CN117903958A - Saccharomyces cerevisiae mutant strain with low yield of fusel and high yield of ester and application thereof - Google Patents

Abstract

The invention discloses a saccharomyces cerevisiae mutant strain with low yield of fusel and high yield of ester and application thereof, belonging to the field of fermentation engineering and biotechnology. The invention provides a mutant strain saccharomyces cerevisiae AR28 with the characteristic of reducing impurity alcohol extraction ester, which can be widely applied to fermented foods and belongs to the field of fermentation engineering and biotechnology. The invention is based on the excellent yeast strain obtained by previous screening in a laboratory, utilizes the ARTP mutagenesis technology and combines GC-MS to analyze the volatile flavor substances generated by the mutagenesis strain, screens the Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and can improve the ester content, improves the genetic stability by purifying the recombinant spore, and provides precious strain resources for solving the pain point problem (easy top after drinking) in the wine industry.

Description

Saccharomyces cerevisiae mutant strain with low yield of fusel and high yield of ester and application thereof

Technical Field

The invention relates to a saccharomyces cerevisiae mutant strain with low yield of fusel and high yield of ester and application thereof, belonging to the field of fermentation engineering and biotechnology.

Background

The higher alcohol in the alcoholic beverage is a byproduct of the fermentation of Saccharomyces cerevisiae, is an important component of the body and flavor of the alcoholic beverage, and is important for the formation of the typical flavor of various wines. The higher alcohols in the alcoholic beverage mainly comprise n-propanol, isobutanol, isoamyl alcohol, phenethyl alcohol and the like. Too high isoamyl alcohol content can cause the taste of the wine body to be bitter; isobutanol has a slight fatty aroma and bitter taste; n-propanol has a similar flavor to ethanol and has bitter and astringent taste. Thus, too high a higher alcohol content can produce an unpleasant off-taste. During fermentation, saccharomyces cerevisiae can produce higher alcohols via 2 pathways: the catabolic pathway of amino acids (Ehrlich pathway) and the de novo synthesis pathway of sugars (Harris pathway). In the catabolic pathway, amino acids are converted to alpha-keto acids via transaminases; in the de novo synthesis pathway, glucose forms pyruvic acid via the glycolytic pathway, and is converted to alpha-keto acid after entering the mitochondria. The α -keto acids produced by these 2 different metabolic pathways undergo decarboxylation and dehydrogenation to form higher alcohols.

The effect of esters is opposite to that of higher alcohols, which can relax nerves, promote metabolism and slow down the influence of key substances affecting comfort. It was found that the ratio of higher alcohols to esters also affects the metabolism of ethanol by humans and thus the sensory experience of alcoholic beverages. The ester in the yellow wine is mainly synthesized by the metabolism of the saccharomyces cerevisiae by using higher alcohol under the action of acetyl transferase, and the difference of the self fermentation performance among strains and the fixed fermentation process lead to smaller difference of the ester content in the yellow wine and relatively lower ester content in the yellow wine because the quantity of the saccharomyces cerevisiae used in the current mechanized production is smaller. Therefore, the breeding of the Saccharomyces cerevisiae with low production of fusel and high production of ester has important significance for the wine industry.

The conventional breeding means include mutation breeding, cell fusion breeding, genetic engineering breeding and the like. Mutation breeding is a relatively mature method for changing the characteristics of saccharomycetes at present, and has the advantages of good effect, simple equipment, simple and convenient operation and the like, for example Li Fan et al obtain a strain with high hyaluronic acid yield through ARTP mutation screening; lin Xianju et al obtained a high-yield cyclosporin A strain by ARTP mutagenesis screening; zhang Lifang et al obtained a strain of highly acid resistant Jiujiu coccus by ARTP mutagenesis screening. Although the transformation of Saccharomyces cerevisiae by genetic engineering means can clearly meet different target demands, whether the transgenic technology is absolutely safe is not clear, so that the transgenic technology is not widely accepted, and particularly in fermented foods such as yellow rice wine and table vinegar, most of metabolites of Saccharomyces cerevisiae often remain in the final product.

Concerning the research on the breeding and application of low-yield and high-yield ester saccharomyces cerevisiae in fermented foods such as white wine, yellow wine, beer, wine and the like, a strain of low-yield and high-yield ester saccharomyces cerevisiae and application thereof are reported in CN113621528B patent. The invention further carries out ARTP mutagenesis and systematic evaluation and breeding based on the ultraviolet mutagenesis strain of the strain to obtain the mutagenesis strain with better effect and the application of the mutagenesis strain in fermented foods, and has important significance for improving the quality, health and safety of alcoholic beverages such as yellow wine and fermented foods such as table vinegar.

Disclosure of Invention

In order to solve the adverse effect caused by too high content of the fusel, the invention provides the Saccharomyces cerevisiae mutagenesis strain AR28 with good fermentation performance and low yield of the fusel and high yield of the ester. The strain is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of CCTCC NO: m20231848, the preservation address is China, the university of Wuhan, and Wuhan.

In one embodiment of the invention, the monoclonal colony of the mutagenized strain AR28 is characterized by a milky white color, oval or elliptical shape, convex, smooth surface, moist and glossy, and clean strain edges.

The invention also provides a microbial preparation, which contains the saccharomyces cerevisiae (Saccharomyces cerevisiae) AR28 or fermentation liquid thereof, or immobilized cells thereof, or lyophilized powder thereof, or extract thereof, or contains living cells of the saccharomyces cerevisiae (Saccharomyces cerevisiae) AR28 and other microorganisms, or contains dry thalli obtained by lyophilization of the saccharomyces cerevisiae (Saccharomyces cerevisiae) AR28 and other microorganisms.

The strain Saccharomyces cerevisiae (Saccharomyces cerevisiae) AR28 with low yield of fusel and high yield of ester has the fermentation characteristics of high yield of alcohol, low yield of fusel, high yield of ester and the like while meeting the requirements of normal fermentation. The highest alcohol content of the fermented alcoholic beverage can reach 24.8% (v/v), and the total alcohol content of the low-alcohol-yield high-ester-yield Saccharomyces cerevisiae mutant strain which is metabolized to generate alcohol with unit alcohol content is 11.69mg/L-14.96mg/L.

In one embodiment of the invention, the microbial agent is a solid or liquid agent.

In one embodiment of the present invention, the microbial agent contains living cells of the Saccharomyces cerevisiae (Saccharomyces cerevisiae) AR28 cells, the Saccharomyces cerevisiae AR28 dry cells obtained by freeze drying, the Saccharomyces cerevisiae AR28 yeast cells obtained by a solidification technique, liquid microbial agents of the Saccharomyces cerevisiae AR28, solid microbial agents of the Saccharomyces cerevisiae AR28, or the Saccharomyces cerevisiae AR28 in any other form.

In one embodiment of the invention, the amount of Saccharomyces cerevisiae AR28 in the microbial preparation is not less than 1X 10 ⁶ CFU/g.

In one embodiment of the invention, the application mode of the strain is that the strain AR28 is compounded with other microorganisms, wherein the other microorganisms comprise Saccharomyces cerevisiae and non-Saccharomyces cerevisiae; the non-Saccharomyces cerevisiae includes, but is not limited to, lactic acid bacteria.

