Saccharomyces cerevisiae FNFH_sc18 and use thereof

Abstract

The present invention belongs to the technical field of microorganisms. Provided are Saccharomyces cerevisiae FNFH_Sc18 and the use thereof. In one aspect, provided is an engineered Saccharomyces cerevisiae strain FNFH_Sc18 for synthesizing immunonutrients β-carotene and astaxanthin, which strain has a deposit number of CGMCC No. 24838. In another aspect, provided is the use of the Saccharomyces cerevisiae. The strain uses soybean meal as a solid-state fermentation substrate, the resulting fermented soybean meal product exhibits significantly increased contents of crude proteins and amino acids, and substantially reduced anti-nutritional factors, such as oligosaccharides and urease. More importantly, the fermented soybean meal is rich in β-carotene and astaxanthin, which imparts new functional immunonutritional properties to a soybean meal protein, thereby improving the overall nutritional value of the soybean meal, and attaining a leading level in the field of developing functional fermented soybean meal by using Saccharomyces cerevisiae.

Description

A brewing yeast FNFH_Sc18 and its application Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a brewer's yeast strain FNFH_Sc18 and its applications. 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, coupled with low production costs and high efficiency, is widely used in the feed industry. Astaxanthin and β-carotene are known functional pigments that enhance immunity and are permitted additives in my country's "List of Feed Additives (2013)". They enable 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. Solid-state fermentation of feed protein raw materials such as soybean meal using carotenoid-producing microorganisms yields fermented feed directly containing carotenoids such as β-carotene and astaxanthin. This not only enhances the nutritional value and flavor of soybean meal protein but also avoids the extraction and additional addition of carotenoids, resulting in a simple process with low production costs. Chinese patent CN118160870A discloses a method and its application for producing natural astaxanthin in poultry compound feed using solid-state fermentation of Pharbitis rubrum. The method uses a Pharbitis rubrum strain P406 obtained from screening to carry out solid-state fermentation of fermentation substrate (60% corn flour, 25% soybean meal, and 15% wheat bran). After fermentation, the astaxanthin content in the feed can reach 17.50±0.31 mg / kg, which significantly improves the egg production quality of laying hens.

[0004] The rapid development of synthetic biology technology has provided a feasible solution for producing functional fermented feed ingredients rich in higher levels of carotenoids. Saccharomyces cerevisiae has always been a dominant strain in traditional food and fermentation industries due to its superior biocompatibility; 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 phytic acid and trypsin inhibitor levels (Hassaan et al., 2015). Chinese patent CN116790703A discloses "a yeast engineered strain for producing astaxanthin and its construction method and application," which, by upregulating MVA pathway-related genes, downregulating the ERG9 gene, and heterologously expressing astaxanthin synthesis genes, achieved a yield of 708 mg / L in a 2L liquid fermenter, with astaxanthin extracted from the fermentation broth. Therefore, developing engineered Saccharomyces cerevisiae using synthetic biology technology, and then producing fermented feed ingredients rich in β-carotene and astaxanthin through solid-state fermentation, is essential for the development and utilization of functional feeds. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a technical solution for Saccharomyces cerevisiae FNFH_Sc18 and its application.

[0006] The first aspect of this invention provides a Saccharomyces cerevisiae strain FNFH_Sc18, which was deposited on May 6, 2022, 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. 24838.

[0007] A second aspect of the present invention provides a microbial agent containing Saccharomyces cerevisiae FNFH_Sc18.

[0008] The third aspect of the present invention provides the application of the above-mentioned brewing yeast FNFH_Sc18 or the above-mentioned inoculum in soybean meal fermentation.

[0009] The fourth aspect of this invention provides the application of the above-mentioned brewing yeast FNFH_Sc18 or the above-mentioned inoculant in improving the nutritional value of soybean meal during soybean meal fermentation.

[0010] Furthermore, improving the nutritional value of soybean meal is specifically manifested in an increase in the content of immune nutrients, an upregulation of crude protein and total amino acid levels, and a decrease in the level of anti-nutritional factors.

[0011] Furthermore, the immunonutrients include astaxanthin and β-carotene, and the antinutritional factors include oligosaccharides and urease.

[0012] The fifth aspect of the present invention provides a method for producing fermented soybean meal rich in astaxanthin and β-carotene, wherein the soybean meal is fermented using the above-mentioned brewer's yeast FNFH_Sc18 or the above-mentioned inoculant.

[0013] The Saccharomyces cerevisiae FNFH_Sc18 of this invention uses soybean meal as a solid-state fermentation substrate. The resulting fermented soybean meal has significantly increased crude protein and amino acid content, and significantly reduced anti-nutritional factors such as oligosaccharides and urease. More importantly, the fermented soybean meal is rich in β-carotene and astaxanthin, which gives the soybean meal protein new functional immune nutrition, thereby improving the overall nutritional value of 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 shows the spectrum of plasmid pJET1.2;

[0020] Figure 7 shows the spectrum of plasmid pRS405;

[0021] Figure 8 shows the spectrum of plasmid pRS313;

[0022] Figure 9 shows before and after photos of fermentation of soybean meal by Saccharomyces cerevisiae FNFH_Sc18 ((A) shake flask fermentation 0h; (B) shake flask fermentation 24h; (C) crushed soybean meal; (D) crushed fermented soybean meal).

