Saccharomyces cerevisiae with high protein yield and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to saccharomyces cerevisiae with high protein yield and application of the saccharomyces cerevisiae. The saccharomyces cerevisiae with high protein yield is a saccharomyces cerevisiae HP-1 strain, the saccharomyces cerevisiae is preserved in the China Center for Type Culture Collection on August 21, 2024, and the preservation number of the saccharomyces cerevisiae is CCTCC NO: M 20241829. The fungicide prepared from the saccharomyces cerevisiae HP-1 strain through a fermentation method has the characteristics of high biomass, high protein and low RNA (Ribonucleic Acid). The Saccharomyces cerevisiae HP-1 strain and the fermented product, the yeast milk, the microbial inoculum and the extract prepared from the strain provided by the invention can be applied to the fields of feeds, foods, health-care products or seasonings.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a high-protein-yielding brewer's yeast and application thereof. Background Art

[0002] Saccharomyces cerevisiae, also known as baker's yeast or budding yeast, is the yeast most widely associated with humans. It can not only be used to make foods such as bread and steamed buns and brew wine, but also used as a eukaryotic model organism in modern molecular and cell biology, which is equivalent to the prokaryotic model organism Escherichia coli. Saccharomyces cerevisiae is the most commonly used biological species in fermentation.

[0003] Yeast protein is a microbial protein made from Saccharomyces cerevisiae through one or more processes such as enzymolysis and extraction, and is used as food or food ingredients. Yeast processed products are products made from yeast for food processing as the main raw material, which are autolyzed or enzymolyzed by the action of yeast's own enzymes or external enzymes, and are separated or not separated, dried or not dried. As an important raw material for yeast protein and yeast processed products, the protein and nucleic acid content of Saccharomyces cerevisiae plays a key role in the quality and cost of yeast protein and yeast processed products.

[0004] Chinese patent CN113862164A discloses a yeast strain obtained by natural screening, which has a protein content of 68.13%, an RNA content of 15.99% and a biomass of 48 g / L, namely, Saccharomyces cerevisiae AMCC 30743. Summary of the invention

[0005] The technical problem solved by the present invention is as follows: In the prior art, the Saccharomyces cerevisiae strain cannot simultaneously have the properties of high biomass, high protein and low RNA.

[0006] In view of the above technical problems, the present invention provides a high-protein brewing yeast and its application. In the present invention, a yeast strain with a protein content of 75.2% is obtained by means of adaptive evolution and directional screening, and its protein content is higher than the level of existing literature or patent research, and the RNA content is 8.28%. The yeast strain has the characteristics of high protein and low RNA.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides a high-protein-producing Saccharomyces cerevisiae strain, wherein the Saccharomyces cerevisiae HP-1 strain is deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20241829.

[0009] Preferably, the ITS sequence of the Saccharomyces cerevisiae HP-1 strain is shown as SEQ ID NO.1.

[0010] In a second aspect, the present invention provides a fermentation product, which is obtained by culturing the Saccharomyces cerevisiae strain.

[0011] In a third aspect, the present invention provides a method for preparing a fermentation product, comprising the following steps: culturing the brewer's yeast strain to obtain the fermentation product.

[0012] Preferably, the strain is inoculated into a culture medium for seed culture and then subjected to fermentation culture.

[0013] More preferably, the strain is first expanded and cultured, then inoculated into a culture medium for seed culture, and then transferred for fermentation culture.

[0014] More preferably, the expanded culture includes test tube culture; further preferably, the culture temperature is 28 to 34° C.; and / or the culture time is 12 to 60 hours;

[0015] And / or, more preferably, the expanded culture includes shake flask culture; further preferably, the culture temperature is 28 to 34° C.; and / or, the culture time is 8 to 48 h.

[0016] More preferably, the volume percentage of the bacterial strain inoculation is 0.8% to 5%.

[0017] More preferably, the temperature of seed culture is 28-34°C; and / or the time of seed culture is 20-60h;

[0018] And / or, more preferably, the fermentation temperature is 28-34° C.; and / or, the fermentation time is 11-15 h.

[0019] Preferably, the culture medium used for the test tube culture, shake flask culture and seed culture comprises a carbon source, a nitrogen source, inorganic salts and trace elements.

[0020] Preferably, in parts by weight, the culture medium comprises 15 to 25 parts of glucose, 3 to 7 parts of (NH4)2SO4, 0.5 to 1.5 parts of MgSO4, 2 to 4 parts of KH2PO4, 0.5 to 1.5 parts of the first mother liquor and 0.5 to 1.5 parts of the second mother liquor.

[0021] More preferably, in parts by weight, each liter of culture medium includes 15 to 25 parts of glucose, 3 to 7 parts of (NH4)2SO4, 0.5 to 1.5 parts of MgSO4, 2 to 4 parts of KH2PO4, 0.5 to 1.5 parts of the first mother liquor and 0.5 to 1.5 parts of the second mother liquor.

[0022] More preferably, the first mother liquor comprises EDTA, ZnSO4, MnCl2, CoCl2, CuSO4, Na2MoO4, CaCl2, FeSO4, H3BO3 and KI.

[0023] More preferably, the first mother liquor comprises, in parts by weight, 10 to 20 parts of EDTA, 2 to 6.5 parts of ZnSO4, 0.6 to 0.95 parts of MnCl2, 0.1 to 0.3 parts of CoCl2, 0.1 to 0.3 parts of CuSO4, 0.1 to 0.5 parts of Na2MoO4, 1 to 5 parts of CaCl2, 1 to 3 parts of FeSO4, 1 to 2 parts of H3BO3 and 0.1 to 0.3 parts of KI.

