Kazachstania unispora strain, microbial agent, fermentation product, and preparation and use thereof

Abstract

Provided are a Kazachstania unispora strain, a microbial agent containing the strain and a preparation method therefor, a fermentation culture product of the strain and a preparation method therefor, and the use of the strain or the microbial agent. The strain is deposited in the China Center for Type Culture Collection with the deposit number of CCTCC M 20231787. The strain can effectively degrade furfural and produce a relatively high content of acetoin and acetic acid.

Description

Single-spored Kazakh yeast strain, bacterial agent, fermentation product, and preparation and application thereof

[0001] Related applications

[0002] This application claims priority to the prior application document with application number 202410302653.9 filed with the State Intellectual Property Office of China on March 15, 2024, and incorporates its entire contents into this document. Technical Field

[0003] The present invention belongs to the field of microorganisms, and in particular relates to a single-spored Kazakh yeast strain, a bacterial agent, a fermentation product, and preparation and application thereof. Background Art

[0004] Kazachstania unispora belongs to the genus Kazachstania and is widely distributed in traditional fermentation scenarios. So far, this strain has been isolated from Tibetan lingzhi mushrooms, traditional yogurt in Inner Mongolia, and fermented camel milk and cheese in Xinjiang. Due to its safe source, this strain is widely used in the production of health factors, such as exopolysaccharides, ACE inhibitory peptides, pyrazines, etc.

[0005] Furfural is primarily produced by the dehydration of pentose sugars in a high-temperature, acidic environment. Corncobs, wheat bran, rice bran, rice husks, and sawdust, all high in pentosans, are prone to producing furfural, primarily in the chemical industry. Heat pretreatment is essential for the high-value utilization of plant biomass. The furfural produced not only affects the subsequent hydrolysis efficiency of cellulase but also severely inhibits bacterial growth and metabolism, negatively impacting animal health.

[0006] Distillers dried grains with solubles (DDGS) are waste biomass produced during the fermentation of cereal grains to produce ethanol. DDGS contains microbial growth inhibitors such as furfural, has high acidity, low content of nitrogen-free extracts (starch and sugars), high lignocellulose content, and poor palatability. Summary of the Invention

[0007] In view of the problems that furfural produced in the process of plant biomass resource utilization in the existing technology not only affects product quality but also leads to low efficiency of cellulase hydrolysis and inhibition of microbial growth in the subsequent deep utilization process, the present invention has screened and obtained a single-spored Kazakh yeast that can not only efficiently degrade furfural to achieve the effect of biomass detoxification, but also produce flavor substances and functional substances, which is used for the resource development of plant biomass such as dry distiller's grains and their solubles.

[0008] Specifically, the present invention provides the following technical solutions:

[0009] Technical solution 1: A Kazakh unispora yeast, characterized in that the Kazakh unispora yeast is Kazakh unispora yeast AMCC 30302 (Kazachstania unispora AMCC 30302), which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCCNO: M 20231787.

[0010] Technical Solution 2: The monosporic Kazakh yeast strain according to Technical Solution 1 is characterized in that the 26S rDNA gene sequence of the monosporic Kazakh yeast strain is shown as SEQ ID NO.1.

[0011] Technical Solution 3: The Kazakhstan monosporic yeast strain according to Technical Solution 1 or 2 is characterized in that the Kazakhstan monosporic yeast strain has the characteristics of increasing niacin content, and / or has the characteristics of degrading furfural content, and / or has the characteristics of producing aromatic substances.

[0012] Technical Solution 4: A fermentation preparation method of a single-spore Kazakh yeast agent, characterized in that the preparation method comprises the following steps:

[0013] (1) amplifying and culturing the monosporic Kazakh yeast strain described in any one of technical solutions 1 to 3 at a culture temperature of 20-42° C.;

[0014] (2) fermenting the monosporic Kazakh yeast strain amplified and cultured in step (1) at a culture temperature of 20-42°C.

[0015] Technical Solution 5: The fermentation preparation method according to Technical Solution 4 is characterized in that the culture time in step (1) is 12-48 hours; and / or the culture time in step (2) is 24-144 hours.

[0016] Technical Solution 6: A single-spore Kazakh yeast agent, characterized in that it is obtained by the fermentation preparation method described in Technical Solution 4.

[0017] Technical Solution 7: The monosporic Kazakhstan yeast agent according to Technical Solution 6 is characterized in that the volatile components in the monosporic Kazakhstan yeast agent include one or more substances selected from the group consisting of acetoin, phenylethanol, acetic acid, citronellol, phenylacetaldehyde, benzaldehyde, 3-ethyl-2,5-dimethylpyrazine, 2,5-dimethylpyrazine, 2-methylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, ethyl phenylacetate, phenylethyl acetate, γ-queolactone, γ-dodecalactone, γ-nonalactone, amyl acetate, phenylethyl propionate, ethyl benzoate, benzyl acetate, phenylethyl isobutyrate and 2-nonanone.

[0018] Technical Solution 8: A yeast fermentation culture, characterized in that it is prepared by a method comprising the following preparation steps: inoculating the single-spored Kazakhstan yeast strain described in any one of Technical Solutions 1-3 or the single-spored Kazakhstan yeast agent described in Technical Solution 6 or 7 into plant biomass, and fermenting and culturing at 30-37°C.

[0019] Technical Solution 9: The yeast fermentation culture according to Technical Solution 8 is characterized in that the yeast fermentation culture contains one or more substances selected from the group consisting of acetoin, phenylethanol, acetic acid and phenylethyl acetate.

[0020] Technical Solution 10: The method for preparing a yeast fermentation culture according to Technical Solution 8 or 9 is characterized in that it comprises the following steps: inoculating the single-spored Kazakhstan yeast strain according to any one of Technical Solutions 1-3 or the single-spored Kazakhstan yeast agent according to Technical Solution 5 into plant biomass, and fermenting and culturing at 30-37°C.

[0021] Technical Solution 11: Use of the Kazakhstan monosporic yeast strain described in any one of Technical Solutions 1-3 or the Kazakhstan monosporic yeast agent described in Technical Solution 6 or 7 in degrading furfural content, and / or in increasing nicotinic acid content, and / or in producing aromatic substances.

