CN111548945A

CN111548945A - Space-mutagenesis saccharomyces cerevisiae ST26-4 and application thereof in brewing beer

Info

Publication number: CN111548945A
Application number: CN202010438569.1A
Authority: CN
Inventors: 郝红炜; 刘慧�; 袁明; 张红星; 谢远红
Original assignee: Fuledun Bioengineering Technology Beijing Co ltd
Current assignee: Fuledun Bioengineering Technology Beijing Co ltd
Priority date: 2020-05-20
Filing date: 2020-05-20
Publication date: 2020-08-18

Abstract

The invention discloses space mutagenesis saccharomyces cerevisiae ST26-4 and application thereof in brewing beer. The preservation number of the space mutation saccharomyces cerevisiae ST26-4 disclosed by the invention in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 19247. The invention screens out a yeast with excellent fermentation performance, namely space mutagenesis saccharomyces cerevisiae ST26-4, which is suitable for brewing beer after space mutagenesis is carried out on high-quality saccharomyces cerevisiae for brewing wine.

Description

Space-mutagenesis saccharomyces cerevisiae ST26-4 and application thereof in brewing beer

Technical Field

The invention relates to the field of biotechnology, and discloses space mutagenesis saccharomyces cerevisiae ST26-4 and application thereof in brewing beer.

Background

The Saccharomyces cerevisiae (Saccharomyces cerevisiae) is a unicellular eukaryotic microorganism which is mainly used for budding propagation, the cell shape is oval, long oval (elliptical), the size of a bacterial colony on a YEPD culture medium plate is 4-5 mm, the surface is convex, smooth and moist, round, neat in edge, sticky in texture, opaque and milky in color. Fermenting in wort liquid culture medium to generate bubbles and foam on the surface, suspending thallus in the culture medium to be turbid, depositing thallus cells at the bottom of the container in the later stage of fermentation, and clarifying the fermentation liquid. The excellent saccharomyces cerevisiae can ferment grape juice at 25-30 ℃ to generate good fruit fragrance and wine fragrance. The optimum pH value for fermentation is 4.0-4.5, the fermentation capacity is high, sugar can be completely fermented, the capacity of resisting high-concentration ethanol and sulfur dioxide (preservative normally added into grape juice) is high, and the coagulation capacity and the rapid sedimentation capacity are high.

Space microorganisms are subjected to mutagenesis effects of space microgravity effect, high vacuum, extreme temperature difference, weak magnetic field, high-energy particle (electron, proton, heavy ion) radiation and the like, so that mutation frequency can be obviously improved, gene mutation is generated, and biological properties (such as individual morphology, colony characteristics, physiological and biochemical characteristics, immunogenicity and the like) and fermentation production performance (such as biomass, product quantity, enzyme activity, titer, fermentation speed and the like) of the space microorganisms are changed. At present, the study of scholars at home and abroad on space microorganisms mainly focuses on space pathogenic bacteria, space corrosion bacteria and microbial pharmacy, and the study of scholars at home on space mutagenesis brewer's yeast brewed beer is not reported. At present, the following fermentation yeast, namely grape juice yeast (S.uvaram), is adopted in China to brew beer, the traditional fermentation period is long, the post-fermentation diacetyl reduction is slow, and the wine age is long, so that the economic benefit of beer enterprises is influenced. The diacetyl content is reduced, the fermentation period is shortened, the production cost of the brewing industry can be reduced, and the method is one of the important concerns of the beer production industry.

Disclosure of Invention

The invention aims to provide a saccharomyces cerevisiae for brewing beer.

The Saccharomyces cerevisiae provided by the invention is obtained by space mutagenesis of ground Saccharomyces cerevisiae GT26, the name of the space mutagenesis Saccharomyces cerevisiae is space mutagenesis Saccharomyces cerevisiae F-H-ST26-4, and the preservation number of the strain in China general microbiological culture Collection center is CGMCC No. 19247.

The invention also provides a microbial inoculum, and the active component of the microbial inoculum is the space mutagenesis Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST 26-4.

The microbial inoculum can be used for brewing beer.

The microbial inoculum may also include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of grass peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the biological material is at least one of straws, pine shells, rice straws, peanut shells, corn flour, bean flour, starch and turf of various crops; the liquid carrier may be water or a medium useful for culturing saccharomyces cerevisiae. The preparation formulation of the microbial inoculum can be various preparation formulations, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules.

According to the requirement, the microbial inoculum can also be added with a surfactant (such as Tween 20, Tween 80 and the like), a binder, a stabilizer (such as an antioxidant), a pH regulator and the like.

The present invention also provides a method of brewing beer, the method comprising: the space mutagenesis Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST26-4 is utilized for fermentation to obtain the beer.

In the above method, beer is obtained by fermenting the space-mutagenized Saccharomyces cerevisiae F-H-ST26-4 in wort.

In the method, the sugar degree of the wort may be 10 to 12 ° Bx. In one embodiment of the invention, the wort has a brix of 10 ° Bx.

In the above method, the space-mutagenized Saccharomyces cerevisiae F-H-ST26-4 may be inoculated into the wort in an amount of 6 × 10⁶～9×10⁶CFU/mL。

In the above method, the fermentation using the space mutagenesis Saccharomyces cerevisiae F-H-ST26-4 may include the following 1) and 2):

1) fermenting the space mutagenesis Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST26-4 in wort at the temperature of 12-17 ℃ to obtain primary fermentation liquor;

2) and continuously fermenting the primary fermentation liquor at 4-5 ℃ to obtain the beer.

In the above method, the temperature of the fermentation in step 1) may be 15 ℃.

The fermentation time in the step 1) can be 6-8 days.

