Saccharomyces cerevisiae with high freeze-thaw tolerance as well as screening method and application of saccharomyces cerevisiae

Abstract

The invention provides saccharomyces cerevisiae with high freeze-thaw tolerance as well as a screening method and application thereof, and relates to the technical field of microbial breeding. Two wild-type saccharomyces cerevisiae and a laboratory saccharomyces cerevisiae with a clear genetic background are selected as parent strains, and the parent strains are repeatedly subjected to the steps of spore production, random mating, repeated freeze thawing and the like to obtain the saccharomyces cerevisiae with high freeze thawing tolerance. The saccharomyces cerevisiae disclosed by the invention has high freeze-thaw tolerance, is intersected with a parent strain, the freeze-thaw tolerance is improved by 7.5 times, and the saccharomyces cerevisiae has ethanol tolerance and high-temperature tolerance. Through random mating of spores of different strains, the genetic diversity of the diploid is increased, and the probability of mutation of the diploid in the directional screening process is increased, so that compared with a parent strain, the freeze-thaw tolerance of the obtained strain is improved by 7.5 times, and the strain can tolerate ethanol with the concentration of 30% and a high-temperature environment of 50 DEG C.

Description

Technical Field

[0001] The invention relates to the technical field of microbial breeding, and in particular to a brewer's yeast with high freeze-thaw tolerance and a screening method and application thereof. Background Art

[0002] Saccharomyces cerevisiae, commonly known as baker's yeast, is the cornerstone of the industrial fermentation process. In the industrial fermentation process, the dough needs to be frozen first to stop the fermentation of the yeast in the dough, thereby increasing the shelf life of the dough; when the dough needs to be fermented, the frozen dough is thawed to allow the yeast in the dough to continue to work. In this process, the freeze-thaw tolerance of Saccharomyces cerevisiae is crucial to the fermentation quality of the dough. The survival rate of Saccharomyces cerevisiae after freezing and thawing directly affects the ability of the dough to expand and maintain the desired flavor and texture characteristics. However, during the freezing and thawing process, ice crystals and oxidative stress will occur in the cells of Saccharomyces cerevisiae. The generation of ice crystals and oxidative stress reactions will seriously damage the cells of Saccharomyces cerevisiae, reducing the activity of Saccharomyces cerevisiae or even inactivating it. Specifically, the freezing process will cause high osmotic pressure and ice crystals in the cells, thereby destroying the cell membrane; and the thawing process will produce reactive oxygen species that are harmful to the cells, thereby affecting the survival of Saccharomyces cerevisiae. Therefore, there is an urgent need for a Saccharomyces cerevisiae that can tolerate repeated freezing and thawing.

[0003] At present, the strategy to improve the freeze-thaw tolerance of Saccharomyces cerevisiae mainly relies on genetic modification of industrial strains to increase glycerol accumulation or enhance the expression of aquaporins, thereby improving the freeze-thaw tolerance of Saccharomyces cerevisiae. Glycerol acts as a cryoprotectant by stabilizing the cell structure at low temperatures, and ε-poly-L-lysine can promote the accumulation of glycerol, trehalose and fatty acids. Therefore, the expression of ε-poly-L-lysine can be enhanced in Saccharomyces cerevisiae to promote the accumulation of glycerol in Saccharomyces cerevisiae cells, thereby enhancing the freezing tolerance of Saccharomyces cerevisiae. In addition, intracellular aquaporins in Saccharomyces cerevisiae, such as Aqy1 and Aqy2, can regulate the transport of intracellular water. Therefore, the formation of intracellular ice crystals can be prevented by overexpressing aquaporins to regulate intracellular water transport, thereby improving the tolerance of Saccharomyces cerevisiae. However, the above methods can only improve the tolerance of Saccharomyces cerevisiae during freezing, and the tolerance improvement is insufficient. The activity of Saccharomyces cerevisiae during repeated freezing and thawing is still greatly affected.

