Saccharomyces cerevisiae FNFHSc18 and application thereof

Abstract

The invention discloses saccharomyces cerevisiae FNFHSc18 and application thereof, and belongs to the technical field of microorganisms. On one hand, the invention provides a saccharomyces cerevisiae engineering strain FNFHSc18 for synthesizing immune nutrients-beta-carotene and astaxanthin, and the preservation number of the saccharomyces cerevisiae engineering strain is CGMCC (China General Microbiological Culture Collection Center) No.24838; on the other hand, the invention provides application of the saccharomyces cerevisiae, the strain takes soybean meal as a solid state fermentation substrate, the content of crude protein and amino acid of the obtained product fermented soybean meal is remarkably increased, anti-nutritional factors such as oligosaccharide and urease are greatly reduced, more importantly, the fermented soybean meal is rich in beta-carotene and astaxanthin, and the content of the beta-carotene and the astaxanthin is greatly reduced. The soybean meal protein obtains new functional immune nutrition, so that the overall nutritional value of the soybean meal is improved, and the soybean meal reaches a leading level in the field of developing functional fermented soybean meal by utilizing saccharomyces cerevisiae.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Saccharomyces cerevisiae FNFH_Sc18 and its application. Background Art

[0002] Fermentation has become a hot topic in the current feed industry. Developing functional feeds has become an industry consensus, and the key is to develop functional feed raw materials. The functional combination of high-quality protein and immune nutrients can systematically improve the body immunity of the breeding objects, which is one of the ideal solutions to the problem of "antibiotic-free" in feeds.

[0003] Fermenting soybean meal by microorganisms can remove the anti-nutritional factors of soybean meal, improve the flavor and nutritional value. Coupled with low production cost and high efficiency, it is widely used in the feed industry. Astaxanthin and β-carotene are pigment immune enhancers with well-known and clear functions. They can enable farm animals to grow faster and have better feed conversion rates, especially with remarkable effects in improving immunity. Currently, the carotenoids consumed by the aquaculture industry exceed 300 million US dollars annually. Using carotenoid-producing strains to perform solid-state fermentation on feed protein raw materials such as soybean meal, the obtained fermented feed directly contains carotenoids such as β-carotene and astaxanthin, which can not only improve the nutritional value and flavor of soybean meal protein, but also avoid the extraction and additional addition processes of carotenoids. The process is simple and the production cost is low. Chinese Patent CN118160870A discloses "A method for producing natural astaxanthin poultry compound feed by solid-state fermentation using Rhodotorula rubra and its application", using a screened Rhodotorula rubra P406 to perform solid-state fermentation on a fermentation substrate (60% corn flour, 25% soybean meal, 15% wheat bran). After fermentation, the astaxanthin content in the feed can reach 17.50 ± 0.31 mg / kg, significantly improving the egg-laying quality of laying hens.

[0004] The rapid development of synthetic biology technology has provided a feasible solution for realizing functional fermented feed ingredients rich in higher levels of carotenoids. Saccharomyces cerevisiae has always been a dominant strain in traditional food and fermentation industries, with higher biosafety. Its live bacteria, inactive components, and cell components are widely used in the aquaculture and feed industries. Research has shown that fermenting soybean meal with Saccharomyces cerevisiae can significantly increase the crude protein and amino acid content of soybean meal and reduce the levels of phytic acid and trypsin inhibitor (Hassaan et al., 2015). Chinese Patent CN116790703A discloses "A yeast engineering strain for producing astaxanthin, its construction method, and application". By upregulating genes related to the MVA pathway, downregulating the ERG9 gene, and heterologously expressing astaxanthin synthesis genes, the engineered Saccharomyces cerevisiae strain constructed has a yield of 708 mg / L in a 2L liquid fermentation tank, and astaxanthin is obtained after extraction from the fermentation broth. Therefore, it is very necessary to develop an engineered Saccharomyces cerevisiae strain using synthetic biology technology and then produce fermented feed ingredients rich in β-carotene and astaxanthin through solid-state fermentation for the development and utilization of functional feeds. Summary of the Invention

[0005] Aiming at the problems existing in the prior art, the purpose of the design of the present invention is to provide a technical solution for a Saccharomyces cerevisiae FNFH_Sc18 and its application.

