Bacillus subtilis and application thereof in rapeseed meal fermentation

Abstract

The invention discloses bacillus subtilis and application thereof in rapeseed meal fermentation. The bacillus subtilis is named as bacillus subtilis H8, and the preservation number of the bacillus subtilis is CCTCC (China Center For Type Culture Collection) NO: M20241745; the 16S rDNA gene sequence of the bacillus subtilis H8 is as shown in SEQ ID No: 1. The strain can effectively degrade the content of antinutritional factors in the rapeseed dregs, and the nutritional characteristics of the rapeseed dregs are improved through solid fermentation of the rapeseed dregs. According to the method, the problem that the fermentation effect of existing feed fermentation raw materials is not ideal is solved, and through multidisciplinary cross research and industrial application and popularization, the rapeseed meal fermentation technology provides important support for resource recycling and agricultural sustainable development.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Bacillus subtilis and an application thereof in rapeseed meal fermentation. Background Art

[0002] Rapeseed meal is a byproduct of rapeseed after oil extraction and is widely used in feed, fertilizer and other aspects. Rapeseed meal is rich in protein, fat, vitamins and minerals and has high nutritional value. However, rapeseed meal contains some anti-nutritional factors, such as glucosinolates, phytic acid and gossypol, which will affect its utilization in feed and limit the wide application of rapeseed meal. Rapeseed meal fermentation technology is a method of treating rapeseed meal by microbial fermentation, which can effectively degrade anti-nutritional factors and improve its nutritional value and feed utilization. During the fermentation process, microorganisms can use the nutrients in rapeseed meal for growth and metabolism, and secrete enzymes to decompose anti-nutritional factors, such as glucose isothiocyanate esterase, phytase and protease. By screening high-efficiency strains, the fermentation efficiency and product quality can be significantly improved. Fermented rapeseed meal feed can not only significantly improve the digestion and absorption rate of animals, but also improve the balance of intestinal microecology and enhance the immunity of animals. The development of rapeseed meal fermentation technology will continue to rely on the progress of biotechnology and the improvement of fermentation technology, and will provide important support for resource recycling and sustainable agricultural development. Summary of the invention

[0003] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a Bacillus subtilis and its application in rapeseed meal fermentation. The present invention screened a strain of Bacillus subtilis H8 from a flower culture base pot (containing rotten plant rhizosphere and sphagnum moss) of Huazhong Agricultural University, and the Bacillus subtilis H8 has a significant degradation effect on anti-nutritional factors in rapeseed meal. The present invention provides theoretical support for the development and utilization of fermented rapeseed meal in aquatic feed by screening strains with stable performance and excellent fermentation effect.

[0004] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0005] The invention provides a Bacillus subtilis H8. The Bacillus subtilis H8 is named as Bacillus subtilis H8 and has a preservation number of CCTCC NO: M 20241745.

[0006] Furthermore, the 16S rDNA sequence of the Bacillus subtilis H8 is shown in SEQ ID NO: 1.

[0007] The present invention also provides an application of the Bacillus subtilis H8 in reducing the content of anti-nutritional factors in rapeseed meal and improving the nutritional value of rapeseed meal.

[0008] The present invention also provides an application of the Bacillus subtilis H8 in preparing a feed containing rapeseed meal.

[0009] The present invention also provides a Bacillus subtilis fermentation agent, wherein the fermentation agent comprises the Bacillus subtilis H8, and the concentration thereof is 2×10 7 CFU / mL.

[0010] The present invention also provides a method for preparing the fermentation agent, comprising the following steps:

[0011] (1) Activating Bacillus subtilis H8, and then inoculating the activated bacteria into liquid LB medium and culturing with shaking to obtain a primary seed solution;

[0012] (2) The first-level seed liquid is inoculated into liquid LB culture medium and cultured with shaking to obtain the second-level seed liquid, which is the Bacillus subtilis fermentation agent.

