Enterococcus faecalis for producing protease and feed fermentation method using the enterococcus faecalis

Abstract

The application discloses a protease-producing Enterococcus faecalis and a production method of a biological fermentation feed using the Enterococcus faecalis, and relates to the technical field of functional microorganism screening and fermentation enzyme hydrolysis of soybean meal. The Enterococcus faecalis is preserved in the China General Microbiological Culture Collection Center (CGMCC) on June 6, 2022, and has a preservation number of CGMCC NO. 25007. The application adopts a domestication screening method to breed functional microorganisms, and obtains an Enterococcus faecalis which has strong protease production capacity, good biomass and good metabolic product activity. The strain can be used for soybean meal fermentation, can greatly degrade macromolecular antigen proteins, can improve the anti-nutritional characteristics of the soybean meal, can produce rich functional small peptides, can improve the protein digestion and utilization rate of the soybean meal, and can improve the conversion rate of the feed, so that the value of reducing cost and increasing efficiency in the era without antibiotics can be played.

Description

Technical Field

[0001] This invention relates to the field of microbiology and fermentation engineering, specifically to a protease-producing Enterococcus faecalis and its applications. Background Technology

[0002] Corn and soybean meal are among the main animal protein feeds in my country. Soybean meal, in particular, has advantages such as high protein content (43%-48%), a more balanced amino acid content (2.5%-2.8%), and a particularly rich lysine content, which can balance the nutrition of poultry and pigs. Anti-nutritional factors in soybean meal mainly include trypsin inhibitors, soybean antigen proteins, oligosaccharides, phytic acid, and lectins. These factors can reduce the body's nitrogen utilization rate, decrease vitamin efficacy, cause intestinal flora imbalance, and affect normal metabolism, thus reducing the feed value of soybean meal. Currently, microbial fermentation can fully utilize the nutritional value of soybean meal. Microbial fermentation of soybean meal can reduce the content of antigen proteins soybean globulin and β-conglycinin by 67% and 62%, respectively, and essentially eliminate trypsin inhibitors, phytic acid, and lectins in soybean meal. Microbial fermentation produces a large number of probiotics, oligopeptides, lactic acid, vitamins, and minerals, which inhibit the growth of harmful intestinal bacteria, reduce the content of neutral and acid detergent fiber, promote the absorption of nutrients by animals, and improve feed utilization. Simultaneously, microbial fermentation of soybean meal significantly increases the content of soybean isoflavones, enhances antioxidant activity, improves the antioxidant capacity of animals, and strengthens the body's immunity. (Feng et al.) [1] Feeding piglets with 24.5% fermented soybean meal significantly increased villus height in various segments of the small intestine, increased duodenal crypt depth, and increased the expression of mucin and tight junction protein genes in the jejunum and ileum of piglets. This indicates that fermented soybean meal can regulate the expression of gut-related genes to increase intestinal immune function, reduce weaning stress, and improve economic value.

[0003] Lactic acid bacteria are an important flora residing in the digestive tracts of humans and animals, playing a crucial role in maintaining intestinal flora balance. Fermenting soybean meal with lactic acid bacteria hydrolyzes the proteins in the soybean meal, increasing the release of free amino acids. The resulting fermented soybean meal has a significantly higher total free amino acid content than regular soybean meal. Simultaneously, fermented soybean meal improves feed flavor and palatability, thus helping to increase feed intake. Furthermore, the abundant lactic acid bacteria and their metabolites in fermented soybean meal can enhance the body's specific immune level and have anti-inflammatory effects. The *Enterococcus faecalis* CGMCC NO.25007 obtained in this invention is a high-protease-producing lactic acid bacteria obtained through gradient selection. In addition to the advantages of fermented soybean meal obtained with lactic acid bacteria mentioned above, it can also produce a large amount of highly active protease, playing a vital role in the degradation of large-molecule anti-nutritional factors in soybean meal. The fermented soybean meal obtained by *Enterococcus faecalis* in this invention has a significant alleviating effect on weaning stress in piglets, increasing feed intake and daily weight gain.