[0004] In addition, traditional genetic improvement strategies often focus on mature industrial yeast strains and ignore the potential of wild Saccharomyces cerevisiae strains. Wild yeast strains evolve under different environmental conditions and may have genetic characteristics that are critical for freeze-thaw tolerance that domesticated strains do not have. The genetic diversity in wild yeast strains can enhance the probability of beneficial mutations in Saccharomyces cerevisiae during directed evolution, thereby improving the freeze-thaw tolerance of Saccharomyces cerevisiae. However, traditional methods rarely combine these strains, so the freeze-thaw tolerance of the resulting Saccharomyces cerevisiae is still low. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problem in the prior art that there is a lack of a brewer's yeast that can tolerate repeated freezing and thawing.

[0006] To solve the above technical problems, the present invention provides a brewer's yeast with high freeze-thaw tolerance and a screening method and application thereof. The present invention selects two wild-type brewer's yeasts and a laboratory brewer's yeast with a clear genetic background as parent strains, extracts spores of the parent strains respectively and places them in the same culture medium for culture, so that the spores from the three brewer's yeasts are randomly mated to obtain diploids, and the obtained diploids are repeatedly frozen and thawed and strains with good growth are screened as first-generation strains, and the obtained first-generation strains are repeated with the above-mentioned "spore production, random mating, repeated freezing and thawing" steps twice to obtain three-generation strains, and the strain with the best freeze-thaw resistance among the three-generation strains is selected as the brewer's yeast with high freeze-thaw tolerance of the present invention. After identification, the brewer's yeast of the present invention has high freeze-thaw tolerance, intersecting with the parent strain, and its freeze-thaw tolerance is increased by 7.5 times, and it has ethanol tolerance and high temperature tolerance. The present invention increases the genetic diversity of diploids by random mating between spores of different strains, thereby increasing the probability of mutation of diploids during directional screening. Therefore, the obtained strain has a freeze-thaw tolerance that is 7.5 times higher than that of the parent strain, and can tolerate 30% ethanol and a high temperature environment of 50°C.

[0007] The first object of the present invention is to provide a brewer's yeast with high freeze-thaw tolerance, wherein the brewer's yeast is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32515.

[0008] The second object of the present invention is to provide an application of the above-mentioned brewer's yeast in preparing fermented food.

[0009] The third object of the present invention is to provide a method for screening the above-mentioned Saccharomyces cerevisiae, comprising the following steps:

[0010] S1. Inoculate several species of Saccharomyces cerevisiae in spore-producing medium, culture them and screen them to obtain sporophytes, inoculate the obtained sporophytes in medium for mixed culture and perform typing identification, and select colonies that are diploid as initial colonies;

[0011] S2, preparing the initial colonies obtained in S1 into a resuspension, subjecting the resuspension to freeze-thaw cycles, applying the resuspension after freeze-thaw cycles to culture medium, and performing matching identification on the colonies after colonies grow out of the culture medium, and screening the colonies identified as diploid as the first generation colonies, wherein one freeze-thaw cycle is to freeze the initial colonies at -30°C to -50°C for 1.5-2 hours and then thaw them at 20-25°C for 0.5-1.5 hours;

[0012] S3, inoculating the first generation colonies in a spore-producing medium for culture and screening to obtain sporophytes, inoculating the obtained sporophytes in a culture medium for mixed culture and performing typing identification on the colonies on the culture medium, screening the colonies that are identified as diploid and repeating the operation of S2 to obtain second generation colonies;

[0013] S4. Repeat the operation of S3 with the second-generation strain to obtain third-generation colonies, and screen out the colonies identified as diploid among the third-generation colonies to obtain the brewer's yeast.

[0014] Furthermore, the number of freeze-thaw cycles in step S1 is 8-15 times.

[0015] Furthermore, the several types of Saccharomyces cerevisiae in step S1 include Saccharomyces cerevisiae CSM WT, Saccharomyces cerevisiae YPS1000 WT, and Saccharomyces cerevisiae DBY12007.

[0016] Furthermore, the step of screening and obtaining sporophytes is to dissolve the colonies collected on the spore-producing culture medium in ultrapure water to prepare a suspension, centrifuge the suspension, remove the residual culture medium components, collect the precipitate in the centrifuge tube and add a lysis solution to lyse the ascosporophyte, and the spores located in the ascosporophyte are lysed and released, and heated to 60-70°C. Since the spores have a higher tolerance temperature than the vegetative cells, they are heated to 60-70°C to cause the vegetative cells to die, and centrifuge again to obtain the sporophyte.