[0013] Furthermore, in the step (1), the activated bacteria are inoculated in a liquid LB culture medium at an inoculation rate of 1-4%, and the culture conditions are shaking culture at 30° C. for 20-24 hours;

[0014] In the step (2), the inoculation amount of the primary seed liquid in the liquid LB culture medium is 5-8%, and the culture condition is shaking culture at 30° C. for 20-24 hours.

[0015] The present invention also provides an application of the Bacillus subtilis fermentation agent in preparing fermented rapeseed meal with low anti-nutritional factor content and high nutritional value.

[0016] A method for preparing fermented rapeseed meal comprises the following steps:

[0017] (1) crushing rapeseed meal, sieving, and sterilizing by high pressure for later use;

[0018] (2) adding the rapeseed meal treated in step (1) to the Bacillus subtilis fermentation agent, mixing, and oscillating to perform solid-state fermentation to obtain fermented rapeseed meal;

[0019] The solid-liquid ratio of rapeseed meal to Bacillus subtilis fermentation agent is 10:0.6-0.8, and the fermentation is carried out at 30°C for 45-50 hours.

[0020] The present invention also provides an application of the fermented rapeseed meal prepared by the method in preparing a largemouth bass breeding feed using a vegetable protein source to replace fish meal.

[0021] Beneficial effects of the present invention:

[0022] 1. The present invention screens out a strain of Bacillus subtilis that has a significant degradation effect on anti-nutritional factors in rapeseed meal, especially reducing the content of glucosinolates in rapeseed meal. The glucosinolate degradation rate of the screened strain H8 is 68.85%, the phytic acid degradation rate is 11.57%, and the gossypol degradation rate is 39.85%, thereby achieving the purpose of detoxification.

[0023] 2. The present invention is used to screen and obtain Bacillus subtilis H8 fermented rapeseed meal, which not only converts the rapeseed meal into other substances, but also improves the nutritional value of the rapeseed meal. The content of total acid, antioxidant performance, crude protein, acid-soluble protein, etc. is increased during the fermentation process, which can improve the nutritional value of the rapeseed meal and increase the proportion of fermented rapeseed meal used in feed.

[0024] 3. During the fermentation process of the present invention, the enzymes and metabolites secreted by the microorganisms can effectively degrade the anti-nutritional factors in the rapeseed meal, including glucosinolates, phytic acid, cottonpol, etc. This not only improves the digestibility of the rapeseed meal, but also reduces its negative impact on the animal digestive system, thereby enhancing the feeding value of the rapeseed meal.

[0025] 4. During the fermentation process of the present invention, microorganisms decompose the complex proteins in rapeseed meal to generate small molecular peptides and amino acids that are easy to digest and absorb. This not only improves the utilization rate of protein, but also enhances the nutritional value of rapeseed meal, providing a more efficient protein source for animal growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a colony morphology diagram of Bacillus subtilis H8 described in Example 1;

[0027] Figure 2 The phylogenetic tree diagram of Bacillus subtilis H8 described in Example 1;

[0028] Figure 3 This is a graph showing the optimum temperature and optimum pH of Bacillus subtilis H8 described in Example 2;

[0029] Figure 4 This is the glucosinolate degradation diagram of the unfermented and fermented rapeseed meal described in Example 6;

[0030] Figure 5 This is the SDS-PAGE picture described in Example 9. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0032] Use and preparation of culture medium in this example

[0033] LB liquid medium / enriched medium: 10 g peptone, 5 g yeast powder, 10 g sodium chloride, distilled water to 1000 mL, pH 7.0 ± 0.2, sterilized at 121 °C for 20 min; used for seed liquid culture.

[0034] LB solid medium: peptone 10g, yeast powder 5g, sodium chloride 10g, agar powder 18g, distilled water to 1000mL, pH 7.0±0.2, sterilized at 121℃ for 20min; used for bacterial preservation.

[0035] Skim milk screening medium: 5g yeast powder, 5g NaCl, 18g agar powder, 10g skim milk powder, add distilled water to 1000mL, sterilize at 121℃ for 20min; used to isolate protease-producing strains.