[0024] Most preferably, in parts by weight, each liter of the first mother liquor includes 10 to 20 parts of EDTA, 2 to 6.5 parts of ZnSO4, 0.6 to 0.95 parts of MnCl2, 0.1 to 0.3 parts of CoCl2, 0.1 to 0.3 parts of CuSO4, 0.1 to 0.5 parts of Na2MoO4, 1 to 5 parts of CaCl2, 1 to 3 parts of FeSO4, 1 to 2 parts of H3BO3 and 0.1 to 0.3 parts of KI.

[0025] Preferably, the second mother solution comprises biotin, p-aminobenzoic acid, niacin, calcium pantothenate, pyridoxine hydrochloride, VB1 and inositol.

[0026] More preferably, in parts by weight, the second mother liquor comprises 0.01-0.1 parts of biotin, 0.1-0.5 parts of p-aminobenzoic acid, 0.5-3 parts of nicotinic acid, 1-2 parts of calcium pantothenate, 1-2 parts of pyridoxine hydrochloride, 1-2 parts of VB1 and 10-25 parts of inositol.

[0027] More preferably, in parts by weight, each liter of the second mother liquor comprises 0.01-0.1 parts of biotin, 0.1-0.5 parts of p-aminobenzoic acid, 0.5-3 parts of niacin, 1-2 parts of calcium pantothenate, 1-2 parts of pyridoxine hydrochloride, 1-2 parts of VB1 and 10-25 parts of inositol.

[0028] Preferably, the culture medium for the fermentation culture comprises a carbon source, a nitrogen source, a phosphorus source and trace elements.

[0029] Preferably, the culture medium for the fermentation culture further comprises a defoaming agent.

[0030] Preferably, the fermentation culture medium comprises 75-120 parts of carbon source, 13-25 parts of nitrogen source, 2.5-5 parts of phosphorus source and 0.01-2 parts of trace elements in parts by weight;

[0031] More preferably, the fermentation culture medium includes 0.01 to 1 parts of trace elements by weight; more preferably, the fermentation culture medium includes 0.01 to 0.5 parts of trace elements by weight; preferably, the fermentation culture medium includes 0.08 to 0.22 parts of trace elements by weight.

[0032] More preferably, the trace elements include 0.01 to 1 part of ZnSO4 and 0.01 to 1 part of CuSO4. More preferably, the trace elements include 0.01 to 0.5 part of ZnSO4 and 0.01 to 0.5 part of CuSO4. More preferably, the trace elements include 0.01 to 0.3 part of ZnSO4 and 0.01 to 0.1 part of CuSO4. More preferably, the trace elements include 0.07 to 0.2 part of ZnSO4 and 0.01 to 0.02 part of CuSO4.

[0033] Preferably, in parts by weight, each liter of the fermentation culture medium includes 75 to 120 parts of carbon source, 13 to 25 parts of nitrogen source, 2.5 to 5 parts of phosphorus source and 0.01 to 2 parts of trace elements.

[0034] More preferably, the culture medium for fermentation culture comprises 0.01 to 1 parts of trace elements per liter of culture medium by weight; more preferably, the culture medium for fermentation culture comprises 0.01 to 0.5 parts of trace elements per liter of culture medium by weight; preferably, the culture medium for fermentation culture comprises 0.08 to 0.22 parts of trace elements per liter of culture medium by weight.

[0035] More preferably, the trace elements include 0.01 to 1 part of ZnSO4 and 0.01 to 5 parts of CuSO4. More preferably, the trace elements include 0.01 to 0.5 parts of ZnSO4 and 0.01 to 0.5 parts of CuSO4. More preferably, the trace elements include 0.01 to 0.3 parts of ZnSO4 and 0.01 to 0.1 parts of CuSO4. More preferably, the trace elements include 0.07 to 0.2 parts of ZnSO4 and 0.01 to 0.02 parts of CuSO4.

[0036] Preferably, a carbon source is added during the fermentation culture process so that the alcohol content is in the range of 0.01% to 0.2%; preferably, a carbon source is added during the fermentation culture process so that the alcohol content is in the range of 0.05% to 0.15%; more preferably, the carbon source includes glucose and / or molasses; further preferably, the mass ratio of glucose to molasses is 0 to 7:3 to 10.

[0037] Preferably, the phosphorus source is fed during the fermentation process at a flow rate of 30 to 60 mL / h; more preferably, the phosphorus source includes diammonium phosphate; and further preferably, the mass percentage of the phosphorus source is 10% to 20%.

[0038] Preferably, the nitrogen source is added in batches during the fermentation culture process; more preferably, the nitrogen source is added in three batches, with the first batch adding 10% to 12% of the total amount, the second batch adding 25% to 30% of the total amount; the third batch adding 58% to 65% of the total amount; and / or, the first batch adding time is 2 to 5 hours, the second batch adding time is 3 to 5 hours, and the third batch adding time is 4 to 7 hours.

[0039] In a fourth aspect, the present invention provides a yeast milk, wherein the yeast milk is prepared by the brewer's yeast strain, the fermented product or the fermented product prepared by the method for preparing the fermented product.

[0040] In a fifth aspect, the present invention provides a bacterial agent, which is prepared by the brewer's yeast strain, the fermentation product, the fermentation product prepared by the method for preparing the fermentation product, or the yeast milk.

[0041] Preferably, the biomass of the bacterial agent is 250-280 g / L.

[0042] Preferably, the intracellular protein content of the bacterial agent is greater than 75wt% of the dry weight of the bacteria.

[0043] Preferably, the intracellular protein content of the bacterial agent is 75wt% to 80wt% of the dry weight of the bacteria.