[0017] Furthermore, the matching identification is to pick the colonies on the culture medium, use primers to perform colony PCR, and the strain that simultaneously amplifies the 544bp and 404bp bands is diploid, wherein the gene sequence of the primers is shown in SEQ ID NO.7-9.

[0018] Furthermore, the HO gene in the brewer's yeast in step S1 is knocked out, and the knockout treatment is as follows: using the pFA6a-kanMX6 plasmid carrying a resistance tag as a template, using primers LoxP-kanMX-F and LoxP-kanMX-R to perform a first round of PCR to amplify the kanMX gene with a LoxP site, i.e., a LoxP-kanMX fragment; then using the kanMX-LoxP fragment as a template, using primers HO-F and HO-R to perform a second round of PCR amplification to amplify the kanMX gene with complementary sequences of upstream and downstream homologous arm sequences of the HO gene to obtain HOL-LoxP-kanMX-HOR, and transforming HOL-LoxP-kanMX-HOR into yeast.

[0019] Furthermore, the HO gene in Saccharomyces cerevisiae can express an endonuclease, which will lead to the conversion of haploid type (type a or type α) during yeast sporulation, thereby combining with surrounding heterospores to restore diploidy. Therefore, it is not easy to separate haploid Saccharomyces cerevisiae with HO gene activity. The present invention constructs an HO gene knockout component (i.e., HOL-LoxP-kanMX-HOR) and knocks out the HO gene of Saccharomyces cerevisiae by homologous recombination, thereby reducing the difficulty of separating haploid Saccharomyces cerevisiae.

[0020] Furthermore, the gene sequence of the primer LoxP-kanMX-F is shown as SEQ ID NO.1, the gene sequence of the primer LoxP-kanMX-F is shown as SEQ ID NO.2, the gene sequence of the primer HO-F is shown as SEQ ID NO.3, and the gene sequence of the primer HO-R is shown as SEQ ID NO.4.

[0021] Furthermore, the spore production medium comprises a carbon source, a nitrogen source, an inorganic salt and metal ions.

[0022] Beneficial effects of the present invention:

[0023] The present invention provides a brewer's yeast with high freeze-thaw tolerance, and a screening method and application thereof. The present invention improves the genetic diversity of the brewer's yeast strain by introducing genes of wild-type strains and random mating between spores of different strains, thereby increasing the probability of beneficial mutations in the directional screening process of continuous freeze-thaw, and finally obtaining the brewer's yeast with high freeze-thaw tolerance of the present invention. The brewer's yeast of the present invention has good freeze-thaw tolerance and still has a high survival rate under extreme freeze-thaw tests. In addition, the brewer's yeast obtained by directional screening in the prior art can tolerate a maximum ethanol concentration of about 18%, and a temperature range that can be tolerated does not exceed 45°C, while the brewer's yeast strain of the present invention still has good tolerance in 30% concentration ethanol and grows well at 50°C.

[0024] Biomaterial Deposit

[0025] Saccharomyces cerevisiae, the brewer's yeast is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit number is CGMCC No.32515, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit date is December 5, 2024. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0027] Figure 1 It is a screening process for brewer's yeast with high freeze-thaw tolerance;

[0028] Figure 2 This is the electrophoresis pattern after HO gene knockout;

[0029] Figure 3 It is the result of colony PCR;

[0030] Figure 4 It is a comparison chart of the growth conditions of the parent strain, the first-generation strain, the second-generation strain, and the third-generation strain before and after the extreme freeze-thaw cycle;

[0031] Figure 5 It is a performance test of parent strains, first-generation strains, second-generation strains and third-generation strains. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0033] Saccharomyces cerevisiae CSM WT, also known as Saccharomyces cerevisiae PGY7, Saccharomyces cerevisiae YPS1000 WT purified from oak secretions, also known as Saccharomyces cerevisiae PGY34; Saccharomyces cerevisiae DBY12007 is a commonly used laboratory strain. For detailed information on the above three strains, please refer to the literature "Anqi Chen, Jeremy R Smith, Hugo Tapia, Patrick A Gibney, Characterizing phenotypic diversity of trehalose biosynthesis mutants in multiple wild strains of Saccharomyces cerevisiae, G3 Genes | Genomes | Genetics, Volume 12, Issue 11, November 2022, jkac196, https: / / doi.org / 10.1093 / g3journal / jkac196".