In one embodiment of the invention, the microbial preparation is prepared as follows: inoculating Saccharomyces cerevisiae AR28 strain into a rice saccharification liquid culture medium, and carrying out shaking culture for 20-24 hours at the culture temperature of 28+/-2 ℃ to obtain primary seed liquid; inoculating the first-stage seed liquid into a new rice saccharification liquid culture medium according to the proportion of 5% -10%, and carrying out shake culture for 36-48 h at the culture temperature of 28+/-2 ℃ to obtain a second-stage seed liquid, thereby obtaining the quick brewing mother preparation with the yeast number of more than or equal to 1 multiplied by 10 ⁷ CFU/mL and the germination rate of more than or equal to 30%.

The invention also provides a composition containing the saccharomyces cerevisiae (Saccharomyces cerevisiae) AR 28.

In one embodiment of the invention, the composition includes, but is not limited to, a microbial inoculant, an enhanced wine agent, or an enhanced malt.

In one embodiment of the invention, the reinforced wine medicine refers to the mutagenesis strain AR28 or the microbial agent in the manufacturing process of the wine medicine, and the reinforced wine medicine is compounded with other microorganisms or added into the wine medicine in any other form, so that the purpose that the fermentation characteristic of the wine medicine can be reinforced when the wine medicine is used is achieved.

In one embodiment of the invention, the reinforced distiller's yeast means that the mutagenesis strain AR28 or the microbial agent is compounded with other microorganisms in the distiller's yeast preparation process, and the reinforced distiller's yeast or other substances are added into the distiller's yeast in any form, so that the purpose of reinforcing the fermentation characteristic of the distiller's yeast when the distiller's yeast is used is achieved.

The invention also provides the use of the Saccharomyces cerevisiae AR28, or the microbial preparation, or the composition, in the production of a fermented product.

In one embodiment of the invention, the fermented product is a fermented food or a fermented beverage.

In one embodiment of the invention, the fermented beverage includes, but is not limited to, a fermented alcoholic beverage or a fermented vinegar.

In one embodiment of the present invention, the fermented alcoholic beverage includes, but is not limited to, wine, cooking wine, rice wine, sweet rice wine, fruit wine, beer or white wine.

In one embodiment of the invention, the fermented vinegar comprises fruit vinegar or table vinegar.

In one embodiment of the invention, the fermented product is a fermented tobacco leaf or a fermented ice cream.

In one embodiment of the invention, the application is for brewing yellow wine, and the strain is used for mutagenesis of Saccharomyces cerevisiae with low yield of fusel and high yield of ester into Saccharomyces cerevisiae AR 28.

In one embodiment of the invention, the fermented alcoholic beverage has harmonious body fragrance, soft taste and good drinking comfort, the fermented vinegar has soft, clean taste and unique flavor, and the cigarette prepared by the fermented tobacco leaf group has soft smoke, low irritation, light fruit fragrance, flower fragrance, pleasant sweet and mellow fragrance and extract special fragrance.

In one embodiment of the invention, the yellow wine brewing is a yellow wine obtained by adding the low-impurity-alcohol high-ester-production saccharomyces cerevisiae mutagenesis strain AR28 serving as a quick brewing yeast into raw materials (rice, millet, corn, millet and the like) which are steamed or gelatinized according to the addition amount of 5-15%, fermenting, squeezing, decocting, ageing, filtering, sterilizing and filling.

In one embodiment of the invention, the application is for cooking wine brewing.

In one embodiment of the invention, the cooking wine brewing is to prepare the cooking wine by using the low-impurity-alcohol-yield high-ester-yield saccharomyces cerevisiae mutagenesis strain AR28 as a quick brewing parent to ferment first to obtain the yellow wine and then using the yellow wine.

In one embodiment of the invention, the application is for rice wine brewing, and the low-yield and high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 is used as a starter.

In one embodiment of the invention, the rice wine brewing is performed by brewing the low-impurity-alcohol-yield high-ester-yield saccharomyces cerevisiae mutagenesis strain AR28 and distiller's yeast serving as a fermenting agent, adding the low-impurity-alcohol-yield high-ester-yield saccharomyces cerevisiae mutagenesis strain AR28 and distiller's yeast into a steamed rice raw material according to the adding amount of 0.5% -1.5%, and performing processes such as steaming, adding yeast (adding the saccharomyces cerevisiae), saccharification, fermentation, squeezing and the like.

In one embodiment of the invention, the application is for brewing sweet rice, and the low-yield and high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 is used as a starter.

In one embodiment of the present invention, the rice wine brewing is performed by brewing the low-impurity-alcohol-yield high-ester-yield saccharomyces cerevisiae mutant strain AR28 and distiller's yeast as a starter, adding the low-impurity-alcohol-yield high-ester-yield saccharomyces cerevisiae mutant strain AR28 and distiller's yeast into steamed raw materials (rice, millet, corn, millet, etc.) according to an adding amount of 0.5% -1.5%, and performing processes such as steaming, starter adding (adding the saccharomyces cerevisiae), saccharification, fermentation, etc.

In one embodiment of the invention, the application is for vinegar brewing.

In one embodiment of the invention, the application is for brewing vinegar by using the low-yield fusel high-yield ester saccharomyces cerevisiae mutagenesis strain as a yeast to ferment first to obtain yellow wine and then using the yellow wine as an acetic acid fermentation raw material.

In one embodiment of the invention, the use is for brewing fruit wine.

In one embodiment of the present invention, the brewed fruit wine includes, but is not limited to, any one of green plum wine, red bayberry wine, kiwi fruit wine, plum wine, hawthorn wine, pomegranate wine, lemon wine, loquat wine.

In one embodiment of the invention, the brewed fruit wine comprises the following processes: and (3) treatment before fermentation, and inoculating the low-yield and high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 into clear juice obtained after juice clarification.

In one embodiment of the invention, the low-impurity-alcohol-yield and high-ester-yield Saccharomyces cerevisiae mutant strain AR28 is inoculated in an amount of 1% (v/v) to 5% (v/v) of the total volume of the clear juice when the fruit wine is brewed.

In one embodiment of the invention, the use is for brewing fruit vinegar.

In one embodiment of the invention, the application is that the low-impurity-alcohol high-ester-yield saccharomyces cerevisiae mutagenesis strain is firstly utilized to ferment to obtain fruit wine, then the fruit wine is utilized as a fermentation raw material to be inoculated with acetic acid strain, and acetic acid fermentation is carried out to brew the fruit vinegar.

In one embodiment of the invention, the use is for brewing beer.

In one embodiment of the invention, the application is used for brewing beer, wherein the low-impurity-alcohol-yield and high-ester-yield saccharomyces cerevisiae mutagenesis strain AR28 is used as pure yeast, and is added into wort according to the addition amount of 0.2% -1% after the amplification, and the beer is obtained through malt treatment, brewing, filling and other processes.

In one embodiment of the invention, the use is for making cigarettes.

In one embodiment of the invention, the application is for making cigarettes, and tobacco flavor is directly prepared from the low-impurity-alcohol-yield high-ester-yield saccharomyces cerevisiae mutagenesis strain AR28 culture solution; or taking pears, grapes, sweet osmanthus, tobacco extracts and the like as culture medium raw materials, inoculating the low-yield fusel high-yield ester saccharomyces cerevisiae AR28 for fermentation to prepare fermented tobacco flavor, and then directly adding the obtained tobacco flavor into a tobacco rolling group according to the adding amount of 1-5% of the mass of the tobacco rolling group.

In one embodiment of the invention, the use is for making a fermented nutritional ice cream.

In one embodiment of the invention, the application is used for preparing the fermented nutritional ice cream, which is obtained by fermenting raw materials (fruit juice, sugar, honey, whole milk powder and the like, sweet potato, purple sweet potato, corn and other starches) with 5-10% of the low-impurity-alcohol high-ester saccharomyces cerevisiae mutagenesis strain AR28 serving as a fermenting agent, adding other food additives, and then mixing, pasteurizing, homogenizing, cooling and aging, freezing and stirring, injection molding, freezing, demolding and other processes.

