Kluyveromyces marxianus strain, microbial agent, and preparation and use thereof

Abstract

The present invention belongs to the field of microorganisms, and specifically relates to a Kluyveromyces marxianus strain, a microbial agent, and preparation and use thereof. The Kluyveromyces marxianus strain comprises Kluyveromyces marxianus AMCC 31690 with a deposit number of CCTCC NO: M 2024246, Kluyveromyces marxianus AMCC 31691 with a deposit number of CCTCC NO: M 2024247, and Kluyveromyces marxianus AMCC 30630 with a deposit number of CCTCC NO: M 20222107. The described Kluyveromyces marxianus provided by the present invention has multiple tolerances of osmotic pressure resistance, acid resistance, bile salt resistance, and artificial gastric and intestinal fluid resistance, and can survive and maintain vitality in artificial gastric and intestinal fluids. In addition, the Kluyveromyces marxianus also has hydrophobicity and self-aggregating ability.

Description

Kluyveromyces martensii strains, inoculants, their preparation and application

[0001] Related applications

[0002] This application claims priority to the earlier application No. 202411652817.7 filed with the China National Intellectual Property Administration on November 18, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of microbiology, specifically relating to Kluyveromyces martensii strains, inoculants, their preparation and application. Background Technology

[0004] Kluyveromyces marxianus is a food-grade yeast, certified by the European Food Safety Authority (EFSA) and the US FDA, and approved as a new food ingredient by the National Health and Family Planning Commission of China. Although the safety and basic growth characteristics of Kluyveromyces marxianus are widely recognized, current technology lacks a Kluyveromyces marxianus variety that possesses superior multi-tolerance capabilities along with excellent hydrophobicity and self-aggregating ability. This hinders the further application of Kluyveromyces marxianus in functional foods and industrial biotechnology. Summary of the Invention

[0005] The technical problem solved by this invention is that there is a lack of Max Kluyveromyces yeast in the prior art that has superior multi-resistance capabilities as well as superior hydrophobicity and self-aggregation capabilities.

[0006] To address the problems existing in the prior art, this invention provides a Kluyveromyces martensii strain, inoculum, its preparation, and its application.

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

[0008] In a first aspect, the present invention provides a Kluyveromyces marxianus, specifically Kluyveromyces marxianus AMCC 31690, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2024246.

[0009] Secondly, the present invention provides a Kluyveromyces marxianus, specifically Kluyveromyces marxianus AMCC 31691, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M 2024247.

[0010] Thirdly, the present invention provides a Kluyveromyces marxianus, specifically Kluyveromyces marxianus AMCC 30630, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M 20222107.

[0011] Preferably, the ITS gene sequence of the Kluyveromyces martensii AMCC 31690 is shown in SEQ ID No. 4.

[0012] Preferably, the ITS gene sequence of the Kluyveromyces martensii AMCC 31691 is shown in SEQ ID No. 5.

[0013] Preferably, the ITS gene sequence of the Kluyveromyces martensii AMCC 30630 is shown in SEQ ID No. 3.

[0014] Preferably, the Kluyveromyces macrocarpa AMCC 31690 or the Kluyveromyces macrocarpa AMCC 31691 is obtained by mutagenesis of the Kluyveromyces macrocarpa AMCC 30630.

[0015] Preferably, the Kluyveromyces martensii has osmotic pressure resistance, and / or acid resistance, and / or bile salt resistance, and / or hydrophobicity, and / or self-aggregation ability, and / or gastric juice resistance, and / or intestinal juice resistance.

[0016] More preferably, the osmotic pressure resistance is: survival in a liquid culture medium containing sodium chloride at a concentration of 3% (W / V) to 7% (W / V).

[0017] More preferably, the acid resistance is: survival in liquid culture medium with a pH of 2.0-3.0.

[0018] More preferably, the bile salt tolerance is: survival in a liquid culture medium containing bile salts at a concentration of 0.2% (W / V) to 0.4% (W / V).

[0019] More preferably, the gastric acid resistance is: the survival rate of the Kluyveromyces martensii AMCC 31690 after gastric acid treatment is greater than or equal to 68%.

[0020] Or, the gastric acid resistance is: the survival rate of the *Kluyveromyces martensii* AMCC 31691 after gastric acid treatment is greater than or equal to 63%.

[0021] Alternatively, the gastric resistance is: the survival rate of the described Kluyveromyces martensii AMCC 30630 after gastric juice treatment is greater than or equal to 58%.

[0022] More preferably, the resistance to gastric and intestinal fluids is such that the survival rate of the *Kluyveromyces martensii* AMCC 31690 after treatment with gastric and intestinal fluids is greater than or equal to 95%.

[0023] Or, the resistance to gastric and intestinal fluids is: the survival rate of the described Kluyveromyces martensii AMCC 31691 after treatment with gastric and intestinal fluids is greater than or equal to 70%.

[0024] Alternatively, the tolerance to gastric and intestinal fluids is: the survival rate of the described Kluyveromyces martensii AMCC 30630 after treatment with gastric and intestinal fluids is greater than or equal to 52%.

[0025] More preferably, the self-agglomeration ability is: the self-agglomeration rate is greater than or equal to 60%; more preferably, the self-agglomeration rate is greater than or equal to 85% and less than or equal to 100%.

[0026] More preferably, the hydrophobicity is: a hydrophobicity to acetone of ≥60% and / or a hydrophobicity to chloroform of ≥40%.

[0027] More preferably, the hydrophobicity is: a hydrophobicity to acetone greater than or equal to 60% and less than or equal to 100%, and / or a hydrophobicity to chloroform greater than or equal to 60% and less than or equal to 100%.

[0028] Fourthly, the present invention provides a fermentation preparation method for Kluyveromyces martensii inoculum, comprising the following steps: amplifying and culturing Kluyveromyces martensii.

[0029] Fifthly, the present invention provides a Max Kluyveromycin inoculum containing Max Kluyveromycin.

[0030] Preferably, the Max Kluyveromycin inoculum is prepared by the fermentation preparation method described above.

[0031] In a sixth aspect, the present invention provides a food fermentation agent containing Kluyveromyces martensii and / or the aforementioned Kluyveromyces martensii inoculum.

[0032] In a seventh aspect, the present invention provides the use of Kluyveromyces martensii, and / or the aforementioned Kluyveromyces martensii inoculum, in food fermentation agents.

[0033] Eighthly, the present invention provides a probiotic product containing Kluyveromyces martensii and / or the aforementioned Kluyveromyces martensii inoculum.

[0034] In a ninth aspect, the present invention provides the use of Kluyveromyces martensii, and / or the aforementioned Kluyveromyces martensii inoculum, in probiotic products.

[0035] In a tenth aspect, the present invention provides a feed additive containing Kluyveromyces martensii and / or the aforementioned Kluyveromyces martensii inoculum.

[0036] In one aspect, the present invention provides the use of Kluyveromyces martensii, and / or the aforementioned Kluyveromyces martensii inoculum, in feed additives.

[0037] Beneficial effects of this invention:

[0038] The *Kluyveromyces martensii* strains AMCC 30630, AMCC 31690, and AMCC 31691 provided by this invention possess multiple tolerances, including resistance to osmotic pressure, acid, bile salts, and artificial gastric and intestinal fluids. They can survive and maintain viability in artificial gastric and intestinal fluids. Furthermore, they exhibit hydrophobicity and self-aggregating ability. These characteristics make these three *Kluyveromyces martensii* strains have significant application potential in the food industry and probiotic research. Among them, *Kluyveromyces martensii* strains AMCC 31690 and AMCC 31691 exhibit better resistance to osmotic pressure, acid, bile salts, and gastrointestinal fluids, as well as higher hydrophobicity and self-aggregating ability. Attached Figure Description

[0039] Figure 1 shows the colony morphology of Kluyveromyces martensii AMCC 30630.

[0040] Figure 2 shows a microscopic observation of Kluyveromyces martensii AMCC 30630.

[0041] Figure 3 shows the colony morphology of Kluyveromyces martensii AMCC 31690.

