K. kodamae and use thereof

Abstract

The application belongs to the technical field of microorganisms and particularly relates to a Pichia kudriavzevii and application thereof. Pichia kudriavzevii The Pichia kudriavzevii is Pichia kudriavzevii AMCC 31367 (Pichia kudriavzevii CCTCC NO: M 20251680). The Pichia kudriavzevii has the characteristics of multi-environment tolerance, broad-spectrum substrate applicability and high protein and amino acid content, and can tolerate high-concentration lactic acid, and the yeast can still grow under the lactic acid concentration of 170 g / L, thereby solving the problems of limited raw material selection, low production efficiency and high cost in the development of microbial cell protein, and the strain has application potential in the fields of biological materials, microbial engineering, environmental governance, feed and food processing.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a type of *Pichia kudrica* and its applications. Background Technology

[0002] Microbial protein, as an emerging protein resource, has attracted much attention compared to traditional plant, animal, and insect proteins due to its advantages such as short production cycle, wide availability of raw materials, environmental friendliness, low production cost, and high nutritional value. Microbial protein has been widely used in food processing, feed manufacturing, pharmaceutical production, and biotechnology.

[0003] In the production of microbial cell protein, microorganisms often face a series of complex environmental stressors, such as temperature fluctuations, pH changes, and osmotic pressure differences. Simultaneously, byproducts such as lactic acid, acetic acid, and ethanol produced by microbial metabolism during the production process or fermentation can exert a stress effect on microorganisms when their concentrations reach a certain threshold, thereby interfering with their normal growth and metabolic activities. Therefore, in order for microorganisms to maintain stable growth and metabolism, the production strains must possess corresponding tolerance to adapt to these specific environmental conditions.

[0004] Kudria zwibichi yeast ( Pichia kudriavzevii As a non-brewing yeast species widely distributed in nature, *Pichia kudriezvichi* has attracted much attention in recent years due to its unique physiological characteristics and potential wide-ranging applications. However, the existing *Pichia kudriezvichi* strain has limited substrate utilization, its tolerance to multiple stress environments needs improvement, and it contains relatively low protein content. Therefore, developing a high-protein *Pichia kudriezvichi* strain that combines multi-substrate utilization and high tolerance to multiple environmental stresses is of great significance for addressing the problems faced in industrial microbial production, such as limited raw material selection, low production efficiency, high costs, and significant environmental impact. Simultaneously, it can effectively address challenges such as reduced strain activity caused by changes in environmental conditions during practical applications, thereby promoting the sustainable development of the microbial protein industry and significantly improving overall production efficiency. Summary of the Invention

[0005] The technical problem solved by this invention is that the existing Kudria zwibbi yeast has limited substrate utilization ability, its tolerance to multiple stress environments needs to be improved, and its protein content is relatively low.

[0006] To address the aforementioned technical problems, this invention provides a strain of *Kudriazwibichthys* and its applications.

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

[0008] In a first aspect, the present invention provides a strain of *Pichia kudriezvichi*, wherein the strain is *Pichia kudriezvichi* AMCC 31367 (… Pichia kudriavzevii AMCC 31367), accession number CCTCC NO: M20251680.

[0009] Preferably, the temperature that the *Pichia kudriezvichi* strain can tolerate is 15-40°C; more preferably, the temperature that the *Pichia kudriezvichi* strain can tolerate is 20-40°C.

[0010] And / or, preferably, the Kudriazwibich yeast strain can tolerate a pH of 2-9; more preferably, the Kudriazwibich yeast strain can tolerate a pH of 3-8.

[0011] And / or, preferably, the Kudria zwibichi strain can tolerate sugar concentrations of 2%-60%.

[0012] And / or, preferably, the *Pichia kudrica* strain can tolerate an ethanol concentration of less than or equal to 10%.

[0013] And / or, preferably, the concentration of phenylethanol that the *Pichia kudrica* strain can tolerate is less than or equal to 4 g / L.

[0014] And / or, preferably, the *Pichia kudrica* strain can tolerate an acetic acid concentration of less than or equal to 8 g / L; and compared to conditions without acetic acid stress, the biomass of the strain increases by 12%-18% under culture conditions containing an acetic acid concentration of 2-8 g / L.

[0015] And / or, preferably, the *Pichia kudrica* strain can tolerate a lactic acid concentration of less than or equal to 170 g / L; a growth inhibition rate of ≤48% at 160 g / L; and a 14%-28% increase in biomass under culture conditions containing 60-140 g / L lactic acid compared to conditions without lactic acid stress.

[0016] And / or, preferably, the carbon sources that the *Pichia kudrica* strain can utilize include one or more substances selected from the group consisting of xylose, fructose, glucose, maltose, sucrose, misobiose, mesotriose, stachyose, inulin, pullulan, xylan, soluble starch, citric acid, succinic acid, malic acid, lactic acid, and glycerol.

[0017] Secondly, the present invention provides a method for preparing a culture of the Kudriazwibich yeast strain, comprising the following steps: culturing the Kudriazwibich yeast strain.

[0018] Preferably, the method for preparing the culture of the *Pichia kudrica* strain includes the following steps:

[0019] Step 1: Inoculate the Kudria zwibich yeast strain into the culture medium for cultivation to obtain seed culture;

[0020] Step 2: Inoculate the seed liquid into a culture medium for culture to obtain a culture of the Kudriazwibich yeast strain.

[0021] And / or, preferably, in step 1, the culture conditions are: temperature of 25-35℃, time of 20-24h, and / or rotation speed of 150-180rpm.

[0022] And / or, preferably, in step 1, the culture medium comprises, by weight, 8-12 parts yeast extract, 18-22 parts glucose, 18-22 parts peptone and 800-1200 parts water.

[0023] And / or, preferably, in step 2, the culture conditions are: temperature of 25-35℃, time of 20-24h, and / or rotation speed of 150-180rpm.

[0024] And / or, preferably, in step 2, the culture medium comprises, by weight, 80-120 parts glucose, 18-22 parts yeast extract, 0.8-1.2 parts MgSO4, 0.8-1.2 parts KH2PO4 and 800-1200 parts water.

[0025] And / or, preferably, in step 2, the pH of the culture medium is 4.6-5.

[0026] And / or, preferably, in step 2, the inoculation amount of seed liquid is 0.5%-1.2% based on the volume of the culture medium.

[0027] Thirdly, the present invention provides a culture of *Kudriazwibichthys* strain, which is prepared by a method for preparing a culture of *Kudriazwibichthys* strain.

[0028] Fourthly, the present invention provides a method for preparing yeast milk, which includes the following steps:

[0029] The culture of the aforementioned Kudriazwibichi yeast strain was subjected to solid-liquid separation to obtain yeast milk;

[0030] Preferably, the solid-liquid separation is centrifugal separation;

[0031] More preferably, the centrifugation conditions are: a rotation speed of 6000-8000 rpm and / or a time of 5-10 min.

[0032] Fifthly, the present invention provides a yeast milk, which is prepared by the yeast milk preparation method.

[0033] Preferably, the moisture content of the yeast milk is 78%-82%.

[0034] And / or, preferably, the yeast milk contains 65%-70% crude protein based on its dry weight; more preferably, the yeast milk contains 65.85%-68.10% crude protein based on its dry weight.

