Kluyveromyces marxianus strain, microbial agent, fermentation broth, fermented food, and preparation and use thereof

Abstract

The present invention belongs to the field of fermentation, and specifically relates to a Kluyveromyces marxianus strain, a microbial agent, a fermentation broth, a fermented food, and a preparation and a use thereof. Provided is Kluyveromyces marxianus AMCC 31586, having the accession number CCTCC NO:M20231793. In one aspect, Kluyveromyces marxianus AMCC 31586 has good substrate utilization ability, being able to fully utilize carbon source substrates such as lactose and galactose, thereby reducing the health burden of people with lactose intolerance or galactosemia. In another aspect, fermented milk obtained by fermentation with Kluyveromyces marxianus AMCC 31586 contains unique flavor substances such as eugenol and anethole, and it also has good utilization ability for carbon source substrates such as organic acids, lactose and galactose. Furthermore, in dairy fermentation, it can improve the quality of fermented dairy products and enrich the flavor of fermented dairy products.

Description

Kluyveromyces marxianus strain, inoculum, fermentation broth, fermented food and preparation and application thereof

[0001] Related applications

[0002] This application claims priority to the prior application filed on June 14, 2024, with the China National Intellectual Property Office, application number 202410772667.7, and incorporates it in its entirety. TECHNICAL FIELD

[0003] The present application belongs to the field of fermentation, and specifically relates to Kluyveromyces marxianus strain, inoculum, fermentation broth, fermented food and preparation and application thereof. BACKGROUND

[0004] Yeast plays an irreplaceable role in the fermentation of traditional dairy products. The growth and reproduction of yeast improve product flavor, increase product quality and nutritional value. Lactose, the only carbon source in milk, is completely utilized after being hydrolyzed into glucose and galactose by β-galactosidase. However, the yeast used in the prior art, Saccharomyces cerevisiae, cannot directly utilize lactose in dairy product fermentation because it does not contain β-galactosidase. However, Kluyveromyces marxianus contains abundant β-galactosidase, which can rapidly ferment lactose in milk to produce a variety of flavor substances. However, not all Kluyveromyces marxianus strains can ferment lactose in milk, and the sensory differences of dairy products obtained by different strains are large, so it is crucial to screen Kluyveromyces marxianus suitable for dairy product fermentation.

[0005] The invention patent with application publication number CN 115895920 A isolated an ester-producing Kluyveromyces marxianus from Tibetan yak milk residue. First, a tolerance test was conducted, including acid tolerance (pH 2.0-pH 4.0), salt tolerance (NaCl concentration 1%-7%), and bile salt tolerance (bovine bile salt concentration 0.1%-0.5%); second, the Kluyveromyces marxianus strain with good tolerance was mixed with lactic acid bacteria to ferment pasteurized milk, and the volatile flavor components were detected and sensory evaluation was conducted; finally, the best-performing Kluyveromyces marxianus strain was selected. The Kluyveromyces marxianus mixed with lactic acid bacteria can ferment commercially available pasteurized milk to produce alcohol-containing fermented milk and a variety of ester flavor substances, but the lactose not utilized during the fermentation of dairy products and the galactose accumulated by lactic acid bacteria fermentation affect the quality of the products and cause health burden to patients with lactose intolerance and galactosemia. SUMMARY

[0006] The technical problem solved by the present application is that the prior art has the problem of incomplete utilization of carbon source substrates such as lactose and galactose during the fermentation of dairy products, which causes health burden to patients with lactose intolerance or galactosemia, and also causes the negative effects of lack of flavor substances and low quality of fermented dairy products.

[0007] The present application provides a Kluyveromyces marxianus strain, a microbial agent, a fermentation broth, a fermented food and preparation and application thereof to solve the problems of the prior art.

[0008] Specifically, the present application provides the following technical solutions.

[0009] In the first aspect, the present application provides a Kluyveromyces marxianus AMCC 31586, characterized in that the Kluyveromyces marxianus AMCC 31586 (Kluyveromyces marxianus AMCC 31586) is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 20231793.

[0010] Preferably, the ITS gene sequence of the Kluyveromyces marxianus AMCC 31586 is shown in SEQ ID No. 3.

[0011] Preferably, the Kluyveromyces marxianus AMCC 31586 has the characteristics of utilizing multiple carbon source substrates.

[0012] Preferably, the carbon source substrate comprises one or more than one selected from the group consisting of monosaccharides, disaccharides and organic acids; more preferably, the carbon source substrate comprises one or more than one selected from the group consisting of sucrose, lactose, galactose, glucose and lactic acid.

[0013] In the second aspect, the present application provides a fermentation preparation method of a Kluyveromyces marxianus microbial agent, which comprises the following steps:

[0014] (1) amplifying and culturing the Kluyveromyces marxianus AMCC 31586;

[0015] (2) adding the Kluyveromyces marxianus AMCC 31586 after amplification and culture into a liquid culture medium and fermenting and culturing at 26-45℃.

[0016] In the third aspect, the present application provides a Kluyveromyces marxianus microbial agent containing the Kluyveromyces marxianus AMCC 31586 or prepared by the fermentation preparation method.

[0017] In the fourth aspect, the present application provides a fermentation broth containing the Kluyveromyces marxianus AMCC 31586 or the Kluyveromyces marxianus microbial agent.

[0018] Preferably, the flavoring substances in the fermentation broth comprise one or more than one selected from the group consisting of ester substances, alcohol substances, aldehyde substances and acid substances.

[0019] Preferably, the flavoring substances in the fermented dairy product include one or more than one substance selected from the group consisting of phenylethyl acetate, isoamyl acetate, δ-dodecalactone, ethyl decanoate, ethyl octanoate, ethyl hexanoate, geraniol, linalool, isoamyl alcohol, phenylethanol, nerolidol, citronellol, phenylacetaldehyde and nonal.

[0020] In a fifth aspect, the present application provides a fermented food product comprising the Kluyveromyces marxianus AMCC 31586 or the Kluyveromyces marxianus inoculum.

[0021] In a sixth aspect, the present application provides use of the Kluyveromyces marxianus AMCC 31586 or the Kluyveromyces marxianus inoculum in a fermented food product.

[0022] Preferably, the fermented food product comprises one or more than one carbon source substrate selected from the group consisting of monosaccharides, disaccharides and organic acids; preferably, one or more than one carbon source substrate selected from the group consisting of lactose, galactose, sucrose, glucose and lactic acid.

[0023] Preferably, the fermented food product comprises one or more than one substance selected from the group consisting of fermented milk, fermented wine, fruit and vegetable beverage, protein beverage, fermented wine, fermented milk, condiment and flour product.

[0024] In a seventh aspect, the present application provides a fermented milk product comprising the Kluyveromyces marxianus AMCC 31586 or the Kluyveromyces marxianus inoculum.

[0025] In an eighth aspect, the present application provides use of the Kluyveromyces marxianus AMCC 31586 or the Kluyveromyces marxianus inoculum in a fermented milk product.