[0004] Currently, fermented soybean meal is widely used in the production of antibiotic-free feed. The main production processes include synergistic fermentation of bacteria and enzymes, aerobic fermentation by Bacillus, and a combination of aerobic fermentation by Bacillus and anaerobic fermentation by lactic acid bacteria and yeast. These processes are energy-intensive, with aerobic fermentation consuming a large amount of energy from the soybean meal, resulting in high production losses. This invention, through the selection and breeding of a high-protease-producing lactic acid bacteria strain, exhibits high efficiency in degrading large-molecule proteins in soybean meal. This strain can synergistically ferment soybean meal with Saccharomyces cerevisiae in a microaerobic environment. Through simple mixing, bagging, and sealing, static fermentation for 3-7 days yields fermentation products rich in organic acids and small peptides. Anti-nutritional factors in soybean meal are also effectively degraded, thereby improving the nutritional value of the soybean meal. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the technical problem to be solved by the present invention is to select and breed Enterococcus faecalis with high protease activity by means of strain domestication, and to use it for soybean meal fermentation and high-value utilization of soybean meal resources.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0007] On the one hand, the present invention provides a strain of Enterococcus faecalis that produces protease, which was deposited on June 6, 2022, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Chaoyang Road, Beijing, with accession number CGMCC NO.25007, and classified as Enterococcus faecalis.

[0008] On the other hand, the present invention provides the application of the above-mentioned Enterococcus faecalis in the fermentation of soybean meal and soybean meal-containing feed.

[0009] The feed fermentation method is as follows:

[0010] S101. Inoculate Enterococcus faecalis into the fermentation medium at a ratio of 2-5% and culture to obtain the fermentation broth;

[0011] S102. Mix the fermentation broth and the fermentation substrate at a weight ratio of (1.5-2):(4.5-5) to obtain a mixed fermentation substrate;

[0012] S103. Add brewer's yeast to the mixed fermentation substrate, adjust the moisture content of the mixed fermentation substrate to 35%-45%, and ferment to obtain the target fermented feed.

[0013] Furthermore, in S101, the fermentation broth is cultured under anaerobic conditions.

[0014] Furthermore, the viable cell count in the fermentation broth is 2.0*10⁻⁶. 8 -1.0*10 10 cfu / g.

[0015] Furthermore, the total bacterial count in the mixed fermentation substrate is 5.0*10⁻⁶. 6 -1.0*10 8 cfu / g.

[0016] Furthermore, the weight ratio of the fermentation broth to the fermentation substrate is 1.6:4.5.

[0017] Furthermore, the fermentation medium is MRS broth medium; the fermentation substrate includes 70-90 parts by weight of soybean meal, 2-5 parts by weight of sugar and 10-30 parts by weight of wheat bran, wherein the sugar is specifically a sugar-containing substance, which may be brown sugar, white sugar, black sugar or yellow sugar.

[0018] Furthermore, the final concentration of the brewing yeast added is 5*10. 6 -1.0*10 7 cfu / g fermentation substrate.

[0019] Furthermore, in S103, the initial moisture content of the mixed fermentation substrate is 35%-40%, and the fermentation method is anaerobic solid-state fermentation.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] 1. In this embodiment of the invention, a strain domestication and screening method is used to obtain a strain of Enterococcus faecalis EF-JF-10 that produces protease and has good biomass and good metabolite activity, which can increase the biomass in feed.

[0022] 2. The enzyme-producing characteristics of this strain can be used for fermentation in soybean meal and feed containing soybean meal. It can effectively degrade glycinin and β-conglycinin, improve the utilization rate of soybean meal, and improve the nutritional composition of soybean meal.

[0023] 3. Experiments have shown that feed containing Enterococcus faecalis can effectively alleviate weaning stress in piglets, increase feed intake and daily weight gain in weaned piglets, and is of great significance for the high-value utilization of soybean meal. Attached Figure Description

[0024] Figure 1 This is an example of the colony morphology of Enterococcus faecalis EF-JF-10 on MRS plates according to an embodiment of the present invention;

[0025] Figure 2 This is a Gram-stained microscopic image of Enterococcus faecalis colonies from an embodiment of the present invention.

[0026] Figure 3 This is a protease screening culture medium plate according to an embodiment of the present invention;

[0027] Figure 3 In the middle: A represents the clear zone of hydrolysis of Enterococcus faecalis EF-JF-10; B and C are replicates; D, E, and F represent the clear zones of hydrolysis of the control strain.

[0028] Figure 4 The graph shows the degradation effect of antigen proteins in soybean meal as determined by SDS-polyacrylamide gel electrophoresis.