[0044] Preferably, the RNA content of the bacterial agent is 6wt% to 9wt% of the dry weight of the bacteria.

[0045] Preferably, the RNA content of the bacterial agent is 8wt% to 9wt% of the dry weight of the bacteria.

[0046] In a sixth aspect, the present invention provides a use of the high-protein-producing Saccharomyces cerevisiae strain in yeast extract.

[0047] Or, use of the fermentation product in yeast extract.

[0048] Or, use of the yeast milk in yeast extract.

[0049] Or, use of the bacterial agent in yeast extract.

[0050] In a seventh aspect, the present invention provides a yeast extract, wherein the yeast extract is prepared by comprising the high-protein-yielding Saccharomyces cerevisiae strain, the fermented product or the fermented product prepared by the method for preparing the fermented product, the fermented milk or the bacterial agent.

[0051] In an eighth aspect, the present invention provides a high-protein-yielding Saccharomyces cerevisiae strain, fermented product, yeast milk or bacterial agent for use in the preparation of feed, food or health products.

[0052] In a ninth aspect, the present invention provides a high-protein-yielding Saccharomyces cerevisiae strain, fermented product, yeast milk or bacterial agent for use in preparing condiments.

[0053] Beneficial effects of the present invention

[0054] (1) The present invention provides a high-protein-producing Saccharomyces cerevisiae strain. Saccharomyces cerevisiae FX-2 is used as a starting strain, and a bacterial solution is obtained by ARTP mutagenesis and adaptive evolution of a protein synthesis inhibitor. After RNA fluorescence staining, high-fluorescence Saccharomyces cerevisiae strains are screened by flow cytometry to obtain high-nucleic acid yeast strains. The intracellular protein content of the screened strains is then screened and verified by the Bradford protein concentration determination method to obtain three high-protein-producing Saccharomyces cerevisiae strains, and then the best high-protein-producing Saccharomyces cerevisiae strain is verified in a 45L fermentation tank. The protein content of the best high-protein-producing Saccharomyces cerevisiae strain reaches 75.2%, which is 26.7% higher than that of the starting strain.

[0055] (2) The brewer's yeast strain provided by the present invention has the characteristics of high biomass, high protein and low RNA, and can be applied to industries such as feed, food, health care and condiments, to achieve sustainable development of industrialization of high-protein hydrolysates.

[0056] (3) The molasses conversion rate in the present invention can reach 93.29%, which has considerable advantages in reducing industrial production costs.

[0057] (4) Low nucleic acid in yeast protein is a control standard. Yeast protein needs to be separated from nucleic acid during production. Excessive residual nucleic acid in yeast protein will lead to the risk of high uric acid after human consumption. Therefore, the high protein and low RNA characteristics of the brewer's yeast strain of the present invention make the above separation more thorough, so that the final yeast protein product has low nucleic acid content, which is more beneficial to the human body.

[0058] Strain deposit information

[0059] The Saccharomyces cerevisiae HP-1 strain described in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on August 21, 2024, with a deposit number of CCTCC NO: M 20241829. The deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: (027) 68754052. DETAILED DESCRIPTION

[0060] As mentioned above, the purpose of the present invention is to provide a high-protein-producing Saccharomyces cerevisiae strain and its application.

[0061] The HP-1 strain of Saccharomyces cerevisiae provided by the present invention uses Saccharomyces cerevisiae FX-2 as the starting strain, obtains a bacterial solution through ARTP mutagenesis and adaptive evolution of protein synthesis inhibitors, screens high-fluorescence Saccharomyces cerevisiae strains by flow cytometry after RNA fluorescence staining to obtain high-nucleic acid yeast strains, and then screens and verifies the intracellular protein content of the screened strains by Bradford protein concentration determination method to obtain three high-protein Saccharomyces cerevisiae strains, and then verifies the best high-protein Saccharomyces cerevisiae strain in a 45L fermentation tank. The protein content of the best high-protein Saccharomyces cerevisiae strain reaches 75.2%, which is 26.7% higher than that of the starting strain.

[0062] The control strain Saccharomyces cerevisiae FX-2 used in the present invention was deposited in China Center for Type Culture Collection (Wuhan University) on August 1, 2016, with the deposit number of CCTCC NO: M 2016418, and has been recorded in CN108220175A.

[0063] High-protein-yielding yeast strains are the key factor in obtaining high-quality protein sources. They can lay the foundation for the industrial production of yeast extracts and meet the rigid demands for comprehensive, balanced and sustainable development of the feed, food and health care industries.

[0064] The yeast milk described in the present invention refers to the fermentation liquid obtained by fermentation, after the supernatant is removed by centrifugation, and then washed, filtered by suction, filtered by pressure, and the bacterial cells are separated and collected to obtain the yeast milk.

[0065] Unless otherwise specified, the various reagents / instruments used in the examples and comparative examples of the present invention are conventional commercially available products. The experimental materials and instrument information used in the present invention are shown in Tables 1 and 2 below:

[0066] Table 1 Experimental materials and manufacturers

[0067]

[0068]

[0069]

[0070] Table 2 Instruments and manufacturers

[0071] instrument model Manufacturer / seller ARTP Mutagenesis Apparatus ARTP-IIS Wuxi Yuanqing Tianmu Biotechnology Co., Ltd. Metal Slide / Wuxi Yuanqing Tianmu Biotechnology Co., Ltd. Flow cytometry BD FACS Aria III BDSciences Analytical Balance ME4002E METTLERTOLEDO pH Meter PB-10 Sartorius Fast Moisture Meter MJ33 METTLERTOLEDO Clean bench SKJH-1109 Shanghai Sukun Industrial Co., Ltd. Constant temperature shaker ZWYR-2102C Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd. Biochemical incubator SPX-158L Ningbo Xinzhi Biotechnology Co., Ltd. Constant temperature water bath HH-2 Jiangsu Guohua Tower Manufacturing Co., Ltd. Centrifuge DL-5200B-II Shanghai Fei Ge Abrasives Co., Ltd. Optical Microscope CX43 OLYMPUS UV-Vis Spectrophotometer UV2310II Hangzhou Aosheng Instrument Co., Ltd. Electric blast drying oven DHG-9070A Shanghai Jinghong Experimental Equipment Co., Ltd.