[0026] Preferably, the flavoring substances in the fermented dairy product include one or more than one substance selected from the group consisting of ester substances, alcohol substances, aldehyde substances and phenol substances.

[0027] Preferably, the flavoring substances in the fermented dairy product include one or both of the substances selected from the group consisting of eugenol and anethole.

[0028] Preferably, the flavoring substances in the fermented dairy product further include one or more than one substance selected from the group consisting of phenylacetaldehyde, isoamyl alcohol, phenylethanol, ethyl undecanoate, isoamyl acetate, ethyl hexanoate, ethyl decanoate, ethyl isoamylate, phenylethyl acetate and ethyl octanoate.

[0029] The application has the following beneficial effects: the Kluyveromyces marxianus AMCC 31586 provided by the application has good substrate utilization capacity, can fully utilize carbon source substrates such as lactose and galactose, reduces the health burden of lactose intolerance or galactosemia population, and on the other hand, the fermented milk fermented by the strain contains unique flavor substances such as eugenol and anethole, and has good utilization capacity for organic acids, lactose, galactose and other carbon source substrates, so that the quality of fermented dairy products is improved, and the flavor of fermented dairy products is enriched. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1 shows a lactase screening medium plate phenotype diagram.

[0031] Figure 2 shows a Kluyveromyces marxianus strain AMCC 31586 colony morphology diagram.

[0032] Figure 3 shows a Kluyveromyces marxianus strain AMCC 31586 microscopic morphology diagram.

[0033] Figure 4 shows a growth curve of Kluyveromyces marxianus AMCC 31586 under the condition that the carbon source substrate is 10% (w / v) sucrose, 4% (w / v) lactose, 4% (w / v) galactose, 4% (w / v) glucose, 0.7% (w / v) lactic acid, 1.1% (w / v) lactic acid or 2% (w / v) lactic acid.

[0034] Figure 5 shows the aroma activity diagram of flavor substances higher than the threshold concentration in the milk fermented by Kluyveromyces marxianus AMCC 31586.

[0035] Figure 6 shows a fermentation broth GC-MS chromatogram of Kluyveromyces marxianus AMCC 31586.

[0036] Strain preservation information

[0037] The Kluyveromyces marxianus AMCC 31586 provided by the application was preserved in the China Center for Type Culture Collection on September 25, 2023, with the preservation number CCTCC NO: M 20231793, the preservation address is Wuhan, China, Wuhan University, the postcode is 430072, and the telephone number is 027-68754052. DETAILED DESCRIPTION

[0038] In order to better understand the above technical solutions, the technical solutions of the present application will be clearly and completely explained and described below in combination with specific embodiments. It should be noted that the contents in the specific embodiments are only the specific implementation and explanation of the technical solutions of the present application, and should not be understood as a limitation on the protection scope of the present application.

[0039] In view of the problem that the existing technology does not completely utilize carbon source substrates such as lactose and galactose in the fermentation process of dairy products, which causes a health burden to patients with lactose intolerance or galactosemia, and also causes the negative effects of lack of flavor substances and low quality of fermented dairy products.

[0040] The present application provides a Kluyveromyces marxianus AMCC 31586 strain, which was preserved in the China Center for Type Culture Collection on September 25, 2023, and the preservation number is CCTCC NO:M 20231793.

[0041] The Kluyveromyces marxianus strain AMCC 31586 is derived from a fermented dairy product made by a herdsman in Tongliao City, Inner Mongolia Autonomous Region, and is a more suitable yeast strain for dairy product fermentation obtained after isolation and screening. The screening includes carbon source substrate screening and fermented milk screening.

[0042] The carbon source substrate includes one or more of monosaccharides, disaccharides and organic acids; preferably, the carbon source substrate includes one or more of sucrose, lactose, galactose, glucose and lactic acid.

[0043] It should be noted that the carbon source selection of the present application is based on: sucrose is the main component in molasses, which is the main raw material for microbial fermentation; lactose is the only carbon source in milk, and galactose and glucose are the hydrolysis products of lactose, which can be completely utilized after being hydrolyzed by β-galactosidase; lactic acid is the main organic acid in yogurt.

[0044] In some embodiments, the raw materials used in the preparation of the culture medium of the present application include yeast extract and yeast peptone, which are mainly present in the culture medium as organic nitrogen sources to provide the necessary nitrogen elements for the growth of microorganisms during fermentation. After the decomposition of yeast extract, yeast peptone and other common organic nitrogen sources in the seed culture medium, amino acids and small molecular peptides are released to become the nitrogen source required for microbial growth. That is, when yeast peptone and yeast extract are used as common organic nitrogen sources to prepare the culture medium of the present application, the source of the present application is not particularly limited, and they can be obtained from any commercially available or prepared by conventional methods. As long as the commercially available or prepared by conventional methods yeast peptone, the total nitrogen content is greater than or equal to 12.5wt%, the amino nitrogen content is greater than or equal to 2.5wt%, which can be used in the present application. As long as the commercially available yeast extract, the total nitrogen content is greater than or equal to 10.0wt%, the amino nitrogen content is greater than or equal to 5.0wt%, which can be used in the present application.

[0045] Preferably, in some embodiments, the yeast extract further includes, 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 nicotinic acid, and 2-4 ug of vitamin B12 per 100 g of yeast extract.

[0046] And / or based on the weight of the yeast extract, 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.

[0047] And / or based on the weight of the yeast extract, based on the weight of the yeast extract, the yeast extract includes 30.3-40.85% of free amino acids and 51-70.5% of hydrolyzed amino acids.

[0048] The free amino acid content includes: 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 leucine, 0.5-1% 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, based on the weight of the yeast extract powder.

[0049] The hydrolyzed amino acid content includes: 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, based on the weight of the yeast extract powder.

[0050] In some embodiments, the percentage content of the carbon source substrate in the liquid medium containing the carbon source substrate refers to the weight content of the carbon source substrate in g in 100 mL of the liquid medium, so as to obtain the percentage content of the carbon source substrate in the liquid medium, and the percentage content unit is % (w / v). The percentage content of the carbon source substrate in the liquid medium is 0.7% (w / v)-10% (w / v), and the carbon source substrate is one or more of sucrose, lactose, galactose, glucose and lactic acid.

[0051] Preferably, in some embodiments, the liquid medium includes 0.65-0.7% (w / v) of YNB medium and 0.7% (w / v)-10% (w / v) of the carbon source substrate, and the rest is water.