Specifically, the fermentation time in step 1) may be 7 days.

The fermentation in step 1) can be carried out with stirring 3 days before fermentation. The rotating speed of fermentation in the step 1) before 3 days of fermentation can be 150-180 r/min. Oxygen can be introduced for 6 times a day before 3 days of fermentation in the step 1).

The fermentation in step 1) may be carried out at rest for 4 days.

The fermentation time in the step 2) can be 9-7 days, such as 8 days.

And (3) reducing the temperature in the fermentation in the step 2) by 2 ℃ to 4-5 ℃ every day, and fermenting for 2-3 days at the temperature of 4-5 ℃ to obtain beer fermentation liquor, thereby obtaining the beer.

The fermentation of the above process may be carried out in a fermentor.

In the above method, the ratio of the volume of wort to the volume of the fermenter can be 2.0 to 3.0L: 5L.

The application of the space mutagenesis Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST26-4 in brewing beer also belongs to the protection scope of the invention.

The application of the microbial inoculum in brewing beer also belongs to the protection scope of the invention.

The strain which can be used for beer fermentation is screened from space-mutagenized wine brewing yeast, namely space-mutagenized wine brewing yeast (Saccharomyces cerevisiae) F-H-ST26-4, and the strain not only has high low-temperature fermentation capacity at 12-17 ℃, but also can avoid the phenomenon of yeast autolysis death caused by high-temperature fermentation. The strain has strong fermentation capacity, is the following beer fermentation yeast, can ferment all raffinose, is suspended in fermentation liquor in beer brewing, is easy to coagulate and precipitate when the fermentation is nearly finished, is quickly coagulated after the fermentation is finished, has compact precipitate and is quickly clarified, and can accelerate the filtration speed of the fermentation liquor after yeast mud deposited at the bottom of a container is collected, shorten the beer clarification time and easily obtain clarified beer with stable biological property; the beer fermentation yeast can ferment 1/3 raffinose (the raffinose of unit mass is only one third of the raffinose), yeast floats in a foam layer on the fermentation liquid surface in beer brewing, the yeast floats on the liquid surface after coagulation when the fermentation is nearly finished, a yeast bubble cover is formed on the liquid surface after the fermentation is finished, the fermentation liquid is clarified slowly, the fermentation liquid filtering speed is relatively slow, and the beer clarification time is relatively prolonged. The beer brewed by the F-H-ST26-4 strain has low diacetyl content, gives pleasant flower and fruit fragrance to the beer, has active fragrance, and has excellent taste, flavor and texture of the beer body, thereby having important significance for improving the quality of the beer body, shortening the wine storage time and improving the economic benefit of beer enterprises. Therefore, the strain has wide application prospect.

Biological material preservation instructions

Classification nomenclature of biological materials: saccharomyces cerevisiae (Saccharomyces cerevisiae)

Strain number of biological material: F-H-ST26-4

Deposit name of biological material: china general microbiological culture Collection center

The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)

Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101

Preservation date of biological material: 12 month and 27 days 2019

Accession number to the collection of biological materials: CGMCC No.19247

Drawings

FIG. 1 shows the results of colony morphology of space-mutagenized s.cerevisiae ST26 (left plate) and ground s.cerevisiae GT26 (right plate).

FIG. 2 shows the cell morphology and the results of one-end budding propagation of the space-mutagenized s.cerevisiae ST26 (left panel, ammonium oxalate crystal violet staining) and the space-mutagenized s.cerevisiae ST26-4 strain (right panel, normal saline dipping).

FIG. 3 shows the result of TTC color development of the space-mutagenized Saccharomyces cerevisiae ST26 colony.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, each set up three replicates, were analyzed using Excel2007 and SPSS 18 software, and the results are expressed as mean ± standard deviation. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

YEPD liquid medium: adding 10g of FM828 Angel yeast extract powder, 20g of peptone, 20g of D (+) -maltose and 1000mL of distilled water, adjusting the pH value to 6.2, and sterilizing at 115 ℃ for 20min for later use.

YEPD solid medium: to YEPD liquid medium (1000mL) was added 17g of the resulting medium of agar powder to prepare YEPD agar plates.

FM828 Angel yeast extract powder: angel Yeast, Inc.

Peptone: beijing Oobo Star Biotechnology, Inc.

D- (+) -maltose: beijing Biotechnology Ltd.

Wort medium: 11Brix wort, sterilized at 115 deg.C for 20 min.

Wort solid medium: 1000mL of 11Brix wort and 17g of agar are sterilized at 115 ℃ for 20min for later use.

Wort: 11Brix (11 ° Bx) wort: beijing five-star brewery.

The Brix unit Brix, expressed by the symbol ° Bx; at 20 ℃ 1 ℃ Bx is 1g maltose in 100g wort, in mass percent concentration (w/w).

TTC medium:

upper-layer TTC culture medium: 0.05g of TTC (color development agent TTC is 2,3, 5-triphenyltetrazolium chloride, red tetrazolium for short), 0.5g of glucose, 1.5g of agar and deionized water to make up to 100mL, and boiling in boiling water for later use;

② lower YEPD culture medium: FM828 Angel yeast extract powder 10g, glucose 20g, peptone 20g, agar 17g, distilled water make up to 1000mL, adjust pH6.2, and sterilize at 115 deg.C for 20 min.

Du's tube fermentation medium: sucking 5mL of 11 DEG Bx wort by using a pipette, filling the Duchen tubule with the wort until the liquid is about to flow out, inverting the Duchen tubule filled with the wort into the test tube along the wall, taking the fact that no bubble exists in the Duchen tubule, injecting the residual wort in the pipette into the test tube along the wall, sucking 5mL of 11 DEG Bx wort again, and sterilizing the wort for 20min at 115 ℃ by high-pressure steam for later use.