[0034] YPD medium: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L.

[0035] Spore-forming medium (Mclary medium): glucose 1 g / L, sodium acetate 8.2 g / L, potassium chloride 1.8 g / L, yeast extract 2.5 g / L, agar 20 g / L.

[0036] Example 1: Screening of spore-forming strains

[0037] The present invention uses wild-type cerevisiae strains PGY4, PGY7, PGY34, PGY46 and cerevisiae strain DBY12007 commonly used in laboratories as strains to be screened.

[0038] First, the strains to be screened were tested for growth. The strains to be screened were inoculated in YPD medium and cultured. It was found that PGY34 showed the greatest growth advantage. 600 The highest and the shortest doubling time indicated that PGY34 showed superior growth adaptability under standard conditions, so PGY34 was selected as the first spore-forming strain.

[0039] Secondly, the freeze-thaw effect test was performed on the strains to be screened. The strains to be screened were placed under extreme freeze-thaw cycle conditions using an 18:6 hour freeze-thaw cycle repeated over 8 days (i.e., freezing the strains to be screened at -80°C for 18 hours and then thawing at 4°C for 6 hours as a cycle, repeated 8 cycles). The results showed that PGY7 maintained a survival rate of about 40% after 8 days, indicating that the PGY7 strain had considerable resistance to repeated freeze-thaw cycles, so PGY7 was selected as the second spore-producing strain.

[0040] In addition, the DBY12007 strain is a commonly used strain in the laboratory with a clear genetic background, so the DBY12007 strain was selected as the third spore-producing strain.

[0041] In summary, the present invention selected the PGY7 strain with the best growth, the PGY34 strain with the best freeze-thaw effect, and the DBY12007 strain with a clear genetic background as the spore-forming strains of the present invention.

[0042] Example 2: Knockout and verification of HO gene

[0043] 1. Construction of HO gene knockout components

[0044] Using X plasmid as template, primer pair kanMX-LoxP-F / kanMX-LoxP-R was used for the first round of PCR to amplify the kanMX gene with a LoxP site, i.e., the kanMX-LoxP fragment. Then, using the fragment as template, primer pair HO-F and HO-R were used for the second round of PCR amplification to amplify the kanMX gene with complementary sequences of the upstream and downstream homologous arm sequences of the HO gene, i.e., HO gene upstream homologous arm-kanMX-LoxP-HO gene downstream homologous arm (HOL-kanMX-LoxP-HOR).

[0045] Table 1 Primers involved in Example 2

[0046]

[0047] 2. Transformation of Saccharomyces cerevisiae and identification of transformants

[0048] The HO knockout assembly HOL-LoxP-kanMX-HOR obtained in step 1 was transformed into the spore-forming strains PGY7, PGY34, and DBY12007 screened in Example 1 respectively by the lithium acetate standard transformation method and identified, comprising the following steps:

[0049] (1) Select spore-forming strains and inoculate them into YPD medium, and culture them at 30°C and 200 r / min overnight to obtain activated seed solution;

[0050] (2) Transfer the seed solution to 50 mL of fresh YPD liquid medium at a volume ratio of 10% and continue culturing at 30°C and 200 r / min until the bacterial solution OD reaches 0. 600 Between 1.2-1.5;

[0051] (3) Collect the cells by centrifugation at 5000 rpm for 5 min and wash them twice with 25 mL of sterile water;

[0052] (4) Resuspend the cells with 8 mL of DTT solution, shake in a 30°C water bath for 30 min, and collect the cells by centrifugation at 5000 rpm for 5 min;

[0053] (5) Resuspend the cells in 15 mL of 4°C precooled 1.2 M sorbitol solution, centrifuge at 5000 rpm for 5 min at 4°C to collect the cells, and repeat this step three times;

