Kluyveromyces marxianus and application thereof

Abstract

The invention provides kluyveromyces marxianus with the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.32249 and application of the kluyveromyces The Kluyveromyces marxianus provided by the invention can produce protein at high yield, and the bacterial powder obtained by the Kluyveromyces marxianus has the characteristics of high protein content, low peculiar smell and light color, is easily applied to food, and better meets the requirements of food processing.

Description

Technical Field

[0001] The present application relates to the field of microorganisms, and in particular to a strain of Kluyveromyces marxianus and applications thereof. Background Art

[0002] Kluyveromyces marxianus is a safe food-grade strain strictly approved and approved by the National Health Commission. It is used as a fermentation bacterium in the food industry for milk beer production. It has the characteristics of vigorous growth, large biomass, short growth cycle, wide range of suitable growth temperature, high protein secretion, etc., and shows great application potential as a protein source for food and feed. In the patent document CN118126853B, a strain of Kluyveromyces marxianus NS127 suitable for high-density culture is disclosed, which can be converted into high-quality protein raw materials using cheap substrates, and can obtain high-density bacteria based on the method of fed-batch culture, with a dry weight of 87.20g / L and a total protein yield of 30.01g / L. In the patent document CN116240121B, a strain of Kluyveromyces marxianus CJA004 with good flavor is disclosed, and the shake flask fermentation yield of total intracellular protein is about 41%.

[0003] However, compared with the mainstream alternative protein products on the market, such as soy protein powder and pea protein powder, which generally have a protein content of more than 80%, the protein content of existing Kluyveromyces marxianus is relatively low and the obtained bacterial powder has a yeasty and bitter taste. At the same time, its cell wall is dense and the protein solubility is poor, which greatly hinders the application of the product. Therefore, it is urgent to find new Kluyveromyces marxianus to solve the problems of low protein content and poor flavor. Summary of the invention

[0004] In view of the problems existing in the prior art, the purpose of this application is to provide a strain of Kluyveromyces marxianus with high protein production and its application. The Kluyveromyces marxianus has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (CGMCC) on October 17, 2024, with a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, zip code 100101, deposit number CGMCC No. 32249, and classification name Kluyveromyces marxianus.

[0005] Specifically, this application involves the following aspects:

[0006] 1. A strain of Kluyveromyces marxianus, whose deposit number is CGMCC No.32249.

[0007] 2. A fermentation and culturing method of Kluyveromyces marxianus according to item 1, comprising:

[0008] The Kluyveromyces marxianus is inoculated into a fermentation medium and fermented in a fermenter.

[0009] 3. The fermentation and culturing method according to item 2, wherein the fermentation nitrogen source is selected from one or more of yeast extract, malt extract, corn steep liquor, peptone, defatted soybean meal, defatted peanut meal, ammonia water, monoammonium phosphate, and diammonium phosphate.

[0010] 4. The fermentation and culturing method according to item 2, wherein the fermentation carbon source is selected from one or more of glucose, molasses, starch and straw.

[0011] 5. The fermentation method according to item 2, wherein the fermentation conditions are: fermentation for 20-72 hours at a temperature of 25-38°C, a rotation speed of 100-800 rpm, and a tank pressure of 0.03-0.09 MPa, with carbon source and nitrogen source added, a constant pH of 4-6, and an RQ of 0.7-1.5.

[0012] 6. A method for preparing bacterial powder, comprising:

[0013] Fermenting and culturing Kluyveromyces marxianus by the fermentation and culturing method described in any one of items 2 to 5 to obtain bacterial cells, and treating the bacterial cells to obtain the bacterial powder;

[0014] The Kluyveromyces marxianus is the Kluyveromyces marxianus described in item 1.

[0015] 7. The preparation method according to item 6, wherein the processing of the bacterial bodies comprises washing and separating the bacterial bodies, vacuum sterilizing, high-pressure homogenizing and vacuum drying.

[0016] 8. A bacterial powder obtained according to the preparation method described in item 6 or 7.

[0017] 9. Use of the Kluyveromyces marxianus according to item 1 in preparing bacterial powder.

[0018] 10. Use of the Kluyveromyces marxianus according to item 1 or the bacterial powder according to item 8 in preparing a food product.