Sterile physiological saline: 8.5g of sodium chloride and 1000mL of distilled water are supplemented, and the mixture is sterilized at 121 ℃ for 15 min.

Sterile tween-saline: 8.5g of sodium chloride, 801 mL of tween-801 and 1000mL of distilled water, and sterilizing at 121 ℃ for 15 min.

Example 1 screening of strains with superior fermentation Performance of space-mutagenized Saccharomyces cerevisiae

1. Bacterial strains

Space-mutagenized Saccharomyces cerevisiae (Saccharomyces cerevisiae) ST 26: space mutation strains returned by ground Saccharomyces cerevisiae GT26 carried by spacecraft of Tiangong No. two and Shenzhou No. eleven are stored in aerospace microorganism strain bank of Fule Tokyo Biotechnology (Beijing) Limited company. In order to distinguish the strain differences before and after space loading, the space mutant strain after space loading of ground Saccharomyces cerevisiae (Saccharomyces cerevisiae) GT26 was labeled as space mutant Saccharomyces cerevisiae (Saccharomyces cerevisiae) ST 26.

2. Separation and purification of space mutation saccharomyces cerevisiae

Placing a ground saccharomyces cerevisiae GT26 glycerol bacteria-protecting tube frozen at-80 ℃ and a space mutagenesis saccharomyces cerevisiae ST26 glycerol bacteria-protecting tube into a 4 ℃ slow freezing chamber, respectively inoculating into 10mL of sterilized malt extract culture medium by the inoculation amount of 2.0 percent by volume in a sterile workbench, culturing for 18h at 28 ℃, after three generations of activation, respectively absorbing 1mL of the culture solution after GT26 and ST26 activation by using a 1mL liquid-transferring gun, transferring into a sterile homogenizing bag containing 99mL of sterile Tween-physiological saline, uniformly beating by using a beating type sterile homogenizer (model SCIENTZ-II, Ningbo New Ganoderma Biotech Co., Ltd.) with 6T/Sec 20min, absorbing 1mL of bacterial solution in 9mL of sterile physiological saline by using the liquid-transferring gun, and diluting to 10 ℃ in a gradient manner^-5、10^-6、10^-7、10^-8The diluted bacterial liquid in a test tube is uniformly oscillated by a vortex oscillator (model QL-901, Ribel company of Haimen city), 100 mu L of the bacterial liquid with 4 dilutions is respectively absorbed in a YEPD agar plate, the plate is coated by a coating rod until the bacterial liquid is completely and uniformly dried, three dilutions are repeated in parallel, the plate is placed in a full-temperature double-layer oscillation incubator (model HZQ-F100, Jiangsu Taicang) for static culture for 72h (screening low-temperature resistant fermentation strains), after the single bacterial strain grows out, the characteristics of the bacterial colony are observed and described, meanwhile, in an aseptic ultra-clean workbench, the single bacterial colony is picked up on a glass slide by an inoculating ring smear in a small amount of physiological saline, the bacterial colony is uniformly mixed, dried and thermally fixed, the single staining is carried out by ammonium oxalate crystal violet staining solution, and the morphology and the budding propagation mode of the yeast are observed under a microscope. Selecting several single colonies of original ground Saccharomyces cerevisiae GT26 and space mutation Saccharomyces cerevisiae ST26, streaking and inoculating in YEPD agar slant test tube, culturing at 28 deg.C for 48 hr, and cold preserving at 4 deg.C for use.

As shown in figure 1, the colony of the original ground saccharomyces cerevisiae GT26 cultured on a YEPD agar plate at 15 ℃ is round, 4-6 mm in size, flat in surface edge, slightly blunted and round in the center, smooth and moist, neat in edge, sticky in texture, opaque, milky in color and typical in wine flavor, the cell morphology of the cell is elliptical, oval and nearly spherical, and one end of the cell buds and grows; the colony of space-mutagenized saccharomyces cerevisiae ST26 cultured on a YEPD agar plate at 15 ℃ is round, 4-6 mm in size, flat in surface edge, slightly round in center and slightly convex in cone shape, smooth and moist, neat in edge, sticky in texture, opaque, milky in color, typical in wine flavor, elliptical, oval and nearly spherical in cell morphology, and budding and breeding at one end (shown in figure 2).

3. Bacterial strain preliminary screening with excellent fermentation performance

3.1 Triphenyltetrazolium chloride (TTC) color test

TTC (2,3, 5-triphenyltetrazolium chloride) is a color developing agent, is colorless in an oxidation state and red or purple in a reduction state, is applied to screening of ethanol-producing yeast, can generate a color reaction on a metabolite of the yeast, and judges the magnitude of the respiratory enzyme activity of the yeast, namely the alcohol-producing capacity of the yeast through color shade observation. On a lower YEPD culture medium plate inoculated and cultured with space mutagenesis saccharomyces cerevisiae ST26, selecting a plate with 10-100 bacterial colonies, pouring the plate into about 12mL of upper TTC culture medium cooled to 45 ℃, covering the original bacterial colonies, culturing for 2-3 h at 28 ℃ in a dark place, judging the activity (alcohol production capacity) of the saccharomyces cerevisiae according to the color of the bacterial colonies, wherein the purplish red color reflects that the stronger the life activity and the higher the alcohol production capacity of the saccharomyces cerevisiae. And selecting a single colony which is dark purple red in color and large in colony size by taking the individual morphology and colony characteristics of the ground saccharomyces cerevisiae GT26 as a control. And inoculating the single colony of the space mutation saccharomyces cerevisiae ST26 dark purple selected for the first time onto a lower YEPD culture medium plate divided into cells by using a sterilized toothpick, covering an upper TTC culture medium after the colony grows out, and repeating the operation. As shown in FIG. 3, 110 spatially mutagenized Saccharomyces cerevisiae ST26 strains having a strong alcohol-producing ability were selected from the dark purple colonies by comparing the shades of the respective colonies in the TTC color test and labeled as ST26-1 to ST 26-110.