The invention also provides a product obtained by distilling, blending or adding the low-yield fusel high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 fermentation product.

The invention also provides a product obtained by distilling or blending a fermentation product prepared by applying the Saccharomyces cerevisiae AR28 or the microbial preparation or the composition fermentation.

The invention also provides a preparation method of the fermented beverage, which is characterized in that the fermented beverage is prepared by adding the Saccharomyces cerevisiae AR28, or the microbial preparation, or the composition into brewing raw materials for brewing.

In one embodiment of the invention, the fermented beverage includes, but is not limited to, fermented alcoholic beverage or fermented vinegar; preferably, the fermented alcoholic beverage includes, but is not limited to: yellow wine, cooking wine, rice wine, sweet rice wine, fruit wine, beer or white wine, preferably, the fermented vinegar comprises fruit vinegar or table vinegar.

In one embodiment of the present invention, the fruit wine includes, but is not limited to, any one of green plum wine, red bayberry wine, kiwi fruit wine, plum wine, hawthorn wine, pomegranate wine, lemon wine, loquat wine.

Advantageous effects

(1) The invention provides a low-impurity-alcohol high-ester-yield saccharomyces cerevisiae AR28 which is excellent in stress resistance and high in ethanol tolerance up to 25% (v/v), and the saccharomyces cerevisiae strain has a good temperature tolerance range, has good growth conditions in the range of 15-45 ℃, and can meet the requirements of different types of fermented foods.

(2) The yellow wine fermentation experiment shows that the AR28 can show good fermentation performance under the fermentation condition that the main fermentation temperature is 20-35 ℃ and the post-fermentation temperature is 10-15 ℃.

(3) When the low-yield and high-yield ester saccharomyces cerevisiae AR28 provided by the invention is used for brewing mechanized yellow wine, compared with the low-yield and hetero-alcohol saccharomyces cerevisiae strain disclosed in the patent CN113621528B, the alcoholicity of the AR28 strain is high, the highest alcoholicity of the yellow wine obtained after fermentation is up to 24.80% (v/v), the total amount of hetero-alcohol is as low as 289.84mg/L (the hetero-alcohol content per unit alcoholicity is 11.68+/-0.71 mg/L, the isobutanol content per unit alcoholicity is 3.15+/-0.23 mg/L, the isoamyl alcohol content per unit alcoholicity is 3.59+/-0.83 mg/L, and the phenylethanol content per unit alcoholicity is 2.33+/-0.62 mg/L). The total value of the OAV esters is increased by about 15% compared with the control, which shows that the contribution degree of the aroma active ingredients to the flavor of the yellow wine is increased. The strain has remarkable fermentation characteristics of low-impurity alcohol and high-ester yield, and the finally obtained yellow wine after squeezing, blending and filtering has softer aroma and taste, good drinking comfort, is not easy to get up after drinking in proper amount and has quick sobering up after drinking.

Preservation of biological materials

Saccharomyces cerevisiae (Saccharomyces cerevisiae) AR28, classified and named Saccharomyces cerevisiae AR28Saccharomyces CEREVISIAE AR, is preserved in China Center for Type Culture Collection (CCTCC) No. M20231848 in 2023, 10 and 9, and has a preservation address of China, university of Wuhan, and Wuhan.

Drawings

FIG. 1 is a graph showing the lethality of an ARTP mutant strain of Saccharomyces cerevisiae (S. Cerevisiae) of the present invention.

FIG. 2 shows the total amount of fusel produced during the re-screening of Saccharomyces cerevisiae (S. Cerevisiae) according to the present invention.

FIG. 3 shows the OAV values of esters produced during the re-screening of Saccharomyces cerevisiae (S. Cerevisiae) of the present invention.

FIG. 4 shows the content of fusel produced by purification of a spore of Saccharomyces cerevisiae (S.cerevisiae) of the present invention.

FIG. 5 shows the content of esters produced by purification of a spore of Saccharomyces cerevisiae (S.cerevisiae) of the present invention.

Detailed Description

Specific embodiments of the present invention are described below with reference to the accompanying drawings. The experimental methods used in the examples are all conventional methods unless otherwise specified; materials, reagents and the like used, unless otherwise indicated, are all commercially available.

Jiangnan1# related to the following examples is described in the chinese patent publication No. CN 113621528B. The "23-1-35" referred to in the examples below was a strain obtained by ultraviolet mutagenesis using jiangnan # 1 (CN 113621528B) as a starting cell.

The following examples relate to the following media:

YPD medium: 10g yeast extract, 20g peptone, 20g glucose, 1000mL of water, 2% agar, and autoclaved at 121 ℃ for 20min, and cooled for later use.

Rice saccharification liquid culture medium: taking proper amount of high-quality rice raw materials, soaking the rice for 30min in a water bath at the constant temperature of 60 ℃ and steaming for 20min under normal pressure, then respectively taking the rice raw materials as a reference, adding 150U/g-300U/g 1%o saccharifying enzyme and 200U/g-400U/g 2%o liquefying enzyme, adding raw wheat starter with the mass of 10% of rice, saccharifying for 4h-5h under the condition of 55-65 ℃ until the sugar degree is above 13Brix, subpackaging, sterilizing at 121 ℃ under high pressure for 15min-20min, and cooling for later use.

Pre-spore production culture medium: 1% KAc,1% yeast extract, 2% peptone, and sterilizing at 121deg.C for 15min.

High-efficiency spore-producing culture medium: 1% KAc,0.1% yeast extract, 0.05% glucose, adenine 50mg/L, histidine 100mg/L, uracil 50mg/L, tryptophan 100mg/L, leucine 100mg/L. If necessary, specific amino acid components can be omitted, and the mixture is sterilized at 121℃for 15min.

Definition of technical terms:

The term "strain" as used herein refers to a microorganism of a particular species having common characteristics. The terms "strain" and "cell" are used interchangeably herein unless indicated to the contrary.

The term "plate" as used herein refers to a plate culture medium, which is the most commonly used form of solid medium used to obtain pure culture of microorganisms, and which is a solid plane of the medium formed by cooling solidified solid medium in a sterile petri dish, often referred to simply as a culture plate, or plate.

The term "medium" as used herein refers to a medium comprising the chemical elements necessary for the growth of the microorganism together with at least one carbon source and one nitrogen source.

The term "culturing" as used herein means culturing the microorganism for a period of time until a desired target is reached.

The term "heteroalcoholic" as used herein: n-propanol, isobutanol, isoamyl alcohol, phenethyl alcohol.

The term "ester" as used herein in the term "ester-producing" refers to: ethyl acetate, ethyl isobutyrate, isoamyl acetate, ethyl caproate, ethyl caprylate, ethyl 2-hydroxy-4-methylpentanoate, ethyl benzoate, ethyl 2-phenylacetate, ethyl acetate, ethyl 3-phenylpropionate, diethyl succinate, ethyl butyrate, and the like.

The term "fermentation broth" as used herein refers to a liquid culture medium into which a microorganism strain is introduced, after a period of time, the microorganism utilizes the nutrients in the culture medium to synthesize bacterial cells and secretion products, and the liquid after metabolism of the microorganism is called a fermentation broth.

The term "strain extract" as used herein refers to an extract obtained by fermentation of a strain of lactobacillus salivarius, which is inoculated into a suitable medium, fermented under conventional fermentation conditions to synthesize and secrete the product into the medium, and then purified.