[0054] (6) Use the lithium acetate standard transformation method to transform the HO knockout component HOL-LoxP-kanMX-HOR into the GY7 strain, PGY34 strain and DBY12007 strain in step (5), respectively, and then inoculate them on YPD medium and culture for 4-6 hours, then spread them on a plate containing G418 drug (300μg / mL), and then pick the clone colonies for lysis first. The lysis solution in this example is 0.5% (v / v) Triton X-100, 1mM EDTA, 0.05M NaOH. In a PCR instrument at 99°C for 25 minutes, take 3μL of the lysis solution and add it to the premixed PCR system for PCR amplification (using HO::KANMX-F and HO::KANMX-R as primers), and then use agarose gel electrophoresis to determine the fragment size to determine the positive transformants in which the HO gene is knocked out. The agarose gel electrophoresis results are as follows Figure 2 shown.

[0055] 5. Isolation and typing identification of haploid spore-forming strains

[0056] (1) Select the positive transformants with HO gene knockout in step 4 and inoculate them into YPD medium, activate them at 30°C and 200 r / min for 24 h, and inoculate them into fresh YPD medium at an inoculum volume ratio of 10% and continue to culture for 24 h;

[0057] (2) centrifuging at 4000 r / min for 8 min to collect yeast slurry, washing it twice with physiological saline, and then piling it on a spore-forming medium and culturing it at 26°C for 5-7 days to produce spores;

[0058] (3) Pick an appropriate amount of bacteria on the spore-forming medium to prepare a spore suspension. Take 1 mL of bacterial suspension, centrifuge at 5000 rpm for 3 minutes in a 1.5 mL centrifuge tube, and collect ascosporophytes. For each milliliter of collected precipitate (about 20 mg wet weight), add 600 μL Snailase Reaction buffer, 50 μL Snailase (dissolved in PBS buffer or water), and 2.4 μL ≥ 96% β-mercaptoethanol in sequence; place the centrifuge tube in a 37°C water bath for 10-12 hours to obtain spores; since spores have a higher tolerance temperature than vegetative cells, increase the temperature to 65°C for 25 minutes to kill the vegetative cells, vortex to break up the spores, and then separate the spores under the action of a centrifuge at 5000 rpm for 3 minutes; resuspend the spore solution with sterile water, wash three times, and then smear the suspension on a YPD plate containing antibiotics (G418), and invert at 30°C for more than two days until obvious colonies grow;

[0059] (4) Pick colonies and perform colony PCR using primers (MAT-R, MAT-a, MAT-α) to identify haploid strains and their matching types. The colony PCR amplification results are as follows: Figure 2 shown.

[0060] Table 2 Confirmation primers used for typing identification

[0061] name sequence Sequence number MAT-R ACTCC ACTTC AAGTAAGAGT TTG SEQ ID NO.7 MAT-a GCACG GAATATGGGACTACT TCG SEQ ID NO.8 MAT-α AGTCACATCAAGATC GTTTA TGG SEQ ID NO.9

[0062] When using primers MAT-R / MAT-a / MAT-α for haploid typing identification, the strain that amplified the 544bp band was type a haploid, the strain that amplified the 404bp band was type α haploid, and the strain that amplified the 544bp and 404bp bands was diploid.

[0063] Example 3: Screening of Saccharomyces cerevisiae with high freeze-thaw tolerance

[0064] 1. The haploid spores of the PGY7 strain, PGY34 strain and DBY12007 strain obtained in Example 2 were inoculated into liquid YPD medium at the same time and cultured overnight. After reaching the stable period, the matching verification was performed. The strains with matching verification as diploid were selected, 1 mL of bacterial solution was centrifuged at 6000 rpm for 3 min to remove the supernatant, washed twice with 1 mL of sterile ddH2O, and then resuspended in sterile ddH2O and then subjected to continuous freeze-thaw cycles. Freezing at -40°C for 2 hours and thawing at room temperature for 1 hour was used as a freeze-thaw cycle. The cycle was repeated at least 10 times within 3 days, and then the bacteria after the continuous freeze-thaw cycle were diluted and spread on solid YPD medium, and inverted and cultured at 30°C for 2 days until obvious colonies were generated. Then, obviously larger colonies were picked, and single colonies were picked for matching verification. The strains verified to be diploid were determined as first-generation colonies and subjected to extreme freeze-thaw performance verification (for extreme freeze-thaw performance verification, see step 5 (1) of this embodiment).