3.2 spatial mutagenesis Saccharomyces cerevisiae Biomass determination

Inoculating the 110 selected from the 110 strains ST26-1 to ST26-110 with strong alcohol production ability in the step 3.1 into test tubes containing 10mL YEPD liquid culture medium according to the inoculation amount of 2%, respectively, culturing at 28 deg.C for 18h with ground Saccharomyces cerevisiae GT26 as reference, and measuring the culture solution at 560nmOD, three determinations per sample were averaged to record turbidity. Comparison of culture OD of each strain_560nmValue size, 45 excellent strains with higher biomass than ground saccharomyces cerevisiae were screened from 110 ST26 strains. Among them, as shown in Table 1, OD of the strain numbered F-H-ST26-4 (i.e., space-time-variant Saccharomyces cerevisiae F-H-ST26-4, hereinafter abbreviated as ST26-4)_560nmThe value is 2.829, which is significantly higher than the ground Saccharomyces cerevisiae GT26 strain (OD)_560nmValue 2.816), indicating that the biomass and growth rate of the strain are significantly higher than those of ground saccharomyces cerevisiae GT26, and showing that the related genes of the strain are positively mutated under the space condition.

TABLE 1 results of biomass measurement of space-mutagenized Saccharomyces cerevisiae ST26 strain

3.3 space mutagenesis Saccharomyces cerevisiae Du's tubule gas production test

And (3) respectively inoculating the 110 selected strains ST26-1 to ST26-110 with strong alcohol production capacity in the step 3.1 into test tubes with Du's tubules and containing 10mL of wort culture medium according to the inoculation amount of 2 percent, placing the test tubes at 15 ℃ for culturing for 48h by taking the ground Saccharomyces cerevisiae GT26 as a control, recording the gas production rate and the liquid level foam gas production height of the strain for culturing for 24h and 48h, and inspecting the fermentation performance of the strain. According to the gas production rate (high and low fermentation speed) and the foam height (foam retentivity of beer), 20 excellent strains with the gas production speed higher than that of ground saccharomyces cerevisiae are screened from 110 ST26 strains, the method comprises the step 3.2 of screening the ST26-4 strains with higher biomass, the gas production rate and the foam height of the strains are obviously higher than that of the ground saccharomyces cerevisiae GT26 (see table 2), namely the gas production rate and the foam height of the strains are higher than those of the ground saccharomyces cerevisiae GT26 strains, and the results show that the space mutagenesis saccharomyces cerevisiae has high gas production speed (strong fermentation starting capability), high growth and propagation speed and strong cohesiveness (the bottom of a Ducheng tubule is provided with bacterial sludge sediment with the size of a thumb cover, and is in a yeast fermentation finished state), show that related genes of the strains have positive mutation under the space condition, and have good beer fermentation capability.

TABLE 2 results of the gas production rate and foam height of the small Du's pipe of the space-mutagenized Saccharomyces cerevisiae ST26 strain

Note: in Table 2, "+" indicates 1/4 with capacity of Duchen, "++" indicates 2/4 with capacity of Duchen, "+++" indicates 3/4 with capacity of Duchen, and "++++" indicates that the capacity of Duchen fills the entire Duchen; "P" means the amount of foam < 2cm, "PP" means the amount of foam 2-4cm, "PPP" means the amount of foam 4-6cm, and "PPPP" means the amount of foam > 6 cm.

4. Rescreening of space-mutagenized saccharomyces cerevisiae

4.1 spatial mutagenesis Saccharomyces cerevisiae genetic stability test

And (3) after the preliminarily screened strains with high gas production speed, high activity, strong alcohol production capacity and high biomass are subcultured for 50 generations in 10mL of wort culture medium, carrying out biomass measurement, a Du's tubule gas production test and a fermentation capacity test according to the steps 3.2 and 3.3, inspecting the genetic stability of the strains, screening out the strains with stable heredity, and carrying out a beer fermentation test.

Measurement of fermentation force: inoculating the preliminarily screened strain with excellent fermentation performance into 10mL YEPD culture medium with an inoculation amount of 2% by adopting a microorganism turbidimetric analyzer (model WBS-100, Beijing pioneer Weifeng technology development company), culturing at 28 ℃ for 48h, and measuring and recording OD of each ST26 strain culture on line_600nmValue and cultivation time as abscissa, OD_600nmThe values are plotted on the ordinate, and a growth curve is automatically drawn on which the time to lag phase (fermentation time) and the time to end of logarithmic growth phase are found. The time of the slow phase can indirectly reflect the fermentation starting time of the strains, the logarithmic growth phase is the most vigorous growth and reproduction phase of the strains, the shorter the time of the slow phase is, the faster the strains can reach the logarithmic growth phase, and the stronger the fermentation capacity is.

As shown in Table 3, the biomass, the gas production speed, the foam height and the fermentation capacity of the space-mutagenized Saccharomyces cerevisiae ST26-4 strain are all higher than those of the ground Saccharomyces cerevisiae GT26 strain after 50 generations of the strain, which indicates that the strain subjected to space mutagenesis has a shorter lag phase, can quickly enter a logarithmic growth phase, accelerates the fermentation speed and has good genetic stability, and the strain is further used for beer fermentation tests.