The term "lysate of a strain" as used herein means a lysate of a strain obtained by inoculating a cultured Saccharomyces cerevisiae into a cell lysate, subjecting the cell lysate to lysis under conventional conditions, and centrifuging and purifying the cell lysate.

The Saccharomyces cerevisiae strains of the present invention also include mutants, variants, and/or progeny of the above Saccharomyces cerevisiae strains.

By "mutant" is meant any microorganism produced by modification of a parent s.cerevisiae strain. For example, the mutant may be a microorganism produced by genetic modification of a strain of Saccharomyces cerevisiae. By "variant" is meant a naturally occurring microorganism derived from a parent s.cerevisiae strain. For example, the variant may be a microorganism produced by Saccharomyces cerevisiae in response to specific cell culture conditions. By "progeny" is meant any microorganism produced by propagation or multiplication of a parent saccharomyces cerevisiae strain or mutant, variant thereof, which itself may be identified as the same or substantially the same strain as the parent strain. It will be appreciated that, in view of the asexual propagation process, the progeny strain is almost identical in gene to the parent s.cerevisiae strain. Thus, the progeny strain is identical in gene to the parent strain and can be considered a "clone" of the parent strain. Or the progeny strain is substantially identical in gene to the parent strain.

The "culture" in the present invention refers to a product obtained by culturing a strain in a medium, and the product may include the strain itself. The lysate refers to a product obtained by treating a strain with enzymes, ultrasound, homogenization and the like. The term "extract" refers to a product obtained by subjecting a strain to a solvent extraction or the like. The term "inactivated product" refers to a product obtained by treating a strain with heat, pressure, or a drug.

The "art mutagenesis" in the present invention is: atmospheric Room Temperature Plasma (ARTP) mutagenesis is a microorganism mutagenesis tool using helium-emitted plasma jet, and compared with conventional chemical mutagenesis and ultraviolet mutagenesis, ARTP can efficiently induce DNA strand breakage at atmospheric pressure and room temperature (25-40 ℃) to generate obvious mutagenesis effect

ARTP is an acronym for atmospheric pressure room temperature plasma (Atmospheric and Room Temperature Plasma) capable of generating a plasma jet at atmospheric pressure at a temperature between 25-40 ℃ with a high concentration of reactive particles (including helium atoms, oxygen atoms, nitrogen atoms, OH radicals, etc. in an excited state).

The detection method involved in the following examples is as follows:

And (3) detecting physical and chemical indexes of the yellow rice wine: the measurement of alcohol content, amino acid nitrogen and total acid is carried out by referring to GB/T13662-2018 yellow wine. The content of organic acid and amino acid is detected by High Performance Liquid Chromatography (HPLC), and volatile flavor substances such as ethyl carbamate, higher alcohol, esters and the like are detected by gas chromatography-mass spectrometry (GC-MS). The determination of the reducing sugar content adopts a DNS method. The concentration of the bacterial liquid is measured by spectrophotometry. The higher alcohol (also called as fusel) in the yellow wine mainly comprises 4 types of n-propanol, isobutanol, isopentyl alcohol and 2-phenethyl alcohol, a dispersion liquid-liquid microextraction technology (DLLME) is adopted, GC-MS detection is utilized, 4-methyl-2-pentanol is used as an internal standard, and an external standard curve is established for quantitatively measuring the fusel content.

The preparation method of the wheat starter related in the following examples comprises the following steps: the Guyue Longshan is prepared with wheat as material and through milling, adding water, mixing, treading, cutting, and swaying to obtain saccharifying agent with rich beneficial microbe, amylase, proteinase, etc.

The preparation method of the wine sweat related in the following examples comprises the following steps: guyue Longshan provides: fermenting the distilled grain after filtering the yellow wine again, and distilling to obtain distilled liquor.

EXAMPLE 1 mutagenesis breeding of Saccharomyces cerevisiae Strain and Breeding of Low-yield hybrid alcohol high-yield ester Saccharomyces cerevisiae mutagenesis Strain

1. Mutagenesis experiment

Fixing the working condition of an ARTP plasma mutagenesis instrument, and carrying out mutagenesis treatment on bacterial liquid by taking mutagenesis time as mutagenesis variable. The specific processing steps are as follows: the thalli in logarithmic phase is centrifugally washed, 10 mu L of bacterial suspension is taken on a sterile stainless steel slide with the diameter of 5mm, helium (He) with the purity of 99.999% is taken as injection gas, the flow rate is QHe =10.0L/min, the power supply power is 100W, the treatment distance is 2mm, and the treatment temperature is lower than 40 ℃. The original strain is subjected to mutagenesis treatment for 0s, 5s, 10s, 15s, 20s, 25s, 30s, 35s, 40s, 45s and 50s respectively under an ARTP instrument, a stainless steel slide is placed in an EP tube containing 490 mu L of sterile physiological saline after each mutagenesis is finished, and the mutagenized bacteria are eluted by shaking and washing for 1min on a whirlpool. Respectively carrying out gradient dilution on the bacterial liquid until the bacterial concentration is 10 ³～10⁴/mL, absorbing 0.1mL of the diluted liquid, coating the diluted liquid on a YPD solid culture medium plate, inversely culturing the bacterial colony in a constant temperature incubator at 28 ℃ for 48 hours, observing and recording the bacterial colony number, calculating the mortality according to a formula, drawing a lethal curve, determining the optimal mutagenesis treatment time, carrying out mutagenesis on a starting bacterial strain under the optimal mutagenesis condition, and picking a single bacterial colony for performance identification after culturing. The bacterial strain mortality (fig. 1) is calculated as follows:

Mortality (%) = (control colony count-ARTP colony count)/control colony count×100.

The Saccharomyces cerevisiae strain after mutagenesis is prepared.

2. Primary screening based on Saccharomyces cerevisiae ARTP mutagenesis strain yellow wine fermentation

In order to select a mutagenesis strain with excellent fermentation performance which can meet the requirements of brewing yellow wine, the obtained ARTP strain is respectively prepared into a quick brewing mother and then fermented with yellow wine, physicochemical indexes such as alcohol degree and the like are measured after fermentation is finished, the strain is initially screened, and the measured impurity and flavor substances with alcohol degree meeting national standards are screened according to different batches.

The method comprises the following specific steps:

(1) The proportion of the raw materials for yellow wine fermentation in the factory selected in the embodiment

100% Glutinous rice (benchmark): 500g;125% water: 625g;12% -15% wheat starter: 60g-75g;10% -15% of quick brewing yeast culture solution: 50mL-75mL; perspiration: 17.15g.

The preparation method of the quick brewing mother culture solution comprises the following steps: the preparation method comprises the steps of performing slant preparation on a Saccharomyces cerevisiae glycerol pipe preservation strain, inoculating the Saccharomyces cerevisiae strain subjected to slant culture in a rice saccharification liquid culture medium, performing shake culture for 24 hours under the condition that the culture temperature is 28+/-2 ℃ and 200r/min to obtain primary seed liquid (bacterial liquid concentration is 10 ⁶-10⁸ CFU/mL), inoculating the primary seed liquid to a new rice saccharification liquid culture medium according to the proportion of 5% -10%, performing shake culture for 36-48 hours under the condition that the culture temperature is 28+/-2 ℃ and 200r/min to obtain secondary seed liquid, and determining the viable count and germination rate of the yeast by using the obtained secondary seed liquid, wherein the germination rate is not less than 30% and not less than 1×10: 10 ⁷, and taking the secondary seed liquid as a quick brewing mother culture liquid for brewing yellow wine.

