CN116355769A - Kluyveromyces marxianus strain and application thereof - Google Patents
Kluyveromyces marxianus strain and application thereof Download PDFInfo
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- CN116355769A CN116355769A CN202310485453.7A CN202310485453A CN116355769A CN 116355769 A CN116355769 A CN 116355769A CN 202310485453 A CN202310485453 A CN 202310485453A CN 116355769 A CN116355769 A CN 116355769A
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- 229940031154 kluyveromyces marxianus Drugs 0.000 title claims abstract description 51
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 title claims abstract description 50
- 235000018368 Saccharomyces fragilis Nutrition 0.000 title claims abstract description 50
- 241000235650 Kluyveromyces marxianus Species 0.000 title claims abstract 10
- 238000000034 method Methods 0.000 claims abstract description 19
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- 238000000855 fermentation Methods 0.000 claims description 46
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- 239000007952 growth promoter Substances 0.000 description 1
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- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- AIHDCSAXVMAMJH-GFBKWZILSA-N levan Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@@H]1[C@@H](O)[C@H](O)[C@](CO)(CO[C@@H]2[C@H]([C@H](O)[C@@](O)(CO)O2)O)O1 AIHDCSAXVMAMJH-GFBKWZILSA-N 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- HAWPXGHAZFHHAD-UHFFFAOYSA-N mechlorethamine Chemical class ClCCN(C)CCCl HAWPXGHAZFHHAD-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1048—Glycosyltransferases (2.4)
- C12N9/1051—Hexosyltransferases (2.4.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01007—Inulinase (3.2.1.7)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
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Abstract
The present application relates to kluyveromyces marxianus strains having a genetic mutation located in one or more genes selected from the group consisting of: inulase coding geneinuProtease encoding genelonOr glucan synthase encoding genebglThe method comprises the steps of carrying out a first treatment on the surface of the The mutant strains have higher total intracellular protein production and better taste.
Description
Technical Field
The application belongs to the technical field of biological fermentation, and particularly relates to a Kluyveromyces marxianus strain and application thereof.
Background
Kluyveromyces marxianus is a food-grade edible microorganism, has passed the safety certification of GRAS (Generally Recognized as Safe) and QPS (Qualified Presumption of Safety) in the United states and Europe, is regarded as a safe microorganism strain, and is also regarded as a safe microorganism strain for preparing food-grade recombinant proteins (enzymes), and is a food-grade yeast approved by European Union, and the expression system thereof has been widely used for expression of recombinant proteins and synthesis of pharmaceutical compounds. It has the characteristics of extremely simple nutrition requirement, vigorous growth, large biomass, wide growth temperature application range and the like. Meanwhile, the novel recombinant protein has the characteristic of high-efficiency secretion of proteins. At present, kluyveromyces marxianus as a chassis cell has been successfully used for producing industrial enzymes: beta-glucosidase, inulase, beta-galactosidase, lipase, etc., are also used to prepare single cell proteins and synthesize aromatic compounds; and the Kluyveromyces marxianus can be utilized to reduce lactose content in food; the method is also used for fermenting alcoholic drinks, including milk beer, whey wine, wine fermentation, agave wine and the like, and has great application prospect and production potential.
Lon is a proteasome, which acts primarily in the cytoplasm of prokaryotes and in the mitochondria and peroxisomes of eukaryotes,lonthe gene expresses ATP-dependent proteolytic enzymes which have a specific cleavage effect on foreign, heterologous proteins and are generally responsible for the cleavage of certain misfolded proteins in the mitochondria of eukaryotic organisms, as well as for the cleavage and destruction of certain structural proteins. Natural naturelonMutant strains, which may reduce protein degradation, may be beneficial for protein accumulation. Inulinase (inulinase) is a class of hydrolytic enzymes capable of hydrolysing beta-2, 1-D levan, widely occurring in microorganisms and plants, especially filamentous fungi and yeasts. The inulase-producing microorganisms mainly include bacteria, fungi andyeasts, wherein the ability of the yeasts to produce inulinase is stronger than fungi and bacteria, in particular Kluyveromyces (Chi Z M et al Appl Microbiol Biotechnol,2009, 82:211-220). Inulase INU is the main extracellular secreted protein of Kluyveromyces marxianus, and mutation of inulase INU is beneficial to intracellular protein accumulation. Beta-1, 3-glucan synthase (beta-1, 3-glucan synthase) degrades glucan, which is the main component of the inner cell wall. Yeast beta-glucan is a polysaccharide derived from yeast cell walls, and is a high polymer polysaccharide taking beta-1, 3-D-glucan as a main chain and beta-1, 6-D-glucan as a branched chain. In Saccharomyces cerevisiae, beta-1, 3-glucan accounts for 80-90% of the inner cell wall, and the cell wall accounts for 20-30% of the dry weight of the cell.