[0019] The protein content of the Kluyveromyces marxianus provided in the present application is greater than 70%. The bacterial powder obtained by using the Kluyveromyces marxianus of the present application has the characteristics of high protein content, low odor and light color, is easy to be used in food, and better meets the needs of food processing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Microscope observations of different strains.

[0021] Figure 2 This is a picture of the colony morphology of Kluyveromyces marxianus.

[0022] Figure 3 The 10% solid content graph of the bacterial powder obtained in Example 2-9. The upper part of the graph represents the results of Example 2-5 from left to right, and the lower part of the graph represents the results of Example 6-9 from left to right. DETAILED DESCRIPTION

[0023] The present application is further described below in conjunction with examples. It should be understood that the examples are only used to further describe and illustrate the present application and are not used to limit the present application.

[0024] Unless otherwise defined, the technical and scientific terms in this specification have the same meaning as those generally understood by those skilled in the art. Although similar or identical methods and materials as described herein may be used in experiments or practical applications, materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, materials, methods and examples are provided for illustration only and are not restrictive. The present application is further described below in conjunction with specific embodiments, but is not intended to limit the scope of the present application.

[0025] The experimental methods used below are conventional methods unless otherwise specified.

[0026] Unless otherwise specified, the materials and reagents used below can be obtained from commercial sources.

[0027] The present application provides a strain of Kluyveromyces marxianus, which was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) on October 17, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, the deposit number is CGMCC No. 32249, and the classification name is Kluyveromyces marxianus.

[0028] The Kluyveromyces marxianus provided in the present application is isolated from old household dough in Inner Mongolia, and is capable of high protein production, and can be applied in the field of alternative proteins.

[0029] The present application provides a fermentation and culture method of the Kluyveromyces marxianus, which comprises: inoculating the Kluyveromyces marxianus into a fermentation medium, and fermenting and culturing the same in a fermenter.

[0030] The "fermentation culture" refers to the process in which Kluyveromyces marxianus produces the bacteria itself, direct metabolites or secondary metabolites through life activities under aerobic conditions.

[0031] The present application does not limit the fermentation culture method, as long as it can meet the culture requirements of Kluyveromyces marxianus provided in the present application. The fermentation culture can be carried out under any suitable conditions and can use any suitable culture medium.

[0032] In a specific embodiment, in the fermentation medium, the fermentation nitrogen source is selected from one or more of yeast extract, malt extract, corn steep liquor, peptone, defatted soybean meal, defatted peanut meal, ammonia water, monoammonium phosphate, and diammonium phosphate. In a specific embodiment, in the fermentation medium, the fermentation nitrogen source is yeast extract. In a specific embodiment, in the fermentation medium, the fermentation nitrogen source is yeast extract and malt extract.

[0033] In a specific embodiment, in the fermentation medium, the fermentation carbon source is selected from one or more of glucose, molasses, starch, and straw. In a specific embodiment, in the fermentation medium, the fermentation carbon source is glucose. In a specific embodiment, in the fermentation medium, the fermentation carbon source is molasses. In a specific embodiment, in the fermentation medium, the fermentation carbon source is starch.

[0034] In a specific embodiment, in the fermentation medium, in addition to the fermentation nitrogen source and the fermentation carbon source, other components are also included: potassium, calcium, phosphorus, magnesium, copper, riboflavin, biotin or pyridoxine. In a specific embodiment, in the fermentation medium, in addition to the fermentation nitrogen source and the fermentation carbon source, other components are also included: (NH4)2SO4, KH2PO4, CaCl2, FeSO4, hydrochloric acid thiamin, biotin, D-calcium pantothenate and inositol. In a specific embodiment, in the fermentation medium, in addition to the fermentation nitrogen source and the fermentation carbon source, other components are also included: 0.2% (NH4)2SO4, 0.3% KH2PO4, 28.8mg / L CaCl2, 14.4mg / L FeSO4, 4.8mg / L hydrochloric acid thiamin, 0.12mg / L biotin, 6.4mg / L D-calcium pantothenate and 120mg / L inositol.

[0035] Among them, the applicant found that the Kluyveromyces marxianus sludge obtained by fermentation culture using the fermentation medium of the present application has a lighter color and does not require an adsorption process to remove pigments.