[0072] In the present invention, the OD value test step is: take an appropriate amount of PBS buffer and mix it evenly with the bacterial solution, put it into a cuvette, and use a spectrophotometer to measure the absorbance value at OD600 nm.

[0073] The culture medium components involved in the embodiment are as follows:

[0074] Slant culture medium: 10 parts sucrose, 2 parts yeast extract powder, 0.1 parts magnesium sulfate and 0.1 parts potassium dihydrogen phosphate, and sulfuric acid is used to adjust the pH to 4.3.

[0075] The fermentation medium formula is: glucose 20 g / L, (NH4)2SO4 5 g / L, MgSO4·7H2O 1 g / L, KH2PO4 3 g / L, ultrapure water 100 mL, 1 g / L first mother liquor, sterilized at 121°C for 20 minutes; the second mother liquor is prepared with sterile water, and the second mother liquor is added to a concentration of 1 g / L under sterile conditions and shaken well.

[0076] Table 3 Formula of the first mother solution

[0077] project Concentration (g / L) EDTA (sodium salt) 10～20 <![CDATA[ZnSO4·7H2O]]> 2～6.5 <![CDATA[MnCl2·2H2O]]> 0.6～0.95 <![CDATA[CoCl2·6H2O]]> 0.1～0.3 <![CDATA[CuSO4·5H2O]]> 0.1～0.3 <![CDATA[Na2MoO4·2H2O]]> 0.1～0.5 <![CDATA[CaCl2·2H2O]]> 1～5 <![CDATA[FeSO4·7H2O]]> 1～3 <![CDATA[H3BO3]]> 1～2 KI 0.1～0.3

[0078] Table 4 Formula of the second mother solution

[0079] project Concentration (g / L) Biotin 0.01～0.1 p-aminobenzoic acid 0.1～0.5 Nicotinic acid 0.5～3 Ca-pantothenate 1～2 Pyridoxine-HCl(Pyridoxine Hydrochloride, VB6) 1～2 Thiamine-HCl(VB1) 1～2 Myo-inositol 10～25

[0080] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.

[0081] Example 1: Acquisition of Saccharomyces cerevisiae

[0082] The starting strain (Saccharomyces cerevisiae FX-2) and other contents stored in a glycerol tube at -80°C were aseptically transferred to a 250 mL shake flask containing 50 mL of slant culture medium, and cultured at 220 rpm and 30°C for 48 hours. Then, 2 to 3 loops of the culture were inoculated under aseptic conditions and streaked into a 5 mL slant culture medium test tube slant containing 2% agar and cultured at 30°C for 48 hours. The resulting test tube slant culture was the activated strain.

[0083] A single colony of the activated Saccharomyces cerevisiae FX-2 strain was picked from the plate and placed in 2 mL of fermentation medium, and cultured at 30° C. and 220 rpm with shaking for 8 to 12 hours until the logarithmic growth phase.

[0084] Take an appropriate amount of the cultured yeast FX-2 bacterial solution, wash it 2-3 times with PBS buffer by centrifugation, and resuspend it in PBS buffer until OD600 = 0.6-0.8. In the sterile environment of the clean bench, take 10 μL of the bacterial solution with an OD600 value of 0.6-0.8 on a metal slide for mutagenesis treatment. The mutagenesis conditions are as follows: the parameters of the ARTP mutagenesis breeding instrument (ARTP-ⅡS) are: power 100W, irradiation distance 2mm and gas flow 10SLM.

[0085] After the starting strain was ARTP-induced for 50 seconds, it was inoculated into fermentation medium containing different types of protein synthesis inhibitors with different initial concentrations and without protein synthesis inhibitors for cultivation. The protein synthesis inhibitor was prepared with sterile water and added to the medium at the corresponding concentration under sterile conditions and shaken well. The induced yeast was cultured in the fermentation medium without inhibitors for 24 hours, and its OD600 was 4.5-5.0. The induced yeast was inoculated into different types of protein synthesis inhibitors with different initial concentrations and cultured for 24 hours, and then the OD600 value was measured. If the OD600 value did not recover to 4.5-5.0, the cultured strain was inoculated into the same concentration of protein synthesis inhibitor according to the inoculation amount of 1% by volume and continued to be cultured for 24 hours to measure the OD600 value. If the OD600 value did not recover to 4.5-5.0, the cultured strain was inoculated into the same concentration of protein synthesis inhibitor according to the inoculation amount of 1% by volume and continued to be cultured for 24 hours, and this was repeated until the OD600 value recovered to 4.5-5.0; then the cultured strain was transferred to the fermentation medium containing a higher concentration of protein synthesis inhibitor according to the inoculation amount of 1% by volume and continued to be cultured, and the above steps were repeated. After the strain grew in the fermentation medium containing a certain concentration of protein synthesis inhibitor for 24 hours, its growth was inhibited. After it was transferred to the fermentation medium containing the same concentration of protein synthesis inhibitor and continued to grow for 24 hours, the OD600 value increased, indicating that it was gradually adapting to the inhibitor concentration. Therefore, after the OD600 value is restored to 4.5-5.0 at the same protein synthesis inhibitor concentration, the concentration of the protein synthesis inhibitor is continued to be increased on the basis of the original concentration, and the number of concentration increases is 5 times to reach the final concentration. After each increase in the concentration of the protein synthesis inhibitor, the OD600 value is detected after culturing for 24 hours. If the OD600 value has not yet recovered to 4.5-5.0, the cultured strain is transferred to the culture medium containing the same concentration of protein synthesis inhibitor according to the inoculation volume percentage of 1% and continued to be cultured for 24 hours. This is repeated until the OD600 value is restored to 4.5-5.0, and then the concentration of the protein synthesis inhibitor is increased again. The above operation is repeated until the OD600 value at the final concentration is 4.5-5.0. Fermentation culture conditions: shaking culture at 30°C and 220rpm, a total of 40-60 days of continuous evolution.