[0035] And / or, preferably, the content of essential amino acids, based on the dry weight of the yeast milk, is 23%-26%; more preferably, the content of essential amino acids, based on the dry weight of the yeast milk, is 23.27%-25.86%; further preferably, the essential amino acids include threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, and tryptophan; more preferably, the threonine content, based on the dry weight of the yeast milk, is 3.3%-3.6%; and / or, the valine content is 3.2%-3.4%; and / or, the methionine content is 0.1%-0.2%; and / or, the isoleucine content is 2.7%-3.3%; and / or, the leucine content is 4.35%-5.2%; and / or, the phenylalanine content is 3.0%-3.2%; and / or, the lysine content is 6.0%-6.3%; and / or, the tryptophan content is 0.62%-0.66%.

[0036] And / or, preferably, the content of non-essential amino acids in the yeast milk is 34%-37% based on the dry weight; more preferably, the content of non-essential amino acids in the yeast milk is 34.55%-36.85% based on the dry weight; even more preferably, the non-essential amino acids include histidine, arginine, aspartic acid, serine, glutamic acid, glycine, alanine, cysteine, tyrosine, and proline; more preferably, the content of histidine is 1.5%-1.7% based on the dry weight of the yeast milk; and / or, the content of arginine is 2%. 80%-3.15%; and / or, aspartic acid content is 6.90%-7.15%; and / or, serine content is 3.1%-3.3%; and / or, glutamic acid content is 8.3%-8.5%; and / or, glycine content is 3.0%-3.2%; and / or, alanine content is 4.60%-4.75%; and / or, cystine content is 0.35%-0.50%; and / or, tyrosine content is 1.8%-2.2%; and / or, proline content is 2.2%-2.4%.

[0037] In a sixth aspect, the present invention provides a microbial agent comprising the said Kudriazwipichia strain, a culture of the said Kudriazwipichia strain, or the yeast milk.

[0038] In a seventh aspect, the present invention provides a yeast extract comprising the said Kudriazwipichia strain, a culture of the said Kudriazwipichia strain, or the yeast milk.

[0039] Eighthly, the present invention provides the application of the said Kudriazwipitch yeast strain, the culture of the said Kudriazwipitch yeast strain, the yeast milk, the yeast agent or the yeast extract in the fermentation of materials under stress conditions;

[0040] Preferably, the stress conditions include one or more substances selected from the group consisting of temperature stress, acid-base stress, sugar stress, ethanol stress, phenylethanol stress, acetic acid stress, and lactic acid stress.

[0041] In a ninth aspect, the present invention provides the use of the aforementioned *Kudriazwibich yeast* strain, a culture of the aforementioned *Kudriazwibich yeast* strain, the yeast milk, the yeast agent, or the yeast extract in the preparation of feed, food, or wastewater treatment products;

[0042] Preferably, the food includes baked goods, brewed foods, or beverages.

[0043] In a tenth aspect, the present invention provides the application of the aforementioned *Pichia kudrica* strain in the preparation of protein products.

[0044] Beneficial effects of the present invention

[0045] (1) The Pichia pastoris strain AMCC 31367 described in this invention has broad substrate applicability. Specifically, it can efficiently utilize organic acids such as citric acid, succinic acid, malic acid and lactic acid, and sugars such as pullulan, xylan and soluble starch. In addition, it can be cultured using glucose, fructose, xylose, stachyose, maltose, melibiose, sucrose, melitriose, inulin or glycerol as the sole carbon source.

[0046] (2) The Kudria zweipichia strain AMCC 31367 described in this invention can tolerate 170 g / L lactic acid; the growth inhibition rate is ≤48% under 160 g / L conditions; and compared with no lactic acid stress conditions, the biomass of the strain increased by 14%-28% under culture conditions containing lactic acid concentration of 60-140 g / L.

[0047] (3) The Kudria zweipichia strain AMCC 31367 described in this invention can tolerate 8 g / L acetic acid; and its biomass can increase by 12%-18% under the condition of a culture medium containing acetic acid concentration of 2-8 g / L.

[0048] (4) The Kudria zweipichia strain AMCC 31367 described in this invention can grow under environmental pressure conditions such as temperature of 15-40℃, pH of 2-9, ethanol concentration of 0%-10%, sugar concentration of 2%-60%, phenylethanol concentration of 0-4 g / L, acetic acid concentration of 0-8 g / L or lactic acid concentration of 0-170 g / L.

[0049] (5) The protein content of the Pichia kudrica strain AMCC 31367 described in this invention is above 66.69%, and the amino acid content is above 61%.

[0050] (6) The *Pichia pastoris* strain AMCC 31367 described in this invention possesses characteristics such as multi-environment tolerance, broad substrate applicability, and high protein and amino acid content, alleviating problems such as limited raw material selection, low production efficiency, and high costs in the development of microbial cell proteins. Furthermore, this strain does not require genetic engineering modification, avoiding the risks and complexities that may arise from genetic engineering technology. Simultaneously, the strain and production method of this application exhibit good stability and reproducibility, making them suitable for large-scale production. Moreover, this strain has application potential in fields such as biomaterials, microbial engineering, environmental remediation, feed, and food processing.

[0051] Strain Preservation Information

[0052] The present invention describes the Kudria zweipichia AMCC 31367 ( Pichia kudriavzevii AMCC31367 was deposited at the China Center for Type Culture Collection (CCTCC) on July 23, 2025, with accession number CCTCC NO: M20251680. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: (027) 68754052. Attached Figure Description

[0053] Figure 1 This is a colony morphology diagram of *Kudriazwibichthys*.

[0054] Figure 2 Microscopic morphology of *Kudriazwibichthys*. Detailed Implementation

[0055] As described above, the purpose of this invention is to provide a Kudriazwibich yeast and its application.

[0056] Specifically, the present invention provides the following technical solution:

[0057] Technical Solution 1: A strain of Pichia kudriezvichia, wherein the strain is Pichia kudriezvichia AMCC 31367 ( Pichia kudriavzeviiAMCC 31367), accession number CCTCC NO: M20251680.

[0058] Technical Solution 2: According to the *Pichia kudriezvichia* strain described in Technical Solution 1, the *Pichia kudriezvichia* strain can tolerate temperatures of 15-40°C; preferably, the *Pichia kudriezvichia* strain can tolerate temperatures of 20-40°C; more preferably, in some specific embodiments, the *Pichia kudriezvichia* strain can tolerate temperatures of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40°C, or temperatures within a range of any two of the above specific values ​​as endpoints;

[0059] And / or, the Kudriazwijki yeast strain can tolerate a pH of 2-9; preferably, the Kudriazwijki yeast strain can tolerate a pH of 3-8; more preferably, in some specific embodiments, the Kudriazwijki yeast strain can tolerate a pH of 2, 3, 4, 5, 6, 7, 8 or 9, or a pH range containing any two of the above specific values ​​as endpoints;

[0060] And / or, the sugar concentration that the *Pichia kudriezvichi* strain can tolerate is 2%-60%; preferably, in some specific embodiments, the sugar concentration that the *Pichia kudriezvichi* strain can tolerate is 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55% or 60%, or contains sugar concentrations within the numerical range formed by any two of the above specific values ​​as endpoints;

[0061] And / or, the *Pichia kudrica* strain can tolerate an ethanol concentration of less than or equal to 10%;

[0062] And / or, the concentration of phenylethanol that the Kudria zwibich yeast strain can tolerate is less than or equal to 4 g / L;

[0063] And / or, the *Pichia kudrica* strain can tolerate an acetic acid concentration of less than or equal to 8 g / L; and compared to conditions without acetic acid stress, the biomass of the strain increases by 12%-18% under culture conditions containing an acetic acid concentration of 2-8 g / L;

[0064] And / or, the *Pichia kudrica* strain can tolerate lactic acid concentrations of less than or equal to 170 g / L; growth inhibition rate of ≤48% at 160 g / L; and compared to no lactic acid stress conditions, the biomass of the strain increased by 14%-28% under culture conditions containing lactic acid concentrations of 60-140 g / L.

