CN115895925A

CN115895925A - Saccharomyces cerevisiae Y28 and application thereof

Info

Publication number: CN115895925A
Application number: CN202211671271.0A
Authority: CN
Inventors: 韩英; 杨埔; 张丽珍; 张鑫; 甄攀
Original assignee: Shanxi University; Shanxi Xinghuacun Fen Wine Factory Co Ltd
Current assignee: Shanxi University; Shanxi Xinghuacun Fen Wine Factory Co Ltd
Priority date: 2022-12-26
Filing date: 2022-12-26
Publication date: 2023-04-04

Abstract

The invention belongs to the technical field of microorganisms and wine brewing, and particularly relates to saccharomyces cerevisiae Y28 and application thereof. The Saccharomyces cerevisiae Y28 is classified and named as Saccharomyces cerevisiae, and the preservation number is as follows: CCTCC M20221741, the Saccharomyces cerevisiae Y28 provided by the invention has stronger pyruvate decarboxylase and alcohol dehydrogenase activity, has high liquor yield after being used as a biological enhancer for simulated fermentation, can be applied to the production practice of fen-flavor liquor, and improves the liquor yield in hot seasons.

Description

Saccharomyces cerevisiae Y28 and application thereof

Technical Field

The invention belongs to the technical field of microorganisms and wine brewing, and particularly relates to saccharomyces cerevisiae Y28 and application thereof.

Background

Chinese white spirit is prepared by fermentation under the combined action of various microorganisms, and is unique to other distilled spirits in the world. The Chinese white spirit has various types of odor, including faint scent type, strong scent type, maotai-flavor type and other odor types, wherein the faint scent type white spirit is deeply loved by consumers in China. The fen-flavor liquor is prepared from grain grains as raw materials, low-temperature Daqu prepared from barley and peas as a leaven, and through solid-state vat fermentation and steaming-cooking brewing processes, and has a sweet, fresh and clean liquor body, mellow and fine taste and long aftertaste. The characteristics of pure and fragrant faint scent of the faint scent type white spirit are formed by the coordination of various esters and acid.

The traditional fen-flavor liquor has a unique brewing process and a microbial community structure, and the production process mainly comprises two stages of hawthorn fermentation and secondary hawthorn fermentation. Generally, in the fermentation process of fen-flavor liquor, the physicochemical properties of fermented grains change along with the fermentation time. The whole trend of the fermented grain temperature in the fermentation process shows the characteristics of 'forward slow, moderate and backward slow falling', and the fermented grain temperature rises after short-time fermentation and then slowly falls. Unlike the temperature, during the fermentation of the white spirit, the acidity of the fermented grains gradually increases along with the fermentation time, and the pH value shows a trend of decreasing. The growth of the saccharomyces cerevisiae during fermentation can be reflected by the change of the alcohol content. In the initial stage of fermentation, the alcohol content in the fermented grains is not greatly changed, then the alcohol content is gradually increased, the alcohol content reaches the maximum in the middle stage of fermentation (about 7-10 days), and then the alcohol content is kept stable.

In the process of fermenting the white spirit in hot seasons, because the temperature is higher, various microorganisms grow vigorously and easily cause mixed bacterium pollution, the acidity of the fermented grains is high, the growth of beneficial bacteria is inhibited, the stagnation of alcohol is caused, and the wine yield is reduced, so that the acid resistance and the high temperature resistance can be improved by further screening the beneficial bacteria, and the wine brewing yield can be improved, and the method is particularly important.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide the saccharomyces cerevisiae Y28 which is acid-resistant and high-temperature-resistant and can improve the wine yield of wine brewing and the application thereof.

In a first aspect, the present invention provides a Saccharomyces cerevisiae Y28 with a deposit number of: CCTCC M20221741.

In a second aspect, the invention provides the use of saccharomyces cerevisiae Y28 for the fermentation of reducing sugars to ethanol.

In a third aspect, the invention provides application of the saccharomyces cerevisiae Y28 as a biological enhancer in brewing, wherein the biological enhancer can increase the utilization efficiency of reducing sugar and improve the liquor yield.

