Saccharomyces cerevisiae MBPSc22 and application thereof

Abstract

The invention relates to saccharomyces cerevisiae MBPSc22 and application thereof, and belongs to the technical field of microorganisms. On one hand, the invention provides a saccharomyces cerevisiae engineering strain MBPSc22 for synthesizing an immune nutrient, namely cantharidin, and the preservation number of the saccharomyces cerevisiae engineering strain MBPSc22 is CGMCC No. 35982; on the other hand, the invention provides application of the saccharomyces cerevisiae, the strain takes soybean meal as a solid-state fermentation substrate, the crude protein content of the obtained product fermented soybean meal is remarkably increased, anti-nutritional factors such as oligosaccharide and urease are greatly reduced, and more importantly, the fermented soybean meal is rich in cantharidin, so that the soybean meal protein obtains new functional immune nutrition; therefore, the whole nutritional value of the soybean meal is improved, and the method reaches a leading level in the field of developing functional fermented soybean meal by using saccharomyces cerevisiae.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a brewer's yeast strain MBP_Sc22 and its applications. Background Technology

[0002] The functional combination of high-quality protein and immune nutrients can systematically improve the immunity of farmed animals, making it one of the ideal solutions to the problem of "antibiotic-free" feed.

[0003] Soybean meal, as a high-quality plant protein source, can be processed using microbial fermentation technology to effectively degrade anti-nutritional factors and improve palatability and nutritional value, thus being widely used in the feed industry. Cantharidin is a known functional pigment-based immune enhancer and is permitted for use 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. Therefore, using cantharidin-producing strains to perform solid-state fermentation of soybean meal and other feed protein raw materials yields fermented feed containing cantharidin directly. This not only enhances the nutritional value and flavor of soybean meal protein but also avoids the extraction and additional addition of cantharidin, offering significant advantages such as simple processing and low production costs. The rapid development of synthetic biology technology provides a feasible solution for developing cantharidin-producing strains and functional fermented feed raw materials rich in cantharidin. Saccharomyces cerevisiae has always been a dominant strain in traditional food and fermentation industries, possessing good biosafety. Its live bacteria, inactive components, and cell components are widely used in the 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 conclusion, developing engineered *Saccharomyces cerevisiae* strains that efficiently synthesize cantharidin using synthetic biology techniques, and producing cantharidin-rich fermented feed ingredients through solid-state fermentation, is an effective pathway for the development and utilization of functional feeds. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of this invention is to provide a technical solution for Saccharomyces cerevisiae MBP_Sc22 and its application.

[0005] The first aspect of this invention provides a brewing yeast ( Saccharomyces cerevisiae MBP_Sc22, this strain was deposited on September 15, 2025 at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 35982.

[0006] A second aspect of the present invention provides a microbial agent containing Saccharomyces cerevisiae MBP_Sc22.

[0007] The third aspect of this invention provides the application of the above-mentioned brewing yeast MBP_Sc22 or the above-mentioned inoculum in soybean meal fermentation.

[0008] The fourth aspect of this invention provides the application of the above-mentioned brewer's yeast MBP_Sc22 or the above-mentioned inoculant in improving the nutritional value of soybean meal during soybean meal fermentation.

[0009] Furthermore, improving the nutritional value of soybean meal is specifically manifested in an increase in the content of immune nutrients, an increase in crude protein content, and a decrease in the level of anti-nutritional factors.

[0010] Furthermore, the immunonutrient is cantharidin, and the antinutritional factors include oligosaccharides and urease.

[0011] The fifth aspect of the present invention provides a method for producing fermented soybean meal rich in canthaxanthin, which uses the above-mentioned brewer's yeast MBP_Sc22 or the above-mentioned inoculum to ferment the soybean meal.