Experimental group: saccharomyces cerevisiae strain obtained by mutagenesis in example 1 was used as a pure yeast, and Saccharomyces cerevisiae was prepared by the above method to obtain a speed-changing Saccharomyces cerevisiae culture solution.

Control group: the strain-jiangnan # and the excellent Saccharomyces cerevisiae 23-1-35 (experiments prove that the strain-jiangnan # has better effect of reducing the impurity alcohol compared with the strain-jiangnan #) obtained by ultraviolet mutagenesis are used as pure yeast, and the quick brewing yeast is prepared according to the method to obtain the quick brewing yeast jiangnan # culture solution and the quick brewing yeast 23-1-35 culture solution respectively.

(2) Traditional yellow wine brewing process

A) Preparation of fermented raw material rice:

The raw rice with the production dosage is added with water to be soaked until the water exceeds the liquid level by more than 10cm, the acidity of the rice slurry of the soaked rice reaches more than 4.5g/L for 3-5 days, the water is drained to obtain wet rice, the wet rice is steamed for 20-30 min at the temperature of 121 ℃ in a rice steaming cabinet until the rice is cooked but not transparent, white cores are not arranged in the rice grains, the rice has sour taste and has rice fragrance, and the rice yield is 140-160%.

B) Blanking and fermenting according to the raw material proportion of the traditional yellow wine fermentation:

The yellow wine fermentation raw material proportion according to the step (1) is used for blanking and fermenting, and the specific steps are as follows:

S1-S2, preparing a quick brewing mother culture solution according to the method of the step (1);

s3, adding raw materials of rice, water and sweat into a sterilized fermentation tank to obtain a mixed fermentation system, respectively adding the quick brewing mother liquor obtained in the steps with the ratio of 10% -15% based on the raw materials of rice into the mixed fermentation system, adding wheat starter accounting for 12% -15% of the mass of the raw materials of rice, completing material mixing at 25 ℃ -28 ℃, standing at 20 ℃ -35 ℃ and performing primary fermentation for 3-5 days;

s4, reducing the temperature of the fermentation tank to 10-15 ℃, and standing for 15-20 days for post fermentation to obtain fermented mash;

S5, squeezing the fermented mash obtained in the step S4 through a plate frame (4 times of feeding, the mash feeding pressure is 0.2-0.6MPa, the filtering area is 100m < 2 >, the filter plate diameter is 1 m), and filtering the filtrate by diatomite (the diatomite adding proportion is 4% -6%, and the pressure is 0.3-0.5 MPa), clarifying to obtain sake, blending the sake, adding 1-3 per mill of caramel according to national standard of yellow wine, and frying the sake to obtain the yellow wine.

A total of 87 ARTP mutant strains (obtained in example 1) were prepared according to the above steps to obtain yellow wine, and the batch containing the mutant strain AR28 was used as an example, and the mutant strain with an alcohol content of 16% (v/v) or more was selected for physicochemical index, impurity and flavor measurement after fermentation. Comprehensive data analysis is carried out according to the flavor and the fusel index of the strain, and the mutagenized strain with low fusel yield and high ester yield is obviously distinguished.

3. Compound sieve based on Saccharomyces cerevisiae ARTP mutant strain yellow wine fermentation

And (3) repeatedly performing re-screening on the strain with excellent fermentation performance obtained in the step (2) to prepare wine (namely repeatedly preparing yellow wine according to the method of the step (2)), and measuring physicochemical indexes (table 1), flavor and fusel (table 2 and figure 2) of the yellow wine after fermentation.

(1) The physicochemical indexes of the yellow wine obtained by the re-screening after the fermentation of the Saccharomyces cerevisiae with excellent low-yield and high-yield miscellaneous alcohols all meet the national standard of the yellow wine, and the results are shown in Table 1.

Table 1: finally, the physical and chemical indexes of the yellow wine at the end of fermentation of the strain of the Saccharomyces cerevisiae are obtained by re-screening

(2) Finally, the impurity alcohol content in the yellow wine at the end of the fermentation of the excellent Saccharomyces cerevisiae mutant strain is obtained by re-screening is shown in table 2, and the impurity alcohol content with unit alcohol degree is listed in the table, and the calculation method is as follows: impurity alcohol content/alcohol content.

Table 2: re-screening to obtain excellent impurity alcohol content at the end of fermentation of Saccharomyces cerevisiae mutant strain

Note that: the content unit of the fusel is mg/L/%vol; values are mean ± standard deviation of at least three independent assays.

(3) The total OAV values obtained by rescreening the resulting good Saccharomyces cerevisiae mutant strains for fermentation to produce esters are shown in Table 3. The detection method of the OAV total value comprises the following steps: concentration/threshold. Wherein, the concentration refers to: ester concentration as determined by GC-MS;

the threshold values in the respective substance threshold references are specifically (units g/m ³): ethyl acetate 7500; ethyl isobutyrate 15; isoamyl acetate 30; ethyl caproate 5; ethyl octanoate 5; ethyl 2-phenylacetate 73; phenethyl acetate 250; ethyl 3-phenylpropionate 125; diethyl succinate 200000; ethyl butyrate 20; ethyl lactate 154000; ethyl isovalerate 3; propionolactone 30; ethyl propionate 2100.

The results are shown in Table 3 and FIG. 3:

TABLE 3 production of the final OAV values for the production of esters by fermentation of the good Saccharomyces cerevisiae mutant strains by re-screening

Note that: values are mean ± standard deviation of at least three independent assays

The comprehensive analysis of the mutant strain Saccharomyces cerevisiae AR28 shows excellent fermentation characteristics of low-yield fusel and high-yield ester, and Saccharomyces cerevisiae AR28 is preserved in China Center for Type Culture Collection (CCTCC) at the preservation number of M20231848 in 10-9 of 2023.

4. Saccharomycetes purification based on Saccharomyces cerevisiae ARTP mutant strain Saccharomyces cerevisiae AR28

The method for purifying the Saccharomyces cerevisiae AR28 spore comprises the following steps:

(1) Pre-spore production treatment: culturing in 100mL YPD liquid to logarithmic phase, centrifuging the bacterial liquid at 10000r/min for 30s, discarding supernatant, washing with sterile water three times, re-suspending with pre-spore-forming medium, and culturing at 22deg.C at 200r/min for 24h.

(2) High-efficiency spore production treatment: centrifuging the bacterial liquid 10000r/min in the pre-spore-producing culture medium for 30s, discarding the supernatant, and re-suspending to 100mL of high-efficiency liquid spore-producing culture medium, and culturing at 22 ℃ at 200 r/min. Sampling every 24 hours, observing the spore production condition by using a carbolic acid multiple red dyeing method, taking three fields of view each time, calculating the spore production rate, and carrying out the next experiment when the spore production rate reaches 90%.

The spore yield calculating method comprises the following steps: and (3) observing ascospores by using an oil lens after gradient dilution, observing and recording the number of ascopes and the number of diploid vegetative cells which do not form ascopes in the visual field, and calculating the spore yield in parallel for 5 times.

Sporulation rate = ascus number/(ascus number + diploid vegetative cell number) ×100%

(3) Inactivation of diploid trophozoites: centrifuging the bacterial liquid 10000r/min in the high-efficiency spore-producing culture medium for 30s, discarding the supernatant, re-suspending and washing with a sodium phosphate buffer solution with PH=7, heating in a water bath at 65 ℃ for 10min, sampling, coating and culturing on a YPD plate to verify that the diploid nutrition strain is completely inactivated, and collecting haploids to obtain the subsequent homozygote diploid.