The wild-type Kluyveromyces marxianus has low protein content during the fermentation production of single cell protein, and can generate bad yeast flavor.
Disclosure of Invention
In view of the problems existing at present, the application provides a Kluyveromyces marxianus strain with mutation so as to increase the yield of intracellular total protein and/or improve the flavor of fermentation products.
In a first aspect the present application provides a kluyveromyces marxianus strain having a genetic mutation located in one or more genes selected from the group consisting of: inulase coding geneinuProtease encoding genelonAnd glucan synthase encoding genesbgl。
In a second aspect, the present application provides a product comprising a kluyveromyces marxianus strain provided in the first aspect of the present application.
A third aspect of the present application provides the use of a kluyveromyces marxianus strain provided in the first aspect of the present application or a product provided in the second aspect of the present application in at least one of the following (1) - (3):
(1) Producing single cell proteins;
(2) The yield of single-cell proteins is improved;
(3) The undesirable flavors are eliminated, preferably, the undesirable flavors include at least one of a yeast flavor and a bitter flavor.
In a fourth aspect, the present application provides a method of fermentation using a kluyveromyces marxianus strain of the first aspect of the present application, comprising the steps of:
inoculating the Kluyveromyces marxianus strain into a fermentation medium, and performing fermentation culture.
In a fifth aspect, the present application provides a single cell protein product prepared according to the method of the fourth aspect of the present application.
The application provides a mutant Kluyveromyces marxianus, wherein the total intracellular protein content can reach 45%, and the single-cell protein yield is obviously increased; the bad flavor is obviously eliminated, and after multiple sensory evaluation, the mutant strain has excellent flavor performance and can be inherited stably. The Kluyveromyces marxianus strain lays a foundation for the application of the Kluyveromyces marxianus strain in the field of yeast single cell protein manufacturing and processing.
Detailed Description
In a first aspect the present application provides a kluyveromyces marxianus strain having a genetic mutation located in one or more genes selected from the group consisting of: inulase coding geneinuProtease encoding genelonOr glucan synthase encoding genebgl。
In some embodiments, the Kluyveromyces marxianus strain has a mutated inulase encoding geneinuMutant protease encoding geneslonAnd mutated glucan synthase encoding genesbgl。
In some embodiments, the kluyveromyces marxianus strain is obtained by mutagenesis;
preferably, the mutagenesis mode comprises one or more of ultraviolet mutagenesis, ARTP mutagenesis, microwave mutagenesis, ion implantation mutagenesis, space mutagenesis or chemical reagent mutagenesis;
preferably, the subject of mutagenesis (i.e., the starting strain) is a wild-type Kluyveromyces marxianus strainKluyveromyces marxianus) CJ3113 (preservation number CCTCC No: m20211265).
More preferably, the chemical agent is selected from the group consisting of one or more of diethyl sulfate, ethyl methylsulfonate, nitrosoguanidine, and nitrogen mustard.
In some embodiments, the Kluyveromyces marxianus strain is classified and named as Kluyveromyces marxianusKluyveromyces marxianus) CJA0005 with a preservation number of CCTCC No. M20211605.
Kluyveromyces marxianus described in the application is preserved in China center for type culture Collection; deposit unit address: university of martial arts; the preservation date is 2021, 12 and 13.
In a second aspect, the present application provides a product comprising a kluyveromyces marxianus strain provided in the first aspect of the present application.
In some embodiments, the product may include food or feed additives and the like.
In some embodiments of the present application, the corresponding substrate may be fermented by a kluyveromyces marxianus strain having a genetic mutation of the present application for preparing a food product; in other embodiments of the present application, the kluyveromyces marxianus strain with genetic mutation or single cell protein produced by the kluyveromyces marxianus strain can be used as a fermentation substrate of other strains, such as lactic acid bacteria, so as to prepare food; in other embodiments of the present application, the kluyveromyces marxianus strain with the genetic mutation of the present application can be added directly to food.
