Kluyveromyces marxianus and application of Kluyveromyces marxianus in production of single-cell protein by taking straw as raw material

Abstract

The invention discloses kluyveromyces marxianus and application of the kluyveromyces marxianus in production of single-cell protein by taking straw as a raw material. The Kluyveromyces marxianus is a ZH-2 bacterial strain which is CGMCC (China General Microbiological Culture Collection Center) No.32295 The strain is obtained from a wild strain in a laboratory adaptive evolution mode, wheat straw and an inorganic nitrogen source can be used as raw materials for efficiently producing single-cell protein, compared with a parent strain, the utilization speed and the utilization rate of biomass, glucose and xylose are remarkably increased, the yield of ZH-2 SCP can reach 0.047 g.g <-1 >, and the yield of ZH-2 SCP can reach 0.045 g.g <-1 >. And the essential amino acid mode basically accords with adult and infant amino acid modes, and can be used as high-quality protein nutrition supply. The Kluyveromyces marxianus ZH-2 strain capable of obtaining single-cell protein by fermenting wheat straw as a raw material is provided from the perspective of utilizing cheap waste resources, so that the production cost of SCP is greatly reduced, and the problem of protein resource shortage is effectively solved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a strain of Kluyveromyces martensii and its application in the production of single-cell protein from straw. Background Technology

[0002] It is estimated that the world population will reach 10 billion by 2050. Limited by land and climate, traditional livestock protein supply methods can no longer meet the ever-increasing protein demand, urgently requiring the search for new alternative proteins. Single-cell protein (SCP), also known as microbial protein, has a protein content of 40%–80%, containing essential amino acids, vitamins, phospholipids, and other bioactive substances, with high bioavailability. Commonly used microorganisms that produce single-cell protein include bacteria, fungi, and microalgae. Compared to bacteria, yeast has characteristics such as large cell volume, low flocculation ability with nucleic acids, and easy separation. Its application in food and feed can be traced back to the 1920s. With continuous technological advancements, in recent years, yeast single-cell protein has been widely used as a novel food ingredient in food flavoring, nutritional supplements, and feed ingredients.

[0003] Approximately 75% of the production cost of yeast SCPs comes from the carbon and nitrogen sources required for cell proliferation. Therefore, using inexpensive carbon and nitrogen sources, such as carbon-containing waste and inorganic ammonia compounds, can significantly reduce the production cost of yeast SCPs. Various industrial wastes, by-products, as well as household garbage, fruit scraps, and food residues can all be used for fermentation to produce yeast SCPs. Gervasia et al. used food residues to cultivate *Saccharomyces cerevisiae* to obtain SCPs, while Tropea et al. successfully obtained *Saccharomyces cerevisiae* SCPs using lemon peels. Lignocellulose is currently the most abundant and inexpensive renewable resource and can also be used as a carbon source for yeast growth. However, lignocellulose cannot be directly utilized by yeast; therefore, pretreatment and enzymatic hydrolysis of lignocellulose are necessary before SCP production to hydrolyze cellulose and hemicellulose into monosaccharides. Zhang et al. studied the pretreatment and saccharification of wheat straw to cultivate the oily yeast *Hylocereus erinaceus* (*Hylocereus erinaceus*). Trichosporon cutaneum Under MP11 conditions, the SCP yield of lignocellulose reached 0.15 g / g. Song et al. used straw to cultivate genetically engineered Yersinia lipolyticis (Yersinia lipolyticis). Yarrowia lipolyticaCellulose in straw is degraded and converted into yeast SCPs, increasing the protein content of wheat straw and corn straw to 16.23% and 14.75%, respectively. During straw treatment, NaOH pretreatment can improve SCP yield, and various enzymes can hydrolyze cellulose and hemicellulose into monosaccharides. However, since glucosidase and xylosidase are rate-limiting enzymes, the hydrolysate from straw contains a large amount of oligosaccharides. Oligosaccharides extracted from rice straw can alleviate fermentation stress and increase ethanol production in Saccharomyces cerevisiae. Oligosaccharides in straw hydrolysate can increase the yield of Saccharomyces cerevisiae SCPs. Therefore, straw pretreatment can provide usable carbon sources for yeast growth, including monosaccharides (glucose, xylose) and oligosaccharides.

[0004] Max Kluyveromycin ( Kluyveromyces marxianus Kluyveromyces martensii is a strictly aerobic, unconventional yeast strain, recognized as generally safe (GRAS) by the U.S. Food and Drug Administration (FDA). Xylose is the most abundant monosaccharide besides glucose in lignocellulose hydrolysate. Unlike Saccharomyces cerevisiae, Kluyveromyces martensii naturally utilizes xylose. my country has a vast amount of lignocellulose resources, with straw production alone reaching 700 million tons annually. Currently, most straw is directly burned or returned to the field, which not only wastes resources but also pollutes the atmosphere, endangers human health, and poses risks to traffic safety. Summary of the Invention

[0005] The technical problem to be solved by this invention is how to produce single-cell proteins using inexpensive and discarded resources.

[0006] To solve the above-mentioned technical problems, the present invention first provides a strain of Kluyveromyces martensii. Kluyveromyces marxianus ZH-2.

[0007] The Kluyveromycin provided by this invention Kluyveromyces marxianus ZH-2 has the accession number CGMCC No. 32295 and is classified as *Kluyveromyces martensii*. Kluyveromyces marxianus It was deposited on October 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China).

