Strain of kluyveromyces marxianus and use thereof

Abstract

The present application relates to the technical field of fermentation, and in particular to a strain of Kluyveromyces marxianus and use thereof. The Kluyveromyces marxianus strain is Kluyveromyces marxianus AMCC 31395, which is preserved in the China Center for Type Culture Collection (CCTCC), under the accession number of CCTCC NO: M20241527. The Kluyveromyces marxianus strain has high tolerance to furfural, phenol, and mixed inhibitors, and can effectively use a low-cost carbon source straw hydrolysate, on the basis of a feeding culture way, to obtain a high-density yeast, which has a total yeast protein yield of up to 51.55 g / L. The Kluyveromyces marxianus strain is a high-quality protein raw material, and can be used for preparing high-protein products such as animal feed and pet food. The present invention uses Kluyveromyces marxianus to efficiently convert low-cost substrates, providing technical support for the production of green, low-consumption yeast proteins, and exhibiting great application prospects in the field of alternative proteins.

Description

A strain of Kluyveromyces Marcius and its applications

[0001] Related applications

[0002] This application claims priority to the earlier application No. 202510123748.9 filed with the China National Intellectual Property Administration on January 26, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This application relates to the field of fermentation technology, specifically to a strain of Kluyveromyces martensii and its applications, particularly to Kluyveromyces martensii and its applications, which exhibits high tolerance to furfural and / or phenol inhibitors and can effectively utilize carbon sources to obtain cell bodies and accumulate cell proteins in biomass containing multiple inhibitors. Background Technology

[0004] The main alternative protein source for feed is microbial protein, also known as single-cell protein (SCP) or microbial protein. This protein resource is obtained through large-scale cultivation of single-celled organisms such as bacteria, yeast, and microalgae, as well as simple multicellular organisms such as molds, using various low-value industrial and agricultural biomass and other nutrients. Compared to animal and plant proteins, microbial protein has advantages such as comprehensive nutrition, clean labeling, and sustainable environmental friendliness. Yeast protein is a typical example among microorganisms, possessing significant potential and development space. Traditional yeast protein strains include Saccharomyces cerevisiae and Candida albicans. Kluyveromyces martensii is a non-traditional yeast that has been included in China's list of edible fungi and can be used in the production of various foods and pharmaceuticals. This strain has a broad substrate spectrum. Compared to Saccharomyces cerevisiae, Kluyveromyces martensii can ferment and metabolize multiple carbon sources in low-value biomass. It also exhibits heat resistance, rapid growth rate, and outstanding flavor. For these reasons, Kluyveromyces martensii is widely recognized as a non-traditional yeast that holds promise for playing a significant role in the industrialization of low-value agricultural biomass resources.

[0005] Low-value biomass such as straw, rice husks, and corn cobs provide raw materials for the production of microbial protein. Therefore, utilizing microbial fermentation to achieve efficient utilization of low-value biomass and increase the added value of inexpensive byproducts is a good way to turn waste into treasure. However, various pretreatments of low-value biomass, such as acid treatment, alkali treatment, and steam explosion, produce inhibitors such as acetic acid, furfural, and phenol, which inhibit the growth and fermentation of microbial strains. Furthermore, a low pH environment is unfavorable for microbial growth, limiting the development of low-value biomass. To maximize the conversion of carbon sources in low-value biomass hydrolysate into microbial cell protein, screening microorganisms with inhibitor tolerance and efficient substrate utilization is the optimal solution to these problems.

[0006] Therefore, developing a strain of *Kluyveromyces martensii* that is resistant to inhibitors and can convert carbon sources such as xylose and acetic acid into microbial protein can maximize the utilization of low-quality biomass resources in the field of microbial feed, obtain microbial protein, and alleviate the current shortage of feed resources in the aquaculture industry.

[0007] Chinese patent application CN113512506A describes a method to improve the tolerance of fermenting microorganisms to inhibitors derived from lignocellulose pretreatment by overexpressing the nitroreductase gene in *Kluyveromyces martensii*. The patent successfully obtained a *Kluyveromyces martensii* mutant strain with enhanced tolerance to lignocellulose biomass-derived inhibitors. This strain showed increased ethanol yield and production rate in the presence of the inhibitors. However, the stability of the recombinant plasmids obtained through overexpression in actual production processes can be affected by various factors such as equipment, environment, and processes, leading to inconsistent performance of superior traits. Furthermore, the patent did not investigate the strain's maximum tolerance to each inhibitor and only applied it to ethanol production. Therefore, the application potential of *Kluyveromyces martensii* in scenarios with higher inhibitor concentrations and other fields is limited.

[0008] Chinese patent application CN114410487A identifies a strain of *Megamiprid* that can grow normally in rice straw saccharification liquid containing growth inhibitors. This strain can fully utilize monosaccharides and disaccharides in the rice straw saccharification liquid, which are unavailable to other common protein-producing yeasts, thus maximizing the utilization of the saccharification liquid and obtaining more cell protein. However, the straw treatment method used in this patent is alkaline treatment combined with enzyme treatment, which is mild and produces fewer inhibitors such as furfural, acetic acid, and phenol compared to the more widely used and lower-cost acid treatment and steam explosion treatment in industry. Therefore, the inhibitor tolerance of this strain has not been investigated, limiting its application scenarios, and its protein production capacity has not been evaluated.

[0009] Chinese patent application CN118126853A describes the isolation and purification of a *Kluyveromyces martensii* strain suitable for high-density culture and high-yield single-cell protein production from traditional fermented dairy products. This strain can efficiently utilize inexpensive carbon sources like molasses and inorganic nitrogen sources, achieving high-density cell density through fed-batch culture. It can be used to prepare high-fiber, high-protein beverages, functional foods, pet foods, and other high-protein products. However, the protein content of this patented strain is less than 50%, which is not conducive to cost reduction and efficiency improvement. Furthermore, with the rising price of molasses, there is an urgent need to find new, inexpensive raw materials for producing single-cell protein. This patent only studies the relevant physiological and biochemical characteristics of the strain during molasses fermentation, such as tolerance to high sugar, ethanol, and low pH. The relevant physiological and biochemical characteristics in fermentation scenarios using other inexpensive raw materials have not yet been evaluated, and its application potential remains unknown.

[0010] Currently, there are no reports on the production of microbial protein from low-value biomass such as straw by Kluyveromyces martensii, and the tolerance of yeast strains that produce microbial protein from low-value biomass such as straw in existing patents is limited. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide a Kluyveromyces martensii yeast that improves tolerance to furfural and / or phenol, and can effectively utilize carbon sources such as xylose and acetic acid in the presence of multiple inhibitors, thereby achieving high-value conversion of low-value biomass and turning low-value biomass into a valuable resource.

[0012] To address the technical problems existing in the prior art, this application uses a culture medium containing straw hydrolysate components as a screening medium to isolate a strain of Kluyveromyces marxianus from yogurt from herders in Zhalute Banner, Tongliao City, Inner Mongolia Autonomous Region, China, and names it Kluyveromyces marxianus AMCC 31395.

[0013] Specifically, this application proposes the following technical solutions.

[0014] Technical Solution 1. A strain of Kluyveromyces marxianus, wherein the strain is Kluyveromyces marxianus AMCC 31395, which is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20241527.

[0015] Technical Solution 2. The *Kluyveromyces martensii* strain according to Technical Solution 1 has a high xylose utilization capacity, wherein, when the *Kluyveromyces martensii* reaches an initial OD... 620nm =0.18-0.22 After culturing in a xylose medium containing 18-22 g / L at 36-38℃ for 46-50 h, the xylose utilization rate of this strain is over 85%.

[0016] Technical Solution 3. The Kluyveromyces martensii strain according to Technical Solution 1 or 2 has the ability to utilize acetic acid; preferably, the concentration of acetic acid that the Kluyveromyces martensii strain can utilize at 37°C and pH=6.0 is 3.3-25 g / L.

[0017] Technical Solution 4. The Kluyveromyces martensii strain according to any one of Technical Solutions 1-3, wherein the Kluyveromyces martensii strain is tolerant to furfural and / or phenol, preferably tolerant to furfural and phenol;

[0018] Preferably, the Kluyveromyces martensii strain tolerates furfural at concentrations of 0.5-4.5 g / L; and / or

[0019] The Kluyveromyces martensii strain tolerates phenol at concentrations of 0.2-2.0 g / L.

[0020] Technical Solution 5. The *Kluyveromyces martensii* strain according to any one of Technical Solutions 1-4, wherein the *Kluyveromyces martensii* strain is tolerant to one or more substances selected from the group consisting of 5-hydroxymethylfurfural, catechol, eugenol, vanillin and 4-hydroxybenzaldehyde, preferably simultaneously tolerant to 5-hydroxymethylfurfural, catechol, eugenol, vanillin and 4-hydroxybenzaldehyde, more preferably the tolerant concentration of 5-hydroxymethylfurfural is 0.3-0.475 g / L, the tolerant concentration of catechol is 0.03-0.0475 g / L, the tolerant concentration of vanillin is 0.03-0.0475 g / L, and the tolerant concentration of 4-hydroxybenzaldehyde is 0.03-0.0475 g / L.

[0021] Technical Solution 6. A bacterial preparation comprising the *Kluyveromyces martensii* strain as described in any one of Technical Solutions 1-5.

[0022] Technical Solution 7. The application of the Kluyveromyces martensii strain described in any one of Technical Solutions 1-5 or the bacterial preparation described in Technical Solution 6 in the high-value conversion of low-value biomass, preferably, the low-value biomass is straw hydrolysate.

[0023] Technical Solution 8. The use of the *Kluyveromyces martensii* strain described in any one of Technical Solutions 1-5 or the cell preparation described in Technical Solution 6 in the preparation of cell protein.

[0024] Technical Solution 9. A method for preparing microbial protein using the *Kluyveromyces martensii* strain described in any one of Technical Solutions 1-5 or the microbial preparation described in Technical Solution 6, comprising fermenting and culturing the *Kluyveromyces martensii* strain described in any one of Technical Solutions 1-5 or the microbial preparation described in Technical Solution 6 using straw hydrolysate as a carbon source.

[0025] Technical Solution 10. According to the method described in Technical Solution 9, the straw hydrolysate includes one or more substances selected from the group consisting of xylose, glucose, fructose and acetic acid; preferably, the straw hydrolysate also contains furfural and / or phenol;

[0026] Preferably, the concentration of xylose in the straw hydrolysate is 30-180 g / L; and / or,

[0027] The concentration of fructose is 10-80 g / L, and / or,

[0028] The glucose concentration is 20-180 g / L, and / or,

[0029] The concentration of acetic acid is 2-20 g / L, and / or,

[0030] The concentration of furfural is 0.5-5 g / L, and / or,

[0031] The concentration of phenol is 0.2-3 g / L.