In a fourth aspect, the invention provides a method for brewing wine by using the saccharomyces cerevisiae Y28, which comprises the following steps:

s1, activating the saccharomyces cerevisiae Y28;

s2, mixing the Daqu and the fermented grains, putting the mixture into a container, adding activated saccharomyces cerevisiae Y28, and fermenting.

Further activating the saccharomyces cerevisiae Y28 by inoculating in YPD medium and culturing for 22-24h at 27-30 ℃. Furthermore, the addition amount of the Daqu is 8-10% of the mass of the fermented grains.

Furthermore, the Daqu is prepared by compounding the stubble cleaning yeast, the red-heart yeast and the after-fire yeast according to the mass ratio of 3.

Further, the final concentration of the saccharomyces cerevisiae Y28 in the mixed fermented grains is 10 ⁵ -10 ⁶ CFU/g。

Furthermore, the fermentation temperature is 18-35 ℃.

The invention has the following beneficial effects:

in the process of white spirit fermentation, fermented grains are used as the basis of microbial growth, and the continuous change of the physical and chemical properties of the fermented grains can change the microbial community structure in the fermented grains and generate ordered growth and degradation change among microbial communities in the fermented grains as the main body of white spirit fermentation. According to the research of the bank and the like and the Jialiyan and the like, the temperature of the fermented grains is increased and then decreased, the acidity is continuously increased along with the continuous accumulation of the fermentation time, the pH value is continuously decreased, and the alcohol content is kept stable after the alcohol content reaches the maximum after the fermented grains are fermented for a certain time. Therefore, the evaluation of the metabolic activity of the saccharomyces cerevisiae in the environment of high temperature, high acid and high alcohol is an important index for screening the saccharomyces cerevisiae with high alcohol yield.

The invention mainly researches the metabolic activity change of the saccharomyces cerevisiae in the environment of high temperature, high acid and high alcohol. As a result, it was found that: the metabolic activities of different saccharomyces cerevisiae growing under the same environmental condition in the primary screening process and the secondary screening process are different, and are mainly reflected in the difference of the enzyme activities of pyruvate decarboxylase and alcohol dehydrogenase. The change of the physicochemical property of the fermented grains can obviously influence the enzyme activities of pyruvate decarboxylase and alcohol dehydrogenase. In the fermentation process of the white spirit, when the content of acetic acid in the fermented grains reaches 0.5 percent, the activity of the two enzymes is greatly influenced, and the activity of the two enzymes can be obviously inhibited; when the content of lactic acid reaches 1%, the pyruvate decarboxylase cannot be greatly influenced, but the activity of the alcohol dehydrogenase can be obviously inhibited; after the ethanol content in the fermented grains is higher than 8%, the activity of pyruvate dehydrogenase can be obviously inhibited. In the middle stage of fermentation (day 7), the content of acetic acid in fermented grains is only 0.1%, and the content of lactic acid is 1%. On the 28 th day of fermentation, the contents of acetic acid and lactic acid in the fermented grains reach 0.17% and 2% respectively, and the organic acid with the concentrations can not inhibit the growth of the saccharomyces cerevisiae. In addition, the alcohol concentration in the white spirit fermentation process is only less than 10%, and the growth of the saccharomyces cerevisiae cannot be influenced by the alcohol with the concentration. Therefore, the stress of high acid and high alcohol in the fermented grains does not influence the growth of the saccharomyces cerevisiae, but influences the metabolic activity of the saccharomyces cerevisiae: in the middle period of white spirit fermentation, after the lactic acid content in the fermented grains reaches 1% and the alcohol content is higher than 8%, key enzyme activity of alcohol production of the saccharomyces cerevisiae is inhibited, so that the yield of the fermented grains is reduced.

Six brewers yeasts with stronger key enzyme activity for producing alcohol are obtained by screening according to a TTC method and a Du's small tube gas production method, and are added into fermented grains before being added into a jar as a biological enhancer, and strains with stronger alcohol production capability in actual production can be screened out by simulating the change of the fermentation temperature in hot seasons. The simulated fermentation results show that: the liquor yield is highest after the Y28 is used as a biological enhancer for simulated fermentation, and is improved by more than 40% compared with the liquor yield without bacteria. The content of reducing sugar is obviously reduced, which shows that the saccharomyces cerevisiae can increase the utilization efficiency of the reducing sugar after being enhanced. The method can be applied to fermentation of fen-flavor liquor, and can improve liquor yield and production efficiency.

