Method for preparing fermented soybean meal by synergistic fermentation of saccharomyces cerevisiae FNFH_sc18 and bacillus subtilis FNFH_BS08, and use thereof

Abstract

Provided is the use of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 in the preparation of high-quality fermented soybean meal enriched with astaxanthin and β-carotene by synergistic fermentation. A method for preparing high-quality fermented soybean meal enriched with astaxanthin and β-carotene, characterized in that the soybean meal is subjected to synergistic fermentation with Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08, wherein the Saccharomyces cerevisiae FNFH_Sc18 has a deposit number of CGMCC No. 24838, and the Bacillus subtilis FNFH_BS08 has a deposit number of CGMCC No. 24837.

Description

Method for preparing fermented soybean meal by co-fermentation of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 and its application Technical Field

[0001] This invention belongs to the field of microbial fermentation technology, specifically relating to a method for preparing fermented soybean meal by co-fermentation of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08, and its application. Background Technology

[0002] Since the ban on antibiotics in livestock, poultry, and aquaculture, higher demands and challenges have been placed on the feed industry. Developing functional feeds has become an industry consensus, and the key lies in developing functional feed ingredients. Among feed protein resources, soybeans and fishmeal are the most important and scarce, representing a bottleneck restricting the development of my country's feed industry. As my country's largest source of plant-based protein, soybean meal has high crude protein content, a reasonable amino acid composition, and is inexpensive. However, it contains various anti-nutritional factors, including a large number of protein allergens, oligosaccharides, trypsin inhibitors, and urease, significantly reducing the digestibility and utilization rate of soybean meal and exacerbating the shortage of feed ingredient resources. Therefore, improving the utilization rate of soybean meal protein and enhancing its immune function are critical issues that the feed industry urgently needs to address.

[0003] Fermentation is currently a hot topic in the feed industry. Utilizing microbial fermentation to process soybean meal, the highly active carbohydrate enzymes and proteases secreted by the microorganisms decompose and destroy anti-nutritional factors in the soybean meal, effectively improving the digestibility of soybean meal protein. Chinese patent CN115197876A discloses "A Bacillus subtilis strain FNFH_BS08 and its application," which can efficiently degrade the main antigenic proteins and non-starch polysaccharides in soybean meal, improving the overall nutritional parameters of the soybean meal. Saccharomyces cerevisiae has always been a superior strain in traditional food and fermentation industries due to its higher biosafety; its live bacteria, inactive components, and cell components are widely used in animal husbandry and feed industries. 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.

[0004] Astaxanthin and β-carotene are known pigment-based immune enhancers with well-defined functions, and are permitted additives in my country's "Catalogue of Feed Additive Varieties (2013)". They can enable farmed animals to achieve faster growth and better feed conversion rates. Chinese patent CN118160870A discloses "a method for producing natural astaxanthin in poultry compound feed using Pharrellis redis fermentation and its application". Using a screened strain of Pharrellis redis P406, a fermentation substrate (60% corn flour, 25% soybean meal, 15% wheat bran) was fermented in a solid state. After fermentation, the astaxanthin content in the feed reached 17.50±0.31 mg / kg, significantly improving the egg quality of laying hens. Therefore, using carotenoid-producing strains to ferment feed protein raw materials such as soybean meal yields fermented feed directly containing carotenoids. This not only enhances the nutritional value and flavor of soybean meal protein but also avoids the extraction and additional addition of carotenoids. The process is simple, has low production costs, and can give soybean meal protein new immune functional properties.

[0005] In summary, utilizing microbial technology to enhance the value of soybean meal has great potential in developing functional, high-quality fermented soybean meal. Summary of the Invention

[0006] To address the problems existing in the prior art, the purpose of this invention is to provide a method and its application for the co-fermentation of brewing yeast FNFH_Sc18 and Bacillus subtilis FNFH_BS08 to prepare fermented soybean meal. This invention is achieved through the following technical solution:

[0007] The first aspect of this invention provides the application of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 in the co-fermentation preparation of high-quality fermented soybean meal rich in astaxanthin and β-carotene.