(4) Haploid separation: adding 1mL of 0.3g/L helicase and sterile glass beads into the bacterial solution in the last step of centrifugation, vibrating for 0.5-1 h at 28 ℃, carrying out enzymolysis on the ascus wall to release haploids, centrifuging for 5min at 5000r/min to collect thalli, washing with a sodium phosphate buffer solution, suspending, dispersing spores by using 12W ultrasonic waves for 60s, diluting the spore suspension for 5 times by using a ten-fold dilution method, taking 100 mu L of coated YPD plates for each gradient, and culturing for 1-2 d at 30 ℃.

(5) Yellow rice wine fermentation screening: and (3) selecting strains with larger bacterial colonies on the YPD flat plate in the last step to perform a yellow wine fermentation experiment (the method in the step 2), and measuring indexes such as the impurity alcohol content, the alcohol degree and the like of the obtained yellow wine after fermentation, wherein each group of the indexes is three in parallel, and the results are shown in tables 4-5 and figures 4-5.

TABLE 4 physicochemical index of the end of fermentation of the purified plant of conidium

TABLE 5 yield per alcohol and acetate content at the end of fermentation of the purification plant of Saccharum sinensis Roxb

Note that: the content unit of the fusel is mg/L/%vol; the content unit of acetate is mg/L; values are mean ± standard deviation of at least three independent assays.

The results show that the AR28 is subjected to haploid separation after sporulation, haploids are combined with surrounding heterotypic spore trophosomes to generate diploid cells in the process of culturing haploids, and low-yield heteroalcohol diploid strains are screened. The two alleles screened into the diploid are identical and are all derived from a haploid containing positive mutant genes, so that the ester-extracting and fusogenic performance is further enhanced and the genetic stability is enhanced.

Example 2: preparation of Saccharomyces cerevisiae mutagenesis strain AR28 starter with low yield of fusel and high yield of ester

(1) Taking the instant brewing yeast as an example, the instant brewing yeast containing the mutagenesis strain AR28 is prepared by brewing yellow wine.

The manufacturing method of the quick brewing master comprises the following steps:

The preparation method comprises the steps of preparing a slope from a Saccharomyces cerevisiae glycerol pipe preservation strain, inoculating a mutagenized strain AR28 strain screened in a slope culture example 1 into a rice saccharification liquid culture medium, carrying out shaking culture for 24 hours under the conditions that the culture temperature is 28+/-2 ℃ and 200r/min to obtain a first-stage seed liquid (the concentration of bacterial liquid is 10 ⁶-10⁸ CFU/mL), inoculating the first-stage seed liquid into a new rice saccharification liquid culture medium according to the proportion of 5% -10%, carrying out shaking culture for 36-48 hours under the conditions that the culture temperature is 28+/-2 ℃ and 200r/min to obtain a second-stage seed liquid, and measuring the viable count and germination rate of yeast by using the obtained second-stage seed liquid to meet the conditions that the yeast count is not less than 1×10 ⁷ CFU/mL and the germination rate is not less than 30% and taking the second-stage seed liquid as a quick brewing yeast (starter) to brew yellow wine.

(2) Preparation of starter for other types of fermented foods by using the mutant strain AR28

And (3) using the instant brewing yeast containing the pure strain prepared in the step (1) for fermentation production of food according to different requirements of fermented food to prepare a fermented alcoholic beverage containing the saccharomyces cerevisiae, wherein the culture medium can select various culture mediums such as rice saccharification liquid, wort, sugar solution and the like meeting the nutrition requirements of food fermentation production C, N to perform activation culture on the mutagenized strain AR28 to reach corresponding colony numbers meeting the requirements.

Example 3: application of Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and high yield of ester in yellow wine industry

(1) Application in traditional yellow wine production

Preparing quick brewing yeast by using a low-yield hybrid alcohol high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 and excellent saccharomyces cerevisiae jiangnan # as experimental strains and control strains respectively according to the method of the step (1) of the example 2;

Meanwhile, the method of the step (2) in the step 2 of the example 1 is used for brewing yellow wine, and after the fermentation is finished, physical and chemical indexes such as alcohol degree, impurity alcohol and flavor and the like in the obtained yellow wine are respectively measured.

The results show that: the alcohol degree in the yellow wine obtained after the fermentation of the mutagenesis strain saccharomyces cerevisiae AR28 is 21.13% (v/v), the content of the fusel is 289.84 +/-0.30 mg/L, and the total value of the ester OAV is as follows: 56.59mg/L;

Compared with strain jiangnan # 1 (19.73% (v/v) alcohol content, 414.48mg/L fusel content and total ester OAV value of 43.31mg/L, fusel yield is reduced by more than 25%, and total ester content is increased by more than 30%.

Therefore, the Saccharomyces cerevisiae AR28 strain has remarkable fermentation characteristics of low-yield and high-yield miscellaneous alcohols, and the finally obtained yellow wine after squeezing, blending and filtering has softer and coordinated aroma and taste, good drinking comfort, is not easy to get up after drinking in proper amount, and can sober up quickly after drinking in excessive amount, and other indexes such as urea, carbamic acid, amino acid, organic acid and the like all conform to the national standard GB/T13662-2018 yellow wine of yellow wine.

(2) Application of mutagenesis strain AR28 in millet wine

The millet wine fermentation raw materials selected in the embodiment are as follows:

millet (benchmark): 500g; water: 500g (mL) -625g (mL); raw wheat starter: 50g-75g; cooked wheat starter: 9g; saccharomyces cerevisiae AR28 fast brewing yeast: 57mL; when Angel brewing high-activity dry yeast and Angel brewing yeast are used, the activating starter replaces raw wheat yeast, cooked wheat yeast and quick brewing yeast.

Experimental group: a strain AR28 was obtained by mutagenesis of Saccharomyces cerevisiae with low yield of fusel and high yield of ester as a stock solution of Saccharomyces cerevisiae in the same manner as in step (1) of example 2.

Control group 1: a quick brewing mother culture broth prepared by using excellent Saccharomyces cerevisiae jiangnan # as a pure yeast was prepared according to the method of the step (1) of the example 2.

Control group 2: 1g of Angel brewing high-activity dry yeast, 2.5g of Angel brewing yeast, and activating Angel brewing high-activity dry yeast and Angel brewing yeast with sugar water containing 20mg/mL glucose at a water temperature of 30-35 ℃ for 20-30 min in advance, wherein the number of yeasts in the activating starter is more than 10 ⁸ CFU/mL.

The millet yellow wine brewing process comprises the following steps:

a) The preparation method of the fermented raw material rice comprises the following steps: soaking millet in water 2-3 times of the weight of the millet raw rice for 1-3 days at normal temperature with acidity higher than 3mg/L, draining water to obtain wet rice, steaming the wet rice in a rice steaming cabinet at 121 ℃ for 30-50 min until the rice is cooked but not transparent, no white core exists in the rice grains, and the rice yield is 140-160%.

B) According to the raw material proportion of millet wine fermentation, cooling an experimental group and a control group to 25-28 ℃ respectively for pre-fermentation: fermenting at 20-35 deg.c for 3-7 days to enter post-fermentation: and (3) after post fermentation for 15-20 days at the temperature of 10-15 ℃ to obtain fermented mash.

C) The fermented mash is subjected to plate and frame squeezing and diatomite filtration in factory production, the obtained filtrate is clarified to obtain sake, the sake is prepared into yellow wine after the sake is decocted, and medlar, red date, honey, astragalus mongholicus, mountain ginseng, preserved plums and the like can be optionally added for blending after ageing. The experimental example is a laboratory scale small system simulated fermentation, which is carried out by adopting a gauze filtration and centrifugation mode.

