Saccharomyces cerevisiae strain MBP_sc21 and use thereof

Abstract

Provided are a Saccharomyces cerevisiae strain MBP_Sc21 and the use thereof, belonging to the technical field of microorganisms. On one hand, provided is the Saccharomyces cerevisiae strain MBP_Sc21 expressing an immunonutrient β-carotene, the accession number of said strain being CGMCC No. 32663; and on the other hand, provided is the use of the Saccharomyces cerevisiae strain. The strain uses soybean meal as a solid fermentation matrix, the obtained fermented soybean meal product has a significantly increased content of crude proteins and greatly decreased contents of antinutritional factors such as oligosaccharides and urease. More importantly, the fermented soybean meal is rich in β-carotene, which endows the soybean meal protein new functional immunonutrition, thereby improving the overall nutritional value of soybean meal.

Description

A brewing yeast MBP_Sc21 and its applications Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a brewer's yeast MBP_Sc21 and its application in soybean meal fermentation. Background Technology

[0002] In 2019, my country's Ministry of Agriculture and Rural Affairs issued Announcement No. 194, requiring a complete ban on growth-promoting drug feed additives. This marked the official entry of my country's livestock, poultry, and aquaculture industries into the "antibiotic-free" era, and also placed higher demands and challenges on the feed industry. Fermentation has become a hot topic in the feed industry, and the development of functional feeds has become an industry consensus. The key is to develop functional feed ingredients. A functional combination of high-quality protein and immune nutrients to systematically improve the immunity of farmed animals is one of the ideal solutions to the "antibiotic-free" problem in feed.

[0003] Soybean meal, as the highest quality plant protein source, is a critical raw material restricting the development of my country's feed industry. Microbial fermentation of soybean meal can remove anti-nutritional factors, improve flavor and nutritional value, and its low production cost and high efficiency have led to its widespread application in the feed industry. β-carotene is a known functional pigment-based immune enhancer and is permitted in my country's "List of Feed Additives (2013)". It enables farmed animals to achieve faster growth and better feed conversion rates, especially in improving immunity. Currently, the livestock industry consumes over $300 million worth of carotenoids annually. Therefore, using β-carotene-producing microorganisms to ferment soybean meal and other feed protein raw materials yields fermented feed directly containing β-carotene. This not only enhances the nutritional value and flavor of soybean meal protein but also avoids the extraction and additional addition of carotenoids. The process is simple, low-cost, and can endow soybean meal protein with new immune functional properties. After solid-state fermentation using *Ulva cylindrica* CCF2412, a filamentous fungus that naturally produces β-carotene and γ-linolenic acid, the resulting product was directly added to broiler feed, significantly improving the biochemical, hematological, and immunological parameters of the broilers. (et al., 2020). The rapid development of synthetic biology technology has provided a feasible solution for realizing functional fermented feed ingredients rich in β-carotene. Saccharomyces cerevisiae has always been a dominant strain in traditional food and fermentation industries due to its higher biosafety; its live bacteria, inactive components, and cellular components are widely used in aquaculture and feed industries. Studies have shown that fermenting soybean meal with Saccharomyces cerevisiae can significantly increase the crude protein and amino acid content of soybean meal while reducing the levels of phytic acid and trypsin inhibitors. In summary, utilizing microbial technology to enhance the value of soybean meal and produce fermented feed ingredients rich in β-carotene is essential for the development and utilization of functional feeds. Summary of the Invention

[0004] In view of the problems in the background art, the purpose of this invention is to design and provide a brewing yeast MBP_Sc21 and its application in soybean meal fermentation.

[0005] This invention is achieved through the following technical solutions:

[0006] The first aspect of the present invention provides a Saccharomyces cerevisiae MBP_Sc21 strain, which was deposited on November 15, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 32663.

[0007] A second aspect of the present invention provides a microbial agent containing Saccharomyces cerevisiae MBP_Sc21.

[0008] The third aspect of the present invention provides the application of the above-mentioned brewer's yeast MBP_Sc21 or the above-mentioned inoculum in soybean meal fermentation.

[0009] The fourth aspect of the present invention provides the application of the above-mentioned brewer's yeast MBP_Sc21 or the above-mentioned inoculum in improving the nutritional value of soybean meal during soybean meal fermentation.