[0012] The Saccharomyces cerevisiae MBP_Sc22 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 canthaxanthin, which gives the soybean meal protein 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

[0013] Figure 1 This is a schematic diagram of gene elements for 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. Figure 2 This is a schematic diagram of gene elements for 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. Figure 3 This is a schematic diagram of gene elements for 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. Figure 4 This is a schematic diagram of gene elements for 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. Figure 5 This is a schematic diagram of gene elements for gene fragment 5; where gal80 LHA and gal80 RHA at both ends represent the upstream and downstream homologous sequences of the yeast gal80 site, respectively. Figure 6 The image shows the pJET1.2 plasmid. Figure 7 Image of plasmid pRS405; Figure 8 Before and after photos of fermentation of soybean meal with Saccharomyces cerevisiae MBP_Sc22 ((A) shake flask fermentation for 0 h; (B) shake flask fermentation for 36 h; (C) crushed soybean meal; (D) crushed fermented soybean meal). Figure 9 The image shows the chromatographic peaks of canthaxanthin in fermented soybean meal products made with Saccharomyces cerevisiae MBP_Sc22 ((A) canthaxanthin standard; (B) canthaxanthin in fermented soybean meal). Detailed Implementation

[0014] The present invention will be further described in detail below with reference to specific embodiments in order to better understand the technical solution.

[0015] Some of the plasmid vectors and strains involved in the embodiments of this invention are commercially available. For example, the pJET1.2 plasmid vector was purchased from Thermo Scientific's CloneJET PCR Cloning Kit, #K1231 (see plasmid map). Figure 6 ); plasmid pRS405 (plasmid map see...) Figure 7 The *Saccharomyces cerevisiae* strain CEN.PK2-1D was purchased from the EUROSCARF of Scientific Research and Development GmbH, Germany; *Saccharomyces cerevisiae* BY4742 was purchased from the National Center for Type Culture Collection.

[0016] 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: 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.

[0017] Gene fragment 2 (SEQ ID NO. 2) containing the CarB and CarRP genes from *Mucor truncatula*: 1-394bp is the 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 the 317bp homologous sequence downstream of the ypl062w site.

[0018] Gene fragment 3 (SEQ ID NO. 3) containing the CrtE gene from Archaeocystis 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 Archaeocystis scintillans; 7255-7654bp is the GPM1 terminator sequence; 7655-7888bp is a 234bp homologous sequence downstream of the gal1 site.

[0019] Gene fragment 4 (SEQ ID NO. 4) containing the CrtW gene from Chlamydomonas reinhardtii: 1-561bp is a 561bp homologous sequence upstream of the LEU2 site; 562-1656bp is the LEU2 marker; 1657-2056bp is the TDH2 terminator sequence; 2057-2513bp is the GAL1 promoter sequence; 2514-3848bp is the CrtW gene from Chlamydomonas reinhardtii; 3849-4067bp is the SpO1 terminator sequence; 4068-4651bp is a 584bp homologous sequence downstream of the LEU2 site.

[0020] Gene fragment 5 (SEQ ID NO. 5) containing the CrtW gene from Chlamydomonas reinhardtii: 1-362bp is a 362bp homologous sequence upstream of the gal80 site; 363-1919bp is the DR-URA3-DR nutrient tag sequence; 1920-2691bp is the GAL1 promoter sequence; 2692-4026bp is the CrtW gene from Chlamydomonas reinhardtii; 4027-4245bp is the SpO1 terminator sequence; 4246-4699bp is a 454bp homologous sequence downstream of the gal80 site.

[0021] 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.

[0022] Example 1

[0023] Explanation of the origin of gene elements The exogenous genes involved in this invention include the gerany pyrophosphate synthase gene CrtE, the bifunctional enzyme gene CarRP (phytoene synthase / lycopene cyclase), the phytoene dehydrogenase gene CarB, the β-carotene hydroxylase gene CrtZ, and the β-carotene ketolase gene CrtW. Among them, CrtE is derived from *Archaeopterygium scintillans* (…). Archaeoglobus fulgidus CarB and CarRP originate from Mucor ( ); Mucor circinelloides ), CrtW originates from Chlamydomonas reinhardtii ( Chlamydomonas reinhardtii All of the above genes were synthesized artificially after codon optimization and appropriate avoidance of commonly used restriction enzyme sites.