[0042] Figure 4 shows a microscopic observation of Kluyveromyces martensii AMCC 31690.

[0043] Figure 5 shows the colony morphology of Kluyveromyces martensii AMCC 31691.

[0044] Figure 6 shows a microscopic observation of Kluyveromyces martensii AMCC 31691.

[0045] Figure 7 shows the OD values ​​of *Kluyveromyces martensii* AMCC31690 suspensions cultured in YPD liquid medium with sodium chloride concentrations of 3% (w / v), 5% (w / v), or 7% (w / v). 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm value.

[0046] Figure 8 shows the OD values ​​of *Kluyveromyces martensii* AMCC31690 suspensions cultured in YPD liquid medium at pH 2.0, pH 2.5, or pH 3.0. 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm value.

[0047] Figure 9 shows the OD values ​​of *Kluyveromyces martensii* AMCC31690 suspensions cultured in YPD liquid medium with bile salt concentrations of 0.2% (W / V), 0.3% (W / V), or 0.4% (W / V). 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm value.

[0048] Figure 10 shows the survival rates of Kluyveromyces macrocarpa AMCC30630, Kluyveromyces macrocarpa AMCC31690, and Kluyveromyces macrocarpa AMCC31691 after being cultured in artificial gastric fluid for 2 hours and then in artificial intestinal fluid for 3 hours (after 5 hours of treatment).

[0049] Figure 11 shows the autoagglutination rates of Kluyveromyces AMCC30630, Kluyveromyces AMCC31690, and Kluyveromyces AMCC31691.

[0050] Figure 12 shows the hydrophobicity of Kluyveromyces AMCC30630, Kluyveromyces AMCC31690 and Kluyveromyces AMCC31691 to chloroform and acetone after standing for 2 hours.

[0051] Figure 13 shows the hydrophobicity of Kluyveromyces AMCC30630, Kluyveromyces AMCC31690 and Kluyveromyces AMCC31691 to chloroform and acetone after standing for 5 hours.

[0052] In Figures 7, 8, 9, 10, 11, 12, and 13, AMCC30630 represents Kluyveromyces macrocarpa AMCC30630, AMCC31690 represents Kluyveromyces macrocarpa AMCC31690, and AMCC31691 represents Kluyveromyces macrocarpa AMCC31691.

[0053] Information on strain preservation

[0054] The Kluyveromyces marxianus AMCC 30630 provided by this invention was deposited at the China Center for Type Culture Collection on December 30, 2022, with accession number CCTCC NO:M 20222107. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: 027-68754052.

[0055] The Kluyveromyces marxianus AMCC 31690 provided by this invention was deposited at the China Center for Type Culture Collection on January 29, 2024, with accession number CCTCC NO:M 2024246. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: 027-68754052.

[0056] The Kluyveromyces marxianus AMCC 31691 provided by this invention was deposited at the China Center for Type Culture Collection on January 29, 2024, with accession number CCTCC NO:M 2024247. The deposit address is: Wuhan University, Wuhan, China, Postcode: 430072; Telephone: 027-68754052. Detailed Implementation

[0057] To better understand the above technical solutions, the technical solutions of the present invention will be clearly and completely explained below in conjunction with specific embodiments. It should be noted that the content of the specific embodiments is only a specific implementation and explanation of the technical solutions of the present invention, and should not be construed as a limitation on the scope of protection of the present invention.

[0058] In some specific embodiments, the present invention provides a Kluyveromyces marxianus yeast, characterized in that the Kluyveromyces marxianus is Kluyveromyces marxianus AMCC 30630, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20222107.

[0059] Kluyveromyces martensii AMCC 30630 was obtained from fermented dairy products obtained in Xilingol League, Inner Mongolia Autonomous Region, after separation and purification.

[0060] Kluyveromyces martensii AMCC 30630 has osmotic pressure resistance, and / or acid resistance, and / or bile salt resistance, and / or hydrophobicity, and / or self-aggregation ability, and / or gastric juice resistance, and / or intestinal juice resistance.

[0061] Among them, the osmotic pressure resistance is as follows: Kluyveromyces martensii AMCC 30630 survives in liquid culture medium containing sodium chloride at a concentration of 3% (w / v) to 7% (w / v).

[0062] The acid resistance is as follows: Kluyveromyces martensii AMCC 30630 survives in liquid culture medium with a pH of 2.0-3.0.

[0063] Bile salt tolerance: Kluyveromyces macrocarpa AMCC 30630 survived in liquid culture medium containing 0.2% (w / v) to 0.4% (w / v) bile salts.

[0064] The gastric juice resistance is as follows: the survival rate of Kluyveromyces martensii AMCC 30630 after gastric juice treatment is greater than or equal to 58%, preferably, the survival rate after 2 hours of gastric juice treatment is greater than or equal to 58%.

[0065] The resistance to gastric and intestinal fluids is as follows: the survival rate of Kluyveromyces martensii AMCC 30630 after treatment with gastric and intestinal fluids is greater than or equal to 52%.

[0066] The self-agglutination ability is as follows: the self-agglutination rate of Kluyveromyces martensii AMCC 30630 is greater than or equal to 60%, preferably greater than or equal to 85% and less than or equal to 100%.

[0067] More preferably, the autoagglutination rate of Kluyveromyces martensii AMCC 30630 after standing for 5 hours is greater than or equal to 90% and less than or equal to 100%.

[0068] Hydrophobicity is defined as follows: Kluyveromyces macrocephala AMCC 30630 has a hydrophobicity to acetone of ≥40% and / or a hydrophobicity to chloroform of ≥40%.

[0069] Preferably, the hydrophobicity to acetone is greater than or equal to 60% and less than or equal to 100%.

[0070] More preferably, after standing for 2 hours, the hydrophobicity of *Kluyveromyces martensii* AMCC 30630 to acetone is greater than or equal to 62% and less than or equal to 100%; and / or after standing for 5 hours, the hydrophobicity of *Kluyveromyces martensii* AMCC 30630 to acetone is greater than or equal to 90% and less than or equal to 100%.

[0071] Preferably, the hydrophobicity to chloroform is greater than or equal to 60% and less than or equal to 100%.

[0072] More preferably, the hydrophobicity of Kluyveromyces macrocarpa AMCC 30630 to chloroform is greater than or equal to 85% and less than or equal to 100% after standing for 2 hours, and / or the hydrophobicity of Kluyveromyces macrocarpa AMCC 30630 to chloroform is greater than or equal to 90% and less than or equal to 100% after standing for 5 hours.

[0073] In some specific embodiments, the present invention provides a Kluyveromyces marxianus yeast, characterized in that the Kluyveromyces marxianus is Kluyveromyces marxianus AMCC 31690, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2024246.

[0074] Kluyveromyces macrocarpa AMCC 31690 was obtained by mutagenesis and screening of Kluyveromyces macrocarpa AMCC 30630.

[0075] Kluyveromyces martensii AMCC 31690 has osmotic pressure resistance, and / or acid resistance, and / or bile salt resistance, and / or hydrophobicity, and / or self-aggregation ability, and / or gastric juice resistance, and / or intestinal juice resistance.

[0076] Among them, the osmotic pressure resistance is as follows: Kluyveromyces martensii AMCC31690 survives in liquid culture medium containing sodium chloride at a concentration of 3% (W / V)-7% (W / V).

[0077] The acid tolerance is as follows: Kluyveromyces martensii AMCC31690 survives in liquid culture medium with a pH of 2.0-3.0.

[0078] Bile salt tolerance: Kluyveromyces martensii AMCC 31690 survived in liquid culture medium containing 0.2% (w / v) to 0.4% (w / v) bile salts.

[0079] The gastric juice resistance is as follows: the survival rate of Kluyveromyces martensii AMCC 31690 after gastric juice treatment is greater than or equal to 68%, preferably, the survival rate after 2 hours of gastric juice treatment is greater than or equal to 68%.