[0032] Technical Solution 11. According to the method described in Technical Solution 9 or 10, the fermentation culture medium further includes a nitrogen source, preferably the nitrogen source includes one or more substances selected from the group consisting of ammonia, ammonium sulfate, yeast extract, peptone and urea, more preferably ammonia and ammonium sulfate.

[0033] Technical Solution 12. According to any one of Technical Solutions 9-11, the fermentation medium further includes a phosphorus source, preferably the phosphorus source includes one or more substances selected from the group consisting of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and ammonium dihydrogen phosphate, more preferably ammonium dihydrogen phosphate.

[0034] Technical Solution 13. The method according to any one of Technical Solutions 9-12, wherein the fermentation medium further comprises growth factors, preferably the growth factors comprising one or more substances selected from the group consisting of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and biotin, more preferably vitamin B1 and vitamin B6.

[0035] Technical Solution 14. The method according to any one of Technical Solutions 9-13, wherein the fermentation temperature is 30-38℃, and the preferred fermentation pH is 4-6.

[0036] Technical Solution 15. The method according to any one of Technical Solutions 9-14, wherein the fermentation is high-density fermentation;

[0037] Preferably, the high-density fermentation is carried out using a continuous feeding batch fermentation method.

[0038] Technical Solution 16. According to the method described in Technical Solution 15, the continuous feeding includes continuously adding a carbon source, a nitrogen source, and a phosphorus source to the fermentation culture medium, wherein the carbon source is straw hydrolysate, the nitrogen source includes ammonia water and ammonium sulfate, and the phosphorus source contains ammonium dihydrogen phosphate.

[0039] Technical Solution 17. The method according to Technical Solution 15 or 16 includes the following steps:

[0040] (1) Activation of strains: The Kluyveromyces martensii strain described in technical solutions 1-5 or the bacterial preparation described in technical solution 6 is inoculated into a liquid activation medium for culture.

[0041] (2) Seed culture: The activated bacterial solution from step (1) is inoculated into a fermenter for continuous feeding and batch fermentation to obtain seed culture solution, wherein carbon source, nitrogen source and phosphorus source are added in a continuous feeding manner;

[0042] (3) Fermentation tank culture: The seed culture solution from step (2) is inoculated into the fermentation tank for continuous feeding and batch fermentation. The carbon source, nitrogen source and phosphorus source are added in a continuous feeding manner. The alcohol concentration of the culture solution is maintained at 0.5-1g / L and the dissolved oxygen is maintained at 10-20%.

[0043] Technical Solution 18. According to the method described in Technical Solution 17, the fermentation temperature in step (3) is 30-38℃, the pH is preferably maintained at 4-6, the fermentation time is more preferably 15-16h, and the inoculum amount is even more preferably 15-19g / L.

[0044] Technical Solution 19. According to the method described in Technical Solution 17 or 18, before inoculating the seed culture solution in step (2) into the fermenter, step (3) includes the step of adding fermentation bottom water to the fermenter, preferably also including adding growth factors to the fermentation bottom water, wherein the growth factors include vitamin B1 and vitamin B6, more preferably, the amount of vitamin B1 added is 5-15 mg / L and the amount of vitamin B6 added is 1-3 mg / L based on the volume of fermentation bottom water.

[0045] Technical Solution 20. The method according to any one of Technical Solutions 17-19, wherein the carbon source is straw hydrolysate, wherein the total sugar content of the straw hydrolysate is 340-400 g / L, preferably 340-350 g / L; preferably, the straw hydrolysate contains 160-180 g / L xylose, 110-130 g / L glucose, 60-80 g / L fructose, 9-11 g / L acetic acid, 4-5 g / L furfural and 2-3 g / L phenol; more preferably, the feeding rate of the straw hydrolysate is 200-1050 mL / h;

[0046] And / or, the nitrogen source contains ammonia and ammonium sulfate, wherein the concentration of ammonia is 150-170 g / L and the concentration of ammonium sulfate is 210-250 g / L; preferably, the feeding rate of the nitrogen source is 45-180 mL / h;

[0047] And / or, the phosphorus source contains ammonium dihydrogen phosphate, wherein the concentration of ammonium dihydrogen phosphate is 110-130 g / L; preferably, the feeding rate of ammonium dihydrogen phosphate is 0-60 mL / h, more preferably 20-60 mL / h.

[0048] Technical Solution 21. The method according to any one of Technical Solutions 17-20, the method further includes the step of centrifuging and collecting the fermentation broth obtained after fermentation in step (3) to obtain yeast milk; preferably, it further includes the step of granulating the yeast milk after vacuum drum filtration, and more preferably, it further includes the step of fluidized bed drying after granulation.

[0049] Technical Solution 22. A bacterial protein comprising a protein prepared by any one of the methods described in Technical Solutions 9-21.

[0050] Technical Solution 23. The bacterial protein according to Technical Solution 22, wherein the bacterial protein has a protein content of ≥58wt%, preferably essential amino acids account for ≥40% of the total amino acids by mass, and more preferably the bacterial protein has a branched-chain amino acid content of ≥5wt%.

[0051] Technical Solution 24. The application of the microbial protein described in Technical Solution 22 or 23 in the feed field, preferably in poultry feed, aquatic feed, pig feed, ruminant feed or pet food, more preferably as a substitute protein in the preparation of poultry feed, aquatic feed, pig feed, ruminant feed or pet food.

[0052] Technical Solution 25. A feed comprising the above-mentioned microbial protein, preferably wherein the amount of microbial protein added accounts for 5-58% of the total weight of the feed.

[0053] Technical Solution 26. The feed according to Technical Solution 25, wherein the feed is one of poultry feed, aquatic feed, pig feed, ruminant feed or pet food.

[0054] The beneficial effects achieved by this application are as follows:

[0055] The Kluyveromyces marxianus AMCC 31395 strain of this application exhibits high tolerance to furfural, phenol, and mixed inhibitors. In the presence of multiple inhibitors, the carbon source conversion rate reaches 94.3% of that without inhibitors. It can effectively utilize carbon sources to obtain cell bodies and accumulate cell proteins in straw hydrolysate containing multiple inhibitors.

[0056] The Kluyveromyces marxianus AMCC 31395 strain of this application can obtain cell protein through high-density fermentation using straw hydrolysate based on a fed-batch method. The protein content after fermentation reaches 60.67%, and the protein yield is 51.55 g / L. Moreover, this strain has a significant advantage in terms of time conversion efficiency even when using low-value straw hydrolysate, and has considerable potential for industrial production.

[0057] The cell protein prepared by this application using Kluyveromyces marxianus AMCC 31395 strain has a complete amino acid profile, with essential amino acids accounting for 42.3% of the total amino acid content; and has a high content of flavor substances, such as glutamic acid, which can increase animal feed intake and has the potential to prepare yeast protein animal feed and pet food.

[0058] Strain Preservation Information

[0059] The strain Kluyveromyces marxianus AMCC 31395 used in this application was deposited on July 9, 2024, at the China Center for Type Culture Collection (CCTCC) with accession number CCTCCNO:M 20241527. The depository address is: Wuhan University, Wuhan, China, 430072, China; telephone: (027) 68754052.

[0060] The Kluyveromyces marxianus strain YZN013 mentioned in this application was deposited on February 5, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 21814. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, 100101, China. This strain has already been described in Chinese patent application No. 202110473879.1 (Publication No. CN113512506A). The purpose of mentioning the Kluyveromyces marxianus strain YZN013 in this application is to compare the technical effects of the Kluyveromyces marxianus strain YZN013 as described in the specification of patent CN113512506A with the technical effects of the Kluyveromyces marxianus strain AMCC 31395 claimed in this application.

[0061] The Kluyveromyces marxianus NS127 strain mentioned in this application was deposited on March 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30034. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing, 100101, China. This strain has been described in Chinese patent application No. 202410576609.7 (Publication No. CN118126853A). The purpose of mentioning the Kluyveromyces marxianus NS127 strain in this application is to compare the technical effects of the Kluyveromyces marxianus NS127 strain described in the specification of patent CN118126853A with the technical effects of the Kluyveromyces marxianus AMCC 31395 strain claimed in this application. Attached Figure Description

[0062] Figure 1 is a schematic diagram of a plate used for screening inhibitor tolerance using straw hydrolysate.

[0063] Figure 2 is a schematic diagram of the colony morphology of Kluyveromyces martensii AMCC 31395.

[0064] Figure 3 is a schematic diagram of the morphology of Kluyveromyces martensii AMCC 31395 under a microscope.

[0065] Figure 4 shows the growth of Kluyveromyces martensii AMCC 31395 at different temperatures.

[0066] Figure 5 shows the growth of Kluyveromyces martensii AMCC 31395 at different pH values.

[0067] Figure 6 shows the growth of Kluyveromyces martensii AMCC 31395 using xylose. (A) is the growth curve of Kluyveromyces martensii AMCC 31395 in a carbon source medium containing xylose; (B) is the curve of Kluyveromyces martensii AMCC 31395 consuming xylose in a carbon source medium containing xylose.

[0068] Figure 7 shows the growth curves of Kluyveromyces martensii AMCC 31395 in media with different concentrations of acetic acid as the single carbon source.

[0069] Figure 8 shows the growth curves of Kluyveromyces martensii AMCC 31395 in media containing different concentrations of furfural inhibitor.

[0070] Figure 9 shows the growth curves of Kluyveromyces martensii AMCC 31395 in media containing different concentrations of phenol inhibitors.

[0071] Figure 10 shows the growth curves of Kluyveromyces martensii AMCC 31395 in multi-inhibitor medium at different temperatures.

[0072] Figure 11 shows the utilization of carbon sources by Kluyveromyces martensii AMCC 31395 during fermentation in straw hydrolysate medium (YPI) containing inhibitors.

[0073] Figure 12 shows the utilization of carbon sources by Kluyveromyces martensii AMCC 31395 during fermentation in straw hydrolysate carbon source medium (YPW). Detailed Implementation

[0074] This application provides a strain of Kluyveromyces marxianus, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241527.

[0075] The *Kluyveromyces marxianus* strain described in this application was isolated from yogurt from herders in Zhalute Banner, Tongliao City, Inner Mongolia Autonomous Region, China, and named *Kluyveromyces marxianus* AMCC 31395. This strain can utilize xylose and acetic acid, exhibits high tolerance to furfural, phenol, and mixed inhibitors, and can effectively utilize carbon sources to obtain cell cells and accumulate cell proteins in low-value biomass (such as straw hydrolysate) containing multiple inhibitors.

[0076] In this application, the ITS sequence of the above-mentioned strain is shown as SEQ ID NO:3 in Table 13.

[0077] In this application, the ITS sequence refers to the spacer sequence between the 5.8S rDNA and 28S rDNA genes in the rDNA gene. Its length and sequence vary considerably. RFLP or sequence analysis of its amplified products can be used for the classification and identification of different biotypes, strains, species, and genera of fungi. It can even be used to distinguish very closely related species.