[0065] Technical Solution 3: The *Pichia kudriezvichi* strain according to Technical Solution 1 or 2, wherein the carbon source that the *Pichia kudriezvichi* strain can utilize includes one or more substances selected from the group consisting of xylose, fructose, glucose, maltose, sucrose, misobiose, mesenteroside, stachyose, inulin, pullulan, xylan, soluble starch, citric acid, succinic acid, malic acid, lactic acid, and glycerol.

[0066] Technical Solution 4: The application of the *Pichia kudrica* strain described in any one of Technical Solutions 1-3 in the preparation of protein products, wherein the protein product is a product containing protein.

[0067] Technical Solution 5: A method for preparing a culture of *Pichia kudriezvichi* strain, comprising the following steps: culturing the *Pichia kudriezvichi* strain described in any one of technical solutions 1-3;

[0068] Preferably, the method for preparing the culture of the *Pichia kudrica* strain includes the following steps:

[0069] Step 1: Inoculate the Kudria zwibich yeast strain into the culture medium for cultivation to obtain seed culture;

[0070] Step 2: Inoculate the seed liquid into a culture medium for culture to obtain a culture of the Kudriazwibich yeast strain.

[0071] Technical Solution 6: The method for preparing the culture of the Kudria zwibbiac yeast strain according to Technical Solution 5, wherein, in step 1, the culture conditions are: temperature of 25-35℃, time of 20-24h, and / or rotation speed of 150-180rpm;

[0072] And / or, in step 1, the culture medium comprises, by weight, 8-12 parts yeast extract, 18-22 parts glucose, 18-22 parts peptone and 800-1200 parts water;

[0073] And / or, in step 2, the culture conditions are: temperature 15-40℃, time 20-150h, and / or, rotation speed 150-180rpm; and / or, in step 2, the culture medium comprises, by weight, 8-12 parts yeast extract, 18-600 parts glucose, 18-22 parts peptone, and 800-1200 parts water; and / or, in step 2, the pH of the culture medium is 2-9; and / or, in step 2, the inoculum volume of the seed culture is 0.5%-1.2% by volume of the culture medium.

[0074] Alternatively, in step 2, the cultivation conditions are: a temperature of 25-35℃, a time of 20-24h, and / or a rotation speed of 150-180rpm; and / or, by weight, the culture medium comprises 80-120 parts glucose, 18-22 parts yeast extract, 0.8-1.2 parts MgSO4, 0.8-1.2 parts KH2PO4, and 800-1200 parts water; and / or, the pH of the culture medium is 4.6-5; and / or, by volume of the culture medium, the inoculum size of the seed culture is 0.5%-1.2%.

[0075] Technical Solution 7: A culture of *Pichia kudriezvichi* strain, which is prepared by the method for preparing a culture of *Pichia kudriezvichi* strain described in Technical Solution 5 or 6.

[0076] Technical Solution 8: A method for preparing yeast milk, comprising the following steps:

[0077] The culture of the Kudriazwibichi yeast strain obtained by technical solution 5 or 6 is subjected to solid-liquid separation to obtain yeast milk;

[0078] Preferably, the solid-liquid separation is centrifugal separation;

[0079] More preferably, the centrifugation conditions are: a rotation speed of 6000-8000 rpm and / or a time of 5-10 min.

[0080] Technical Solution 9: A yeast milk, which is prepared by the yeast milk preparation method described in Technical Solution 8;

[0081] Preferably, the moisture content of the yeast milk is 78%-82%;

[0082] And / or, preferably, the crude protein content, based on the dry weight of the yeast milk, is 65%-70%; more preferably, the crude protein content, based on the dry weight of the yeast milk, is 65.85%-68.10%.

[0083] And / or, preferably, the content of essential amino acids in the yeast milk is 23%-26% based on the dry weight; more preferably, the content of essential amino acids in the yeast milk is 23.27%-25.86% based on the dry weight; further preferably, the essential amino acids include threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, and tryptophan; even more preferably, the threonine content is 3.3%-3.6% based on the dry weight of the yeast milk; and / or, the valine content is 3.2%-3.4%; and / or, the methionine content is 0.1%-0.2%; and / or, the isoleucine content is 2.7%-3.3%; and / or, the leucine content is 4.35%-5.2%; and / or, the phenylalanine content is 3.0%-3.2%; and / or, the lysine content is 6.0%-6.3%; and / or, the tryptophan content is 0.62%-0.66%;

[0084] And / or, preferably, the content of non-essential amino acids in the yeast milk, based on its dry weight, is 34%-37%; more preferably, the content of non-essential amino acids in the yeast milk, based on its dry weight, is 34.55%-36.85%; further preferably, the non-essential amino acids include histidine, arginine, aspartic acid, serine, glutamic acid, glycine, alanine, cysteine, tyrosine, and proline; even more preferably, the content of histidine in the yeast milk, based on its dry weight, is 1.5%-1.7%; and / or, the content of arginine is... 2.80%-3.15%; and / or, aspartic acid content of 6.90%-7.15%; and / or, serine content of 3.1%-3.3%; and / or, glutamic acid content of 8.3%-8.5%; and / or, glycine content of 3.0%-3.2%; and / or, alanine content of 4.60%-4.75%; and / or, cystine content of 0.35%-0.50%; and / or, tyrosine content of 1.8%-2.2%; and / or, proline content of 2.2%-2.4%.

[0085] Technical Solution 10: A microbial agent comprising the *Pichia kudriazinon* strain described in any one of Technical Solutions 1-3, a culture of the *Pichia kudriazinon* strain described in Technical Solution 7, or the yeast milk described in Technical Solution 9.

[0086] Technical Solution 11: A yeast extract comprising the *Kudriazwibich yeast* strain described in any one of Technical Solutions 1-3, a culture of the *Kudriazwibich yeast* strain described in Technical Solution 7, or the yeast milk described in Technical Solution 9.

[0087] Technical Solution 12: The application of any one of the following technical solutions 1-3: the strain of *Pichia kudriazina*, the culture of the strain of *Pichia kudriazina* described in technical solution 7, the yeast milk described in technical solution 9, the inoculant described in technical solution 10, or the yeast extract described in technical solution 11 in the fermentation of materials under stress conditions.

[0088] Preferably, the stress conditions include one or more substances selected from the group consisting of temperature stress, acid-base stress, sugar stress, ethanol stress, phenylethanol stress, acetic acid stress, and lactic acid stress.

[0089] Technical Solution 13: The application of any one of the following technical solutions 1-3: the *Kudriazwipitch* strain, the culture of the *Kudriazwipitch* strain of technical solution 7, the yeast milk of technical solution 9, the microbial agent of technical solution 10, or the yeast extract of technical solution 11 in the preparation of feed, food, or wastewater treatment products;

[0090] Preferably, the food includes baked goods, brewed foods, or beverages.

[0091] This invention does not limit the source of peptone; any commercially available or prepared yeast extract can be used. Preferably, the inventors have found that commercially available or prepared peptone with a total nitrogen content ≥12% and / or an amino nitrogen content ≥2% can be used in this invention. More preferably, the total nitrogen content of the peptone is 12%~17%, and / or the amino nitrogen content is 2%~5%. Even more preferably, the peptone content is 30%~40%.

