CN111592950B

CN111592950B - Application of space mutagenesis saccharomyces cerevisiae ST26-22 in brewing beer

Info

Publication number: CN111592950B
Application number: CN202010499227.0A
Authority: CN
Inventors: 郝红炜; 刘慧�; 袁明; 张红星; 谢远红
Original assignee: Fullarton Bioengineering Technology Beijing Co ltd
Current assignee: Fullarton Bioengineering Technology Beijing Co ltd
Priority date: 2020-06-04
Filing date: 2020-06-04
Publication date: 2022-03-08
Anticipated expiration: 2040-06-04
Also published as: CN111592950A

Abstract

The invention discloses application of space mutagenesis saccharomyces cerevisiae ST26-22 in brewing beer. The preservation number of the space mutation saccharomyces cerevisiae ST26-22 disclosed by the invention in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 19246. The invention screens the saccharomycetes with excellent fermentation performance suitable for brewing beer, namely space mutagenesis saccharomyces cerevisiae ST26-22, after adopting high-quality saccharomyces cerevisiae for brewing the wine to carry out space mutagenesis.

Description

Application of space mutagenesis saccharomyces cerevisiae ST26-22 in brewing beer

Technical Field

The invention relates to the application of space mutagenesis saccharomyces cerevisiae ST26-22 in brewing beer in the field of biotechnology.

Background

Saccharomyces cerevisiae is a unicellular eukaryotic microorganism mainly based on budding propagation, and the somatic cells under a microscope are elliptical or oval, single-grown, one-end budding and facultative anaerobes. The colony on the YEPD medium plate is circular, the size is 4-6 mm, the surface is convex, smooth and moist, the edge is neat, the texture is sticky, the colony is opaque, the color is milky white, and the colony has typical wine fragrance. The yeast can perform aerobic respiration under aerobic conditions, generate carbon dioxide and water through an EMP way and release energy, and has the advantages of fast growth and metabolism and vigorous propagation. And can be fermented under the condition of no oxygen to produce alcohol. The saccharomyces cerevisiae for producing beer with excellent fermentation performance not only has fast propagation speed, can tolerate higher sugar degree and alcoholic strength and lower temperature and pH, but also has the performances of fast fermentation speed and low diacetyl production. The optimal growth temperature of the saccharomyces cerevisiae for brewing beer is 25-28 ℃, the fermentation temperature is 12-18 ℃, and the optimal growth pH4.5-5.5. Flocculation is the characteristic of yeast sedimentation in the beer fermentation process, and is beneficial to the clarification of beer and the post-treatment of beer.

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. The diacetyl has a very low taste threshold (less than or equal to 0.15mg/L) and when it exceeds 0.15mg/L, it produces an unpleasant rancid taste.

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, is named as space mutagenesis Saccharomyces cerevisiae F-H-ST26-22, and has the preservation number of CGMCC No.19246 in China general microbiological culture collection center.

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 26-22.

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-ST26-22 is utilized for fermentation to obtain the beer.

In the above method, beer is obtained by fermenting the space-mutagenized Saccharomyces cerevisiae F-H-ST26-22 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-ST26-22 may be inoculated in the wort in an amount of 6X 10⁶～9×10⁶CFU/mL。

In the above method, the fermentation using the space mutagenesis Saccharomyces cerevisiae F-H-ST26-22 may include the following 1) and 2):

1) fermenting the space mutagenesis Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST26-22 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-ST26-22 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 yeasts, namely space-mutagenized wine brewing yeast (Saccharomyces cerevisiae) F-H-ST26-22, 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-ST26-22 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-ST26-22

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.19246

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 ST26-22 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 ground Saccharomyces cerevisiae GT26 glycerol conservation tube frozen at-80 deg.C and space mutagenesis Saccharomyces cerevisiae ST26 glycerol conservation tube into 4 deg.C, slowly freezing, respectively inoculating into 10mL sterilized malt juice culture medium with inoculum size of 2.0 vol%, and culturing at 28 deg.CCulturing for 18h, activating for the third generation, respectively sucking 1mL of the culture solution after GT26 and ST26 activation by using 1mL of pipette, transferring into a sterile homogenizing bag containing 99mL of sterile Tween-physiological saline, beating uniformly by using a beating type sterile homogenizer (model SCIENTZ-II, Ningbo New Ganoderma Biotech Co., Ltd.) with 6T/Sec 20min, sucking 1mL of bacterial solution into 9mL of sterile physiological saline by using the pipette, and diluting to 10% in gradient^-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

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 containing 10mL of YEPD liquid culture medium according to the inoculation amount of 2%, taking the ground Saccharomyces cerevisiae GT26 as a reference, measuring the OD value of the culture solution at the wavelength of 560nm after culturing for 18h at 28 ℃, measuring three times of the average value of each sample, and recording the 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. Wherein, as shown in Table 1, the number is FOD of-H-ST 26-22 strain (i.e., space-time-mutagenized Saccharomyces cerevisiae F-H-ST26-22, hereinafter abbreviated as ST26-22)_560nmThe value is 2.823, 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 ST26-22 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 strains are shown to have high gas production rate (high fermentation starting capacity), high growth and reproduction speed and strong cohesiveness (bacterial sludge sediment with the size of a thumb cover at the bottom of a Duchen tubule is in a fermentation yeast ending state), show that related genes of the strains have positive mutation under the space condition and have good beer fermentation capacity.