[0024] 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, TDH2 terminator, SpO1 terminator, ERG10 gene and ERG10 terminator, and the truncated 3-hydroxy-3-methylglutaryl-CoA reductase gene (tHMGR1), were obtained by PCR amplification using the genome of *Saccharomyces cerevisiae* strain BY4742 as a template, with suitable primers designed and synthesized. The upstream homologous sequence of LEU2 and the LEU2 marker were also obtained by PCR amplification from plasmid pRS405. The DR-KlURA3-DR nutrient tag sequence was obtained by PCR amplification using plasmid pWJ1042 (full genome sequence shown in SEQ ID NO:6) as a template.

[0025] 1. Construction of gene fragments

[0026] (1) Construction of gene fragment 1 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 (plasmid map shown). Figure 6 ), and the integrated plasmid of gene fragment 1 was obtained, denoted as: pJET-TRP1-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0027] 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.

[0028] 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.

[0029] (2) Construction of gene fragment 2 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 the fragment ypl062w LHA-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-ypl062w RHA, which contains PmeI restriction sites at both ends. This fragment was then ligated into the vector pJET1.2 to obtain the gene fragment 2 integrated plasmid, denoted as: pJET-ypl062w-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0030] 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.

[0031] 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.

[0032] (3) Construction of gene fragment 3 A 426bp 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 234bp homologous sequence downstream of the yeast gal1 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends. gal7LHA-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1-gal1 RHA. Then, it was ligated into the vector pJET1.2 to obtain the gene fragment 3 integration plasmid, denoted as: pJET-gal-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1.

[0033] 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.

[0034] After verification, the fragment was cut with PmeI restriction endonuclease to obtain gene fragment 3, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0035] (4) Construction of gene fragment 4 A 561bp homologous sequence upstream of the yeast LEU2 site, the LEU2 marker, the TDH2 terminator, the GAL1 promoter, the CrtW gene, the SpO1 terminator, and a 584bp homologous sequence downstream of the yeast LEU2 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: LEU2 LHA-LEU2-PGAL1-CrtW-TSPO1-LEU2 RHA. This fragment was then ligated into the vector pJET1.2 to obtain the gene fragment 4 integrated plasmid, denoted as pJET-LEU2-PGAL1-CrtW-TSPO1.

[0036] 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.

[0037] 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.

[0038] (5) Construction of gene fragment 5 A 362bp homologous sequence upstream of the yeast gal80 site, the DR-URA3-DR nutrient tag sequence, the GAL1 promoter, the CrtW gene, the SpO1 terminator, and a 454bp homologous sequence downstream of the yeast gal80 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: gal80 LHA-DR-URA3-DR-PGAL1-CrtW-TSPO1-gal80 RHA. This fragment was then ligated into the vector pJET1.2 to obtain a gene fragment 5 integrated plasmid, denoted as: pJET-gal80-DR-URA3-DR-PGAL1-CrtW-TSPO1.

[0039] 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.

[0040] 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.

[0041] The schematic diagrams of gene fragments 1-5 are shown below. Figures 1-5 .

[0042] 2. Construction of Saccharomyces cerevisiae MBP_Sc22

[0043] 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.

[0044] 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 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.

[0045] 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 powder). 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.

[0046] 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_Sc17.

[0047] Gene fragment 5 was transformed into *Saccharomyces cerevisiae* FNFH_Sc17 using the lithium acetate method. It integrated into the yeast genome through homologous recombination between the upstream and downstream homologous sequences of gal80 and the gal80 site. After transformation, the transformants were screened using SD-TRP-LEU-URA agar plates (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking tryptophan, leucine, 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 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 MBP_Sc22.

[0048] This strain was deposited on September 15, 2025, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession number is CGMCC No. 35982, and the suggested name is *Saccharomyces cerevisiae*. Saccharomyces cerevisiae ).

[0049] Example 2

[0050] Application of Saccharomyces cerevisiae MBP_Sc22 in soybean meal fermentation

[0051] Experimental materials: Strain: Saccharomyces cerevisiae MBP_Sc22.

[0052] YPD medium: 20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract.