[0008] To solve the above-mentioned technical problems, the present invention further provides the above-mentioned Kluyveromycin. Kluyveromyces marxianus Applications of ZH-2.

[0009] This invention provides the above-mentioned Kluyveromyces martensii. Kluyveromyces marxianus Applications of ZH-2 in the production of single-cell proteins or in the preparation of products that produce single-cell proteins.

[0010] To address the aforementioned technical problems, the present invention also provides a product for producing single-cell proteins.

[0011] The active ingredient in the single-cell protein production product provided by this invention is the aforementioned Kluyveromyces martensii. Kluyveromyces marxianus ZH-2.

[0012] The production of single-cell protein described above refers to the production of single-cell protein using straw as raw material.

[0013] The straw can be straw from various plant sources, such as rice straw, wheat straw, corn straw, sorghum straw, etc.

[0014] In some implementations, the straw is wheat straw.

[0015] To address the aforementioned technical problems, the present invention ultimately provides a method for producing single-cell proteins.

[0016] The method for producing single-cell protein provided by this invention includes using the above-mentioned Kluyveromyces martensii. Kluyveromyces marxianus The steps involved in the production of single-cell proteins using ZH-2.

[0017] Furthermore, the method includes the following steps: using straw as raw material and utilizing the above-mentioned Kluyveromyces martensii. Kluyveromyces marxianus ZH-2 produces single-cell proteins.

[0018] Furthermore, the straw can be straw from various plant sources, such as rice straw, wheat straw, corn straw, sorghum straw, etc.

[0019] In some implementations, the straw is wheat straw.

[0020] Furthermore, the method of using straw as raw material and utilizing the aforementioned Kluyveromyces martensii yeast... Kluyveromyces marxianus The method for producing single-cell protein from ZH-2 may include the following steps: pretreating and enzymatically hydrolyzing straw sequentially to obtain a reaction solution containing glucose and xylose; then adding Kluyveromyces martensii... Kluyveromyces marxianus ZH-2 was inoculated into a fermentation medium containing the reaction solution for fermentation culture to obtain single-cell protein.

[0021] In some embodiments, the pretreatment method may include the following steps: drying the straw to constant weight, crushing it, and sieving it to obtain crushed material; adding NaOH solution to the crushed material to react, and filtering the solution after reaction to obtain solid material; adding HCl to the solid material to adjust the pH to neutral, then rinsing it with water until the water becomes clear, and drying the solid material to constant weight to obtain pretreated straw.

[0022] In some specific implementations, the sieving may be through a 30-mesh sieve.

[0023] In some specific implementations, the pulverized material can be added to a NaOH solution at a solid-liquid ratio of 1:20.

[0024] In some specific implementations, the reaction conditions may be a reaction in a 50°C water bath shaker for 8 hours.

[0025] In some embodiments, the enzymatic hydrolysis method may include the following steps: first adding citrate-sodium citrate buffer to pretreated straw, then adding cellulase, hydrolyzing to obtain a reaction solution containing glucose and xylose.

[0026] In some specific embodiments, the citrate-sodium citrate buffer solution may have a pH of 5.0 and a concentration of 0.1 mol·L⁻¹. -1 .

[0027] In some specific implementations, the pretreated straw can be added to a citric acid-sodium citrate buffer solution at a solid-liquid ratio of 1:10.

[0028] In some specific embodiments, the concentration of the cellulase in the hydrolysis system may be 90 U·g. -1 .

[0029] In some specific implementations, the hydrolysis conditions may be hydrolysis at 50°C for 72 h.

[0030] In some embodiments, the reaction solution is concentrated and diluted to a certain concentration before being used to prepare a fermentation medium, such that the glucose concentration in the fermentation medium is 20 g / L and the xylose concentration is 3.58 g / L.

[0031] In some specific embodiments, the solvent of the fermentation medium is water, and the solutes and their concentrations are as follows: 20 g / L glucose (derived from the reaction solution), 3.58 g / L xylose (derived from the reaction solution), 5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 3 g / L KH2PO4, 15 mg / L EDTA, 4.5 mg / L ZnSO4·7H2O, 1 mg / L MnCl2·7H2O, 0.3 mg / L CoCl2·6H2O, 0.3 mg / L CuSO4·5H2O, 0.4 mg / L Na2MoO4·2H2O, 4.5 mg / L CaCl2·2H2O, 3 mg / L FeSO4·7H2O, 1 mg / L H3BO3, 0.1 mg / L KI, 0.1 mg / L biotin, 1 mg / L calcium pantothenate, and 1 mg / L nicotinic acid.

[0032] In some implementations, the fermentation culture conditions may be 20-30 hours at 30°C.

[0033] In some embodiments, the fermentation culture may further include a step of spray drying the fermentation product.

[0034] In some specific implementations, the spray drying conditions may be: material concentration: 10-50%, inlet air temperature: 80-120°C, and outlet air temperature: 50-70°C.