[0010] Furthermore, the improvement in the nutritional value of soybean meal is specifically manifested in the large accumulation of β-carotene, the increase in crude protein content, and the decrease in the content of anti-nutritional factors.

[0011] Furthermore, the anti-nutritional factors include oligosaccharides and urease.

[0012] The fifth aspect of the present invention provides a method for producing fermented soybean meal rich in β-carotene, wherein the method uses Saccharomyces cerevisiae MBP_Sc21 or an inoculum containing Saccharomyces cerevisiae MBP_Sc21 to ferment the soybean meal.

[0013] The Saccharomyces cerevisiae MBP_Sc21 of this invention uses soybean meal as a solid-state fermentation substrate. The resulting fermented soybean meal has a significantly increased crude protein content and a substantial reduction in anti-nutritional factors such as oligosaccharides and urease. More importantly, the fermented soybean meal is rich in β-carotene, which endows the soybean meal protein with new functional immune nutrition, thereby improving the overall nutritional value of the soybean meal. This invention represents a leading achievement in the field of developing functional fermented soybean meal using Saccharomyces cerevisiae. Attached Figure Description

[0014] Figure 1 is a schematic diagram of gene elements of gene fragment 1; where TRP1 LHA and TRP1 RHA at both ends represent the upstream and downstream homologous sequences of the yeast TRP1 site, respectively.

[0015] Figure 2 is a schematic diagram of gene elements of gene fragment 2; where ypl062w LHA and ypl062w RHA at both ends represent the upstream and downstream homologous sequences of the yeast ypl062w site, respectively.

[0016] Figure 3 is a schematic diagram of gene elements of gene fragment 3; where gal7 LHA and gal1 RHA at both ends represent the upstream homologous sequence of the yeast gal7 site and the downstream homologous sequence of the gal1 site, respectively.

[0017] Figure 4 is a schematic diagram of gene elements of gene fragment 4; where LEU2 LHA and LEU2 RHA at both ends represent the upstream and downstream homologous sequences of the yeast LEU2 site, respectively.

[0018] Figure 5 is a schematic diagram of gene elements of gene fragment 5; where HIS3 LHA and HIS3 RHA at both ends represent the upstream and downstream homologous sequences of the yeast HIS3 site, respectively.

[0019] Figure 6 is a schematic diagram of gene elements of gene fragment 6; where rox1 LHA and rox1 RHA at both ends represent the upstream and downstream homologous sequences of the yeast rox1 site, respectively.

[0020] Figure 7 shows the spectrum of plasmid pJET1.2;

[0021] Figure 8 shows the spectrum of plasmid pRS405;

[0022] Figure 9 shows the spectrum of plasmid pRS313;

[0023] Figure 10 shows before and after photos of fermentation of soybean meal by Saccharomyces cerevisiae MBP_Sc21 ((A) shake flask fermentation for 0 h; (B) shake flask fermentation for 24 h; (C) crushed soybean meal; (D) crushed fermented soybean meal).

[0024] Figure 11 shows the HPLC peaks of β-carotene in fermented soybean meal products by Saccharomyces cerevisiae MBP_Sc21 ((A) β-carotene standard; (B) Carotenoids contained in fermented soybean meal products). Detailed Implementation

[0025] To fully disclose the Saccharomyces cerevisiae strain of the present invention and its applications, the following examples are provided, but this does not imply any limitation on the present invention.

[0026] Some of the plasmid vectors and strains involved in this invention are commercially available. For example, the pJET1.2 plasmid vector was purchased from Thermo Scientific's CloneJET PCR Cloning Kit, #K1231 (plasmid map shown in Figure 7); plasmid pRS405 (plasmid map shown in Figure 8); plasmid pRS313 (plasmid map shown in Figure 9); the Saccharomyces cerevisiae strain CEN.PK2-1D was purchased from the EUROSCARF of Scientific Research and Development GmbH, Germany; and Saccharomyces cerevisiae BY4742 was purchased from the National Center for Type Culture Collection.