[0078] The Kluyveromyces martensii AMCC 31395 described in this application has high xylose utilization capacity, wherein, when the Kluyveromyces martensii has an initial OD... 620nm =0.18-0.22, after culturing in a xylose medium containing 18-22 g / L at 36-38°C for 46-50 h, the xylose utilization rate of this strain is above 85%. Preferably, when the said *Kluyveromyces martensii* has an initial OD of 0.18-0.22, the xylose utilization rate is above 85%. 620nm =0.2, after being cultured at 37°C for 48 hours in a medium containing 20 g / L xylose, the xylose utilization rate of this strain was over 85%.

[0079] The Kluyveromyces marxianus AMCC 31395 strain described in this application has the ability to utilize acetic acid. Preferably, the Kluyveromyces marxianus strain can utilize acetic acid at a concentration of 3.3-25 g / L under conditions of 36-38°C and pH = 5.8-6.2; more preferably, the Kluyveromyces marxianus strain can utilize acetic acid at a concentration of 3.3-25 g / L under conditions of 37°C and pH = 6.0.

[0080] The Kluyveromyces martensii AMCC 31395 described in this application exhibits high tolerance to inhibitors containing furfural and / or phenol. In some embodiments, the mixed inhibitor contains furfural and phenol.

[0081] In this application, tolerance refers to the growth of a strain in the presence of a certain substance. For example, tolerance to furfural refers to the growth of *Kluyveromyces martensii* in the presence of furfural. 。

[0082] In some embodiments of this application, the Kluyveromyces martensii yeast has a furfural tolerance concentration of 0.5-4.5 g / L; and / or

[0083] The Kluyveromyces martensii yeast is tolerant to phenol at concentrations below 2.5 g / L, preferably 0.2-2 g / L.

[0084] For example, the Kluyveromyces martensii yeast can tolerate furfural concentrations of 0.5 g / L, 1.0 g / L, 1.5 g / L, 2.0 g / L, 2.5 g / L, 3.0 g / L, 3.5 g / L, 4.0 g / L, and 4.5 g / L.

[0085] The tolerance concentration of phenol to the *Kluyveromyces martensii* can be 0.2 g / L, 0.4 g / L, 0.6 g / L, 0.8 g / L, 1.0 g / L, 1.2 g / L, 1.4 g / L, 1.6 g / L, 1.8 g / L, 2.0 g / L, etc.

[0086] The *Kluyveromyces martensii* strain of this application is tolerant to one or more substances selected from the group consisting of 5-hydroxymethylfurfural, catechol, eugenol, vanillin, and 4-hydroxybenzaldehyde. Preferably, it is tolerant to 5-hydroxymethylfurfural, catechol, eugenol, vanillin, and 4-hydroxybenzaldehyde simultaneously. More preferably, the tolerant concentrations of 5-hydroxymethylfurfural are 0.3-0.475 g / L, the tolerant concentrations of catechol are 0.03-0.0475 g / L, the tolerant concentrations of vanillin are 0.03-0.0475 g / L, and the tolerant concentrations of 4-hydroxybenzaldehyde are 0.03-0.0475 g / L.

[0087] The strain described in this application can grow using xylose and acetic acid and has high tolerance to furfural, phenol and mixed inhibitors. It can ferment low-value agricultural and forestry biomass containing inhibitors, converting it into microbial cells to obtain microbial protein, thereby alleviating the current shortage of feed resources in the aquaculture industry.

[0088] This application provides a bacterial preparation comprising the aforementioned Kluyveromyces martensii strain.

[0089] This application provides the application of the above-mentioned Kluyveromyces martensii strain or bacterial preparation in the high-value conversion of low-value biomass, preferably, the low-value biomass is straw hydrolysate.

[0090] It should be noted that the low-value biomass mentioned in this application refers to agricultural and forestry waste and / or agricultural and forestry product processing waste and their hydrolysis products. Among them, agricultural and forestry waste includes straw, firewood, bark or branches, and agricultural and forestry product processing waste includes corn cobs, rice husks, bagasse, nut shells and fruit peels, etc.

[0091] Straw hydrolysate refers to the hydrolysis products of straw, which can be obtained by treating straw using one or more methods, including acid hydrolysis, alkaline hydrolysis, and steam explosion.

[0092] This application provides the use of the above-mentioned Kluyveromyces martensii strain or cell preparation in the production of cell protein.

[0093] This application provides a method for preparing cell protein using the above-mentioned Kluyveromyces martensii strain or cell preparation, which includes fermenting the above-mentioned Kluyveromyces martensii strain or cell preparation with straw hydrolysate as a fermentation medium to obtain cell protein.

[0094] In some embodiments of this application, the straw hydrolysate includes one or more substances selected from the group consisting of xylose, glucose, fructose and acetic acid; preferably, the straw hydrolysate also contains furfural and / or phenol.

[0095] In some embodiments of this application, the concentration of xylose in the straw hydrolysate is 30-180 g / L, preferably 160-180 g / L.

[0096] In some embodiments of this application, the concentration of fructose in the straw hydrolysate is 10-80 g / L, preferably 60-80 g / L.

[0097] In some embodiments of this application, the concentration of glucose in the straw hydrolysate is 20-180 g / L, preferably 20-130 g / L, and more preferably 110-130 g / L.

[0098] In some embodiments of this application, the concentration of acetic acid in the straw hydrolysate is 2-20 g / L, preferably 9-11 g / L.

[0099] In some embodiments of this application, the concentration of furfural in the straw hydrolysate is 0.5-5 g / L, preferably 4-5 g / L.

[0100] In some embodiments of this application, the concentration of phenol in the straw hydrolysate is 0.2-3 g / L, preferably 2-3 g / L.

[0101] In some embodiments of this application, the fermentation culture medium further includes a nitrogen source in the above-described method for preparing bacterial protein. Preferably, the nitrogen source includes one or more substances selected from the group consisting of ammonia, ammonium sulfate, yeast extract, peptone, and urea. More preferably, it includes ammonia and ammonium sulfate.

[0102] In some embodiments of this application, in the above-described method for preparing bacterial protein, the fermentation medium further includes a phosphorus source, preferably the phosphorus source includes one or more substances selected from the group consisting of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and ammonium dihydrogen phosphate, more preferably containing ammonium dihydrogen phosphate.

[0103] In some embodiments of this application, the fermentation culture medium further includes growth factors in the above-described method for preparing bacterial protein. Preferably, the growth factors include one or more substances selected from the group consisting of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and biotin, and more preferably include vitamin B1 and vitamin B6.

[0104] In some embodiments of this application, the fermentation temperature in the above-described method for preparing bacterial protein is 30-38°C, and in some embodiments, the pH is maintained at 4-6.

[0105] In some embodiments of this application, the fermentation in the above-described method for preparing bacterial protein is high-density fermentation.

[0106] It should be noted that the high-density fermentation described in this application refers to a fermentation technology that is an improvement on traditional fermentation technology. Its core is to ensure that the microorganisms in the fermentation system have sufficient nutrients and that there are no growth inhibitors present or accumulated.

[0107] In some embodiments of this application, high-density fermentation is carried out using a continuous fed-batch fermentation method; wherein, continuous feeding includes continuously adding a carbon source, a nitrogen source and a phosphorus source to the culture medium, wherein the carbon source is straw hydrolysate, the nitrogen source includes ammonia water and ammonium sulfate, and the phosphorus source contains ammonium dihydrogen phosphate.

[0108] Specifically, this application provides a method for preparing cell protein using the above-mentioned Kluyveromyces martensii strain or cell preparation through high-density fermentation, which includes the following steps:

[0109] (1) Activation of strains: The above-mentioned Kluyveromyces martensii strain or bacterial preparation is inoculated into liquid activation medium for culture.

[0110] (2) Seed culture: The activated bacterial solution from step (1) is inoculated into a fermenter for continuous feeding and batch fermentation, wherein the carbon source, nitrogen source and phosphorus source are added in a continuous feeding manner;

[0111] (3) Fermentation tank culture: Add fermentation bottom water to the fermentation tank, and inoculate the seed culture solution from step (2) into the fermentation tank for continuous feeding and batch fermentation. The carbon source, nitrogen source and phosphorus source are added in a continuous feeding manner. The alcohol concentration of the culture solution is maintained at 0.5-1g / L and the dissolved oxygen is maintained at 10-20%.

[0112] In this application, there are no particular limitations on the liquid activation culture medium, as long as it can activate the strain. YPD liquid culture medium is preferred, and more preferably, the solid culture medium contains 5-15 parts by weight of yeast extract, 10-30 parts by weight of glucose, 10-30 parts by weight of peptone and 10-30 parts by weight of agar.

[0113] In some embodiments of this application, the yeast extract contains a total nitrogen content ≥10%, an amino nitrogen content ≥5%, an ash content ≤15%, a NaCl content ≤2%, and a moisture content ≤6%.

[0114] In some embodiments of this application, the free amino acid content in the yeast extract is 30-40 wt%, preferably 35-40 wt%. Specifically, in some embodiments, the yeast extract contains one or more of the following: 1-2 wt% aspartic acid, 2-3 wt% threonine, 1-2 wt% serine, 6-7 wt% glutamic acid, 1-2 wt% glycine, 4-5 wt% alanine, 0.1-0.5 wt% cysteine, 2-3 wt% valine, 0.5-1 wt% methionine, 2-3 wt% isoleucine, 3-4 wt% leucine, 0.5-1 wt% tyrosine, 1-2 wt% phenylalanine, 2-3 wt% lysine, 0.1-1 wt% histidine, 1.5-2.5 wt% arginine, and 0.5-1.0 wt% proline. In some embodiments, the yeast extract further contains 2-3 ppm of vitamin B1, 30-40 ppm of vitamin B2, 100-150 ppm of vitamin B5, 10-20 ppm of vitamin B6, 5-10 ppm of vitamin B7, 20-30 ppm of vitamin B9, 0.001-0.005 ppm of vitamin B12, 3000-4000 ppm of choline, 1000-2000 ppm of inositol, and 300-400 ppm of niacin, or two or more of these components. In some embodiments, each kg of the yeast extract contains 30000-40000 mg of potassium, 5000-6000 mg of sodium, 300-400 mg of calcium, 2000-3000 mg of magnesium, 50-100 mg of zinc, and 50-100 mg of iron, or two or more of these components.

[0115] There are no restrictions on the source of the aforementioned yeast extract powder; it can be commercially purchased, such as yeast extract powder FM888 purchased from Angel Yeast Co., Ltd.

[0116] In some embodiments of this application, the peptone contains a total nitrogen content ≥12.5%, an amino nitrogen content ≥2.5%, an ash content ≤15%, a NaCl content ≤5%, a moisture content ≤6%, and a peptone content ≥30%.