[0053] Test method: 1. Soybean meal pretreatment: Weigh 150 g of soybean meal and place it in a 2 L sterilized shake flask. Seal the flask opening with gauze and sealing film, autoclave at 100℃ for 30 min, and cool to room temperature.

[0054] 2. Seed preparation: Saccharomyces cerevisiae MBP_Sc22 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.

[0055] 3. Solid-state fermentation: A 5% (v / m) inoculum of *Saccharomyces cerevisiae* MBP_Sc22 seed culture, cultured to the logarithmic growth phase, was mixed with a certain volume of sterile water and evenly sprinkled into the pretreated soybean meal (total volume of the mixed seed culture was 150 mL, meaning the initial moisture content of the soybean meal for fermentation was 50%). After thorough mixing, the shake flask was placed in a humidity-controlled shaker and fermented at 30℃ and 150 rpm for 48 h.

[0056] 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 such as canthaxanthin were tested in the fermented soybean meal and the raw soybean meal.

[0057] Cantharidin 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 detect carotenoid content by HPLC. Specifically, a Hypersil GOLD™ C18 column (150×4.6 mm, 5 μm) was used, with methanol-acetonitrile-dichloromethane (21:21:8, v / v) as the mobile phase, flow rate 1.0 mL / min, column temperature 30 ℃, and cantharidin detection wavelength 470 nm.

[0058] Crude protein: Detected using the Kjeldahl method (GB / T 6432-2018).

[0059] Crude fiber: Tested by filtration method (GB / T 6434-2006).

[0060] Crude fat: Detected using Soxhlet extraction method (GB / T 6433-2006).

[0061] Ash content: Tested by the ignition method (GB / T 6438-2007).

[0062] Stachyose and raffinose: Detected by high performance liquid chromatography (Appendix A of NY-T2218-2012).

[0063] Urease: Detected by spectrophotometry (GB / T 8622-2006).

[0064] Experimental results: After fermenting soybean meal with Saccharomyces cerevisiae MBP_Sc22 for 36 hours, the resulting fermented soybean meal was orange-red in color, with a light, sour aroma, a delicate texture, and no graininess, exhibiting good sensory quality. Figure 8 ).

[0065] The resulting fermented soybean meal showed a significant increase in crude protein content and a significant decrease in anti-nutritional factors. More importantly, the product exhibited a substantial accumulation of cantharidin. HPLC analysis revealed that the peak area of ​​cantharidin in the fermented soybean meal was 1498.94 mAU·min, close to that of the standard cantharidin (1674.11 mAU·min), and the cantharidin content reached 101.41 mg / kg (Table 1). Figure 9 Compared to raw soybean meal, fermented soybean meal showed a 7.7% increase in crude protein and a 100% decrease, 75.7% decrease, 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_Sc22 significantly improves the overall nutritional parameters of the soybean meal, primarily through a substantial accumulation of the immunonutrient canthaxanthin, 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 immunonutrition.

[0066] Table 1. Analysis of key components of soybean meal before and after fermentation with brewer's yeast MBP_Sc22 .

Claims

1. A type of brewing yeast ( Saccharomyces cerevisiae MBP_Sc22, with accession number CGMCC No.35982, was deposited on September 15, 2025.

2. A microbial agent containing the Saccharomyces cerevisiae MBP_Sc22 as described in claim 1.

3. The application of the brewing yeast MBP_Sc22 as described in claim 1 or the inoculum as described in claim 2 in soybean meal fermentation.

4. The application of the brewing yeast MBP_Sc22 as described in claim 1 or the inoculant as described in claim 2 in improving the nutritional value of soybean meal during soybean meal fermentation.

5. The application as described in claim 4, characterized in that, The improvement in the nutritional value of soybean meal is specifically manifested in the increased content of immune nutrients, the increased crude protein content, and the decreased level of anti-nutritional factors.

6. The application as described in claim 5, characterized in that, The immunonutrient is cantharidin, and the antinutritional factors include oligosaccharides and urease.

7. A method for producing fermented soybean meal rich in canthaxanthin, characterized in that, Soybean meal is fermented using the brewing yeast MBP_Sc22 described in claim 1 or the inoculum described in claim 2.

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