[0052] wherein the YNB medium comprises, in parts by weight, ammonium sulfate 4900-5100 parts, myo-inositol 1-3 parts, nicotinic acid 0.2-0.5 parts, thiamine hydrochloride 0.2-0.5 parts, copper sulfate 0.02-0.05 parts, potassium phosphate monobasic 950-1050 parts, boric acid 0.4-0.6 parts, pyridoxal hydrochloride 0.3-0.5 parts, calcium pantothenate 0.3-0.5 parts, p-aminobenzoic acid 0.3-0.5 parts, magnesium sulfate 490-510 parts, manganese sulfate 0.3-0.5 parts, zinc sulfate 0.3-0.5 parts, ferric chloride 0.1-0.3 parts, riboflavin 0.1-0.3 parts, calcium chloride 95-105 parts, potassium iodide 0.05-0.15 parts, sodium molybdate 0.1-0.3 parts, biotin 0.001-0.003 parts, folic acid 0.001-0.003 parts, and sodium chloride 95-105 parts per liter.

[0053] Preferably, after the YNB medium is mixed with water and made up to 1 L, the nutritional composition comprises, per L, ammonium sulfate 4900-5100 mg, myo-inositol 1-3 mg, nicotinic acid 0.2-0.5 mg, thiamine hydrochloride 0.2-0.5 mg, copper sulfate 0.02-0.05 mg, potassium phosphate monobasic 950-1050 mg, boric acid 0.4-0.6 mg, pyridoxal hydrochloride 0.3-0.5 mg, calcium pantothenate 0.3-0.5 mg, p-aminobenzoic acid 0.3-0.5 mg, magnesium sulfate 490-510 mg, manganese sulfate 0.3-0.5 mg, zinc sulfate 0.3-0.5 mg, ferric chloride 0.1-0.3 mg, riboflavin 0.1-0.3 mg, calcium chloride 95-105 mg, potassium iodide 0.05-0.15 mg, sodium molybdate 0.1-0.3 mg, biotin 0.001-0.003 mg, folic acid 0.001-0.003 mg, and sodium chloride 95-105 mg.

[0054] In some embodiments, the growth rate (OD600nm) of K. marxianus AMCC 31586 cells after being cultured in a liquid medium containing a carbon source substrate (0.7% (w / v) - 10% (w / v) of the carbon source substrate) is 310% - 1065%.

[0055] Preferably, in some embodiments, the growth rate (OD600nm) of K. marxianus AMCC 31586 cells after being cultured in a liquid medium containing 9-11% (w / v) sucrose is 740% - 745%.

[0056] Preferably, in some embodiments, the growth rate (OD600nm) of K. marxianus AMCC 31586 cells after being cultured in a liquid medium containing 3-5% (w / v) glucose is 555% - 560%.

[0057] Preferably, in some embodiments, the growth rate (OD600nm) of the K. marxianus AMCC 31586 cells after cultivation in a liquid medium containing 3-5% (w / v) galactose is 510-515%.

[0058] Preferably, in some embodiments, the growth rate (OD600nm) of the K. marxianus AMCC 31586 cells after cultivation in a liquid medium containing 3-5% (w / v) galactose is 510-515%.

[0059] Preferably, in some embodiments, the growth rate (OD600nm) of the K. marxianus AMCC 31586 cells after cultivation in a liquid medium containing 0.6-0.8% (w / v) lactic acid is 310-315%.

[0060] Preferably, in some embodiments, the growth rate (OD600nm) of the K. marxianus AMCC 31586 cells after cultivation in a liquid medium containing 1-1.2% (w / v) lactic acid is 470-475%.

[0061] Preferably, in some embodiments, the growth rate (OD600nm) of the K. marxianus AMCC 31586 cells after cultivation in a liquid medium containing 1.9-2.1% (w / v) lactic acid is 245-250%.

[0062] Preferably, in some embodiments, the growth rate (OD600nm) of the K. marxianus AMCC 31586 cells after cultivation in a liquid medium containing 0.6-2.1% (w / v) lactic acid is 310-250%.

[0063] More preferably, in some embodiments, the growth rate (OD600nm) of the K. marxianus AMCC 31586 cells after cultivation in a liquid medium containing 0.7-2% (w / v) lactic acid is 310-250%.

[0064] In some embodiments, the K. marxianus AMCC 31586, after 24h cultivation in a liquid medium containing 3-5% (w / v) galactose, the K. marxianus AMCC 31586 has a lactose degradation rate in the liquid medium of 88.8-100%.

[0065] In some embodiments, the Kluyveromyces marxianus AMCC 31586 has a lactose degradation rate of 22.5-25% in a liquid medium containing 3-5% (w / v) lactose after being cultured in the liquid medium for 12 h.

[0066] In some embodiments, the Kluyveromyces marxianus AMCC 31586 has a lactose degradation rate of 12.5-15% in a liquid medium containing 3-5% (w / v) lactose after being cultured in the liquid medium for 8 h.

[0067] In some embodiments, the Kluyveromyces marxianus AMCC 31586 has a lactose degradation rate of 5-10% in a liquid medium containing 3-5% (w / v) lactose after being cultured in the liquid medium for 4 h.

[0068] In some embodiments, the present application provides a fermentation broth containing the Kluyveromyces marxianus AMCC 31586 or the Kluyveromyces marxianus inoculum.

[0069] Preferably, the flavor substances in the fermentation broth include one or more than one selected from the group consisting of ester substances, alcohol substances, aldehyde substances, and acid substances.

[0070] More preferably, the flavor substances in the fermented dairy product include one or more than one selected from the group consisting of phenethyl acetate, isoamyl acetate, δ-dodecalactone, ethyl decanoate, ethyl octanoate, ethyl hexanoate, geraniol, linalool, isoamyl alcohol, phenethyl alcohol, nerolidol, citronellol, phenylacetaldehyde, and nonanal.

[0071] Most preferably, the aroma activity value of phenethyl acetate is 54-57, and or the aroma activity value of isoamyl acetate is 845-850, and or the aroma activity value of δ-dodecalactone is 90-95, and or the aroma activity value of ethyl decanoate is 10-15, and or the aroma activity value of ethyl octanoate is 1-2, and or the aroma activity value of ethyl hexanoate is 1-2, and or the aroma activity value of geraniol is 55-60, and or the aroma activity value of linalool is 40-45, and or the aroma activity value of isoamyl alcohol is 10-15, and or the aroma activity value of phenethyl alcohol is 8-10, the aroma activity value of nerolidol is 1-2, and or the aroma activity value of citronellol is 1-2, and or the aroma activity value of phenylacetaldehyde is 2-5, and or the aroma activity value of nonanal is 45-50, wherein the aroma activity value is the ratio of the concentration of the aroma component (μg / L) to the threshold value of the aroma component (μg / L).

[0072] In some embodiments, the present application provides a fermented milk product, wherein the flavoring substances in the fermented milk product comprise one or more than one substance selected from the group consisting of ester substances, alcohol substances, aldehyde substances, and phenol substances.

[0073] Preferably, the flavoring substances in the fermented milk product comprise one or both of the substances selected from the group consisting of eugenol and anethole.

[0074] More preferably, the aroma activity value of eugenol is 2-3, and / or the aroma activity value of anethole is 1-2, wherein the aroma activity value is the ratio of the concentration of the aroma component (μg / L) to the threshold value of the aroma component (μg / L).