[0022] Technical Solution 12: Use of the single-spored Kazakh yeast strain described in any one of Technical Solutions 1-3 or the single-spored Kazakh yeast agent described in Technical Solution 6 or 7 to degrade furfural content in plant biomass fermentation; and / or to increase niacin content in plant biomass fermentation; and / or to produce aromatic substances in plant biomass fermentation.

[0023] Technical Solution 13: Use of the single-spored Kazakh yeast strain described in any one of Technical Solutions 1-3 or the single-spored Kazakh yeast agent described in Technical Solution 6 or 7 in feed additives or food additives.

[0024] Technical Solution 14: Use of the single-spored Kazakh yeast strain according to any one of Technical Solutions 1-3 or the single-spored Kazakh yeast agent according to Technical Solution 6 or 7 in the preparation of food or feed.

[0025] Technical Solution 15: A feed additive or food additive, characterized in that the feed additive or food additive contains the single-spored Kazakhstan yeast strain described in any one of Technical Solutions 1-3 or the single-spored Kazakhstan yeast agent described in Technical Solution 6 or 7.

[0026] Technical Solution 16: A food or feed, characterized in that the food or feed contains the single-spored Kazakhstan yeast strain described in any one of Technical Solutions 1-3 or the single-spored Kazakhstan yeast agent described in Technical Solution 6 or 7.

[0027] Beneficial effects of the present invention:

[0028] 1. The Kazakh yeast strain provided by the present invention can effectively degrade furfural and produce compounds such as acetoin and acetic acid at higher contents.

[0029] 2. The feed fermented by the Kazakh yeast strain provided by the present invention mainly produces a sensory characteristic of sour and fragrant flavor, which can stimulate the appetite of animals and improve the palatability of the feed.

[0030] 3. The feed fermented by the Kazakh yeast strain provided by the present invention can produce organic acids, which is beneficial to maintaining the health of the animal's intestinal environment; at the same time, it also produces a large amount of niacin, which can improve feed conversion rate and promote animal growth and development. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 shows the colony morphology of the monosporic Kazakh yeast;

[0032] FIG2 shows a microscopic observation of the single-spore Kazakh yeast;

[0033] FIG3 shows the result of the hydrolysis zone formed by the monosporic Kazakh yeast AMCC30302 on the phytase screening plate after being cultured at 30° C. for 7 days;

[0034] FIG4 shows the gas chromatograms of the fermentation samples of the experimental group and the control group;

[0035] FIG5 shows the niacin content of fermented samples of the experimental group and the control group;

[0036] FIG6 shows the reducing sugar content in the fermentation samples of the experimental group and the control group;

[0037] Culture collection information

[0038] The Kazakh unispora yeast AMCC 30302 (Kazachstania unispora AMCC 30302) provided by the present invention was deposited in the China Center for Type Culture Collection on September 25, 2023, with a deposit number of CCTCC NO: M 20231787, and a deposit address of Wuhan University, Wuhan, China, with a postal code of 430072; and a telephone number of 027-68754052. DETAILED DESCRIPTION

[0039] In order to better understand the technical solution of the present invention, the technical solution of the present invention is clearly and completely explained below in conjunction with the specific implementation methods. It should be noted that the content in the specific implementation methods is only a specific implementation and explanation of the technical solution of the present invention and should not be understood as limiting the scope of protection of the present invention.

[0040] In view of the problems that furfural produced in the process of plant biomass resource utilization in the existing technology not only affects product quality but also leads to low efficiency of cellulase hydrolysis and inhibition of microbial growth in subsequent deep utilization, the present invention provides the following specific implementation methods.

[0041] In some specific embodiments, the present invention provides a Kazakh unispora yeast strain, wherein the Kazakh unispora yeast strain is Kazakh unispora yeast AMCC 30302 (Kazachstania unispora AMCC 30302), which was deposited in the China Center for Type Culture Collection on September 25, 2023, with a deposit number of CCTCC M 20231787, and a deposit address of Wuhan University, Wuhan, China, with a postal code of 430072; and a telephone number of 027-68754052.

[0042] The monosporic Kazakh yeast strain AMCC 30302 provided herein was isolated from a fermented milk sample. Individual colonies are round, white, with a raised center, neat edges, and a smooth, opaque surface. Microscopically, they are oval, with cells measuring approximately 4 x 5 μm, and microscopic observations indicate budding reproduction. The 26S rDNA gene sequence of the monosporic Kazakh yeast strain AMCC 30302 is shown in SEQ ID NO. 1.

[0043] The monosporic Kazakh yeast AMCC 30302 provided by the present invention has the characteristics of high-efficiency furfural degradation, high nicotinic acid content, and production of acetoin and acetic acid, can reduce the toxicity of furfural, and increase the acid flavor.

[0044] It should be noted that Kazakhstan monosporus yeast has been included in the second edition of the "List of Microbial Species Used in Traditional Chinese Fermented Foods." This indicates that Kazakhstan monosporus yeast can be used in traditional Chinese fermented foods, thus proving that a specific strain of Kazakhstan monosporus yeast, namely Kazakhstan monosporus yeast AMCC 30302, can also be used in traditional Chinese fermented foods.

[0045] In some specific embodiments, the present invention also provides a method for preparing a monosporic Kazakh yeast agent, and the specific technical scheme is as follows:

[0046] 1.1. A fermentation method for preparing a monosporic Kazakh yeast agent, characterized in that the preparation method comprises the following steps:

[0047] (1) amplifying and culturing the monosporic Kazakh yeast strain at a culture temperature of 20-42° C.;

[0048] (2) fermenting the monosporic Kazakh yeast strain amplified and cultured in step (1) at a culture temperature of 20-42°C.

[0049] 1.2. The fermentation preparation method according to technical solution 1.1 is characterized in that the culture time in step (1) is 12-48 hours; and / or the culture time in step (2) is 24-144 hours.