[0027] The gene elements used in constructing the recombinant yeast strain of this invention, such as amino acid markers, tags, endogenous genes, and exogenous genes, are all well-known in the art, and their specific sequences are known to those skilled in the art. To facilitate understanding of this invention, the gene elements in each gene fragment are described below:

[0028] Gene fragment 1 (SEQ ID NO.1) containing the CarB and CarRP genes from *Mucor truncatula*: 1-631bp is the 631bp homologous sequence upstream of the TRP1 site; 632-886bp is the CYC1 terminator sequence; 887-2626bp is the CarB gene from *Mucor truncatula*; 2627-3294bp is the GAL10-GAL1 bidirectional promoter sequence; 3295-5139bp is the CarRP gene from *Mucor truncatula*; 5140-5414bp is the PGK1 terminator sequence; 5415-6147bp is the 733bp homologous sequence downstream of the TRP1 site.

[0029] Gene fragment 2 (SEQ ID NO. 2) containing the CarB and CarRP genes from *Mucor truncatula*: 1-394bp is a 394bp homologous sequence upstream of the ypl062w site; 395-1951bp is the DR-URA3-DR nutrient tag sequence; 1952-2206bp is the CYC1 terminator sequence; 2207-3946bp is the CarB gene from *Mucor truncatula*; 3947-4614bp is the GAL10-GAL1 bidirectional promoter sequence; 4615-6459bp is the CarRP gene from *Mucor truncatula*; 6460-6734bp is the PGK1 terminator sequence; 6735-7051bp is a 317bp homologous sequence downstream of the ypl062w site.

[0030] Gene fragment 3 (SEQ ID NO. 3) containing the CrtE gene from Archaeococcus scintillans: 1-426bp is a 426bp homologous sequence upstream of the gal7 site; 427-1983bp is the DR-URA3-DR nutrient tag sequence; 1984-3330bp is the ERG10 gene and its terminator sequence; 3331-3836bp is the GAL7 promoter sequence; 3837-4123bp is the ACT1 terminator sequence; 4124-5632bp is the truncated HMG-CoA reductase gene tHMGR1; 5633-6300bp is the GAL10-GAL1 bidirectional promoter sequence; 6301-7254bp is the CrtE gene from Archaeococcus scintillans; 7255-7654bp is the GPM1 terminator sequence; 7655-7888bp is a 234bp homologous sequence downstream of the gal1 site.

[0031] Gene fragment 4 (shown in SEQ ID NO.4): 1-561bp is the 561bp homologous sequence upstream of the LEU2 site; 562-1656bp is the LEU2 marker; 1657-2056bp is the TDH2 terminator sequence; 2057-3682bp is the ERG13 gene and its terminator sequence; 3683-4188bp is the GAL7 promoter sequence; 4189-5984bp is the truncated HMG-CoA reductase gene tHMGR1; 5985-6652bp is the GAL10-GAL1 bidirectional promoter sequence; 6653-8134bp is the ERG12 gene and its terminator sequence; 8135-8718bp is the 584bp homologous sequence downstream of the LEU2 site.

[0032] Gene fragment 5 (shown in SEQ ID NO. 5): 1-312bp is the 312bp homologous sequence upstream of the HIS3 site; 313-975bp is the HIS3 marker; 976-1375bp is the ENO2 terminator; 1376-2392bp is the IDI1 gene and its terminator sequence; 2393-3049bp is the GAL7 promoter sequence; 3050-4240bp is the ERG19 gene and its terminator sequence; 4241-4908bp is the GAL10-GAL1 bidirectional promoter sequence; 4909-6414bp is the ERG8 gene and its terminator sequence; 6415-6992bp is the 578bp homologous sequence downstream of the HI3 site.

[0033] Gene fragment 6 (SEQ ID NO. 6) containing the Vgb gene from *Vibrio hygroscopicus* contains the following sequences: 1-244 bp is a 244 bp homologous sequence upstream of the rox1 site; 245-1022 bp is the PGK1 promoter sequence; 1023-1463 bp is the Vgb gene from *Vibrio hygroscopicus*; 1464-1750 bp is the ACT1 terminator sequence; 1751-3307 bp is the DR-URA3-DR nutrient tag sequence; and 3308-3618 bp is a 311 bp homologous sequence downstream of the rox1 site.

[0034] Once the specific sequences of the aforementioned gene elements are known, those skilled in the art can perform amplification and OE-PCR assembly according to conventional primer design principles. Furthermore, the SD medium used in this invention is a commonly used medium in the field of yeast screening. Target strains are screened by intentionally removing one or more components from the basic medium based on the specific gene defects present in the yeast.