[0006] In the first aspect of the present invention, a Saccharomyces cerevisiae FNFH_Sc18 is provided. This strain was deposited at the China General Microbiological Culture Collection Center (CGMCC) on May 6, 2022. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its deposit number is: CGMCC No. 24838.

[0007] In the second aspect of the present invention, a bacterial agent containing Saccharomyces cerevisiae FNFH_Sc18 is provided.

[0008] In the third aspect of the present invention, the above-mentioned Saccharomyces cerevisiae FNFH_Sc18 or the above-mentioned bacterial agent is provided for use in the fermentation of soybean meal.

[0009] In the fourth aspect of the present invention, the above-mentioned Saccharomyces cerevisiae FNFH_Sc18 or the above-mentioned bacterial agent is provided for use in improving the nutritional value of soybean meal during the fermentation of soybean meal.

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

[0011] Furthermore, the immune nutrients include astaxanthin and β-carotene, and the anti-nutritional factors include oligosaccharides and urease.

[0012] The fifth aspect of the present invention provides a production method of fermented soybean meal rich in astaxanthin and β-carotene, which ferments soybean meal by using the above-mentioned Saccharomyces cerevisiae FNFH_Sc18 or the above-mentioned microbial agent.

[0013] The Saccharomyces cerevisiae FNFH_Sc18 of the present invention uses soybean meal as a solid fermentation substrate. The crude protein and amino acid contents of the obtained product, fermented soybean meal, are significantly increased, and anti-nutritional factors such as oligosaccharides and urease are greatly reduced. More importantly, the fermented soybean meal is rich in β-carotene and astaxanthin, endowing the soybean meal protein with new functional immune nutrition, thereby enhancing the overall nutritional value of soybean meal and reaching the leading level in the field of developing functional fermented soybean meal by using Saccharomyces cerevisiae. Brief Description of the Drawings

[0014] Figure 1 It is a gene element pattern diagram of gene fragment 1; wherein, the two ends TRP1LHA and TRP1RHA respectively represent the upstream and downstream homologous sequences of the yeast TRP1 locus;

[0015] Figure 2 It is a gene element pattern diagram of gene fragment 2; wherein, the two ends ypl062w LHA and ypl062w RHA respectively represent the upstream and downstream homologous sequences of the yeast ypl062w locus;

[0016] Figure 3 It is a gene element pattern diagram of gene fragment 3; wherein, the two ends gal7LHA and gal1RHA respectively represent the upstream homologous sequence of the yeast gal7 locus and the downstream homologous sequence of the gal1 locus;

[0017] Figure 4 It is a gene element pattern diagram of gene fragment 4; wherein, the two ends LEU2LHA and LEU2RHA respectively represent the upstream and downstream homologous sequences of the yeast LEU2 locus;

[0018] Figure 5 It is a gene element pattern diagram of gene fragment 5; wherein, the two ends HIS3LHA and HIS3RHA respectively represent the upstream and downstream homologous sequences of the yeast HIS3 locus;

[0019] Figure 6 It is a map of plasmid pJET1.2;

[0020] Figure 7 It is a map of plasmid pRS405;

[0021] Figure 8 It is a map of plasmid pRS313;

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

[0023] Figure 10 The carotenoid content of the fermented soybean meal product by Saccharomyces cerevisiae FNFH_Sc18 is shown ((A) Fermented soybean meal before crushing; (B) HPLC peak map of fermented soybean meal before crushing; (C) Fermented soybean meal after crushing; (D) HPLC peak map of fermented soybean meal after crushing; (E) Carotenoid content in fermented soybean meal before and after crushing). Detailed implementation manners

[0024] The following further describes the present invention in detail with specific embodiments to better understand the technical solution.