[0080] The resistance to gastric and intestinal fluids is as follows: the survival rate of Kluyveromyces martensii AMCC 31690 after treatment with gastric and intestinal fluids is greater than or equal to 95%, preferably, the survival rate after treatment with gastric fluid for 2 hours and culture in artificial intestinal fluid for 3 hours is greater than or equal to 95%.

[0081] The self-agglutination ability is as follows: the self-agglutination rate of Kluyveromyces martensii AMCC31690 is greater than or equal to 60%, preferably greater than or equal to 85% and less than or equal to 100%.

[0082] More preferably, the autoagglutination rate of Kluyveromyces martensii AMCC 31690 after standing for 5 hours is greater than or equal to 90% and less than or equal to 100%.

[0083] Hydrophobicity is defined as follows: Kluyveromyces macrocarpa AMCC 31690 exhibits a hydrophobicity of ≥40% to acetone, and / or Kluyveromyces macrocarpa AMCC 31690 exhibits a hydrophobicity of ≥40% to chloroform.

[0084] Preferably, the hydrophobicity to acetone is greater than or equal to 60% and less than or equal to 100%.

[0085] More preferably, the hydrophobicity of Kluyveromyces macrocarpa AMCC 31690 to acetone is greater than or equal to 62% and less than or equal to 100% after standing for 2 hours, and / or the hydrophobicity of Kluyveromyces macrocarpa AMCC 31690 to acetone is greater than or equal to 90% and less than or equal to 100% after standing for 5 hours.

[0086] Preferably, the hydrophobicity to chloroform is greater than or equal to 60% and less than or equal to 100%.

[0087] More preferably, the hydrophobicity of Kluyveromyces macrocarpa AMCC 31690 to chloroform is greater than or equal to 85% and less than or equal to 100% after standing for 2 hours, and / or the hydrophobicity of Kluyveromyces macrocarpa AMCC 31690 to chloroform is greater than or equal to 90% and less than or equal to 100% after standing for 5 hours.

[0088] In some specific embodiments, the present invention provides a Kluyveromyces marxianus yeast, characterized in that the Kluyveromyces marxianus is Kluyveromyces marxianus AMCC 31691, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2024247.

[0089] Kluyveromyces macrocarpa AMCC 31691 was obtained by mutagenesis and screening of Kluyveromyces macrocarpa AMCC 30630.

[0090] Kluyveromyces martensii AMCC 31691 has osmotic pressure resistance, and / or acid resistance, and / or bile salt resistance, and / or hydrophobicity, and / or self-aggregation ability, and / or gastric juice resistance, and / or intestinal juice resistance.

[0091] Among them, the osmotic pressure resistance was as follows: Kluyveromyces martensii AMCC31691 survived in liquid culture medium containing sodium chloride at a concentration of 3% (W / V)-7% (W / V).

[0092] The acid resistance is as follows: Kluyveromyces martensii AMCC31691 survives in liquid culture medium with a pH of 2.0-3.0.

[0093] Bile salt tolerance: Kluyveromyces martensii AMCC 31691 survived in liquid culture medium containing 0.2% (w / v) to 0.4% (w / v) bile salts.

[0094] The gastric juice resistance is as follows: the survival rate of Kluyveromyces martensii AMCC 31691 after gastric juice treatment is greater than or equal to 63%, preferably, the survival rate after 2 hours of gastric juice treatment is greater than or equal to 63%.

[0095] The resistance to gastric and intestinal fluids is as follows: the survival rate of Kluyveromyces martensii AMCC 31691 after treatment with gastric and intestinal fluids is greater than or equal to 70%, preferably, the survival rate after treatment with gastric fluid for 2 hours and culture in artificial intestinal fluid for 3 hours is greater than or equal to 70%.

[0096] The self-agglutination ability is as follows: the self-agglutination rate of Kluyveromyces martensii AMCC31691 is greater than or equal to 60%, preferably greater than or equal to 85% and less than or equal to 100%.

[0097] More preferably, the autoagglutination rate of Kluyveromyces martensii AMCC 31691 after standing for 5 hours is greater than or equal to 90% and less than or equal to 100%.

[0098] Hydrophobicity is defined as follows: Kluyveromyces macrocarpa AMCC 31691 has a hydrophobicity of 40% or more to acetone, and / or Kluyveromyces macrocarpa AMCC 31691 has a hydrophobicity of 40% or more to chloroform.

[0099] Preferably, the hydrophobicity to acetone is greater than or equal to 60% and less than or equal to 100%.

[0100] More preferably, after standing for 2 hours, the hydrophobicity of *Kluyveromyces martensii* AMCC 31691 to acetone is greater than or equal to 62% and less than or equal to 100%, and / or after standing for 5 hours, the hydrophobicity of *Kluyveromyces martensii* AMCC 31691 to acetone is greater than or equal to 90% and less than or equal to 100%.

[0101] Preferably, the hydrophobicity to chloroform is greater than or equal to 60% and less than or equal to 100%.

[0102] More preferably, after standing for 2 hours, the hydrophobicity of Kluyveromyces macrocarpa AMCC 31691 to chloroform is greater than or equal to 85% and less than or equal to 100%, and / or after standing for 5 hours, the hydrophobicity of Kluyveromyces macrocarpa AMCC 31691 to chloroform is greater than or equal to 90% and less than or equal to 100%.

[0103] The preparation method of the liquid culture medium with a bile salt concentration of 0.2% (W / V)-0.4% (W / V) is as follows: 2-4g of bovine bile salt, 5-15g of yeast extract, 15-25g of glucose, 15-25g of peptone, and distilled water are mixed and brought to a final volume of 1000mL before sterilization to obtain the liquid culture medium with a bile salt concentration of 0.2% (W / V)-0.4% (W / V).

[0104] The preparation method of liquid culture medium with sodium chloride concentration of 3% (W / V)-7% (W / V) is as follows: Mix 30-70g of sodium chloride, 5-15g of yeast extract, 15-25g of glucose, 15-25g of peptone and distilled water, and make up to 1000mL before sterilization to obtain liquid culture medium with sodium chloride concentration of 3% (W / V)-7% (W / V).

[0105] The preparation method of liquid culture medium with pH 2.0-3.0 is as follows: Mix 5-15g of yeast extract, 15-25g of glucose, 15-25g of peptone and distilled water, and make up to 1000mL. Then adjust the pH to 2.0-3.0 and sterilize to obtain liquid culture medium with pH 2.0-3.0.

[0106] In some specific embodiments, the raw materials used in preparing any of the above-mentioned liquid culture media include peptone and yeast extract. Peptone and yeast extract primarily exist as organic nitrogen sources in the culture medium, providing the necessary nitrogen element for microbial growth during fermentation. Common organic nitrogen sources such as yeast extract and peptone decompose in the culture medium, releasing amino acids and small peptides, which then become nitrogen sources required for microbial growth. That is, when using peptone and yeast extract as common organic nitrogen sources to prepare the culture medium, this invention does not particularly limit their source; they can be commercially available or prepared using conventional methods. Preferably, commercially available yeast extract with a total nitrogen content of ≥10.0 wt% and an amino nitrogen content of ≥5.0 wt% can be used in this invention; commercially available peptone with a total nitrogen content of ≥12.5 wt%, an amino nitrogen content of ≥2.5 wt%, and a peptone content of ≥30.0 ​​wt% can be used in this invention.

[0107] Preferably, in some specific embodiments, the yeast extract powder, by weight, further comprises: 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 μg of vitamin B12 per 100 g of yeast extract powder.

[0108] And / or, by weight of the yeast extract, the content of 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.

[0109] And / or, based on the weight of the yeast extract, the yeast extract comprises: 30.3-40.85% free amino acids and 51-70.5% hydrolyzed amino acids.

[0110] The free amino acid content includes, based on the weight of the yeast extract, 1-2% free aspartic acid, 2-3% free threonine, 1.5-2% free serine, 6.5-7% free glutamic acid, 1-1.5% free glycine, 4-5% free alanine, 0.1-0.15% free cysteine, 2-3% free valine, 0.5-1% free methionine, 2-2.5% free isoleucine, 3.3-3.7% free tyrosine, 1.5-2% free phenylalanine, 2-2.5% free lysine, 0.1-1% free histidine, 1.5-2.5% free arginine, and 0.5-1% free proline.