[0117] There are no restrictions on the source of the peptone mentioned above; it can be commercially purchased, such as peptone FP328 purchased from Angel Yeast Co., Ltd.

[0118] In this application, there are no particular restrictions on the carbon source in the seed culture, as long as it allows the strain to grow normally. Preferably, it is straw hydrolysate. More preferably, the total sugar content of the straw hydrolysate is 340-400 g / L, of which xylose content is 160-180 g / L, glucose content is 110-130 g / L, fructose content is 60-80 g / L, acetic acid content is 9-11 g / L, furfural content is 4-5 g / L and phenol content is 2-3 g / L.

[0119] In some embodiments of this application, the fermentation temperature in step (3) is 30-38°C, the pH is preferably maintained at 4-6, the fermentation time is more preferably 15-16h, the inoculum amount is more preferably 15-19g / L, and the amount of fermentation bottom water added is preferably 8-10L.

[0120] It should be noted that the above inoculation amount refers to the initial dry weight of the cell mass after inoculation and fermentation.

[0121] In some embodiments of this application, the above method further includes the step of adding growth factors to the fermentation bottom water, wherein the growth factors include vitamin B1 and vitamin B6, and more preferably, the amount of vitamin B1 added is 5-15 mg / L and the amount of vitamin B6 added is 1-3 mg / L based on the volume of the fermentation bottom water. For example, if the amount of fermentation bottom water added is 8-10 L, the amount of vitamin B1 added is 0.04-0.15 g, and the amount of vitamin B6 added is 0.008-0.03 g.

[0122] In some embodiments of this application, during the fermentation process described above, dissolved oxygen is associated with stirring and aeration, and the stirring speed is preferably 400-800 rpm.

[0123] In some embodiments of this application, the carbon source in step (3) is straw hydrolysate, wherein the total sugar content of the straw hydrolysate is 340-400 g / L, preferably 340-350 g / L; preferably, the straw hydrolysate includes xylose content of 160-180 g / L, glucose content of 110-130 g / L, fructose content of 60-80 g / L, acetic acid content of 9-11 g / L, furfural content of 4-5 g / L and phenol content of 2-3 g / L; more preferably, the feeding rate of the straw hydrolysate is 200-1050 mL / h.

[0124] In some embodiments of this application, the nitrogen source in step (3) contains ammonia and ammonium sulfate, wherein the concentration of ammonia is 150-170 g / L and the concentration of ammonium sulfate is 210-250 g / L; preferably, the feeding rate of the nitrogen source is 45-180 mL / h.

[0125] In some embodiments of this application, the phosphorus source in step (3) contains ammonium dihydrogen phosphate, wherein the concentration of ammonium dihydrogen phosphate is 110-130 g / L; preferably, the feeding rate of ammonium dihydrogen phosphate is 0-60 mL / h, more preferably 20-60 mL / h.

[0126] In some embodiments of this application, the above method further includes separation and drying steps after fermentation in step (3). Specifically, after fermentation, the cells are centrifuged, washed with sterile water, and the yeast milk collected by centrifugation after fermentation is then granulated by vacuum drum filtration and then dried in a fluidized bed to obtain cell protein.

[0127] This application also provides a bacterial protein prepared by the above method.

[0128] In some embodiments of this application, the protein content of the bacterial protein is greater than or equal to 58 wt%, and in some embodiments, the essential amino acids account for more than or equal to 40% of the total amino acids by mass.

[0129] In some embodiments of this application, the protein content in the bacterial protein can be 58-65 wt%, 58-63 wt%, 58-61 wt%, 58-59 wt%, 59-63 wt%, 59-61 wt%, 60-65 wt%, 60-63 wt%, 61-65 wt%, 61-63 wt%, 62-65 wt%, 62-64 wt%, or 63-65 wt%.

[0130] In some embodiments of the application, the essential amino acid may account for 40-45%, 41-45%, 42-45%, 43-45%, 44-45%, 41-44%, or 42-44% of the total amino acid mass.

[0131] It should be noted that essential amino acids are those that the human body or animals need but cannot synthesize on their own and must obtain through diet.

[0132] In some embodiments of this application, the essential amino acid includes one or more of the group consisting of threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine, and tryptophan. In some embodiments, the essential amino acid includes threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine, and tryptophan.

[0133] In some embodiments of this application, the bacterial protein contains one or more of the following substances: 2-3 wt% threonine, 2-3 wt% valine, 2-3 wt% isoleucine, 3-4 wt% leucine, 2-3 wt% phenylalanine, 3.5-4.5 wt% lysine, 0.5-1.5 wt% histidine, 0.5-1 wt% methionine, 1.5-2.5 wt% arginine, 4.5-5.5 wt% aspartic acid, 2-3 wt% serine, 6-7 wt% glutamic acid, 1.5-2.5 wt% proline, 2-3 wt% glycine, 4-5 wt% alanine, 0.5-1.5 wt% tyrosine, and 0.1-0.5 wt% cysteine.

[0134] In some embodiments, the branched-chain amino acid content in the bacterial protein is greater than or equal to 5 wt%.

[0135] In some embodiments of the application, the mass percentage of branched-chain amino acids in the bacterial protein can be 5-10%, 5-9%, 5-8%, 5-7%, 5-6%, 6-10%, 7-10%, 8-10%, or 9-10%.

[0136] It should be noted that, in this application, branched-chain amino acids refer to a class of amino acids with aliphatic side chains. The three most common branched-chain amino acids in proteins are leucine, valine, and isoleucine. In some embodiments, the branched-chain amino acids in the bacterial protein include one or more of the substances composed of leucine, valine, and isoleucine. In some embodiments, the branched-chain amino acids in the bacterial protein include leucine, valine, and isoleucine. Supplementing with branched-chain amino acids after exercise can improve athletic performance and delay fatigue.

[0137] In some embodiments of this application, the bacterial protein contains 20-30 wt% of flavor-enhancing amino acids. In some embodiments, the content of flavor-enhancing amino acids in the bacterial protein can be 20-28 wt%, 20-26 wt%, 20-24 wt%, 22-30 wt%, 22-28 wt%, or 22-26 wt%.

[0138] In some embodiments of this application, the flavor-enhancing amino acids include one or more of the group consisting of glutamic acid, aspartic acid, phenylalanine, alanine, glycine, and tyrosine. In some embodiments, the flavor-enhancing amino acids include glutamic acid, aspartic acid, phenylalanine, alanine, glycine, and tyrosine.

[0139] This application also provides the application of the above-mentioned microbial protein in the feed field, preferably in poultry feed, aquatic feed, pig feed, ruminant feed, or pet food, and more preferably as a protein substitute in the preparation of poultry feed, aquatic feed, pig feed, ruminant feed, or pet food. This application provides a feed containing the above-mentioned microbial protein. In some embodiments, the amount of microbial protein added accounts for 5-60% of the total weight of the feed. In some embodiments, the amount of microbial protein added as a percentage of the total weight of the feed can be 5-60%, 5-50%, 5-40%, 5-30%, 5-20%, 5-10%, 10-60%, 20-60%, 30-60%, 40-60%, or 50-60%.

[0140] Feed is generally defined as food used to feed animals. The microbial protein produced in this application can be used as an ingredient in various feeds. More specifically, although not limited thereto, it is anticipated that the microbial protein produced in this application can be used in poultry feed, aquatic feed, pig feed, ruminant feed, or pet food. Pet food is a product used to feed pets such as dogs, cats, birds, reptiles, rodents, etc.; these products may include grains (e.g., corn, wheat flour, oats, etc.), nutrients (e.g., vitamins), and seasonings (e.g., salt, chicken fat), etc. Ruminant feed is used to feed animals with ruminant digestion such as cattle and sheep; poultry feed is used to feed poultry such as chickens and ducks; pig feed is used to feed piglets, fattening pigs, etc. These feeds may include grains (corn, wheat bran, etc.), legumes (soybean meal), nutrients (e.g., amino acids, vitamins, minerals, choline chloride, etc.), and seasonings (e.g., sodium chloride and soybean oil), etc. Aquatic feed is a product used in aquaculture. These products can include grains (flour, corn starch, etc.), nutrients (such as amino acids, vitamins, minerals, choline chloride, etc.), and seasonings (such as soybean oil).

[0141] This application also provides a poultry feed, which comprises the following components by weight: 40-60 parts of the above-mentioned microbial protein, 500-550 parts of corn, 250-300 parts of soybean meal, 80-100 parts of wheat bran, 0-12 parts of dicalcium phosphate, 6-8 parts of lysine, 3-5 parts of methionine, 2-4 parts of sodium chloride, 2-2.5 parts of choline chloride, 12-16 parts of limestone powder, 2-3 parts of mineral premix, 0.2-0.5 parts of vitamin premix, and 25-30 parts of soybean oil;

[0142] This application also provides a ruminant feed, which comprises the following components by weight: 60-80 parts of the above-mentioned microbial protein, 600-650 parts of corn, 120-180 parts of soybean meal, 100-120 parts of wheat bran, 12-17 parts of dicalcium phosphate, 3-7 parts of sodium chloride, 10-15 parts of limestone powder, 8-12 parts of mineral premix and 1-3 parts of vitamin premix;

[0143] This application also provides a pig feed, which comprises the following components by weight: 60-80 parts of the above-mentioned microbial protein, 580-620 parts of corn, 200-250 parts of soybean meal, 30-70 parts of wheat bran, 10-30 parts of corn starch, 4-6 parts of dicalcium phosphate, 1-3 parts of calcium carbonate, 7-10 parts of lysine, 2-4 parts of methionine, 4-6 parts of sodium chloride, 1-3 parts of choline chloride, 3-5 parts of mineral premix, and 0.1-0.5 parts of vitamin premix;

[0144] This application also provides an aquatic feed, which comprises the following components by weight: 280-320 parts of the above-mentioned microbial protein, 230-270 parts of flour, 150-200 parts of casein, 80-120 parts of corn starch, 40-60 parts of cellulose, 10-30 parts of sodium carboxymethyl cellulose, 10-30 parts of lysine, 10-30 parts of calcium dihydrogen phosphate, 2-4 parts of choline chloride, 3-7 parts of mineral premix, 0.5-2 parts of vitamin premix, 40-60 parts of soybean oil, and 0.3-0.7 parts of ethoxyquinoline;

[0145] This application also provides a pet food comprising the following components in parts by weight: 550-600 parts of the above-mentioned microbial protein, 100-150 parts of corn, 80-120 parts of wheat flour, 150-200 parts of oats, 5-10 parts of sodium chloride, 50-70 parts of compound vitamins and 30-50 parts of chicken fat;

[0146] The complex vitamin contains, by weight percentage, 10-20% vitamin A, 10-20% vitamin B1, 10-20% vitamin D2, 10-20% vitamin D3 and 30-40% vitamin E.