[0036] Protease fermentation medium: glucose 5g, peptone 10g, KH 2 PO 4 0.5 g, MgSO 4 0.3g, (NH4) 2 SO 4 1.0g, CaCl 2 1g, NaCl 1g, pH 7.2, add distilled water to 1000mL, sterilize at 121℃ for 20min; used for shake flask fermentation.

[0037] Rapeseed meal solid fermentation medium: 30g sterilized feed, 18mL distilled water, 2.4mL strain; fermented feed is used for the determination of relevant nutrients.

[0038] Example 1

[0039] Isolation and Identification of Bacillus subtilis H8

[0040] 1. Isolation of Bacillus subtilis H8

[0041] (1) Source: Wastewater from the flower culture base of Huazhong Agricultural University, containing rotten plant rhizospheres and sphagnum moss.

[0042] (2) A method for isolating a Bacillus subtilis strain, comprising the following steps:

[0043] Step S1: strain H8 was sampled from wastewater in a flower cultivation base of Huazhong Agricultural University, which contained rotten plant rhizosphere and sphagnum moss;

[0044] Step S2: Weigh 5 mL of the sewage obtained in step S1 and place it in a 100 mL conical flask containing 45 mL of sterile water, shake it at 160 r / min for 30 min, and then let it stand; take 2 mL of the supernatant and place it in a 250 mL conical flask containing 45 mL of enrichment culture medium, and shake and culture it at 160 r / min for 2 days;

[0045] Step S3: dilute the bacterial culture solution obtained in step S2 to 10 -3 , 10 -4 , 10 -5 times, 30 μL of each gradient was spread on skim milk screening medium and incubated at 30°C for 2 days;

[0046] Step S4: According to the size of the transparent zone of the colonies of different shapes on the plate, record the diameter of the hydrolysis transparent zone d1 and the diameter of the single bacterial colony d2, calculate the ratio of d1 / d2, select the single colony with strong protease transparent zone activity and streak it with LB solid medium for preservation. Through the initial screening and observation of the size of the hydrolysis zone, the hydrolysis zone of strain H8 on the skim milk powder plate is 3.04±0.27cm.

[0047] 2. Identification of Bacillus subtilis H8

[0048] (1) Identification of colony morphology and physiological and biochemical characteristics

[0049] The bacterial strain H8 isolated in Example 1 was subjected to colony morphology observation and physiological and biochemical characteristics identification.

[0050] like Figure 1 Shown: The strain is dirty white and shiny, with a rough, flat surface and neat edges.

[0051] (2) Molecular Biological Identification

[0052] The strain H8 isolated in Example 1 was subjected to 16S rDNA molecular biological identification. The genomic DNA of the single colony culture was extracted as a template, and PCR amplification was performed using the bacterial 16S universal primer 27F / 1492R. The product was sent to Qingke Biological Co., Ltd. for sequencing, and the sequence was compared with the known sequences in the GenBank database by BLAST analysis, and a phylogenetic tree was constructed.

[0053] According to the nucleotide and amino acid comparison results of strain H8, strain H8 has a high degree of match with Bacillus subtilis, and its physiological characteristics are highly similar to Bacillus subtilis. Figure 2 , named it Bacillus subtilis H8, and deposited it on August 5, 2024. Its deposit number is CCTCC NO: M 20241745. The depository is China Center for Type Culture Collection. The deposit address is Wuhan University, Wuhan, China, and the postal code is 430072.

[0054] Example 2

[0055] Determination of the Optimal Growth Temperature and pH of Bacillus subtilis H8

[0056] 1. Optimum growth temperature: Prepare LB medium, add 25 mL LB medium into a 500 mL Erlenmeyer flask, add 10% Bacillus subtilis H8 bacterial solution, and culture in a shaker at 30°C, 40°C, and 50°C, respectively, with a rotation speed of 160 r / min. Measure OD600 every 3 hours, and perform 3 replicates at each temperature.

[0057] 2. Optimal growth pH: Prepare LB medium and adjust the pH to 1, 3, 5, 7, 9, and 11 with HCl, with a total of six gradients and 3 parallels each. Add 25 mL of LB medium to each 500 mL Erlenmeyer flask and inoculate 10% of Bacillus subtilis H8 bacterial solution. The temperature is 30°C and the rotation speed is 160 r / min. Measure once every 8 hours.