[0035] Example 1

[0036] Explanation of the origin of gene elements

[0037] The exogenous genes involved in this invention include the geranylidene pyrophosphate synthase gene CrtE, the bifunctional enzyme gene CarRP (lycopene synthase / lycopene cyclase), the lycopene dehydrogenase gene CarB, and the hemoglobin-coding gene Vgb. CrtE is derived from *Archaeoglobus fulgidus*; CarB and CarRP are derived from *Mucor circinelloides*; and Vgb is derived from *Vitreoscilla stercoraria*. All of these genes were synthesized artificially after codon optimization and appropriate avoidance of commonly used restriction enzyme sites.

[0038] Promoters, terminators, endogenous genes, and related upstream and downstream homologous sequences in *Saccharomyces cerevisiae*, including the CYC1 terminator, GAL10 promoter, GAL1 promoter, PGK1 terminator, ACT1 terminator, GPM1 terminator, GAL7 promoter, ERG10 gene and its terminator, truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMGR1), ERG12 gene and its terminator, ERG19 gene and its terminator, ERG13 gene and its terminator, ERG8 gene and its terminator, and IDI1 gene and its terminator, were amplified by PCR using the genome of *Saccharomyces cerevisiae* strain BY4742 as a template and appropriate primers were designed and synthesized. The upstream homologous sequence of LEU2 and the LEU2 marker were amplified together from plasmid pRS405, and the upstream homologous sequence of HIS3 and the HIS3 marker were amplified together from plasmid pRS313. The DR-Kl URA3-DR nutrient tag sequence was obtained by PCR amplification using plasmid pWJ1042 (the full genome sequence is shown in SEQ ID NO:7) as a template.

[0039] 1. Construction of gene fragments

[0040] (1) Construction of gene fragment 1

[0041] A 631bp homologous sequence upstream of the yeast TRP1 site, the CYC1 terminator, the CarB gene, the GAL10 promoter, the GAL1 promoter, the CarRP gene, the PGK1 terminator, and a 733bp homologous sequence downstream of the yeast TRP1 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: TRP1 LHA-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-TRP1 RHA. This fragment was then ligated into the vector pJET1.2 (full gene sequence shown in SEQ ID NO.7, plasmid map shown in Figure 7) to obtain the gene fragment 1 integrated plasmid, denoted as [insert plasmid name here].

[0042] pJET-TRP1-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0043] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0044] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 1, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0045] (2) Construction of gene fragment 2

[0046] The following homologous sequences were amplified: a 394 bp upstream of the yeast ypl062w site, the DR-URA3-DR nutrient tag sequence, the CYC1 terminator, the CarB gene, the GAL10 promoter, the GAL1 promoter, the CarRP gene, the PGK1 terminator, and a 317 bp downstream of the yeast ypl062w site. These sequences were then sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends.

[0047] The gene fragment ypl062wLHA-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-ypl062w RHA was then ligated into the vector pJET1.2 to obtain the integrative plasmid containing gene fragment 2, denoted as ypl062wLHA-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-ypl062w RHA.

[0048] pJET-ypl062w-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0049] The integrative plasmid was transformed into competent E. coli DH5α cells, and colony PCR was used for screening. The plasmid was extracted and verified by enzyme digestion and sequencing to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0050] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 2, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0051] (3) Construction of gene fragment 3

[0052] The following sequences were amplified: a 426 bp homologous sequence upstream of the yeast gal7 site, the DR-URA3-DR nutrient tag sequence, the ERG10 gene and its terminator, the GAL7 promoter, the ACT1 terminator, the tHMGR1 gene, the GAL10 promoter, the GAL1 promoter, the CrtE gene, the GPM1 terminator, and a 234 bp homologous sequence downstream of the yeast gal1 site. These sequences were then sequentially spliced ​​together using overlap extension PCR to obtain fragments containing PmeI restriction sites at both ends.

[0053] The gene fragment is gal7LHA-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1-gal1 RHA. This fragment is then ligated into the vector pJET1.2 to obtain the integrative plasmid containing gene fragment 3, denoted as [insert plasmid name here].

[0054] pJET-gal-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PG AL1-CrtE-TGPM1.