[0111] The hydrolyzed amino acid content includes, based on the weight of the yeast extract, 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.

[0112] The present invention will be further illustrated below with reference to embodiments. It should be noted that the following embodiments are merely more specific implementations and explanations of the technical solutions of the present invention, and should not be construed as limiting the scope of protection of the present invention.

[0113] Unless otherwise stated, all reagents / instruments used in the embodiments of this invention are conventional commercially available products. The sources of experimental materials used in this invention are shown in Table 1, and the sources of information on experimental instruments used in this invention are shown in Table 2.

[0114] Table 1. Reagent Information Table

[0115] Table 2. Instrument Information Sheet

[0116] The composition of the culture medium involved in the examples is shown below:

[0117] (1) YPD solid medium: Mix 10g yeast extract, 20g glucose, 20g peptone, 20g agar and distilled water and bring the volume to 1000mL. Sterilize at 115℃ for 20min to obtain YPD solid medium.

[0118] (2) YPD liquid culture medium: Mix 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Sterilize at 115℃ for 20min to obtain YPD liquid culture medium.

[0119] (3) YPD liquid culture medium with a bile salt concentration of 0.2% (W / V): Mix 2g of bovine bile salt, 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Sterilize at 115℃ for 20min to obtain YPD liquid culture medium with a bile salt concentration of 0.2% (W / V).

[0120] (4) YPD liquid culture medium with a bile salt concentration of 0.3% (W / V): Mix 3g of bovine bile salt, 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Sterilize at 115℃ for 20min to obtain YPD liquid culture medium with a bile salt concentration of 0.3% (W / V).

[0121] (5) YPD liquid culture medium with a bile salt concentration of 0.4% (W / V): Mix 4g of bovine bile salt, 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Sterilize at 115℃ for 20min to obtain YPD liquid culture medium with a bile salt concentration of 0.4% (W / V).

[0122] (6) YPD liquid culture medium with sodium chloride concentration of 3% (W / V): Mix 30g sodium chloride, 10g yeast extract, 20g glucose, 20g peptone and distilled water and bring the volume to 1000mL. Sterilize at 115℃ for 20min to obtain YPD liquid culture medium with sodium chloride concentration of 3% (W / V).

[0123] (7) YPD liquid culture medium with a sodium chloride concentration of 5% (W / V): Mix 50g of sodium chloride, 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Sterilize at 115℃ for 20min to obtain YPD liquid culture medium with a sodium chloride concentration of 5% (W / V).

[0124] (8) YPD liquid culture medium with sodium chloride concentration of 7% (W / V): Mix 70g of sodium chloride, 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Sterilize at 115℃ for 20min to obtain YPD liquid culture medium with sodium chloride concentration of 7% (W / V).

[0125] (9) YPD liquid culture medium with pH 2.0: Mix 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Adjust the pH to 2.0 and sterilize at 115℃ for 20min to obtain YPD liquid culture medium with pH 2.0.

[0126] (10) YPD liquid culture medium with pH 2.5: Mix 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Adjust the pH to 2.5 and sterilize at 115℃ for 20min to obtain YPD liquid culture medium with pH 2.5.

[0127] (11) YPD liquid culture medium with pH 3.0: Mix 10g of yeast extract, 20g of glucose, 20g of peptone and distilled water and bring the volume to 1000mL. Adjust the pH to 3.0 and sterilize at 115℃ for 20min to obtain YPD liquid culture medium with pH 3.0.

[0128] The trace elements contained in the yeast extract powder (model: FM888) used in the examples are 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 (ug / 100g), choline 3206.0 ppm, inositol 1577.7 ppm, and niacin 328.0 ppm.

[0129] 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.

[0130] The yeast extract (model: FM888) used in the examples contained 35.1% free amino acids and 61.21% hydrolyzed amino acids.

[0131] The free amino acid content is as follows, based on the weight of the yeast extract: 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%.

[0132] The hydrolyzed amino acid content is as follows: based on the weight of the yeast extract, 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%.

[0133] Example 1: Isolation and Identification of Strains

[0134] Fermented dairy products obtained from Xilingol League, Inner Mongolia Autonomous Region, were dissolved in sterile water and mixed thoroughly. The bacterial suspension was then serially diluted 10-fold to prepare 10... -5 10 -6 Bacterial suspensions were spread onto YPD solid agar plates and incubated at 30°C for 48 h. Then, all single colonies from the incubated YPD agar plates were picked and transferred to YPD liquid agar, incubated at 180 rpm and 30°C for 24 h to obtain bacterial suspensions. These suspensions were then inoculated at 1% (v / v) into 96-well plates containing 0.3% (w / v) YPD liquid agar, and incubated at 180 rpm and 37°C for 24 h before OD was measured.600nm Value, select OD 600nm The bacterial suspension with the highest value was the optimal tolerant strain suspension. The optimal tolerant strain suspension was spread onto YPD solid agar plates and incubated at 30°C for 48 hours. Single colonies were then picked for purification. The purification method was as follows: a single colony was picked and placed in 5 mL of YPD liquid agar, incubated at 180 rpm and 30°C for 24 hours, then streaked onto YPD solid agar plates and incubated for another 24 hours. Single colonies were then purified twice using the same method before being inoculated onto YPD solid agar slants and stored at 4°C.

[0135] The purified bacterial suspension was spread onto YPD solid medium and incubated at 30°C for 24 hours. The colony morphology was then observed. A single colony was picked and placed in 5 mL of YPD liquid medium and incubated at 180 rpm and 30°C for 24 hours. Then, 10 μL of the bacterial suspension was transferred to a glass slide and the bacterial morphology was observed under an optical microscope at 400x magnification.

[0136] A bacterial strain was obtained. The colonies had a cheese-like texture, were milky white in color, and had a smooth surface. Microscopically, they were elliptical, indicating budding reproduction. The genome of this strain was extracted. Using ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3', SEQ ID No. 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID No. 2) as primers, a PCR program of 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 45 s, and 72℃ extension for 90 s, for 30 cycles, followed by a final extension at 72℃ for 10 min, was used to amplify the yeast internal transcribed spacer (ITS) sequence. After 1% gel electrophoresis and sequencing, BLAST analysis was performed to compare the sequence with sequences in GenBank. The sequence similarity was greater than 99%, indicating the same species. The ITS sequence of this strain, SEQ ID No. 3, is shown below:

[0137] Based on morphological analysis and molecular identification, this strain was identified as *Kluyveromyces marxianus* AMCC 30630, deposited on December 30, 2022, at the China Center for Type Culture Collection (CCTCC), accession number CCTCCNO: M 20222107, address: Wuhan University, Wuhan, China, 430072, China; telephone: 027-68754052. Figure 1 shows the colony morphology of *Kluyveromyces marxianus* AMCC 30630. Figure 2 shows a microscopic observation of *Kluyveromyces marxianus* AMCC 30630.

[0138] Example 2: Observation and identification of colony morphology and microscopic morphology of strains obtained after ARTP mutagenesis treatment.

[0139] Kluyveromyces martensii AMCC30630 was inoculated at a 1% inoculum into 300 mL of LYPD liquid medium and cultured at 30°C and 180 rpm for 18 h to obtain a concentration of 3 × 10⁻⁶. 8 A CFU / mL suspension of *Kluyveromyces martensii* AMCC30630 was centrifuged at 5000g for 2 min, the supernatant was discarded, and the bacterial precipitate was retained and resuspended to obtain the target bacterial suspension. Then, 10 μL of the target bacterial suspension was evenly spread onto a sterile metal slide, which was then placed in an ARTP mutagenesis instrument for atmospheric and room temperature plasma (ARTP) mutagenesis. The ARTP mutagenesis conditions were as follows: helium as the working gas, power set at 120 W, gas flow rate at 10 SLM, distance between the plasma source and the bacterial suspension on the iron slide at 2 mm, and mutagenesis times set to 0 s, 10 s, 15 s, 20 s, 25 s, and 30 s.