[0058] like Figure 3 As shown, Bacillus subtilis H8 has strong tolerance to temperature and pH, and grows best and fastest at 30°C, and still grows when the temperature reaches 40°C. However, it hardly grows when the temperature rises to 50°C. In addition, Bacillus subtilis H8 also has good tolerance to pH. When the pH is 1, the strain stops growing, but the strain grows well at pH 3-11, and the optimal growth pH is 5-7.

[0059] Example 3

[0060] Preparation of Bacillus subtilis fermentation agent

[0061] Step S1: First, scrape 2-3 loops of a single Bacillus subtilis H8 preserved in LB solid culture medium with an inoculation loop, inoculate into 15 mL of liquid LB culture medium placed in a 50 mL centrifuge tube, and culture at 30° C. with shaking for 24 hours to obtain a primary seed solution.

[0062] Step S2: The first-level seed solution was inoculated into the liquid LB medium at an inoculum size of 5%, and cultured at 30°C with shaking for 24 hours to obtain the second-level seed solution, i.e., the Bacillus subtilis fermentation agent, wherein the concentration of Bacillus subtilis H8 was 2×10 7 CFU / mL.

[0063] Example 4

[0064] Identification of enzyme activity in fermentation broth

[0065] Step S1: Inoculate Bacillus subtilis H8 into 4.5 mL of protease fermentation medium in a 15 mL centrifuge tube, and culture at 30°C and 2000 r / min for 12 to 16 hours. Inoculate the cultured seed liquid with 10% inoculation into 6 mL of protease fermentation medium in a 15 mL centrifuge tube and culture for 24 hours. Centrifuge at 4°C and 10000 r / min for 15 to 20 minutes, remove the bacteria and precipitate, and absorb the supernatant to obtain the fermentation supernatant.

[0066] Step S2: Take 3 test tubes, number them 1, 2, and 3 respectively, add 2 mL of fermentation supernatant into each tube, preheat it in a 40°C water bath for 2 min, then add 1 mL of the preheated 20 g / L casein solution, and keep the temperature precisely for 10 min.

[0067] Step S3: After the incubation is completed, 2 mL of 0.4 mol / L trichloroacetic acid is immediately added to each tube to terminate the reaction. The incubation is continued in a water bath for 20 min to precipitate the residual protein, and the tube is centrifuged at 4000 r for 20 min.

[0068] Step S4: Take another 3 test tubes, numbered 1, 2, and 3, add 1 mL of the corresponding numbered supernatant, then add 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of Folin reagent, shake well, keep in a 40°C water bath for 20 min to develop color, take out and cool to room temperature.

[0069] Step S5: Determine OD680. For the blank control, add 2 mL of trichloroacetic acid before adding the substrate casein solution to inactivate the enzyme. Determine the OD680 value of the sample, calculate the corresponding tyrosine amount (μg) according to the standard curve, and then calculate the enzyme activity.

[0070] Protease activity (U / g) = sample tyrosine content (μg) * dilution factor / 10min;

[0071] The protease activity in the fermentation broth of Bacillus subtilis H8 was calculated by repeated screening to be 140.58±0.40U / g.

[0072] Example 5

[0073] Fermentation of rapeseed meal

[0074] Step S1: crush the rapeseed meal with a crusher, pass it through a 60-mesh sieve, and sterilize it under high pressure at 120° C. for 20 min for later use.

[0075] Step S2: Add 2.4 mL of Bacillus subtilis fermentation agent and 30 g of rapeseed meal to each fermentation glass bottle, mix well, and solid-state ferment for 48 hours at 30° C. in an oscillating incubator. Put the fermented rapeseed meal into an oven for drying, crush it with a grinder, and pass it through a 40-mesh sieve. Store the fermented rapeseed meal in a dryer.

[0076] Example 6

[0077] Detection of anti-nutritional factors in fermented rapeseed meal

[0078] 1. Determination of glucosinolates:

[0079] Step S1: Accurately weigh 0.1 g of crushed and sieved fermented rapeseed meal into a 10 mL test tube, add an equal amount of 80% ethanol solution, shake well, place on a 40° C. shaker for 4 h, and centrifuge at 4000 r / min for 5 min.