[0086] Geneticin G418: Initial concentration is 100-200 mg / L. When the OD600 value is restored to 4.5-5.0, the concentration is increased by 200 mg / L. When the OD600 value is restored to 4.5-5.0, the concentration is increased by 200 mg / L. Repeat this process 5 times to the final concentration. Rapamycin: Initial concentration is 3-5 mg / L. When the OD600 value is restored to 4.5-5.0, the concentration is increased by 2 mg / L. When the OD600 value is restored to 4.5-5.0, the concentration is increased by 2 mg / L. Repeat this process 5 times to the final concentration of 13-15 mg / L. Cycloheximide: The initial concentration is 100-200 μg / L. When the OD600 value is restored to 4.5-5.0, the concentration is increased by 200 μg / L. When the OD600 value is restored to 4.5-5.0, the concentration is increased by 200 μg / L. This process is repeated 5 times to a final concentration of 1100-1200 μg / L. Hygromycin B: The initial concentration is 100-200 mg / L. When the OD600 value is restored to 4.5-5.0, the concentration is increased by 400 mg / L. When the OD600 value is restored to 4.5-5.0, the concentration is increased by 400 mg / L. This process is repeated 5 times to a final concentration of 2100-2200 mg / L.

[0087] Table 5 Initial and final concentrations of protein synthesis inhibitors

[0088] Types of protein synthesis inhibitors Initial concentration Final concentration Geneticin G418 100~200mg / L 1100~1200mg / L Rapamycin 3~5mg / L 13~15mg / L Cycloheximide 100~200μg / L 1100~1200μg / L Hygromycin B 100~200mg / L 2100~2200mg / L

[0089] Take an appropriate amount of yeast suspension that has been screened by adaptive evolution of different concentrations of different types of protein synthesis inhibitors, centrifuge and wash, and resuspend with PBS buffer solution. Take 0.5mL of bacterial solution and add it to the EP tube, add an equal amount of 5μmol / L RNA fluorescent dye and mix evenly, culture at 30℃ for 30min, centrifuge and wash, resuspend the bacteria, and obtain RNA-stained bacteria. Use flow cytometry for sorting, and the specific parameters are set to an excitation wavelength of 490nm and an emission wavelength of 530nm. The proportion of sorted yeast cells accounts for about 1% to 2% of yeast cells with high fluorescence intensity. The sorted yeast cells are transferred to YPD medium, incubated at 30℃ for 2 hours, placed on a YPD solid plate, and cultured for 24 hours to obtain a single colony.

[0090] The above single colonies were selected and inoculated in 96-well plates, each culture medium contained 200 μL fermentation medium, and cultured at 30°C, 1200 rpm for 48 hours. Then, an appropriate amount of fermentation broth was taken and diluted appropriately, and OD600 was measured, OD600 = 0.2-0.4. The sample was then centrifuged and treated with 0.05 mol / L sodium hydroxide at 100°C for 20 minutes to release intracellular proteins. The total protein content was determined using the Bradford protein assay kit (Sangon Biotech, Shanghai, China). The protein concentration was calculated using the standard protein curve, and the ratio of protein concentration to OD600 was used to express the protein content in yeast cells. Finally, the three Saccharomyces cerevisiae strains with the highest protein concentration were selected and named strain 1, strain 2 and strain 3.

[0091] Expanded culture: Three high-protein concentration Saccharomyces cerevisiae strains and the control strain (starting strain) were inoculated into a test tube containing 2 mL of fermentation medium and cultured at 220 rpm and 30°C for 12 to 18 hours. Subsequently, they were transferred to a shake flask containing 50 mL of fermentation medium and cultured at 220 rpm and 30°C for 48 hours. The cells were centrifuged under sterile conditions, sterilized, resuspended in 30% glycerol, and stored frozen.