[0147] In some embodiments of this application, each kilogram of the above-mentioned mineral premix contains 80-120 mg of ferrous sulfate, 5-10 mg of copper sulfate, 80-120 mg of zinc sulfate, 100-150 mg of manganese sulfate, 0.5-1 mg of potassium iodide, and 0.1-0.5 mg of sodium selenite.

[0148] In some embodiments of this application, each kilogram of the vitamin premix contains 8000-12000 IU of vitamin A, 800-1200 IU of vitamin D3, 10-30 IU of vitamin E, 0.2-0.8 mg of vitamin K3, 1-3 mg of vitamin B1, 5-10 mg of vitamin B2, 8-12 mg of pantothenic acid, 30-40 mg of niacin, 2-5 mg of vitamin B6, 0.01-0.1 mg of biotin, 0.3-0.7 mg of folic acid, and 0.01-0.05 mg of vitamin B12.

[0149] To better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments. Unless otherwise stated, all raw materials / reagents / instruments used in the embodiments of the present invention are conventional commercially available products. The manufacturers of the raw materials and equipment used in this embodiment, as well as the equipment and analytical methods used for product analysis, are described below. Unless otherwise specified, the chemical substances mentioned are all chemically pure grades of conventional reagents. Table 1 is a table of reagent information used in the embodiments, and Table 2 is a table of instrument information used in the embodiments.

[0150] Table 1 Reagent Information Table

[0151] Table 2 Instrument Information Table

[0152] Example 1: Determination of the composition and content of wheat straw hydrolysate obtained after sulfuric acid and steam explosion treatment.

[0153] Wheat straw was pretreated with 1.5 wt% sulfuric acid and then subjected to steam explosion treatment at a steam pressure of 1.5 MPa and a steam temperature of 150 °C to obtain wheat straw hydrolysate. Since the hydrolysate still contained a large amount of dry matter, the supernatant was collected by centrifugation at 8000 × g (equivalent to 9100 rpm) for 3 min. After membrane filtration, the carbon source components and inhibitor content in the supernatant were determined by high-performance liquid chromatography (HPLC). The results are shown in Table 3.

[0154] Table 3 Composition of wheat straw hydrolysate

[0155] The carbon source was determined to be mainly composed of xylose, glucose, and fructose, with xylose having the highest proportion. The main inhibitors were acetic acid, furfural, and phenol. However, because the wheat straw hydrolysate is black, its use in fermentation culture makes it impossible to determine the growth performance indicators of the strain. Furthermore, the wheat straw hydrolysate contains a large number of impurities, which cannot be completely removed by centrifugation alone in the laboratory stage, thus affecting the determination of cell and cell protein indicators. Therefore, to facilitate the evaluation of the growth performance of the strain, the characteristics of the strain and shake-flask fermentation were conducted using an aqueous solution containing the above-mentioned components to simulate the straw hydrolysate. Additionally, because the pH of the wheat straw hydrolysate after sulfuric acid pretreatment and steam explosion treatment is 4.6, which is lower than the pKa value of acetic acid (4.8), acetic acid exists in molecular form and can diffuse freely across the cell membrane into the cell, disrupting the intracellular environment and affecting normal intracellular metabolism. Therefore, in subsequent experiments related to the straw hydrolysate culture medium, the pH needs to be adjusted to ensure cell viability.

[0156] Example 2: Isolation and Identification of Strains

[0157] 1. Preparation of straw hydrolysate solid culture medium:

[0158] Weigh 10g of yeast extract FM888, 20g of peptone FP328, 34g of glucose, 20g of fructose, 19g of xylose, 1.5g of furfural, and 20g of agar, add them to 1000mL of water, sterilize at 115℃ for 20min, then add 3.3g of acetic acid and 0.8g of phenol, adjust the pH to 6.0 with sterile NaOH solution, pour the culture medium into petri dishes and cool to obtain straw hydrolysate solid culture medium.

[0159] 2. Preparation of YPD solid culture medium:

[0160] Weigh 10g of yeast extract FM888, 20g of glucose, 20g of peptone FP328 and 20g of agar, add them to 1000mL, sterilize at 115℃ for 20min, pour the culture medium into a petri dish and cool to obtain YPD solid culture medium.

[0161] 3. Screening, isolation, and identification of bacterial strains

[0162] Yogurt collected from herders in Zhalute Banner, Tongliao City, Inner Mongolia Autonomous Region, China, was dissolved in sterile water and mixed thoroughly. The bacterial suspension was then serially diluted 10 times to prepare 10... -5 10 -6 The bacterial suspension was spread onto the above-mentioned straw hydrolysate solid medium and incubated at 30℃ for 24-48 hours. Colony size was then observed, as shown in Figure 1. The strain with the largest colony diameter was selected, representing the superior strain capable of utilizing straw hydrolysate and tolerating inhibitors. After streaking the selected colonies twice onto the above-mentioned YPD solid medium plates, the colonies were washed with 30% glycerol, and 100 μL was spread onto YPD solid medium. After incubation at 30℃ for 24 hours, single colonies were obtained, as shown in Figure 2. The colonies of this strain had a cheese-like texture, were milky white in color, and had a smooth surface. Single colonies were prepared and observed under a microscope to examine their morphology, while also observing the characteristics of single colonies on the plates.

[0163] The microscopic morphology observed under an optical microscope is shown in Figure 3. It is elliptical in shape and measures 3.0 × 6.5 μm. Microscopic observation indicates that it is a budding reproductive process. The genome of this strain was extracted. Using ITS1 (5'-TCCGTAGGTGAACCTGCGG-3', SEQ ID NO:1) and ITS4 (5'-TCCTCCGCTTATTGATATGC-3', SEQ ID NO:2) as primers, the PCR program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 45 s, 72℃ extension for 90 s, 30 cycles, and a final extension at 72℃ for 10 min, to amplify the yeast ITS rDNA sequence. After 1% gel electrophoresis and sequencing, BLAST analysis was performed to compare the sequence with sequences in GenBank. The sequence similarity was greater than 99%, indicating that it belonged to the same species. Combined with morphological analysis and molecular identification, this strain was identified as *Kluyveromyces marxianus*, strain number AMCC 31395, where AMCC is the abbreviation for Angel Microbiological Culture Collection Center. ， Its ITS rDNA sequence is shown in Table 13 as SEQ ID NO:3. This strain was deposited at the China Center for Type Culture Collection on July 9, 2024, with accession number CCTCC No: M 20241527.

[0164] Example 3: Analysis of Optimal Growth Conditions

[0165] 1. Preparation of YPI (Yellow Inhibitor) Straw Hydrolysate Culture Medium

[0166] Weigh 10g of yeast extract FM888, 20g of peptone FP328, 34g of glucose, 20g of fructose, 45.7g of xylose and 1.5g of furfural, add them to 1000mL of water, sterilize at 115℃ for 20min, then add 3.3g of acetic acid and 0.8g of phenol, and adjust the pH with sterile NaOH solution.

[0167] 2. Optimal temperature and pH analysis

[0168] Kluyveromyces martensii AMCC 31395, preserved in glycerol, was inoculated at a rate of 2 v / v% into test tubes containing 5 mL of YPD liquid medium at pH 6.5. The cells were cultured at 30°C and 180 rpm for 20 h. After centrifugation at 8000 × g for 3 min, the cells were collected and resuspended in an appropriate volume of double-deionized water (ddH2O). The cells were then inoculated at a rate of 2 v / v% into 100-well plates containing 300 μL of YPI liquid medium at pH 6.5. Bioscreen analysis was performed using the following parameters: temperature 30°C, time 24 h, wavelength 600 nm. Data were measured every 30 min, and the absorbance at 600 nm over 24 h was recorded as the OD value. 600nm To represent the growth process of the strain, the specific growth rate is calculated using the following formula:

[0169] OD corresponding to OD1—t1 600nm value;

[0170] OD corresponding to OD2—t2 600nm value;

[0171] t1: The start time of the logarithmic growth phase;

[0172] t2: End time of logarithmic growth phase;

[0173] The experimental procedures for the growth of the strain at incubation temperatures of 37℃ and 42℃ were the same as above, the difference being that the incubation temperatures were adjusted to 37℃ and 42℃ respectively. The results are shown in Table 4.

[0174] The experimental procedures for the growth of the strains under different pH conditions were the same as above, except that 300 μL of YPD liquid medium with pH 6.5 was used instead of YPI liquid medium with different pH values ​​(pH = 4.0, 4.8, 5.0, 5.5, 6.0). The experiments were conducted at the optimum temperature of 37℃, and the results are shown in Table 5.

[0175] Table 4. Highest OD of *Kluyveromyces martensii* at different temperatures 600nm and maximum specific growth rate

[0176] Table 5. Highest OD of Kluyveromyces martensii at different pH values 600nm and maximum specific growth rate

[0177] Finding the optimal growth conditions for the strain is beneficial for the efficient conversion of multiple carbon sources in straw hydrolysate. As shown in Tables 4 and 5 and Figures 4 and 5, the optimal biomass (OD) of strain AMCC 31395 is achieved at 37℃ and pH 6.5. 600nmThe maximum specific growth rate was higher than that at 30℃ and 42℃, and the time to reach the stationary phase was shortened by 4 hours compared to 30℃. Under pH 6.0 conditions, the biomass and maximum specific growth rate of the strain were higher than those at other pH values. Therefore, 37℃ and pH 6.0 were determined to be the optimal growth conditions for strain AMCC 31395.

[0178] Example 4: Substrate utilization characteristics analysis

[0179] 1. Preparation of YPD liquid culture medium

[0180] Weigh 10g of yeast extract FM888, 20g of peptone FP328 and 20g of glucose, add them to 1000mL of water, sterilize at 115℃ for 20min, and adjust to the selected pH with sterile NaOH solution for later use.

[0181] 2. Preparation of carbon source culture medium containing xylose

[0182] Weigh 10g of yeast extract FM888, 20g of peptone FP328 and 20g of xylose, add them to 1000mL of water, sterilize at 115℃ for 20min, and set aside.

[0183] 3. Preparation of acetic acid-containing carbon source culture medium

[0184] Weigh 10g of yeast extract FM888 and 20g of peptone FP328, add them to 1000mL of water, sterilize at 115℃ for 20min, then add 3.3g, 4.0g, 7.0g, 10g, 13g, 15g, 20g, 25g and 30g of acetic acid respectively, adjust the pH to 6.0 with sterile NaOH solution to obtain carbon source culture media containing different concentrations of acetic acid (calculated as acetate ions).