[0029] Figure 4 In the middle section: A and B are duplicate samples, which are soybean meal raw materials (unfermented); C and D are duplicate samples, which are the degradation of macromolecular antigen proteins in solid fermented soybean meal; E and F are two duplicate samples, which are the degradation of macromolecular antigen proteins after 10% soybean meal liquid fermentation. Detailed Implementation

[0030] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0031] The *Enterococcus faecalis* strain of this invention was deposited on June 6, 2022, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCCNO.25007, and classified as *Enterococcus faecalis*. In practical use, it was found that the *Enterococcus faecalis* strain of this invention can effectively degrade daidzein (11S) and β-congaidzein (7S) in soybean meal, improving the palatability of soybean meal.

[0032] The application of Enterococcus faecalis in the preparation of fermented feed according to this embodiment of the invention is as follows: Enterococcus faecalis is inoculated into a fermentation medium at a ratio of 2-5% and cultured at 37°C for 48-60 hours to obtain a fermentation broth; the fermentation broth is mixed with a fermentation substrate at a weight ratio of (1.5-2):(4.5-5) to obtain a mixed fermentation substrate; Saccharomyces cerevisiae is added to the mixed fermentation substrate, the moisture content of the mixed fermentation substrate is adjusted to 35%-45%, and fermentation is carried out at 37°C for 3-7 days.

[0033] Preferably, the fermentation broth is cultured at 37°C under anaerobic conditions for 48-60 hours.

[0034] More preferably, the viable cell count of the fermentation broth is 2.0*10⁻⁶. 8 -1.0*10 10 cfu / g.

[0035] Preferably, the total number of colonies in the mixed fermentation substrate is 1.0*10⁻⁶. 7 -1.0*10 8 cfu / g.

[0036] Preferably, the weight ratio of the fermentation broth to the fermentation substrate is 1.6:4.5.

[0037] Preferably, the fermentation medium is MRS broth medium.

[0038] Preferably, the fermentation substrate comprises 80% soybean meal, 5% corn and 15% wheat bran.

[0039] Preferably, the final concentration of the brewing yeast added is 25-30 g / t.

[0040] Preferably, the initial moisture content of the mixed fermentation substrate is 40%-45%, the fermentation temperature is 37°C, the fermentation method is anaerobic solid-state fermentation, and the fermentation time is 4-5 days.

[0041] Examples 1-4 are provided to illustrate the application process of the embodiments of the present invention.

[0042] Example 1: Screening and domestication of Enterococcus faecalis protease

[0043] 1. Experimental materials

[0044] Microbial strain source: antibiotic-free poultry manure from farms

[0045] Enrichment medium: containing 100g of crushed soybean meal (passed through an 80-mesh sieve), 20g of glucose, and water to 1000mL, stirred thoroughly, pH 6.0-6.5, and sterilized at 110℃ for 20min;

[0046] Initial screening medium: containing 3g beef extract, 10g peptone, 5g sodium chloride, 15g agar, 15g skim milk, add water to 1000mL, stir thoroughly and mix well, sterilize the medium at 110℃ for 15min;

[0047] MRS medium (seed culture medium): consists of 10g peptone, 5g beef meal, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, and 1000ml distilled water.

[0048] Slant culture medium: composed of 10g peptone, 5g beef meal, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, and 1000ml distilled water.

[0049] Basic fermentation medium: 40g soybean meal, 30g corn flour, 30g wheat bran, 0.3g KH2PO4, 4g Na2HPO4-12H2O, add water to 1000mL, stir thoroughly and sterilize the medium at 110℃ for 15min.

[0050] 2. Strain screening

[0051] Enrichment process: Add 10g of the collected sample to a 100mL / 250mL Erlenmeyer flask containing sterile water with glass beads, and incubate on a shaker at 180rpm and 37℃ for 30min to mix the sample thoroughly. Take 1mL of the mixture and add it to a 100mL Erlenmeyer flask containing 100mL of water, and incubate statically at 37℃ for 48h.

[0052] Initial screening process: The enriched bacterial solution was serially diluted to different multiples (10-fold) using the tenfold dilution method. -1 -10 -8 Take 10 -5 10 -6 10 -7 and 10 -8Five dilution gradients were used, and 100 μL of bacterial culture from each dilution was spread onto a protease screening medium plate. Each gradient was performed in triplicate. After anaerobic incubation at 37°C for 24 hours, a clear proteolysis zone appeared (see...). Figure 3 Using an inoculation loop, pick up a single colony of a strain with a clear hydrolysis zone and streak it. Measure the diameter of the hydrolysis zone and the colony using calipers. Select strains with a larger ratio of hydrolysis zone diameter to colony diameter (H / C) as the initial screening protease-producing strains. Preserve the selected strains on test tube slant and store them at 4°C.