[0050] 1.3. The fermentation preparation method according to technical solution 1.1 or 1.2 is characterized in that the components of the culture medium used for amplification culture include, by weight, 15-25 parts of yeast extract powder, 15-25 parts of glucose, 15-25 parts of yeast peptone, and 15-25 parts of agar.

[0051] 1.4. The fermentation preparation method according to any one of technical solutions 1.1 to 1.3 is characterized in that the components of the culture medium used for fermentation culture include, by weight, 5-15 parts of yeast peptone, 5-10 parts of beef extract, 1-5 parts of yeast extract powder, 10-25 parts of glucose, 0.5-1.5 parts of Tween 80, 1-5 parts of anhydrous dipotassium hydrogen phosphate, 1-10 parts of anhydrous sodium acetate, 1-5 parts of triammonium citrate, 0.1-0.5 parts of anhydrous magnesium sulfate, 0.01-0.1 parts of manganese sulfate monohydrate, 0.1-1 parts of L-cysteine, and 15-25 parts of lactose.

[0052] 1.5. A monosporic Kazakh yeast agent, characterized in that it is obtained by the fermentation preparation method described in any one of technical solutions 1.1-1.4.

[0053] 1.6. The monosporic Kazakh yeast agent according to technical solution 1.5 is characterized in that the volatile components in the monosporic Kazakh yeast agent include one or more substances selected from the group consisting of acetoin, phenylethyl alcohol, acetic acid, citronellol, phenylacetaldehyde, benzaldehyde, 3-ethyl-2,5-dimethylpyrazine, 2,5-dimethylpyrazine, 2-methylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, ethyl phenylacetate, phenylethyl acetate, γ-queolactone, γ-dodecalactone, γ-nonalactone, amyl acetate, phenylethyl propionate, ethyl benzoate, benzyl acetate, 2-phenylethyl isobutyrate and 2-nonanone,

[0054] Preferably, the volatile components in the monosporic Kazakh yeast agent include one or more substances selected from the group consisting of acetoin, 3-ethyl-2,5-dimethylpyrazine, phenylacetaldehyde, ethyl phenylacetate, phenylethanol, γ-nonalactone, γ-decalactone and γ-dodecalactone.

[0055] 1.7. The monosporic Kazakh yeast agent according to technical solution 1.6 is characterized in that, per mL of the monosporic Kazakh yeast agent, the content of acetoin is greater than 0 and less than or equal to 48.099 ng, and / or phenylethanol 1556-9998 ng, and / or acetic acid, and / or citronellol greater than 0 and less than or equal to 6.801 ng, and / or phenylacetaldehyde 11.06-45.4 ng, and / or benzaldehyde 241.3-794 ng, and / or 3-ethyl-2,5-dimethylpyrazine 281.6-522.8 ng, and / or 2,5-dimethylpyrazine 26.27-106 ng, and / or 2-ethyl-6-methylpyrazine 49.14-316.8 ng, and / or 2-ethyl-3,5-dimethylpyrazine 2 81.6-522.8ng, and / or ethyl phenylacetate 35.91-156.5ng, and / or phenylethyl acetate 46.2-140.1ng, and / or γ-quinoline lactone 1.983-143.8ng, and / or γ-dodecalactone 1.53-58.18ng, and / or γ-nonalactone 1.394-18.45ng, and / or amyl acetate greater than 0 and less than or equal to 21.55ng, and / or phenylethyl propionate 6.553-12.88ng, and / or ethyl benzoate greater than and less than or equal to 0.4901ng, and / or benzyl acetate 6.925-10.17ng, and / or 2-phenylethyl isobutyrate 1.426-146.29ng, and / or 2-nonanone 16.2-21.94ng.

[0056] In some specific embodiments, the present invention further provides a yeast fermentation culture (i.e., a fermentation culture of Kazakhstan Saccharomyces unispora), and the specific technical scheme is as follows:

[0057] 2.1. A yeast fermentation culture, characterized in that it is prepared by a method comprising the following steps: inoculating the monosporic Kazakhstan yeast or the monosporic Kazakhstan yeast inoculum into plant biomass, and fermenting and culturing at 30-37°C.

[0058] 2.2. The fermentation culture according to technical solution 2.1 is characterized in that the initial viable bacteria count in the plant biomass is 3×10 7 -5×10 7 cfu / g.

[0059] 2.3. The fermentation culture according to technical solution 2.3 is characterized in that the furfural content in each gram of the yeast fermentation culture is less than or equal to 13 ng.

[0060] 2.4. The fermentation culture according to any one of technical solutions 2.1 to 2.3, characterized in that the yeast fermentation culture contains one or more substances selected from the group consisting of acetoin, phenylethanol, acetic acid and phenylethyl acetate.

[0061] 2.5. The fermentation culture according to technical solution 2.4 is characterized in that the content of acetoin in each gram of the yeast fermentation culture is 1300-3300 ng, and / or the content of phenylethanol is 1260-3700 ng, and / or the content of acetic acid is 40-1100 ng, and / or the content of phenylethyl acetate is 10-150 ng.

[0062] 2.6. The fermentation culture according to technical solution 2.4 or 2.5 is characterized in that, after one day of fermentation culture, the acetoin content in each g of the yeast fermentation culture is 1990-2000 ng, and / or the phenylethanol content is 3300-3500 ng, and / or the acetic acid content is 1000-1100 ng.

[0063] 2.7. The fermentation culture according to technical solution 2.4 or 2.5 is characterized in that, after 3 days of fermentation culture, the content of acetoin per g of the yeast fermentation culture is 3000-3300 ng, and / or the content of phenylethanol is 3500-3700 ng, and / or the content of acetic acid is 700-750 ng, and / or the content of phenylethyl acetate is 100-150 ng.

[0064] 2.8. The fermentation culture according to technical solution 2.4 or 2.5, characterized in that, after 5 days of fermentation culture, the acetoin content in each g of the yeast fermentation culture is 2400-2500 ng, and / or the phenylethanol content is 2700-2800 ng, and / or the acetic acid content is 355-365 ng, and / or the phenylethyl acetate content is 60-65 ng.