[0092] In some specific embodiments, the present invention does not limit the source of the yeast extract powder; any commercially available or prepared yeast extract powder can be used in the present invention. Preferably, the inventors have found that as long as the total nitrogen content of the commercially available or prepared yeast extract powder is ≥10.0% and / or the amino nitrogen content is ≥5.0%, it can be used in the present invention. More preferably, the total nitrogen content of the yeast extract powder is 10%~15%, and / or the amino nitrogen content is 5%~8%.

[0093] More preferably, the yeast extract contains 40,700-40,900 mg / kg of trace elements by weight; even more preferably, the trace elements contain 31,906-31,916 mg / kg potassium, 5,733-5,743 mg / kg sodium, 350-360 mg / kg calcium, 2,667-2,677 mg / kg magnesium, 75-85 mg / kg zinc and / or 75-85 mg / kg iron; still more preferably, the trace elements contain 31,911.66 mg / kg potassium, 5,738.91 mg / kg sodium, 355.25 mg / kg calcium, 2,673.59 mg / kg magnesium, 80.94 mg / kg zinc and / or 80.21 mg / kg iron.

[0094] And / or, more preferably, the yeast extract contains 5100-5500 ppm of vitamins by weight; even more preferably, the yeast extract contains 2-3 ppm of vitamin B1, 35-42 ppm of vitamin B2, 110-120 ppm of vitamin B5, 15-21 ppm of vitamin B6, 5-11 ppm of vitamin B7, 24-30 ppm of vitamin B9, 0.17-0.27 μg / 100g of vitamin B12, 3150-3250 ppm of choline, and inositol content... The content of vitamin B1 is 1520~1620ppm, and / or the content of niacin is 300~350ppm; more preferably, the yeast extract powder includes: vitamin B1 content of 2.3ppm, vitamin B2 content of 38.8ppm, vitamin B5 content of 115.0ppm, vitamin B6 content of 18.0ppm, vitamin B7 content of 7.9ppm, vitamin B9 content of 26.7ppm, vitamin B12 content of 0.23μg / 100g, choline content of 3206ppm, inositol content of 1577.7ppm, and / or niacin content of 328ppm;

[0095] And / or, more preferably, the yeast extract comprises 58% to 64% hydrolyzed amino acids by weight; even more preferably, the yeast extract comprises 61.21% hydrolyzed amino acids.

[0096] The method for detecting vitamin B1 content refers to the People's Republic of China National Standard GB5009.84-2016 National Food Safety Standard - Determination of Vitamin B1 in Food - Method I - High Performance Liquid Chromatography.

[0097] The method for detecting vitamin B2 content refers to the People's Republic of China National Standard GB5009.85—2016 National Food Safety Standard - Determination of Vitamin B2 in Food - Method I - High Performance Liquid Chromatography.

[0098] The method for detecting vitamin B5 content refers to the People's Republic of China National Standard GB5009.210-2023, National Food Safety Standard, Determination of Pantothenic Acid in Food, Method III, Microbiological Method.

[0099] The method for detecting vitamin B6 content refers to the People's Republic of China National Standard GB5009.154-2023, National Food Safety Standard, Determination of Vitamin B6 in Food, Method III, High Performance Liquid Chromatography-Fluorescence Detection.

[0100] The method for detecting vitamin B7 content refers to the People's Republic of China National Standard GB5009.259-2023, National Food Safety Standard, Determination of Biotin in Food, Method II, Microbiological Method.

[0101] The method for detecting vitamin B9 content refers to the National Standard of the People's Republic of China GB5009.211-2022, National Food Safety Standard, Determination of Folic Acid in Food.

[0102] The method for detecting vitamin B12 content refers to the People's Republic of China National Standard GB5009.285-2022 National Food Safety Standard - Determination of Vitamin B12 in Food - Method I - Liquid Chromatography.

[0103] The method for detecting choline content refers to the People's Republic of China National Standard GB5413.20-2022 Food Safety National Standard for the Determination of Choline in Infant Foods and Dairy Products, Method III: Liquid Chromatography-Tandem Mass Spectrometry.

[0104] The method for detecting inositol content refers to the People's Republic of China National Standard GB5009.270-2023, National Food Safety Standard, Determination of Inositol in Food, Method I: Gas Chromatography.

[0105] The method for detecting niacin content refers to the People's Republic of China National Standard GB5009.89-2023, National Food Safety Standard, Determination of Niacin and Nicotinamide in Food, Method I, High Performance Liquid Chromatography.

[0106] The method for detecting total nitrogen content refers to the National Standard of the People's Republic of China GB5009.5-2016, National Food Safety Standard – Determination of Protein in Food – Method I – Kjeldahl Nitrogen Method.

[0107] The method for detecting amino acid nitrogen content refers to the People's Republic of China National Standard GB5009.235-2016, National Food Safety Standard – Determination of Amino Acid Nitrogen in Food – Method I – Acidity Meter Method.

[0108] The method for detecting ash content refers to the People's Republic of China National Standard GB5009.4-2016, National Food Safety Standard – Determination of Ash in Food – Method I – Determination of Total Ash in Food.

[0109] The method for detecting sodium chloride content refers to the People's Republic of China National Standard GB5009.44-2016, National Food Safety Standard, Determination of Chloride in Food, Method I: Potentiometric Titration.

[0110] The method for detecting moisture content refers to the People's Republic of China National Standard GB5009.3-2016 Food Safety National Standard - Determination of Moisture in Food - Method I - Direct Drying Method.

[0111] The detection method for peptone content refers to Angel Yeast Co., Ltd.'s enterprise standard Q / YB.J02.148-2017 Biological Preparations Peptone. The peptone content was determined according to the method specified in Appendix A of GB / T22492-2008, and the proportion of the peak area with a relative molecular mass in the range of 500-3000 Da to the sum of all peak areas was calculated using the peak area normalization method.

[0112] Unless otherwise stated, all reagents / instruments used in the embodiments and comparative examples of this invention are conventional commercially available products. Information on the experimental materials and instruments used in this invention is shown in the table below:

[0113] Table 1 Experimental Materials / Instruments and Manufacturers

[0114]

[0115]

[0116] Table 2 Instruments and Manufacturers

[0117]

[0118] In this invention, the culture medium components involved in the embodiments are as follows:

[0119] The preparation method of YPD solid culture medium includes the following steps: 10 g of yeast extract powder (model FM888), 20 g of glucose, 20 g of peptone (model FP328), 20 g of agar and water are mixed and the volume is adjusted to 1000 mL. The mixture is then sterilized at 115℃ for 20 min to obtain YPD solid culture medium.

[0120] The preparation method of YPD liquid culture medium includes the following steps: take 10g of yeast extract powder (model FM888), 20g of glucose, 20g of peptone (model FP328) and water, mix them and make up to 1000 mL, sterilize at 115℃ for 20 min to prepare YPD liquid culture medium.

[0121] The preparation method of the expansion culture medium includes the following steps: take 100 g of glucose, 20 g of yeast extract (model FM888), 1 g of MgSO4, 1 g of KH2PO4 and 1000 mL of water, mix them, adjust the pH to 4.8, sterilize at 115℃ for 20 min to prepare the expansion culture medium.