Drawings

FIG. 1 shows the basic principle for determining the alcohol dehydrogenase activity and pyruvate dehydrogenase activity of Saccharomyces cerevisiae.

FIG. 2 shows TTC scoring criteria.

FIG. 3 is the sum of TTC scores for Saccharomyces cerevisiae.

FIG. 4 shows the gas production of 20 strains of Saccharomyces cerevisiae cultured at 30 ℃.

FIG. 5 is the total score of 20 strains of Saccharomyces cerevisiae by gas production method.

FIG. 6 is the pH of fermented grains after simulated fermentation.

FIG. 7 shows the acidity of fermented grains after simulated fermentation.

FIG. 8 is the alcohol yield after simulated fermentation.

FIG. 9 shows the reducing sugar content of fermented grains after simulated fermentation.

Detailed Description

The invention is described in detail below with reference to the figures and the specific embodiments, but the invention should not be construed as being limited thereto. The technical means used in the following examples are conventional means well known to those skilled in the art, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1: separation and screening of Saccharomyces cerevisiae

The invention obtains 64 strains of saccharomyces cerevisiae from fermented grains of fen wine by a conventional method, wherein one strain with the best alcohol production capacity is selected for preservation, the name of the strain is Y28, and the preservation description is as follows:

the strain name is as follows: saccharomyces cerevisiae;

latin name: saccharomyces cerevisiae;

the preservation organization: china center for type culture Collection (CCTCC for short);

the preservation date is as follows: 11/2022, 08/11/d;

the preservation number is: CCTCC M20221741.

Example 2: saccharomyces cerevisiae fermentation performance test and application of saccharomyces cerevisiae fermentation performance test and application as biological enhancer in brewing

1. Materials and reagents

Experimental materials: 64 s.cerevisiae isolated from fermented Fenjiu and 63 s.cerevisiae except Y28 were all stored in the institute of Life sciences of Shanxi university.

Culture medium and reagents:

YPD medium: 20g of glucose, 10g of yeast powder, 20g of maltose, 20g of peptone and 1L of water, and the pH value is 5.5. 15g/L agar was added to the solid medium.

TTC upper medium: 5g of glucose, 15g of agar, 0.5g of TTC, 1L of water and pH 5.5.

TTC lower layer medium: 20g of glucose, 10g of yeast powder, 20g of maltose, 20g of peptone, 8g of agar and 1L of water, and the pH value is 5.5.

Acetic acid, lactic acid, ethanol, analytical pure, bio-engineering (Shanghai) Co., ltd.

The reducing sugar content detection kit is purchased from Beijing Solaibao science and technology Limited.

2. Principle and method of experiment

2.1 principle of the experiment

As shown in figure 1, during the fermentation process of Saccharomyces cerevisiae, glucose is converted into pyruvate through EMP pathway, and then converted into acetaldehyde and carbon dioxide under the action of pyruvate decarboxylase; acetaldehyde is converted to ethanol by an alcohol dehydrogenase. Therefore, the alcohol production capacity of the saccharomyces cerevisiae can be preliminarily judged by detecting the activities of the pyruvate decarboxylase and the alcohol dehydrogenase of the saccharomyces cerevisiae. In this process, ethanol dehydrogenase can convert

colorless triphenyltetrazolium

2,3,5-chloride (TTC) to red triphenylformazan (TTF). When the high-yield alcohol saccharomyces cerevisiae is screened, the activity of the alcohol dehydrogenase can be judged according to the red shade (TTC method), and the activity of the pyruvate dehydrogenase can be judged according to the volume of carbon dioxide generated in a Duchenne tubule (gas production method).