[0065] 2.9. The fermentation culture according to technical solution 2.4 or 2.5 is characterized in that, after 7 days of fermentation culture, the content of acetoin per g of the yeast fermentation culture is 1300-1350 ng, and / or the content of phenylethanol is 1200-1250 ng, and / or the content of acetic acid is 40-45 ng, and / or the content of phenylethyl acetate is 10-15 ng.

[0066] 2.10. The fermentation culture according to any one of technical solutions 2.1 to 2.9, characterized in that the niacin content in every 100 g of the fermentation culture is 60,000-65,000 μg.

[0067] 2.11. The fermentation culture according to any one of technical solutions 2.1 to 2.10, characterized in that the Kazakh monosporic yeast is Kazakh monosporic yeast AMCC 30302.

[0068] In some specific embodiments, the sources of the YPD medium, MRS medium, phytase screening medium, and phytase production medium used in the culture of Kazakhstan monosporic yeast are not particularly limited in the present invention; they can be any commercially available medium or prepared by conventional methods. As long as the components of the commercially available or conventionally prepared medium can function for culture and / or screening, they can be used in the present invention.

[0069] For example, in parts by weight, the YPD culture medium includes: 15-25 parts of yeast extract powder, 15-25 parts of glucose, 15-25 parts of yeast peptone and 15-25 parts of agar.

[0070] For example, the MRS medium includes, by weight: 5-15 parts of yeast peptone, 5-10 parts of beef extract, 1-5 parts of yeast extract powder, 10-25 parts of glucose, 0.5-1.5 parts of Tween 80, 1-5 parts of anhydrous dipotassium hydrogen phosphate, 1-10 parts of anhydrous sodium acetate, 1-5 parts of triammonium citrate, 0.1-0.5 parts of anhydrous magnesium sulfate, 0.01-0.1 parts of manganese sulfate monohydrate, 0.1-1 parts of L-cysteine, and 15-25 parts of lactose.

[0071] For example, the phytase screening medium includes, by weight, 1-10 parts of calcium phytate, 1-5 parts of ammonium sulfate, 0.1-1 parts of magnesium sulfate, 0.1-1 parts of potassium chloride, 0.005-0.01 parts of ferrous sulfate, 0.005-0.01 parts of manganese sulfate, 10-20 parts of glucose and 15-25 parts of agar.

[0072] For example, in parts by weight, the phytase production culture medium includes: 1-5 parts of sodium phytate, 0.1-1 parts of potassium chloride, 0.01-0.05 parts of manganese sulfate, 0.1-1 parts of magnesium sulfate, 0.01-0.05 parts of ferrous sulfate, 1-10 parts of ammonium nitrate, and 25-35 parts of glucose.

[0073] In some specific embodiments, the raw materials used in preparing the culture medium of the present invention include yeast peptone and yeast extract. Yeast peptone and yeast extract primarily serve as organic nitrogen sources in the culture medium, providing the nitrogen necessary for microbial growth during fermentation. Common organic nitrogen sources, such as yeast extract and yeast peptone, decompose in the seed culture medium, releasing amino acids and small peptides, which serve as nitrogen sources for microbial growth. In other words, when yeast peptone and yeast extract are used as common organic nitrogen sources in preparing the culture medium of the present invention, their sources are not particularly limited; they can be commercially available or prepared by conventional methods. Commercially available yeast peptone or yeast extract prepared by conventional methods with a total nitrogen content of 12.5% ​​or greater and an amino nitrogen content of 2.5% or greater can be used in the present invention. Commercially available yeast extract with a total nitrogen content of 10.0% or greater and an amino nitrogen content of 5.0% or greater can be used in the present invention.

[0074] Preferably, in some specific embodiments, the yeast extract further comprises, based on the weight of the yeast extract, 2-2.5 ppm of vitamin B1, 37-40 ppm of vitamin B2, 113-116 ppm of vitamin B5, 15-20 ppm of vitamin B6, 6-10 ppm of vitamin B7, 25-28 ppm of vitamin B9, 3205-3210 ppm of choline, 1575-1580 ppm of inositol, 325-330 ppm of niacin, and 2-4 ug of vitamin B12 per 100 g of yeast extract.

[0075] and / or based on the weight of the yeast extract powder, potassium is 31910-31912 mg / kg, sodium is 5735-5740 mg / kg, calcium is 354-357 mg / kg, magnesium is 2670-2675 mg / kg, zinc is 77-83 mg / kg and iron is 77-83 mg / kg.

[0076] And / or based on the weight of the yeast extract powder, the yeast extract powder comprises: 30.3-40.85% free amino acids and 51-70.5% hydrolyzed amino acids.

[0077] The free amino acid content includes, based on the weight of the yeast extract powder, 1-2% of free aspartic acid, 2-3% of free threonine, 1.5-2% of free serine, 6.5-7% of free glutamic acid, 1-1.5% of free glycine, 4-5% of free alanine, 0.1-0.15% of free cysteine, 2-3% of free valine, 0.5-1% of free methionine, 2-2.5% of free isoleucine, 3.3-3.7% of free leucine, 0.5-1% of free tyrosine, 1.5-2% of free phenylalanine, 2-2.5% of free lysine, 0.1-1% of free histidine, 1.5-2.5% of free arginine and 0.5-1% of free proline.

[0078] The hydrolyzed amino acid content includes, based on the weight of the yeast extract powder, 6-6.5% hydrolyzed aspartic acid, 2-3% hydrolyzed threonine, 2-3% hydrolyzed serine, 10-15% hydrolyzed glutamic acid, 2-3% hydrolyzed glycine, 5-6% hydrolyzed alanine, 0.5-1% hydrolyzed cysteine, 3-4% hydrolyzed valine, 0.5-1% hydrolyzed methionine, 3-4% hydrolyzed isoleucine, 4-5% hydrolyzed leucine, 1-2% hydrolyzed tyrosine, 2-3% hydrolyzed phenylalanine, 4-5% hydrolyzed lysine, 1-2% hydrolyzed histidine, 3-4% hydrolyzed arginine and 2-3% hydrolyzed proline.

[0079] 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.