[0122] YNB culture medium comprises, by weight: 4900-5100 parts ammonium sulfate, 0.001-0.003 parts biotin, 0.001-0.003 parts calcium pantothenate, 0.001-0.003 parts folic acid, 1-3 parts inositol, 0.2-0.5 parts niacin, 0.1-0.3 parts para-aminobenzoic acid, 0.3-0.5 parts pyridoxine hydrochloride, 0.1-0.3 parts riboflavin, and 0.2 parts thiamine hydrochloride. -0.5 parts, boric acid 0.4-0.6 parts, copper sulfate 0.02-0.05 parts, potassium iodide 0.05-0.15 parts, ferric chloride 0.1-0.3 parts, manganese sulfate 0.3-0.5 parts, sodium molybdate 0.1-0.3 parts, zinc sulfate 0.3-0.5 parts, potassium dihydrogen phosphate 950-1050 parts, magnesium sulfate 490-510 parts, sodium chloride 95-105 parts, and calcium chloride 95-105 parts.

[0123] The method for detection using an ELISA reader includes the following steps: Place the culture plate containing the bacterial solution to be tested into the inlet of the ELISA reader, set the detection OD to 600nm, and the OD can be detected. 600 value.

[0124] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0125] Example 1: Isolation, purification and identification of yeast strains

[0126] A sample of homemade rice wine lees from Qiaopi Village, Tiandeng County, Chongzuo City, Guangxi Zhuang Autonomous Region, was dissolved in sterile water and mixed thoroughly to obtain a bacterial suspension. The bacterial suspension was then diluted 10-fold by volume to prepare 10% concentrations of the original solution. -5 and 10 -6 The bacterial suspension (1 mL of bacterial suspension is added to 9 mL of sterile water and mixed well, which is one dilution, resulting in a 10⁻⁶ dilution). -1Each subsequent dilution is based on a 10-fold volume dilution of the sample from the previous dilution gradient, and so on. The sample is then spread onto YPD solid medium and incubated at 30°C for 24-48 h. Single colonies are picked, streaked for purification, and inoculated onto YPD solid medium. The samples are then stored at 4°C.

[0127] Observation revealed that the colonies of a selected yeast strain were white, round, entire, viscous, dry, and opaque; the colonies were easy to pick up. Figure 1 As shown. The microscopic morphology observed under a microscope is that the cells are oval, elliptical, or elongated, single or paired, without mycelium, and measuring (2.2-4.5) × (5.3-7.5) μm (width × length). They reproduce through multipolar budding, such as... Figure 2 As shown.

[0128] The genome of the yeast strain was extracted and molecularly identified using a PCR instrument. ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', sequence shown in SEQ ID NO. 2) was used as the upstream primer, and ITS4 (5'-TCCTCC GCTTAT TGATATGC-3', sequence shown in SEQ ID NO. 3) was used as the downstream primer for PCR amplification. The PCR reaction system consisted of: 25 μL of Mix (2xTaq PCRMix, sold by Tiangen Biotech (Beijing) Co., Ltd.); 3 μL of sample; 2 μL of NL1; 2 μL of NL4; and 18 μL of ddH2O. The PCR amplification program conditions were: 94℃ pre-denaturation for 5 min; then, 94℃ denaturation for 30 s, 55℃ annealing for 45 s, and 72℃ extension for 90 s, repeated for 30 cycles; finally, a final extension at 72℃ for 10 min. After passing PCR product testing, the product was identified by ITS sequencing using 1% gel electrophoresis. The sequence is shown as SEQ ID NO.1 in Table 12. Homology comparison analysis was performed in the NCBI-GenBank database. Combined with morphological analysis and molecular identification, the result showed that it is *Pichia kudrica* (Kudela stolonifera). Pichia Kudriavzevii ).

[0129] The strain was named Pichia kudrica, AMCC 31367. Pichia kudriavzevii The *Pichia kudrica* AMCC 31367 was deposited at the China Center for Type Culture Collection (CCTCC) on July 23, 2025, with accession number CCTCCNO: M 20251680.

[0130] Example 2: Substrate utilization characteristics analysis of Pichia pastoris strain AMCC 31367

[0131] The substrate utilization characteristics analysis of Pichia pastoris strain AMCC 31367 included the following steps:

[0132] Step 1: Inoculate the Kudria zwibbiac yeast AMCC 31367 strain into a test tube containing 5m LYPD liquid medium and culture it at 30℃ and 180rpm for 20h to prepare the seed culture.

[0133] Step 2: Centrifuge the seed culture at 12000 rpm for 3 minutes, remove the supernatant, resuspend in an equal volume of sterile purified water (the same volume as the removed supernatant), then centrifuge again at 12000 rpm for 3 minutes, remove the supernatant, and add another equal volume of sterile purified water. The purpose of this step is to wash away any components from the original culture medium.

[0134] Step 3: Based on the volume of the culture medium, inoculate the resuspended seed solution into a 96-well cell culture plate containing 200 μL of liquid culture medium with different carbon sources at an inoculation rate of 1%. Incubate at 30℃ and 180 rpm for 48 h to obtain the bacterial culture.

[0135] The preparation method of culture media containing different carbon sources includes the following steps: Take 0.67g of YNB culture medium, add 100mL of distilled water and 2g of carbon source, and filter to remove bacteria. The carbon source can be xylose, fructose, glucose, maltose, sucrose, mesquite, stachyose, inulin, pullulan, xylan, soluble starch, citric acid, succinic acid, malic acid, lactic acid, or glycerol. For example, the preparation of a culture medium using fructose as a carbon source includes the following steps: Take 0.67g of YNB culture medium, add 100mL of distilled water and 2g of fructose, and filter to remove bacteria.

[0136] YNB medium was purchased from Beijing Solarbio Science & Technology Co., Ltd., and is a white or near-white powder. The YNB medium comprises: 5g ammonium sulfate, 2μg biotin, 2μg calcium pantothenate, 2μg folic acid, 2000μg inositol, 400μg niacin, 200μg para-aminobenzoic acid, 400μg pyridoxine hydrochloride, 200μg riboflavin, 400μg thiamine hydrochloride, 500μg boric acid, 40μg copper sulfate, 100μg potassium iodide, 200μg ferric chloride, 400μg manganese sulfate, 200μg sodium molybdate, 400μg zinc sulfate, 1g potassium dihydrogen phosphate, 0.5g magnesium sulfate, 0.1g sodium chloride, and 0.1g calcium chloride.

[0137] The OD (optical density) value of the bacterial suspension obtained by measuring it at 600 nm using an ELISA reader is called OD. 600 The OD value was measured in the bacterial culture immediately after inoculation with the seed culture in step 3, i.e., after 0 hours of culture. 600The value is recorded as OD1, and the OD of the bacterial culture cultured for 48 hours in step 3 is measured. 600 The value is denoted as OD2, OD 600 A growth rate exceeding 100% is considered growth. Among these, OD... 600 The formula for calculating the growth rate of the value is as follows:

[0138]

[0139] In the formula: OD1 represents the OD of the bacterial culture immediately after inoculation with the seed culture in step 3, i.e., after 0 hours of culture. 600 value;

[0140] OD2 represents the OD of the bacterial culture obtained after 48 hours of cultivation in step 3. 600 value;

[0141] OD 空白 OD of liquid culture medium containing carbon source without added bacterial solution 600 value.

[0142] Table 3 Substrate utilization characteristics analysis

[0143]

[0144] In Table 3, "+" indicates OD. 600 A growth rate exceeding 100% is considered growth.