[0023] Figure 10 shows the carotenoid content of fermented soybean meal products made with Saccharomyces cerevisiae FNFH_Sc18 ((A) fermented soybean meal before grinding; (B) HPLC peak chromatogram of fermented soybean meal before grinding; (C) fermented soybean meal after grinding; (D) HPLC peak chromatogram of fermented soybean meal after grinding; (E) Carotenoid content in fermented soybean meal before and after grinding). Detailed Implementation

[0024] The present invention will be further described in detail below with reference to specific embodiments in order to better understand the technical solution.

[0025] 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 (plasmid map shown in Figure 6); plasmid pRS405 (plasmid map shown in Figure 7); plasmid pRS313 (plasmid map shown in Figure 8); 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.

[0026] 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:

[0027] Gene fragment 1 (SEQ ID NO.1) containing the CarB and CarRP genes from *Mucor truncatula*: 1-631 bp is the 631 bp homologous sequence upstream of the TRP1 site; 632-886 bp is the CYC1 terminator sequence; 887-2626 bp is the CarB gene from *Mucor truncatula*; 2627-3294 bp is the GAL10-GAL1 bidirectional promoter sequence; 3295-5139 bp is the CarRP gene from *Mucor truncatula*; 5140-5414 bp is the PGK1 terminator sequence; 5415-6147 bp is the 733 bp homologous sequence downstream of the TRP1 site.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] Gene fragment 5 (SEQ ID NO. 5) containing the CrtZ gene from Paracoccus: 1-312bp is a 312bp homologous sequence upstream of the HIS3 site; 313-975bp is the HIS3 marker; 976-1375bp is the ENO2 terminator; 1376-1832bp is the GAL1 promoter sequence; 1833-2321bp is the CrtZ gene from Paracoccus; 2322-2521bp is the HIS5 terminator sequence; 2522-3099bp is a 578bp homologous sequence downstream of the HI3 site.

[0032] 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.

[0033] Example 1

[0034] Explanation of the origin of gene elements

[0035] The exogenous genes involved in this invention include the geranylide pyrophosphate synthase gene CrtE, the bifunctional enzyme gene CarRP (lycopene synthase / lycopene cyclase), the lycopene dehydrogenase gene CarB, the β-carotene hydroxylase gene CrtZ, and the β-carotene ketolase gene CrtW. CrtE is derived from *Archaeoglobus fulgidus*; CarB and CarRP are derived from *Mucor circinelloides*; CrtZ is derived from *Paracoccus sp. N81106*; and CrtW is derived from *Chlamydomonas reinhardtii*. All of these genes were synthesized artificially after codon optimization and appropriate avoidance of commonly used restriction enzyme sites.

[0036] 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, ENO2 terminator, SpO1 terminator, HIS5 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 and with appropriately designed and synthesized primers. 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:6) as a template.

[0037] 1. Construction of gene fragments

[0038] (1) Construction of gene fragment 1

[0039] 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. 6, plasmid map shown in Figure 6) to obtain the gene fragment 1 integrated plasmid, denoted as:

[0040] pJET-TRP1-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0041] 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.

[0042] 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.

[0043] (2) Construction of gene fragment 2

[0044] 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.

[0045] 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.

[0046] 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.

[0047] (3) Construction of gene fragment 3

[0048] 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.

[0049] 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:

[0050] pJET-gal-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PG AL1-CrtE-TGPM1.

[0051] 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.

[0052] 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.

[0053] (4) Construction of gene fragment 4

[0054] 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.

[0055] 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.

[0056] 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.

[0057] (5) Construction of gene fragment 5

[0058] A 312bp homologous sequence upstream of the yeast HIS3 site, the HIS3 marker, the ENO2 terminator, the GAL1 promoter, the CrtZ gene, the HIS5 terminator, and a 578bp homologous sequence downstream of the yeast HI3 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain a fragment containing PmeI restriction sites at both ends: HIS3 LHA-HIS3-PGAL1-CrtZ-THIS5-HIS3 RHA. This fragment was then ligated into the vector pJET1.2 to obtain the gene fragment 4 integrated plasmid, denoted as pJET-HIS3-PGAL1-CrtZ-THIS5.

[0059] 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.

[0060] 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.

[0061] The schematic diagrams of gene fragments 1 to 5 are shown in Figures 1 to 5, respectively.

[0062] 2. Construction of Saccharomyces cerevisiae FNFH_Sc18

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] Gene fragment 5 was transformed into *Saccharomyces cerevisiae* FNFH_Sc17 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_Sc18.