Results of biomass, gas production speed, foam height and fermentation capacity of space mutation saccharomyces cerevisiae ST26 strain with passage of 350 generations

Note: in Table 3, "+" indicates 1/4 with capacity of Duchen, "++" indicates 2/4 with capacity of Duchen, "+++" indicates 3/4 with capacity of Duchen, and "++++" indicates that the capacity of Duchen fills the entire Duchen; "P" means the amount of foam < 2cm, "PP" means the amount of foam 2-4cm, "PPP" means the amount of foam 4-6cm, and "PPPP" means the amount of foam > 6 cm.

4.2 determination of inoculum size and cultivation time of space mutagenesis Saccharomyces cerevisiae

And (3) inoculating the screened space mutagenesis saccharomyces cerevisiae ST26-4 strain with better genetic stability into a YEPD liquid culture medium according to 2%, culturing for 18h at 28 ℃, after continuous three-generation activation, respectively inoculating the space mutagenesis saccharomyces cerevisiae ST26-4 strain into 10mL of YEPD liquid culture medium according to 2%, 3%, 4%, 5% and 10%, culturing for 24h at 28 ℃, measuring and recording OD values (optical density value/turbidity) of bacterial suspensions cultured for 10h, 12h, 14h, 16h, 18h, 20h and 24h under the wavelength of 560nm, and measuring and averaging for three times.

As can be seen from Table 4, the ST26-4 strain was cultured for 24h at inoculum sizes of 2%, 3%, 4%, 5%, and 10%, the cell concentration was proportional to the optical density value, the OD value showed a tendency of increasing first and then decreasing with the increase of the culture time, and the result showed that the turning point (end stage) of the logarithmic growth phase could be reached at 16h, i.e., the maximum OD value was 2.043; and when the inoculation amount is 3 percent, the turning point of the logarithmic growth phase can be reached as fast as 16 hours, the maximum OD value is 2.044, the difference with the maximum OD value of 2 percent is not large, and the inoculation amount is finally determined to be 2 percent in consideration of the cost of later-stage industrial production. And (4) conclusion: the optimal inoculation amount of the space mutagenesis saccharomyces cerevisiae ST26-4 strain is determined to be 2%, and the culture time is 16 h.

TABLE 4 results of biomass (OD value) of optimum inoculum size and cultivation time of space-mutagenized Saccharomyces cerevisiae ST26-4 strain

4.3 space mutagenesis Saccharomyces cerevisiae fermentation wort sugar reduction test

Inoculating the selected space-mutagenized Saccharomyces cerevisiae ST26-4 strain glycerol seed-preserving tube with good genetic stability into 10mL of sterile wort [ Huarun snow beer (China) Co., Ltd.) according to the inoculation amount of 2%]After culturing at 28 ℃ for 16h and activating two generations, the number of viable bacteria in the culture solution of the ST26-4 strain reaches 3.0 × 10⁸CFU/mL, again by 2% (6.0 × 10)⁶CFU/mL) inoculum size is inoculated into a 500mL triangular flask (marked as fermentation 0d) containing 200mL sterile wort, the flask is placed in a full-temperature double-layer shaking incubator for fermentation for 7d at 15 ℃, wherein the first 3d is subjected to shake flask fermentation for 3d at 180r/min, the second 4d is subjected to standing fermentation, the wort sugar degree of the fermentation liquor fermented for 0d and the sugar degrees of the fermentation liquors fermented for 3d, 5d and 7d are respectively measured, the average value is obtained by measuring four times, and the fermentation speed of the space mutagenesis yeast strain brewing is detected according to the sugar reduction result on the 7 th day. Ground Saccharomyces cerevisiae GT26 and Saccharomyces cerevisiae ST28-61 in Chinese patent application 201810811754.3 (published 2018-11-30) were used as controls. Wherein, the steps of measuring the sugar degree of the wort by using the handheld refractometer are as follows: (1) and correcting the zero point. A plurality of drops of distilled water are taken and placed on the detection prism, the zero adjusting bolt is screwed, the boundary is adjusted to the position of 0% of the scale, and then the detection prism is carefully wiped off by using soft cloth. (2) And (4) sample determination. Opening the cover plate of the refractometer, taking a plurality of drops of the fermented wort to be detected at 20 ℃, placing the drops on the detection prism, slightly closing the cover plate to avoid bubbles, and enabling the solution to be distributed on the surface of the prism. The light inlet plate of the instrument is aligned to a light source or a bright place, eyes observe a view field through an ocular lens, and an ocular lens adjusting hand wheel is rotated to enable a blue-white boundary of the view field to be clear. The scale value of the dividing line is the sugar degree (. degree.Bx) of the wort.

The blood sugar reduction speed and the fermentation speed in the wort fermentation process of the yeast are positively correlated. As can be seen from Table 5, the sugar content of the wort gradually decreased with the increase of the fermentation time, the sugar content of the wort was decreased faster in the 0-5 d high-bubble period, the ST26 strain decreased at the sugar content of 1.0-1.5 Bx every day, and the sugar content of the fermented wort by the 7 th day was in the order of: ST26-4 < GT26 < ST 28-61. Compared with the ground GT26 strain, the blood sugar reducing index of the ST26-4 strain is 1.30 times that of the GT26 strain; compared with the strain ST28-61 of saccharomyces cerevisiae, the glucose reduction index of the strain ST26-4 is 1.43 times of that of the strain ST 28-61. Therefore, the space mutation saccharomyces cerevisiae ST26-4 is an excellent strain with higher sugar-reducing speed and fermentation speed for fermenting wort.