[0025] Some plasmid vectors and strains involved in the embodiments of the present invention are commercially available. For example, the pJET1.2 plasmid vector is purchased from CloneJET PCR Cloning Kit of Thermo Scientific, #K1231 (plasmid map is shown in Figure 6 ); plasmid pRS405 (plasmid map is shown in Figure 7 ); plasmid pRS313 (plasmid map is shown in Figure 8 ); The Saccharomyces cerevisiae strain CEN.PK2-1D is purchased from EUROSCARF of Scientific Research and Development GmbH, Germany; the Saccharomyces cerevisiae BY4742 is purchased from the National Collection of Type Cultures.

[0026] For each gene element used in the construction of the recombinant yeast strain of the present invention, such as amino acid markers, tags, endogenous genes, exogenous genes, etc., are well-known in the art, and those skilled in the art know their specific sequences. For the convenience of understanding the present invention, the present invention describes each gene element in each gene fragment:

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

[0028] Gene fragment 2 containing the CarB and CarRP genes from Mucor circinelloides (shown in SEQ ID NO.2): 1-394bp is the 394bp homologous sequence upstream of the ypl062w locus; 395-1951bp is the DR-URA3-DR nutritional label sequence; 1952-2206bp is the CYC1 terminator sequence; 2207-3946bp is the CarB gene from Mucor circinelloides; 3947-4614bp is the GAL10-GAL1 bidirectional promoter sequence; 4615-6459bp is the CarRP gene from Mucor circinelloides; 6460-6734bp is the PGK1 terminator sequence; 6735-7051bp is the 317bp homologous sequence downstream of the ypl062w locus.

[0029] Gene fragment 3 containing the CrtE gene from Archaeoglobus fulgidus (shown in SEQ ID NO.3): 1-426bp is the 426bp homologous sequence upstream of the gal7 locus; 427-1983bp is the DR-URA3-DR nutritional label 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 Archaeoglobus fulgidus; 7255-7654bp is the GPM1 terminator sequence; 7655-7888bp is the 234bp homologous sequence downstream of the gal1 locus.

[0030] Gene fragment 4 containing the CrtW gene from Chlamydomonas reinhardtii (shown in SEQ ID NO.4): 1-561bp is the 561bp homologous sequence upstream of the LEU2 locus; 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 the 584bp homologous sequence downstream of the LEU2 locus.

[0031] Gene fragment 5 containing the CrtZ gene from Paracoccus sp. (shown in SEQ ID NO.5): 1-312bp is the 312bp homologous sequence upstream of the HIS3 locus; 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 sp.; 2322-2521bp is the HIS5 terminator sequence; 2522-3099bp is the 578bp homologous sequence downstream of the HI3 locus.

[0032] After knowing the specific sequences of the above gene elements, those skilled in the art can perform amplification and OE-PCR splicing according to the conventional primer design principles. At the same time, the SD medium used in the present invention is a commonly used medium in the field of yeast screening. By deliberately deleting one or several components from the basic medium according to the gene defects of yeast, the target strain can be screened out.

[0033] Example 1

[0034] Source description of gene elements

[0035] The exogenous genes involved in the present invention include geranyl pyrophosphate synthase gene CrtE, bifunctional enzyme gene CarRP of phytoene synthase / lycopene cyclase, phytoene dehydrogenase gene CarB, β-carotene hydroxylase gene CrtZ, and β-carotene ketolase gene CrtW. Among them, the source of CrtE is Archaeoglobus fulgidus; the sources of CarB and CarRP are Mucor circinelloides, the source of CrtZ is Paracoccus sp. N81106, and the source of CrtW is Chlamydomonas reinhardtii. The above genes are all obtained by artificial synthesis after codon optimization and appropriate avoidance of common restriction enzyme cleavage sites.