The final millet fermented mash obtained by fermenting the control strain AR28 and the experimental strain AR28 after the fermentation is completed is detected, and the results are shown in Table 6.

TABLE 6 main index of millet wine fermentation

The result shows that the final alcohol degree is 12% (v/v) -16% (v/v), the content of the bacterial strain AR28 fusel is lower than 350mg/L, the content of the fusel per unit alcohol degree is reduced by more than 20% compared with that of the control group 1 and the control group 2, other indexes all accord with national standards of yellow wine, the fermented millet yellow wine body is transparent, the color is bright, the taste is fresh, the final millet yellow wine obtained by fermenting the mutagenesis bacterial strain AR28 has fruit fragrance and ester fragrance, and the requirements of consumers on low fusel, high ester, nutrition and health of alcoholic beverages can be met.

(4) Application of mutagenesis strain AR28 in reduction of red rice yellow wine

The red yeast rice wine production application is carried out according to the method in the step (2) of the embodiment 1, wherein the difference is that red yeast is used for replacing wheat yeast in the raw material in the step S3, fermentation is carried out for 15-20 days in the step S5, fermented mash is subjected to plate-bin frame squeezing and diatomite filtering, and the obtained filtrate is clarified to obtain sake and fried to obtain red yeast rice wine (no caramel color is added).

A quick brewing yeast was prepared by using a low-impurity-alcohol-yield high-ester-yield Saccharomyces cerevisiae mutagenesis strain AR28 as an experimental strain in the manner of example 3 (3), quick brewing yeast was replaced by Angel brewing high-activity dry yeast and Angel brewing yeast, red yeast and quick brewing yeast were used as control groups to prepare activation ferment agents, red yeast yellow wine fermentation was performed according to the raw material ratio, and physicochemical properties such as alcohol content, flavor, urea content, urethane, amino acid, organic acid and the like were measured after the fermentation was completed, and the results are shown in Table 7.

Table 7: main index of red rice yellow wine fermentation

The results show that the alcohol degree of the red rice yellow wine obtained by fermenting the control group and the experimental group after the fermentation is finished is 16% (v/v) -18% (v/v), the red rice yellow wine body obtained by fermenting is brown or dark brown, the taste is fresh and free of peculiar smell, the wine body is coordinated, and the red rice yellow wine obtained by fermenting the mutagenesis strain AR28 has stronger fruit fragrance and ester fragrance.

Compared with a control group, the content of the fusel per unit alcohol in the yellow wine obtained by the low-yield fusel high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 group is 18.5mg/L, the fusel per unit alcohol is reduced by more than 20%, the fusel reducing effect is obvious, and other indexes meet national standards of the yellow wine.

Example 4 application of Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and high yield of ester in rice wine

Taking rice as a main raw material, washing with clear water, soaking for about 4 hours at normal temperature until water is fully absorbed, and kneading the rice with hands after soaking until the rice is fragile and has no hard rice core; draining the soaked rice grains, and cooking for about 30min under normal pressure until no white core exists in the rice; steaming rice, cooling to 25-28deg.C with cold boiled water, adding sweet wine yeast nest with mass of 0.4-0.8% of rice, saccharifying at 28deg.C+ -2deg.C for 36-42 h, inoculating 5-10% of low-yield hybrid alcohol high-yield ester AR 28-speed brewing yeast prepared according to example 3 (3), stirring, adding drinking water with mass 1-1.5 times of raw material rice, fermenting at 26-34 deg.C for 3-5 days, reducing temperature to 10-15deg.C, fermenting for 10-15 days, squeezing, and filtering to obtain rice wine. The rice wine obtained by fermentation is rich in rice aroma, has low higher alcohol content, has the impurity alcohol content of less than 20mg/L per unit alcohol degree, and is not easy to drunk after drinking.

Example 5 application of Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and high yield of ester in sweet rice brewing

Taking rice as a main raw material, washing with clear water, soaking for about 4 hours at normal temperature until water is fully absorbed, and kneading the rice with hands after soaking until the rice is fragile and has no hard rice core; draining the soaked rice grains, and cooking for about 30min under normal pressure until no white core exists in the rice; pouring cold boiled water into rice to 25-28 ℃ after the rice is steamed, transferring the rice to a fermentation tank, adding Angel sweet distiller's yeast with the mass of 0.4-0.8% of that of raw rice, inoculating low-yield miscellaneous alcohol and high-yield ester Saccharomyces cerevisiae AR28 prepared in the embodiment 3 (3), uniformly stirring, and digging a groove in the center of the rice to ensure a certain dissolved oxygen amount; adding 1-1.5 times of drinking water, fermenting at 26-34 deg.C for 36-72 hr, and providing wine fragrance. The brewing accuracy of the obtained sweet rice is 2% -4% (v/v), the aroma is coordinated, the sweet rice is rich, the texture is uniform, and the taste is sour and sweet and palatable.

Example 6 application of Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and high yield of ester in vinegar

Acetic fermentation was carried out using yellow wine obtained in the method described in (1) of example 3 as a raw material.

The vinegar brewing adopts a solid state fermentation process: mixing yellow wine, bran and bran according to the mass ratio of 10:4:1, inoculating vinegar grains with the total system mass of 3% -8%, turning over the materials, keeping the fermentation temperature at 35-40 ℃, and carrying out material turning over the materials for the first 2 days. Turning over the fermented grains from top to bottom to the bottom of the material for 2-8 days, and cooling from bottom to top for 8-12 days. Pouring vinegar after fermentation to obtain raw vinegar, sterilizing, aging in jar in open air, sterilizing at 85deg.C for 30min before filling different year of vinegar, and hot-pipe filling. After fermentation, the physical and chemical indexes of the obtained vinegar are normal, the acetic acid content is 50-80g/L, the ethyl carbamate content is low, and the safety is improved.

Example 7 application of Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and high yield of ester in fruit wine

Taking green plums as raw materials, sequentially cleaning, removing cores, draining, crushing, adding a fructose and glucose syrup solution with the mass 2-3 times of the raw materials, obtaining 100L mixed materials, subpackaging in a 150L fermentation tank, adding 20mg/L-40mg/L pectase (enzyme activity 20000U/g) and 40mg/L-80mg/L potassium metabisulfite, respectively inoculating 1x10 ⁷ CFU/mL low-yield fusel high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 culture expanding liquid in the amount of 1% (v/v) -5% (v/v), carrying out main fermentation for 10-15 days at 20-25 ℃ and carrying out post fermentation for 5-10 days at the temperature of 5-8 ℃ to obtain the fermented green plums wine. There are 2 parallel fermentors per group. The alcohol content of the fermented green plums is 10.5% (v/v) -12% (v/v), and the total sugar (calculated by glucose) is <40g/L. Sensory evaluation results show that the brewing of the green plum wine by adding the low-impurity-alcohol-yield high-ester saccharomyces cerevisiae mutagenesis strain AR28 is coordinated in wine body, soft in taste and better in quality of fruit wine. Other indexes of the fermented green plum wine accord with the specification of the general technical requirements of GB 2758-2012 and QB/T5476-2020 fruit wine.