[0036] In a specific embodiment, the fermentation medium is sterilized by high pressure sterilization at a temperature of 115-150°C for 2s-30min. In a specific embodiment, the fermentation medium is sterilized by direct steam injection at a temperature of 115-150°C for 2s-30min.

[0037] Among them, the applicant has found that by performing high-pressure sterilization on the fermentation medium (such as steam direct injection sterilization), the sterilization efficiency can be improved while effectively removing the volatile flavor in the fermentation medium, thereby greatly reducing the generation of odor during fermentation culture.

[0038] In a specific embodiment, the fermentation culture conditions are: fermentation culture for 20-72 hours at a temperature of 25-38°C, a rotation speed of 100-800 rpm, and a tank pressure of 0.03-0.09 MPa, with carbon and nitrogen sources added, a constant pH of 4-6, and an RQ of 0.7-1.5. In a specific embodiment, the fermentation culture conditions are: fermentation culture for 30-60 hours at a temperature of 28-35°C, a rotation speed of 200-600 rpm, and a tank pressure of 0.05-0.08 MPa, with carbon and nitrogen sources added, and feeding is started from 8-12 hours of fermentation culture, with a constant residual sugar content of 3-8 g / L, a constant pH of 4.5-5.5, and an RQ of 0.8-1.2.

[0039] In a specific embodiment, the fermentation and cultivation method of the above-mentioned Kluyveromyces marxianus comprises the following steps: sterilizing the fermentation medium by direct steam injection for 2s-30min at a temperature of 115-150°C; inoculating the Kluyveromyces marxianus into the fermentation medium after sterilization; and fermenting and culturing in a fermenter for 30-60h at a temperature of 28-35°C, a rotation speed of 200-600rpm, and a tank pressure of 0.05-0.08mpa, adding carbon source and nitrogen source, and starting feeding from 8-12h of fermentation and cultivation, with a constant residual sugar content of 3-8g / L, a constant pH of 4.5-5.5, and an RQ of 0.8-1.2. Wherein, in the fermentation medium, the fermentation nitrogen source is yeast extract powder, or yeast extract powder and malt extract; the fermentation carbon source is glucose, molasses or starch; the fermentation medium also includes other components: 0.2% (NH4)2SO4, 0.3% KH2PO4, 28.8mg / L CaCl2, 14.4mg / LFeSO4, 4.8mg / L ammonium sulfoxide hydrochloride, 0.12mg / L biotin, 6.4mg / L D-calcium pantothenate and 120mg / L inositol.

[0040] The present application provides a method for preparing bacterial powder, the preparation method comprising: fermenting and culturing Kluyveromyces marxianus by any of the fermentation and culturing methods described above to obtain bacterial bodies, and treating the bacterial bodies to obtain the bacterial powder; wherein the Kluyveromyces marxianus is the above-mentioned Kluyveromyces marxianus.

[0041] The present application does not limit the preparation method of bacterial powder, and the bacterial bodies can be processed by conventional methods in the art to obtain the bacterial powder.

[0042] In a specific embodiment, the processing of the bacteria includes washing and separating the bacteria, vacuum sterilizing, high-pressure homogenizing and vacuum drying.

[0043] In a specific embodiment, the cleaning and separation conditions are: using a batch vertical centrifuge or a continuous disc centrifuge, at a temperature of 2-25°C and a rotation speed of 3000-6000rpm, the bacterial cells are mixed with water in a volume ratio of (1:3)-(1:12), and washed with water 1-5 times. For example, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12. In a specific embodiment, the cleaning and separation conditions are: using a continuous disc centrifuge, at a temperature of 2-15°C and a rotation speed of 3000-6000rpm, the bacterial cells are mixed with water in a volume ratio of (1:4)-(1:8), and washed with water 2-3 times.

[0044] Among them, the applicant has found that low-temperature and continuous centrifugation treatment of bacteria during the cleaning and separation process can reduce the activity of the bacteria, shorten the processing time, and reduce the impact of enzyme activity on flavor.