[0080] Step S2: Take 0.5 mL of the supernatant after centrifugation into a 10 mL test tube, add 2 mL of 0.1% sodium carboxymethyl cellulose solution and 1 mL of 0.7 mg / mL palladium chloride color development solution, shake well and place in a 24°C constant temperature box for 1.5 to 2 hours for color development reaction.

[0081] Step S3: using a spectrophotometer to measure the absorbance of the reaction solution at a wavelength of 540 nm, and obtaining the content of glucosinolates through conversion.

[0082] 2. Detection of free gossypol content:

[0083] Step S1: Accurately weigh 1 g of fermented rapeseed meal into a 250 mL stoppered conical flask, add glass beads, accurately add 50 mL of reagent A (500 mL of isopropanol-n-hexane mixed solution with a volume ratio of isopropanol to n-hexane of 6:4, add 2 mL of aminopropanol, 8 mL of glacial acetic acid, 50 mL of distilled water, and then make up to 1000 mL with isopropanol-n-hexane mixed solution), shake and extract at room temperature for 3 hours, filter with filter paper, and obtain the filtrate as the sample to be tested.

[0084] Step S2: Pipette two equal portions of 10 mL of the sample to be tested into 25 mL brown volumetric flasks a and b, and take two equal portions of 10 mL of solvent A into 25 mL volumetric flask a. 0 , b 0 In the solution, a and a are fixed to volume with isopropanol-n-hexane mixed solution. 0 to the scale as a reference solution, b, b 0 Add 2 mL of aniline, boil in water for 30 min, cool to room temperature, and dilute to scale with isopropanol-n-hexane mixed solution. Measure the absorbance values ​​at 440 nm with an ELISA reader, and then calculate according to the calculation formula in the national standard method.

[0085] 3. Determination of phytic acid:

[0086] Step S1: Weigh 0.5 g of sample, add 10 mL of 2.4% HCl solution, extract at 200 rpm / min at room temperature for 16 h, centrifuge for 20 min and take the supernatant.

[0087] Step S2: Add 1.0 g NaCl to the supernatant, shake and dissolve, treat at -20°C for 20 min, centrifuge for 20 min, take the supernatant and dilute, mix 3 mL of the diluted sample with 1 mL of modified Wade reagent (0.03% FeCl 3 6H 2The mixture was mixed with 1% sulfosalicylic acid (0.05% ethanol) and centrifuged for 10 min after thorough mixing in a vortex.

[0088] Step S3: Using sodium phytate as a raw material, prepare a standard product containing 0.00, 1.12, 2.24, 3.36, 5.60, 7.84 and 11.20 mg / L of phytic acid, and use absorbance as the ordinate and phytic acid content as the abscissa to prepare a standard curve. Use a spectrophotometer to read the absorbance value at 500 nm, and substitute it into the formula to obtain the phytic acid content.

[0089] The anti-nutritional factors related to fermented and unfermented rapeseed meal were determined. The results are shown in Table 1. The degradation rate of glucosinolates, phytic acid and gossypol in fermented rapeseed meal was 68.85%, 11.57% and 39.80%, respectively. Figure 4 As shown, 1 is unfermented rapeseed meal, 2 is fermented rapeseed meal without adding bacteria, and 3 is fermented rapeseed meal. It was observed that Bacillus subtilis H8 has an excellent performance in degrading glucosinolates.

[0090] Table 1 Anti-nutritional factor content of rapeseed meal

[0091]

[0092] Example 7

[0093] Determination of Nutritional Indexes of Fermented Rapeseed Meal

[0094] 1. The crude protein content is determined according to GB / T 6432-2018.

[0095] 2. The determination of acid-soluble protein is carried out according to NY / T 3801-2020. The operation steps are as follows:

[0096] Weigh 2g of fermented rapeseed meal into a 50mL centrifuge tube, add 40mL of 20% trichloroacetic acid solution, shake at 150r / min for 30min, let stand for 5min, and centrifuge at 4000r / min for 5min. Take 10mL of filtrate and add it to a digestion tube, add a catalyst, and then determine the crude protein content according to the crude protein content determination method.