[0145] Table 3 shows that *Pichia kudriezvichirosa* strain AMCC 31367 can utilize 17 carbon sources, indicating its broad substrate applicability. The strain can efficiently utilize various organic acids such as citric acid, succinic acid, malic acid, and lactic acid, demonstrating its ability to use organic acids as carbon or energy sources, thus enabling it to function in different industrial environments. *Pichia kudriezvichirosa* strain AMCC 31367 can also effectively utilize xylose, soluble starch, pullulan, and xylan (in flour, xylan is a major non-starch polysaccharide, usually existing in an insoluble form, which affects flour processing properties and baking quality. Degrading xylan can improve dough processing properties and optimize the quality and nutritional value of baked goods), indicating the strain's application potential in biomaterials, microbial engineering, environmental remediation, feed, and fermented foods.

[0146] Example 3: Tolerance analysis of Pichia pastoris AMCC 31367 strain

[0147] Preparation of seed culture: The strain of Pichia kudrica was inoculated into a test tube containing 5 ml of LYPD liquid medium and cultured at 30℃ and 180 rpm for 20 h to obtain the seed culture.

[0148] Temperature tolerance test

[0149] Based on the volume of the culture medium, the seed culture was inoculated into a 96-well cell culture plate containing 200 μL of LYPD liquid culture medium at an inoculation rate of 1%. The culture speed was 180 rpm, and the culture was carried out at temperatures of 15℃, 20℃, 25℃, 30℃, 35℃ and 40℃ for 48 h to obtain the bacterial culture.

[0150] The OD value of the bacterial culture is measured at 600 nm using an ELISA reader; this value is the OD value. 600 Values. The test results are shown in Table 4 below.

[0151] Table 4. Growth of strains at different temperatures

[0152]

[0153] As shown in Table 4, Pichia kudrica AMCC 31367 can grow in a culture temperature range of 15-40℃, and its biomass is relatively high in the range of 20-40℃, indicating a wide suitable temperature range for growth.

[0154] pH tolerance test

[0155] Preparation of the culture medium used in the pH tolerance test: The pH value of the YPD liquid culture medium was controlled by adding hydrochloric acid or sulfuric acid. Eight different pH values ​​were prepared, specifically, the pH values ​​of the culture media were 2, 3, 4, 5, 6, 7, 8, or 9.

[0156] Take 200 μL of each of the eight different pH culture media and place them into a 96-well cell culture plate. Then, inoculate each culture medium with 1% (based on the total volume of the culture medium) of seed culture and culture at 30℃ and 180 rpm for 48 h to obtain bacterial culture.

[0157] The OD value of the bacterial culture is measured at 600 nm using an ELISA reader; this value is the OD value. 600 Values. The test results are shown in Table 5 below.

[0158] Table 5. Growth of strains at different pH levels

[0159]

[0160] As shown in Table 5, the Pichia kudrica strain AMCC31367 can grow in a pH range of 2-9, and its biomass is relatively high in a pH range of 3-8. It has a wide suitable pH range for growth and strong acid and alkali tolerance.

[0161] Sugar tolerance test

[0162] Preparation of the culture medium used in the glucose tolerance test: Different amounts of glucose were added to YPD liquid medium to prepare YPD liquid medium with different sugar concentrations. Seven YPD mediums with different sugar concentrations were prepared, specifically, based on the volume of YPD liquid medium, the mass-volume percentage of sugar in the medium was 2%, 10%, 20%, 30%, 40%, 50%, or 60%, respectively.

[0163] Take 200 μL of each of the seven YPD culture media containing different sugar concentrations and place them into a 96-well cell culture plate. Then, inoculate each culture medium with 1% (based on the total volume of the culture medium) of seed culture and culture at 30℃ and 180 rpm for 48 h to obtain bacterial culture.

[0164] The OD value of the bacterial culture is measured at 600 nm using an ELISA reader; this value is the OD value. 600 Values. The test results are shown in Table 6 below.

[0165] Table 6. Growth of strains at different sugar concentrations

[0166]

[0167] As shown in Table 6, the Pichia kudrica strain AMCC 31367 can still grow at a sugar concentration of 60%, indicating that it can tolerate a sugar concentration of 60%.

[0168] Ethanol tolerance test

[0169] Preparation of the culture medium used in the ethanol tolerance test: Different amounts of anhydrous ethanol were added to the above-mentioned YPD liquid culture medium to prepare YPD liquid culture media containing different concentrations of ethanol. Five YPD culture media with different ethanol concentrations were prepared; specifically, based on the volume of the YPD liquid culture medium, the volume percentage of ethanol in the culture medium was 4%, 6%, 8%, 10%, or 12%, respectively.

[0170] 200 μL of YPD liquid medium (0% ethanol concentration) and 5 YPD media containing different ethanol concentrations were placed into 96-well cell culture plates. Then, 1% (based on the total volume of the medium) of seed culture was inoculated into each medium. The plates were cultured at 30℃ and 180 rpm for 48 h to obtain bacterial culture.

[0171] The OD value of the bacterial culture is measured at 600 nm using an ELISA reader; this value is the OD value. 600 Values. The test results are shown in Table 7 below.

[0172] Table 7. Growth of strains at different ethanol concentrations

[0173]

[0174] As shown in Table 7, the Pichia kudriezvich AMCC 31367 strain could hardly grow at an ethanol concentration of 12%, and the maximum ethanol concentration that this strain could tolerate was 10%. Within a certain range, the strain grew vigorously with increasing ethanol concentration; however, when the ethanol concentration increased further, the growth of Pichia kudriezvich AMCC 31367 was significantly inhibited.

[0175] Phenylephethanol tolerance test

[0176] Preparation of culture medium for the phenylethanol tolerance test: Different concentrations of phenylethanol were prepared by adding different amounts of phenylethanol to YPD liquid culture medium. Specifically, five YPD culture media with different phenylethanol concentrations were prepared, with concentrations of phenylethanol of 2 g / L, 3 g / L, 4 g / L, 5 g / L, or 6 g / L per volume of YPD liquid culture medium.

[0177] 200 μL of YPD liquid medium (phenylethanol concentration of 0 g / L) and 5 YPD media containing different concentrations of phenylethanol were placed into 96-well cell culture plates. Then, 1% (based on the total volume of the medium) of seed culture was inoculated into each medium and cultured at 30℃ and 180 rpm for 48 h to obtain bacterial culture.

[0178] The OD value of the bacterial culture is measured at 600 nm using an ELISA reader; this value is the OD value. 600 Values. The test results are shown in Table 8 below.

[0179] Table 8. Growth of strains at different concentrations of phenylethanol

[0180]

[0181] As shown in Table 8, the growth of Pichia kudrica strain AMCC 31367 was severely inhibited at a concentration of 5 g / L phenylethanol, but it could tolerate 4 g / L phenylethanol.

[0182] Acetic acid tolerance test

[0183] Preparation of the culture medium used in the acetic acid tolerance test: YPD liquid culture medium containing different concentrations of acetic acid can be prepared by adding different amounts of acetic acid to the YPD liquid culture medium. Specifically, five YPD culture media with different acetic acid concentrations were prepared, with acetic acid concentrations of 2 g / L, 4 g / L, 6 g / L, 8 g / L, or 10 g / L per volume of the YPD liquid culture medium.

[0184] Take 200 μL of the above YPD liquid medium (acetic acid concentration of 0 g / L) and 5 YPD media containing different acetic acid concentrations and put them into 96-well cell culture plates. Then, inoculate each medium with 1% (based on the total volume of the medium) of seed culture and culture at 30℃ and 180 rpm for 48 h to obtain bacterial culture.

[0185] The OD value of the bacterial culture is measured at 600 nm using an ELISA reader; this value is the OD value. 600 Values. The test results are shown in Table 9 below.