[0045] In a specific embodiment, the vacuum sterilization conditions are: using a vacuum sterilization tank, at a temperature of 75-120°C, inactivation for 10-200 minutes. In a specific embodiment, the vacuum sterilization conditions are: at a temperature of 85-95°C, a vacuum degree of -0.05 MPa-0.1 MPa, inactivation for 30-120 minutes by intermittent vacuum and high shear agitator.

[0046] Among them, the applicant has found that during the vacuum sterilization process, intermittent vacuum operation and high shear agitator are used to treat the cleaned and separated bacteria, which is beneficial to the separation and rupture of the bacterial cell wall, and the odor can be continuously removed in this process.

[0047] In a specific embodiment, the high-pressure homogenization condition is: using a high-pressure homogenizer, homogenizing 2-6 times under the condition of a homogenizing pressure of 60-200 MPa. In a specific embodiment, the high-pressure homogenization condition is: using a high-pressure jet homogenizer, homogenizing 2-4 times under the condition of a homogenizing pressure of 90-170 MPa.

[0048] In a specific embodiment, the vacuum drying is vacuum microwave drying, vacuum belt drying, vacuum puffing drying or vacuum freeze drying.

[0049] Among them, the applicant has found that high-pressure homogenization and vacuum drying can effectively break up the bacteria and increase the solubility of the protein.

[0050] In a specific embodiment, the preparation method of bacterial powder comprises: fermenting and culturing Kluyveromyces marxianus by any of the fermentation and culturing methods described above to obtain bacterial bodies, washing and separating the bacterial bodies, vacuum sterilizing, high-pressure homogenizing and vacuum drying the bacterial bodies to obtain the bacterial powder; wherein the Kluyveromyces marxianus is the above-mentioned Kluyveromyces marxianus. Wherein, the conditions for the washing and separation are: using a continuous disc centrifuge, mixing the bacterial bodies with water at a volume ratio of (1:4)-(1:8) at a temperature of 2-15°C and a speed of 3000-6000rpm, and washing with water 2-3 times. The conditions for the vacuum sterilization are: inactivating for 30-120min by intermittent vacuum and high shear agitator at a temperature of 85-95°C and a vacuum degree of -0.05mpa-0.1mpa. The conditions for the high-pressure homogenization are: using a high-pressure jet homogenizer, homogenizing 2-4 times at a homogenization pressure of 90-170mpa. The vacuum drying is vacuum microwave drying, vacuum belt drying, vacuum puffing drying or vacuum freeze drying.

[0051] The protein content of the Kluyveromyces marxianus provided by the present application is greater than 70%; the bacterial powder obtained by the Kluyveromyces marxianus of the present application has good sensory properties and a high soluble protein content, showing great application prospects in the food industry. Compared with the prior art, the Kluyveromyces marxianus of the present application can obtain bacterial powder with high protein content, better flavor and solubility, and suitable for food processing without adding processing aids such as enzymes and acids and alkalis.

[0052] The present application provides a bacterial powder obtained according to the above-mentioned preparation method.

[0053] The present application provides the use of the above-mentioned Kluyveromyces marxianus in preparing bacterial powder.

[0054] The present application also provides the use of the above-mentioned Kluyveromyces marxianus or the above-mentioned bacterial powder in preparing food products.

[0055] The following examples of the present application are only used to illustrate the specific implementation methods of the present application, and these implementation methods cannot be understood as limiting the present application. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present application are deemed to be equivalent replacement methods and fall within the scope of protection of the present application.

[0056] Example

[0057] Example 1 Isolation and Obtaining Kluyveromyces marxianus

[0058] (1) Treatment of bacterial strains

[0059] Multiple yeast strains from old household dough in Inner Mongolia were diluted with 0.9% saline to a concentration of 1:100.

[0060] (2) Streak separation of bacterial strains

[0061] Prepare a yeast screening medium, including: 10 g / L yeast extract, 20 g / L glucose, 20 g / L peptone, and 20 g / L agar, and adjust the pH to 6.0; inoculate the diluted bacterial sample into the screening medium, streak it using the plate streak method, and then culture it at 30°C for 48 hours until the colonies appear.

[0062] (3) Identification of bacterial species

[0063] Select suspected yeast species in the plate for identification. The suspected yeast is round or oval in shape and about 3-5 μm in size ( Figure 1 ); Pick a single colony and extract genomic DNA, use specific primers (ITS sequence) for amplification and sequencing, and use BLAST comparison analysis to determine whether it is Kluyveromyces marxianus. If the sequence similarity is greater than 99%, it is the same species.