[0053] Secondary screening: Five single colonies with the largest ratio of hydrolysis zone diameter to colony size from the initial screening were inoculated into 250 mL Erlenmeyer flasks containing 100 mL of seed culture medium and cultured at 37 °C for 18 h on a shaker at 100 rpm. The seed culture medium was then added at a 4% inoculation rate to 500 mL Erlenmeyer flasks containing 200 mL of basal fermentation medium. Fermentation was carried out at 37 °C for 48 h on a shaker at 100 rpm. The supernatant was collected by centrifugation, and protease activity was measured to screen for the strain with the highest protease activity.

[0054] 3. Strain identification

[0055] Colony morphology observation and Gram staining were performed: the strain with the highest protease production activity was picked with an inoculation loop and streaked on a primary screening agar plate. The shape, color and size of single colonies were observed. Gram staining was performed using a Gram staining kit (purchased from Shanghai Sangon Biotech) and examined under a microscope.

[0056] 16S rDNA assay: Using single colonies on solid plates as templates, PCR amplification of bacterial 16S rDNA was performed using universal primers synthesized by Shanghai Sangon Biotech. The PCR amplification products were detected by agarose gel electrophoresis and then sent to Hunan Qingke Biotechnology Co., Ltd. for sequencing.

[0057] 5. Experimental Results

[0058] In Example 1 of this invention, a high-protease-producing strain was obtained through multiple enrichment and screening isolation processes. The protease activity in the supernatant of shake-flask fermentation was 72 U / mL. Its colony morphology and microscopic cell morphology are shown in the figure. Figure 1 , Figure 2 As shown in the image. This selected strain was inoculated onto skim milk agar plates, exhibiting distinct protein hydrolysis zones, as shown in the image. Figure 3As shown. The 16S rDNA sequence of this strain, through sequence alignment in the GenBank database (see sequence listing for details), showed the highest similarity (99.86%) to *Enterococcus faecalis* strain H4. Based on its physiological and biochemical characteristics, the strain of Example 1 of this invention was identified as *Enterococcus faecalis*, named EF-JF-10. The specific sequence is shown in the table below. The *Enterococcus faecalis* obtained in Example 1 of this invention has been deposited at the China Microbiological Culture Collection Center (CGMCC) with the accession number CGMCCNO.25007.

[0059]

[0060]

[0061] Example 2: Preparation of Lactic Acid Bacteria Liquid Fermentation of Soybean Meal

[0062] Implementation steps:

[0063] Preparation of S1 10% soybean meal solution: Weigh 100g of crushed (passed through an 80-mesh sieve) soybean meal powder, resuspend it in 900g of tap water, add 2g of glucose, shake well, and sterilize at 110℃ for 20min.

[0064] Preparation of S2 Enterococcus faecalis seed culture: Take a single colony from the EF-JF-10 slant of Enterococcus faecalis and inoculate it into sterile MRS broth medium. Place it in a shaker and culture for 16 h at 37 ℃ and 100 r / min.

[0065] S3 Liquid Fermented Soybean Meal Preparation: Enterococcus faecalis seed culture was inoculated into a 10% soybean meal solution at a 4% inoculation rate. After fermentation at 37℃ and 100rpm for 48 hours, the content of small peptides and the degradation of antigen proteins were detected.

[0066] Experimental results:

[0067] Soybean meal, after liquid fermentation with *Enterococcus faecalis* according to this invention, yields a solution rich in small peptides, accounting for over 40% of the total protein. The antigenic proteins in soybean meal are mainly composed of β-globulin (7S) and glycinin (11S). The 7S protein consists of three subunits: α (approximately 67 kDa), α' (approximately 71 kDa), and β (approximately 50 kDa), while the 11S protein consists of an acidic subunit (approximately 40 kDa) and a basic subunit (approximately 20 kDa). The degradation of antigenic proteins in the fermented soybean meal solution was determined using SDS-polyacrylamide gel electrophoresis. The results showed that after fermentation, over 85% of the 7S and 11S proteins were degraded into small peptide molecules with a molecular weight below 20,000 Daltons. Figure 4 As shown in E and F.