[0186] Table 9. Growth of strains at different acetic acid concentrations

[0187]

[0188] As shown in Table 9, the growth of Pichia kudrica strain AMCC 31367 was severely inhibited at a concentration of 10 g / L acetic acid, while the biomass increased by 12%-18% at a concentration of 2-8 g / L acetic acid (i.e., the increase in OD value was 12%-18% under the condition of 2-8 g / L acetic acid compared to 0 g / L acetic acid), and it could tolerate 8 g / L acetic acid.

[0189] Lactic acid tolerance test

[0190] Preparation of culture medium for lactate tolerance test: Different amounts of lactate were added to YPD liquid culture medium to prepare YPD liquid culture medium with different lactate concentrations. Specifically, six YPD culture media with different lactate concentrations were prepared, with lactate concentrations of 60 g / L, 110 g / L, 140 g / L, 150 g / L, 160 g / L, and 170 g / L, based on the volume of the YPD liquid culture medium.

[0191] Take 200 μL of the above-mentioned YPD liquid medium (lactate concentration of 0 g / L) and 6 YPD media containing different lactate concentrations and put them into 96-well cell culture plates. Then, inoculate each medium with 1% (based on the total volume of the medium) of seed culture and culture at 30℃ and 180 rpm for 144 h to obtain bacterial culture.

[0192] The OD value of the bacterial culture is measured at 600 nm using an ELISA reader; this value is the OD value. 600 Values. The test results are shown in Table 10 below.

[0193] Table 10 Growth of strains at different lactic acid concentrations

[0194]

[0195] Table 10 shows that the biomass of *Pichia goudryana* strain AMCC 31367 increased at lactic acid concentrations of 60, 110, and 140 g / L, with an inhibition rate of 48% at 160 g / L lactic acid (this inhibition rate represents a 48% reduction in OD value compared to 0 g / L lactic acid). It could still grow at a lactic acid concentration of 170 g / L. Furthermore, compared to conditions without lactic acid stress, the biomass of the strain increased by 14%-28% under culture conditions containing lactic acid concentrations of 60-140 g / L (i.e., a 14%-28% increase in OD value compared to 0 g / L lactic acid). This characteristic indicates that this strain has the potential for application in the production of biomaterials such as polylactic acid (PLA).

[0196] Example 4: Protein and amino acid content analysis of Pichia pastoris AMCC 31367

[0197] The *Pichia pastoris* strain AMCC 31367 was inoculated into a test tube containing 5 mL of LYPD liquid medium and cultured for 20 h at 30 °C and 180 rpm to prepare a seed culture. Using 0.5% of the culture medium volume, the seed culture was inoculated into an Erlenmeyer flask containing 300 mL of expansion medium and cultured for 20 h at 30 °C and 180 rpm. After centrifugation, the heavy phase, consisting of cell precipitate and water, was obtained, referred to as yeast milk. The centrifugation conditions were: 8000 rpm for 5 min and 4 °C. The moisture content, protein content, and hydrolyzed amino acid content of the yeast milk were then measured.

[0198] (1) Determination of moisture content in yeast milk: The moisture content was determined according to the direct drying method of the National Food Safety Standard of the People's Republic of China GB 5009.3-2016, Determination of Moisture in Food. The moisture content in yeast milk was 78%-82%.

[0199] (2) Protein content determination:

[0200] The specific operating steps are as follows:

[0201] 1.1.1. Principle

[0202] The proteins in yeast decompose with concentrated sulfuric acid under the action of a catalyst to produce ammonia. The ammonia reacts with sulfuric acid to produce ammonium sulfate. The ammonium sulfate is heated in an alkaline solution to release ammonia, which is received by boric acid solution and then titrated with a standard sulfuric acid solution, using methyl red-bromocresol green as an indicator.

[0203] 1.1.2. Reagents and Solutions

[0204] (1) Digestive powder Se:K2SO4 = 0.1 g:100;

[0205] (2) Sulfuric acid: analytical grade;

[0206] (3) 30% sodium hydroxide solution: Weigh 30 g of analytical grade sodium hydroxide, add an appropriate amount of distilled water to dissolve, cool, dilute to 100 mL, and mix well;

[0207] (4) 3% boric acid solution: Weigh 3 g of analytical grade boric acid, add an appropriate amount of distilled water to dissolve it, dilute to 100 mL, and mix well;

[0208] (5) Methyl red-bromocresol green mixed indicator:

[0209] ① Weigh 0.1 g of bromocresol green, dissolve it in 95% ethanol, and dilute it to 100 mL with 95% ethanol;

[0210] ② Weigh 0.2 g of methyl red and dissolve it in 95% ethanol, then dilute it to 100 mL with 95% ethanol;

[0211] ③ Mix bromocresol green indicator and methyl red indicator at a volume ratio of 3:1 and shake well.

[0212] (6) Sulfuric acid standard solution: Prepared according to GB / T 601-2016;

[0213] 1.1.3. Operating Procedures

[0214] Pour a small amount of well-mixed yeast milk into a 100mL dry beaker. Accurately weigh 1.5g of yeast milk (to the nearest 0.0002g) into a digestion tube using the subtraction method. Add 2.5g of digestion powder. Slowly add 10-12mL of concentrated sulfuric acid along the tube wall. Place a small funnel at the opening of the digestion tube and then place it on a digestion apparatus. Digest for about 3 hours until there is no smoke, the solution becomes clear, and it turns pale yellow. Continue heating for 10 minutes.

[0215] Remove the digestion tube, let it cool, rinse the tube wall with about 30 mL of distilled water, cool it again, and then transfer the digestion solution to a 100 mL volumetric flask through a small funnel. Rinse the digestion tube and funnel three times with a small amount of water, pour all of the solution into the volumetric flask, add water to make up to the mark, shake well, and the digestion solution is ready for use.

[0216] Accurately pipette 25 mL of digestive fluid into a digestion tube and place it on a distillation apparatus.

[0217] Take 25 mL of boric acid solution into an Erlenmeyer flask, add 4-6 drops of methyl red-bromocresol green mixed indicator, and place it at the outlet of the distillate as the receiving solution.

[0218] Add 25 mL of sodium hydroxide solution to the digestion tube, turn on the switch, and start distillation. Stop distilling when the receiving liquid is about 200-250 mL. The distillation time is about 6-8 minutes. Use pH test paper to check whether the last distillate is neutral to determine whether the distillation can be stopped. If it is neutral, it means that the nitrogen has been distilled.

[0219] Titrate the receiving solution with a 0.05 mol / L sulfuric acid standard solution until the color changes from green to slightly red, indicating the endpoint.

[0220] Perform a blank test simultaneously using the same method.

[0221] 1.1.5. Result Calculation

[0222] The protein content in the sample is calculated using the following formula:

[0223]

[0224] Where: X6—protein content in the sample, g / 100g; C—concentration of sulfuric acid standard solution, mol / L; V1—volume of sulfuric acid standard solution consumed in titrating the sample, mL; V2—volume of sulfuric acid standard solution consumed in titrating the blank, mL; 0.014—mass of nitrogen in grams equivalent to 1.00 mL of sulfuric acid [C(1 / 2H2SO4)=1.000mol / L] standard solution, g / mmol; W—mass of the sample, g; Ds—percentage of dry matter in yeast milk - oven drying method, %; 6.25—conversion factor between nitrogen and crude protein.

[0225] After testing, the crude protein content of Pichia pastoris AMCC 31367 yeast milk was found to be 66.69 g / 100g, or 66.69%.