[0092] Example 2

[0093] The strains 1 to 3 obtained in Example 1 were inoculated into a shake flask containing 1L fermentation medium at an inoculum volume percentage of 1%, and cultured at 30°C, 220rpm for 24 hours to obtain a seed culture. 9LH2O and 1mL defoamer were added to a 45L fermenter, the amount of ZnSO4 added to each liter of culture medium was 0.1g, and the amount of CuSO4·5H2O added to each liter of culture medium was 0.012g, and then sterilized at 121°C for 30 minutes. Then all the seed cultures were added to a 45L fermenter to make the initial volume reach 10L, and the initial pH was adjusted to 4.0 to 4.8. The carbon source was 30% mass concentration (30g of carbon source was added to water and fixed to 100mL), consisting of glucose and molasses in a mass ratio of 1:1. The nitrogen source was 20% mass concentration of ammonium sulfate (20g of nitrogen source was added to water and fixed to 100mL). The phosphorus source is ammonium dihydrogen phosphate with a mass concentration of 10% (10g of phosphorus source is added with water to a constant volume of 100mL). The above raw materials are added to the fermentation tank during the fermentation process, and the conditions for the addition are as follows: the carbon source addition rate is controlled to keep the alcohol content in the range of 0.05-0.15% during the fermentation process, and the amount of carbon source added per liter of culture medium is 90g; the phosphorus source is added evenly every hour for the first 10h after the start of fermentation, and the flow rate is 30-60mL / h, and the amount of phosphorus source added per liter of culture medium is 3.5g; the nitrogen source is added in the first 3h after the start of fermentation, 10-12% of the total amount is added; 25-30% of the total amount is added in the 4th to 6th hours; 58-65% of the total amount is added in the 7th to 12th hours, and the amount of nitrogen source added per liter of culture medium is 15g. The pH value of ammonia water is maintained at 5.0, and the fermentation process is ventilated and stirred to control the dissolved oxygen to not less than 8-15%. After fermenting at 28-34°C for 12 hours, the fermentation liquid was taken out and centrifuged at 5000 rpm for 5 minutes. The supernatant was discarded and the heavy phase was taken. Pure water with the same volume of the supernatant was added for washing and continued centrifugation. This was repeated twice to obtain the heavy phase called washed yeast milk, and indicators such as biomass, protein and RNA were measured.

[0094] Table 6 Physiological index data of yeast milk

[0095] project Protein content% Biomass g / L RNA % strain 1 61.4 252 6.92 strain 2 75.2 254 8.28 strain 3 63.7 278 7.38 Starting strain 59.32 249 7.02

[0096] As can be seen from the table above, the protein content of strain 2 increased by 26.7% compared with the starting strain; in terms of biomass, strain 3 was more advantageous, and strains 1 and 2 exceeded the starting strain. In terms of RNA, strain 2 was higher than the starting strain and other strains. It can be seen that the high-protein-yielding strain obtained by the present invention has the characteristics of high protein and low RNA.

[0097] The biomass detection method includes the following steps: accurately taking 10 mL of fermentation liquid into a weighed centrifuge tube, taking it out immediately after centrifugation at 5000 rpm for 5 minutes, pouring out the supernatant, weighing the heavy phase, and obtaining the biomass unit in g / L.

[0098] The method for detecting protein content includes the following steps: accurately weigh 1g yeast milk (weighed to 0.0002g) into a digestive tube, add 2.5g digestion powder, slowly add 10mL concentrated sulfuric acid along the tube wall, and then place it on a digestion device. Digest for about 3 hours until smoke is gone, the solution becomes clear and light yellow, and then continue to heat for 10 minutes. Take out the digestive tube, let it cool, rinse the tube wall with about 30mL distilled water, cool it again, and then transfer it to a 100mL volumetric flask, rinse the digestive tube with a small amount of water 3 times, pour it all into the volumetric flask, add water to the scale, shake well, and the digestive solution is obtained. Accurately draw 25mL of digestive solution into the digestive tube and place it on a distillation device. Take 25mL of boric acid solution in a triangular flask, add 4 to 6 drops of methyl red-bromocresol green mixed indicator as the receiving solution, and place it on the receiving table. Open the circulating water valve, add 25mL of sodium hydroxide solution to the digestion tube, turn on the steam switch, lift the receiving platform, immerse the receiving tube in the receiving liquid, and stop distilling when the receiving liquid is 150-200mL. Put down the receiving platform, turn off the steam and circulating water, rinse the receiving tube mouth with distilled water, and take out the receiving bottle. Titrate the receiving solution with 0.05mol / L sulfuric acid standard solution. The color changes from green to slightly red, which is the end point. Perform a blank test at the same time according to the above method. The protein content is calculated according to the formula:

[0099] X=[c×(V1-V2)×0.01401]÷(W×Ds×25÷100)×100%×6.25

[0100] Where: X-the percentage of protein in the sample, %;

[0101] c-concentration of sulfuric acid standard solution, mol / L;

[0102] V1-volume of sulfuric acid standard solution consumed by titration sample, mL;

[0103] V2-volume of sulfuric acid standard solution consumed in titration blank, mL;

[0104] 0.01401 - the mass of nitrogen in grams equivalent to 1.00 mL of sulfuric acid [c(1 / 2H2SO4)=1.000 mol / L] standard solution;

[0105] W-mass of the sample, g;

[0106] Ds- dry matter content of sample, %;

[0107] 25-volume of sample digestion solution, mL;

[0108] 6.25-Nitrogen to crude protein conversion factor.

[0109] Among them, the RNA detection method includes the following steps: weighing 0.4-0.8g yeast milk sample into a centrifuge tube and weighing it, adding 8mL of cold (0-4°C) 0.25mol / L HClO4, oscillating and mixing, and immediately placing it in 4°C cold water, standing for 15 minutes; centrifuging at 4000rpm for 10 minutes, discarding the supernatant, adding 5mL of 0.5mol / L HClO4 to the precipitate, oscillating and mixing, placing the centrifuge tube in a 70°C water bath, keeping warm for 15 minutes, and oscillating every 5 minutes; centrifuging at 4000rpm for 10 minutes, absorbing 1mL of supernatant, making up the volume to 100mL with distilled water, and mixing.

[0110] Rinse the cuvette with the sample to be tested, fill the cuvette and place it in the spectrophotometer. Wipe the surface clean, measure the absorbance at 260nm, use distilled water as a blank, and record the absorbance. The formula is as follows:

[0111] RNA(%)=(A×100×0.03365×5)÷(m×Ds)×100%

[0112] Where: A-absorbance of sample solution;

[0113] m-the mass of the sample weighed, mg;

[0114] Ds- dry matter content of the sample, %;

[0115] 5-the volume of the solution after adding 0.5mol / L HClO4;

[0116] 0.03365-corresponds to the RNA content in the test solution when the absorbance is 1, mg / mL;

[0117] 100 - dilution factor.