[0035] This invention uses Kluyveromyces martensii (Kluyveromyces masculinus) Kluyveromyces marxianus ZH-1 was the starting strain, and Kluyveromyces martensii was obtained through adaptive laboratory evolution (ALE). K. marxianus ZH-2 was deposited and archived under CGMCC No. 32295. Experiments demonstrated that culturing with wheat straw hydrolysate and ammonium sulfate... K. marxianus ZH-2, when cultured in a 3 L fermenter for 16 h, can achieve a biomass of 11.30 ± 0.90 g·L. -1 Biomass, maximum specific growth rate, cell yield, volumetric glucose uptake rate, and volumetric xylose uptake rate Qs are respectively... K. marxianus The fermentation time was shortened by 10 hours for ZH-1 at 1.54, 2.33, 1.54, 1.88, and 2.55 times the concentration. Ultimately... K. marxianus The yield of ZH-2 SCP is 0.047 g·g. -1 That is, 21.88 tons of wheat straw can yield 1 ton K. marxianus ZH-2 single-cell protein. This invention further addresses...K. marxianus Nutritional composition and amino acid composition analysis were performed on ZH-2 SCP. The results showed that: K. marxianus ZH-2 SCP has a crude protein content of 53.9%, a fat content of 3.6%, a carbohydrate content of 26.6%, a moisture content of 5.51%, and an ash content of 10.4%. K. marxianus The total amino acid (TAA) content of ZH-2SCP is 38.91±2.86%, the essential amino acid content is 18.03±1.57%, and the EAA / NEAA ratio is 0.86±0.02. The essential amino acid pattern basically conforms to the amino acid patterns of adults and infants, making it a suitable source of high-quality protein nutrition. This invention, from the perspective of utilizing inexpensive waste resources, provides a strain of *Kluyveromyces martensii* that can be fermented from wheat straw to obtain single-cell protein. K. marxianus ZH-2 not only effectively reduces the production cost of SCP, but also effectively solves the problem of protein resource shortage.

[0036] Preservation Instructions Strain name: Kluyveromyces martensii Latin name: Kluyveromyces marxianus Strain number: ZH-2 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Collection institution abbreviation: CGMCC Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Deposit date: October 21, 2024 CGMCC Registration Number: CGMCC No. 32295 Attached Figure Description Figure 1 20g·L -1 Fermentation performance of yeast in xylose chemical synthesis medium. A is Kluyveromyces martensii. K. marxianus ZH-1. B is Kluyveromyces martensii. K. marxianus ZH-2.

[0037] Figure 2 40 g·L -1 Fermentation performance of yeast in xylose chemical synthesis medium. A is Kluyveromyces martensii. K. marxianus ZH-1. B is Kluyveromyces martensii. K. marxianus ZH-2.

[0038] Figure 3 20 g·L -1 Fermentation performance of yeast in a glucose chemical synthesis medium. A is *Kluyveromyces martensii*.K. marxianus ZH-1. B is Kluyveromyces martensii. K. marxianus ZH-2.

[0039] Figure 4 40 g·L -1 Fermentation performance of yeast in a glucose chemical synthesis medium. A is *Kluyveromyces martensii*. K. marxianus ZH-1. B is Kluyveromyces martensii. K. marxianus ZH-2.

[0040] Figure 5 20 g·L -1 glucose and 20 g·L -1 Fermentation performance of yeast in xylose chemical synthesis medium. A is Kluyveromyces martensii. K. marxianus ZH-1. B is Kluyveromyces martensii. K. marxianus ZH-2.

[0041] Figure 6 40 g·L -1 glucose and 20 g·L -1 Fermentation performance of yeast in xylose chemical synthesis medium. A is Kluyveromyces martensii. K. marxianus ZH-1. B is Kluyveromyces martensii. K. marxianus ZH-2.

[0042] Figure 7 The fermentation performance of yeast in an inorganic synthesis medium containing wheat straw hydrolysate was evaluated. A represents *Kluyveromyces martensii*. K. marxianus ZH-1. B is Kluyveromyces martensii. K. marxianus ZH-2.

[0043] Figure 8 Kluyveromyces maculae K. marxianus The adult amino acid scoring pattern of ZH-2 single-cell protein. Orange represents the human amino acid pattern, and blue represents the amino acid pattern of Kluyveromyces martensii ZH-2 single-cell protein.

[0044] Figure 9 Kluyveromyces maculae K. marxianus Infant amino acid scoring pattern for ZH-2 single-cell protein. Orange represents the human amino acid pattern, and blue represents the amino acid pattern of Kluyveromyces martensii ZH-2 single-cell protein. Detailed Implementation

[0045] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0046] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0047] The solvent for the chemically synthesized culture medium (pH 5.0) in the following examples is water, and the solute and its concentration are 5 g·L⁻¹. -1 (NH4)2SO4, 0.5 g·L -1 MgSO4·7H2O, 3 g·L -1 KH2PO4, 15 mg·L -1 EDTA, 4.5 mg·L - 1 ZnSO4·7H2O, 1 mg·L -1 MnCl2·7H2O, 0.3 mg·L -1 CoCl2·6H2O, 0.3 mg·L -1 CuSO4·5H2O, 0.4 mg·L -1 Na₂MoO₄·2H₂O, 4.5 mg·L⁻¹ -1 CaCl₂·2H₂O, 3 mg·L⁻¹ -1 FeSO 4· 7H2O, 1 mg·L -1 H3BO3, 0.1 mg·L -1 KI, 0.1 mg·L -1 Biotin, 1 mg·L -1 Calcium pantothenate, 1 mg·L -1 niacin.

[0048] CDX in the following embodiments 20 The chemically synthesized culture medium contains 20 g·L -1 Culture medium for the chemical synthesis of xylose.

[0049] CDX in the following embodiments 40 The chemically synthesized culture medium contains 40 g·L -1 Culture medium for the chemical synthesis of xylose.

[0050] CDG in the following embodiments 20 The chemically synthesized culture medium contains 20 g·L-1 Culture medium for the chemical synthesis of glucose.