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 ST26-22 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 ST26-22 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 ST26-22 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.

And (4) conclusion: the optimal inoculation amount of the space mutagenesis saccharomyces cerevisiae ST26-22 strain is determined to be 2%, and the culture time is 16 h.

4.3 space mutagenesis Saccharomyces cerevisiae fermentation wort sugar reduction test

Inoculating the selected space-mutagenized Saccharomyces cerevisiae ST26-22 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 ST26-22 strain reaches 3.0 multiplied by 10⁸CFU/mL, again by 2% (6.0X 10)⁶CFU/mL) was inoculated into a 500mL Erlenmeyer flask containing 200mL of sterile wort (denoted as fermentation 0d), and placed in a full-temperature double-layer shaking incubator for fermentation 7d at 15 deg.C, wherein the first 3d was 180 dAnd (3) r/min shaking flask fermentation for 3d, standing and fermenting for 4d, respectively measuring the sugar degree of the wort of the fermentation liquor fermented for 0d and the sugar degrees of the fermentation liquors fermented for 3d, 5d and 7d, measuring four times, taking an average value, and detecting the fermentation speed of the space mutation saccharomyces cerevisiae strain 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 4, 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: ST26-22 < GT26 < ST 28-61. Compared with the ground GT26 strain, the blood sugar reducing index of the ST26-22 strain is 1.27 times that of the GT26 strain; compared with the strain ST28-61 of saccharomyces cerevisiae, the glucose-reducing index of the strain ST26-22 is 1.40 times of that of the strain ST 28-61. Therefore, the space mutation saccharomyces cerevisiae ST26-22 is an excellent strain with higher sugar-reducing speed and fermentation speed of the fermented wort.

TABLE 4 determination of the sugar degree (. degree.Bx) of the wort at different fermentation times for the ST26-22 and ST28-61 strains

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

Inoculating the screened space mutagenesis saccharomyces cerevisiae ST26-22 glycerol seed-preserving pipe with better genetic stability into 10mL of sterile 11-degree Bx wort according to the inoculation amount of 2%, culturing for 16h at 28 ℃ for two generations, then inoculating into 100mL of sterile 11-degree Bx wort according to the inoculation amount of 2%, performing shake-flask culture at 28 ℃ for 180r/min for 16h to obtain the viable count of 3.0 multiplied by 10⁸CFU/mL of yeast (yeast seed liquid). Adding yeast 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. And (5) detecting the pH, the alcoholic strength and the diacetyl content of the beer fermentation liquor at 15d, measuring four times, taking an average value, and simultaneously carrying out sensory scoring, wherein the detection method refers to a beer analysis method of GB 4927-2008 and GB/T4928-2008. 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＝A₃₃₅X 1.2 … … … … formula (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 5. According to the requirements of the beer sensory evaluation standard in the table 5, 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: by using

Analysis of beer flavor composition by gas chromatography ion mobility spectrometry (model: FlavourSpec, G.A.S. Germany). Test group samples: numbered ST26-22, spatial mutagenesis of beer fermentation liquor fermented for 15d by saccharomyces cerevisiae ST26-22 strain; control group samples: no. GT26, ground original Saccharomyces cerevisiae GT26 fermented 15d beer fermentation broth. Processing a sample: sample 1mL was placed on top of 20mLEmpty bottles were to be analyzed. Analysis conditions of the gas phase-ion mobility spectrometry unit: the analysis time is 30 min; the type of the chromatographic column FS-SE-54-CB-115 m ID is 0.53 mm; the column temperature is 60 ℃; carrier/drift gas N₂(ii) a The IMS temperature was 45 ℃. Fourthly, analyzing conditions of the automatic headspace sample introduction unit: the incubation temperature is 40 ℃; incubation time 15 min; the sample injection volume is 500 mu L; the temperature of a sample injection needle is 85 ℃; the hatching rotation speed is 500 r/min. Analysis software: the analysis software matched with the instrument comprises LAV (laboratory Analytical viewer) and three plugins as well as GC x IMS Library Search, and can be used for analyzing samples from different angles respectively. Measuring three times, taking an average value, 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 5 beer sensory Scoring criteria