Example 8 application of Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and high yield of ester in fruit vinegar

Acetic fermentation was performed using acetic acid from the fruit wine obtained in example 7. The fruit vinegar brewing adopts a liquid fermentation process to inoculate 1% -3% of acetic acid bacteria, acetic acid fermentation is carried out for 16 days at 34 ℃, vinegar spraying, glue discharging, clarification, degassing and sterilization are carried out, and the finished fruit vinegar is obtained. The acidity of the vinegar in the obtained fruit vinegar is 3% -8% (g/100 mL), other physical and chemical indexes are normal, the national standard of apple vinegar beverage GB/T30884-2014 is satisfied, and the fruit vinegar has rich fruit vinegar taste, soft and clean taste and unique flavor.

Example 9 application of Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and high yield of ester in beer

The preparation method comprises the steps of taking barley malt (15 kg-20 kg), hops (50 g-70 g) and water (100L-120L) as main raw materials, specifically adopting a multi-step soaking sugar-out method, taking wort as a culture medium, preparing a low-yield and high-yield fusel high-ester mutagenesis strain Saccharomyces cerevisiae AR28 pure strain Saccharomyces cerevisiae by the method of the step (3) in the example 3, adding the low-yield and high-yield fusel high-ester mutagenesis strain Saccharomyces cerevisiae AR28 pure strain into the wort according to the adding amount of 0.2% -1%, crushing the malt, saccharifying, filtering, boiling the wort, fermenting and the like, adopting the above fermentation yeast, pre-fermenting at 18-23 ℃ for 2-3 days under the fermentation pressure of 0.15Mpa, reducing the sugar degree per day to 5 ℃ when the sugar degree is reduced below 5Brix, and storing. The beer has 3% -5% (v/v) of alcohol content, total sugar (calculated by glucose) of <50g/L, prominent fragrance, no strong fragrance, malt fragrance and fruit fragrance, impurity alcohol content lower than 120mg/L, and normal other physical and chemical indexes.

Example 10 application of Saccharomyces cerevisiae AR28 with low yield of fusel and high yield of ester in cigarette

The tobacco leaves referred to in this embodiment are processed tobacco shred samples, and the tobacco extract is an aqueous extract or an alcohol extract, and is obtained by commercial purchase.

The low-impurity-alcohol high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 is subjected to shaking culture in a YPD culture medium for 24 hours at the culture temperature of 28+/-2 ℃ to obtain primary seed liquid with the concentration of 10 ⁶CFU/mL-10⁸ CFU/mL, then is inoculated into a seed culture medium containing pear juice, grape juice, osmanthus fragrans or tobacco extracts for fermentation culture according to the proportion of 5% -10%, the saccharomyces cerevisiae culture liquid obtained after 48 hours of culture is subjected to culture, the concentration of 10 ⁸CFU/mL-10⁹ CFU/mL is added with spice for cigarettes, the obtained added spice for cigarettes is directly added into the tobacco leaves according to the adding amount of 1% -5% of the mass of the tobacco leaves, water is supplemented to 10% -20%, then the tobacco leaves are cultured for 4 hours to 37 ℃, finally, moisture is balanced after baking, cigarettes are manufactured, or the added spice for cigarettes is dissolved in 75% of alcohol to prepare spice (0.1 g/mL-0.5 g/mL), and the cigarettes are manufactured after being uniformly sprayed into the treated tobacco samples according to the proper proportion. The obtained cigarette has soft smoke, low irritation, light fruit fragrance, light flower fragrance, pleasant sweet fragrance and extract special fragrance, can improve the smoking quality of cigarettes, and is suitable for improving the special fragrance and quality of cigarettes and novel tobacco products.

Example 11 application of Saccharomyces cerevisiae mutagenesis strain AR28 with low yield of fusel and high yield of ester in fermentation type nutritional ice cream

Mixing drinking water, squeezed juice, sugar, honey and the like as main raw materials in a proportion of (2-4) (0.05-0.2) (0.01-0.05), blending until the sugar concentration is 10Brix-20Brix, regulating the pH to 4-5 by using food-grade lactic acid, inoculating a low-impurity alcohol high-yield ester saccharomyces cerevisiae mutagenesis strain AR28 with the concentration of 5-10% for malt juice culture at 10 ⁶-10⁸ CFU/mL, carrying out shaking culture for 24-48 h in a constant temperature incubator at 30+/-2 ℃, centrifuging after fermentation, adding 30-50% of whole milk powder, 1-5% of thickener, 1-5% of emulsifier, 1-5% of puffing agent, 5-10% of vegetable fat powder and the like in the proportion by using supernatant or yellow wine obtained in the example 3 instead of drinking water until the total dry matter is 20-30%, carrying out pasteurization after mixing, homogenizing, then carrying out low-temperature cooling aging for about 1h at 4 ℃, freezing and stirring the mixed material, and packaging after the mixed material is carried out the nutrition mould, and the nutrition mould is obtained after the fermentation. The obtained ice cream has light sweet fragrance and flower and fruit fragrance, and soft taste while relieving summer heat, and can reduce the irritation and sweet feeling of ice cream.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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.

Claims (15)

1. Saccharomyces cerevisiae (Saccharomyces cerevisiae) AR28 was deposited at China center for type culture Collection, accession number CCTCC NO: m20231848, the preservation address is Wuhan, university of Wuhan, china.

2. A microbial preparation comprising the Saccharomyces cerevisiae AR28 or a fermentation broth thereof, or immobilized cells thereof, or lyophilized powder thereof, or an extract thereof according to claim 1; or living cells containing Saccharomyces cerevisiae AR28 with other microorganisms; or contains Saccharomyces cerevisiae AR28 and other microorganisms, and freeze-drying to obtain dry thallus.

3. The microbial preparation according to claim 2, wherein the number of cells of Saccharomyces cerevisiae AR28 in the microbial preparation is not less than 1X 10 ⁷ CFU/mL and the germination rate is not less than 30%.

4. A composition comprising saccharomyces cerevisiae AR28 according to claim 1.

5. The composition of claim 4, wherein the composition includes, but is not limited to, microbial agents, wines or wheat starter.

6. Use of Saccharomyces cerevisiae AR28 of claim 1, or the microbial preparation of claim 2 or 3, or the composition of claim 4 or 5, in the manufacture of a fermented product.

7. The use according to claim 6, wherein the fermented product is a fermented food or a fermented beverage.

8. The use according to claim 7, wherein the fermented beverage comprises, but is not limited to, fermented alcoholic beverage, fermented vinegar.

9. The use according to claim 8, wherein the fermented alcoholic beverage includes, but is not limited to: yellow wine, cooking wine, rice wine, sweet rice wine, grape wine, fruit wine, beer or white wine.

10. The use according to claim 7, wherein the fermented vinegar comprises fruit vinegar or table vinegar.

11. The use according to claim 6 or 7, wherein the fermented product is also a fermented tobacco leaf or a fermented ice cream.

12. A product obtained by distillation or blending of a fermented product prepared by fermenting a microbial preparation according to claim 1, or a microbial preparation according to claim 2 or 3, or a composition according to claim 4 or 5.

13. A method for producing a fermented drink, characterized by comprising adding the Saccharomyces cerevisiae AR28 according to claim 1, the microbial preparation according to claim 2 or 3, or the composition according to claim 4 or 5 to a brewing material and brewing.

14. The method of claim 13, wherein the fermented beverage includes, but is not limited to, fermented alcoholic beverage or fermented vinegar; preferably, the fermented alcoholic beverage includes, but is not limited to: yellow wine, cooking wine, rice wine, sweet rice wine, fruit wine, beer or white wine, preferably, the fermented vinegar comprises fruit vinegar or table vinegar.

15. The method of claim 14, wherein the fruit wine includes, but is not limited to, any one of green plum wine, red bayberry wine, kiwi fruit wine, plum wine, hawthorn wine, pomegranate wine, lemon wine, loquat wine.