[0068] Example 3: Preparation of Fermented Soybean Meal

[0069] The fermented soybean meal in Example 3 of this invention is produced by solid-state fermentation of soybean meal with the addition of fermentation agents. The specific fermentation agents include Enterococcus faecalis EF-JF-10 and Saccharomyces cerevisiae (purchased from Angel Yeast Co., Ltd. in this embodiment of the invention).

[0070] S1: Preparation of Enterococcus faecalis EF-JF-10 bacterial suspension:

[0071] (1) Seed culture preparation: Take purified Enterococcus faecalis EF-JF-10 slant and inoculate it into sterile MRS broth medium. Place it in a shaker and culture for 16 h at 37℃ and 100 rpm.

[0072] (2) Liquid fermentation: Add MRS liquid fermentation medium and 0.05% organosilicon defoamer to the fermenter for sterilization. Inoculate the cultured seed liquid into the fermenter at a volume ratio of 2% and culture for 24 hours. Fermentation conditions: tank pressure 0.01 MPa, aeration rate 0 L / min, rotation speed 125 rpm, temperature 37℃.

[0073] S2: Preparation of soybean meal solid fermentation feed

[0074] The EF-JF-10 Enterococcus faecalis bacterial solution of S1 was mixed with the soybean meal solid fermentation substrate at a weight ratio of (1.5-2):(4.5-5) (the solid fermentation substrate includes 90% soybean meal, 2% brown sugar and 8% wheat bran). In this embodiment of the invention, the EF-JF-10 Enterococcus faecalis bacterial solution and the soybean meal solid fermentation substrate were mixed at a weight ratio of 1.5:5 to obtain a mixed fermentation substrate. Saccharomyces cerevisiae (20g / t) was added to the mixed fermentation substrate, and the moisture content of the mixed fermentation substrate was adjusted to 40%. The mixed fermentation substrate was packed into fermentation bags with breathing valves, 10kg per bag, and the air was purged. The mixture was sealed and fermented at 37℃ for 5 days. After the fermentation was terminated, samples were taken to test the moisture, pH, number of viable lactic acid bacteria, crude protein content, acid-soluble protein content and total acid, etc.

[0075] The control group consisted of soybean meal samples with added microbial agents. Clean, sterile tap water was mixed with the soybean meal solid-state fermentation substrate at a weight ratio of (1.5-2):(4.5-5) (the solid-state fermentation substrate consisted of 90% soybean meal, 2% brown sugar, and 8% wheat bran), packaged according to S2, fermented, and then sampled for testing.

[0076] The experimental results are shown in the table below:

[0077]

[0078] Example 4: Effects of Fermented Soybean Meal on Weaned Piglets

[0079] 1. Experimental Design

[0080] CK: Basal Ration

[0081] T1: Basal diet + 6% soybean meal (unfermented)

[0082] T2: Basal ration + 6% fermented soybean meal

[0083] The experiment selected 126 weaned piglets (approximately 30 days old) with an initial weight of 5.66±0.52 kg. They were divided into 3 groups according to the principle of uniform weight, with 6 pens in each group and 7 piglets in each pen. The experiment lasted for 9 days. The nutritional ratio and basal diet feeding amount were implemented in accordance with the People's Republic of China Agricultural Industry Standard for Pig Feeding 《NY / T65-2004》.

[0084] 2. Experimental Methods

[0085] Before the experiment, the pigsty and all feeding equipment were cleaned and fumigated (formalin / potassium permanganate ratio 1:15), and then ventilated and air-dried for one week before use. CK, T1, and T2 pigs were housed in separate pens, with the pigs kept in the same building on the floor. They had free access to feed and water. The temperature was maintained between 28±2℃, and the humidity between 65±5%. The pigsty was kept well-ventilated, dry, and clean.

[0086] 3. Evaluation Indicators

[0087] Growth performance includes: initial weight, final weight, daily weight gain, daily feed intake, feed conversion ratio, and diarrhea rate.

[0088] On the morning of day 1 and the last day of the experiment, fasting weights were measured, and feed consumption, average daily gain (ADG), average daily feed intake (ADFI), and feed / gain (F / G) ratio were calculated.

[0089] Diarrhea rate = (Number of animals with diarrhea × Number of days with diarrhea) / (Number of animals in the test × Number of days in the test) × 100%

[0090] Appearance indicators: coat color score.