[0064] Among them, the specific primer sequences are as follows:

[0065] The forward primer (ITS1) was 5'-TCCGTAGGTGAACCTGCGG-3'; the reverse primer (ITS4)

[0066] is 5'-TCCTCCGCTTATTGATATGC-3'.

[0067] (4) Preservation of strains

[0068] After identification, a strain of Kluyveromyces marxianus was finally obtained, which was white, round, smooth, with convex surfaces and neat edges in YPD medium ( Figure 2). The Kluyveromyces marxianus was deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration (CGMCC) on October 17, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101, the deposit number is CGMCC No. 32249, and the classification name is Kluyveromyces marxianus.

[0069] Example 2 Cultivation of Kluyveromyces marxianus and preparation of bacterial powder

[0070] (1) Activation of bacteria

[0071] Kluyveromyces marxianus was inoculated into YPD medium (10 g / L yeast extract, 20 g / L glucose, 20 g / L peptone) for activation at 30°C for 24 h to obtain a purified strain for later use.

[0072] (2) Preparation of seed solution

[0073] The activated Kluyveromyces marxianus was inoculated into YPD medium for cultivation at a temperature of 30° C. for 24 h and a shaking speed of 200 rpm to obtain seed solution for later use.

[0074] (3) Fermentation tank culture

[0075] Before inoculation, the fermentation medium is prepared and placed in a fermenter, and sterilized by direct steam spray at 115°C for 30 minutes; the seed liquid is inoculated into the fermenter at an inoculation amount of 10% for fermentation culture, the culture temperature is 28°C, the rotation speed is 300rpm, the tank pressure is 0.05mpa, and the culture time is 40h; during the yeast fermentation culture process, the carbon source and the nitrogen source are supplemented by a flow addition method, and the feed is started from 10h of the fermentation culture, the carbon source is supplemented to keep the residual sugar content of the culture medium at 5g / L, the nitrogen source is supplemented to keep the pH at 5.0, and the RQ value is kept at 1.0.

[0076] The fermentation medium includes: 2% glucose, 1.5% yeast extract powder, 0.2% (NH4)2SO4, 0.3% KH2PO4, 28.8mg / L CaCl2, 14.4mg / L FeSO4, 4.8mg / L thiocyanate, 0.12mg / L biotin, 6.4mg / L D-calcium pantothenate, and 120mg / L inositol.

[0077] (4) Preparation of bacterial powder

[0078] After the fermentation culture is completed, the bacteria are collected, the solids of the bacteria are mixed with water in a volume ratio of 1:4, poured into a disc centrifuge, the rotation speed is 4000rpm, and the bacteria are washed and separated twice at 15°C; the washed and separated bacteria are vacuum sterilized to inactivate the bacteria, the inactivation temperature is 85°C, the inactivation time is 120min, and the high shear is continuously 3000rpm. Four vacuum cycles are performed during the inactivation process, and in each cycle, the vacuum degree is first adjusted to -0.09mpa and maintained for 20min, and then adjusted to normal pressure and maintained for 10min; the inactivated bacteria are placed in a high-pressure jet homogenizer for homogenization 4 times to obtain yeast milk, and the homogenization pressure is 90mpa; the homogenized yeast milk is vacuum puffed and dried, the drying temperature is 80°C, and the vacuum degree is alternately adjusted from 30pa to 600pa every 10min during the drying process, and the drying is continued until the moisture content is less than 8%, thereby obtaining bacterial powder.