[0080] Unless otherwise specified, the various reagents / instruments used in the examples of the present invention are conventional commercial products. The sources of experimental materials and instrument information used in the present invention are shown in Table 1:

[0081] Table 1. Reagent information

[0082] Table 2. Instrument information

[0083] The vitamin content of the yeast extract powder (Model: FM888) used in the examples is as follows: vitamin B1 2.3 ppm, vitamin B2 38.8 ppm, vitamin B5 115.0 ppm, vitamin B6 18.0 ppm, vitamin B7 7.9 ppm, vitamin B9 26.7 ppm, vitamin B12 2.3 μg / 100 g, choline 3206.0 ppm, inositol 1577.7 ppm, and niacin 328.0 ppm.

[0084] The trace elements contained in the yeast extract powder (model: FM888) used in the examples are as follows: potassium 31911.66 mg / kg, sodium 5738.91 mg / kg, calcium 355.25 mg / kg, magnesium 2673.59 mg / kg, zinc 80.94 mg / kg, and iron 80.21 mg / kg.

[0085] The yeast extract powder (model: FM888) used in the examples contained 35.1% free amino acids and 61.21% hydrolyzed amino acids.

[0086] The free amino acid content is specifically as follows: based on the weight of the yeast extract powder, free aspartic acid 1.6%, free threonine 2.1%, free serine 1.7%, free glutamic acid 6.7%, free glycine 1.2%, free alanine 4.2%, free cysteine ​​0.1%, free valine 2.7%, free methionine 0.8%, free isoleucine 2.2%, leucine 3.5%, free tyrosine 0.9%, free phenylalanine 1.8%, free lysine 2.3%, free histidine 0.5%, free arginine 2.0%, and free proline 0.8%.

[0087] The hydrolyzed amino acid content is specifically as follows: based on the weight of the yeast extract powder, the following: hydrolyzed aspartic acid 6.23%, hydrolyzed threonine 2.71%, hydrolyzed serine 2.73%, hydrolyzed glutamic acid 12.33%, hydrolyzed glycine 2.74%, hydrolyzed alanine 5.17%, hydrolyzed cysteine ​​0.61%, hydrolyzed valine 3.84%, hydrolyzed methionine 0.84%, hydrolyzed isoleucine 3.65%, hydrolyzed leucine 4.72%, hydrolyzed tyrosine 1.65%, hydrolyzed phenylalanine 2.68%, hydrolyzed lysine 4.63%, hydrolyzed histidine 1.19%, hydrolyzed arginine 3.30%, and hydrolyzed proline 2.19%.

[0088] The culture medium components involved in the embodiment are as follows:

[0089] YPD medium: Mix 20 g yeast extract powder, 20 g glucose, 20 g yeast peptone, 20 g agar, and 1000 mL water, and sterilize at 115°C for 20 min.

[0090] MRS medium: Mix 10 g yeast peptone, 8 g beef extract, 4 g yeast extract powder, 20 g glucose, 1.08 g Tween 80, 2 g anhydrous dipotassium hydrogen phosphate, 5 g anhydrous sodium acetate, 2 g triammonium citrate, 0.2 g anhydrous magnesium sulfate, 0.05 g manganese sulfate monohydrate, 0.5 g L-cysteine, 20 g lactose, and 1000 mL distilled water, and sterilize at 115°C for 20 min.

[0091] Phytase screening medium: Mix 5 g of calcium phytate, 2.5 g of ammonium sulfate, 0.5 g of magnesium sulfate, 0.5 g of potassium chloride, 0.01 g of ferrous sulfate, 0.01 g of manganese sulfate, 15 g of glucose, 20 g of agar, and 1000 mL of water, and sterilize at 115°C for 20 min.

[0092] Phytase production medium: Mix 2 g of sodium phytate, 0.5 g of potassium chloride, 0.03 g of manganese sulfate, 0.5 g of magnesium sulfate, 0.03 g of ferrous sulfate, 5 g of ammonium nitrate, 30 g of glucose and 1000 mL of water, adjust the pH to 5.5, and sterilize at 115°C for 20 min.

[0093] Example 1 Isolation and Identification of Yeast Strains in the Present Invention

[0094] The strain of the present invention is derived from fermented milk, and its preparation method is to naturally ferment fresh milk. The fermented milk sample is diluted in sterile water to prepare a bacterial suspension, and the bacterial suspension is diluted in 10-fold gradients to prepare 10 -5 , 10 -6 The bacterial suspension was spread on YPD medium and cultured at 30℃ for 7 days. A single colony was picked and streaked on YPD medium for purification and cultured for 3 days. After multiple purifications, it was inoculated on YPD slant medium, stored at 4℃ and numbered.

[0095] The purified strain was streaked onto YPD solid culture medium and cultured at 30°C for 2 days before observing the colony morphology. A single colony was picked and inoculated into 5 mL YPD liquid culture medium and cultured at 180 rpm and 30°C for 24 hours. 10 μL was aspirated onto a glass slide and the bacterial morphology was observed under an optical microscope at a magnification of 400 times.

[0096] A strain of yeast was obtained. Its colonies were round, white, with a raised center, neat edges, and a smooth, opaque surface. Microscopically, they were oval, with individual cells approximately 4 × 5 μm in size and budding. The yeast genome was extracted and the yeast 26S rDNA sequence was amplified using primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG-3', SEQ ID NO. 2) and NL4 (5'-GGTCCGTGTTTCAAGACGG-3', SEQ ID NO. 3). The PCR procedure consisted of 94°C pre-denaturation for 5 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 45 s, and extension at 72°C for 90 s, with a final extension at 72°C for 10 min. Detection by 1% gel electrophoresis and sequencing revealed the following 26S rDNA sequence: SEQ ID NO. 1:

[0097] Combined morphological analysis and molecular identification identified the strain as Kazakhstan yeast AMCC30302 (Kazachstania unispora AMCC30302), which was deposited with the China Center for Type Culture Collection on September 25, 2023, with the accession number CCTCC M 20231787. Figure 1 shows the colony morphology of Kazakhstan yeast AMCC30302. Figure 2 shows a microscopic image of Kazakhstan yeast AMCC30302.

