Bacillus paralicheniformis and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus paralicheniformis and application thereof. The preservation number of the bacillus parlicheniformis is CGMCC (China General Microbiological Culture Collection Center) No. 34409. The application comprises the steps of degrading glucosinolate, improving the quality of rapeseed dregs and improving the growth performance and immune performance of fish organisms. The bacillus paralicheniformis with glucosinolate degradation capacity is obtained through screening, the content of antinutritional factors in the rapeseed dregs can be effectively reduced, the quality of the rapeseed dregs is improved, and then the bacillus paralicheniformis can be better applied to feed of fish organisms. The bacillus paralicheniformis provided by the invention can effectively improve the growth performance of fish organisms and improve the liver health of the fish organisms, and has an important value in the field of aquaculture.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a Bacillus paralichrysogenus and its applications. Background Technology

[0002] Rapeseed meal, as an important by-product of oil crops, is rich in crude protein and is cheaper than soybean meal, giving it an economic advantage in feed selection. However, anti-nutritional factors such as glucosinolates in rapeseed meal can adversely affect animal production performance, inhibiting growth and health, thus significantly limiting its widespread application in the feed industry.

[0003] Microbial fermentation technology offers an effective solution to this problem. Through microbial fermentation, the large molecular nutrients in rapeseed meal can be effectively broken down into smaller molecules that are more easily digested and absorbed by animals. This process not only significantly improves the absorption efficiency of nutrients in rapeseed meal by animals but also enhances their immunity to some extent, promoting healthy growth. Simultaneously, the beneficial metabolites produced during fermentation further enhance the nutritional value of rapeseed meal, optimize its flavor, and make it more palatable, thereby stimulating animals' appetite and increasing feed intake.

[0004] Microorganisms that can effectively degrade anti-nutritional factors such as glucosinolates still need further development. Summary of the Invention

[0005] To address the problems existing in the prior art, the present invention provides a Bacillus paralichrysum and its applications.

[0006] In a first aspect, the present invention provides a Bacillus paralichrysum ( Bacillus paralicheniformis The accession number of Bacillus paralichrysiformis CP34 is: CGMCC No. 34409.

[0007] This invention screened a strain of *Bacillus paralichrysiformis* capable of degrading glucosinolates through glucosinolate degradation experiments combined with ultraviolet mutagenesis, and then preserved it biologically. The preservation information is as follows: Accession number: CGMCC No. 34409; Classification: Bacillus paralichrysiformis Bacillus paralicheniformis Depository Institution: China General Microbiological Culture Collection Center; Depository Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing; Deposit Date: April 30, 2025.

[0008] Secondly, the present invention provides a microbial agent comprising the aforementioned Bacillus paralicheniformis CP34 or its fermentation product.

[0009] Further, the bacterial agent is a solid bacterial agent, a liquid bacterial agent, or a microbial bacterial agent, wherein the total viable count of *Bacillus paralichrysiformis* CP34 in the bacterial agent is 1 × 10⁻⁶. 7-10 cfu / g.

[0010] Thirdly, the present invention provides a method for preparing the aforementioned microbial agent, comprising: All microorganisms are placed in a fermentation system for fermentation culture.

[0011] Furthermore, the fermentation substrate of the fermentation system is rapeseed meal, and the cultivation conditions include a temperature of 35~40℃.

[0012] Fourthly, the present invention provides a product comprising the aforementioned Bacillus paralichrysiformis CP34 or the aforementioned bacterial agent; the product is feed, desulfurizer, fertilizer or plant growth promoter.

[0013] Fifthly, the present invention provides the application of the aforementioned Bacillus paralichrysiformis CP34, or the aforementioned bacterial agent, in any of the following: (1) Degradation of glucosinolates; (2) Preparation of reagents for degrading glucosinolates.

[0014] Sixthly, the present invention provides the application of the aforementioned Bacillus paralichrysiformis CP34, or the aforementioned bacterial agent, in any of the following: (1) Enhance the nutritional value of rapeseed meal; (2) Prepare a reagent for improving the nutritional value of rapeseed meal.

[0015] Furthermore, the enhancement of the nutritional value of rapeseed meal includes: Increase the content of crude protein, crude fat, neutral detergent fiber, and acid detergent fiber in rapeseed meal, and reduce the content of glucosinolates.