[0226] (3) Determination of hydrolyzed amino acid content: The content of threonine, valine, methionine, isoleucine, leucine, phenylalanine and lysine was determined by acid extraction method in the People's Republic of China National Standard GB / T 18246—2019 Determination of Amino Acids in Feed; the content of tryptophan was determined by high performance liquid chromatography (arbitration method) in the People's Republic of China National Standard GB / T15400-2018. The test results are shown in Table 11 below.

[0227] Table 11 Amino acid content of Pichia pastoris AMCC 31367 cells

[0228]

[0229] As shown in Table 11, the amino acid content, based on dry weight, is 61%. Among these, lysine, the first limiting amino acid in feed, accounts for 6.25%. Other essential amino acids include leucine (5.1%), threonine (3.5%), valine (3.3%), isoleucine (3.2%), phenylalanine (3.1%), tryptophan (0.65%), and methionine (0.15%). Furthermore, the EAA / TAA and EAA / NEAA ratios are 0.41 and 0.71, respectively, meeting the ideal protein conditions proposed by the World Health Organization / Food and Agriculture Organization of the United Nations, indicating high nutritional value. In addition, the contents of the umami amino acids glutamic acid and aspartic acid are 8.4% and 7.05%, respectively, while the contents of the sweet amino acids glycine and alanine are 3.1% and 4.65%, respectively. Therefore, this bacterium has great application potential in feed and food processing.

[0230] The Kudria zweipichia AMCC 31367 described in this invention can utilize a variety of carbon sources, has high tolerance, high protein content, and is rich in amino acids. It has great industrialization market prospects in the development of single-cell proteins from a variety of low-value carbon sources.

[0231] Table 12 Sequence List

[0232]

[0233] While the specific embodiments of the present invention have been described above, they are not intended to limit the scope of protection of the present invention. Based on the technical solutions of the present invention, various modifications or variations that can be made by those skilled in the art without creative effort are still within the scope of protection of the present invention.

Claims

1. A strain of *Pichia kudrica*, characterized in that, The strain is *Pichia kudrica* AMCC 31367 (…). Pichia kudriavzevii AMCC 31367), accession number CCTCC NO: M 20251680.

2. The *Pichia kudrica* strain according to claim 1, wherein, The specified Kudria zweibichi yeast strain can tolerate temperatures of 15-40℃; And / or, the Kudriazwibich yeast strain can tolerate pH 2-9; And / or, the *Pichia kudrica* strain can tolerate sugar concentrations of 2%-60%; And / or, the *Pichia kudrica* strain can tolerate an ethanol concentration of less than or equal to 10%; And / or, the concentration of phenylethanol that the Kudria zwibich yeast strain can tolerate is less than or equal to 4 g / L; And / or, the *Pichia kudrica* strain can tolerate an acetic acid concentration of less than or equal to 8 g / L; and compared to conditions without acetic acid stress, the biomass of the strain increases by 12%-18% under culture conditions containing an acetic acid concentration of 2-8 g / L; And / or, the *Pichia kudrica* strain can tolerate lactic acid concentrations of less than or equal to 170 g / L; growth inhibition rate of ≤48% at 160 g / L; and compared to no lactic acid stress conditions, the biomass of the strain increased by 14%-28% under culture conditions containing lactic acid concentrations of 60-140 g / L.

3. The *Pichia kudrica* strain according to claim 1, wherein, The carbon sources that the *Pichia kudrica* strain can utilize include one or more substances selected from the group consisting of xylose, fructose, glucose, maltose, sucrose, mesquite, maltodextrin, stachyose, inulin, pullulan, xylan, soluble starch, citric acid, succinic acid, malic acid, lactic acid, and glycerol.

4. A method for preparing a culture of *Pichia kudriezvichirosa* strain, characterized in that, The process includes the following steps: culturing the *Pichia kudrica* strain as described in any one of claims 1-3. The culture includes culturing the *Pichia kudrica* strain to obtain a seed culture, wherein the culture conditions are: temperature 25-35℃, time 20-24h; and the culture medium used for the culture, by weight, comprises 8-12 parts yeast extract, 18-22 parts glucose, 18-22 parts peptone, and 800-1200 parts water. The seed culture was inoculated into a culture medium for cultivation to obtain a culture of *Pichia kudriezvichi* strain. The cultivation conditions were as follows: temperature 25-35℃, time 20-24h; the inoculation amount of seed culture was 0.5%-1.2% by volume of the culture medium; the culture medium comprised 80-120 parts glucose, 18-22 parts yeast extract, 0.8-1.2 parts MgSO4, 0.8-1.2 parts KH2PO4, and 800-1200 parts water by weight; and the pH of the culture medium was 4.6-5.

5. The method for preparing a culture of the *Pichia kudriezvichi* strain according to claim 4, wherein, The rotation speed for cultivation is 150-180 rpm.

6. A method for preparing yeast milk, characterized in that, Includes the following steps: The culture of the *Kudriazwibichthys* strain prepared by the method of claim 4 or 5 is subjected to solid-liquid separation to obtain yeast milk.

7. A yeast milk, characterized in that, The yeast milk is prepared by the yeast milk preparation method described in claim 6.

8. The yeast milk according to claim 7, wherein, The moisture content of yeast milk is 78%-82%; And / or, based on the dry weight of the yeast milk, the yeast milk comprises a crude protein content of 65%-70%; And / or, based on the dry weight of the yeast milk, the yeast milk contains 23%-26% essential amino acids; And / or, the yeast milk includes essential amino acids, including threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, and tryptophan; And / or, based on the dry weight of the yeast milk, the yeast milk comprises 3.3%-3.6% threonine; and / or 3.2%-3.4% valine; and / or 0.1%-0.2% methionine; and / or 2.7%-3.3% isoleucine; and / or 4.35%-5.2% leucine; and / or 3.0%-3.2% phenylalanine; and / or 6.0%-6.3% lysine; and / or 0.62%-0.66% tryptophan. And / or, based on the dry weight of the yeast milk, the yeast milk contains 34%-37% non-essential amino acids; And / or, the yeast milk includes non-essential amino acids, including histidine, arginine, aspartic acid, serine, glutamic acid, glycine, alanine, cystine, tyrosine, and proline. And / or, based on the dry weight of the yeast milk, the yeast milk comprises the following contents: histidine content of 1.5%-1.7%; and / or arginine content of 2.80%-3.15%; and / or aspartic acid content of 6.90%-7.15%; and / or serine content of 3.1%-3.3%; and / or glutamic acid content of 8.3%-8.5%; and / or glycine content of 3.0%-3.2%; and / or alanine content of 4.60%-4.75%; and / or cystine content of 0.35%-0.50%; and / or tyrosine content of 1.8%-2.2%; and / or proline content of 2.2%-2.4%.

9. A microbial agent, characterized in that, The microbial agent comprises the *Kudriazwibichthys* strain according to any one of claims 1-3 or the yeast milk according to claim 7 or 8.

10. A yeast extract, characterized in that, The yeast extract comprises the *Kudriazwibichthys* strain as described in any one of claims 1-3 or the yeast milk as described in claim 7 or 8.

11. The use of any one of the Kudriazwibich yeast strains according to claims 1-3, the yeast milk according to claim 7 or 8, the inoculum according to claim 9, or the yeast extract according to claim 10 in the fermentation of materials under stress conditions.

12. The use of any one of the *Pichia kudrica* strains according to claims 1-3, the yeast milk according to claim 7 or 8, the inoculum according to claim 9, or the yeast extract according to claim 10 in the preparation of feed, food, or wastewater treatment products.

13. The use of any one of the *Pichia kudrica* strains according to claims 1-3 in the preparation of protein products.

Images