[0075] Preferably, the flavoring substances in the fermented milk product further comprise one or more than one substance selected from the group consisting of phenylacetaldehyde, isoamyl alcohol, phenylethanol, ethyl undecanoate, isoamyl acetate, ethyl hexanoate, ethyl decanoate, ethyl isoamylate, phenylethyl acetate, and ethyl octanoate.

[0076] More preferably, the aroma activity value of phenylacetaldehyde is 5-8, and / or the aroma activity value of isoamyl alcohol is 10-15, and / or the aroma activity value of phenylethanol is 5-8, and / or the aroma activity value of ethyl undecanoate is 4-7, and / or the aroma activity value of isoamyl acetate is 40-45, and / or the aroma activity value of ethyl hexanoate is 8-10, and / or the aroma activity value of ethyl decanoate is 1-3, and / or the aroma activity value of ethyl isoamylate is 230-235, and / or the aroma activity value of phenylethyl acetate is 7-10, and / or the aroma activity value of ethyl octanoate is 10-15, wherein the aroma activity value is the ratio of the concentration of the aroma component (μg / L) to the threshold value of the aroma component (μg / L).

[0077] The present application will be further described in conjunction with the following examples. It should be noted that the following examples are only more specific implementations and explanations of the technical solutions of the present application, and should not be understood as limiting the scope of protection of the present application.

[0078] The various reagents / instruments used in the examples of the present application are all conventional commercially available products, unless otherwise specified.

[0079] Table 1. Reagent information table

[0080] Table 2. Instrument information table

[0081] 1. The medium component in the examples is as follows:

[0082] YPD solid medium: 10 g of yeast extract powder, 20 g of glucose, 20 g of yeast peptone, 20 g of agar, and 1000 mL of distilled water were mixed and sterilized at 115°C for 30 min.

[0083] YPD liquid medium: 10 g of yeast extract powder, 20 g of glucose, 20 g of agar, and 1000 mL of distilled water were mixed and sterilized at 115°C for 30 min.

[0084] Lactase screening medium: 10 g of yeast extract powder, 10 g of lactose, 10 g of yeast peptone, 20 g of agar, and 1000 mL of distilled water were mixed and sterilized at 115°C for 15 min, and 0.1% X-gal solution was added after cooling to 50°C or less.

[0085] 2. Liquid medium containing carbon source substrate involved in the examples is as follows:

[0086] Liquid medium containing 10% (w / v) sucrose, i.e., 10 g of sucrose in 100 mL of liquid medium, was prepared by mixing 10 g of sucrose, 0.67 g of YNB medium, and distilled water and adjusting the volume to 100 mL, and sterilized by filtration using a 0.22 μm filter.

[0087] Liquid medium containing 4% (w / v) glucose, i.e., 4 g of glucose in 100 mL of liquid medium, was prepared by mixing 4 g of glucose, 0.67 g of YNB medium, and distilled water and adjusting the volume to 100 mL, and sterilized by filtration using a 0.22 μm filter.

[0088] Liquid medium containing 4% (w / v) galactose, i.e., 4 g of galactose in 100 mL of liquid medium, was prepared by mixing 4 g of galactose, 0.67 g of YNB medium, and distilled water and adjusting the volume to 100 mL, and sterilized by filtration using a 0.22 μm filter.

[0089] Liquid medium containing 4% (w / v) lactose, i.e., 4 g of lactose in 100 mL of liquid medium, was prepared by mixing 4 g of lactose, 0.67 g of YNB medium, and distilled water and adjusting the volume to 100 mL, and sterilized by filtration using a 0.22 μm filter.

[0090] Liquid medium containing 0.7% (w / v) lactic acid, i.e., 0.7 g of lactic acid in 100 mL of liquid medium, was prepared by mixing 0.7 g of lactic acid, 0.67 g of YNB medium, and distilled water and adjusting the volume to 100 mL, and sterilized by filtration using a 0.22 μm filter.

[0091] A liquid medium containing 1.1% (w / v) of lactic acid, i.e., 1.1 g of lactic acid in 100 mL of the liquid medium, was prepared by mixing 1.1 g of lactic acid, 0.67 g of YNB medium, and distilled water and then filtering the mixture through a 0.22 μm filter to sterilize the mixture.

[0092] A liquid medium containing 2% (w / v) of lactic acid, i.e., 2 g of lactic acid in 100 mL of the liquid medium, was prepared by mixing 2 g of lactic acid, 0.67 g of YNB medium, and distilled water and then filtering the mixture through a 0.22 μm filter to sterilize the mixture.

[0093] 3. The yeast extract powder (model: FM888) used in the examples contained vitamins as follows: vitamin Bl 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 nicotinic acid 328.0 ppm.

[0094] The yeast extract powder (model: FM888) used in the examples contained trace elements 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.

[0095] The yeast extract powder (model: FM888) used in the examples contained 35.1% of free amino acids and 61.21% of hydrolyzed amino acids.

[0096] Among the free amino acids, the contents of the free amino acids were as follows: 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%, based on the weight of the yeast extract powder.

[0097] The hydrolyzed amino acid content is specifically as follows: 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%, based on the weight of the yeast extract powder.

[0098] Source and identification of the strain of example 1

[0099] 1 mL of the fermented milk collected from the herdsmen's home in the sixth team of the Luobei Town of the Zhalute Banner of the Tongliao City of the Inner Mongolia Autonomous Region was mixed with 9 mL of sterile water, and then diluted by 10 times in a serial gradient to obtain 10 -5 -6 mL of bacterial suspensions. 200 μL of the bacterial suspensions of different gradients were taken and spread on the lactase screening medium plates, which were cultured at 30°C for 48 h. As shown in FIG. 1, the lactase-producing strain had a blue hydrolysis circle phenotype in the lactase screening medium. Then, the single colonies with the blue hydrolysis circle were picked from the lactase screening medium, and then streaked and purified on the YPD medium plates after culture for 2 d. The streaking and purification was repeated for 3 times, and then inoculated on the YPD slant medium and stored at 4°C.

[0100] In addition, the purified strain was inoculated on the YPD solid medium, and the colony morphology was observed after being cultured at 30°C for 2 d. In addition, the single colony was inoculated in 200 μL of YPD liquid medium, and the bacterial suspension was obtained by culturing at 180 rpm and 30°C for 16 h. 20 μL of the bacterial suspension was taken and placed on a glass slide, and the bacterial morphology was observed under an optical microscope at 400 times magnification.