[0051] CDG in the following embodiments 40 The chemically synthesized culture medium contains 40 g·L -1 Culture medium for the chemical synthesis of glucose.

[0052] CDG in the following embodiments 20 X 20 The chemical synthesis medium contains 20 g·L -1 glucose and 20 g·L -1 Culture medium for the chemical synthesis of xylose.

[0053] CDG in the following embodiments 40 X 20 The chemically synthesized culture medium contains 40 g·L -1 glucose and 20 g·L -1 Culture medium for the chemical synthesis of xylose.

[0054] The solvent for the wheat straw sugar fermentation medium (pH 5.0) in the following examples was water, and the solutes and their concentrations were as follows: 20 g / L glucose (obtained from wheat straw hydrolysis), 3.58 g / L xylose (obtained from wheat straw hydrolysis), 5 g / L (NH4)2SO4, 0.5 g / L MgSO4·7H2O, 3 g / L KH2PO4, 15 mg / L EDTA, 4.5 mg / L ZnSO4·7H2O, 1 mg / L MnCl2·7H2O, 0.3 mg / L CoCl2·6H2O, 0.3 mg / L CuSO4·5H2O, 0.4 mg / L Na2MoO4·2H2O, 4.5 mg / L CaCl2·2H2O, 3 mg / L FeSO4·7H2O, 1 mg / L H3BO3, 0.1 mg / L KI, 0.1 mg / L biotin, 1 mg / L calcium pantothenate, 1 mg / L nicotinic acid.

[0055] Example 1: Kluyveromyces martensii K. marxianus Isolation, Identification and Preservation of ZH-2 Strains I. Isolation of strain ZH-2 Max Kluyveromycin K. marxianus ZH-1 is a strain isolated from the environment. Its whole genome sequence aligned 95.68% with the reference genome of *Kluyveromyces martensii* (18S rRNA sequence shown in Sequence 1). It exhibits the morphological and physiological / biochemical characteristics of *Kluyveromyces martensii*, and has been identified as *Kluyveromyces martensii*. Kluyveromyces marxianus .

[0056] Max Kluyveromycin K. marxianus ZH-1 was inoculated on CDX 20 The cells were cultured in a chemically synthesized medium at 30°C and 150 rpm for 7 days. After culture, the cells were centrifuged at 12,000 rpm for 10 min to remove the medium and then transferred to a new CDX culture medium. 20 In a chemically synthesized culture medium, after culturing at 30°C and 150 rpm for 7 days, the above steps were repeated. After 20 consecutive subculturings, a significant increase in cell concentration was observed compared to the parent strain. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) K. marxianus The ZH-1 culture was streaked to isolate the bacteria, and the resulting single colony was named strain ZH-2.

[0057] II. Whole genome sequencing and sequence analysis of strain ZH-2 ZH-2 strain is *Kluyveromyces martensii*. K. marxianus ZH-1 was obtained through adaptive laboratory evolution. Through whole-genome sequencing and sequence analysis, ZH-2 was identified as *Kluyveromyces martensii*. Kluyveromyces marxianus And its 18S rRNA sequence is similar to K. marxianus ZH-1 is exactly the same.

[0058] To obtain genomic variation information of strain ZH-2, the whole genome sequences of strains ZH-1 and ZH-2 were analyzed. Single nucleotide polymorphism (SNP) detection and small fragment insertion and deletion (InDel) detection were used to identify variation information between the two strains, which was then compared with... K. marxianus The FIM1 reference genome sequence (https: / / www.ncbi.nlm.nih.gov / datasets / genome / GCA_001854445.2 / ) and gene locations were integrated to ultimately screen for the mutated gene. This was then compared with the parental strain. K. marxianus Compared to ZH-1, some genes in the MAPK signaling pathway and cell cycle regulation pathway in the ZH-2 genome have been mutated (Table 1). These mutated genes may have led to a shortened cell cycle in the strain, resulting in an increased growth rate phenotype.

[0059] Table 1 K. marxianus Information on some mutated genes in the ZH-2 genome

[0060] III. Preservation of ZH-2 strain The ZH-2 strain was classified as *Kluyveromyces martensii*. Kluyveromyces marxianusThis strain was deposited on October 21, 2024, at the China General Microbiological Culture Collection Center (CGMCC, address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China), with accession number CGMCC No. 32295.

[0061] Example 2: Kluyveromycin K. marxianus Application of ZH-2 strain one, K. marxianus ZH-1 and K. marxianus ZH-2 utilization of xylose at the shake-flask level For comparison K. marxianus ZH-1 and K. marxianus ZH-2's ability to utilize xylose in chemically synthesized culture media will... K. marxianus ZH-2 was inoculated on CDX 20 Chemically synthesized culture medium and CDX 40 Chemically synthesized culture medium, cultured at 30℃ for 72 h, while simultaneously using K. marxianus ZH-1 was used as the control strain, and the same procedures were performed. Fermentation kinetic parameters were calculated according to the formulas in the literature "SHARMA et al., 2017", as follows: .