2.2 Experimental methods

2.2.1TTC test

A total of 18 environmental conditions are set, wherein the three environmental conditions comprise a temperature condition, an organic acid condition and an alcohol condition. Primary screening of high-yield alcohol saccharomyces cerevisiae under the temperature condition: and (2) dibbling the single saccharomyces cerevisiae colony obtained by separation and purification to a TTC lower-layer culture medium, culturing for 48 hours under the conditions of 15 ℃,20 ℃,25 ℃,30 ℃,35 ℃ and 5, pouring the colony into the TTC upper-layer culture medium after the colony grows out, completely covering the colony, culturing for 3 hours at the original temperature, observing the color of the colony, recording the color depth of the colony of each strain, and assigning values according to the scoring standard (figure 2) of the TTC method, wherein the deeper the red color is, the higher the assignment is. Each strain was done in 3 replicates.

The organic acid condition comprises two organic acids of acetic acid and lactic acid with different concentrations. The specific operation is as follows: 0.125%,0.25%,0.5% (v/v) acetic acid or 0%,1%,2%,4% (v/v) lactic acid was added to TTC lower layer medium, mixed well and poured into disposable sterile plates. And (3) after the culture medium is cooled and solidified, inoculating a single saccharomyces cerevisiae colony on the surface of the culture medium, culturing for 48h at 30 ℃, adding TTC upper-layer culture medium with organic acid with the same concentration as the lower-layer culture medium, pouring the TTC upper-layer culture medium into the culture medium to completely cover the colony, continuously culturing for 3h at 30 ℃, observing the color of the colony, assigning the color of the colony, and carrying out assignment on the color of the colony. Each strain was done in 3 replicates.

Under the condition of alcohol: adding alcohol with the concentration of 4%,8%,12%,16%,20% and (v/v) into TTC lower layer culture medium respectively, mixing uniformly, and pouring into a disposable sterile plate. Inoculating a single saccharomyces cerevisiae colony obtained by separation and purification into TTC lower-layer culture media added with alcohol with different concentrations, culturing for 48 hours at 30 ℃, then pouring TTC upper-layer culture media added with alcohol with the same concentration as the lower-layer culture media into the ttC lower-layer culture media to completely cover the colony, continuously culturing for 3 hours at 30 ℃, observing the color of the colony, and assigning the color of the colony, wherein the assigning method is the same as the above method. Each strain was done in 3 replicates.

And scoring and adding according to a scoring rule. And then according to the scoring result, selecting the 20 strains with the highest scores for subsequent experiments.

2.2.2 gas production experiment

15 environmental conditions are set, and the temperature, the organic acid and the alcohol are also included.

Re-screening the high-yield alcohol saccharomyces cerevisiae under the temperature condition: inoculating 20 strains of saccharomyces cerevisiae into a YPD culture medium, activating for 24 hours at 28 ℃, filling a Duchenne tubule with the YPD liquid culture medium, then reversing and buckling the small duchenne tubule in a test tube of the YPD liquid culture medium for sterilization, transferring the activated saccharomyces cerevisiae into the test tube according to the inoculation amount of 1% (v/v), uniformly mixing, placing the mixture in the test tube under the conditions of 15 ℃,20 ℃,25 ℃,30 ℃,35 ℃ and 5, observing the gas production condition in the Duchenne tubule, and measuring and recording the length of the gas part in the Duchenne tubule. Each strain was done in 5 replicates.

The re-screening of the saccharomyces cerevisiae for producing alcohol with high yield under the organic acid condition also comprises two organic acids of acetic acid and lactic acid with different concentrations. The specific operation is as follows: activating 20 strains of saccharomyces cerevisiae for 24 hours, respectively adding acetic acid with the concentration of 0.125 percent, 0.25 percent, 0.5 percent (v/v) or lactic acid with the concentration of 0.5 percent, 1 percent, 2 percent and 4 percent (v/v) into a YPD liquid culture medium, transferring the activated saccharomyces cerevisiae into a test tube with the inoculation amount of 1 percent (v/v) after sterilization is finished, uniformly mixing, placing the test tube at 30 ℃ for culture, observing the gas production condition in a Duchenne tubule, and measuring and recording the length of a gas part in the Duchenne tubule. Each strain was done in 5 replicates.