[0065] 2. Inoculate the first generation colony in a spore-forming medium to produce spores and screen the spores. Inoculate the obtained spores in a liquid YPD medium for culture and perform matching verification. Screen the diploid strains for continuous freeze-thaw cycles. Freeze for 2 hours at -40°C and thaw at room temperature for 1 hour as a freeze-thaw cycle. Repeat at least 10 cycles within 3 days. Then dilute the bacteria after the continuous freeze-thaw cycle and spread them on a solid YPD medium. Invert and culture at 30°C for 2 days until obvious colonies are generated. Then pick a significantly larger colony, pick a single colony for extreme freeze-thaw performance verification and matching verification. The strain verified as diploid is determined as the second generation colony. The strain verified as diploid is determined as the second generation colony and the extreme freeze-thaw performance is verified (for extreme freeze-thaw performance verification, see step 5 (1) of this embodiment).

[0066] 3. Inoculate the second-generation colonies in the spore-forming medium to produce spores and screen the spores. Inoculate the obtained spores in the liquid YPD medium for culture and perform matching verification. Screen the diploid strains for continuous freeze-thaw cycles. Take 2 hours of freezing at -40°C and thawing at room temperature for 1 hour as a freeze-thaw cycle. Repeat at least 10 cycles within 3 days, then dilute the bacteria after the continuous freeze-thaw cycle and spread them on the solid YPD medium. Invert and culture at 30°C for 2 days until obvious colonies are generated. Then pick the obviously larger colonies and pick single colonies for matching verification. The strains verified to be diploid are determined as the third-generation colonies.

[0067] 4. Performance identification and preservation of strains

[0068] Three diploid colonies were randomly selected from the first-generation, second-generation, and third-generation colonies and inoculated into YPD medium for culture (i.e. Figure 4The parent colonies PGY7 strain, PGY34 strain and DBY12007 strain were used as control groups.

[0069] (1) Verification of the extreme freeze-thaw performance of strains

[0070] Part of the strains were picked out from the culture medium inoculated with first-generation-1, first-generation-2, first-generation-3, second-generation-1, second-generation-2, second-generation-3, third-generation-1, third-generation-2, and third-generation-3, resuspended in sterile water, and subjected to extreme freeze-thaw cycle treatment. The extreme freezing cycle treatment was frozen at -80°C for 16 hours, and then thawed at 4°C for 8 hours for one cycle, and the number of cycles was 8 times. The results of extreme freeze-thaw cycle treatment are shown in Figure 4 As shown, third-generation-1, third-generation-2, and third-generation-3 still maintain good tolerance under extreme freeze-thaw cycle treatments.

[0071] (2) 30% ethanol tolerance test

[0072] Single colonies were picked from the culture media inoculated with third-generation-1, third-generation-2, and third-generation-3, respectively, and inoculated into new culture media while adding 30% ethanol for 15 minutes, then graded diluted and inoculated into YPD culture media to observe the growth of the colonies. Figure 5 As shown, the third-generation colonies had good tolerance to 30% ethanol compared with the parent colonies.

[0073] (3) 50℃ high temperature verification

[0074] Single colonies were picked from the culture media inoculated with third-generation-1, third-generation-2, and third-generation-3, respectively, and inoculated into new culture media, respectively, and subjected to high temperature treatment at 50°C for 30 minutes, and the growth of the colonies was observed. Figure 5 As shown, the third-generation colonies had better tolerance to high temperature of 50°C compared with the parent colonies.

[0075] The results are as follows Figure 4 and Figure 5 As shown, the third-generation-1, third-generation-2, and third-generation-3 colonies have high freeze-thaw tolerance, and compared with the parent strain, their freeze-thaw tolerance is improved by 7.5 times (freeze-thaw tolerance is calculated by counting the CFU before and after freeze-thaw and calculating Viability = CFU2 / CFU1 according to this formula, where CFU1 represents the CFU before freeze-thaw, and CFU2 represents the CFU after freeze-thaw, and a 7.5-fold improvement is obtained by comparing with the control group), and can tolerate 30% ethanol and a high temperature of 50°C. A single colony of third-generation-2 was selected for preservation to obtain the strain of the present invention.