TABLE 5 results of determination of the sugar degree (. degree.Bx) of wort at different fermentation times for ST26-4 and ST28-61 strains

4.4 space mutagenesis Saccharomyces cerevisiae 5L fermentation tank beer fermentation test

Inoculating the screened space mutagenesis saccharomyces cerevisiae ST26-4 glycerol seed preservation tube with better genetic stability into 10mL of sterile 11 DEG Bx wort according to the inoculation amount of 2%, culturing for 16h at 28 ℃ for two generations, then inoculating into 100mL of sterile 11 DEG Bx wort according to the inoculation amount of 2%, performing shake culture at 28 ℃ for 180r/min for 16h to obtain the viable count of 3.0 × 10⁸CFU/mL yeast (yeast seed solution) by 3% (9.0 × 10)⁶CFU/mL) was inoculated into a container containing 2.5L of sterile wort at 10 ° Bx [10 ° Bx wort: huarun snow beer (China) Co., Ltd]5L fermenter [ model: 5L/DM9000A, Demg Biotechnology (Shanghai) Ltd]Controlling the fermentation at 15 ℃ for 7 days, wherein oxygen is introduced for 6 times every day for the first 3 days, the stirring speed is 150-180 r/min, and standing for fermentation for the later 4 days to obtain primary fermentation liquor; and 7d, measuring the sugar degree of the wort in the primary fermentation liquid, sealing the tank, continuing fermenting the primary fermentation liquid for 8d, reducing the temperature of the fermentation liquid at 2 ℃ by using a fermentation tank cooling system every day, and finally reducing the temperature to 4-5 ℃, and maintaining the fermentation for 3d at the temperature to obtain the beer fermentation liquid. Detecting pH, alcohol content and diacetyl content of beer fermentation broth at 15d, measuring for four times, averaging, simultaneously performing sensory evaluation, and detectingThe method refers to GB 4927-2008 and GB/T4928-2008 beer analysis methods. Ground Saccharomyces cerevisiae GT26 was used as a control.

(1) Alcohol content determination: the alcohol content was measured by referring to the bottle density method (first method) of the national standard GB/T4928-2008, and the result was calculated by the formula (1).

Wherein:

-relative density of sample alcohol distillate (20 ℃);

m₂-mass in grams (g) of density bottles and distillate;

m is the mass of the density bottle in grams (g);

m₁mass of density bottles and water in grams (g).

According to relative density

And examining GB/T4928-2008 appendix A to obtain the volume fraction of the alcohol content of the distillate of the sample, namely the alcoholic strength of the sample.

(2) And (3) measuring diacetyl: the diacetyl was determined by reference to the method of national standard GB/T4928-2008 and the results were calculated using equation (2).

X₅＝A₃₃₅× 1.2.2.............. equation (2)

Wherein: x₅The diacetyl content of the sample in milligrams per liter (mg/L);

A₃₃₅-absorbance of the sample at wavelength 335nm is measured using a 20mm quartz cuvette;

1.2-conversion coefficient of absorbance to diacetyl content using a 20mm quartz cuvette.

(3) And (3) sugar degree measurement: the measurement is carried out by using a handheld refractometer in the same manner as in 4.3.

(4) And (3) pH measurement: measured by a PHS-3B pH meter.

(5) And (3) beer quality grading: 10 professional tasters are invited to conduct sensory scoring, the evaluation method refers to the national standard GB 4927-2008, and the sensory beer scoring standard is shown in Table 6. According to the requirements of the beer sensory evaluation standard in the table 6, the beer fermentation liquor fermented for 15d is subjected to comprehensive sensory evaluation in four aspects of color, foam, taste and aroma.

(6) Detection of floral and fruity compounds: using FlavourSpec

Analysis of beer flavor and substance composition by gas chromatography ion mobility spectrometer (model: FlavourSpec, Germany G.A.S. company). ① test group sample No. ST26-4 beer fermentation liquid fermented by space mutagenesis saccharomyces cerevisiae ST26-4 strain, control group sample No. GT26 beer fermentation liquid fermented by ground original saccharomyces cerevisiae GT26 for 15d, ② sample treatment, wherein 1mL of sample is put in 20mL of headspace bottle to be analyzed, and ③ gas-ion mobility spectrometry unit analysis condition is that analysis time is 30min, chromatographic column type FS-SE-54-CB-115 m ID is 0.53mm, column temperature is 60 ℃, and carrier gas/drift gas N is₂The automatic headspace sample injection unit comprises IMS (ion exchange resin) temperature of 45 ℃ and ④, analysis conditions of an incubation temperature of 40 ℃, an incubation time of 15min, a sample injection volume of 500 muL, a sample injection needle temperature of 85 ℃, an incubation rotation speed of 500r/min, ④ analysis software, and analysis software matched with the apparatus, wherein the analysis software comprises LAV (laboratory Analytical viewer), three plug-ins and GC × IMSLibrary Search, and can be used for analyzing samples from different angles respectively, measuring ⑤ for three times, averaging and performing data processing and analysis.

The analysis software includes (i) an LAV: used for checking and analyzing a spectrogram, wherein each point in the spectrogram represents a volatile organic compound; after a standard curve is established, quantitative analysis can be carried out; secondly, a Reporter plug-in: directly comparing the spectral differences between samples (two-dimensional top view and three-dimensional spectra); ③ Gallery plug-in: comparing the fingerprints, and visually and quantitatively comparing the difference of the volatile organic compounds among different samples; dynamic PCA plug-in: the dynamic principal component analysis is used for clustering and analyzing the samples and quickly determining the types of unknown samples; GC × IMS Library Search: the NIST database and the IMS database built in the application software can perform qualitative analysis on the substances.