[0036] Promoters, terminators, endogenous genes in Saccharomyces cerevisiae, and related upstream and downstream homologous sequences, including 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, truncated 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (tHMGR1), etc., were designed and synthesized with appropriate primers using the genome of Saccharomyces cerevisiae strain BY4742 as a template and obtained by PCR amplification. The LEU2 upstream homologous sequence and the LEU2 marker were amplified by PCR from plasmid pRS405 together, and the HIS3 upstream homologous sequence and the HIS3 marker were amplified by PCR from plasmid pRS313 together. The DR-KlURA3-DR nutritional tag sequence was obtained by PCR amplification using plasmid pWJ1042 (the full gene 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] Amplify the 631 bp homologous sequence upstream of the yeast TRP1 locus, CYC1 terminator, CarB gene, GAL10 promoter, GAL1 promoter, CarRP gene, PGK1 terminator, and the 733 bp homologous sequence downstream of the yeast TRP1 locus, and splice them together sequentially by the overlap extension PCR method to obtain the fragment TRP1LHA-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-TRP1RHA containing PmeI restriction sites at both ends. Then ligate it into vector pJET1.2 (the full gene sequence is shown in SEQ ID NO.6, and the plasmid map is shown in Figure 6 ), to obtain the gene fragment 1 integration plasmid, denoted as:

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

[0041] The integration plasmid was transformed into Escherichia coli competent DH5α, screened by colony PCR, and the plasmid was extracted for restriction enzyme digestion verification and sequencing verification to ensure that the target fragment was ligated correctly and the base sequence did not mutate.

[0042] After correct verification, it was cut with PmeI restriction endonuclease to obtain gene fragment 1, and the nucleotide sequence is shown in SEQ ID NO.1.

[0043] (2) Construction of gene fragment 2

[0044] Amplify the 394 bp homologous sequence upstream of the yeast ypl062w locus, the DR-URA3-DR nutritional label sequence, the CYC1 terminator, the CarB gene, the GAL10 promoter, the GAL1 promoter, the CarRP gene, the PGK1 terminator, and the 317 bp homologous sequence downstream of the yeast ypl062w locus, and splice them sequentially by the overlap extension PCR method to obtain the fragment ypl062wLHA-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-yp l062w RHA containing PmeI restriction sites at both ends. Then ligate it into the vector pJET1.2 to obtain the gene fragment 2 integration plasmid, denoted as: pJET-ypl062w-DR-URA3-DR-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0045] Transform the integration plasmid into the competent Escherichia coli DH5α, screen by colony PCR, extract the plasmid for restriction enzyme digestion verification and sequencing verification to ensure that the target fragment is ligated correctly and the base sequence has not mutated.

[0046] After verification, cut with the PmeI restriction enzyme to obtain gene fragment 2, and the nucleotide sequence is shown in SEQ ID NO.2.

[0047] (3) Construction of gene fragment 3

[0048] Amplify the 426 bp homologous sequence upstream of the yeast gal7 locus, the DR-URA3-DR nutritional label 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 the 234 bp homologous sequence downstream of the yeast gal1 locus, and splice them sequentially by the overlap extension PCR method to obtain the fragment: gal7LHA-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1-gal1RHA containing PmeI restriction sites at both ends. Then ligate it into the vector pJET1.2 to obtain the gene fragment 3 integration plasmid, denoted as:

[0049] pJET-gal-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1.

[0050] The integrated plasmid was transformed into competent Escherichia coli DH5α, and colonies were screened by PCR. The plasmid was extracted for restriction enzyme digestion verification and sequencing verification to ensure that the target fragment was ligated correctly and the base sequence did not mutate.

[0051] After verification, it was digested with the PmeI restriction enzyme to obtain Gene Fragment 3, and the nucleotide sequence was as shown in SEQ ID NO.3.

[0052] (4) Construction of Gene Fragment 4

[0053] The 561bp homologous sequence upstream of the yeast LEU2 locus, the LEU2 marker, the TDH2 terminator, the GAL1 promoter, the CrtW gene, the SPO1 terminator, and the 584bp homologous sequence downstream of the yeast LEU2 locus were amplified and sequentially spliced by the overlap extension PCR method to obtain the fragment LEU2LHA-LEU2-PGAL1-CrtW-TSPO1-LEU2RHA containing PmeI restriction enzyme sites at both ends. Then it was ligated into the vector pJET1.2 to obtain the integrated plasmid of Gene Fragment 4, denoted as: pJET-LEU2-PGAL1-CrtW-TSPO1.

[0054] The integrated plasmid was transformed into competent Escherichia coli DH5α, and colonies were screened by PCR. The plasmid was extracted for restriction enzyme digestion verification and sequencing verification to ensure that the target fragment was ligated correctly and the base sequence did not mutate.