[0185] 4. Substrate utilization characteristics analysis

[0186] The glycerol-preserved *Kluyveromyces martensii* AMCC 31395 strain was inoculated at a rate of 2 v / v% into 5 mL of YPD liquid medium at pH 6.5 and cultured at 30°C and 180 rpm for 20 h. The cells were collected by centrifugation, resuspended in an appropriate volume of dd H2O, and then cultured according to the initial OD value. 620nm =0.2% inoculum was inoculated into Erlenmeyer flasks containing 100 mL of xylose-containing carbon source medium. The flasks were incubated at 37℃ and 180 rpm for 48 h on a shaker. Samples were taken at 4 h, 8 h, 12 h, 24 h, and 48 h. The uninoculated carbon source medium served as a negative control. The absorbance (OD) at 620 nm was measured using a microplate reader. 620nm The growth process of the strain was represented by the method of collecting fermentation supernatant, and the xylose consumption was determined by high performance liquid chromatography to calculate the xylose utilization rate. Xylose medium without inoculation was used as a control. The results are shown in Figure 6.

[0187] C1—Xylose content in the control culture medium;

[0188] C t —Xylose content in culture media at different fermentation times;

[0189] Fermentation experiments of Kluyveromyces martensii AMCC 31395 strain were conducted using carbon source culture media containing different concentrations of acetic acid, following the method described above. The results of the production process of the strain under different acetic acid concentrations are shown in Figure 7.

[0190] As shown in Figures 6 and 7, Kluyveromyces martensii AMCC 31395 can utilize xylose or acetic acid as a carbon source for growth. After 48 hours of fermentation, the OD... 620nm The xylose utilization rate reached 5.99, indicating that the strain utilized 85.2% of xylose. The strain could grow normally when the acetic acid concentration was 0-25 g / L. However, when the concentration was higher than 25 g / L, acetate entered the cell via transport proteins, inhibiting the cell activity of the strain. Therefore, Kluyveromyces macrocarpa AMCC 31395 can convert xylose and acetic acid (3.3-25 g / L) into cell volume.

[0191] Example 5: Tolerance analysis of a single inhibitor

[0192] 1. Prepare culture media containing furfural inhibitors at different concentrations:

[0193] Weigh 10g of yeast extract FM888, 20g of peptone FP328, 20g of glucose, and a certain amount of furfural, add them to 1000mL of water, and sterilize at 115℃ for 20min. The furfural concentration in the culture medium containing furfural inhibitor is 0, 0.5g / L, 1g / L, 1.5g / L, 2g / L, 2.5g / L, 3g / L, 3.5g / L, 4g / L, 4.5g / L, and 5g / L, respectively.

[0194] 2. Prepare culture media containing phenol inhibitors at different concentrations:

[0195] Weigh 10g of yeast extract FM888, 20g of peptone FP328 and 20g of glucose, add them to 1000mL of water, sterilize at 115℃ for 20min, and then add a certain amount of phenol. The concentration of phenol in the phenol-containing inhibitor culture medium is 0, 0.2g / L, 0.4g / L, 0.6g / L, 0.8g / L, 1g / L, 1.2g / L, 1.4g / L, 1.6g / L, 1.8g / L, 2g / L and 2.5g / L, respectively.

[0196] 3. Preparation of YPD liquid culture medium

[0197] Prepare according to the method in Example 4.

[0198] 4. Strain tolerance analysis

[0199] The glycerol-preserved *Kluyveromyces martensii* AMCC 31395 strain was inoculated at a 2 v / v inoculation rate into test tubes containing 5 mL of YPD liquid medium at pH 6.5. The culture was incubated at 30 °C and 180 rpm for 20 h. After centrifugation and resuspending, the culture was then inoculated at a 1 v / v inoculation rate into 100-well plates containing 300 μL of furfural inhibitor medium or phenol inhibitor medium. The absorbance was measured using a Bioscreen C instrument with the following parameters: temperature 37 °C, time 24 h, wavelength 600 nm. Data were measured every 30 min, and the absorbance at 600 nm over 24 h was recorded as the OD value. 600nm The growth process of the strain was represented by YPD liquid medium with a pH of 6.5 as a control. The tolerance of the strain to different inhibitor concentrations was calculated, and the formula for calculating tolerance is as follows:

[0200] The OD1-0h value of the strain in the medium containing the inhibitor was... 600nm value;

[0201] The OD2-24h value of the strain in the medium containing the inhibitor 600nm value;

[0202] The OD3-0h strain corresponds to the OD in YPD liquid medium. 600nm value;

[0203] OD4-24h strains in YPD liquid medium correspond to OD 600nm value;

[0204] The strain was cultured using the method described above, and the OD values ​​of the strain at different time points were observed under different inhibitors and concentrations. 600nm As shown in Figures 8 and 9, the obtained furfural tolerance and phenol tolerance are shown in Tables 6 and 7.

[0205] Table 6. Growth parameters of strain AMCC 31395 in culture medium containing furfural inhibitor.

[0206]

[0207] Table 7 Growth parameters of strain AMCC 31395 in phenol-containing inhibitor medium

[0208] As shown in Table 6, strain AMCC 31395 exhibits furfural tolerance, with a maximum tolerated concentration of 4.5 g / L (as OD).600nm (A growth rate exceeding 50% is considered growth), which is 4.5 times the furfural tolerance concentration of *Kluyveromyces martensii* in existing patent CN113512506A. When the furfural concentration in the culture medium is 0-0.5 g / L, the growth of the strain is basically the same as in the medium without inhibitors. When the concentration is increased to 2.0 g / L, the highest OD of the strain after 24 hours is... 600nm There was no significant reduction; furfural's inhibitory effect on the growth of the strain was manifested in the prolongation of the lag phase and the reduction of the growth rate.

[0209] Table 7 shows that the strain has good tolerance to phenol, with a maximum tolerated concentration of 2.0 g / L. At this concentration, the strain's tolerance remains at 90%. The effects of phenol on the strain are growth rate and biomass (maximum OD). 600nm The study investigated the triple inhibition of phenol and formic acid in lignocellulose hydrolysate, including the lag phase. Literature reports that phenol and formic acid are the most toxic of all inhibitors. Currently, no patents have studied the phenol tolerance of strains. Existing patent CN113512506A, concerning Kluyveromyces martensii YZN013, only evaluated the strain's tolerance to phenolic compounds such as catechol and eugenol, with the highest phenol concentration being 0.19 g / L. 。 Therefore, the strain AMCC 31395 of this application has excellent phenol tolerance and has good application prospects in the field of microbial feed protein.

[0210] Example 6: Tolerance analysis of multiple inhibitors

[0211] 1. Prepare a culture medium containing multiple inhibitors:

[0212] A liquid culture medium was prepared containing 0.67 wt% yeast nitrogen source basal (YNB), 2 wt% glucose, and a mixture of inhibitors. The inhibitor mixture consisted of 1.9 g / L acetic acid, 0.95 g / L furan derivatives (0.475 g / L each of furfural and 5-hydroxymethylfurfural), and 0.19 g / L phenolic compounds (0.0475 g / L each of catechol, eugenol, vanillin, and 4-hydroxybenzaldehyde).

[0213] 2. Preparation of YPD liquid culture medium

[0214] Prepare according to the method in Example 4.

[0215] 3. Assessment of strain tolerance to multiple inhibitors

[0216] The glycerol-preserved *Kluyveromyces martensii* strain AMCC 31395 was inoculated at a rate of 2 v / v into a test tube containing 5 mL of YPD liquid medium at pH 6.5. The culture was incubated at 30°C and 180 rpm for 20 h. After centrifugation and resuspending, the culture was inoculated into a conical flask containing 30 mL of multiple inhibitor medium at an initial OD of 0.3. The flask was then incubated in a shaker (37°C or 42°C, 250 rpm). The growth status of the strain was observed, and samples were taken at 4, 6, 8, 12, 16, 20, 24, 36, and 48 h to measure the OD. 600nm A blank control was prepared using uninoculated culture medium containing multiple inhibitors. The results are shown in Table 8 and Figure 10.

[0217] Table 8. Growth parameters of strain AMCC 31395 in media containing multiple inhibitors.

[0218]

[0219] As shown in Table 8 and Figure 10, strain AMCC 31395 can grow normally in a medium containing multiple inhibitors, and its lag phase is shorter at the optimal temperature of 37℃, resulting in a higher final OD. 600nm Higher, and at 42℃, due to the combined pressure of high temperature and inhibitors, the lag phase is prolonged, and the biomass (maximum OD) is lower. 600nm The tolerance of strain AMCC 31395 to multiple inhibitors decreased, but still showed an increase of over 70% compared to the strain YZN013 in Example 3 of the specification in patent CN113512506A, which is Kluyveromyces martensii YZN013. The tolerance assessment conditions for multiple inhibitors were the same as in Example 6 of this application. Therefore, the tolerance of AMCC 31395 to multiple inhibitors is beneficial for its accumulation of cell bodies and cell proteins in relevant low-value biomass products.

[0220] Example 7: Shake-flask fermentation experiment of simulated straw hydrolysate culture medium

[0221] 1. Preparation of YPD liquid culture medium

[0222] Weigh 10g of yeast extract FM888, 20g of glucose and 20g of peptone FP328, add them to 1000mL of water, sterilize at 115℃ for 20min, and adjust to the selected pH with sterile NaOH solution for later use.

[0223] 2. Preparation of YPI (Yellow Inhibitor) Straw Hydrolysate Culture Medium

[0224] Prepare according to the method in Example 3.

[0225] 3. Preparation of YPW (Yellow Powdered Water) medium containing carbon source from straw hydrolysate.

[0226] Weigh 10g of yeast extract FM888, 20g of peptone FP328, 34g of glucose, 20g of fructose and 45.7g of xylose, add them to 1000mL of water, sterilize at 115℃ for 20min, then add 3.3g of acetic acid, and adjust the pH to 6.0 with sterile NaOH solution to obtain the straw hydrolysate carbon source culture medium.

[0227] 4. Evaluation of the ability of the strain to produce cell mass and cell protein during shake-flask fermentation

[0228] The glycerol-preserved *Kluyveromyces martensii* AMCC 31395 strain was inoculated at a 2 v / v% inoculation rate into test tubes containing 5 mL of YPD liquid medium at pH 6.5. The culture was carried out at 30℃ and 180 rpm for 20 h. After centrifugation and resuspending, the culture was inoculated at a 1 v / v% inoculation rate into 1 L of straw hydrolysate medium (YPI) containing inhibitors and straw hydrolysate carbon source medium (YPW). After incubation at 37℃ in a shaker for 48 h, the yeast milk and fermentation supernatant were collected by centrifugation. Biomass, moisture, yeast protein, and total nitrogen (Kjeldahl method) were determined. Simultaneously, lactic acid and acetic acid contents were determined using high-performance liquid chromatography (HPLC) (BIO-RAD Aminex HPX-87H column, 300 mm × 7.8 mm, with 0.005 mol / L sulfuric acid as the mobile phase). The results are shown in Table 9. The formulas for calculating the carbon source or acetic acid balance (%) are as follows, and the results are shown in Figures 11 and 12.