TABLE 6 beer sensory Scoring criteria

TABLE 7 physicochemical index results of beer fermentation broth in ST 26-45L fermenter

TABLE 8 physical and chemical requirements of light beer (beer GB 4927-2008 national standard)

TABLE 9 organoleptic evaluation of ST 26-45L fermenter beer fermentation broths

A plurality of physicochemical indexes need to be examined for brewing beer, wherein when the threshold value of the diacetyl content exceeds 0.15mg/L, the quality of the beer is influenced, the flavor is deteriorated, and the foul smell is generated. As can be seen from tables 7 and 8, the diacetyl content in the beer fermentation broth of ST26-4 was significantly lower than that of GT26 beer fermentation broth (0.027 mg/L for ST26-4 beer fermentation broth, 0.190mg/L for GT26 beer fermentation broth); compared with a ground strain GT26, the diacetyl content in the beer fermentation liquor of ST26-4 is lower than the requirement (less than or equal to 0.15mg/L) of national standard GB 4927-2008 first-grade beer and is also lower than the requirement (less than or equal to 0.10mg/L) of national standard GB 4927-2008 top-grade beer; ST26-4 beer fermentation liquor with significant alcohol contentThe content of the GT26 beer fermentation liquid is 2.5 times of GT26 beer fermentation liquid (4.79% (v/v) for ST26-4 beer fermentation liquid and 1.95% (v/v) for GT26 beer fermentation liquid)]. As can be seen from Table 9, the beer fermentation broth ST26-4 has an organoleptic score of more than 95 points, golden yellow, clear and transparent color, no suspended matter, pure white foam, fine and smooth foam, good durability, hop aroma of beer, pure, clean, delicious and mellow taste, no rancid taste, pleasant floral and fruity aroma, active aroma and good taste, flavor and texture of the beer body. Using FlavourSpec

The gas chromatography ion mobility spectrometry technology is used for determining the composition of beer flavor compounds, and the result shows that the content of 8 ester compounds of ethyl propionate, isobutyl acetate, ethyl butyrate, ethyl isobutyrate, ethyl hexanoate, methyl hexanoate, butyl hexanoate and DL-2-hydroxy-4-methyl ethyl valerate in beer fermentation liquor of space mutation saccharomyces cerevisiae ST26-4 is obviously higher than that of GT26 beer fermentation liquor. Wherein butyl caproate has pineapple flavor and its content is 4.5 times of GT26 beer (ST26-4 beer fermentation liquid peak volume is 17573.68, GT26 beer fermentation liquid peak volume is 3876.19); DL-2-hydroxy-4-methylpentanoic acid ethyl ester has fresh blackberry smell, and the content of the substance is 3.4 times of that of GT26 beer (the peak volume of ST26-4 beer fermentation liquid is 255.86, and the peak volume of GT26 beer fermentation liquid is 74.67); ethyl caproate has a sweet fruit pineapple flavour and is 1.5 times as rich as GT26 beer (ST26-4 beer fermentation peak volume of 16942.46, GT26 beer fermentation peak volume of 11625.48); ethyl isobutyrate had a sweet fruity flavor with a content of 1.3 times that of GT26 beer (peak volumes of ST26-4 beer fermentation broth 1781.12, GT26 beer fermentation broth 1373.26); isobutyl acetate has a ripe fruit aroma, and its content is 1.2 times of GT26 beer (ST26-4 beer fermentation peak volume is 5732.56, GT26 beer fermentation peak volume is 4675.21); methyl caproate has pineapple ether taste, and its content is 1.8 times of GT26 beer (ST26-4 beer fermentation liquid peak volume is 352.62, GT26 beer fermentation liquid peak volume is 192.29); the ethyl butyrate has banana and pineapple smells, the content of the ethyl butyrate is 1.2 times of that of GT26 beer (the peak volume of ST26-4 beer fermentation liquid is 5086.39,GT26 beer fermentation broth peak volume 4406.67) these ester compounds can impart ST26-4 beer with sweet fruit, pineapple, banana, fresh blackberry and other sweet fruit notes (see table 10) furthermore, linalool and β -phenylethyl alcohol are also significantly higher in content than GT26 beer linalool has citrus and rose scent with 1.6 times the content of GT26 beer (ST26-4 beer fermentation broth peak volume 269.87, GT26 beer fermentation broth peak volume 171.23), β -phenylethyl alcohol has sweet and floral notes with rose-like honey scent with 1.3 times the content of GT26 beer with rose scent (ST26-4 beer fermentation broth peak volume 368.82, GT26 beer broth peak volume 280.83) these ester compounds can impart ST 6-4 beer citrus, rose and honey with rose scent with a substantial sweet scent such as ST 3510, ST26, ST 73727, ST26 shows a substantial sensory fruity notes superior to GT 9627 beer.

TABLE 10 odor of different flavor Compounds

And (4) conclusion: ST26-4 fermented wort has high sugar-reducing speed and fermentation speed, high alcohol content in beer fermentation liquor, high beer sensory score, pleasant flower and fruit fragrance, active fragrance, good taste, flavor and texture of the beer body, and diacetyl content in the beer fermentation liquor lower than the requirement (less than or equal to 0.10mg/L) of national standard GB 4927-2008 top grade beer, and can become a good strain for brewing beer.

4.5 Excellent Strain identification

The selected excellent strain ST26-4 is streaked and inoculated to a YEPD culture medium plate, cultured for 2d at 28 ℃, and the colony characteristics and the cell morphology and the budding propagation mode are observed by a physiological saline solution immersion method. Inoculating a single colony into a 10mLYEPD liquid culture medium, culturing at 28 ℃ for 16h, centrifuging at 4 ℃ at 7000r/min for 2min, removing supernatant, taking bacterial sludge, extracting DNA of yeast by using a fungal genome DNA rapid extraction kit (Beijing engine science and technology Co., Ltd.), performing PCR amplification by using fungal 18SrDNA universal primers, namely upstream ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and downstream ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), sending out a sequencing product to sequence [ engineering biology (Shanghai) engineering Co., Ltd ], performing BLAST homology sequence comparison analysis on a sequencing result in a NCBI database, and determining the genus position of the strain.