[0097] 3. Determination of total acid:

[0098] Accurately weigh 1g of fermented rapeseed meal in distilled water, shake well and filter with filter paper, add 2 drops of phenolphthalein solution to the filtrate, titrate with a standardized sodium hydroxide solution until the initial pink color appears and does not fade, which is the titration end point, record the volume of sodium hydroxide used, and calculate the total acid content.

[0099] After the rapeseed meal was fermented by strain H8 microorganism, the nutritional index results were shown in Table 2. The crude protein, acid-soluble protein and total acid contents were significantly increased by 19.25%, 490.75% and 174.7% respectively.

[0100] Table 2 Nutritional indicators of rapeseed meal

[0101]

[0102] Example 8

[0103] Determination of Antioxidant Activity of Fermented Rapeseed Meal

[0104] 1. Determination of DPPH free radical scavenging ability:

[0105] Step S1: Take 1 g of fermented rapeseed meal and mix and shake it in 10 mL of distilled water at 100 r / min for 30 min, dilute it by 1 times, take 2 mL of the diluted sample and mix it in 2 mL of 0.2 mmol / L DPPH-anhydrous ethanol solution, mix it evenly to obtain a sample dilution, and place it in a dark place for 30 min.

[0106] Step S2: Determine the absorbance of the sample diluent at 517 nm (A0); mix 2 mL of the sample diluent with 2 mL of 95% ethanol solution and determine the absorbance (A1); mix 2 mL of 95% ethanol solution with 2 mL of DPPH-anhydrous ethanol solution and determine the absorbance (A2); calculate the DPPH free radical scavenging rate Y1, the formula is as follows:

[0107]

[0108] 2. Determination of hydroxyl free radical scavenging ability:

[0109] Step S1: Take 1g of fermented rapeseed meal and mix it in 10mL of distilled water at 100r / min for 30min, dilute it by 1 times, and take 1mL of dilution, 1mL of ferrous sulfate solution (9mmol / L, diluted 2 times before use), 1mL of 0.3% H 2 O 2 The solution and 1 mL of salicylic acid-ethanol solution (9 mmol / L, diluted 2 times before use) were mixed to obtain a sample reaction solution, which was allowed to stand at 37°C for 0.5 h.

[0110] Step S2: Determine the absorbance value (A0) of the sample reaction solution at 510 nm, replace the diluent with distilled water, and measure the absorbance value (A1) with other conditions unchanged; the diluent, ferrous sulfate, and salicylic acid remain unchanged, and no color developer H is added. 2 O 2 Determine the absorbance value (A). Calculate the hydroxyl radical scavenging rate Y using the following formula:

[0111]

[0112] The scavenging ability of DPPH free radicals and hydroxyl free radicals represent the antioxidant capacity of substances. The antioxidant performance is shown in Table 3. After fermentation, rapeseed meal eliminates the influence of active oxygen in the feed, and the oxidation performance is significantly improved (P<0.05). Compared with the control group, the scavenging rate of DPPH free radicals of fermented rapeseed meal increased by 42.56%, and the scavenging rate of hydroxyl free radicals of fermented rapeseed meal increased by 68.57%.

[0113] Table 3 Antioxidant properties of rapeseed meal

[0114]

[0115] Example 9

[0116] SDS-PAGE detection of fermented rapeseed meal

[0117] Step S1: Take 1 g of fermented rapeseed meal, extract with 10 mL of 0.03 mol / L Tris-HCl buffer (PH=8.0) by shaking for 2 h, then centrifuge at 5000 r, 4°C for 10 min to obtain the supernatant, then centrifuge at 10000 r, 4°C for 5 min, aspirate the supernatant, and store at 4°C.

[0118] Step S2: stain with Coomassie Brilliant Blue R250, prepare separation gel and stacking gel, mix the protein sample to be tested with SDS sample buffer, and heat the sample at 95° C. for 5 to 10 minutes to denature the protein.