TABLE 6 physicochemical index results of beer fermentation broth in ST 26-225L fermenter

TABLE 7 physical and chemical requirements of light beer (beer GB 4927-2008 national standard)

TABLE 8 organoleptic evaluation of ST 26-225L 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 6 and 7, the diacetyl content in the beer fermentation broth of ST26-22 was significantly lower than that of GT26 beer fermentation broth (0.058 mg/L for ST26-22 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 ST26-22 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; the alcohol content in the ST26-22 beer fermentation liquid is obviously higher than that of the GT26 beer fermentation liquid, and the alcohol content is 2.5 times of that of the GT26 beer fermentation liquid [ the ST26-22 beer fermentation liquid is 4.77% (v/v), the GT26 beer fermentation liquid is 1.95% (v/v)]. As can be seen from Table 8, the ST26-22 beer fermentation broth has an organoleptic score of more than 88, golden yellow, clear and transparent color, no suspended matter, pure white foam, fine and smooth foam, good durability, good hop aroma of beer, pure, clean, delicious, mellow and without rancid taste, pleasant floral and fruity aroma, and has active aroma and good taste, flavor and texture of the beer body. By using

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 ST26-22 is obviousHigher than GT26 beer fermentation broth. Wherein butyl caproate has pineapple flavor and its content is 4.4 times of GT26 beer (ST26-22 beer fermentation liquid peak volume is 17136.52, 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 3.0 times of that of GT26 beer (ST26-22 beer fermentation liquid peak volume is 224.67, GT26 beer fermentation liquid peak volume is 74.67); ethyl caproate has a sweet fruit pineapple flavour and is 1.4 times as high as GT26 beer (ST26-22 beer fermentation peak volume of 16725.12, GT26 beer fermentation peak volume of 11625.48); ethyl isobutyrate had a sweet fruity flavor with a content of 1.2 times that of GT26 beer (ST26-22 peak beer broth volume of 1632.18, GT26 peak beer broth volume of 1373.26); isobutyl acetate has a ripe fruit aroma, and its content is 1.2 times of GT26 beer (ST26-22 beer fermentation peak volume is 5491.76, GT26 beer fermentation peak volume is 4675.21); methyl caproate has pineapple ether taste, and its content is 1.7 times of GT26 beer (ST26-22 beer fermentation liquid peak volume is 328.32, GT26 beer fermentation liquid peak volume is 192.29); the ethyl butyrate has banana and pineapple flavors, and the content of the ethyl butyrate is 1.1 times of that of GT26 beer (the peak volume of ST26-22 beer fermentation liquid is 4926.15, and the peak volume of GT26 beer fermentation liquid is 4406.67). These ester compounds can impart sweet fruit, pineapple, banana, and fresh blackberry flavors to ST26-22 beer (see Table 9). In addition, the alcohol compound content of linalool and beta-phenylethyl alcohol is also significantly higher than that of GT26 beer. Linalool has a citrus fragrance and rosa banksiae, and is 1.4 times as high as GT26 beer (peak volume of ST26-22 beer fermentation broth is 246.19, peak volume of GT26 beer fermentation broth is 171.23); beta-phenylethyl alcohol has a sweet and floral aroma and a rosette honey note, and is 1.2 times as high as GT26 beer (ST26-22 peak beer broth volume of 338.86, GT26 peak beer broth volume of 280.83). These alcoholic compounds impart the ST26-22 beer with a citrus flower aroma, rosa rugosa and a sweet floral aroma such as a rosette honey aroma (see table 9). In conclusion, the ST26-22 beer contains a large amount of esters, alcohols and other substances, which bring flower fragrance and fruit fragrance to the ST26-22 beer, and the substances are obvious in sensory evaluation according to the sensory evaluationSuperior to the material basis of GT26 beer.

TABLE 9 odor of different flavor Compounds

And (4) conclusion: ST26-22 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 ST26-22 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 10mL YEPD 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 Optimalaceae, Biotechnology, Inc.), performing PCR amplification by using fungal 18S rDNA universal primers, namely upstream ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and downstream ITS4 (5'-TCCTCCGCTTATTGATATGC-3'), sending the obtained PCR product out for sequencing [ Producer organism (Shanghai) engineering, Inc. ], performing BLAST homology sequence comparison analysis on the sequencing result in a NCBI database, and determining the genus status of the strain.

The sequence of the PCR product of the ST26-22 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 ST26-22 strain can be determined to be Saccharomyces cerevisiae (Saccharomyces cerevisiae), and the identification result is shown in Table 10.