[0068] This strain was deposited on May 6, 2022, 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. 24838. It is recommended to name it Saccharomyces cerevisiae.

[0069] Example 2

[0070] Application of brewer's yeast FNFH_Sc18 in soybean meal fermentation

[0071] Experimental materials:

[0072] Strain: Saccharomyces cerevisiae FNFH_Sc18.

[0073] Culture medium:

[0074] YPD medium: 20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract.

[0075] Test method:

[0076] 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.

[0077] 2. Seed preparation: Saccharomyces cerevisiae FNFH_Sc18 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.

[0078] 3. Solid-state fermentation: A 5% (v / m) inoculum of *Saccharomyces cerevisiae* FNFH_Sc18 cultured to 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 the mixed seed culture was 20 mL, i.e., 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.

[0079] 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, as well as carotenoids such as astaxanthin and β-carotene in the fermented soybean meal and raw soybean meal were tested respectively.

[0080] 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), column temperature 25℃. Mobile phase A: acetonitrile:water = 9:1; mobile phase B: methanol:isopropanol = 3:2. Chromatographic conditions: initial conditions 0% B phase, 0-13min 0-90% B phase, 13-23min 90% B phase, 23-28min 90-0% B phase, 28-35min 0% B phase. Astaxanthin detection wavelength is 470nm, β-carotene detection wavelength is 450nm.

[0081] Crude protein: Detected using the Kjeldahl method (GB / T 6432-2018).

[0082] Crude fiber: Tested by filtration method (GB / T 6434-2006).

[0083] Crude fat: Detected by Soxhlet extraction (GB / T 6433-2006).

[0084] Ash content: Tested by the ignition method (GB / T 6438-2007).

[0085] Stachyose and raffinose were detected by high performance liquid chromatography (Appendix A of NY-T2218-2012).

[0086] Urease: Detected by spectrophotometry (GB / T 8622-2006).

[0087] Amino acids: detected by conventional acid hydrolysis method (GB / T 18246-2019).

[0088] Experimental results:

[0089] After fermenting soybean meal with brewer's yeast FNFH_Sc18 for 48 hours, the resulting fermented soybean meal was brownish-red, with a light and sour aroma, a delicate texture, no graininess, and good sensory quality (Figure 9).

[0090] The resulting fermented soybean meal showed significantly increased protein and amino acid content, and significantly decreased anti-nutritional factors. More importantly, the product exhibited substantial accumulation of astaxanthin and β-carotene (Table 1). In the dried, uncrushed soybean meal fermented with *Saccharomyces cerevisiae* FNFH_Sc18, the astaxanthin and β-carotene contents reached 51.20 mg / kg and 96.13 mg / kg, respectively (Figure 10). After drying and pulverizing the fermented soybean meal, the astaxanthin and β-carotene contents showed varying degrees of loss, with astaxanthin and β-carotene contents of 40.48 mg / kg and 25.59 mg / kg, respectively (Figure 10, Table 1). Furthermore, compared to the raw soybean meal, the fermented soybean meal showed a 7.8% increase in crude protein, a 20.6% increase in total amino acid content, and a 100% decrease in the contents of anti-nutritional factors such as stachyose, raffinose, and urease, respectively (Table 1). This demonstrates that fermenting soybean meal with Saccharomyces cerevisiae FNFH_Sc18 significantly improves the overall nutritional parameters of soybean meal, mainly manifested in the substantial accumulation of immunonutrients such as astaxanthin and β-carotene, the upregulation of crude protein and total amino acid levels, and the decrease of anti-nutritional factors such as oligosaccharides and urease. Therefore, it helps to enhance the digestibility and utilization of soybean meal protein raw materials and its functional immunonutrients.

[0091] Table 1. Analysis of key components in soybean meal before and after fermentation with Saccharomyces cerevisiae FNFH_Sc18

Claims

1. A type of Saccharomyces cerevisiae, FNFH_Sc18, with accession number CGMCC No. 24838 and accession date of May 6, 2022.

2. A microbial agent containing the Saccharomyces cerevisiae FNFH_Sc18 as described in claim 1.

3. The application of the brewing yeast FNFH_Sc18 as described in claim 1 or the inoculum as described in claim 2 in soybean meal fermentation.

4. The application of the brewing yeast FNFH_Sc18 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 5, characterized in that, The improvement in the nutritional value of soybean meal is specifically manifested in the increased content of immune nutrients, the increased levels of crude protein and total amino acids, and the decreased levels of anti-nutritional factors.

6. The application as described in claim 5, characterized in that, The immunonutrients include astaxanthin and beta-carotene, and the antinutritional factors include oligosaccharides and urease.

7. A method for producing fermented soybean meal rich in astaxanthin and β-carotene, characterized in that, Soybean meal is fermented using the brewing yeast FNFH_Sc18 described in claim 1 or the inoculum described in claim 2.

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