[0119] Step S3: Install the gel in the electrophoresis tank and add electrophoresis running buffer. Perform electrophoresis. After the electrophoresis is completed, take out the gel and stain it in Coomassie Brilliant Blue staining solution for 30 to 60 minutes. After staining, place the gel in a destaining solution to destain until the protein bands are clear and the background is clear. Figure 5 As shown, protein bands were observed on the gel, C was unfermented rapeseed meal, and F was fermented rapeseed meal.

[0120] The results are as follows Figure 5 As shown in the figure, compared with the control group, all 25-35kDa bands of fermented rapeseed meal are completely invisible to the naked eye, and the protein bands of protein molecules below 15kDa are darker than those of the control group, indicating that the complex proteins after rapeseed meal fermentation produce small molecular proteins that are easy to digest and absorb.

[0121] In summary, the present invention provides a Bacillus subtilis and its application in rapeseed meal fermentation, the strain can increase the protein content of fermented rapeseed meal, significantly reduce the content of anti-nutritional factors, improve antioxidant properties, convert complex proteins in rapeseed meal into small molecule proteins that are easy to digest and absorb, provide a more efficient protein source for animal growth, and significantly improve the nutritional value of the fermented rapeseed meal prepared. The present invention solves the problem that the fermentation effect of existing feed fermentation strain raw materials is not ideal, and has great application potential for improving the nutritional quality of rapeseed meal and the development of feed resources.

[0122] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A Bacillus subtilis H8, characterized in that: The Bacillus subtilis H8 is named as Bacillus subtilis H8, and the preservation number is CCTCC NO: M 20241745.

2. The Bacillus subtilis H8 according to claim 1, characterized in that: The 16S rDNA sequence of the Bacillus subtilis H8 is shown in SEQ ID NO:

1.

3. Use of the Bacillus subtilis H8 as claimed in claim 1 in reducing the content of anti-nutritional factors in rapeseed meal and improving the nutritional value of rapeseed meal.

4. Use of the Bacillus subtilis H8 according to claim 1 in preparing a feed containing rapeseed meal.

5. A Bacillus subtilis fermentation agent, characterized in that: The fermentation agent comprises the Bacillus subtilis H8 as claimed in claim 1, and its concentration is 2×10 7 CFU / mL.

6. A method for preparing the fermentation agent as claimed in claim 5, characterized in that: The following steps are involved: (1) activating Bacillus subtilis H8, and then inoculating the activated bacteria into liquid LB medium and culturing with shaking to obtain a primary seed solution; (2) The first-level seed liquid is inoculated into liquid LB culture medium and cultured with shaking to obtain the second-level seed liquid, which is the Bacillus subtilis fermentation agent.

7. The preparation method according to claim 6, characterized in that: In the step (1), the activated bacteria are inoculated in a liquid LB culture medium at an inoculation rate of 1-4%, and the culture conditions are shaking culture at 30° C. for 20-24 hours; In the step (2), the inoculation amount of the primary seed liquid in the liquid LB culture medium is 5-8%, and the culture condition is shaking culture at 30° C. for 20-24 hours.

8. Use of the Bacillus subtilis fermentation agent as claimed in claim 5 in preparing fermented rapeseed meal with low anti-nutritional factor content and high nutritional value.

9. A method for preparing fermented rapeseed meal with low anti-nutritional factor content and high nutritional value, characterized in that: The following steps are involved: (1) crushing rapeseed meal, sieving, and sterilizing by high pressure for later use; (2) adding the rapeseed meal treated in step (1) to the Bacillus subtilis fermentation agent, mixing, and oscillating to perform solid-state fermentation to obtain fermented rapeseed meal; The solid-liquid ratio of rapeseed meal to Bacillus subtilis fermentation agent is 10:0.6-0.8, and the fermentation is carried out at 30°C for 45-50 hours.

10. Use of the fermented rapeseed meal prepared by the method according to claim 9 in preparing a largemouth bass aquaculture feed as a vegetable protein source to replace fish meal.

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