[0055] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0056] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 3, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0057] (4) Construction of gene fragment 4

[0058] The following sequences were amplified: a 561 bp homologous sequence upstream of the yeast LEU2 site, the LEU2 marker, the TDH2 terminator, the ERG13 gene and its terminator, the GAL7 promoter, the ACT1 terminator, the tHMGR1 gene, the GAL10-GAL1 bidirectional promoter, the ERG12 gene and its terminator, and a 584 bp homologous sequence downstream of the yeast LEU2 site. These sequences were then sequentially spliced ​​using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: LEU2LHA-LEU2-TERG13-ERG13-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-ERG12-TERG12-LEU2 RHA. This fragment was then ligated into the vector pJET1.2 to obtain gene fragment 4 integrated plasmid, denoted as […].

[0059] pJET-LEU2-TERG13-ERG13-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-ERG12-TERG12.

[0060] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0061] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 4, the nucleotide sequence of which is shown in SEQ ID NO.4.

[0062] (5) Construction of gene fragment 5

[0063] The following sequences were amplified: a 312 bp homologous sequence upstream of the yeast HIS3 site, the HIS3 marker, the ENO2 terminator, the IDI1 gene and its terminator, the GAL7 promoter, the ERG19 gene and its terminator, the GAL10-GAL1 bidirectional promoter, the ERG8 gene and its terminator, and a 578 bp homologous sequence downstream of the yeast HI3 site. These sequences were then sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: HIS3LHA-HIS3-TIDI1-IDI1-PGAL7-TERG19-ERG19-PGAL10-PGAL1-ERG8-TERG8-HIS3RHA. This fragment was then ligated into the vector pJET1.2 to obtain a gene fragment 5 integrated plasmid, designated pJET-HIS3-TIDI1-IDI1-PGAL7-TERG19-ERG19-PGAL10-PGAL1-ERG8-TERG8.

[0064] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0065] After verification, the gene fragment was cut with PmeI restriction endonuclease to obtain gene fragment 5, the nucleotide sequence of which is shown in SEQ ID NO.5.

[0066] (6) Construction of gene fragment 6

[0067] A 244bp homologous sequence upstream of the yeast rox1 site, the HIS3 marker, the PGK1 promoter, the Vgb gene, the ACT1 terminator, the DR-URA3-DR nutrient tag sequence, and a 311bp homologous sequence downstream of the yeast rox1 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain the fragment rox1 LHA-PPGK1-Vgb-TACT1-DR-URA3-DR-rox1 RHA containing PmeI restriction sites at both ends. This fragment was then ligated into the vector pJET1.2 to obtain the integrative plasmid containing gene fragment 6, denoted as [insert fragment name here].

[0068] pJET-rox1-PPGK1-Vgb-TACT1-DR-URA3-DR.

[0069] The integrative plasmid was transformed into competent E. coli DH5α cells, colony PCR was used for screening, and the plasmid was extracted for enzyme digestion and sequencing verification to ensure that the target fragment was correctly ligated and that the base sequence had not been mutated.

[0070] After verification, the gene fragment 6 was obtained by cutting with PmeI restriction endonuclease, and its nucleotide sequence is shown in SEQ ID NO.6.

[0071] The schematic diagrams of gene fragments 1 to 6 are shown in Figures 1 to 6, respectively.

[0072] 2. Construction of Saccharomyces cerevisiae MBP_Sc21

[0073] Gene fragment 1 was transformed into *Saccharomyces cerevisiae* CEN.PK2-1D using the lithium acetate method. The fragment integrated into the yeast genome through homologous recombination between the upstream and downstream homologous sequences of TRP1 and the trp1 site. After transformation, the transformants were screened using SD-TRP solid plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan, and 2% agar powder). The transformed strains were streaked and purified, and the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were preserved as glycerol culture and named FNFH_Sc11.