[0062] In CDX 20 In chemically synthesized culture media, K. marxianus ZH-1 fermentation at 72 hours OD 600nm The value was 2.41 ± 0.02, and the cell dry weight was 0.93 ± 0.01 g·L. -1 ( Figure 1 A), and K. marxianus ZH-2 fermentation at 72 h OD 600nm The mean value was 8.96 ± 0.47, and the cell dry weight was 4.19 ± 0.23 g·L. -1 ( Figure 1 B) P <0.05), compared to K. marxianus ZH-1 biomass increased by 350.54%. After fermentation... K. marxianus ZH-1 utilizes 6.56±1.16 g·L -1 xylose, and K. marxianus ZH-2 utilized 16.65±0.47 g·L -1 Xylose was increased, and xylose utilization was improved by 50.44%, with a volumetric xylose uptake rate Q. s An increase of 155.56% ( Figure 1 ).also, K. marxianusZH-1's maximum specific growth rate μ max It is 0.02 ± 0.01 h -1 Xylose cell production Y X / S It is 0.14 ± 0.02 g·g -1 The rate of xylose consumption q s It is 0.13 ± 0.02 g·g -1 ·h -1 ; K. marxianus ZH-2's maximum specific growth rate μ max It is 0.07±0.01h -1 Xylose cell production Y X / S It is 0.25 ± 0.01 g·g -1 The rate of xylose consumption q s It is 0.23 ± 0.01 g·g -1 ·h -1 (Table 2), all showed significant differences ( P <0.05), which are respectively K. marxianus 3.58 times, 1.77 times and 2.02 times that of ZH-1.

[0063] In CDX 40 In chemically synthesized culture media, the two yeast strains showed similar growth trends. K. marxianus ZH-1 fermentation at 72 hours OD 600nm The value was 1.90 ± 0.75, and the cell dry weight was 0.75 ± 0.01 g·L. -1 ( Figure 2 A), K. marxianus ZH-2 fermentation at 72 h OD 600nm The mean value was 7.09 ± 0.16, and the cell dry weight was 3.88 ± 0.15 g·L. -1 ( Figure 2 B), compared to K. marxianus ZH-1 biomass increased by 417.33% ( P <0.05). After fermentation is complete. K. marxianus ZH-1 utilizes 8.85±1.76 g·L -1 xylose, and K. marxianus ZH-2 utilized 26.03±1.30 g·L -1 The xylose utilization rate was increased by 42.97%, and the volumetric xylose uptake rate Q... s Increase by 200.00% Figure 2 ).also, K. marxianus ZH-1's maximum specific growth rate m maxIt is 0.02 ± 0.01 h -1 Cell yield Y X / S It is 0.08 ± 0.01 g·g -1 The rate of xylose consumption q s It is 0.27 ± 0.02 g·g -1 ·h -1 ;and K. marxianus ZH-2's maximum specific growth rate m max It is 0.07±0.01h -1 Xylose cell production Y X / S It is 0.14 ± 0.01 g·g -1 The rate of xylose consumption q s It is 0.48 ± 0.01 g·g -1 ·h -1 (Table 2), all showed significant differences ( P <0.05), which are respectively K. marxianus 3.43 times, 1.95 times, and 1.76 times that of ZH-1.

[0064] When the concentration of xylose is from 20 g·L -1 Increase to 40 g·L -1 , K. marxianus ZH-1 and K. marxianus ZH-2's xylose uptake rate Q s They increased by 107.77% and 81.13% respectively, but biomass decreased by 24% and 7.99% respectively. P <0.05). During fermentation. K. marxianus ZH-2 produces xylitol, and the xylitol concentration increases significantly with increasing xylose concentration. The volumetric xylitol production rate Qs increases by 142.56%, and the specific xylitol formation rate qs increases by 109.36%. P <0.05 (Table 2).

[0065] Table 2. Fermentation kinetics of yeast in xylose chemical synthesis medium under shake-flask conditions

[0066] Note: Different lowercase letters indicate that the fermentation kinetic parameters of different strains in shake flasks are significantly different at the P<0.05 level.

[0067] two, K. marxianus ZH-1 and K. marxianus ZH-2 glucose utilization at the shake-flask level For comparison K. marxianus ZH-1 and K. marxianus The ability of ZH-2 to utilize glucose in chemically synthesized culture media was investigated. K. marxianus ZH-1 and K. marxianus ZH-2 was inoculated into CDG 20 Chemically synthesized culture medium and CDG 40 Yeast fermentation performance was observed after culturing in a chemically synthesized culture medium at 30°C for 72 h, as follows: Figure 3 and Figure 4 As shown. The results show that as the concentration of glucose in the chemically synthesized medium increases, K. marxianus ZH-1 and K. marxianus The rate of glucose consumption by ZH-2 increased accordingly, but the biomass did not change significantly. P >0.05).

[0068] In CDG 20 In chemically synthesized culture media, K. marxianus ZH-1 fermentation at 72 hours OD 600nm The value was 5.44 ± 0.11, and the cell dry weight was 2.44 ± 0.05 g·L. -1 ( Figure 3 A), K. marxianus ZH-2 fermentation at 72 h OD 600nm The mean value was 6.89 ± 0.18, and the cell dry weight was 3.16 ± 0.09 g·L. -1 ( P <0.05)( Figure 3 B), biomass compared to K. marxianus ZH-1 increased by 29.70%. After fermentation... K. marxianus ZH-1 utilized 19.72±0.09 g·L -1 glucose, and K. marxianus ZH-2 had completely consumed all the glucose in the culture medium by the 20th hour of fermentation, with a glucose uptake rate of Q. s An increase of 270.37%. Figure 3 ).also, K. marxianus ZH-1's maximum specific growth rate m max It is 0.13 ± 0.01 h. -1 cellular glucose production Y G / S It is 0.12 ± 0.01 g·g -1 The rate of glucose consumption q s It is 0.61 ± 0.01 g·g -1 ·h -1 ; K. marxianus ZH-2's maximum specific growth rate m max It was 0.18 ± 0.03 h. -1 cellular glucose production Y G / SIt is 0.16 ± 0.01 g·g -1 The rate of glucose consumption q s It is 1.13 ± 0.03 g·g -1 ·h -1 (Table 3) showed significant differences in all cases. P <0.05), which are respectively K. marxianus 2.36 times, 1.28 times, and 1.84 times that of ZH-1.