[0098] Example 2 Enzyme system screening and enzyme activity determination of Kazakhstan yeast AMCC30302

[0099] 1. Enzyme system screening

[0100] The Kazakh yeast AMCC30302 was spotted onto enzyme screening medium plates and cultured at 30°C for 7 days. The results are shown in Figure 3, which shows the hydrolysis zone formed by the Kazakh yeast AMCC30302 on the phytase screening plate after 7 days of culture at 30°C. This result demonstrates that the Kazakh yeast AMCC30302 can produce phytase, which served as the basis for initial screening and also indicates that the Kazakh yeast AMCC30302 has the potential to ferment plant biomass.

[0101] It should be noted that the phytase screening plate was used to screen for the Kazakh monosporic yeast AMCC30302, which has the potential to ferment plant biomass. Phytic acid is widely present in plants. Its most notable characteristic is its strong complexation with metal ions. It easily combines with divalent and trivalent ions and proteins in plants to form insoluble complexes, resulting in negative anti-nutritional effects. This prevents plant nutrients, such as protein, from being absorbed and utilized by animals. However, phytase can alleviate or even eliminate these negative anti-nutritional effects of phytic acid, allowing them to be fully absorbed and utilized by animals, thereby increasing their nutritional value.

[0102] 2. Enzyme Activity Determination

[0103] (1) Obtaining crude enzyme solution

[0104] The monosporic Kazakh yeast AMCC30302 was inoculated onto YPD medium and cultured at 30°C for 1-2 days until a single colony formed. A loopful of liquid was scraped from the culture and inoculated into a test tube containing 5 mL of YPD liquid medium. The culture was shaken at 30°C and 180 rpm for 48 hours to obtain a seed solution. Subsequently, 100 μL of this seed solution was inoculated into a test tube containing 5 mL of phytase production medium. After shaken culture, the supernatant was centrifuged and used as the crude enzyme solution to be tested. The shake culture parameters were: 30°C, 24 hours, and 180 rpm. The centrifugation parameters were: 4°C, 5 minutes, and 8000 rpm.

[0105] (2) Phytase activity determination

[0106] Phytase activity was determined according to GB / T 18634-2009 Spectrophotometric method for determination of enzymatic activity of feed phytase. Sodium phytate was used as a reaction substrate, the crude enzyme solution was added and mixed evenly, and the mixture was reacted at 37° C. for 30 min. After color development with a color developing solution, the absorbance was measured at a wavelength of 415 nm using a UV-visible photometer. The phytase activity in the fermentation supernatant of the monosporic Kazakh yeast AMCC30302 was calculated to be 0.84 U / mL.

[0107] Example 3 Analysis of DDGS Fermentation by Kazakhstan Saccharomyces cerevisiae

[0108] (1) Preparation of solid-state fermentation materials

[0109] For each test, 300 g of DDGS raw material was accurately weighed, 197 g of distilled water and 1 mL of 10 M NaOH were added and mixed evenly, and then 100 g / portion was accurately weighed. The mixture was sterilized at 121°C for 20 min and cooled for use to obtain solid-state fermentation material.

[0110] (2) Preparation of single-spore Kazakh yeast liquid

[0111] The monosporic Kazakh yeast AMCC30302 was inoculated on YPD medium and cultured at 30°C for 1-2 days until a single colony grew. A loop of the yeast was scraped with an inoculating loop and inoculated into a test tube containing 5 mL of YPD liquid medium. The culture was shaken at 30°C and 180 rpm for 48 hours to obtain a seed solution. Subsequently, 1 mL of the seed solution was inoculated into a 250 mL conical flask containing 50 mL of YPD liquid medium and shaken to obtain a monosporic Kazakh yeast solution (viable count of 5×10 8 cfu / mL). Shaking culture parameters were as follows: temperature 30°C, time 24 h, and rotation speed 180 rpm.

[0112] (3) Preparation of fermentation samples

[0113] Experimental group: 10 mL of the monosporic Kazakh yeast liquid obtained in step (2) was inoculated into 100 g of the solid-state fermentation material obtained in step (1), mixed evenly, and allowed to ferment at 30°C for 1 day to obtain a fermentation sample. Three replicate groups were set up.

[0114] Control group: 10 mL of sterile water was inoculated into 100 g of the solid fermentation material obtained in step (1), mixed evenly, and then allowed to ferment at 30° C. for 1 day to obtain a fermentation sample. Three replicate groups were set up.

[0115] (4) Analysis of furfural and volatile flavor substances

[0116] Weigh 4 g of each fermentation sample from the experimental and control groups into a solid-phase extraction headspace vial. Add 1 μL of internal standard solution (o-dichlorobenzene) and mix thoroughly. Seal the vial with a cap. Equilibrate at 50°C for 30 min. GC-MS analysis is performed. The results are shown in Figure 4 and Table 3.

[0117] The gas chromatography analysis conditions were as follows: chromatographic column: DB-HeavyWAX, 30 m × 0.25 mm × 0.25 μm, injection port temperature 250 °C, carrier gas: He; temperature program: 40 °C for 3 min, increase to 200 °C at a rate of 5 °C / min, and maintain for 0 min; increase to 250 °C at a rate of 10 °C / min, and maintain for 3 min.

[0118] Figure 4 shows the gas chromatograms of the fermentation samples from the experimental and control groups. Table 3 shows the relative contents of the main volatile components in the fermentation samples from the experimental and control groups. The results show that the main volatile substances in the control group were furfural and phenylethanol, while the main volatile substances in the experimental group were acetoin, acetic acid, and phenylethanol. The relative content of furfural in the control group was 7061 ng / g, while that in the experimental group was 11.73 ng / g. This indicates that the furfural degradation rate reached 99.83% after one day of fermentation with the Kazakhstan monosporic yeast AMCC30302, and the levels of acetoin and acetic acid produced were much higher than those in the control group. Furthermore, fermentation with the Kazakhstan monosporic yeast AMCC30302 produced a predominantly sour and fragrant flavor, which can stimulate animal appetite and improve palatability. Furthermore, feed fermented with the Kazakhstan monosporic yeast AMCC30302 produces a large amount of organic acid, namely acetic acid, which is beneficial for maintaining a healthy intestinal environment in animals.