[0229] Table 9. Fermentation Indicators of Straw Hydrolysate Culture Medium in Shake Flasks Note: Protein yield (g) = dry weight × protein content

[0230] Carbon source conversion rate (g dry weight / g sugar) = Dry weight × Fermentation volume / Carbon source consumption (calculated based on the initial carbon source and the remaining carbon source at the end of fermentation)

[0231] Relative carbon source conversion rate (%) = (YPI medium cell yield / YPW medium cell yield) × 100%

[0232] As shown in Table 9, Figure 11, and Figure 12, strain AMCC 31395 can simultaneously utilize various carbon sources to generate cell bodies and cell proteins in both culture media. Due to the presence of multiple high-concentration inhibitors, the fermentation start-up time in YPI medium is longer than that in YPW medium, but the dry weight after fermentation is basically the same as that in YPW medium. The carbon source conversion rate is 0.104 (g / g), reaching 94.3% of that in YPW medium. Therefore, this strain can still achieve efficient conversion of carbon sources in straw hydrolysate even in the presence of inhibitors. However, due to limiting factors such as insufficient dissolved oxygen and alcohol accumulation in shake flasks, further fed-batch fermentation experiments are needed to explore the true application potential of the strain.

[0233] Example 8: High-density fermentation culture using straw hydrolysate in a 45L fermenter.

[0234] 1. Preparation of straw hydrolysate (fermentation carbon source)

[0235] The straw hydrolysate obtained in Example 1 was evaporated and concentrated to about one-third of its original volume and collected for later use. The total sugar concentration in the concentrated straw hydrolysate was 349 g / L, including 164 g / L xylose, 117 g / L glucose, and 68 g / L fructose; the concentrations of acetic acid, furfural, and phenol were 10 g / L, 4.7 g / L, and 2.8 g / L, respectively.

[0236] 2. Preparation of nitrogen source for fermentation

[0237] The nitrogen source for fermentation is a mixed solution of ammonia and ammonium sulfate, with the concentration of ammonia being 159 g / L and the concentration of ammonium sulfate being 238 g / L.

[0238] 3. Preparation of phosphorus source for fermentation

[0239] The phosphorus source for fermentation was ammonium dihydrogen phosphate, with a concentration of 128 g / L.

[0240] 4. High-density fermentation culture of Kluyveromyces martensii AMCC 31395

[0241] 1) Strain activation:

[0242] The Kluyveromyces martensii AMCC 31395 preserved in glycerol tubes was inoculated at a rate of 0.5% into an Erlenmeyer flask containing 500 mL of YPD liquid medium. The pH was adjusted to 6.5, and the flask was incubated at 37°C for 24 hours. The YPD liquid medium was prepared as follows: 5 g of yeast extract FM888, 10 g of glucose, 10 g of peptone FP328, and 500 mL of water were weighed, sterilized at 115°C for 20 minutes, and then poured into plates for later use.

[0243] 2) Seed culture:

[0244] Add 9L of bottom water (containing 0.1g of vitamin B1) to a 45L fermenter, add 200mL of activated inoculum for fermentation culture, and culture temperature 37℃. Adjust the pH to 6.0 with soda ash. During yeast fermentation culture, the above-mentioned fermentation carbon source, fermentation nitrogen source and fermentation phosphorus source are added in a continuous feeding manner. The feeding rate of carbon source is 1-71mL per hour, the feeding rate of nitrogen source is 8-20mL per hour, and the feeding rate of phosphorus source is 8-20mL per hour. During fermentation, the stirring speed is maintained at 250-550rpm until the fermentation is completed in 24 hours.

[0245] 3) Fermentation tank cultivation

[0246] Add 10L of bottom water (containing 0.1g vitamin B1 and 0.02g vitamin B6) to a 45L fermenter. Then, inoculate the seed culture from step 2) into the fermenter. After inoculation, the dry weight of the cells should be 17.5g / L (dry weight is the weight of the yeast milk after centrifugation and dehydration). Start fermentation culture at 37℃, adjusting the pH to 6.0 with sodium carbonate. During yeast fermentation, the above-mentioned fermentation carbon source, nitrogen source, and phosphorus source are added using a continuous feed method to maintain the alcohol content in the fermenter at 0.5-1g / L and the dissolved oxygen at 10%-20%. The agitator speed is 400-800rpm, and dissolved oxygen is correlated with agitation and aeration rate until fermentation ends after 16 hours. The feed rates of the fermentation carbon source, nitrogen source, and phosphorus source are shown in Table 10.

[0247] Table 10 Feeding rates of fermentation carbon source, fermentation nitrogen source, and fermentation phosphorus source

[0248] 4) Separation and drying

[0249] After fermentation, the bacterial cells in the fermenter were collected by centrifugation, and the biomass yield and protein yield were measured. The carbon source utilization efficiency and nitrogen source conversion efficiency were calculated, and the results are shown in Table 11. Subsequently, the bacterial cells were granulated by vacuum drum filtration and then dried in a fluidized bed to obtain bacterial protein.

[0250] Table 11 Results of fermentation dry weight, total protein content and conversion rate Note: Dry weight is the result after deducting the dry weight of the seed culture medium added at the time of inoculation;

[0251] Total protein content (g / L) = Dry weight × Protein content;

[0252] Carbon source conversion rate (g dry weight / g sugar) = dry weight × tank volume / carbon source consumption;

[0253] Nitrogen source conversion rate (g nitrogen / g nitrogen) = Total protein content × Tank volume / Nitrogen source consumption;

[0254] Biomass conversion efficiency (g / L / h) = dry weight / fermentation time;

[0255] Protein conversion efficiency (g / L / h) = Total protein content / Fermentation time.

[0256] The Kluyveromyces macrocarpa AMCC 31395 of this application can utilize straw hydrolysate as a carbon source for fermentation. After high-density fermentation for 16 hours in a 45L fermenter using a continuously fed culture method, the cell dry weight was 84.97 g / L, the total protein content was 51.55 g / L, the cell protein content reached 60.67%, and it can utilize inorganic nitrogen source. The carbon source conversion rate was 0.373 g dry weight / g total sugar, the nitrogen source conversion rate reached 0.77 g / g, the biomass conversion efficiency was 5.31 g / L / h, and the protein conversion efficiency was 3.22 g / L / h. The Kluyveromyces martensii NS127 (accession number CGMCC No. 30034) disclosed in Example 6 of patent CN118126853A, after high-density fermentation with molasses for 30 hours, achieved a cell dry weight of 87.2 g / L, but a total protein yield of only 30.01 g / L, with a biomass conversion efficiency of 2.81 g / L / h and a protein conversion efficiency of 1.00 g / L / h. Compared with this disclosed patent, the cell dry weight of this application is similar, and the total protein content reaches 172% of that strain's level. The biomass conversion efficiency and protein conversion efficiency are increased by 82% and 222%, respectively. This indicates that the strain protected in this application can achieve high-density cultivation and efficient protein conversion under straw hydrolysate substrate, and has a higher protein content.

[0257] Example 9: Amino acid content in biomass

[0258] The quality of a protein source also depends on the abundance and variety of its amino acids. The amino acid composition of the bacterial protein obtained in Example 8 is shown in Table 12.

[0259] Table 12 Amino acid composition and content of AMCC 31395 bacterial protein ("*" indicates essential amino acids)

[0260] Table 12 shows that the Kluyveromyces martensii AMCC 31395 cell protein contains nine essential amino acids, including threonine, valine, isoleucine, leucine, phenylalanine, lysine, histidine, methionine, and tryptophan, accounting for 42.3% of the total amino acids by weight. The weight ratio of essential to non-essential amino acids is 73.23%, both close to the ideal protein pattern specified by FAO / WHO standards. Furthermore, the branched-chain amino acids (leucine, valine, and isoleucine) in the cell protein account for 7.99% by weight, higher than the branched-chain amino acid content (5.81%) in the cell protein obtained using the Kluyveromyces martensii NS127 strain disclosed in patent CN118126853. Branched-chain amino acids can enhance the immune function of animals and are of great significance for ensuring animal health.

[0261] These data demonstrate that the microbial protein of this application is an edible protein source with high added value. Furthermore, the biomass of the microbial protein of this application provides sufficient protein and a complete amino acid profile, meeting the nutritional requirements of dog and cat foods as stipulated by AAFCO. In addition, the microbial protein of this application contains 22 wt% of flavor-enhancing amino acids (glutamic acid, aspartic acid, phenylalanine, alanine, glycine, and tyrosine), which can increase animal feed intake, thereby improving animal growth performance.

[0262] Application Example 1: Yeast Protein Broiler Feed

[0263] 50g of the bacterial protein raw material obtained in Example 8, 530g of corn, 260g of soybean meal, and 90g of wheat bran were crushed, and then 10g of dicalcium phosphate, 7.5g of lysine, 3.5g of methionine, 3g of sodium chloride, 2.2g of choline chloride, 14g of limestone powder, 2.5g of mineral premix, and 0.3g of vitamin premix were added and transported to a mixing hopper for thorough mixing. 27g of soybean oil was added and mixed evenly, and then broiler feed was obtained by pelleting.

[0264] Each kilogram of the mineral premix contains: 100 mg ferrous sulfate, 8.0 mg copper sulfate, 100 mg zinc sulfate, 120 mg manganese sulfate, 0.7 mg potassium iodide, and 0.3 mg sodium selenite. Each kilogram of the vitamin premix contains: 10,000 IU vitamin A, 1,000 IU vitamin D3, 20 IU vitamin E, 0.5 mg vitamin K3, 2.0 mg vitamin B1, 8.0 mg vitamin B2, 10.0 mg pantothenic acid, 35.0 mg niacin, 3.5 mg vitamin B6, 0.05 mg biotin, 0.55 mg folic acid, and 0.01 mg vitamin B12.

[0265] Application Example 2: Yeast Protein Calf Concentrate

[0266] 70g of the bacterial protein prepared in Example 8, 620g of corn, 150g of ordinary soybean meal, 115g of wheat bran were crushed, and then 15g of dicalcium phosphate, 13g of limestone powder, 10g of mineral premix (same as in Application Example 1), 5g of sodium chloride, and 2g of vitamin premix (same as in Application Example 1) were added to a mixer and mixed evenly. After pelleting, calf concentrate feed was obtained.

[0267] Application Example 3: Yeast Protein Piglet Feed

[0268] 70g of the bacterial protein raw material obtained in Example 8, 600g of corn, 230g of soybean meal, 50g of wheat bran and 20g of corn starch were pulverized, and then 5g of dicalcium phosphate, 2g of calcium carbonate, 5g of sodium chloride, 4g of mineral premix (same as in Application Example 1), 0.3g of vitamin premix (same as in Application Example 1), 2g of choline chloride, 8.5g of lysine and 3.2g of methionine were added to a mixer and mixed evenly. After granulation, piglet feed was obtained.