[0055] After verification, it was digested with the PmeI restriction enzyme to obtain Gene Fragment 4, and the nucleotide sequence was as shown in SEQ ID NO.4.

[0056] (5) Construction of Gene Fragment 5

[0057] The 312bp homologous sequence upstream of the yeast HIS3 locus, the HIS3 marker, the ENO2 terminator, the GAL1 promoter, the CrtZ gene, the HIS5 terminator, and the 578bp homologous sequence downstream of the yeast HI3 locus were amplified and sequentially spliced by the overlap extension PCR method to obtain the fragment HIS3LHA-HIS3-PGAL1-CrtZ-THIS5-HIS3RHA containing PmeI restriction enzyme sites at both ends. Then it was ligated into the vector pJET1.2 to obtain the integrated plasmid of Gene Fragment 4, denoted as: pJET-HIS3-PGAL1-CrtZ-THIS5.

[0058] The integrated plasmid was transformed into competent Escherichia coli DH5α, and colonies were screened by PCR. The plasmid was extracted for restriction enzyme digestion verification and sequencing verification to ensure that the target fragment was ligated correctly and the base sequence did not mutate.

[0059] After verification, it was digested with the PmeI restriction endonuclease to obtain Gene Fragment 5, and the nucleotide sequence is as shown in SEQ ID NO.5.

[0060] The gene element pattern diagrams of the above Gene Fragment 1 - Gene Fragment 5 are shown in Figures 1 - 5 .

[0061] 2. Construction of Saccharomyces cerevisiae FNFH_Sc18

[0062] Gene Fragment 1 was transformed into Saccharomyces cerevisiae CEN.PK2-1D by the lithium acetate method. It was integrated into the genome through homologous recombination between the upstream and downstream homologous sequences of TRP1 and the trp1 locus on the yeast genome. After transformation, it was screened using an SD-TRP solid plate (6.7 g / L yeast nitrogen source, 20 g / L glucose, 2 g / L mixed amino acid powder lacking only tryptophan, 2% agar powder). The obtained transformants were streaked and subcultured, and then the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were preserved as glycerol stocks and named FNFH_Sc11.

[0063] Gene Fragment 2 was transformed into Saccharomyces cerevisiae FNFH_Sc11 by the lithium acetate method. It was integrated into the genome through homologous recombination between the upstream and downstream homologous sequences of ypl062w and the ypl062w locus on the yeast genome. After transformation, it was screened using an SD-TRP-URA solid plate (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). The obtained transformants were subcultured, and then the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were cultured in YPD liquid medium (20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract powder), and then a small amount of the bacterial solution was spread on a 5-fluoroorotic acid (5-FOA) solid plate (because there are 143 bp direct repeat sequences DR at both ends of the DR-URA3-DR nutritional label, and the yeast itself will use these two identical sequences to undergo homologous recombination to delete the URA3 gene and one of the DRs. Strains containing URA3 can convert 5-FOA into a substance toxic to cells, making them unable to grow on a medium containing 5-FOA, thus screening out the strains with the URA3 gene deleted). Single colonies were picked and subcultured, and then the genome was extracted for PCR verification to screen the correct strains with the URA3 gene deleted through spontaneous recombination between the DR sequences. The correctly verified recombinant strains were preserved as glycerol stocks and named FNFH_Sc12.

[0064] The gene fragment 3 was transformed into Saccharomyces cerevisiae FNFH_Sc12 by the lithium acetate method. It was integrated into the genome through homologous recombination with the gal7 and gal1 loci on the yeast genome by the upstream and downstream homologous sequences of gal7 respectively. After transformation, SD-TRP-URA solid plates (yeast nitrogen source 6.7 g / L, glucose 20 g / L, mixed amino acid powder lacking tryptophan and uracil 2 g / L, 2% agar powder) were used for screening. The obtained transformants were purified by subculture, and then the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were cultured in YPD liquid medium, and then a small amount of bacterial liquid was spread on 5-FOA solid plates. Single colonies were picked and purified by subculture, and then the genome was extracted for PCR verification to screen the correct strains in which the URA3 gene was deleted by spontaneous recombination between the DR sequences. The correctly verified recombinant strains were preserved as glycerol bacteria and named FNFH_Sc13.