In some embodiments, the product may be a feed additive, and in some embodiments, the product may further comprise at least one of a growth promoter, a nutrient, a nutritional supplement, a storage stabilizer, and a coating agent.
A third aspect of the present application provides the use of a kluyveromyces marxianus strain provided in the first aspect of the present application or a product provided in the second aspect of the present application in at least one of the following (1) - (3):
(1) Producing single cell proteins;
(2) The yield of single-cell proteins is improved;
(3) The undesirable flavors are eliminated, preferably, the undesirable flavors include at least one of a yeast flavor and a bitter flavor.
In a fourth aspect, the present application provides a method of fermentation using a kluyveromyces marxianus strain of the first aspect of the present application, comprising the steps of:
inoculating the Kluyveromyces marxianus strain into a fermentation culture medium, and performing fermentation culture;
the culture medium is a culture medium commonly used in the art, wherein the fermentation nitrogen source is selected from one or more of corn steep liquor, ammonium sulfate, whey powder, tryptone and semi-complete protein, and the fermentation carbon source is selected from one or more of glucose, glycerol, sucrose, lactose, xylose and molasses.
The inventor finds that the fermentation of the Kluyveromyces marxianus strain in the culture medium is beneficial to the growth and high-density propagation of strains.
"fermentation" in the present application refers to a process in which Kluyveromyces marxianus prepares a cell itself or a direct metabolite or a secondary metabolite by vital activity under aerobic conditions.
In some embodiments, the fermentation method may further include collecting thalli, where the method for collecting thalli is not limited, and conventional methods in the art, such as centrifugation and filtration, may be used; in some embodiments, the single cell protein may be separated and purified, and the separation and purification method is not limited herein, and conventional methods in the art, such as precipitation, membrane separation, filtration, flocculation, and the like, may be used.
In some embodiments, the fermentation nitrogen source is selected from one or more of corn steep liquor, ammonium sulfate, whey powder, tryptone, and semi-complete protein.
In some embodiments, the fermentation carbon source is selected from one or more of glucose, glycerol, sucrose, lactose, xylose, molasses.
In some embodiments, the fermentation conditions are: fermenting at 25-45 deg.c and 100-600 rpm for 24-120 h deg.c. The inventors found that too low a fermentation temperature, slow fermentation speed, and too high a fermentation temperature affect the activity of the cells, and found that when the fermentation temperature is within the above temperature range, it is advantageous to improve the fermentation speed and the quality of the fermentation product. In addition, the fermentation time is too short, the yield of the fermentation product is low, the fermentation time is too long, and the quality of the fermentation product is affected due to aging of thalli.
In a fifth aspect, the present application provides a single cell protein product produced by the fermentation process of the fourth aspect of the present application. The inventors have found that the single cell protein products obtained using the methods of the present application exhibit better taste, such as a yeast taste and/or a less bitter taste.
The term "single cell protein" as used herein has its ordinary meaning and may refer to a cytoplasmic mass composed of a mixture of proteins, fats, carbohydrates, nucleic acids, and nitrogen-containing compounds, vitamins, and inorganic compounds that are not proteins, etc., produced by fermentation of the Kluyveromyces marxianus strain of the present application. The term "fermentation product" as used herein refers to a product produced by a process including a fermentation step using a fermenting organism, and may include single cell proteins composed of kluyveromyces marxianus cells and/or cytoplasmic masses as used herein.
Embodiments of the present application are described in detail below. The embodiments described below are exemplary only for the purpose of illustrating the present application and are not to be construed as limiting the present application.