[0008] Furthermore, the Saccharomyces cerevisiae FNFH_Sc18 mentioned in this application has the preservation number CGMCC No.24838; and the Bacillus subtilis FNFH_BS08 has the preservation number CGMCC No.24837.

[0009] The second aspect of this invention provides a method for preparing high-quality fermented soybean meal rich in astaxanthin and β-carotene, which uses Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 to co-ferment soybean meal. The Saccharomyces cerevisiae FNFH_Sc18 has the preservation number CGMCC No. 24838; the Bacillus subtilis FNFH_BS08 has the preservation number CGMCC No. 24837.

[0010] Furthermore, the method specifically includes the following steps:

[0011] 1) Soybean meal pretreatment: After high-pressure sterilization, the soybean meal is cooled to room temperature;

[0012] 2) Seed preparation: Saccharomyces cerevisiae FNFH_Sc18 was inoculated into YPD medium and Bacillus subtilis FNFH_BS08 was inoculated into GYP medium. After overnight activation culture, they were transferred to fresh YPD medium and GYP medium at OD=0.5 and cultured to the logarithmic growth phase to obtain Saccharomyces cerevisiae FNFH_Sc18 seed liquid and Bacillus subtilis FNFH_BS08 seed liquid, respectively.

[0013] 3) Solid-state fermentation: At an inoculation rate of 5% (v / m), the seed culture of Saccharomyces cerevisiae FNFH_Sc18 cultured to the logarithmic growth phase was mixed with 1 mL of sterile D-(+) galactose solution and a certain volume of sterile water and then evenly sprinkled into the pretreated soybean meal. After thorough mixing, the shake flask was placed in a constant humidity shaker for fermentation. Subsequently, the Bacillus subtilis FNFH_BS08 seed culture obtained in step 2) was evenly sprinkled into the fermenting soybean meal at an inoculation rate of 5% (v / m). After thorough mixing, the culture conditions of the constant humidity shaker were adjusted to continue fermentation, and high-quality fermented soybean meal was obtained.

[0014] Furthermore, in step 2), the YPD medium formula is: 20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract; the GYP medium formula is: 10 g / L glucose, 8 g / L yeast extract, 2 g / L soybean peptone, pH 7.0.

[0015] Furthermore, in step 3), the concentration of the D-(+) galactose solution is 100 g / L, and the amount of a certain volume of sterile water used is such that the initial moisture content of the soybean meal fermentation is 50%.

[0016] Furthermore, in step 3), the fermentation conditions for both Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 are 30℃, 150rpm, and 24h.

[0017] The third aspect of the present invention provides a high-quality fermented soybean meal rich in astaxanthin and β-carotene prepared by the above-described preparation method.

[0018] The fourth aspect of this invention provides the application of the above-mentioned high-quality fermented soybean meal as aquaculture feed.

[0019] This invention prepares fermented soybean meal through the co-fermentation of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08. This method offers advantages such as a short fermentation cycle, high efficiency, and low cost. The resulting fermented soybean meal is rich in β-carotene and astaxanthin, with significantly increased crude and soluble protein content, and a substantial reduction in anti-nutritional factors such as antigenic proteins, oligosaccharides, and urease, thus comprehensively enhancing the nutritional value of the soybean meal. Furthermore, replacing 30% of fishmeal protein with fermented soybean meal can significantly improve the final weight, weight gain rate, and specific growth rate of largemouth bass, thereby enhancing the growth performance of farmed animals. Attached Figure Description

[0020] Figure 1 shows before and after photos of the co-fermentation of soybean meal by Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 ((A,B) shake flask fermentation for 0h; (C) crushed raw soybean meal; (D,E) shake flask fermentation for 48h; (F) crushed fermented soybean meal).

[0021] Figure 2 shows the HPLC peak chromatogram of fermented soybean meal obtained by co-fermentation of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08.

[0022] Figure 3 shows the protein detection results of polyacrylamide gel electrophoresis (SDS-PAGE);

[0023] Figure 4 shows photographs of whole largemouth bass, liver, and intestines after 8 weeks of culture on an isonitrogenous and isolipidic compound feed formulated with fermented soybean meal replacing fishmeal protein ((A) fermented soybean meal replacing 0% fishmeal protein; (B) fermented soybean meal replacing 30% fishmeal protein). Detailed Implementation

[0024] To fully disclose the method and application of the present invention for preparing fermented soybean meal by co-fermentation of brewing yeast and Bacillus subtilis, the following examples are provided, but these do not imply any limitation on the present invention.