[0065] The gene fragment 4 was transformed into Saccharomyces cerevisiae FNFH_Sc13 by the lithium acetate method. It was integrated into the genome through homologous recombination with the leu2 locus on the yeast genome by the upstream and downstream homologous sequences of LEU2. After transformation, SD-TRP-LEU solid plates (yeast nitrogen source 6.7 g / L, glucose 20 g / L, mixed amino acid powder lacking tryptophan and leucine 2 g / L, 2% agar powder) were used for screening. The obtained transformants were purified by streaking and subculture, and then the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were preserved as glycerol bacteria and named FNFH_Sc17.

[0066] The gene fragment 5 was transformed into Saccharomyces cerevisiae FNFH_Sc17 by the lithium acetate method. It was integrated into the genome through homologous recombination with the his3 locus on the yeast genome by the upstream and downstream homologous sequences of HIS3. After transformation, SD-TRP-LEU-HIS solid plates (yeast nitrogen source 6.7 g / L, glucose 20 g / L, mixed amino acid powder lacking tryptophan, leucine and histidine 2 g / L, 2% agar powder) were used for screening. The obtained transformants were purified by streaking and subculture, and then the yeast genome was extracted for PCR verification. The correctly verified recombinant strains were preserved as glycerol bacteria and named FNFH_Sc18.

[0067] This strain was deposited in the China General Microbiological Culture Collection Center (CGMCC) on May 6, 2022. Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its deposit number is: CGMCC No. 24838. It is proposed to be named Saccharomyces cerevisiae.

[0068] Example 2

[0069] Application of Saccharomyces cerevisiae FNFH_Sc18 in soybean meal fermentation

[0070] Test materials:

[0071] Strain: Saccharomyces cerevisiae FNFH_Sc18.

[0072] Culture medium:

[0073] YPD medium: 20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract powder.

[0074] Test method:

[0075] 1. Pretreatment of soybean meal: Weigh 20 g of soybean meal and put it into a 250 mL sterilized shaking flask. Seal the bottle mouth with gauze and sealing film, sterilize at 100 °C under high pressure for 30 min, and cool to room temperature.

[0076] 2. Seed preparation: Inoculate Saccharomyces cerevisiae FNFH_Sc18 into 5 mL of YPD medium and carry out overnight activation culture at 30 °C and 250 rpm. Transfer to fresh 50 mL of YPD medium at OD = 0.5 and culture at 30 °C and 250 rpm until the logarithmic growth phase.

[0077] 3. Solid-state fermentation: Mix the seed liquid of Saccharomyces cerevisiae FNFH_Sc18 cultured to the logarithmic growth phase with 1 mL of sterilized D-(+)-galactose solution with a concentration of 100 g / L and a certain volume of sterile water according to an inoculation amount of 5% (v / m), and then evenly sprinkle it into the pretreated soybean meal (the total volume of the mixed seed liquid is 20 mL, that is, the initial water content of the soybean meal for fermentation is 50%). After fully stirring, put the shaking flask into a constant humidity shaking table and ferment at 30 °C and 150 rpm for 48 h.

[0078] 4. Drying and detection: After the fermentation is completed, dry the soybean meal at 50 °C, crush it through a 60-mesh sieve, and detect the contents of crude protein, crude fiber, crude fat, ash, stachyose, raffinose, urease, as well as the contents of carotenoids such as astaxanthin and β-carotene in the fermented soybean meal and raw material soybean meal respectively.

[0079] Carotenoid content: Take 1 g of the sample, add an appropriate amount of quartz sand and acetone, shake and extract, centrifuge to collect the organic phase, and repeat the extraction step until the bacteria in the sample turn white. Combine all the organic phases, filter, and detect the carotenoid content by HPLC. The chromatographic column is a C18 column (4.6×150 mm, 5 μm), and the column temperature is 25 °C. Mobile phase A: acetonitrile: water = 9:1; Mobile phase B: methanol: isopropanol = 3:2. Chromatographic conditions: initial condition 0% B phase, 0 - 13 min 0 - 90% B phase, 13 - 23 min 90% B phase, 23 - 28 min 90 - 0% B phase, 28 - 35 min 0% B phase. The detection wavelength of astaxanthin is 470 nm, and the detection wavelength of β-carotene is 450 nm.