[0069] In CDG 40 In chemically synthesized culture media, K. marxianus ZH-1 fermentation at 72 hours OD 600nm The value was 5.16 ± 0.07, and the cell dry weight was 2.30 ± 0.4 g·L. -1 ( Figure 4 A); K. marxianus ZH-2 fermentation at 72 h OD 600nm The mean value was 7.09 ± 0.16, and the cell dry weight was 3.26 ± 0.08 g·L. -1 ( P <0.05)( Figure 4 B), biomass compared to K. marxianus ZH-1 increased by 41.74%. After fermentation... K. marxianus ZH-1 utilized 27.99±0.59 g·L -1 glucose, and K. marxianus ZH-2 completely consumed the glucose in the culture medium by the 24th hour of fermentation, increasing glucose utilization by 30.03% and achieving a volumetric glucose uptake rate Q. s An increase of 412.28% ( Figure 4 ).also, K. marxianus ZH-1's maximum specific growth rate m max It is 0.07 ± 0.01 h -1 The rate of glucose consumption q s It is 0.82 ± 0.01 g·g -1 ·h -1 ; K. marxianus ZH-2's maximum specific growth rate m max It is 0.13 ± 0.03 h -1 The rate of glucose consumption q s It is 1.62 ± 0.04 g·g -1 ·h -1 (Table 3) showed significant differences in all cases. P <0.05), which are respectively K. marxianusThere was no significant difference in biomass yield between the 1.80-fold and 9.43-fold increases in ZH-1.

[0070] K. marxianus ZH-2 produced significantly higher ethanol yields during fermentation compared to other methods. K. marxianus ZH-1 ( P <0.05, and the ethanol concentration increased significantly with increasing glucose concentration ( P <0.05)( Figure 4 B). When the glucose concentration changes from 20 g·L⁻¹ -1 Increase to 40 g·L -1 , K. marxianus ZH-1 and K. marxianus ZH-2 specific glucose consumption rate q s They increased by 44.44% and 100% respectively, but biomass did not increase significantly and even decreased. P >0.05). Both yeast strains produced ethanol during fermentation, but K. marxianus ZH-2 showed a significant increase in ethanol concentration with increasing glucose concentration. P <0.05), K. marxianus ZH-2 at 20 g·L -1 and 40 g·L -1 Volumetric ethanol production rate Q of glucose s They are K. marxianus ZH-1 was 3.68 times and 21.18 times higher than the ethanol formation rate q. s They are K. marxianus 6.69 times and 9.43 times that of ZH-1 ( P <0.05 (Table 3).

[0071] Table 3. Fermentation kinetics of yeast in glucose chemical synthesis medium in shake flasks

[0072] Note: Different lowercase letters represent the fermentation kinetic parameters of different bacterial strains in shake flasks. P The difference was statistically significant at levels <0.05.

[0073] three, K. marxianus ZH-1 and K. marxianus ZH-2 utilization of mixed glucose and xylose carbon sources at the shake-flask level For comparison K. marxianus ZH-1 and K. marxianus The utilization ability of ZH-2 of a mixed carbon source of glucose and xylose in a chemically synthesized culture medium was compared with that of [other materials]. K. marxianus ZH-1 and K. marxianus ZH-2 was inoculated into CDG 20 X 20Chemically synthesized culture medium and CDG 40 X 20 The growth curve is as follows: Cultured at 30℃ for 72 h in a chemically synthesized medium. Figure 5 and Figure 6 As shown in the figure. The results showed that the rapid growth period of the two yeast strains was from 0 to 24 hours during fermentation, and the growth rate slowed down and gradually entered the plateau phase from 24 to 36 hours.

[0074] In CDG 20 X 20 In chemically synthesized culture media, K. marxianus ZH-1 fermentation at 72 hours OD 600nm The mean value was 5.48 ± 0.08, and the cell dry weight was 2.46 ± 0.04 g·L. -1 ( Figure 5 A), utilizing 12.48±0.10 g·L -1 glucose; K. marxianus ZH-2 fermentation at 72 h OD 600nm The value was 7.94 ± 0.20, and the cell dry weight was 3.68 ± 0.10 g·L. -1 ( Figure 5 B), glucose was consumed in the 20th hour of fermentation ( P <0.05), biomass compared to K. marxianus ZH-1 increased by 49.87%, glucose utilization increased by 32.58%, and the volumetric glucose uptake rate Q... s An increase of 319.05%. K. marxianus ZH-1 produced a peak concentration of 1.03 ± 0.11 g·L⁻¹ at 24 h of fermentation. -1 Ethanol; K. marxianus ZH-2 produced its highest concentration of 6.21 ± 0.05 g·L⁻¹ at 28 h of fermentation. -1 Ethanol was produced at a peak concentration of 0.13 ± 0.08 g·L⁻¹ at 36 h of fermentation. -1 Xylitol ( P <0.05).