[0079] Example 3

[0080] The same process as in Example 2 of the present application was used for fermentation culture and bacterial powder preparation. The difference between Example 3 and Example 2 is that:

[0081] (1) Fermentation tank culture

[0082] Fermentation medium: increase wort by 6%;

[0083] Sterilization of culture medium: 125℃ steam direct spray sterilization for 15min;

[0084] Fermentation culture parameters: culture temperature 30°C, rotation speed 400rpm, tank pressure 0.06mpa, culture time 50h, carbon source supplementation to keep the residual sugar content of the culture medium at 4g / L, nitrogen source supplementation to keep the pH at 5.5, and RQ value at 0.9;

[0085] (2) Preparation of bacterial powder

[0086] Washing and separation: Mix the bacterial solids with water at a volume ratio of 1:6, rotate at 4600 rpm, and wash and separate three times at 10°C;

[0087] Inactivation: The inactivation temperature was 95°C, the inactivation time was 30 min, the high shear was continued at 3500 rpm, and two vacuum cycles were performed. In each cycle, the vacuum degree was first adjusted to -0.07 MPa for 10 min, and then adjusted to normal pressure for 5 min;

[0088] High pressure homogenization: homogenization pressure is 120mpa, homogenization 3 times;

[0089] Drying: vacuum belt drying, drying temperature is 120℃, during the drying process, the vacuum degree is alternately adjusted from 1000Pa to 3000Pa every 20min.

[0090] Example 4

[0091] The same process as in Example 2 of the present application was used for fermentation culture and bacterial powder preparation. The difference between Example 4 and Example 2 is that:

[0092] (1) Fermentation tank culture

[0093] Fermentation medium: Replace the carbon source with molasses 60 g / L;

[0094] Sterilization of culture medium: 150℃ steam direct spray sterilization for 2s;

[0095] Fermentation culture parameters: culture temperature 32 ° C, rotation speed 500 rpm, tank pressure 0.08 MPa, culture time 55 h, feed supplementation started from 12 h of fermentation culture, carbon source was supplemented to keep the residual sugar content of the culture medium at 6 g / L, nitrogen source was supplemented to keep the pH at 5.2, and RQ value was kept at 1.1;

[0096] (2) Preparation of bacterial powder

[0097] Washing and separation: Mix the bacterial solids with water at a volume ratio of 1:8, rotate at 4200 rpm, and wash and separate at 6°C for 3 times;

[0098] Inactivation: The inactivation temperature was 90°C, the inactivation time was 60 min, the high shear was continued at 3500 rpm, and 3 vacuum cycles were performed. In each cycle, the vacuum degree was first adjusted to -0.07 MPa for 15 min, and then adjusted to normal pressure for 5 min;

[0099] High pressure homogenization: homogenization pressure is 170mpa, homogenization is performed twice;

[0100] Drying: vacuum microwave drying, the drying temperature is 100°C, and the vacuum degree is alternately adjusted from 100Pa to 800Pa every 15min during the drying process.

[0101] Example 5

[0102] The same process as in Example 2 of the present application was used for fermentation culture and bacterial powder preparation. The difference between Example 5 and Example 2 is that:

[0103] (1) Fermentation tank culture

[0104] Fermentation medium: Replace the carbon source with 60 g / L starch hydrolysate;

[0105] Sterilization of culture medium: 142℃ steam direct spray sterilization for 10s;

[0106] Fermentation culture parameters: culture temperature 34 ° C, rotation speed 600 rpm, tank pressure 0.05 MPa, culture time 45 h, feed supplementation started from 12 h of fermentation culture, carbon source was supplemented to keep the residual sugar content of the culture medium at 4 g / L, nitrogen source was supplemented to keep the pH at 5.5, and RQ value was kept at 0.8;

[0107] (2) Preparation of bacterial powder

[0108] Washing and separation: Mix the bacterial solids with water at a volume ratio of 1:7, rotate at 3500 rpm, and wash and separate twice at 2°C;

[0109] Inactivation: The inactivation temperature was 90°C, the inactivation time was 90 min, the high shear was continued at 2500 rpm, and 3 vacuum cycles were performed. In each cycle, the vacuum degree was first adjusted to -0.07 MPa for 20 min, and then adjusted to normal pressure for 10 min;

[0110] High pressure homogenization: homogenization pressure is 170mpa, homogenization is performed twice;

[0111] Drying: Vacuum freeze drying, place the yeast milk in a tray with a thickness of 3 cm and freeze it to -25℃, then transfer it to a vacuum drying oven with a temperature of 60℃. The vacuum degree is alternately adjusted from 50Pa to 400Pa every 25min.