[0101] ​A strain was obtained, the colony texture of which was cheese-like, the color of which was milk white, the surface of which was smooth, the edge of which was neat, the microscopic morphology of which was oval, and the budding reproduction of which was observed. The genome of the strain was extracted, and the ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', referred to as SEQ ID No. 1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', referred to as SEQ ID No. 2) primers were used for PCR amplification, and the PCR program was as follows: 94°C pre-denaturation for 5 min, 94°C denaturation for 30 s, 55°C annealing for 45 s, 72°C extension for 90 s, 30 cycles, and finally 72°C extension for 10 min. The ITS gene sequence of the yeast was amplified, and after 1% gel electrophoresis detection and sequencing, the sequence was analyzed and compared with the sequences in GenBank. The sequence similarity of more than 99% was the same species. The ITS sequence of the strain was SEQ ID No. 3, as shown below:

[0102] In combination with morphological analysis and molecular identification, the strain was Kluyveromyces marxianus AMCC 31586 (Kluyveromyces marxianus AMCC 31586), which was preserved in the China Center for Type Culture Collection on September 25, 2023, and the preservation number was CCTCC NO: M 20231793. Figure 2 shows the microscopic morphology of the Kluyveromyces marxianus strain AMCC 31586; and Figure 3 shows the colony morphology of the Kluyveromyces marxianus strain AMCC 31586.

[0103] Example 2: Multi-carbon source utilization ability of Kluyveromyces marxianus AMCC 31586

[0104] (1) The steps for determining the carbon source substrate utilization ability are as follows:

[0105] (1.1) Determination of growth curve:

[0106] Kluyveromyces marxianus AMCC 31586 was inoculated into YPD liquid medium at an inoculation amount of 2% by volume, and cultured at 30°C and 180 rpm for 24 h to obtain a viable cell count of 10 8The bacterial suspension was centrifuged and washed, and the centrifugation and washing were performed as follows: 1 mL of the culture solution was centrifuged at 8000 rpm for 2 min, the supernatant was discarded, and the bacterial pellet was reserved. The bacterial pellet was resuspended with 1 mL of sterile water, and the bacterial pellet was centrifuged and washed twice. Then, the bacterial pellet was resuspended with 1 mL of sterile water, and was inoculated into a 100-hole culture plate containing 300 μL of liquid medium containing a carbon source substrate at an inoculation amount of 1% (v / v), and a growth curve was determined and plotted by using a Bioscreen C instrument. The carbon source substrate was 10% (w / v) sucrose, 4% (w / v) lactose, 4% (w / v) galactose, 4% (w / v) glucose, 0.7% (w / v) lactic acid, 1.1% (w / v) lactic acid, or 2% (w / v) lactic acid. The Bioscreen C instrument was set as follows: temperature, 30°C; time, 24 h; wavelength, 600 nm; and absorbance was measured every 30 min. The results are shown in Table 3 and FIG. 4.

[0107] The growth information calculation formula is as follows:

[0108] OD 600nm The growth rate calculation formula is as follows:

[0109] OD1—initial OD 600nm value;

[0110] OD2—final OD 600nm value;

[0111] The maximum specific growth rate (μ max ) calculation formula is as follows:

[0112] The doubling time is the time required for the bacterial amount to double in the exponential growth phase.

[0113] As shown in Table 3 and FIG. 4, FIG. 4 is a growth curve of the K. marxianus strain AMCC 31586 in the presence of a carbon source substrate of 10% (w / v) sucrose, 4% (w / v) lactose, 4% (w / v) galactose, 4% (w / v) glucose, 0.7% (w / v) lactic acid, 1.1% (w / v) lactic acid, or 2% (w / v) lactic acid. The results show that the maximum specific growth rate (μ max ) of the K. marxianus AMCC 31586 in the 10% (w / v) sucrose substrate is 0.4, and the bacterial growth is much better than that in other carbon source substrates. Sucrose is the main component in molasses, and 10% (w / v) sucrose is the carbon source content in high-density fermentation. Higher biomass is the basis for fermentation production, and on this basis, μ maxThe greater the biomass of the bacteria, the shorter the time to reach the biomass, the faster the growth rate, and thus the fermentation time can be shortened, energy consumption can be reduced, and a higher input-output ratio can be obtained, which is of guiding significance for production.

[0114] Further, since the lactose content in each 100 mL of conventional commercially available milk is about 4 g, the present application cultured the K. marxianus AMCC 31586 in a liquid medium containing 4% (w / v) lactose, and the results are shown in Table 3 and FIG. 4. The results showed that the highest OD value (OD max ) of the K. marxianus AMCC 31586 cultured in the liquid medium containing 4% (w / v) lactose within 24 h was 1.561, the maximum specific growth rate (μ max ) was 0.333, and the OD 600nm growth rate was 1063.11%.

[0115] Further, since galactose and glucose are hydrolysis products of lactose, one molecule of lactose is hydrolyzed to generate one molecule of galactose and one molecule of glucose. Therefore, the present application cultured the K. marxianus AMCC 31586 in a liquid medium containing 4% (w / v) galactose, and cultured the K. marxianus AMCC 31586 in a liquid medium containing 4% (w / v) glucose, and the results are shown in Table 3 and FIG. 4. The results showed that the highest OD value (OD max ) of the K. marxianus AMCC 31586 cultured in the liquid medium containing 4% (w / v) galactose within 24 h was 1.215, the maximum specific growth rate (μ max ) was 0.163, and the OD 600nm growth rate was 511.18%; the highest OD value (OD max ) of the K. marxianus AMCC 31586 cultured in the liquid medium containing 4% (w / v) glucose within 24 h was 1.775, the maximum specific growth rate (μ max ) was 0.234, and the OD 600nm growth rate was 557.47%.

[0116] Table 3. Growth information table of K. marxianus AMCC 31586

[0117] It should be noted that according to different processes, the yeast bacteria will also be compounded with lactic acid bacteria in the fermentation of dairy products, and through the mixed fermentation effect, the excessive lactic acid produced by the lactic acid bacteria is consumed, the growth inhibition of lactic acid bacteria caused by lactic acid accumulation is relieved, the growth of lactic acid bacteria is promoted, and the number of probiotics in fermented milk is increased. The lactic acid bacteria fermented dairy product is mainly yogurt, and the main organic acid in yogurt is lactic acid. The national standard (GB2746-1999) of yogurt stipulates that the acidity of the product should be ≥70 °T (1 °T: indicates that 100 mL of yogurt contains 0.01 g of lactic acid), and when the acidity range is 80-110 °T, the flavor of yogurt is the best, and the customer satisfaction is the highest. However, with the extension of storage time, the accumulation of lactic acid produced by lactic acid bacteria, the acidity rises, the pH value decreases, and the taste is greatly affected. Therefore, the present application will culture the Kluyveromyces marxianus AMCC 31586 in the liquid medium containing 0.7% (w / v) lactic acid, the liquid medium containing 1.1% (w / v) lactic acid and the liquid medium containing 2% (w / v) lactic acid. The results are shown in Table 3 and Figure 4, and the results show that the highest OD value (OD max ) of the Kluyveromyces marxianus AMCC 31586 cultured in the liquid medium containing 0.7% (w / v) lactic acid within 24h is 0.959, the maximum specific growth rate (μ max ) is 0.205, and the OD 600nm growth rate is 313.36%; the highest OD value (OD max ) of the Kluyveromyces marxianus AMCC 31586 cultured in the liquid medium containing 1.1% (w / v) lactic acid within 24h is 1.107, the maximum specific growth rate (μ max ) is 0.202, and the OD 600nm growth rate is 473.58%.