Alcohol conditions: activating 20 strains of saccharomyces cerevisiae for 24 hours, respectively adding alcohol with the concentration of 4 percent, 8 percent, 12 percent, 16 percent and 20 percent (v/v) into a YPD liquid culture medium, transferring the activated saccharomyces cerevisiae into a test tube according to the inoculation amount of 1 percent (v/v) after sterilization is finished, uniformly mixing, placing the test tube at 30 ℃ for culture, observing the gas production condition in a Duchenne tubule, and measuring and recording the length of a gas part in the Duchenne tubule. Each strain was done in 5 replicates.

And (3) measuring the gas production of 20 strains of saccharomyces cerevisiae in the Duchenne tubules under different environments, calculating the sum, and selecting 6 strains with the highest gas production to perform subsequent experiments.

2.2.3 simulated fermentation experiments

The following experiments involved the yeast for making fen wine (the mass ratio of the koji for cleaning stubble, the red heart koji and the post fire koji is 3Supplied by parts Ltd. Performing simulated fermentation experiment on 6 Saccharomyces cerevisiae, setting 6 experimental groups, respectively mixing 6 activated Saccharomyces cerevisiae strains for 24h with fermented grains with increased yeast (10% of addition, w/w) before entering jar to obtain final concentration of 10% ⁶ CFU/g.1 negative control, no exogenous saccharomyces cerevisiae was added, but an equal volume of sterile water was added as a control. Putting the fermented grains into a 500mL triangular flask, then placing the flask into an incubator, simulating the temperature change of natural fermentation in hot seasons, adjusting the temperature of the incubator (table 1) along with the temperature change, taking out the fermented grains after fermenting for 28 days, and detecting the physical and chemical properties of the fermented grains. Each strain was done in 5 replicates.

TABLE 1 simulated fermentation temperatures

The detection of physical and chemical properties of the fermented grains mainly comprises the following steps of measuring alcohol content, acidity, pH, reducing sugar content and organic acid type.

And (3) determination of alcohol content: and after fermentation is finished, uniformly stirring the fermented grains, weighing 50g of the fermented grains by using a beaker, accurately weighing the fermented grains to 0.01g, then transferring the fermented grains into a 250mL distillation flask, washing the beaker and the fermented grains attached to the wall of the flask into the distillation flask by using 100mL of water for multiple times, heating the distilled grains on an electric heating sleeve, continuously heating the distilled grains for 1 hour after the distilled grains are boiled, receiving the distillate by using a 100mL triangular flask, taking down the triangular flask, and detecting the alcohol content after the distillate is subjected to constant volume of 50 mL. The alcohol content is determined by the density bottle method in GB 509.225-2016.

Measurement of pH: weighing 10g of the uniformly mixed fermented grains into a 250mL conical flask, then weighing 90mL of water, adding the water into the conical flask, placing the conical flask on a magnetic stirrer, stirring the conical flask for 20min, uniformly mixing the conical flask with the water, then directly detecting the pH value of the solution by using a pH meter, and recording data. An average was taken of 3 measurements per sample.

Measurement of acidity: and (3) measuring the acidity of the fermented grains by referring to an acid-base titration method in GB 5009.239-2016.

The determination of reducing sugar refers to the instruction of the Solebao reducing sugar content detection kit for detection.

2.3 results of the experiment

2.3.1TTC test

Under the same environmental conditions, the activity of the alcohol dehydrogenase of 64 strains of saccharomyces cerevisiae shows a remarkable difference. The ethanol dehydrogenase activity of the strains J24, Y59, Y201, etc. is strong, while the ethanol dehydrogenase activity of the strains J6, J9, J16, etc. is weak when cultured at 30 ℃. Most strains hardly detected the ethanol dehydrogenase activity when cultured in the presence of 0.5% acetic acid, while strains Y57, Y127, etc. still exhibited weak ethanol dehydrogenase activity. Most of the strains lost almost all of the alcohol dehydrogenase activity when cultured in the presence of 4% lactic acid or 20% alcohol, and only very few strains such as Y57 had weak alcohol dehydrogenase activity. According to the results of TTC scoring (FIG. 3), the 20 strains with the highest total score were selected for further study.