[0025] Example 1

[0026] 1. Construction of gene fragments

[0027] (1) Construction of gene fragment 1

[0028] A 631 bp 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 733 bp homologous sequence downstream of the yeast TRP1 site were amplified and sequentially spliced ​​together using overlap extension PCR to obtain the fragment TRP1 LHA-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1-TRP1 RHA containing PmeI restriction sites at both ends. This fragment was then ligated into the vector pJET1.2 (full gene sequence shown in SEQ ID NO. 6) to obtain the gene fragment 1 integrated plasmid, denoted as pJET-TRP1-TCYC1-CarB-PGAL10-PGAL1-CarRP-TPGK1.

[0029] 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.

[0030] 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.

[0031] (2) Construction of gene fragment 2

[0032] 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.

[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 2, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0035] (3) Construction of gene fragment 3

[0036] The following homologous sequences were amplified: a 426 bp 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 downstream of the yeast gal1 site. These sequences were then sequentially spliced ​​together using overlap extension PCR to obtain the fragment gal7 LHA-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1-gal1 RHA, which contains PmeI restriction sites at both ends. Then, it was ligated into the vector pJET1.2 to obtain the gene fragment 3 integrated plasmid, denoted as pJET-gal-DR-URA3-DR-TERG10-ERG10-PGAL7-TACT1-tHMGR1-PGAL10-PGAL1-CrtE-TGPM1.

[0037] 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.

[0038] 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.

[0039] (4) Construction of gene fragment 4

[0040] 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.

[0041] 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.

[0042] 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.

[0043] (5) Construction of gene fragment 5

[0044] 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 HIS3 LHA-HIS3-PGAL1-CrtZ-THIS5-HIS3 RHA containing PmeI restriction sites at both ends. 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.

[0045] 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.

[0046] 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.

[0047] 2. Construction of Saccharomyces cerevisiae FNFH_Sc18

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] Example 2: Preparation of fermented soybean meal by co-fermentation of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08

[0055] Experimental materials:

[0056] Strains: Saccharomyces cerevisiae FNFH_Sc18, Bacillus subtilis FNFH_BS08.

[0057] Culture medium:

[0058] YPD medium: 20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract;

[0059] GYP medium: 10 g / L glucose, 8 g / L yeast extract, 2 g / L soybean peptone, pH 7.0.

[0060] Test method:

[0061] 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 then cool to room temperature.

[0062] 2. Seed preparation:

[0063] Saccharomyces cerevisiae FNFH_Sc18 was inoculated into 5 mL of YPD medium and activated overnight at 30°C and 250 rpm. It was then transferred to 50 mL of fresh YPD medium at OD = 0.5 and cultured at 30°C and 250 rpm until the logarithmic growth phase.

[0064] Bacillus subtilis FNFH_BS08 was inoculated into 5 mL of GYP medium and activated overnight at 37°C and 250 rpm. Then, it was transferred to 30 mL of fresh GYP medium at OD=0.5 and cultured at 37°C and 250 rpm until the logarithmic growth phase.

[0065] 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 mixed seed solution 20 mL, i.e., the initial moisture content of the soybean meal for fermentation is 50%). After thorough mixing, the shake flask was placed in a humidity-controlled shaker and fermented for 24 h at 30℃ and 150 rpm. At the 25th h, a 5% (v / m) inoculum of *Bacillus subtilis* FNFH_BS08 cultured to the logarithmic growth phase was evenly sprinkled into the fermenting soybean meal. After thorough mixing, the culture conditions of the humidity-controlled shaker were adjusted to 30℃ and 150 rpm, and fermentation continued for another 24 h to obtain fermented soybean meal.