[0075] In CDG 40 X 20 In chemically synthesized culture media, K. marxianus ZH-1 fermentation at 72 hours OD 600nm The mean value was 5.06 ± 0.04, and the cell dry weight was 2.25 ± 0.02 g·L. -1 ( Figure 6 A), utilizing 20.30±0.15 g·L -1 glucose; K. marxianus ZH-2 fermentation at 72 h OD 600nm The cell count was 8.59 ± 0.16, and the cell dry weight was 4.01 ± 0.08 g·L. -1 , ( Figure 6 B) P <0.05%, glucose was completely consumed in the 60th hour of fermentation ( P <0.05), biomass compared to K. marxianus ZH-1 increased by 51.36%, glucose utilization increased by 49.24%, and the volumetric glucose uptake rate Q... s An increase of 139.29%. K. marxianus ZH-1 produced its highest concentration of 1.25 ± 0.02 g·L⁻¹ at 28 h of fermentation. -1 Ethanol; K. marxianus ZH-2 produced its highest concentration of 12.05 ± 0.12 g·L⁻¹ at 48 h of fermentation. -1 Ethanol was produced at a peak concentration of 0.21 ± 0.02 g·L⁻¹ at 60 h of fermentation. -1 Xylitol ( P <0.05).

[0076] Similar to the growth trend in CDX and CDG chemically synthesized media, K. marxianus The volumetric glucose uptake rate Q during ZH-2 fermentation s , glucose consumption rate q s and volumetric ethanol production rate Q s All of these increased with increasing glucose concentration, and were significantly higher than [the previous values]. K. marxianus ZH-1 ( P <0.05). As time progressed, the cells utilized some of the ethanol and xylitol, causing their concentrations to gradually decrease. It is worth noting that... K. marxianus Under these conditions, ZH-1 consumes zero xylose and produces no xylitol, while K. marxianus ZH-2 consumed 2.03±0.24 g·L⁻¹ -1 and 1.95±0.03 g·L -1 Xylose.

[0077] In summary, K. marxianus ZH-2 compared to K. marxianus ZH-1 was cultured in shake flasks using chemically synthesized media containing different concentrations of glucose, xylose, and a mixture of glucose and xylose. The resulting biomass and the utilization rates of glucose and xylose during fermentation both significantly increased. P <0.05).

[0078] Four, K. marxianus ZH-1 and K. marxianus ZH-2 Utilization of Wheat Straw Hydrolysate at the Fermentation Tank Level 1. Preparation of hydrolyzed sugar from wheat straw Wheat straw was dried to constant weight in a 65℃ oven, pulverized, and passed through a 30-mesh sieve to obtain wheat straw pulverized material. 5% (mass-volume ratio) NaOH solution was added to the wheat straw pulverized material at a solid-liquid ratio of 1:20, and the mixture was reacted in a 50℃ water bath shaker for 8 h to obtain a reaction solution. The reaction solution was filtered, and the pH of the solid was adjusted to neutral by adding HCl. The solid was then washed with water until the water became clear. The solid was dried to constant weight to obtain the pretreated and dried wheat straw material. The pretreated and dried wheat straw material was then mixed with 0.1 mol·L⁻¹ -1 Prepare a reaction solution using a citrate-sodium citrate buffer (pH 5.0) at a solid-liquid ratio of 1:10, then add 90 U·g. -1 Cellulase was used to hydrolyze the solution at 50°C for 72 h to obtain the final reaction solution. The glucose content in the final reaction solution was determined to be 18.51 g·L⁻¹ using HPLC. -1 The xylose content is 4.00 g·L. -1 That is, the glucose yield is 0.081 g·g -1 The xylose yield was 0.017 g·g. -1 The final reaction solution was concentrated and diluted to a certain concentration before being used to prepare a wheat straw sugar fermentation medium, so that the glucose concentration in the wheat straw sugar fermentation medium reached 20 g / L and the xylose concentration reached 3.58 g / L.

[0079] 2. K. marxianus ZH-1 and K. marxianus ZH-2 Utilization of Wheat Straw Hydrolysate at the Fermentation Tank Level For comparison K. marxianus ZH-1 and K. marxianus The utilization of wheat straw hydrolysate CDT by ZH-2 in a fermenter was investigated. K. marxianus ZH-1 and K. marxianus ZH-2 was inoculated into wheat straw sugar fermentation medium and cultured at 30℃ for 30 h. The fermentation performance of the two strains is as follows: Figure 7 As shown. The results show: K. marxianus ZH-1 fermentation at 28 h OD 600nm The value was 15.29 ± 0.20, and the cell dry weight was 7.34 ± 0.10 g·L. -1 ( Figure 7 A), and K. marxianus 18 hours after ZH-2 fermentation OD 600nm The value was 23.24 ± 1.80, and the cell dry weight was 11.30 ± 0.90 g·L. -1 ( Figure 7 B) ,K. marxianus ZH-2 ratio K. marxianus ZH-1 biomass increased by 53.91% ( P <0.05). K. marxianus ZH-1 depletes the glucose in the culture medium during fermentation from 18 to 20 hours, and begins to consume xylose from the culture medium from 16 to 18 hours of fermentation. The consumption of xylose gradually stops after 24 hours. Figure 7 A), K. marxianus ZH-2 fermentation consumes all glucose in the culture medium after 10 hours and begins to consume xylose. After 14 hours, xylose consumption gradually stops. Figure 7 B). K. marxianus ZH-2's volumetric glucose uptake rate Q s Compared K. marxianus ZH-1 increased by 88.68% ( P <0.05), xylose uptake rate Q s Compared K. marxianus ZH-1 increased by 155.19% ( P <0.05).