[0269] Application Example 4: Yeast Protein Omnivorous Fish Feed

[0270] 300g of the bacterial protein raw material prepared in Example 8, 250g of flour, 180g of casein, 100g of corn starch, 50g of cellulose, 50g of soybean oil, 20g of calcium dihydrogen phosphate, 20g of sodium carboxymethyl cellulose, 20g of lysine, 3.5g of choline chloride, 5g of mineral premix (same as in Application Example 1), 1g of vitamin premix (same as in Application Example 1), and 0.5g of ethoxyquin were mixed evenly in a mixer. The mixture was then processed through a granulator to obtain omnivorous fish feed.

[0271] Application Example 5: Yeast Protein Pet Food

[0272] 580g of the microbial protein raw material prepared in Example 8, 120g of corn flour, 100g of wheat flour, 180g of oats, 220g of water, 8g of edible salt, 60g of compound vitamins (including 10g of vitamin A, 10g of vitamin B1, 10g of vitamin D2, 10g of vitamin D3 and 20g of vitamin E), and 40g of chicken oil were transported to a mixing silo. The mixture was processed through a pellet mill to obtain pellet feed, and then dried to control the moisture content to be below 10% to obtain yeast protein pet food.

[0273] In summary, the Kluyveromyces marxianus AMCC 31395 strain of this application exhibits high tolerance to furfural, phenol, and mixed inhibitors. The carbon source conversion rate reaches 94.3% in the presence of multiple inhibitors, which is the same as that without inhibitors. It can effectively utilize carbon sources to obtain cell bodies and accumulate cell proteins in straw hydrolysate containing multiple inhibitors.

[0274] The Kluyveromyces marxianus AMCC 31395 strain of this application can obtain cell protein through high-density fermentation using straw hydrolysate based on a fed-batch method. The protein content after fermentation reaches 60.67%, and the protein yield is 51.55 g / L. Moreover, this strain has a significant advantage in terms of time conversion efficiency even when using low-value straw hydrolysate, and has considerable potential for industrial production.

[0275] The cell protein prepared by this application using Kluyveromyces marxianus AMCC 31395 strain has a complete amino acid profile, with essential amino acids accounting for 42.3% of the total amino acid content; and has a high content of flavor substances, such as glutamic acid, which can increase animal feed intake and has the potential to prepare yeast protein animal feed and pet food.

[0276] Table 13 Sequence List

[0277] The above description is merely a preferred embodiment of this application and does not constitute any limitation on this application. All modifications, equivalent substitutions, and improvements made within the spirit and principles of this application shall be included within the protection scope of this application.

Claims

1. A strain of Kluyveromyces martensii, in which, The strain of Kluyveromyces marxianus is AMCC 31395, which is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20241527.

2. The Kluyveromyces martensii strain according to claim 1, characterized in that, It has high xylose utilization capacity, wherein, when the Kluyveromyces martensii uses an initial OD... 620nm =0.18-0.22, after being cultured at 36-38℃ for 46-50h in a xylose medium containing 18-22g / L, the xylose utilization rate of this strain is over 85%.

3. The *Kluyveromyces martensii* strain according to claim 1 or 2, characterized in that, It has the ability to utilize acetic acid; preferably, the concentration of acetic acid that the Kluyveromyces martensii strain can utilize under the conditions of 36-38℃ and pH=5.8-6.2 is 3.3-25g / L.

4. The *Kluyveromyces martensii* strain according to any one of claims 1-3, characterized in that, The Kluyveromyces macrocarpa strain is tolerant to furfural and / or phenol, preferably to furfural and phenol; Preferably, the Kluyveromyces martensii strain tolerates furfural at concentrations of 0.5-4.5 g / L; and / or The Kluyveromyces martensii strain tolerates phenol at concentrations of 0.2-2.0 g / L.

5. The *Kluyveromyces martensii* strain according to any one of claims 1-4, characterized in that, The *Kluyveromyces martensii* strain is tolerant to one or more substances selected from the group consisting of 5-hydroxymethylfurfural, catechol, eugenol, vanillin, and 4-hydroxybenzaldehyde. Preferably, it is tolerant to 5-hydroxymethylfurfural, catechol, eugenol, vanillin, and 4-hydroxybenzaldehyde simultaneously. More preferably, the tolerant concentration of 5-hydroxymethylfurfural is 0.3-0.475 g / L, the tolerant concentration of catechol is 0.03-0.0475 g / L, the tolerant concentration of vanillin is 0.03-0.0475 g / L, and the tolerant concentration of 4-hydroxybenzaldehyde is 0.03-0.0475 g / L.

6. A bacterial preparation, characterized in that, It contains the *Kluyveromyces martensii* strain according to any one of claims 1-5.

7. The application of the *Kluyveromyces martensii* strain according to any one of claims 1-5 or the bacterial preparation according to claim 6 in the high-value conversion of low-value biomass, preferably, the low-value biomass is straw hydrolysate.

8. The use of the *Kluyveromyces martensii* strain according to any one of claims 1-5 or the cell preparation according to claim 6 in the preparation of cell protein.

9. A method for preparing cell protein using the *Kluyveromyces martensii* strain according to any one of claims 1-5 or the cell preparation according to claim 6, characterized in that, It includes fermenting and culturing the *Kluyveromyces martensii* strain according to any one of claims 1-5 or the inoculum agent according to claim 6 using straw hydrolysate as a carbon source.

10. The method according to claim 9, characterized in that, The straw hydrolysate includes one or more substances selected from the group consisting of xylose, glucose, fructose and acetic acid; preferably, the straw hydrolysate also contains furfural and / or phenol. Preferably, the concentration of xylose in the straw hydrolysate is 30-180 g / L; and / or, The concentration of fructose is 10-80 g / L, and / or, The glucose concentration is 20-180 g / L, and / or, The concentration of acetic acid is 2-20 g / L, and / or, The concentration of furfural is 0.5-5 g / L, and / or, The concentration of phenol is 0.2-3 g / L.

11. The method as described in claim 9 or 10, characterized in that, The fermentation medium also includes a nitrogen source, preferably one or more substances selected from the group consisting of ammonia, ammonium sulfate, yeast extract, peptone and urea, more preferably ammonia and ammonium sulfate.

12. The method according to any one of claims 9-11, characterized in that, The fermentation medium also includes a phosphorus source, preferably one or more substances selected from the group consisting of dipotassium hydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate and ammonium dihydrogen phosphate, more preferably ammonium dihydrogen phosphate.

13. The method according to any one of claims 9-12, characterized in that, The fermentation medium further includes growth factors, preferably including one or more substances selected from the group consisting of vitamin B1, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and biotin, more preferably vitamin B1 and vitamin B6.

14. The method according to any one of claims 9-13, characterized in that, The fermentation temperature is 30-38℃, and the preferred fermentation pH is 4-6.

15. The method according to any one of claims 9-14, characterized in that, The fermentation is a high-density fermentation; Preferably, the high-density fermentation is carried out using a continuous feeding batch fermentation method.

16. The method according to claim 15, characterized in that, The continuous feeding includes continuously adding a carbon source, a nitrogen source, and a phosphorus source to the fermentation culture medium, wherein the carbon source is straw hydrolysate, the nitrogen source includes ammonia and ammonium sulfate, and the phosphorus source contains ammonium dihydrogen phosphate.

17. The method according to claim 15 or 16, characterized in that, It includes the following steps: (1) Activation of strain: The Kluyveromyces martensii strain described in claims 1-5 or the bacterial preparation described in claim 6 is inoculated into a liquid activation medium for culture. (2) Seed culture: The activated bacterial solution from step (1) is inoculated into a fermenter for continuous feeding and batch fermentation to obtain seed culture solution, wherein carbon source, nitrogen source and phosphorus source are added in a continuous feeding manner; (3) Fermentation tank culture: The seed culture solution from step (2) is inoculated into the fermentation tank for continuous feeding and batch fermentation. The carbon source, nitrogen source and phosphorus source are added in a continuous feeding manner. The alcohol concentration of the culture solution is maintained at 0.5-1g / L and the dissolved oxygen is maintained at 10-20%.

18. The method according to claim 17, characterized in that, Step (3) The fermentation temperature is 30-38℃, the pH is preferably maintained at 4-6, the fermentation time is more preferably 15-16h, and the inoculum amount is even more preferably 15-19g / L.

19. The method according to claim 17 or 18, characterized in that, Before inoculating the seed culture solution from step (2) into the fermenter, step (3) includes adding fermentation bottom water to the fermenter, preferably also adding growth factors to the fermentation bottom water, wherein the growth factors include vitamin B1 and vitamin B6, more preferably, the amount of vitamin B1 added is 5-15 mg / L and the amount of vitamin B6 added is 1-3 mg / L based on the volume of fermentation bottom water.

20. The method according to any one of claims 17-19, characterized in that, The carbon source is straw hydrolysate, wherein the total sugar content of the straw hydrolysate is 340-400 g / L, preferably 340-350 g / L; preferably, the straw hydrolysate contains 160-180 g / L xylose, 110-130 g / L glucose, 60-80 g / L fructose, 9-11 g / L acetic acid, 4-5 g / L furfural and 2-3 g / L phenol; more preferably, the feeding rate of the straw hydrolysate is 200-1050 mL / h. And / or, the nitrogen source contains ammonia and ammonium sulfate, wherein the concentration of ammonia is 150-170 g / L and the concentration of ammonium sulfate is 210-250 g / L; preferably, the feeding rate of the nitrogen source is 45-180 mL / h; And / or, the phosphorus source contains ammonium dihydrogen phosphate, wherein the concentration of ammonium dihydrogen phosphate is 110-130 g / L; preferably, the feeding rate of ammonium dihydrogen phosphate is 0-60 mL / h, more preferably 20-60 mL / h.

21. The method according to any one of claims 17-20, characterized in that, The method further includes a step of centrifuging the fermentation broth obtained after fermentation in step (3) to obtain yeast milk; preferably, it also includes a step of granulating the yeast milk after vacuum drum filtration, and more preferably, it also includes a step of fluidized bed drying after granulation.

22. A bacterial protein, characterized in that, It includes being prepared by the method described in any one of claims 9-21.

23. The bacterial protein according to claim 22, characterized in that, The bacterial protein contains a protein content of 58 wt% or more, preferably, the essential amino acids in the bacterial protein account for 40% or more of the total amino acids by mass, and more preferably, the branched-chain amino acid content in the bacterial protein is 5 wt% or more.

24. The use of the microbial protein of claim 22 or 23 in the field of feed, preferably in poultry feed, aquatic feed, pig feed, ruminant feed or pet food, more preferably as a substitute protein in the preparation of poultry feed, aquatic feed, pig feed, ruminant feed or pet food.

25. A feed, characterized in that, It contains the microbial protein as described in claim 22 or 23, preferably the amount of microbial protein added accounts for 5-58% of the total weight of the feed.

26. The feed according to claim 25, wherein, The feed is one of poultry feed, aquatic feed, pig feed, ruminant feed, or pet food.

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