[0066] Verification Example

[0067] Drying and testing: After fermentation, the soybean meal was taken out and dried at 60℃, then pulverized and passed through a 60-mesh sieve. The contents of crude protein, water-soluble protein, crude fiber, crude fat, ash, phosphorus, β-carotene and astaxanthin in the fermented soybean meal and raw soybean meal were tested. The degree of protein degradation and molecular weight distribution of the soybean meal were also tested. The contents of major anti-nutritional factors in soybean meal, such as soybean globulin, β-conglobulin, stachyose, raffinose, urease, and trypsin inhibitor, were also tested.

[0068] Crude protein: Detected using the Kjeldahl method (GB / T 6432-2018).

[0069] Water-soluble protein: Weigh 1g of sample and dissolve it in 40mL of double-distilled water. Vortex for 2 hours, then centrifuge at 1500rpm for 10 minutes and collect the supernatant. Quantify the supernatant according to the method for determining crude protein content, namely the Kjeldahl method (GB / T 6432-2018).

[0070] Protein solubility = water-soluble protein content / crude protein content × 100%.

[0071] Crude fiber: Tested by filtration method (GB / T 6434-2006).

[0072] Crude fat: Detected using Soxhlet extraction method (GB / T 6433-2006).

[0073] Ash content: Tested by the ignition method (GB / T 6438-2007).

[0074] Phosphorus: Detected by spectrophotometry (GB / T 6437-2018).

[0075] 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.

[0076] Soybean meal protein degradation degree: Weigh 1g of sample and dissolve it in 5mL of 8M urea solution, vortex for 30min, then centrifuge at 4℃ and 8000rpm for 5min to separate the supernatant. Quantify the supernatant using the BCA protein quantification kit, calculate the sample loading amount based on 30μg protein content, and then load it onto a polyacrylamide gel for SDS-PAGE analysis.

[0077] Soy globulin: The assay was performed using an enzyme-linked immunosorbent assay (ELISA) kit purchased from Beijing Longke Fangzhou Biotechnology Co., Ltd., following the product instructions.

[0078] β-Consigmaglobulin: The assay was performed using an enzyme-linked immunosorbent assay (ELISA) kit purchased from Beijing Longke Ark Biotechnology Co., Ltd., following the product instructions.

[0079] Stachyose and raffinose were detected by high performance liquid chromatography (Appendix A of NY-T2218-2012).

[0080] Urease: Detected by spectrophotometry (GB / T 8622-2006).

[0081] Trypsin inhibitors: detected by spectrophotometry (GB 5009.224-2016).

[0082] Verification results:

[0083] Fermented soybean meal prepared by co-fermentation of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 for 48 hours was brownish-red in color, had a light and sour aroma, a soft and delicate texture, no grainy feel, and good sensory quality (Figure 1).

[0084] The fermented soybean meal obtained showed a significant increase in crude protein and water-soluble protein content, a significant decrease in the content of anti-nutritional factors such as antigenic proteins, and was rich in astaxanthin and β-carotene (Table 1). HPLC analysis showed that the contents of astaxanthin and β-carotene reached 42.17 mg / kg and 32.58 mg / kg, respectively (Figure 2). Furthermore, compared to the raw soybean meal, the fermented soybean meal showed a 16.6% increase in crude protein content, an 800.7% increase in water-soluble protein content, and a corresponding 671.2% increase in protein solubility; the degradation rates of soybean globulin and β-conglobulin were 91.6% and 82.4%, respectively; the contents of anti-nutritional factors such as stachyose, raffinose, and urease decreased by 100%, and the contents of trypsin inhibitors decreased by 81.5% (Table 1). This demonstrates that the overall nutritional parameters of soybean meal are comprehensively improved after co-fermentation with Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08. This is mainly reflected in the significant accumulation of astaxanthin and β-carotene, the upregulation of total protein and soluble protein levels, and the decrease in the levels of anti-nutritional factors such as antigen proteins. Therefore, it helps to improve the digestibility and utilization rate of soybean meal protein raw materials and its functional immune nutrition.

[0085] Table 1. Analysis of key components in fermented soybean meal

[0086] Figure 3 shows the degree of total protein degradation in soybean meal before and after co-fermentation using *Saccharomyces cerevisiae* FNFH_Sc18 and *Bacillus subtilis* FNFH_BS08. Compared with the raw soybean meal, the fermented soybean meal obtained through co-fermentation showed that soybean globulin (30-45 kDa) and β-conglobulin (50-100 kDa) were almost completely degraded. Most of the large, poorly soluble proteins in the soybean meal were degraded into small, soluble proteins and peptides (<25 kDa), thus helping to improve the bioavailability of soybean meal protein.