[0074] Gene fragment 2 was transformed into *Saccharomyces cerevisiae* FNFH_Sc11 using the lithium acetate method. The fragment integrated into the genome through homologous recombination between the upstream and downstream homologous sequences of ypl062w and the ypl062w site on the yeast genome. After transformation, the transformants were screened on SD-TRP-URA solid plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan and uracil, 2% agar powder). Transformants were purified and cultured, and yeast genomes were extracted for PCR verification. Correctly verified recombinant strains were cultured on YPD liquid medium (20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract). A small amount of the culture was then spread onto 5-fluoroorotic acid (5-FOA) solid plates (because the DR-URA3-DR nutrient tag has 1...). The yeast itself utilizes two identical 43bp repetitive sequences (DRs) to undergo homologous recombination, deleting the URA3 gene and one of the DRs. Strains containing URA3 can convert 5-FOA into a cytotoxic substance, preventing growth on media containing 5-FOA, thus screening for strains with deleted URA3. Single colonies are isolated, cultured, and their genomes are extracted for PCR verification to screen for the correct strains that have deleted the URA3 gene through spontaneous recombination between DR sequences. The verified recombinant strains are preserved as glycerol culture and named FNFH_Sc12.

[0075] Gene fragment 3 was transformed into *Saccharomyces cerevisiae* FNFH_Sc12 using the lithium acetate method. It integrated into the yeast genome through homologous recombination with the upstream and downstream homologous sequences of gal7 and gal1, respectively. After transformation, the transformants were screened using SD-TRP-URA agar plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan and uracil, and 2% agar). Transformants were purified, cultured, and their genomes were extracted for PCR verification. Correctly verified recombinant strains were cultured in YPD liquid medium, and a small amount of the culture was spread onto 5-FOA agar plates. Single colonies were picked, purified, and their genomes were extracted for PCR verification. Strains that correctly deleted the URA3 gene through spontaneous recombination between DR sequences were screened. The correctly verified recombinant strains were preserved as glycerol culture and named FNFH_Sc13.

[0076] Gene fragment 4 was transformed into *Saccharomyces cerevisiae* FNFH_Sc13 using the lithium acetate method. The fragment integrated into the genome through homologous recombination between the upstream and downstream homologous sequences of LEU2 and the leu2 site on the yeast genome. After transformation, the transformants were screened using SD-TRP-LEU solid plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan and leucine, and 2% agar powder). The transformed strains were streaked and purified, and the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were preserved as glycerol culture and named FNFH_Sc14.

[0077] Gene fragment 5 was transformed into *Saccharomyces cerevisiae* FNFH_Sc14 using the lithium acetate method. The fragment integrated into the genome through homologous recombination between the upstream and downstream homologous sequences of HIS3 and the hiss3 site on the yeast genome. After transformation, the transformants were screened using SD-TRP-LEU-HIS solid plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan, leucine, and histidine, and 2% agar powder). The transformed strains were streaked and purified, and the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were preserved as glycerol culture and named FNFH_Sc15.

[0078] Gene fragment 6 was transformed into *Saccharomyces cerevisiae* FNFH_Sc15 using the lithium acetate method. It integrated into the genome through homologous recombination between the upstream and downstream homologous sequences of rox1 and the rox1 site on the yeast genome. After transformation, the transformants were screened using SD-TRP-LEU-HIS-URA solid plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan, leucine, histidine, and uracil, 2% agar powder). Transformants were purified, and yeast genomes were extracted for PCR verification. Correctly verified recombinant strains were cultured in YPD liquid medium, and a small amount of the culture was spread onto 5-FOA solid plates. Single colonies were picked, purified, and their genomes were extracted for PCR verification and screening. Strains that correctly deleted the URA3 gene through spontaneous recombination between DR sequences were selected. The correctly verified recombinant strains were preserved as glycerol culture and named MBP_Sc21.

[0079] Example 2: Application of Saccharomyces cerevisiae MBP_Sc21 in soybean meal fermentation

[0080] Experimental materials:

[0081] Strain: Saccharomyces cerevisiae MBP_Sc21.

[0082] Culture medium:

[0083] YPD medium: 20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract.

[0084] Test method:

[0085] 1. Soybean meal pretreatment: Weigh 20g of soybean meal and put it into a 250mL sterilized shake flask. Seal the flask opening with gauze and sealing film, autoclave at 100℃ for 30min, and cool to room temperature.

[0086] 2. Seed preparation: Saccharomyces cerevisiae MBP_Sc21 was inoculated into 5 mL of YPD medium and activated overnight at 30℃ and 250 rpm. It was then transferred to 50 mL of fresh YPD medium at OD = 0.5 and cultured at 30℃ and 250 rpm until the logarithmic growth phase.