[0112] Example 6

[0113] The same process as in Example 2 of the present application was used for fermentation culture and bacterial powder preparation. The difference between Example 6 and Example 2 is that:

[0114] (1) Preparation of yeast powder

[0115] Inactivation: 100℃ steam heating for 20min, no vacuum;

[0116] Drying: spray drying.

[0117] Example 7

[0118] The same process as in Example 2 of the present application was used for fermentation culture and bacterial powder preparation. The difference between Example 7 and Example 2 is that:

[0119] (1) Fermentation tank culture

[0120] Fermentation medium: Replace the carbon source with 60 g / L starch hydrolysate;

[0121] Sterilization of culture medium: 142℃ steam direct spray sterilization for 10s;

[0122] Fermentation culture parameters: culture temperature 34 ° C, rotation speed 600 rpm, tank pressure 0.05 MPa, culture time 45 h, feed supplementation started from 12 h of fermentation culture, carbon source was supplemented to keep the residual sugar content of the culture medium at 4 g / L, nitrogen source was supplemented to keep the pH at 5.5, and RQ value was kept at 0.8;

[0123] (2) Preparation of bacterial powder

[0124] No high pressure homogenization was performed.

[0125] Example 8

[0126] The same process as in Example 2 of the present application was used for fermentation culture and bacterial powder preparation. The difference between Example 8 and Example 2 is that:

[0127] (1) Fermentation tank culture

[0128] Fermentation medium: replace the nitrogen source with 20 g / L corn steep liquor;

[0129] (2) Preparation of bacterial powder

[0130] Washing and separation: Mix the bacterial solids with water at a volume ratio of 1:6, rotate at 4600 rpm, and wash and separate three times at 10°C;

[0131] Inactivation: 90℃ steam heating for 40min, no vacuum;

[0132] High pressure homogenization: homogenization pressure is 120mpa, homogenization 3 times;

[0133] Drying: vacuum belt drying, drying temperature is 120℃, during the drying process, the vacuum degree is alternately adjusted from 1000Pa to 3000Pa every 20min.

[0134] Example 9

[0135] The same process as in Example 2 of the present application was used for fermentation culture and bacterial powder preparation. The difference between Example 9 and Example 2 is that:

[0136] (1) Fermentation tank culture

[0137] Fermentation medium: Replace the carbon source with molasses 60 g / L;

[0138] Sterilization of culture medium: 150℃ steam direct spray sterilization for 2s;

[0139] Fermentation culture parameters: culture temperature 32 ° C, rotation speed 500 rpm, tank pressure 0.08 MPa, culture time 72 h, feed supplementation started from 12 h of fermentation culture, carbon source was supplemented to keep the residual sugar content of the culture medium at 6 g / L, nitrogen source was supplemented to keep the pH at 5.2, and RQ value was kept at 1.1;

[0140] (2) Preparation of bacterial powder

[0141] Washing and separation: Mix the bacterial solids with water at a volume ratio of 1:6, rotate at 4600 rpm, and wash and separate three times at 10°C;

[0142] Inactivation: 90℃ steam heating for 40min, no vacuum;

[0143] High pressure homogenization: homogenization pressure is 120mpa, homogenization 3 times;

[0144] Drying: spray drying.

[0145] Experimental Example 1 Determination of bacterial powder protein content and sensory evaluation

[0146] The bacterial powder obtained in the above-mentioned Example 2-9 was subjected to the following tests:

[0147] (1) Determination of crude protein content: The crude protein content of bacterial powder was determined by Kjeldahl method;

[0148] (2) Determination of soluble protein content: The bacterial powder was prepared into a suspension with a solid concentration of 10% using purified water, and the supernatant was obtained by centrifugation at 1500 g / 10 min. The soluble protein content was determined by the Kjeldahl method.

[0149] (3) Odor evaluation: The bacterial powder was prepared into a suspension with a solid concentration of 10% using purified water, sterilized at 90°C for 5 min, and the odor was scored after cooling. Based on Example 6, those with lower odor than Example 6 were given positive scores, the lower the score, and the score range was 0.5 to 5; those with higher odor than Example 6 were given negative scores, the higher the score, and the score range was -0.5 to -5.

[0150] (4) Color evaluation: Observe the color of the 10% solid suspension. The lighter the color, the higher the acceptability.