The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present application. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
1. Ultraviolet mutagenesis
(1) Kluyveromyces marxianus CJ3113 (preservation number CCTCC No: M20211265, hereinafter abbreviated as CJ 3113) is taken as a starting strain, and the starting strain is inoculated on a sterilized YPD liquid culture medium and subjected to rejuvenation culture at 30 ℃ for 2 generations; inoculating the strain with stable growth into a sterilized liquid YPD culture medium for 2 generations, so that the strain reaches a normal growth period for later use:
(2) Preparation of starting Strain suspension
Taking 2 ml of bacterial liquid of CJ3113 in logarithmic phase in the step (1), and centrifuging for 5 min at 3000 r/min; pouring out the supernatant, adding sterilized normal saline 3 ml, slightly shaking to wash the thalli, centrifuging at 3000 r/min for 5 min, and repeating the steps for 2 times; after removing the supernatant, vortex and mix the thalli evenly to prepare the bacterial suspension. Regulating cell concentration to 10 by microscopy 8 -10 9 /ml。
(3) Ultraviolet mutagenesis and intermediate culture of bacterial cells
Sucking 3 ml of the prepared monospore suspension, and transferring the monospore suspension into a sterile plate with the diameter of 9cm, wherein the thickness of the liquid layer reaches about 0.4 cm; performing ultraviolet mutagenesis, wherein the power of an ultraviolet lamp used for the ultraviolet mutagenesis is 18W, the irradiation time is 120 s, and the irradiation distance is 25 cm; under the condition of avoiding light, the bacterial liquid 1 ml irradiated by an ultraviolet lamp is sucked, YPD liquid culture medium is added for intermediate culture, and shaking flask culture is carried out at 30 ℃ for 16 h. Taking out the bacteria liquid after intermediate culture, diluting the bacteria liquid by adopting a ten-fold dilution method, and sucking the 10 4 ～10 6 Three dilutions of 0.2. 0.2 ml were applied to YPD solid plates and incubated at 30℃for 2 days. Performing primary screening, picking 10 colonies with larger phenotypes, respectively inoculating the colonies into YPD liquid culture medium, and re-screening by shaking flask fermentation. The mutant strain with large colony and quick growth in shake flask is selected, and the strain can be stably passaged for more than 3 generations, the steps are repeated for screening, the strain with degenerated strain and reduced growth speed is removed, and the primary mutant strain with quick growth speed and stable inheritance is reserved.
2. Microwave mutagenesis
Performing activation culture on primary mutagenesis strain obtained by ultraviolet mutagenesis in YPD liquid culture medium, collecting yeast with OD=0.6-0.8, and diluting to 10 -3 、10 -4 And (3) suspending the bacterial liquid in a sterile culture dish, and respectively mutagenizing for 15s, 20s, 30s and 40s under different intensities, wherein the thermal effect of microwaves is eliminated by using an ice bath for 10s every 5s, the mortality is calculated by microscopic examination, and the bacterial liquid with the mortality of 70-80% is selected for the coating culture of YPD solid culture medium. The microwave mutagenesis procedure is shown in Table 1. Mortality (%) = (total number of non-mutagenized bacteria-number of viable bacteria after mutagenesis treatment)/total number of non-mutagenized bacteria×100.
TABLE 1 microwave mutagenesis procedure
Coating YPD solid culture medium, culturing at 30deg.C for 2 days, picking 10 colonies with large phenotype, inoculating into YPD liquid culture medium, shake-flask fermenting, and re-screening. The mutagenic strain with large colony is selected, and the strain can be stably passaged for more than 3 generations, the steps are repeated for screening, the strain with degenerated strain and reduced growth speed is removed, and the second-generation mutagenic strain with fast growth speed and stable inheritance is reserved.
3. Atmospheric Room Temperature Plasma (ARTP) mutagenesis
(1) The second generation mutant strain obtained by microwave mutagenesis is activated and cultured in YPD liquid culture medium, centrifuged at 3000 rpm for 5 min, and the supernatant is discarded, washed twice with sterile physiological saline and then added with buffer solution for suspension.
(2) Each buffer was dropped onto a sterilized slide, subjected to ARTP mutagenesis using an ARTP mutagenic instrument, set power 100W, gas flow 10SLM, and select 0s, 10s, 20s, 30s, 40s as gradient times for sequential mutagenesis. After mutagenesis, the slide glass is put into sterile physiological saline with the concentration of 1 mL to be diluted, and then is coated on YPD solid culture medium to be cultured for 2 days at 30 ℃.
(3) Inoculating single colony obtained in step (2) into YPD liquid culture medium, culturing at 30deg.C for 18-28 h, and flavoringScreening (including yeast taste and bitter taste) to obtain a strain with optimal taste (the yeast taste and the bitter taste are the least), named CJA0005, and preserved in China center for type culture Collection (CCTCC for short, china, wuhan university, and the Wuhan university) at the 12 th month 13 of 2021, wherein the strain is the strain preservation number CCTCC No. M20211605. The mutation of the following genes relative to the wild type was determined by sequencing: inulase coding geneinuProtease encoding genelonAnd glucan synthase encoding genesbgl。
In the embodiment, kluyveromyces marxianus with improved growth rate is obtained through screening by a comprehensive iterative mutagenesis mode of ultraviolet mutagenesis, microwave mutagenesis and ARTP mutagenesis, and the time of growing to the highest biomass in a shake flask is 4 hours earlier than that of a starting bacterium CJ 3113; the CJA0005 strain obtained by the composite mutation screening has obviously improved flavor (yeast taste and bitter taste).