[0118] The dry matter detection method includes the following steps: the sample is directly dried in an oven at 103±2°C, and after losing water, the percentage of the mass after losing water to the mass of the sample is the dry matter percentage.

[0119] Example 3: Sugar conversion rate content analysis

[0120] As one of the main sources of cost for industrial production, the conversion rate of molasses greatly affects the cost control of the production process. The method for determining the sugar conversion rate includes the following steps:

[0121] Take out all the fermentation liquid in the 45L fermenter, centrifuge at 5000rpm for 5min, wash twice to obtain the heavy phase yeast milk and weigh it, which is m3, in g. Measure the dry matter percentage of the yeast milk and multiply it by m3. The weight obtained is the dry weight of the yeast milk m4, in g. The volume of the carbon source before the addition is calculated as V1, in mL, and the volume of the carbon source after the addition is calculated as V2, in mL. Subtract the two (V2 minus V1) to get the volume of the carbon source consumed in the fermentation process. The formula for sugar conversion rate is as follows.

[0122]

[0123] In the formula, the correction factor is 6.67. According to records, theoretically, about 2 g of carbohydrates (i.e., the carbon source) are required for every 1 g of yeast dry solids (i.e., the dry weight of the yeast milk) to grow (see "Yeast Production and Application Manual" by Yu Jingzhi, page 53). The mass concentration of the carbon source in Example 2 is 30%, so the correction factor is 6.67.

[0124] Table 7 Protein content of four high protein concentration strains

[0125] project Sugar conversion rate % strain 1 80.15 strain 2 93.29 strain 3 84.14 Starting strain 79.97

[0126] As can be seen from the table above, strain 2 consumes less carbon source to achieve the same number of cells, and its sugar conversion rate is 13.32% higher than that of the starting strain. The sugar conversion rate of saccharomyces cerevisiae recorded in the prior art is generally around 80%, and the sugar conversion rate of strain 2 is higher.

[0127] Example 4: Strain Sequencing and Identification

[0128] After activating the strain 2 stored in the glycerol tube, the strain was cultured in a shake flask and centrifuged at 5000 rpm for 5 minutes under sterile conditions to obtain the bacterial cells. The morphological characteristics of the bacterial cells: the strain 2 was an oval single cell, some of which had budding; the single colony on the solid culture medium was milky white, shiny, flat, and had neat edges.

[0129] After the genomic DNA of strain 2 was crudely extracted by DNA extraction kit (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., catalog number DP307), PCR amplification was performed using ITS1: 5′-TCCGTAGGTGAACCTGCGG-3′ (SEQ ID NO.2) and ITS4: 5′-TCCTCCGCTTATTGATATGC-3′ (SEQ ID NO.3) as upstream and downstream primers. The PCR reaction parameters were: 25 μL PCR Mix; 1 μL sample; 2 μL ITS1 (10 μM); 2 μL ITS4 (10 μM); 20 μL ddH20; reaction conditions were: 98°C pre-denaturation for 5 min; 98°C denaturation for 10 s; 57°C annealing for 10 s; 72°C extension for 30 s; after 35 cycles; 72°C extension for another 5 min, and storage at 4°C. After the PCR product was qualified, ITS sequencing was performed for identification. The sequence was shown in SEQ ID NO: 1. Homology comparison analysis was performed in the NCBI-GenBank database to identify the species of the sample. The result showed that it was Saccharomyces cerevisiae.

[0130] Strain 2 was named Saccharomyces cerevisiae HP-1 and deposited. The Saccharomyces cerevisiae HP-1 was deposited in the China Center for Type Culture Collection (CCTCC) on August 21, 2024, with a deposit number of CCTCC M20241829.

[0131] Table 8 Sequence Listing

[0132]

[0133]

[0134] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.

Claims

1. A high protein producing yeast strain, characterized in that: The saccharomyces cerevisiae strain is: The Saccharomyces cerevisiae HP-1 strain is deposited in China Center for Type Culture Collection with a deposit number of CCTCCNO: M 20241829.

2. The high protein-producing Saccharomyces cerevisiae strain according to claim 1, wherein: The ITS sequence of the Saccharomyces cerevisiae HP-1 strain is shown in SEQ ID NO.

1.

3. A fermented product, characterized in that The fermented product is obtained by culturing the brewer's yeast strain according to claim 1 or 2.

4. A method for preparing the fermented product according to claim 3, characterized in that: The method comprises the following steps: culturing the brewer's yeast strain according to claim 1 or 2 to obtain the fermentation product; Preferably, the strain is inoculated into a culture medium for seed culture and then fermented; More preferably, the volume percentage of the bacterial strain inoculation is 0.8% to 5%; More preferably, the temperature of seed culture is 28-34°C; and / or the time of seed culture is 20-60h; And / or, more preferably, the fermentation temperature is 28-34° C.; and / or, the fermentation time is 11-15 h.