[0076] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from them are still within the protection scope of the invention.

Claims

1. A brewer's yeast with high freeze-thaw tolerance, characterized in that: The brewer's yeast is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No.32515.

2. Use of the brewer's yeast according to claim 1 in preparing fermented food.

3. A method for screening brewer's yeast with high freeze-thaw tolerance, characterized in that: The following steps are involved: S1. Inoculate several species of Saccharomyces cerevisiae in spore-producing medium, culture them and screen them to obtain sporophytes, inoculate the obtained sporophytes in medium for mixed culture and perform typing identification, and select colonies that are diploid as initial colonies; S2, preparing the initial colonies obtained in S1 into a resuspension, subjecting the resuspension to freeze-thaw cycles, applying the resuspension after freeze-thaw cycles to culture medium, and performing matching identification on the colonies after colonies grow out of the culture medium, and screening the colonies identified as diploid as the first generation colonies, wherein one freeze-thaw cycle is to freeze the initial colonies at -30°C to -50°C for 1.5-2 hours and then thaw them at 20-25°C for 0.5-1.5 hours; S3, inoculating the first generation colonies in a spore-producing medium for culture and screening to obtain sporophytes, inoculating the obtained sporophytes in a culture medium for mixed culture and performing typing identification on the colonies on the culture medium, screening the colonies that are identified as diploid and repeating the operation of S2 to obtain second generation colonies; S4. Repeat the operation of S3 with the second-generation strain to obtain third-generation colonies, and screen out the colonies identified as diploid from the third-generation colonies to obtain the brewer's yeast with high freeze-thaw tolerance.

4. The screening method according to claim 3, characterized in that The number of freeze-thaw cycles in step S2 is 8-15 times.

5. The screening method according to claim 3, characterized in that The several types of Saccharomyces cerevisiae described in step S1 include Saccharomyces cerevisiae CSM WT, Saccharomyces cerevisiae YPS1000 WT, and Saccharomyces cerevisiae DBY12007.

6. The screening method according to claim 3, characterized in that The step of screening and obtaining sporophytes is to prepare a suspension from the colonies collected on the spore-producing medium, centrifuge the suspension, collect the precipitate in the centrifuge tube and add a lysis solution, heat it to 60-70° C., and centrifuge again to obtain the sporophytes.

7. The screening method according to claim 3, characterized in that The typing identification is to pick the colonies on the culture medium, use primers to perform colony PCR, and the strain that simultaneously amplifies two bands is a diploid, wherein the gene sequence of the primers is shown in SEQ ID NO.7-9.

8. The screening method according to claim 3, characterized in that In step S1, the HO gene of the Saccharomyces cerevisiae is subjected to a knockout treatment, and the knockout treatment is as follows: using a plasmid carrying a resistance tag kanMX6 as a template, using primers LoxP-kanMX-F and LoxP-kanMX-R to perform a first round of PCR, amplifying a kanMX gene with a LoxP site, i.e., a LoxP-kanMX fragment; then using the kanMX-LoxP fragment as a template, using primers HO-F and HO-R to perform a second round of PCR amplification, amplifying a kanMX gene with complementary sequences of upstream and downstream homologous arm sequences of the HO gene, obtaining a HOL-LoxP-kanMX-HOR fragment, and transforming the HOL-LoxP-kanMX-HOR fragment into the Saccharomyces cerevisiae.

9. The screening method according to claim 8, characterized in that The gene sequence of the primer LoxP-kanMX-F is shown in SEQ ID NO.1, the gene sequence of the primer LoxP-kanMX-F is shown in SEQ ID NO.2, the gene sequence of the primer HO-F is shown in SEQ ID NO.3, and the gene sequence of the primer HO-R is shown in SEQ ID NO.

4.

10. The screening method according to claim 3, characterized in that The spore production medium comprises a carbon source, a nitrogen source, an inorganic salt and metal ions.

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