[0119] Table 3. Information on the main volatile components of fermentation samples in the experimental and control groups

[0120] (5) Niacin

[0121] Niacin content was determined according to the second method in the "GB 5009.89-2016 National Food Safety Standard - Determination of Nicotinic Acid and Nicotinamide in Foods." The results are shown in Figure 5. Figure 5 shows the niacin content of fermented samples from the experimental and control groups. The results show that no niacin was detected in the control group, while the experimental group contained 62,970.87 μg of niacin per 100 g of sample. Niacin, a widely used feed additive, can effectively improve feed protein utilization. Furthermore, since growing and finishing pigs require approximately 7.5 to 20 mg of niacin per kilogram of feed, and lactating dairy cows require approximately 5 to 13 grams of niacin per day, the niacin content in the experimental group fermented with the Kazakh yeast AMCC30302 has the potential to meet the daily niacin requirements of animals. Niacin can also influence animal cellular metabolic pathways, improve feed conversion efficiency, promote growth and development, and enhance meat, milk, and egg production in livestock and poultry.

[0122] (6) Reducing sugar

[0123] Experimental group: 10 mL of the monosporic Kazakh yeast liquid obtained in step (2) was inoculated into 100 g of the solid-state fermentation material obtained in step (1), mixed evenly, and allowed to ferment at 30°C for 1 day to obtain the fermentation sample of the experimental group. Three replicate groups were set up.

[0124] Control group: 10 mL of the monosporic Kazakh yeast liquid obtained in step (2) was inoculated into 100 g of the solid-state fermentation material obtained in step (1), and the mixture was evenly mixed to obtain a fermentation sample of the control group. Three replicate groups were set up.

[0125] The reducing sugar assay method involves weighing 1g of each fermentation sample from the test and control groups, placing them into a 250mL Erlenmeyer flask, adding 50mL of distilled water, and boiling for 5 minutes. The sample is then transferred to a 50mL centrifuge tube and centrifuged at 4000 rpm for 5 minutes. The supernatant is collected and transferred to a 100mL volumetric flask. After washing the precipitate several times, the washes are transferred to a 100mL volumetric flask to obtain a 100-fold diluted reducing sugar test solution. The reducing sugar assay system consists of 1mL of the reducing sugar test solution, 1mL of distilled water, and 1.5mL of DNS colorimetric solution. The blank correction system consists of 2mL of distilled water and 1.5mL of DNS colorimetric solution. The mixture is boiled at 100°C for 5 minutes. After cooling, the absorbance is measured at 540nm using a UV-visible spectrophotometer. A standard curve is constructed using glucose standard solutions at 0, 0.2, 0.4, 0.6, 0.8, and 1mg / mL, and the reducing sugar content in the test solution is calculated. The results are shown in Figure 6.

[0126] Figure 6 shows the reducing sugar content in fermentation samples from the experimental and control groups. The results show that the reducing sugar content in the control group was 0.96 wt %, while that in the experimental group was 1.13 wt %. This indicates that fermentation with the Kazakhstan monosporic yeast AMCC30302 increased the reducing sugar content by 0.17 wt %. This indicates that the cellulase and amylase enzymes produced by the Kazakhstan monosporic yeast AMCC30302 during its growth process effectively degrade cellulose and starch in the substrate, releasing reducing sugars and increasing the reducing sugar content.

[0127] Example 4 Flavor characteristics of DDGS fermented by Kazakhstan yeast

[0128] (1) According to the method of Example 3, solid-state fermentation materials and a single-spore Kazakh yeast liquid were prepared.

[0129] (2) Preparation of fermentation sample (fermentation culture of Kazakhstan monosporic yeast AMCC30302): 10 mL of the Kazakhstan monosporic yeast culture obtained in step (1) was inoculated into 100 g of the solid-state fermentation material obtained in step (1), mixed evenly, and allowed to ferment at 30°C. 4 g of the culture was sampled on the 3rd, 5th, and 7th day of fermentation and transferred to a solid phase extraction headspace vial. The flavor components were analyzed by GC-MS using the same gas chromatography conditions as in Example 3. Three replicates were set up.

[0130] As shown in Table 4, the results indicate that, compared to the fermentation sample after one day of fermentation in Example 3, the relative content of flavor compounds in the fermentation culture of Kazakhstan monosporous yeast AMCC30302 initially increased and then decreased with fermentation time. The flavor compound content of Kazakhstan monosporous yeast AMCC30302 reached its highest level on day 3 of fermentation, with the peak relative content of acetoin at 3289 ng / g, acetic acid at 741 ng / g, phenylethanol at 4486 ng / g, and phenylethyl acetate at 101 ng / g. The primary flavor characteristic of the fermentation culture of Kazakhstan monosporous yeast AMCC30302 is a sour aroma, characterized by the creamy aroma of acetoin, the sour taste of acetic acid, and the rose aroma of phenylethanol.

[0131] Table 4. Changes in flavor substances in fermentation samples

[0132] Unit: ng / g

[0133] Example 5 Flavor of Kazakhstan yeast fermentation broth

[0134] Preparation of fermentation broth of Kazakhstan monosporus yeast AMCC30302: Inoculate Kazakhstan monosporus yeast AMCC30302 into MRS liquid culture medium at an inoculum rate of 2% (v / v), and culture with shaking at 30° C. for 72 h to obtain fermentation broth of Kazakhstan monosporus yeast AMCC30302.

[0135] The volatile components of the fermentation broth of Kazakh yeast AMCC30302 were determined as follows: The fermentation broth was centrifuged to obtain the supernatant. 5 mL of the supernatant was transferred to a headspace vial, and 1.5 g of sodium chloride and 1 μL of internal standard (o-dichlorobenzene) were added and mixed. The mixture was equilibrated at 50°C for 30 min, and then analyzed for volatile components. Gas chromatography conditions included: DB-HeavyWAX column, 30 m × 0.25 mm × 0.25 μm, inlet temperature 250°C, carrier gas He; temperature program: 40°C for 3 min, then increase to 200°C at a rate of 5°C / min, hold for 0 min; then increase to 250°C at a rate of 10°C / min, hold for 3 min. The experiment was repeated three times. The results are shown in Table 5.