[0118] The highest OD value (OD max ) of the Kluyveromyces marxianus AMCC 31586 cultured in the liquid medium containing 2% (w / v) lactic acid within 24h is 0.841, the maximum specific growth rate (μ max ) is 0.123, and the OD 600nm growth rate is 246.09%. It can be seen that the growth of the Kluyveromyces marxianus AMCC 31586 in the liquid medium containing 1.1% (w / v) lactic acid is the best, which shows that the Kluyveromyces marxianus AMCC 31586 can fully utilize lactic acid as a single carbon source for growth, and can effectively consume the excess lactic acid produced by lactic acid bacteria.

[0119] (1.2) Lactose degradation rate detection

[0120] Kluyveromyces marxianus AMCC 31586 was inoculated into liquid medium containing 4% (w / v) lactose at an inoculum of 2% (v / v) and incubated at 30°C at 180 rpm. At 4h, 8h, 12h and 24h of fermentation, 20 mL of culture was centrifuged at 8000 rpm for 2 min, and the supernatant was retained. The residual lactose content in the supernatant was determined by HPLC according to GB 5009.8-2023, and the results are shown in Table 4. The lactose degradation rate was calculated according to the residual lactose content in the supernatant, and the results are shown in Table 5. The test was repeated 3 times.

[0121] As shown in Table 4, the lactose content in the fermentation supernatant after 24h of fermentation ranged from 0 to 0.45g / 100g, and the lactose content in lactose-free milk was required to be less than 0.5g / 100g. This indicates that Kluyveromyces marxianus AMCC 31586 has the potential to fully consume lactose in milk and can be used to produce lactose-free milk.

[0122] Table 4 Residual lactose content of Kluyveromyces marxianus AMCC 31586 in lactose fermentation broth

[0123] As shown in Table 5, the results show that the lactose degradation rate of Kluyveromyces marxianus AMCC 31586 in liquid medium containing 4% (w / v) lactose was 22.5% at 12h of fermentation, and the lactose degradation rate of Kluyveromyces marxianus AMCC 31586 in liquid medium containing 4% (w / v) lactose was 88.8%-100% at 24h of fermentation. This again demonstrates that Kluyveromyces marxianus AMCC 31586 can fully utilize lactose substrate.

[0124] Table 5 Lactose degradation rate of Kluyveromyces marxianus AMCC 31586

[0125] Example 3 Effect analysis of Kluyveromyces marxianus AMCC 31586 fermentation of milk

[0126] (1) The steps for Kluyveromyces marxianus AMCC 31586 to make fermented milk are as follows:

[0127] (1.1) Seed preparation: inoculate AMCC 31586 into a test tube containing 5 mL of YPD liquid medium at an inoculum of 2% by volume, and incubate at 30°C at 180 rpm for 24h to obtain a viable cell count of 10 8CFU / mL, 1 mL of the bacterial suspension was centrifuged at 8000 rpm for 2 min, the supernatant was discarded and the bacterial pellet was retained. The bacterial pellet was resuspended with 1 mL of sterile water and the bacterial pellet was centrifuged for washing twice. Then the bacterial pellet was resuspended with 1 mL of sterile water, and the viable cell count was ensured to reach 2 x 10 8 CFU / mL, to obtain the seed liquid.

[0128] (1.2) Preparation of sterilized milk: commercially available sterilized pure milk of Mongolian cow was taken and divided into 250 mL conical flasks, and the liquid volume in each conical flask was 100 mL.

[0129] (1.3) Fermentation: the seed liquid obtained in step (1.1) was inoculated into the conical flask containing 100 mL of commercially available sterilized pure milk of Mongolian cow at a inoculation amount of 2% by volume, so that the viable cell count in 100 mL of commercially available sterilized pure milk of Mongolian cow was 10 6 CFU / mL, and the fermentation was carried out under the conditions of fermentation temperature of 30°C, shaking speed of 100 r / min, and culture time of 36 h to obtain fermented milk.

[0130] (1.4) The evaluation criteria of fermented milk are as follows: the sensory evaluation of fermented milk is taken as the first evaluation criterion, and experienced personnel are organized to conduct sensory evaluation of fermented milk. The specific sensory evaluation criteria are as follows: the comprehensive score of the aroma and sweetness of the fermented milk is taken as the screening criterion. The aroma score interval is 1-10, the sweetness score interval is 1-10, and the higher the score represents the more significant the aroma and sweetness.

[0131] Kluyveromyces marxianus AMCC 31586 obtained 10 points in the aroma and sweetness evaluation.

[0132] (2) Experienced personnel were organized to conduct sensory evaluation of the fermented milk prepared by fermentation of Kluyveromyces marxianus AMCC 31586, and the results are shown in Table 6.

[0133] (3) The fermented milk prepared by fermentation of Kluyveromyces marxianus AMCC 31586 was detected for the index content of fermented milk, and the results are shown in Table 7. Among them, the indexes of fermented milk include: lactose, ethanol, protein, acetic acid, acidity and PH. The commercially available sterilized pure milk of Mongolian cow was taken as the blank control.

[0134] Among them, the determination of lactose adopts GB 5009.8-2016;

[0135] The determination of ethanol adopts GB 5009.225-2023;

[0136] The determination of protein adopts GB 5009.5-2016;

[0137] The determination of acetic acid adopts GB 5009.157-2016;

[0138] The determination of acidity adopts GB 5009.239-2016;

[0139] The determination of pH adopts GB 5009.237-2016.

[0140] (4) The fermented milk prepared by fermenting the Kluyveromyces marxianus AMCC 31586 was subjected to flavor substance detection, and the results are shown in Figure 5. The flavor substance detection steps are as follows: 5 mL of milk fermentation broth was taken into a headspace bottle, 1.5 g of sodium chloride and 1 μL of internal standard solution (o-dichlorobenzene) were added, and mixed, 50°C was balanced for 30 min, and volatile component analysis was performed. Gas chromatography conditions: chromatographic column: DB-HeavyWAX, 30 m x 0.25 mm x 0.25 μm, injection port temperature 250°C, carrier gas: He; temperature program: 40°C for 3 min, 5°C / min rate to 200°C, hold for 0 min; 10°C / min rate to 250°C, hold for 3 min. The odor activity value (OVA) of the volatile substance was used to characterize the aroma contribution of the aroma substance to the fermented milk, and the calculation formula is as follows:

[0141] OAVs = aroma component concentration / aroma component threshold.

[0142] OAVs value greater than or equal to 1 means reaching the threshold range that can be smelled by human olfactory, which can be smelled.