[0087] Example 3: Application of fermented soybean meal as a substitute for fishmeal in largemouth bass farming trials

[0088] (1) Fermented feed preparation

[0089] Fermented soybean meal prepared by co-fermentation of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 replaced 0%, 10%, 20%, 30%, 40%, and 50% of fishmeal protein, respectively, to formulate six groups of isonitrogenous (48% crude protein) and isolipid (12% crude fat) compound feeds, named Group 1, Group 2, Group 3, Group 4, Group 5, and Group 6. Group 1, with fishmeal content accounting for 40% of the feed dry weight, served as the control group. The feed formulation is shown in Table 2. All feed ingredients were pulverized using an ultrafine grinder and passed through a 60-mesh sieve. All ingredients were then weighed according to the formulation, gradually mixed, and a certain proportion of ultrapure water was added. Fish oil and soybean oil, among other fat sources, were added and mixed thoroughly. The mixture was then granulated using a twin-screw extruder to form pellets with a particle size of 2mm-4mm, and subsequently dried in a 45℃ oven for 24 hours until the feed weight no longer decreased. Store the feed in a -20°C freezer until needed.

[0090] Table 2. Feed Formulation Composition (%, Dry Weight)

[0091] 1 Multivitamins (g / kg): Vitamin B12, 0.020; Vitamin H, 0.225; Vitamin B9, 0.337; Vitamin D3, 1.370; Vitamin A, 2.250; Vitamin B1, 2.390; Vitamin K, 8.900; Vitamin E, 9.900; Vitamin B5, 10.200; Vitamin B3, 10.960; Vitamin B6, 12.800; Vitamin B2, 16.000; Vitamin B8, 118.150; Vitamin C, 19.100; Fiber, 787.400.

[0092] 2 Multiple minerals (g / kg): NaSeO3, 0.038; MnSO4·H2O, 1.040; CoCl2·6H2O, 2.217; CuSO4·5H2O, 9.260; ZnSO4·7H2O, 11.770; MgSO4, 12.800; KCl, 20.800; CaCO3, 24.180; NaCl, 90.720; FeSO4·7H2O, 118.370; Ca(H2PO4)2·H2O, 330.100; Cellulose, 378.705.

[0093] (2) Aquaculture trials and sampling

[0094] Juvenile largemouth bass used in the experiment were purchased from Chia Tai Aquatic Products (Huzhou) Co., Ltd. The culture experiment was conducted in the Aquatic Animal Nutrition and Feed Laboratory of Zhejiang Ocean University. After 14 days of acclimatization, the juvenile largemouth bass adapted to the culture environment. Subsequently, 540 healthy, vigorous, and uniformly sized juvenile largemouth bass (initial weight: 5.77±0.06g) were randomly divided into 18 culture tanks (volume: 750L), with 30 fish in each tank. Each treatment had three biological replicates. Apparently satiated fish were fed at 8:00 AM and 5:00 PM daily. One hour after feeding, feces and uneaten feed were removed by siphon. The culture period was 8 weeks. During the culture experiment, the aquatic environment was maintained within the optimal range for largemouth bass growth (dissolved oxygen above 8 mg / L, ammonia nitrogen below 0.05 mg / L, water temperature maintained at 26±2℃, and pH maintained between 7.4 and 8.4).

[0095] After the aquaculture trial, all fish were starved for 24 hours. The number of surviving fish, total weight, and feed intake weight of each tank were then recorded to calculate survival rate, weight gain rate, specific growth rate, and feed efficiency. Six fish were taken from each tank, and their liver, muscle, and intestinal tissues were collected, flash-frozen in liquid nitrogen, and then stored at -80°C for later use.