[0140] 10 μL of target bacterial suspensions with mutagenesis times of 0 s, 10 s, 15 s, 20 s, 25 s, and 30 s were mixed with 1 mL of physiological saline and vortexed for 1 min. After serial dilution, 200 μL of the target bacterial suspension was spread onto the corresponding YPD solid culture medium plates. Each mutagenesis time corresponded to one YPD solid culture medium plate. All YPD solid culture plates were incubated at 30℃ for 24 h, and the colony counts on all YPD solid culture plates were counted to calculate the lethality rate. The lethality rate was calculated as follows: (1 - number of colonies on the plate after N s of mutagenesis treatment / number of colonies on the plate after 0 s of mutagenesis treatment) x 100%, where N represents 10 s, 15 s, 20 s, 25 s, or 30 s. Next, single colonies with larger diameters were picked from plates with a lethality rate of over 95%, and these single colonies were inoculated into YPD liquid medium and cultured at 30°C and 180 rpm for 18 hours, resulting in 90 mutant strains. These mutant strains were then screened based on a comprehensive evaluation of osmotic pressure tolerance, acid tolerance, and bile salt tolerance, and the OD values ​​for each of these parameters were determined. 600nm All values ​​are above 80%, and are based on a comprehensive OD value considering osmotic pressure resistance, acid resistance, and bile salt resistance. 600nmTen strains were selected from highest to lowest value. These ten strains were then further screened based on their resistance to gastric and intestinal fluids, autoagglutination rate, and hydrophobicity. Two strains with survival rates, autoagglutination rates, and hydrophobicities all exceeding 60% in gastric and intestinal fluids were selected from the highest combined values ​​for these three parameters. These two strains were then inoculated at a 1% inoculum into 300 ml of LYPD liquid medium and cultured at 30°C and 180 rpm for 18 hours to obtain a concentration of 3 × 10⁻⁶. 8 CFU / mL target bacterial suspension A and target bacterial suspension B.

[0141] The target bacterial suspension A was spread on YPD solid medium and incubated at 30°C for 24 hours before observing the colony morphology. A single colony was picked and placed in 5 mL of YPD liquid medium and incubated at 180 rpm and 30°C for 24 hours. Then, 10 μL of the bacterial suspension was transferred to a glass slide and observed under an optical microscope at 400x magnification for the morphology of the cells.

[0142] A bacterial strain was obtained. The colonies of this strain were round, with a cheese-like texture, milky white color, and a smooth surface. Microscopically, they were elliptical, indicating budding reproduction. The genome of this strain was extracted. Using ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3', SEQ ID No. 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID No. 2) as primers, a PCR program of 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 45 s, and 72℃ extension for 90 s, for 30 cycles, followed by a final extension at 72℃ for 10 min, was used to amplify the yeast internal transcribed spacer (ITS) sequence. After 1% gel electrophoresis and sequencing, BLAST analysis was performed with sequences from GenBank. The sequence similarity was greater than 99%, indicating the same species. The ITS sequence of this strain, SEQ ID No. 4, is shown below:

[0143] Based on morphological analysis and molecular identification, this strain was identified as *Kluyveromyces marxianus* AMCC 31690, deposited on January 29, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 2024246, address: Wuhan University, Wuhan, China, 430072, China; telephone: 027-68754052. Figure 3 shows the colony morphology of *Kluyveromyces marxianus* AMCC 31690. Figure 4 shows a microscopic observation of *Kluyveromyces marxianus* AMCC 31690.

[0144] The target bacterial suspension B was spread on YPD solid medium and incubated at 30°C for 24 hours before observing the colony morphology. A single colony was picked and placed in 5 mL of YPD liquid medium and incubated at 180 rpm and 30°C for 24 hours. Then, 10 μL of the bacterial suspension was transferred to a glass slide and observed under an optical microscope at 400x magnification for the morphology of the cells.

[0145] A bacterial strain was obtained. The colonies of this strain were round, with a cheese-like texture, milky white color, and a smooth surface. Microscopically, they were elliptical, indicating budding reproduction. The genome of this strain was extracted. Using ITS5 (5'-GGAAGTAAAAGTCGTAACAAGG-3', SEQ ID No. 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID No. 2) as primers, a PCR program of 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 45 s, and 72℃ extension for 90 s, for 30 cycles, followed by a final extension at 72℃ for 10 min, was used to amplify the yeast internal transcribed spacer (ITS) sequence. After 1% gel electrophoresis and sequencing, BLAST analysis was performed with sequences from GenBank. The sequence similarity was greater than 99%, indicating the same species. The ITS sequence of this strain, SEQ ID No. 5, is shown below:

[0146] Based on morphological analysis and molecular identification, this strain was identified as *Kluyveromyces marxianus* AMCC 31691, deposited on January 29, 2024, at the China Center for Type Culture Collection (CCTCC), accession number CCTCCNO: M 2024247, address: Wuhan University, Wuhan, China, 430072, China; telephone: 027-68754052. Figure 5 shows the colony morphology of *Kluyveromyces marxianus* AMCC 31691. Figure 6 shows a microscopic observation of *Kluyveromyces marxianus* AMCC 31691.

[0147] Example 3: Tolerance analysis of Kluyveromyces martensii strain

[0148] The osmotic pressure resistance, acid resistance, bile salt resistance, and gastric and intestinal fluid resistance of *Kluyveromyces martensii* AMCC31690, AMCC31691, and AMCC30630 (control strain) were analyzed. The screening criteria for osmotic pressure resistance, acid resistance, bile salt resistance, self-aggregation ability, and hydrophobicity of *Kluyveromyces martensii* AMCC31690 and AMCC31691 were as follows: OD of *Kluyveromyces martensii* AMCC31690... 600nm Value, or OD of Kluyveromyces maculae AMCC31691 600nm The survival rate of either *Kluyveromyces masculinus* AMCC30630 (control strain) reached 80% (i.e., greater than or equal to 80%). The screening criteria for resistance to gastric and intestinal fluids were as follows: if the survival rate of *Kluyveromyces masculinus* AMCC31690 or *Kluyveromyces masculinus* AMCC31691 exceeded the survival rate of *Kluyveromyces masculinus* AMCC30630 (control strain) and was greater than or equal to 70%, then *Kluyveromyces masculinus* AMCC31690 or *Kluyveromyces masculinus* AMCC31691 was considered a superior strain with multiple resistance.

[0149] (1) Resistance to osmotic pressure

[0150] Kluyveromyces martensii AMCC31690, AMCC31691, and AMCC30630 were inoculated at 1% (v / v) into YPD liquid medium containing 3%, 5%, and 7% (w / v) sodium chloride, respectively. After culturing at 180 rpm and 37°C for 24 h, OD was measured. 600nm The value is shown in Figure 7.

[0151] As shown in Figure 7, Figure 7 shows the OD of the *Kluyveromyces martensii* AMCC31690 bacterial suspension cultured in YPD liquid medium with a sodium chloride concentration of 3% (w / v). 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm Value; OD value of *Kluyveromyces martensii* AMCC31690 bacterial suspension cultured in YPD liquid medium with a sodium chloride concentration of 5% (w / v). 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nmValue; and OD of *Kluyveromyces martensii* AMCC31690 suspension cultured in YPD liquid medium with a sodium chloride concentration of 7% (w / v). 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm value.

[0152] The results showed that when the sodium chloride concentration was 3% (w / v), the osmotic pressure tolerance of *Kluyveromyces martensii* AMCC31690 and AMCC30630 was not significantly different, while the osmotic pressure tolerance of AMCC31691 was 19.2% higher than that of AMCC30630. When the sodium chloride concentration increased to 7% (w / v), both *Kluyveromyces martensii* AMCC31690 and AMCC31691 exhibited higher osmotic pressure tolerance, with increases of 19.9% ​​and 24.7% respectively compared to AMCC30630.