The sequence of the PCR product of the ST26-4 strain is the sequence 1 in the sequence table, and the identity with the saccharomyces cerevisiae is 99%. The cell shape of the strain is elliptical, oval and approximately spherical, one end of the strain buds and grows (shown in figure 2), a bacterial colony grown on a YEPD culture medium plate at 28 ℃ is circular, the size of the bacterial colony is 5-6 mm, the surface edge is flat, the center of the bacterial colony is slightly conical and convex, the bacterial colony is smooth and moist, the edge is neat, the texture is sticky, the bacterial colony is opaque, the color is milky white, and the bacterial colony has typical wine fragrance.

Through morphology and 18S rDNA sequencing analysis and identification, the ST26-4 strain can be determined to be Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the identification result is shown in Table 11.

TABLE 11 identification of superior strains screened by space mutagenesis of Saccharomyces cerevisiae ST26

Space-mutagenesis Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST26-4 has been deposited in China general microbiological culture Collection center (CGMCC) at 27 months in 2019 with the preservation number of CGMCC No. 19247. Space-mutagenized Saccharomyces cerevisiae F-H-ST26-4 was obtained at 2018 for 7 months.

<110> Fuleton biotechnology and technology (Beijing) Ltd

<120> a space-mutagenic Saccharomyces cerevisiae ST26-4 and its use for brewing beer

<160>1

<170>PatentIn version 3.5

<210>1

<211>1433

<212>DNA

<213> Saccharomyces cerevisiae

<400>1

caagggatac tattttgctt atggattttt ttgttttggc gagagcatga gagcttttac 60

tgggcaagaa gacaagagat ggaaagtcca gccgggcctg cgcttaaatg ctcggtcttg 120

ctaggcttgt aagtttcttt cttgctattc caaacggaga aagatttctg tgcttttgtt 180

ataggacagt taaaaccgta tcactacgac acactgagga gttttcatat ctttgcaact 240

ttttctttgg gcattcgagc aatcggggcc cagaggtaac aaacccaaac aattttatct 300

attcattaaa tttttgtcaa aaacaagaat tttcgtaact ggaaatttta aaatattaaa 360

aactttcaac aacggatctc ttggttctcg catctatgaa gaacgcagcg aaatgcgata 420

cgtaatgtga attgcggaat tccgtgaatc atcgaatctt tgaacgcaca ttgcgcccct 480

tggaattcca gggggcatgc ctggtttgag aatcatttcc ttctcaaaca ttctgtttgg 540

tagtgagtga tactctttgg agttaacttg aaattgctgg ccttttcatt ggatgttttt 600

tttccaaaga gaggtttctc tgcgtgcttg aggtataatg caagtacggg tcgttttagg 660

ttttaccaac tgcggctaat ctttttttta taactgagcg tattggaaac gttttcgact 720

ctacctgatt tgaggtcaga gtttaagaac tttgttctgc taaacgctct cttcttatct 780

ataacgttcc gatccgctca atataataac acattacccg catgtggtaa aacctaaatc 840

taccgtagtg gcctattaca tctcgcacac gcacaaaccc ctctttggaa gaaaacctac 900

caatgaaaag gccctgattt gagttaactc caatcaccgg aattccctac agcgcaaaat 960

gcttgaaaag aaactgagat tcactgaggt ctgccccccg gaatacctag gggcgctcgg 1020

tgcgttcttc aactctatga ttcccggaat tctgcaattc acattacgta tcgcatttca 1080

ctgcgtcctt cttcaataca aaaaccaaaa aatcctatgc gaattgtttt taatatttta 1140

aaatttccac ttgcgaaaat gcttgttttt gacaaactat taaggaaaac actaatgtgc 1200

cttcgtttgg tcccccttgg ggacggattg ctcgaatgcc caaagaaaaa gttgcaaaga 1260

tatgaaaact ccacagtgtg ttgtattgaa acggttttaa ttgtcctata acaaaagcac 1320

agaaatctct caccgtttgg aatagcaaga aagaaactta cagccttagc caagaccgcg 1380

cacttaagcg caggcccggc tggactctcc atctcttgtc ttcttgccca gta 1433

Claims

1. Saccharomyces cerevisiae F-H-ST26-4 with the preservation number of CGMCC No. 19247.

2. A microbial preparation comprising the Saccharomyces cerevisiae F-H-ST26-4 of claim 1 as an active ingredient.

3. The microbial inoculum of claim 2, wherein: the microbial inoculum is used for brewing beer.

4. A method of brewing beer comprising: a beer is obtained by fermenting Saccharomyces cerevisiae F-H-ST26-4 of claim 1.

5. The method of claim 4, wherein: beer is obtained by fermenting Saccharomyces cerevisiae F-H-ST26-4 according to claim 1 in wort.

6. The method of claim 5, wherein: the sugar degree of the wort is 10-12 degrees Bx.

7. The method according to claim 4 or 5, wherein the Saccharomyces cerevisiae F-H-ST26-4 is inoculated into the wort in an amount of 6 × 10⁶～9×10⁶CFU/mL。

8. The method according to claim 4 or 5, characterized in that: the fermentation includes the following 1) and 2):

1) fermenting the Saccharomyces cerevisiae F-H-ST26-4 in wort at 12-17 ℃ to obtain primary fermentation liquor;

9. Use of the Saccharomyces cerevisiae F-H-ST26-4 according to claim 1 for brewing beer.

10. Use of the microbial inoculum according to claim 2 for brewing beer.