[0096] (3) Test Results

[0097] After the rearing period, the growth performance of largemouth bass was shown in Table 3 and Figure 4. The survival rate of all treatment groups was 100%, and the weight gain rate was higher than 600%, indicating the success of the rearing experiment. Compared with the control group, with the increase of the proportion of fermented soybean meal replacement, the final body weight, weight gain rate, and specific growth rate of largemouth bass all showed a trend of first increasing and then decreasing, exhibiting a significant quadratic relationship. The final body weight, weight gain rate, and specific growth rate reached their highest values ​​in group 4 (fermented soybean meal replacing 30% of fishmeal protein), with the weight gain rate reaching 703.17%, an increase of 96.55% compared to group 1 (fermented soybean meal replacing 0% of fishmeal protein). These results indicate that replacing 30% of fishmeal protein with fermented soybean meal can significantly improve the growth performance of largemouth bass.

[0098] Table 3. Growth performance of juvenile largemouth bass (mean ± standard deviation, n = 3)

[0099]

Claims

1. Application of Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 in the co-fermentation preparation of high-quality fermented soybean meal rich in astaxanthin and β-carotene.

2. The application as described in claim 1, characterized in that, The brewing yeast FNFH_Sc18 has the accession number CGMCC No. 24838; the Bacillus subtilis FNFH_BS08 has the accession number CGMCC No. 24837.

3. A method for preparing high-quality fermented soybean meal rich in astaxanthin and β-carotene, characterized in that, Soybean meal was co-fermented using Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08. The Saccharomyces cerevisiae FNFH_Sc18 has the preservation number CGMCC No. 24838, and the Bacillus subtilis FNFH_BS08 has the preservation number CGMCC No. 24837.

4. The method for preparing high-quality fermented soybean meal rich in astaxanthin and β-carotene as described in claim 3, characterized in that, Specifically, the steps include: 1) Soybean meal pretreatment: After high-pressure sterilization, the soybean meal is cooled to room temperature; 2) Seed preparation: Saccharomyces cerevisiae FNFH_Sc18 was inoculated into YPD medium and Bacillus subtilis FNFH_BS08 was inoculated into GYP medium. After overnight activation culture, they were transferred to fresh YPD medium and GYP medium at OD=0.5 and cultured to the logarithmic growth phase to obtain Saccharomyces cerevisiae FNFH_Sc18 seed liquid and Bacillus subtilis FNFH_BS08 seed liquid, respectively. 3) Solid-state fermentation: At an inoculation rate of 5% (v / m), the seed culture of Saccharomyces cerevisiae FNFH_Sc18 cultured to the logarithmic growth phase was mixed with 1 mL of sterile D-(+) galactose solution and a certain volume of sterile water and then evenly sprinkled into the pretreated soybean meal. After thorough mixing, the shake flask was placed in a constant humidity shaker for fermentation. Subsequently, the Bacillus subtilis FNFH_BS08 seed culture obtained in step 2) was evenly sprinkled into the fermenting soybean meal at an inoculation rate of 5% (v / m). After thorough mixing, the culture conditions of the constant humidity shaker were adjusted to continue fermentation, and high-quality fermented soybean meal was obtained.

5. The method for preparing high-quality fermented soybean meal rich in astaxanthin and β-carotene as described in claim 1, characterized in that, In step 2), the YPD medium formula is: 20 g / L peptone, 20 g / L peptone, 10 g / L yeast extract; the GYP medium formula is: 10 g / L glucose, 8 g / L yeast extract, 2 g / L soybean peptone, pH 7.

0.

6. The method for preparing high-quality fermented soybean meal rich in astaxanthin and β-carotene as described in claim 1, characterized in that, In step 3), the concentration of the D-(+) galactose solution is 100 g / L, and the amount of a certain volume of sterile water used is such that the initial moisture content of the soybean meal fermentation is 50%.

7. The method for preparing high-quality fermented soybean meal rich in astaxanthin and β-carotene as described in claim 1, characterized in that, In step 3), the fermentation conditions for both Saccharomyces cerevisiae FNFH_Sc18 and Bacillus subtilis FNFH_BS08 are 30℃, 150rpm, and 24h.

8. High-quality fermented soybean meal rich in astaxanthin and β-carotene prepared by any of the preparation methods described in claims 3-7.

9. The application of the high-quality fermented soybean meal as described in claim 8 as aquaculture feed.

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