[0153] (2) Acid resistance

[0154] Kluyveromyces martensii AMCC31690, AMCC31691, and AMCC30630 were inoculated at 1% (v / v) into 96-well plates of YPD liquid medium at pH 2.0, 2.5, and 3.0, respectively. After incubation at 180 rpm and 37°C for 24 h, OD values ​​were measured. 600nm The value is shown in Figure 8.

[0155] Figure 8 shows the OD values ​​of the *Kluyveromyces martensii* AMCC31690 suspension obtained by culturing in YPD liquid medium at pH 2.0. 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm Value; OD value of *Kluyveromyces martensii* AMCC31690 bacterial suspension cultured in YPD liquid medium at pH 2.5. 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm Value; and OD of Kluyveromyces martensii AMCC31690 suspension cultured in YPD liquid medium at pH 3.0. 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nmOD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm value.

[0156] The results showed that when cultured in YPD liquid medium at pH 2.0, the acid tolerance of Kluyveromyces AMCC31690, AMCC31691, and AMCC30630 was not significantly different. When cultured in YPD liquid medium at pH 2.5 and pH 3, the acid tolerance of Kluyveromyces AMCC31690 and AMCC31691 was slightly lower than that of AMCC30630, but both reached more than 80% of that of AMCC30630 (i.e., greater than or equal to 80%).

[0157] (3) tolerance to bile salts

[0158] Kluyveromyces martensii AMCC31690, AMCC31691, and AMCC30630 were inoculated at 1% (v / v) into 96-well plates containing YPD liquid medium at concentrations of 0.2% (w / v), 0.3% (w / v), and 0.4% (w / v) bile salts, respectively. After incubation at 37°C and 180 rpm for 24 h, OD values ​​were measured. 600nm The values ​​are shown in Figure 9.

[0159] Figure 9 shows the OD values ​​of the *Kluyveromyces martensii* AMCC31690 suspension cultured in YPD liquid medium with a bile salt concentration of 0.2% (w / v). 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm Value; OD value of *Kluyveromyces martensii* AMCC31690 bacterial suspension cultured in YPD liquid medium with a bile salt concentration of 0.3% (w / v). 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm Values; and the OD values ​​of *Kluyveromyces martensii* AMCC31690 suspension cultured in YPD liquid medium with a bile salt concentration of 0.4% (w / v). 600nm Value, OD of Kluyveromyces macrocephala AMCC31691 suspension 600nm OD values ​​of Kluyveromyces macrocephala AMCC30630 bacterial suspension 600nm value.

[0160] The results showed that with increasing bile salt concentration in YPD liquid medium, the difference in bile salt tolerance between *Kluyveromyces martensii* AMCC31690 and AMCC31691 and *Kluyveromyces martensii* AMCC30630 gradually increased. *Kluyveromyces martensii* AMCC31690 and AMCC31691 exhibited significantly higher bile salt tolerance than *Kluyveromyces martensii* AMCC30630. When the bile salt concentration in YPD liquid medium was 0.4% (w / v), the OD value of the bacterial suspension of *Kluyveromyces martensii* AMCC30630 was significantly higher. 600nm The levels of *Kluyveromyces martensii* AMCC31690 and *Kluyveromyces martensii* AMCC31691 were increased by 206% and 134%, respectively.

[0161] (4) Resistance to gastric and intestinal fluids

[0162] Real gastrointestinal digestion is a complex and continuous process involving multiple biochemical steps and the synergistic effects of various digestive enzymes, hormones, and neural regulation. Studies indicate that gastric digestion takes approximately 1 to 3 hours, while small intestinal digestion takes approximately 2 to 3 hours. Based on this, the static model of simulated gastrointestinal digestion designed in this invention sets the gastric digestion stage to 2 hours and the intestinal digestion stage to 3 hours. The in vitro digestion results of this static model are highly correlated with the in vivo digestion results in humans, more accurately simulating the human digestive process. At the same time, it overcomes the limitations of long experimental cycles and the impact of reduced digestive enzyme activity on experimental results caused by prolonged digestion. Therefore, this invention selects the survival rates at 2 hours and 5 hours as evaluation indicators for the resistance of Kluyveromyces martensii strain to gastric and intestinal fluids.

[0163] (4.1) Preparation of artificial gastric juice: Pepsin was dissolved in physiological saline (the concentration of physiological saline was 0.9% (W / V)) to obtain a mixture. The pH of the mixture was adjusted to 2.0 with 1 mol / L HCl solution. The mixture was then filtered through a 0.22 μm filter membrane to obtain a pepsin solution with a final concentration of 3 g / L, which is the artificial gastric juice.

[0164] (4.2) Preparation of artificial intestinal fluid: 3.4 g of KH2PO4 was dissolved in 250 mL of distilled water to obtain a mixture. The pH of the mixture was adjusted to 6.8 with 1 mol / L NaOH solution, and water was added to make up to 500 mL. Then, 0.3% (w / v) bile salts and 1% (w / v) trypsin were added to the mixture. After mixing and dissolving, the mixture was filtered through a 0.22 μm filter membrane to obtain artificial intestinal fluid.

[0165] (4.3) Preparation of bacterial suspensions without artificial gastric or intestinal fluid treatment (i.e., untreated bacterial suspensions): Kluyveromyces macrocarpa AMCC31690, AMCC31691, and AMCC30630 were inoculated into YPD liquid medium at a 1% (v / v) inoculum. After culturing at 30°C and 180 rpm for 24 h, the supernatant was discarded by centrifugation at 8000 g for 5 min, and the bacterial pellet was retained. The pellet was washed twice with sterile physiological saline and resuspended to obtain untreated AMCC31690, AMCC31691, and AMCC30630 bacterial suspensions, respectively. These were then diluted and plated onto YPD solid medium plates. Plates with colony counts between 30 and 300 were selected for viable cell counting (i.e., untreated viable cell counts). The results are shown in Table 3.

[0166] Preparation of bacterial suspensions treated in artificial gastric fluid for 2 hours (i.e., bacterial suspensions after 2 hours of treatment): 1 mL of untreated *Kluyveromyces martensii* AMCC31690 bacterial suspension, 1 mL of untreated *Kluyveromyces martensii* AMCC31691 bacterial suspension, and 1 mL of untreated *Kluyveromyces martensii* AMCC30630 bacterial suspension were mixed with 9 mL of the artificial gastric fluid and cultured at 37°C and 80 rpm for 2 hours to obtain *Kluyveromyces martensii* AMCC31690, *Kluyveromyces martensii* AMCC31691, and *Kluyveromyces martensii* AMCC30630 bacterial suspensions after 2 hours of treatment. 0.5 mL of each suspension was added to 4.5 mL of sterile physiological saline, and then added sequentially at 10... -5 10 -6 10 -7 The bacteria were serially diluted and plated. Plates with colony counts between 30 and 300 were selected for viable count (i.e., viable count after 2 hours of treatment). The results are shown in Table 3.

[0167] Preparation of a bacterial suspension after treatment in artificial gastric fluid for 2 hours and then in artificial intestinal fluid for 3 hours (i.e., the bacterial suspension after 5 hours of treatment): Take 1 mL of the bacterial suspension-artificial gastric fluid mixture, centrifuge at 5000g for 10 min, discard the supernatant and retain the bacterial precipitate, then resuspend the bacterial precipitate in 1 mL of physiological saline to obtain a bacterial suspension. Add the bacterial suspension to 9 mL of artificial intestinal fluid and incubate at 37℃ and 80 rpm for 3 hours to obtain Kluyveromyces martensii AM after 5 hours of treatment. The following bacterial suspensions were used: CC31690, AMCC31691 (treated for 5 hours), and AMCC30630 (treated for 2 hours). 1 mL of each suspension was serially diluted and spread onto YPD solid agar plates. Plates with colony counts between 30 and 300 were selected for viable cell count (i.e., viable cell count after 5 hours of treatment). The results are shown in Table 3. The survival rate was calculated using the formula shown below, and the results are shown in Figure 10. The survival rate calculation formula is: Survival rate (%) = (Viable cell count after 5 hours of treatment / Viable cell count before treatment) × 100%.