[0143] Table 6 Sensory evaluation results

[0144] As shown in Table 6, the results show that the sensory experience of the fermented milk prepared by fermenting the Kluyveromyces marxianus AMCC 31586 is quite different from that of pure milk. The Kluyveromyces marxianus AMCC 31586 fermented milk has a pleasant aroma and a sweet and clear flavor, providing a more rich fermented flavor for dairy products.

[0145] Table 7 Index content of fermented milk

[0146] As shown in Table 7, the results show that the pH of the Kluyveromyces marxianus AMCC 31586 fermented milk is reduced by 0.6, the acidity is increased by 215%, no lactose and galactose is detected, indicating that the lactose and lactose decomposition product galactose in the milk can be completely utilized, the protein content is increased by 5.8%, and the ethanol content is less than 2.5% vol, meeting the low alcohol standard (GB / T 17204-2008).

[0147] As shown in Figure 5, Figure 5 is a diagram of the aroma activity of flavor substances above the threshold concentration in milk fermented by Kluyveromyces marxianus AMCC 31586. The OAVs of flavor substances in milk fermented by Kluyveromyces marxianus AMCC 31586 increased significantly, and the greater the OAVs value, the stronger the aroma. In milk fermentation, the substances with an OAV higher than 1 are the core aroma contributors. There are 12 core aroma substances in milk fermented by Kluyveromyces marxianus AMCC 31586, including eugenol, phenylacetaldehyde, anethole, isoamyl alcohol, phenylethanol, ethyl undecanoate, isoamyl acetate, ethyl hexanoate, ethyl decanoate, ethyl iso-valerate, phenyl ethyl acetate and ethyl octanoate, including 7 ester substances, 2 alcohol substances, 1 phenol substance, 1 aldehyde substance and 1 ether substance. Among them, ethyl iso-valerate is the main aroma contributor of Kluyveromyces marxianus AMCC 31586, with a concentration of 2.328 ng / mL, and the odor threshold of this substance is only 0.01 ng / mL, so its OAVs value is as high as 232.80, and the aroma characteristic of this substance is sweet apple aroma.

[0148] In addition, in milk fermented by Kluyveromyces marxianus AMCC 31586, isoamyl acetate contributes to banana aroma, with an aroma activity value of 43.54; ethyl octanoate contributes to brandy aroma, with an aroma activity value of 13.46; phenyl ethyl acetate contributes to peach aroma, with an aroma activity value of 9.14; ethyl hexanoate contributes to apple-pineapple fruit aroma, with an aroma activity value of 9.26; ethyl palmitate contributes to coconut aroma, with an aroma activity value of 1.08; ethyl undecanoate also contributes to coconut aroma, with an aroma activity value of 5.68; phenylethanol contributes to rose aroma, with an aroma activity value of 6.79; isoamyl alcohol contributes to white wine aroma, with an aroma activity value of 13.44; phenylacetaldehyde contributes to elegant tulip fragrance, with an aroma activity value of 6.79.

[0149] Among the core aroma substances, two substances have not been reported so far, which are eugenol and anethole. Eugenol contributes to dry sweet floral aroma, with an aroma activity value of 2.27; anethole contributes to sweet anise aroma, with an aroma activity value of 1.26.

[0150] Example 4 Flavor of Kluyveromyces marxianus AMCC 31586 strain fermentation broth

[0151] Kluyveromyces marxianus AMCC 31586 strain was inoculated into YPD medium, and cultured at 30°C for 24h to obtain the fermentation broth of Kluyveromyces marxianus AMCC 31586. The volatile substance composition in the fermentation broth was detected, and the results are shown in Figure 6 and Table 8. The specific detection steps are as follows: 5mL of fermentation supernatant was taken into a headspace bottle, 1.5g of sodium chloride and 1μL of internal standard solution (o-dichlorobenzene) were added, and the mixture was mixed, equilibrated at 50°C for 30min, and then analyzed for volatile components, wherein the gas chromatography conditions were as follows: chromatographic column: DB-HeavyWAX, 30m x 0.25mm x 0.25μm, injection port temperature 250°C, carrier gas: He; temperature program: 40°C for 3min, 5°C / min rate to 200°C, hold for 0min; 10°C / min rate to 250°C, hold for 3min.

[0152] The aroma components were qualitatively determined in scan mode, and the information of chromatographic retention time and mass spectrum was retrieved in NIST 17 standard library. The aroma components with standards were quantified by standard curve, and the aroma components without standards were quantified by similar substances with chemical structure, and the aroma activity value was calculated. The components with aroma activity value greater than 1 were analyzed for aroma characteristics, and the results are shown in Table 9, wherein the formula for calculating the aroma activity value is as follows:

[0153] Aroma activity value = aroma component concentration (μg / L) / aroma component threshold value (μg / L).

[0154] As shown in Figure 6, Figure 6 is the GC-MS chromatogram of the fermentation broth of Kluyveromyces marxianus AMCC 31586. As analyzed from Figure 6, 20 ester substances, 17 alcohol substances, 8 acid substances, 4 ketone and phenol substances were produced in the fermentation broth of Kluyveromyces marxianus AMCC 31586. As shown in Table 8, Table 8 is the volatile substances in the fermentation broth of Kluyveromyces marxianus AMCC 31586. Among the volatile substances, the total ester accounts for as high as 51.78%, followed by the total alcohol, accounting for 28.84%.

[0155] Table 8 Volatile substances in the fermentation broth of Kluyveromyces marxianus AMCC 31586

[0156] As shown in Table 9, Table 9 is the activity value and aroma characteristics of flavor substances in the fermentation broth of Kluyveromyces marxianus AMCC 31586. From the calculation results of the aroma activity value, it can be known that in the fermentation broth of Kluyveromyces marxianus AMCC 31586 strain, the components with aroma activity value higher than 1 are the core fermentation aroma, a total of 14 kinds, including 6 kinds of esters such as phenethyl acetate, isoamyl acetate, δ-dodecalactone, ethyl decanoate, ethyl octanoate and ethyl hexanoate, 6 kinds of alcohols such as geraniol, linalool, isoamyl alcohol, phenethyl alcohol, nerolidol and citronellol, and 2 kinds of aldehydes such as phenylacetaldehyde and nonanal. The dominant aroma characteristics are the banana fruit aroma of isoamyl acetate with an aroma activity value of 848.67, followed by δ-dodecalactone with an aroma activity value of 93.07, which contributes the aroma of coconut or butter to the fermentation broth, in addition, there are rose aroma contributed by geraniol with an aroma activity value of 58.07, sweet peach aroma contributed by phenethyl acetate with an aroma activity value of 55.29, sweet orange / oily aroma contributed by nonanal with an aroma activity value of 48.65, green tea wood green aroma contributed by linalool with an aroma activity value of 42.16, coconut aroma contributed by ethyl decanoate with an aroma activity value of 13.66, and the above core fermentation aroma and the fruit aroma contributed by other substances together add various pleasant flavors to the fermentation broth.