[0080] also, K. marxianus ZH-1's maximum specific growth rate m max It was 0.17 ± 0.01 h. -1 Cell yield Y G&X / S It is 0.35 ± 0.01 g·g -1 The rate of xylose consumption q s It is 0.17 ± 0.01 g·g -1 ·h -1 The rate of glucose consumption q s It is 1.06 ± 0.01 g·g -1 ·h -1 ; K. marxianus ZH-2's maximum specific growth rate m max It is 0.40 ± 0.02 h -1 Cell yield Y G&X / S It is 0.53 ± 0.04 g·g -1 The rate of xylose consumption q s It is 0.25 ± 0.03 g·L -1 ·h -1 The rate of glucose consumption q s It is 2.50 ± 0.01 g·L -1 ·h -1 (Table 4), with K. marxianus Significant differences were observed in ZH-1 ( P <0.05), which are respectively K. marxianusThe levels of ZH-1 were 2.33 times, 1.54 times, 1.88 times, and 2.55 times higher.

[0081] Neither yeast strain produced xylitol during fermentation. K. marxianus ZH-2 does not produce ethanol during fermentation, but K. marxianus A large amount of ethanol is produced during the fermentation of ZH-1. As glucose is consumed, K. marxianus ZH-1 produces an increased concentration of ethanol. When glucose is depleted, xylose is also consumed, and ethanol, as a carbon source, is also... K. marxianus ZH-1 is utilized and all the ethanol produced is consumed after fermentation is complete.

[0082] Table 4. Fermentation kinetics of yeast in wheat straw hydrolysate chemical synthesis medium in a fermenter

[0083] Note: Different lowercase letters indicate fermentation kinetic parameters of different bacterial strains in the fermenter. P The difference was statistically significant at levels <0.05.

[0084] 3. Nutritional composition and amino acid analysis right K. marxianus ZH-2 was fermented in wheat straw sugar fermentation medium for 20 hours. The fermentation product was then spray-dried (spray drying conditions were as follows: material concentration: 10-50%, inlet air temperature: 80-120℃, outlet air temperature: 50-70℃) to obtain... K. marxianus ZH-2 SCP was analyzed for its nutritional components, amino acid composition, and amino acid score. The amino acid score was calculated based on the 2007 WHO / FAO / UNU requirements for protein and amino acids in human nutrition (Joint WHO / FAO / UNU Expert Consultation, 2007) and compared with the amino acid patterns of adults and infants.

[0085] Nutritional analysis results show that: K. marxianus ZH-2 SCP contains 53.90% protein, 3.60% fat, 26.6% carbohydrates, 5.51% moisture, and 10.40% ash.

[0086] Amino acid composition analysis results showed that: K. marxianusZH-2 SCP contains 18 common amino acids, including the 8 essential amino acids required by the human body and histidine, which is essential for infants and young children, as well as 9 non-essential amino acids (Table 5). Aspartic acid has the highest content at 5.69±0.01%, while tryptophan has the lowest at 0.05±0.01%. Except for methionine, tryptophan, and cysteine, the content of all other amino acids is above 1%. The total amino acid content (TAA) is 38.91±2.86%, the essential amino acid content (EAA) is 17.98±1.57%, and the EAA / NEAA ratio is 0.86±0.02.

[0087] The results of amino acid scoring and amino acid pattern comparison showed that: K. marxianus In the adult amino acid profile of ZH-2 SCP, except for a slightly lower leucine content, it conforms to the adult amino acid composition pattern. The contents of histidine, isoleucine, methionine, phenylalanine, tyrosine, and threonine are all more than three times the adult requirements. Figure 8 ).exist K. marxianus In the infant amino acid score pattern of ZH-2 SCP, except for low levels of isoleucine and threonine, the rest conform to the infant amino acid composition pattern. Figure 9 ).

[0088] In summary, K. marxianus ZH-2 SCP is rich in amino acids, and its essential amino acid profile basically matches that of adults and infants, making it a high-quality protein supplement.

[0089] Table 5. Amino acid composition of yeast single-cell proteins

[0090] Note: "*" indicates essential amino acids.

[0091] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A strain of Kluyveromyces martensii Kluyveromyces marxianus ZH-2, with accession number CGMCCNo.32295.

2. The Kluyveromyces martensii as described in claim 1 Kluyveromyces marxianus Applications of ZH-2 in the production of single-cell proteins or in the preparation of products that produce single-cell proteins.

3. The application according to claim 2, characterized in that: The production of single-cell protein involves using straw as a raw material.

4. The application according to claim 3, characterized in that: The straw in question is wheat straw.

5. A product for producing single-cell protein, wherein the active ingredient is *Kluyveromyces martensii* as described in claim 1. Kluyveromyces marxianus ZH-2.

6. The product according to claim 5, characterized in that: The production of single-cell protein involves using straw as a raw material.

7. The product according to claim 6, characterized in that: The straw in question is wheat straw.

8. A method for producing single-cell protein, comprising using *Kluyveromyces martensii* as described in claim 1. Kluyveromyces marxianus The steps involved in the production of single-cell proteins using ZH-2.

9. The method according to claim 8, characterized in that: The method includes the following steps: using straw as raw material and utilizing the Kluyveromycin described in claim 1. Kluyveromyces marxianus ZH-2 produces single-cell proteins.

10. The method according to claim 9, characterized in that: The straw in question is wheat straw.

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