The Kluyveromyces marxianus strain with mutation obtained in example 1 of the present application and Kluyveromyces marxianus strain CJ3113 (as a comparative strain) were each subjected to culture at 30℃for 48 hours in a spread culture on YPD solid medium;
picking the single colony obtained in the step 1), and carrying out activation culture by using 5 ml of YPD liquid culture medium, and carrying out culture at 30 ℃ for 36 hours;
performing expansion culture on the bacterial liquid obtained in the step 2) by adopting 50 ml of YPD liquid culture medium, and culturing at 30 ℃ for 36 hours;
inoculating the bacterial liquid obtained in the step 3) into a new YPD culture medium, and carrying out shake flask fermentation, wherein the inoculum size is 2%, and the liquid loading amount is 200mL/L; shaking culture at 30 deg.c and 220 rpm for 36 hr; measuring the fermentation yield of the shake flask;
and collecting the bacterial cells.
1. By examining the biomass during fermentation, the strain of example 1 was found to have a higher cell density during fermentation than the wild strain, and thus the culture period could be shortened.
2. Shake flask fermentation yield of the total protein of the strain of example 1 was measured by kjeldahl method, wherein the total protein content of the composite mutagenesis strain of example 1 was about 45% and the bacterial protein of CJ3113 was about 38%; the strain of the application is demonstrated to have higher intracellular total protein production.
3. After fermentation, the single cell protein spray dried bacterial powder 1 g of example 1 and comparative strain was dissolved in 4. 4 g water, 10 volunteers were randomly selected to taste, and the color and taste thereof were scored, and the results are shown in table 2. Wherein the yeast taste is 1-5, the bitter taste is 1-5, the lower the score is, the higher the quality is, and the better the taste is.
TABLE 2
The results show that the taste of the composite mutagenesis strain CJA0005 strain of the application is obviously improved compared with that of the wild type original strain CJ 3113. Without being bound by any theory, the inventors believe that the metabolic enzymes change after mutagenesis of the Kluyveromyces marxianus strain of the present application, resulting in a change in the product and thus a change in the taste of the product.
From the results, the Kluyveromyces marxianus strain CJA0005 provided by the application has faster growth rate and higher single-cell protein yield; in addition, it has a better taste.
Although embodiments of the present application have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the application, and that variations, modifications, alternatives, and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the application.
Claims (9)
1. A kluyveromyces marxianus strain characterized by having a genetic mutation located in one or more genes selected from the group consisting of: inulase coding geneinuProtease encoding genelonAnd glucan synthase encoding genesbgl，
The classification of the Kluyveromyces marxianus strain is named as Kluyveromyces marxianus @Kluyveromyces marxianus) CJA0005 with a preservation number of CCTCC No. M20211605.
2. Kluyveromyces marxianus strain according to claim 1, characterized in that it is obtained by means of mutagenesis.
3. A product comprising the kluyveromyces marxianus strain of claim 1 or 2.
4. Use of a kluyveromyces marxianus strain as defined in claim 1 or 2 or a product as defined in claim 3 in at least one of the following (1) - (3):
(1) Producing single cell proteins;
(2) The yield of single-cell proteins is improved;
(3) Eliminating bad flavor.
5. A method of fermentation using the kluyveromyces marxianus strain of claim 1 or 2, comprising the steps of:
inoculating the Kluyveromyces marxianus strain into a fermentation medium, and performing fermentation culture.
6. The method of claim 5, wherein the fermentation nitrogen source is selected from one or more of corn steep liquor, ammonium sulfate, whey powder, tryptone.
7. The method of claim 5, wherein the fermentation carbon source is selected from one or more of glucose, glycerol, sucrose, lactose, xylose, and molasses.
8. The method according to any one of claims 5-7, wherein the fermentation conditions are: fermenting at 25-45 deg.c and 100-600 rpm for 24-120 h deg.c.
9. A single cell protein product prepared according to the method of any one of claims 5-8.
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