[0157] Table 9. Core flavor substances in the fermentation broth of Kluyveromyces marxianus AMCC 31586

[0158] Kluyveromyces marxianus AMCC 31586 strain utilizes a wide range of carbon source substrates, and the flavor of the fermentation broth of Kluyveromyces marxianus AMCC 31586 is pleasant, which indicates that Kluyveromyces marxianus AMCC 31586 has application potential in fermented foods.

[0159] It should be noted that the application of Kluyveromyces marxianus AMCC 31586 in fermented foods does not have special limitations on the types of fermented foods, preferably, the types of fermented foods are fruit and vegetable beverages, protein beverages, fermented wine, fermented milk, condiments, and flour products.

[0160] The type of fermented wine is not particularly limited, preferably, the type of fermented wine is grape wine, beer, fruit wine, yellow rice wine, white wine, rice wine and sake;

[0161] The type of fermented milk is not particularly limited, preferably, the type of fermented milk is yogurt, fermented buttermilk, milk wine and sour cream;

[0162] The type of condiment is not particularly limited, preferably, the type of condiment is vinegar, sauce, soy sauce, sweetener and flavor enhancer;

[0163] The kind of the flour product is not particularly limited, and preferably, the kind of the flour product is bread, biscuit, cake and steamed bun.

[0164] It should be noted that the application of the Kluyveromyces marxianus AMCC 31586 to the fermented food is not particularly limited to the kind of the fermented food, and preferably, the kind of the fermented food is fruit and vegetable beverage, protein beverage, fermented wine, fermented milk, condiment, flour product.

[0165] The kind of the fermented wine is not particularly limited, and preferably, the kind of the fermented wine is grape wine, beer, fruit wine, yellow rice wine, white wine, rice wine and sake.

[0166] The kind of the fermented milk is not particularly limited, and preferably, the kind of the fermented milk is yogurt, fermented buttermilk, milk wine and sour cream.

[0167] The kind of the condiment is not particularly limited, and preferably, the kind of the condiment is vinegar, sauce, soy sauce, sweetener and flavor enhancer.

[0168] The kind of the flour product is not particularly limited, and preferably, the kind of the flour product is bread, biscuit, cake and steamed bun.

[0169] The above examples are only for further elaboration and understanding of the technical solutions of the application, and are not a limitation of the application. Any improvement made by a person skilled in the art on the basis of the above examples, which does not have any outstanding substantial features and is not a significant progress, shall belong to the protection scope of the application.

Claims

1. A strain of Kluyveromyces marxianus AMCC 31586, characterized in that, The Kluyveromyces marxianus AMCC 31586, which is preserved in the China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC NO: M 20231793.

2. The K. marxianus AMCC 31586 of claim 1, wherein, The ITS gene sequence of the Kluyveromyces marxianus AMCC 31586 is shown in SEQ ID No.

3.

3. The K. marxianus AMCC 31586 according to any one of claims 1 or 2, characterized in that, It has the characteristics of utilizing multiple carbon source substrates.

4. The K. marxianus AMCC 31586 of claim 3, wherein, The carbon source substrates include one or more than one selected from the group consisting of monosaccharides, disaccharides and organic acids; preferably, the carbon source substrates include one or more than one selected from the group consisting of sucrose, lactose, galactose, glucose and lactic acid.

5. A fermentation production method of a Kluyveromyces marxianus inoculum, characterized in that, The method comprises the following steps: (1) amplifying the Kluyveromyces marxianus AMCC 31586 according to any one of claims 1-4; (2) adding the Kluyveromyces marxianus AMCC 31586 after amplification into a liquid culture medium and carrying out fermentation culture at 26-45°C.

6. A Kluyveromyces marxianus inoculum, characterized in that, The Kluyveromyces marxianus AMCC 31586 according to any one of claims 1-4 or prepared by the fermentation preparation method according to claim 5.

7. A fermentation broth, characterized in that, The Kluyveromyces marxianus AMCC 31586 according to any one of claims 1-4.

8. The fermentation broth of claim 7, wherein, The flavor substances in the fermentation broth include one or more than one selected from the group consisting of ester substances, alcohol substances, aldehyde substances and acid substances.

9. The fermentation broth according to claim 7 or 8, characterized in that, The flavor substances in the fermented dairy product include one or more than one selected from the group consisting of phenethyl acetate, isoamyl acetate, δ-dodecalactone, ethyl decanoate, ethyl octanoate, ethyl hexanoate, geraniol, linalool, isoamyl alcohol, phenethyl alcohol, nerolidol, citronellol, phenylacetaldehyde and nonanal.

10. A fermented food, characterized by, The Kluyveromyces marxianus AMCC 31586 according to any one of claims 1-4 or the Kluyveromyces marxianus inoculant according to claim 6 or the fermentation broth according to any one of claims 7-9.

11. The Kluyveromyces marxianus AMCC 31586 according to any one of claims 1-4 or the Kluyveromyces marxianus inoculant according to claim 6 or the fermentation broth according to any one of claims 7-9 for use in fermented food.

12. The fermented food according to claim 10 or use according to claim 11, wherein The fermented food contains one or more than one carbon source substrate selected from the group consisting of monosaccharides, disaccharides and organic acids; preferably, it contains one or more than one carbon source substrate selected from the group consisting of lactose, galactose, sucrose, glucose and lactic acid.

13. The fermented food of claim 10 or the use of claim 11 or 12, characterized in that, The fermented food includes one or more than one selected from the group consisting of fermented milk, fermented wine, fruit and vegetable beverage, protein beverage, fermented wine, fermented milk, condiment and flour product.

14. A fermented milk, characterized in that, The Kluyveromyces marxianus AMCC 31586 according to any one of claims 1-4 or the Kluyveromyces marxianus inoculant according to claim 6 or the fermentation broth according to any one of claims 7-9.

15. Use of the Kluyveromyces marxianus AMCC 31586 of any one of claims 1-4 or the Kluyveromyces marxianus inoculum of claim 6 or the fermentation broth of any one of claims 7-9 in a fermented milk.

16. The fermented milk product according to claim 14 or use according to claim 15, c h a r a c t e r i s e d i n that The flavor substances in the fermented milk product include one or more than one substance selected from the group consisting of ester substances, alcohol substances, aldehyde substances, and phenol substances.

17. The fermented milk product according to claim 14 or 16 or the use according to claim 15 or 16, characterized in that, The flavor substances in the fermented milk product include one or both of the substances selected from the group consisting of eugenol and anethole.

18. The fermented milk product according to claim 14 or 16 or 17 or the use according to claim 15 or 16 or 17, characterized in that, The flavor substances in the fermented milk product further include one or more than one substance selected from the group consisting of phenylacetaldehyde, isoamyl alcohol, phenylethanol, ethyl undecanoate, isoamyl acetate, ethyl hexanoate, ethyl decanoate, ethyl iso-valerate, phenyl ethyl acetate, and ethyl octanoate.

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