[0168] Table 3. Viable bacterial counts in bacterial suspensions before and after treatment with artificial gastric and intestinal fluids.

[0169] As shown in Table 3, Table 3 shows the viable cell counts of Kluyveromyces AMCC30630, Kluyveromyces AMCC31690, and Kluyveromyces AMCC31691 before and after treatment with artificial gastric fluid and artificial intestinal fluid; as shown in Figure 10, Figure 10 shows the survival rates of Kluyveromyces AMCC30630, Kluyveromyces AMCC31690, and Kluyveromyces AMCC31691 after being cultured in artificial gastric fluid for 2 hours and then in artificial intestinal fluid for 3 hours (after 5 hours of treatment). The results showed that the survival rates of *Kluyveromyces martensii* AMCC31690 and AMCC31691 were 96.78% and 70.23%, respectively. *Kluyveromyces martensii* AMCC31690 and AMCC31691 showed significantly higher tolerance to gastrointestinal fluids than *Kluyveromyces martensii* AMCC30630. Furthermore, compared to the survival rate of *Kluyveromyces martensii* AMCC30630, the survival rates of *Kluyveromyces martensii* AMCC31690 and AMCC31691 were increased by 168.9% and 93.95%, respectively.

[0170] Example 4: Surface activity determination of Kluyveromyces martensii strain

[0171] (1) Determination of self-agglomeration rate

[0172] (1.1) Determination of the initial OD of Kluyveromyces martensii AMCC31690 suspension 600nm Values: Kluyveromyces martensii AMCC31690 was inoculated into YPD liquid medium at a 1% (v / v) inoculum and cultured at 30°C and 180 rpm for 24 h. After centrifugation at 5000 g for 2 min, the supernatant was discarded, and the cell pellet was retained. 1 mL of PBS buffer was added to the cell pellet for resuspending to obtain a Kluyveromyces martensii AMCC31690 suspension. This process was repeated once. The OD value of the Kluyveromyces martensii AMCC31690 suspension was then measured. 600nm Value, i.e., the initial OD value of the Kluyveromyces macrocephala AMCC31690 bacterial suspension. 600nm value.

[0173] Initial OD of Kluyveromyces martensii AMCC31691 600nm Values ​​and initial OD of Kluyveromyces maculae AMCC30630 600nm The method for determining the value is the same as that for Kluyveromyces martensii AMCC31690.

[0174] (1.2) After the *Kluyveromyces martensii* AMCC31690 suspension was allowed to stand at 37°C for 2 h and 5 h, 200 μL of the upper layer of *Kluyveromyces martensii* AMCC31690 suspension was aspirated. The OD value of the upper layer of *Kluyveromyces martensii* AMCC31690 suspension was then measured. 600nm Value, namely the OD value of the Kluyveromyces martensii AMCC31690 bacterial suspension after standing for 2 hours and 5 hours. 600nm value.

[0175] OD values ​​of Kluyveromyces martensii AMCC31691 suspension after standing for 2 hours and 5 hours 600nm The values, and the OD values ​​of Kluyveromyces martensii AMCC30630 bacterial suspensions after standing for 2 hours and 5 hours. 600nm The method for determining the value is the same as that for Kluyveromyces martensii AMCC31690.

[0176] OD was measured using PBS buffer as a control. 600nm This value is used to eliminate the influence of factors such as instrument error and light source fluctuation on the measurement results. The self-agglomeration rate is calculated according to the formula shown below, and the results are shown in Figure 11:

[0177] Self-agglutination rate (%) = 1 - A t / A0×100%,

[0178] Wherein, A0 represents the initial OD of *Kluyveromyces martensii* AMCC31690, *Kluyveromyces martensii* AMCC31691, or *Kluyveromyces martensii* AMCC30630 suspensions. 600nm Value, At, represents the OD value of *Kluyveromyces cerevisiae* AMCC31690, *Kluyveromyces cerevisiae* AMCC31691, or *Kluyveromyces cerevisiae* AMCC30630 suspensions after standing for 2 h and 5 h, respectively. 600nm value.

[0179] Figure 11 shows the autoaggregation rates of *Kluyveromyces martensii* strains AMCC30630, AMCC31690, and AMCC31691. The results indicate that all three strains possess autoaggregation ability, and during static autoaggregation, this ability continuously increases with time. After standing for 5 hours, the autoaggregation rates of *Kluyveromyces martensii* AMCC30630, AMCC31690, and AMCC31691 reached their highest levels, all greater than or equal to 90%, with no significant difference. Among them, the autoaggregation rates of *Kluyveromyces martensii* AMCC30630, AMCC31690, and AMCC31691 were 96.08%, 97.53%, and 97.04%, respectively.

[0180] (2) Hydrophobicity determination

[0181] (2.1) Determination of the initial OD of Kluyveromyces martensii AMCC31690 suspension 600nm Values: Kluyveromyces martensii AMCC31690 was inoculated into YPD liquid medium at a 1% (v / v) inoculum and cultured at 30°C and 180 rpm for 24 h. After centrifugation at 5000 g for 2 min, the supernatant was discarded, and the cell pellet was retained. 1 mL of PBS buffer was added to the cell pellet to resuspend the pellet, obtaining a Kluyveromyces martensii AMCC31690 suspension. This process was repeated once. The OD value of the Kluyveromyces martensii AMCC31690 suspension was then measured. 600nm Value, i.e., the initial OD value of the Kluyveromyces macrocephala AMCC31690 bacterial suspension. 600nm value.

[0182] Initial OD of Kluyveromyces martensii AMCC31691 600nm Values ​​and initial OD of Kluyveromyces maculae AMCC30630 600nmThe method for determining the value is the same as that for Kluyveromyces martensii AMCC31690.

[0183] (2.2) 500 μL of acetone and chloroform were added to 1.5 mL of Kluyveromyces martensii AMCC31690 bacterial suspension and mixed thoroughly to obtain a Kluyveromyces martensii AMCC31690 mixture. The mixture was then allowed to stand at 37 °C for 2 h and 5 h to separate into layers. The aqueous phase of the Kluyveromyces martensii AMCC31690 mixture was collected, and the OD of the aqueous phase was measured. 600nm The value is calculated, and the hydrophobicity is determined.

[0184] OD of the aqueous phase of a mixture of Kluyveromyces martensii AMCC31691 600nm The OD value of the aqueous phase of the Kluyveromyces macrocephala AMCC30630 mixture. 600nm The method for determining the value is the same as that for Kluyveromyces martensii AMCC31690.

[0185] OD was measured using PBS buffer as a control. 600nm This value is used to eliminate the influence of factors such as instrument error and light source fluctuation on the measurement results. The hydrophobicity is calculated according to the formula shown below, and the result is shown in Figure 12:

[0186] Hydrophobicity (%) = [(A0 - A t ) / A0]×100%,

[0187] Wherein, A0 represents the initial OD of *Kluyveromyces martensii* AMCC31690, *Kluyveromyces martensii* AMCC31691, or *Kluyveromyces martensii* AMCC30630 suspensions. 600nm Value, At, represents the OD value of the aqueous phase of a Kluyveromyces macrocephala AMCC31690 mixture. 600 nm OD values ​​of aqueous phase of Kluyveromyces macrocephala AMCC31691 mixture 600nm OD value of aqueous phase of Kluyveromyces macrocephala AMCC30630 mixture 600nm value.

[0188] It should be noted that when evaluating the hydrophobicity of bacterial surfaces, the ability of the strain to adhere to hydrocarbons is usually considered an important indicator. In this experiment, the adhesion ability of the strain to acetone and chloroform was used as the main evaluation indicator. A strain with a hydrophobicity greater than 60% is considered a highly hydrophobic strain, a strain with a hydrophobicity between 40% and 60% can be considered a moderately hydrophobic strain, and a strain with a hydrophobicity less than 40% is considered a hydrophilic strain.