[0087] 3. Solid-state fermentation: A 5% (v / m) inoculum of *Saccharomyces cerevisiae* MBP_Sc21 seed culture in the logarithmic growth phase was mixed with 1 mL of 100 g / L sterile D-(+) galactose solution and a certain volume of sterile water, and then evenly sprinkled into the pretreated soybean meal (total volume of mixed seed culture 20 mL, i.e., the initial moisture content of the soybean meal for fermentation is 50%). After thorough mixing, the shake flask was placed in a humidity-controlled shaker and fermented at 30℃ and 150 rpm for 48 h.

[0088] 4. Drying and testing: After fermentation, the soybean meal was dried at 50℃, pulverized and passed through a 60-mesh sieve, and the contents of crude protein, crude fiber, crude fat, ash, stachyose, raffinose, urease and carotenoids in the fermented soybean meal and raw soybean meal were tested respectively.

[0089] Carotenoid content: Take 1g of sample, add appropriate amount of quartz sand and acetone, shake to extract, centrifuge to collect organic phase, repeat the extraction step until the bacterial cells in the sample turn white. Combine all organic phases, filter, and determine carotenoid content by HPLC. The chromatographic column is a C18 column (4.6×150mm, 5μm), the column temperature is 30℃, the mobile phase is acetonitrile:methanol:dichloromethane = 21:21:8, the flow rate is 1mL / min, and the detection wavelength is 452nm.

[0090] Crude protein: Detected using the Kjeldahl method (GB / T 6432-2018).

[0091] Crude fiber: Tested by filtration method (GB / T 6434-2006).

[0092] Crude fat: Detected using Soxhlet extraction method (GB / T 6433-2006).

[0093] Ash content: Tested by the ignition method (GB / T 6438-2007).

[0094] Stachyose and raffinose were detected by high performance liquid chromatography (Appendix A of NY-T2218-2012).

[0095] Urease: Detected by spectrophotometry (GB / T 8622-2006).

[0096] Experimental results:

[0097] After fermenting soybean meal with Saccharomyces cerevisiae MBP_Sc21 for 24 hours, the resulting fermented soybean meal was orange-red, with a light and sour aroma, a delicate texture, no graininess, and good sensory quality (Figure 10).

[0098] The fermented soybean meal yielded significantly higher crude protein content and significantly lower anti-nutritional factor content. More importantly, it achieved a substantial accumulation of β-carotene, reaching 299.11 mg / kg (Table 1, Figure 11). Compared to the raw soybean meal, the fermented soybean meal showed a 7.9% increase in crude protein and a 100% decrease in the content of anti-nutritional factors such as stachyose, raffinose, and urease, respectively (Table 1). This demonstrates that fermenting soybean meal using Saccharomyces cerevisiae MBP_Sc21 significantly improves the overall nutritional parameters of the soybean meal, primarily through a substantial accumulation of the immunonutrient β-carotene, an increase in crude protein content, and a decrease in the levels of anti-nutritional factors such as oligosaccharides and urease. Therefore, it helps enhance the digestibility and utilization of soybean meal protein and improves its functional immunonutritional properties.

[0099] Table 1. Analysis of key components in soybean meal before and after fermentation with Saccharomyces cerevisiae MBP_Sc21

Claims

1. Saccharomyces cerevisiae MBP_Sc21, with a preservation number of CGMCC No. 32663 and a preservation date of November 15, 2024.

2. An inoculant containing the Saccharomyces cerevisiae MBP_Sc21 of claim 1.

3. Use of the Saccharomyces cerevisiae MBP_Sc21 of claim 1 or the inoculant of claim 2 in the fermentation of soybean meal.

4. Use of the Saccharomyces cerevisiae MBP_Sc21 of claim 1 or the inoculant of claim 2 in the fermentation of soybean meal to improve the nutritional value of the soybean meal.

5. The use according to claim 5, characterized in that, The improvement in the nutritional value of the soybean meal is manifested by the accumulation of a large amount of β-carotene, an increase in the content of crude protein, and a decrease in the content of anti-nutritional factors.

6. The use according to claim 5, wherein the compound is ###0002### The anti-nutritional factors include oligosaccharides and urease.

7. A process for the production of a β-carotene enriched fermented soybean meal, characterized by, The Saccharomyces cerevisiae MBP_Sc21 of claim 1 or the inoculant of claim 2 is used to ferment soybean meal.

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