg cggcataggc ggcgttcagc tcatttgcat taccagccca 1560

tctcaatcca tctacctcgt aaatcttgtc caatagggac aagttgaagt cacctggtag 1620

cccaaaaatg gtgttaacat taacttgctt caatctttca aataagtatt ttccaagagt 1680

gatttcagac at 1692

<210> 9

<211> 416

<212> DNA

<213> Saccharomyces cerevisiae

<400> 9

actccaagct gcctttgtgt gcttaatcac gtatactcac gtgctcaata gtcaccaatg 60

ccctccctct tggccctctc cttttctttt ttcgaccgaa ttcttaatcg gcaaaaaaag 120

aaaagctccg gatcaagatt gtacgtaagg tgacaagcta tttttcaata aagaatatct 180

tccactactg ccatctggcg tcataactgc aaagtacaca tatattacga tgctgttcta 240

ttaaatgctt cctatattat atatatagta atgtcgttaa ccgttgattt ttactgattg 300

gtctctgccg tgcgggtgag cggcgcgtcc agacgggcgg gtgcacgttt ctttttctga 360

ccatttccct gtcagctctt ttagatcggg gctgccgttt gaaaagtttt atcatc 416

<210> 10

<211> 1005

<212> DNA

<213> Saccharomyces cerevisiae

<400> 10

atggccagca agactttgag ggttcttttt ctgggtccca aaggtacgta ttcccatcaa 60

gctgcattac aacaatttca atcaacatct gatgttgagt acctcccagc agcctctatc 120

ccccaatgtt ttaaccaatt ggagaacgac actagtatag attattcagt ggtaccgttg 180

gaaaattcca ccaatggaca agtagttttt tcctatgatc tcttgcgtga taggatgatc 240

aaaaaagccc tatccttacc tgctccagca gatactaata gaattacacc agatatagaa 300

gttatagcgg agcaatatgt acccattacc cattgtctaa tcagcccaat ccaactacca 360

aatggtattg catcccttgg aaattttgaa gaagtcataa tacactcaca tccgcaagta 420

tggggccagg ttgaatgtta cttaaggtcc atggcagaaa aatttccgca ggtcaccttt 480

ataagattgg attgttcttc cacatctgaa tcagtgaacc aatgcattcg gtcatcaacg 540

gccgattgcg acaacattct gcatttagcc attgctagtg aaacagctgc ccaattgcat 600

aaggcgtaca tcattgaaca ttcgataaat gataagctag gaaatacaac aagattttta 660

gtattgaaga gaagggagaa cgcaggcgac aatgaagtag aagacactgg attactacgg 720

gttaacctac tcacctttac tactcgtcaa gatgaccctg gttctttggt agatgttttg 780

aacatactaa aaatccattc actcaacatg tgttctataa actctagacc attccatttg 840

gacgaacatg atagaaactg gcgatattta tttttcattg aatattacac cgagaagaat 900

accccaaaga ataaagaaaa attctatgaa gatatcagcg acaaaagtaa acagtggtgc 960

ctgtggggta cattccccag aaatgagaga tattatcaca aataa 1005

<210> 11

<211> 1455

<212> DNA

<213> Saccharomyces cerevisiae

<400> 11

atgtctgtgc acgctgcaac aaacccaatc aataagcatg tggttctaat tgacaactac 60

gattccttta cctggaacgt ttacgagtac ttgtgccagg agggcgccaa agtgagcgtc 120

taccgtaacg atgcaattac agttccagaa attgccgcct tgaatcccga cacattgctt 180

atctcgcctg gaccaggcca cccaaagaca gattctggca tttcaagaga ctgtatccgg 240

tactttactg ggaaaattcc tgtatttgga atctgtatgg gccagcaatg catgtttgac 300

gtatttggtg gtgaagttgc ctacgctggt gagattgtcc acggtaaaac gtccccaatc 360

tctcacgaca actgtggaat tttcaagaac gtgccgcaag gtattgctgt gaccagatac 420

cattcattgg ccgggacaga atcgtcccta ccatcctgct tgaaggttac tgcgagtacc 480

gaaaatggaa ttatcatggg tgtaagacac aagaagtaca ctgtagaagg tgtgcaattt 540

catccggaat ccatcttgac cgaggaaggt catctgatga tcaggaacat tttaaacgtc 600

agtggaggca cttgggagga aaacaaatca tctccttcaa attctatttt ggaccgtatc 660

tatgctcggc gtaaaataga cgtcaatgag cagtctaaaa tcccaggttt cacctttcaa 720

gacttacaat ctaactatga tttaggtctt gccccaccgt tacaggattt ctacacggtg 780

ttgtcatcat cccataaaag agccgttgtt cttgccgaag tcaagcgtgc ctctccatcg 840

aagggaccca tttgtttaaa agctgttgct gctgaacagg ctctcaaata cgcagaggct 900

ggtgcatccg ccatttccgt attgaccgaa cctcattggt ttcacggttc gttacaggat 960

ttagtaaatg tgaggaaaat cctagatttg aaatttcctc ccaaggaaag gccttgtgtt 1020