[0151] The crude protein content, soluble protein content and odor score of the bacterial powder are shown in Table 1; the color score is shown in Figure 3 shown.

[0152] Table 1 Test results of Kluyveromyces marxianus powder prepared in this application

[0153]

[0154]

[0155] From the data in Table 1 and Figure 3 The results show that the protein content of the Kluyveromyces marxianus of the present application can reach more than 70%; and the bacterial powder prepared according to the method of the present application can obtain a bacterial powder with high protein, low odor and light color.

[0156] Example 10 Shake flask fermentation of Kluyveromyces marxianus

[0157] (1) Preparation of seed solution

[0158] Pick a single colony with good growth from the plate and inoculate it into a shake flask seed culture medium, wherein the shake flask seed culture medium includes: 10 g / L yeast extract, 20 g / L glucose, and 20 g / L peptone; adjust the pH to 6.0, the culture temperature to 30°C, the culture time to 24 h, and the shaker speed to 200 rpm to obtain seed solution for later use.

[0159] (2) Shake flask culture

[0160] The seed liquid was inoculated into a shake flask fermentation medium at a 5% inoculation rate for fermentation culture. The culture temperature was 30° C., the rotation speed was 200 rpm, and the culture time was 48 h. Carbon and nitrogen sources were supplemented at 24 h of fermentation culture.

[0161] The fermentation medium includes: 3-10% carbon source, 1-2% nitrogen source, 0.2% (NH4)2SO4, 0.3% KH2PO4, 0.5% MgSO4, 28.8mg / L CaCl2, 14.4mg / L FeSO4, 4.8mg / L thiourea hydrochloride, 0.12mg / L biotin, 6.4mg / L D-calcium pantothenate, and 120mg / L inositol. The carbon source includes glucose, molasses, and starch hydrolysate, and the nitrogen source includes yeast extract powder and corn steep liquor. According to the selection of carbon source and nitrogen source, a total of 10 groups of experiments were carried out. After the culture was completed, the bacterial sludge was obtained by centrifugation, and the protein content was detected by Kjeldahl method. The results of the shake flask fermentation are shown in Table 2.

[0162] Table 2 Results of shake flask fermentation of Kluyveromyces marxianus in this application

[0163]

[0164]

[0165] It can be seen from the data in Table 2 that after shake flask fermentation, the Kluyveromyces marxianus of the present application has a relatively high protein content, which can reach more than 70%.

Claims

1. A strain of Kluyveromyces marxianus, whose deposit number is CGMCC No.32249.

2. A fermentation and culturing method of Kluyveromyces marxianus according to claim 1, comprising: The Kluyveromyces marxianus is inoculated into a fermentation medium and fermented in a fermenter.

3. The fermentation culture method according to claim 2, wherein: The fermentation nitrogen source is selected from one or more of yeast extract, malt extract, corn steep liquor, peptone, defatted soybean meal, defatted peanut meal, ammonia water, monoammonium phosphate and diammonium phosphate.

4. The fermentation culture method according to claim 2, wherein: The fermentation carbon source is selected from one or more of glucose, molasses, starch and straw.

5. The fermentation culture method according to claim 2, wherein: The fermentation culture conditions are: fermentation culture for 20-72 hours at a temperature of 25-38°C, a rotation speed of 100-800rpm, a tank pressure of 0.03-0.09mpa, feeding of carbon source and nitrogen source, a constant pH of 4-6, and an RQ of 0.7-1.

5.

6. A method for preparing bacterial powder, comprising: Fermenting Kluyveromyces marxianus by the fermentation and culturing method according to any one of claims 2 to 5 to obtain bacterial cells, and treating the bacterial cells to obtain the bacterial powder; The Kluyveromyces marxianus is the Kluyveromyces marxianus described in claim 1.

7. The preparation method according to claim 6, wherein: The processing of the bacterial bodies includes washing and separating the bacterial bodies, vacuum sterilizing, high-pressure homogenizing and vacuum drying.

8. A bacterial powder obtained according to the preparation method according to claim 6 or 7.

9. Use of the Kluyveromyces marxianus according to claim 1 in preparing bacterial powder.

10. Use of the Kluyveromyces marxianus according to claim 1 or the bacterial powder according to claim 8 in preparing food products.

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