[0189] As shown in Figures 12 and 13, Figure 12 shows the hydrophobicity of *Kluyveromyces martensii* AMCC30630, AMCC31690, and AMCC31691 to chloroform and acetone after 2 hours of standing. Figure 13 shows the hydrophobicity of these three yeasts to chloroform and acetone after 5 hours of standing. The results indicate that after 2 hours of standing, the hydrophobicity of *Kluyveromyces martensii* AMCC30630, AMCC31690, and AMCC31691 to acetone is greater than or equal to 60%, and the hydrophobicity of chloroform to chloroform is greater than or equal to 85%.

[0190] Among them, after standing for 2 hours, the hydrophobicity of Kluyveromyces AMCC30630, Kluyveromyces AMCC31690 and Kluyveromyces AMCC31691 to acetone was 62.94%, 64.72% and 67.60%, respectively.

[0191] After standing for 2 hours, the hydrophobicity of Kluyveromyces macrocarpa AMCC30630, Kluyveromyces macrocarpa AMCC31690 and Kluyveromyces macrocarpa AMCC31691 to chloroform was 88.69%, 91.27% and 92.20%, respectively.

[0192] Meanwhile, after standing for 5 hours, the hydrophobicity of *Kluyveromyces martensii* AMCC30630, AMCC31690, and AMCC31691 to acetone and chloroform was all greater than or equal to 90%, with no significant difference.

[0193] Among them, after standing for 5 hours, the hydrophobicity of Kluyveromyces macrocarpa AMCC30630, Kluyveromyces macrocarpa AMCC31690 and Kluyveromyces macrocarpa AMCC31691 to acetone was 95.69%, 96.37% and 94.79%, respectively.

[0194] After standing for 5 hours, the hydrophobicity of Kluyveromyces macrocarpa AMCC30630, Kluyveromyces macrocarpa AMCC31690 and Kluyveromyces macrocarpa AMCC31691 to chloroform was 93.14%, 96.97% and 95.81%, respectively.

[0195] Therefore, Kluyveromyces macrocarpa AMCC30630, Kluyveromyces macrocarpa AMCC31690 and Kluyveromyces macrocarpa AMCC3169 are all highly hydrophobic strains.

[0196] In summary, the Kluyveromyces macrocarpa AMCC30630, AMCC31690, or AMCC31691 provided by this invention possess multiple tolerances, can survive in the gastrointestinal tract, maintain vitality, and exhibit high hydrophobicity and self-aggregation ability. Therefore, Kluyveromyces macrocarpa AMCC30630, AMCC31690, or AMCC31691 can be applied to the preparation of food starter cultures, probiotic products, and feed additives.

[0197] The above embodiments are only for further explanation and understanding of the technical solution of the present invention, and are not intended to limit the present invention. Any improvements made by those skilled in the art on this basis that do not highlight substantive features or make significant progress should fall within the protection scope of the present invention.

Claims

1. A Kluyveromyces marxianus, characterized in that, The Kluyveromyces marxianus mentioned is AMCC 31690, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2024246.

2. A Kluyveromyces marxianus, characterized in that, The Kluyveromyces marxianus mentioned is Kluyveromyces marxianus AMCC 31691, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 2024247.

3. A Kluyveromyces marxianus, characterized in that, The Kluyveromyces marxianus mentioned is AMCC 30630, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO:M 20222107.

4. The K. marxianus of claim 1, wherein, The ITS gene sequence of the Kluyveromyces martensii AMCC 31690 is shown in SEQ ID No.

4.

5. The K. marxianus of claim 2, wherein, The ITS gene sequence of the Kluyveromyces martensii AMCC 31691 is shown in SEQ ID No.

5.

6. The K. marxianus of claim 3, wherein, The ITS gene sequence of the Kluyveromyces martensii AMCC 30630 is shown in SEQ ID No.

3.

7. The K. marxianus of claim 1 or 4 or the K. marxianus of claim 2 or 5, characterized in that, Obtained by mutagenesis of Kluyveromyces masculinus as described in claim 3 or 6.

8. The Kluyveromyces marxianus of any one of claims 1-7, wherein, It possesses osmotic pressure resistance, and / or acid resistance, and / or bile salt resistance, and / or hydrophobicity, and / or self-aggregation ability, and / or gastric juice resistance, and / or intestinal juice resistance.

9. The K. marxianus of claim 8, characterized in that, Its osmotic pressure resistance is: it can survive in liquid culture medium containing 3% (w / v) to 7% (w / v) sodium chloride.

10. The K. marxianus of claim 8, wherein, Its acid resistance is: it can survive in liquid culture medium with a pH of 2.0-3.

0.

11. The K. marxianus of claim 8, wherein, Its bile salt tolerance is: survival in liquid culture medium containing bile salt concentrations of 0.2% (W / V) to 0.4% (W / V).

12. The K. marxianus of claim 8, wherein, The gastric acid resistance is as follows: the survival rate of the Kluyveromyces martensii AMCC 31690 after gastric acid treatment is greater than or equal to 68%. Or, the gastric resistance is: the survival rate of the *Kluyveromyces martensii* AMCC 31691 after gastric juice treatment is greater than or equal to 63%. Alternatively, the gastric resistance is: the survival rate of the described Kluyveromyces martensii AMCC 30630 after gastric juice treatment is greater than or equal to 58%.

13. The K. marxianus of claim 8, wherein, The resistance to gastric and intestinal fluids is as follows: the survival rate of the described Kluyveromyces martensii AMCC 31690 after treatment with gastric and intestinal fluids is greater than or equal to 95%. Or, the resistance to gastric and intestinal fluids is: the survival rate of the described Kluyveromyces martensii AMCC 31691 after treatment with gastric and intestinal fluids is greater than or equal to 70%. Alternatively, the resistance to gastric and intestinal fluids is: the survival rate of the described Kluyveromyces martensii AMCC 30630 after treatment with gastric and intestinal fluids is greater than or equal to 52%.

14. The K. marxianus of claim 8, wherein, The self-agglomeration ability is: the self-agglomeration rate is greater than or equal to 60%; preferably, the self-agglomeration rate is greater than or equal to 85% and less than or equal to 100%.

15. The K. marxianus of claim 8, wherein, Hydrophobicity is defined as follows: hydrophobicity to acetone is greater than or equal to 60%, and / or hydrophobicity to chloroform is greater than or equal to 40%.

16. The K. marxianus of claim 15, characterized in that, Hydrophobicity is defined as follows: hydrophobicity to acetone is greater than or equal to 60% and less than or equal to 100%, and / or hydrophobicity to chloroform is greater than or equal to 60% and less than or equal to 100%.

17. A method for the fermentative production of a Kluyveromyces marxianus inoculum, characterized in that, It includes the following steps: amplifying and culturing *Kluyveromyces martensii* according to any one of claims 1-16.

18. A K. marxianus inoculum, characterized in that, Contains *Kluyveromyces martensii* as described in any one of claims 1-16.

19. The inoculant of claim 18, wherein, It is prepared by the fermentation preparation method described in claim 17.

20. A food fermenting agent, characterized by, Contains *Kluyveromyces martensii* as described in any one of claims 1-16, and / or the inoculum as described in claim 18 or 19.

21. The use of *Kluyveromyces martensii* according to any one of claims 1-16, and / or the inoculum according to claim 18 or 19, in a food fermentation agent.

22. A probiotic product, characterized in that, Contains *Kluyveromyces martensii* as described in any one of claims 1-16, and / or the inoculum as described in claim 18 or 19.

23. The use of Kluyveromyces martensii according to any one of claims 1-16, and / or the bacterial agent according to claim 18 or 19, in probiotic products.

24. A feed additive, characterized in that, Contains *Kluyveromyces martensii* as described in any one of claims 1-16, and / or the inoculum as described in claim 18 or 19.

25. The use of Kluyveromyces martensii according to any one of claims 1-16, and / or the microbial agent according to claim 18 or 19, in feed additives.

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