TABLE 10 identification of superior strains screened by space-mutagenesis of Saccharomyces cerevisiae ST26

Space-mutagenesis Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST26-22 has been deposited in China general microbiological culture Collection center (CGMCC) at 27 months 12 and 2019 with the preservation number of CGMCC No. 19246. Space-mutagenized Saccharomyces cerevisiae F-H-ST26-22 was obtained at 2018 for 7 months.

<110> Fuleton biotechnology and technology (Beijing) Ltd

<120> use of space mutagenesis of Saccharomyces cerevisiae ST26-22 for brewing beer

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 1434

<212> DNA

<213> Saccharomyces cerevisiae

<400> 1

caggaaattt ataattttga aatggatttt tttgttttgg caagagcatg agagctttta 60

ctgggcaaga agacaagaga tggaaagtcc agccgggcct gcgcttaagt gcgcggtctt 120

gctaggcttg taagtttctt tcttgctatt ccaaacggtg agagatttct gtgcttttgt 180

tataggacaa ttaaaaccgt ttcaatacaa cacactgtgg agttttcata tctttgcaac 240

tttttctttg ggcattcgag caatcggggc ccagaggtaa caaacacaaa caattttatc 300

tattcattaa atttttgtca aaaacaagaa ttttcgtaac tggaaatttt aaaatattaa 360

aaactttcaa caacggatct cttggttctc gcatcgatga agaacgcagc gaaatgcgat 420

acgtaatgtg aattgcagaa ttccgtgaat catcgaatct ttgaacgcac attgcgcccc 480

ttggtattcc agggggcatg cctgtttgag cgtcatttcc ttctcaaaca ttctgtttgg 540

tagtgagtga tactctttgg agttaacttg aaattgctgg ccttttcatt ggatgttttt 600

tttccaaaga gaggtttctc tgcgtgcttg aggtataatg caagtacggt cgttttaggt 660

tttaccaact gcggctaatc tttttttata ctgagcgtat tggaacgtta tcgataagaa 720

ggactctacc tgatttgagg tcaactttaa gaacattgtt cgcctagacg ctctcttctt 780

atcgataacg ttccaatacg ctcagtataa aaaaaaatta cccgcagtgg gtaaaaccta 840

aaacaaccgt acttgcttta tacctcaagc acgcaaaaaa acctctcttt gaaaaaaaaa 900

catccaatga aaaggccagc aatttcaagt taactccaaa gagtatcact ccctaccaaa 960

caaaatgttt gaaaaggaaa tgacgctcaa acagggatgc cccctggaat accaaggggg 1020

gcaatgggcg ttcaaaaatt cgatgattcc cggaattctg caattcacat tacgtatcgc 1080

atttcgctgc gttcttcatc gatgcgaaaa ccaaaaaatc cgttgttgaa agtttttaat 1140

attttaaaat ttccagttac gaaaattctt gtttttgaca aaaatttaat gaataaataa 1200

aattgtttgg gtttggtacc cctgggcccc gattgctcga atgcccaaaa aaaaagttgc 1260

aaaaaaatga aaactcccca gggggttgta ttgaaacggt tttaattgtc ctataacaaa 1320

agcacagaaa tctctcaccg tttggaatag caaaaaagaa acttacaagc ctagcaagac 1380

cgcgcactta agcgcaggcg cggctggact ctccatctct tgtcttcttg ccca 1434

Claims

1. Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST26-22, wherein the preservation number of the general microbiological center of China Committee for culture Collection of microorganisms is CGMCC No. 19246.

2. A microbial agent comprising the Saccharomyces cerevisiae(s) according to claim 1 as an active ingredientSaccharomyces cerevisiae）F-H-ST26-22。

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

4. A method of brewing beer comprising: use of the Saccharomyces cerevisiae (Saccharomyces cerevisiae) according to claim 1Saccharomyces cerevisiae) F-H-ST26-22 is fermented to obtain beer.

5. The method of claim 4, wherein: beer produced by fermenting the Saccharomyces cerevisiae(s) (see claim 1)Saccharomyces cerevisiae) F-H-ST26-22 was obtained by fermentation in wort.

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

7. The method of claim 5, wherein: said Saccharomyces cerevisiae (A), (B)Saccharomyces cerevisiae) The inoculum size of F-H-ST26-22 in the wort was 6X 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) mixing said Saccharomyces cerevisiae (Saccharomyces cerevisiae) F-H-ST26-22 is fermented in wort at the temperature of 12-17 ℃ to obtain primary fermentation liquor;

9. Saccharomyces cerevisiae (Saccharomyces cerevisiae) (Saccharomyces cerevisiae, and Saccharomyces cerevisiae, andSaccharomyces cerevisiae) Use of F-H-ST26-22 for brewing beer.

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