ttgagaaaag aatttatttt cagcaagtat caaatactag aagcaagatt agctggagct 1080

gacactgtcc ttcttatagt caagatgcta tctcaaccct tattgaagga actgtacagc 1140

tacagtaaag atttgaacat ggaacctctc gttgaggtga actccaaaga ggaattacaa 1200

agggctctag aaattggtgc taaagttgta ggtgtcaata atagggacct gcactcattc 1260

aacgtagacc taaataccac cagtaacttg gtagaatcta ttccaaagga tgttcttcta 1320

attgctctat cgggaattac caccagggac gatgctgaaa aatacaaaaa agaaggtgtc 1380

catggatttt tagtgggtga agccctaatg aaatcaaccg atgtgaagaa gttcattcat 1440

gaattatgcg aataa 1455

<210> 12

<211> 1143

<212> DNA

<213> Saccharomyces cerevisiae

<400> 12

atgtccgagg cgactttgtt atcttacact aagaaattat tggcttctcc gccgcaattg 60

agtagcacag acctacacga tgcgttgctg gttatattaa gtcttttgca aaaatgtgat 120

acaaatagcg atgagagtct ttccatctat accaaagttt cgagttttct cacggctttg 180

agagttacta aacttgatca taaggctgaa tacattgcgg aagctgcaaa ggctgtgctc 240

agacattccg accttgttga tctaccttta cccaagaagg acgaattaca cccggaagat 300

ggaccagtaa tcttagatat tgtaggtact ggtggtgacg gacagaatac ttttaatgtt 360

tccacgtctg ctgctatcgt tgcctccgga attcagggcc taaaaatttg taagcacggt 420

ggtaaagctt ctacatccaa tagtggagct ggtgacctaa ttggaacttt aggctgtgat 480

atgttcaagg ttaattcatc gacagtgccc aaactttggc cggataatac gttcatgttt 540

ctacttgctc ctttttttca tcatggaatg ggccacgttt ctaagatacg caaatttctt 600

ggaattccga ctgttttcaa cgtactggga ccacttctac atccagttag ccacgtcaac 660

aagagaatat tgggcgttta ctcaaaggaa cttgcgcctg aatatgccaa ggcagccgct 720

ttggtatatc caggaagcga aacctttata gtttggggac atgttgggtt agacgaagta 780

tcacctatag gcaaaactac tgtctggcat attgacccga catcgtccga acttaaattg 840

aagaccttcc aattagaacc ttctatgttt ggtttagaag aacacgagtt gtcgaagtgt 900

gcttcatacg gccctaaaga gaatgcgaga attctaaaag aagaagtctt gtccggcaag 960

taccaccttg gcgacaataa tcctatttat gactacatct tgatgaacac cgccgtgtta 1020

tattgtttaa gccaaggtca ccagaactgg aaggaaggga tcattaaggc agaagaaagc 1080

atacattctg gtaatgcatt acgttcttta gaacacttta tagatagtgt gagctccttg 1140

tag 1143

<210> 13

<211> 2064

<212> DNA

<213> Saccharomyces cerevisiae

<400> 13

atgtcagaac aactcagaca aacatttgtt aacgctaaaa aagaaaacag gaacgccttg 60

gtcacattta tgaccgcagg ttacccaaca gtcaaagaca ctgtccctat tctcaagggt 120

ttccaggatg gtggtgtaga tatcatcgaa ttgggtatgc ccttctctga tccaattgca 180

gatggtccta caattcaatt atctaatact gtggctttgc aaaacggtgt taccttgcct 240

caaactctag aaatggtctc ccaagctaga aatgaaggtg ttaccgtacc cataatccta 300

atgggttact ataaccctat tctaaactac ggtgaagaaa gatttattca ggacgctgcc 360

aaggctggtg ctaatggttt tatcatcgtc gatttgccac cagaggaggc gttgaaggtt 420

agaaactacg tcaatgataa tggtttgagc ctgatcccac tagtggctcc ttctaccacc 480

gatgaaagat tggaattact atcgcatatt gccgattcgt ttgtctacgt tgtgtctaga 540

atgggtacta ctggtgttca aagttctgtg gccagtgatt tggatgaact catctctaga 600

gtcagaaagt acaccaagga tactcctttg gccgttgggt ttggtgtctc taccagagaa 660

catttccaat cagttggtag tgttgctgac ggtgtagtga ttggttccaa aatcgtcaca 720

ttatgtggag atgctccaga gggcaaaagg tacgacgttg ctaaggaata tgtagaggga 780

attctaaatg gtgctaagca taaggttctg tccaaggacg aattctttgc ctttcaaaaa 840

gagtccttga agtccgcaaa cgttaagaag gaaatactgg acgaatttga tgaaaatcac 900

aagcacccaa ttagatttgg ggactttggt ggtcagtatg tcccagaagc tcttcatgca 960

tgtctaagag agttggaaaa gggttttgat gaagctgtcg ctgatcccac attctgggaa 1020