2.3.2 gas production experiment

As shown in FIG. 4, the growth time was the same, and the difference in gas production by the different strains was large. Under different environmental conditions, the carbon dioxide generated by the saccharomyces cerevisiae has different time for filling the Duchenne tubules, and the shorter the time is, the stronger the pyruvate decarboxylase activity is. The result shows that the pyruvate decarboxylase has the strongest activity at the temperature of 30-35 ℃, and the activity is weakened along with the increase and decrease of the temperature; pyruvate decarboxylase has a high pyruvate decarboxylase activity at low concentrations of acetic acid, lactic acid, and alcohol, and its activity decreases as the concentrations of acetic acid, lactic acid, and alcohol increase. When the concentration of acetic acid reaches 1%, the concentration of lactic acid reaches 4% and the concentration of ethanol reaches 16%, the activity of pyruvate decarboxylase is seriously inhibited, and almost no gas is generated within 48 hours.

In the experiment, the gas production rate of 20 strains of saccharomyces cerevisiae in a Duchenne tubule under different environments is measured, and after the sum of the gas production rates is calculated, the pyruvate decarboxylase activity difference among different strains is found to be large (figure 5). 6 strains with the highest gas production are selected for carrying out simulated fermentation experiments.

2.3.3 simulated fermentation experiments

Adding 6 strains of Saccharomyces cerevisiae Y28, Y201, Y57, J20, J17 and Y59 as biological enhancer into fermented grains before vat entry, performing simulated fermentation simultaneously with blank control of the same amount of sterile water, and detecting physicochemical properties and yield of fermented grains. The result shows that the pH value of the fermented grains of the blank control is the minimum and reaches 3.69 +/-0.03 (figure 6); the acidity of the fermented grains is the highest and reaches 20.24 +/-1.40 DEG T (figure 7); the yield of alcohol is the lowest, and is 9.01 plus or minus 0.39ml/100g of fermented grains (figure 8); the content of reducing sugar in fermented grains is 47.56 + -9.54 mg/g fermented grains (FIG. 9). The acidity of fermented grains with Y201 as a biological enhancer is the minimum and is 9.73 +/-0.95 DEG T (figure 7); pH 4.69 + -0.13 (FIG. 6); the alcohol yield reaches 11.09 + -0.73 ml per 100g of fermented grains (FIG. 8). The fermented grain with Y28 as biological enhancer has the highest yield of 12.67 +/-0.66 ml/100g of fermented grain (figure 8); the pH value is maximum and reaches 4.71 +/-0.03 (figure 6); the content of reducing sugar in fermented grains is 28.73 +/-4.37 mg/g (FIG. 9).

It should be noted that when the following claims refer to numerical ranges, it should be understood that both ends of each numerical range and any numerical value between the two ends can be selected, and the preferred embodiments of the present invention are described for the purpose of avoiding redundancy.

While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.

It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.

Claims

1. Saccharomyces cerevisiae Y28 with the deposit number: CCTCC M20221741.

2. Use of the saccharomyces cerevisiae Y28 according to claim 1 for the fermentation of reducing sugars into ethanol.

3. The use of saccharomyces cerevisiae Y28 as a biological enhancer in brewing wine as claimed in claim 1, wherein said biological enhancer is capable of increasing the efficiency of reducing sugar utilization and improving the wine yield.

4. A method for brewing wine by using the Saccharomyces cerevisiae Y28 of claim 1, which comprises the following steps:

s1, activating the saccharomyces cerevisiae Y28;

5. The method according to claim 4, wherein the activation is carried out by inoculating Saccharomyces cerevisiae Y28 in YPD medium and culturing at 27-30 ℃ for 22-24h.

6. The method according to claim 5, wherein the addition amount of the Daqu is 8-10% of the mass of fermented grains.

7. The method according to claim 6, wherein the Daqu is prepared by compounding the green koji, the red koji and the post-fire koji according to the mass ratio of 3.

8. The method of claim 7, wherein the final concentration of Saccharomyces cerevisiae Y28 in the mixed fermented grains is 10 ⁵ -10 ⁶ CFU/g。

9. The method of claim 8, wherein the fermentation temperature is 18-35 ℃.