[0136] As shown in Table 5, the results indicate that 24 flavor compounds with aroma characteristics were detected in the volatile components, eight of which exceeded the detection threshold, indicating that they are detectable in the fermentation broth. These eight compounds are: acetoin, 3-ethyl-2,5-dimethylpyrazine, phenylacetaldehyde, ethyl phenylacetate, phenylethanol, γ-nonalactone, γ-decalactone, and γ-dodecalactone. This combination of eight compounds imparts a unique, floral, fruity, nutty, chocolate-like aroma to the fermentation broth of the Kazakh monosporic yeast AMCC30302. Among these compounds, 3-ethyl-2,5-dimethylpyrazine had the highest relative content and corresponding detection threshold, indicating that 3-ethyl-2,5-dimethylpyrazine possesses the most prominent flavor characteristics in the fermentation broth, with aromas primarily reminiscent of roasted nuts or roasted meat.

[0137] Table 5. Information on the main volatile components in the fermentation broth of Kazakhstan monosporic yeast AMCC30302

[0138] In summary, the monosporic Kazakh yeast AMCC30302 has a high efficiency in furfural degradation, its fermentation broth and fermentation culture have a unique flavor, and can increase the niacin content in plant biomass. It has broad application prospects in the application fields of plant biomass fermentation, production of aroma substances, food and food additives, and feed and feed additives.

[0139] The above embodiments are only for further explanation and understanding of the technical solutions of the present invention, and are not intended to limit the present invention. Any non-prominent substantial features and non-significant improvements made by those skilled in the art on this basis should fall within the scope of protection of the present invention.

Claims

1. A strain of Kazakh yeast, characterized in that: The Kazakh unispora yeast is Kazakh unispora yeast AMCC 30302 (Kazachstania unispora AMCC 30302), which is deposited in the China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 20231787.

2. The monosporic Kazakh yeast strain according to claim 1, characterized in that The 26S rDNA gene sequence of the monosporic Kazakh yeast strain is shown in SEQ ID NO.

1.

3. The monosporic Kazakh yeast strain according to claim 1 or 2, characterized in that The Kazakhstan monosporic yeast strain has the characteristics of increasing nicotinic acid content, and / or having the characteristics of degrading furfural content, and / or having the characteristics of producing aromatic substances.

4. A fermentation method for preparing a monosporic Kazakhstan yeast agent, characterized in that: The preparation method comprises the following steps: (1) amplifying and culturing the monosporic Kazakh yeast strain according to any one of claims 1 to 3 at a culture temperature of 20-42° C.; (2) fermenting the monosporic Kazakh yeast strain amplified and cultured in step (1) at a culture temperature of 20-42°C.

5. The fermentation preparation method according to claim 4, characterized in that: The culturing time in step (1) is 12-48 hours; and / or the culturing time in step (2) is 24-144 hours.

6. A single-spore Kazakh yeast agent, characterized in that It is obtained by the fermentation preparation method according to claim 4.

7. The monosporic Kazakh yeast agent according to claim 6, characterized in that The volatile components in the monosporic Kazakh yeast agent include one or more substances selected from the group consisting of acetoin, phenylethyl alcohol, acetic acid, citronellol, phenylacetaldehyde, benzaldehyde, 3-ethyl-2,5-dimethylpyrazine, 2,5-dimethylpyrazine, 2-methylpyrazine, 2-ethyl-6-methylpyrazine, 2-ethyl-3,5-dimethylpyrazine, ethyl phenylacetate, phenylethyl acetate, γ-quinolide, γ-dodecalactone, γ-nonalactone, amyl acetate, phenylethyl propionate, ethyl benzoate, benzyl acetate, phenylethyl isobutyrate and 2-nonanone.

8. A yeast fermentation culture, characterized in that The yeast is prepared by a method comprising the following steps: inoculating the single-spored Kazakh yeast strain according to any one of claims 1 to 3 or the single-spored Kazakh yeast agent according to claim 6 or 7 into plant biomass, and fermenting and culturing the mixture at 30-37°C.

9. The yeast fermentation culture according to claim 8, characterized in that The yeast fermentation culture contains one or more substances selected from the group consisting of acetoin, phenylethanol, acetic acid and phenylethyl acetate.

10. The method for preparing a yeast fermentation culture according to claim 8 or 9, characterized in that: The method comprises the following steps: inoculating the single-spored Kazakh yeast strain according to any one of claims 1 to 3 or the single-spored Kazakh yeast agent according to claim 5 into plant biomass, and fermenting and culturing at 30-37°C.

11. Use of the Kazakhstan monosporic yeast strain according to any one of claims 1 to 3 or the Kazakhstan monosporic yeast agent according to claim 6 or 7 in degrading furfural content, and / or in increasing nicotinic acid content, and / or in producing aromatic substances.

12. Use of the Kazakhstan monosporic yeast strain according to any one of claims 1 to 3 or the Kazakhstan monosporic yeast agent according to claim 6 or 7 for degrading furfural content in plant biomass fermentation; and / or for increasing nicotinic acid content in plant biomass fermentation; and / or for producing aromatic substances in plant biomass fermentation.

13. Use of the Kazakhstan monosporic yeast strain according to any one of claims 1 to 3 or the Kazakhstan monosporic yeast agent according to claim 6 or 7 in a feed additive or a food additive.

14. Use of the Kazakhstan monosporic yeast strain according to any one of claims 1 to 3 or the Kazakhstan monosporic yeast agent according to claim 6 or 7 in the preparation of food or feed.

15. A feed additive or food additive, characterized in that: The feed additive or food additive contains the single-spored Kazakhstan yeast strain according to any one of claims 1 to 3 or the single-spored Kazakhstan yeast agent according to claim 6 or 7.

16. A food or feed, characterized in that The food or feed contains the Kazakhstan monosporic yeast strain according to any one of claims 1 to 3 or the Kazakhstan monosporic yeast agent according to claim 6 or 7.

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