[0091] Table 1. Evaluation Criteria for Fur Appearance

[0092] very good 5 better 4 Ordinary (ordinary) 3 Difference 2 Very bad 1

[0093] 4. Experimental Results

[0094] The experimental results are shown in the table below, which illustrates the growth performance of weaned piglets fed with creep feed made from different raw materials.

[0095]

[0096]

[0097] As shown in Table 2, the feed of the present invention has the following advantages:

[0098] 1. T2 can effectively improve the growth performance of weaned piglets. After using the product of this invention, the daily weight gain of weaned piglets can be significantly increased, the feed conversion ratio can be reduced, and the diarrhea rate can be significantly reduced.

[0099] 2. When weaned piglets are fed using the embodiments of the present invention, the daily weight gain reaches about 285g, which is 5.95% higher than CK and 5.17% higher than T1, showing a significant improvement effect.

[0100] 3. Regarding the prevention and control of diarrhea, the diarrhea rate of weaned piglets fed with the present invention was low, at 1.87%, which was 63.55% lower than that of the control group (CK) and 54.61% lower than that of the control group (T1).

[0101] 4. Compared with the control group, the feed conversion ratio of group T2 in this embodiment of the invention was reduced by 4.03%, and the coat color of weaned piglets was also improved. This shows that the embodiment of the invention can improve the palatability of the diet for weaned piglets and has a significant effect on alleviating weaning stress.

[0102] In summary, the purpose of this invention is to utilize the protease-producing characteristics of Enterococcus faecalis to ferment and degrade anti-nutritional factors in soybean meal, thereby improving the application effect of soybean meal in weaned piglets. The added microorganisms are only lactic acid bacteria and Saccharomyces cerevisiae, without the need to add Bacillus. In addition, the fermentation method for preparing soybean meal in this embodiment of the invention is anaerobic solid-state fermentation. This fermentation method can fully utilize the characteristics of Enterococcus faecalis while better preserving the nutrients in the fermented material, which is different from the aerobic fermentation of the prior art.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that after reading this application specification, they can still modify or make equivalent substitutions to the specific implementation of the present invention, but these modifications or changes do not depart from the protection scope of the pending claims of the present invention.

Claims

1. A feed fermentation method, wherein Enterococcus faecalis is added to the feed fermentation method, and the Enterococcus faecalis has the preservation number CGMCC NO. 25007.

2. The feed fermentation method according to claim 1, characterized in that, The feed fermentation method is as follows: S101. Inoculate Enterococcus faecalis at a ratio of 2-5% into the fermentation medium and culture to obtain the fermentation broth; S102. Mix the fermentation broth and the fermentation substrate at a weight ratio of (1.5-2):(4.5-5) to obtain a mixed fermentation substrate; S103. Add brewer's yeast to the mixed fermentation substrate, adjust the moisture content of the mixed fermentation substrate to 35%-45%, and ferment for 3-7 days to obtain the target fermented feed.

3. The feed fermentation method according to claim 2, characterized in that, In S101, the fermentation broth is cultured under anaerobic conditions.

4. The feed fermentation method according to claim 3, characterized in that, The viable cell count in the fermentation broth was 2.0*10⁻⁶. 8 -1.0*10 10 cfu / g.

5. The feed fermentation method according to claim 2, characterized in that, The total bacterial count in the mixed fermentation substrate was 5.0*10⁻⁶. 6 -1.0*10 8 cfu / g.

6. The feed fermentation method according to claim 2, characterized in that, The weight ratio of the fermentation broth to the fermentation substrate is 1.6:4.

5.

7. The feed fermentation method according to claim 2, characterized in that, The fermentation medium is MRS broth medium; the fermentation substrate includes 70-90 parts by weight of soybean meal, 2-5 parts by weight of sugar and 10-30 parts by weight of wheat bran, wherein the sugar is a sugar-containing substance, which may be brown sugar, white sugar, black sugar or yellow sugar.

8. The feed fermentation method according to claim 3, characterized in that, The final concentration of the brewing yeast added is 5*10. 6 -1.0*10 7 CFU / g fermentation substrate.

9. The feed fermentation method according to claim 2, characterized in that, In S103, the initial moisture content of the mixed fermentation substrate is 35%-40%, and the fermentation method is anaerobic solid-state fermentation.

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