gacttcaaat ccttgtattc ttatattggc cgtccttctt cactacacaa agctgagaga 1080

ttaactgagc attgtcaagg tgctcaaatc tggttgaaga gagaagatct taaccacacg 1140

ggatctcaca agatcaacaa tgctttagca caagttcttc tagctaaaag attaggcaag 1200

aagaacgtta ttgctgaaac cggtgctggt caacacggtg ttgccactgc cactgcatgt 1260

gctaaatttg gcttaacctg tactgtgttc atgggtgcag aagatgttcg tcgccaagct 1320

ttaaacgtct tcagaatgag aattctcggt gctaaagtaa ttgctgttac taatggtaca 1380

aagactctaa gagacgctac ttcagaggca ttcagatttt gggttactaa cttgaaaact 1440

acttactacg tcgtcggttc tgccattggt cctcacccat atccaacttt ggttagaact 1500

ttacctgacg cagttgttgc atgtgttggg ggtggttcca actctacagg tatgttttca 1560

ccatttgagc acgatacttc cgttaagtta ttgggtgtgg aagccggtgg tgatggtgta 1620

gatacaaagt tccactctgc tactctaact gccggtagac ctggtgtctt ccatggtgtc 1680

aagacttatg tcttgcaaga tagtgatggt caagtccatg atactcattc tgtttctgct 1740

gggttagact acccaggtgt cggtccagaa ttggcatatt ggaaatctac tggccgtgct 1800

caattcattg cagctactga cgctcaggct ctgcttggct ttaaattatt atctcaatta 1860

gaaggtatta ttcccgcttt ggaatcttct catgctgttt atggtgcttg cgaattggct 1920

aagacgatga agcctgatca acatttggtt atcaatattt ctggtagagg tgataaagat 1980

gtccaaagtg tcgctgaagt cttgccgaaa ttaggtccaa agataggttg ggatttgaga 2040

ttcgaagaag acccatctgc ctaa 2064

Claims

1. A method for improving the contents of tyrosol and tryptophol in yellow wine is characterized in that the gene expression level of the Alisma way of yellow wine yeast is improved; the ehrlichia pathway comprises: transaminase encoding gene BAT2^11-1Aromatic decarboxylase encoding gene ARO10¹¹ ^-1Pyruvate decarboxylase gene PDC5^11-1The gene ADH1 encoding alcohol dehydrogenase^11-1、ADH4^11-1And a gene coding for tyrosinase-related protein TRP1^11-1、TRP2^11-1、TRP3^11-1、TRP4^11-1、TRP5^11-1At least one gene of (1).

2. The method of claim 1, wherein the yellow wine yeast is saccharomyces cerevisiae 11-1 or saccharomyces cerevisiae BY 4743.

3. The method according to claim 1 or 2, wherein the gene expression level of the Alisma orientale pathway of the yellow wine yeast is improved by adding acid protease into a yellow wine yeast fermentation environment; the fermentation environment contains protein or protein-containing material.

4. The method according to claim 3, characterized in that the acid protease is added in the blanking stage or in the fermentation stage.

5. The method according to claim 4, wherein the amount of the acidic protease added is 6 to 30U/g.

6. The method according to claim 4, characterized in that 6-30U/g acid protease is added in the blanking stage; or adding no acid protease in the blanking stage and adding 6-30U/g protease in the fermentation stage.

7. A method for preparing yellow wine with high aromatic alcohol content comprises soaking rice, steaming rice, spreading for cooling, adding yeast, blanking and fermenting; adding acid protease in blanking or fermentation stage; the addition amount of the acidic protease is 6-30U/g.

8. The method according to claim 7, characterized in that 6-30U/g acid protease is added in the blanking stage; or adding no acid protease in the blanking stage and adding 6-30U/g protease in the fermentation stage.

9. Yellow wine prepared by the method of claim 7 or 8.

10. Use of the method according to any one of claims 1 to 8 in the field of fermentation.