5. The method for preparing a fermented product according to claim 4, wherein: The culture medium used for seed culture comprises a carbon source, a nitrogen source, inorganic salts and trace elements; Preferably, the culture medium comprises, by weight, 15 to 25 parts of glucose, 3 to 7 parts of (NH4)2SO4, 0.5 to 1.5 parts of MgSO4, 2 to 4 parts of KH2PO4, 0.5 to 1.5 parts of the first mother solution and 0.5 to 1.5 parts of the second mother solution; More preferably, the first mother liquor comprises EDTA, ZnSO4, MnCl2, CoCl2, CuSO4, Na2MoO4, CaCl2, FeSO4, H3BO3 and KI; further preferably, the first mother liquor comprises, by weight, 10 to 20 parts of EDTA, 2 to 6.5 parts of ZnSO4, 0.6 to 0.95 parts of MnCl2, 0.1 to 0.3 parts of CoCl2, 0.1 to 0.3 parts of CuSO4, 0.1 to 0.5 parts of Na2MoO4, 1 to 5 parts of CaCl2, 1 to 3 parts of FeSO4, 1 to 2 parts of H3BO3 and 0.1 to 0.3 parts of KI; And / or, the second mother liquor comprises biotin, p-aminobenzoic acid, nicotinic acid, calcium pantothenate, pyridoxine hydrochloride, VB1 and inositol; more preferably, in parts by weight, the second mother liquor comprises 0.01-0.1 parts of biotin, 0.1-0.5 parts of p-aminobenzoic acid, 0.5-3 parts of nicotinic acid, 1-2 parts of calcium pantothenate, 1-2 parts of pyridoxine hydrochloride, 1-2 parts of VB1 and 10-25 parts of inositol.

6. The method for preparing a fermented product according to claim 4 or 5, wherein: The fermentation culture medium includes a carbon source, a nitrogen source, a phosphorus source and trace elements; Preferably, the fermentation culture medium includes 75-120 parts of carbon source, 13-25 parts of nitrogen source, 2.5-5 parts of phosphorus source and 0.01-2 parts of trace elements in parts by weight; more preferably, the trace elements include 0.01-1 parts of ZnSO4 and 0.01-1 parts of CuSO4; And / or, preferably, a carbon source is added during the fermentation culture process to make the alcohol content in the range of 0.01% to 0.2%; more preferably, the carbon source includes glucose and / or molasses; further preferably, the mass ratio of glucose to molasses is 0 to 7:3 to 10; And / or, preferably, during the fermentation culture process, a phosphorus source is added at a flow rate of 30 to 60 mL / h; more preferably, the phosphorus source comprises ammonium dihydrogen phosphate; further preferably, the mass percentage of the phosphorus source is 10% to 20%; And / or, preferably, the nitrogen source is added in batches during the fermentation culture process; more preferably, the nitrogen source is added in three batches, with the first batch adding 10% to 12% of the total amount, the second batch adding 25% to 30% of the total amount; and the third batch adding 58% to 65% of the total amount; and / or, the first batch adding time is 2 to 5 hours, the second batch adding time is 3 to 5 hours, and the third batch adding time is 4 to 7 hours.

7. A yeast milk, characterized in that: The yeast milk is prepared by the brewer's yeast strain according to claim 1 or 2, the fermented product according to claim 3, or the fermented product prepared by the preparation method of any one of claims 4 to 6.

8. A bacterial agent, characterized in that The bacterial agent is prepared by the brewer's yeast strain according to claim 1 or 2, the fermented product according to claim 3, the fermented product prepared by the method for preparing the fermented product according to any one of claims 4 to 6, or the yeast milk according to claim 7.

9. The bacterial agent according to claim 8, wherein The biomass of the bacterial agent is 250-280 g / L; And / or, the intracellular protein content of the bacterial agent is greater than 75wt% of the dry weight of the bacterial cells; preferably, the intracellular protein content of the bacterial agent is 75wt% to 80wt% of the dry weight of the bacterial cells; And / or, the RNA content of the bacterial agent is 6wt% to 9wt% of the dry weight of the bacteria; preferably, the RNA content of the bacterial agent is 8wt% to 9wt% of the dry weight of the bacteria.

10. A high-protein-yielding Saccharomyces cerevisiae strain, fermented product, yeast milk or bacterial agent used in yeast extract, characterized in that: The high-protein-producing Saccharomyces cerevisiae strain is the high-protein-producing Saccharomyces cerevisiae strain according to claim 1 or 2; The fermented product is the fermented product according to claim 3 or the fermented product prepared by the method for preparing the fermented product according to any one of claims 4 to 6; The fermented milk is the fermented milk according to claim 7; and The bacterial agent is the bacterial agent according to claim 8 or 9.

11. A yeast extract, characterized in that: The yeast extract is prepared by comprising the high-protein brewer's yeast strain of claim 1 or 2, the fermented product of claim 3 or the fermented product prepared by the method for preparing the fermented product of any one of claims 4 to 6, the fermented milk of claim 7 or the bacterial agent of claim 8 or 9.

12. Use of a high-protein-yielding Saccharomyces cerevisiae strain, fermented product, yeast milk or bacterial agent in the preparation of feed, food or health products, characterized in that: The high-protein-producing Saccharomyces cerevisiae strain is the high-protein-producing Saccharomyces cerevisiae strain according to claim 1 or 2; The fermented product is the fermented product according to claim 3 or the fermented product prepared by the method for preparing the fermented product according to any one of claims 4 to 6; The fermented milk is the fermented milk according to claim 7; and The bacterial agent is the bacterial agent according to claim 8 or 9.

13. Use of a high-protein-yielding Saccharomyces cerevisiae strain, fermented product, yeast milk or bacterial agent in the preparation of condiments, characterized in that: The high-protein-producing Saccharomyces cerevisiae strain is the high-protein-producing Saccharomyces cerevisiae strain according to claim 1 or 2; The fermented product is the fermented product according to claim 3 or the fermented product prepared by the method for preparing the fermented product according to any one of claims 4 to 6; The fermented milk is the fermented milk according to claim 7; and The bacterial agent is the bacterial agent according to claim 8 or 9.

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