[0080] Crude protein: Detected by the Kjeldahl method (GB / T 6432-2018).

[0081] Crude fiber: Detected by the filtration method (GB / T 6434-2006).

[0082] Crude fat: Detected by the Soxhlet extraction method (GB / T 6433-2006).

[0083] Ash: Detected by the ignition method (GB / T 6438-2007).

[0084] Stachyose and raffinose: Detected by high performance liquid chromatography (Appendix A in NY-T2218-2012).

[0085] Urease: Detected by the spectrophotometer method (GB / T 8622-2006).

[0086] Amino acids: Detected by the conventional acid hydrolysis method (GB / T 18246-2019).

[0087] Test results:

[0088] After fermenting soybean meal with Saccharomyces cerevisiae FNFH_Sc18 for 48 h, the obtained fermented soybean meal product was brownish red, with a delicate fragrance and sour taste, delicate texture and no granular feeling, and had good sensory quality ( Figure 9 ).

[0089] The protein and amino acid contents of the obtained fermented soybean meal product were both significantly increased, and the contents of anti-nutritional factors were significantly decreased. More importantly, a large amount of astaxanthin and β-carotene were newly accumulated in the product (Table 1). In the dried and uncrushed soybean meal fermented by Saccharomyces cerevisiae FNFH_Sc18, the contents of astaxanthin and β-carotene reached 51.20 mg / kg and 96.13 mg / kg respectively ( Figure 10 ). After the fermented soybean meal was dried and crushed, the contents of astaxanthin and β-carotene showed varying degrees of loss, and the contents of astaxanthin and β-carotene were 40.48 mg / kg and 25.59 mg / kg respectively ( Figure 10, (Table 1). In addition, compared with the raw soybean meal, the crude protein content of the fermented soybean meal increased by 7.8%, the total amino acid content increased by 20.6%, and the contents of anti-nutritional factors such as stachyose, raffinose, and urease decreased by 100%, 67.4%, and 100% respectively (Table 1). This proves that the overall nutritional parameters of soybean meal are significantly improved after fermentation with Saccharomyces cerevisiae FNFH_Sc18, mainly manifested as a large accumulation of immune nutrients - astaxanthin and β-carotene, an increase in the levels of crude protein and total amino acids, and a decrease in the levels of anti-nutritional factors such as oligosaccharides and urease. Therefore, it helps to enhance the digestibility and functional immunity nutrition of soybean meal protein raw materials.

[0090] Table 1. Analysis of key components of soybean meal before and after fermentation with Saccharomyces cerevisiae FNFH_Sc18

[0091]

Claims

1. A brewer's yeast (Saccharomyces cerevisiae) FNFH_Sc18, whose preservation number is: CGMCC No.24838, and the preservation date is May 6, 2022.

2. A bacterial agent containing the brewer's yeast FNFH_Sc18 according to claim 1.

3. Use of the saccharomyces cerevisiae FNFH_Sc18 as described in claim 1 or the bacterial agent as described in claim 2 in soybean meal fermentation.

4. Use of the saccharomyces cerevisiae FNFH_Sc18 according to claim 1 or the microbial agent according to claim 2 in improving the nutritional value of soybean meal during soybean meal fermentation.

5. The use according to claim 5, characterized in that The improvement of the nutritional value of soybean meal is specifically manifested in the increase of the content of immunonutrients, the increase of crude protein and total amino acid levels, and the decrease of anti-nutritional factor levels.

6. The use according to claim 5, characterized in that The immunonutrients include astaxanthin and beta-carotene, and the anti-nutritional factors include oligosaccharides and urease.

7. A method for producing fermented soybean meal rich in astaxanthin and β-carotene, characterized in that: The soybean meal is fermented using the brewer's yeast FNFH_Sc18 described in claim 1 or the bacterial agent described in claim 2.

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