[0016] In a seventh aspect, the present invention provides the use of the aforementioned Bacillus paralichrysiformis CP34, or the aforementioned bacterial agent, in any of the following: (1) Improve the growth performance of fish organisms; (2) Prepare feed to improve the biological growth performance of fish; (3) Improves liver health in fish organisms; (4) Prepare feed for improving the liver health of fish organisms.

[0017] The present invention has the following beneficial effects: This invention obtains a strain of Bacillus paralicheniformis through screening and mutagenesis, which has the ability to degrade glucosinolates. After fermentation of rapeseed meal with it, the quality of rapeseed meal can be significantly improved. The product obtained can be used as fish feed to significantly improve the growth performance and immune performance of fish, which has important application value in the field of aquaculture. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is the macroscopic morphology of strain CP34 provided in Example 1 of the present invention.

[0020] Figure 2 This is the microscopic morphology of strain CP34 provided in Example 1 of the present invention.

[0021] Figure 3 This is the result of the 16S rDNA sequence comparison of strain CP34 provided in Example 1 of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0023] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0024] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0025] Example 1 1. This invention is based on a laboratory-preserved bacterial library. Through strain screening and mutagenesis, bacteria with specific degradation function of glucosinolates were obtained and named CP34.

[0026] 2. The present invention identifies the physicochemical properties of strain CP34. 2.1 Morphological observation Macroscopic form: such as Figure 1 As shown, strain CP34 was cultured on TSA medium at 37°C for 18 hours. The colonies were light yellow, irregular in shape, moist, opaque, and had neat edges.

[0027] Micromorphology: such as Figure 2 As shown, strain CP34 was cultured in TSA medium at 37℃ for 18 h. The cells were rod-shaped, 0.5-0.7 μm × 1.6-9.8 μm, arranged singly or in pairs, and were Gram-positive.

[0028] 2.2 Physiological and Biochemical Detection The physiological and biochemical characteristics of strain CP34 showed that it was positive for β-xylosidase, phenylalanine aromatic aminoaminase, alanine-phenylalanine-proline aromatic aminoaminase, inositol, D-mannose, alanine aromatic aminoaminase, cyclodextrin, methyl-α-D-glucopyranoside, maltotriose, β-glucosidase, α-glucosidase, and D-glucose. Specific results are shown in the table below.

[0029] Table 1 Physicochemical properties of strain CP34

[0030] Symbol explanation: "+", positive; "w", weak positive; "-", negative.

[0031] 2.3 16S rDNA alignment In this embodiment, the gene sequence of strain CP34 was obtained by 16S rDNA sequencing, and BLAST sequence comparison was performed using NCBI. After comparison, as shown... Figure 3 As shown, strain CP34 is Bacillus paralicheniformis.

[0032] Example 2 This embodiment verifies the function of strain CP34 involved in Example 1, and the process is as follows: 1. Preparation of fermented rapeseed meal Using a sterilized inoculation loop, pick an appropriate amount of CP34 colonies and inoculate them into a shaker containing 3 mL of liquid culture medium. Place the shaker in a 37 ℃ incubator and incubate overnight with shaking at 180 r / min to obtain primary seed culture. Take 1 mL of primary seed culture and inoculate it into a conical flask containing 50 mL of liquid culture medium. Incubate at 37 ℃ with shaking at 180 r / min for 24 h to obtain secondary seed culture. Using uninoculated liquid culture medium as a blank control, measure the OD value of the secondary seed culture at 600 nm. Adjust the OD600 of the secondary seed culture to 0.8. Inoculate it at a 10% inoculation rate (m / v) into rapeseed meal solid fermentation medium (containing only rapeseed meal). Ferment at 37 ℃ for 7 days, stirring the rapeseed meal fermentation medium every 12 h. After 7 days of fermentation, dry the fermented rapeseed meal in a 55 ℃ oven to constant weight, pulverize it, and store it in a sealed bag.

[0033] Further, Bacillus paralicheniformis CP34 was selected and fermented using the same fermentation method as described above.

[0034] 2. Determination of nutritional components of fermented rapeseed meal Fermented and unfermented rapeseed meal were pulverized and passed through a 40-mesh sieve, then packaged in sealed plastic bags for later use. The contents of crude protein (CP), crude fat (CF), acid detergent fiber (ADF), and neutral detergent fiber (NDF) in the samples were determined according to the testing methods GB / T 6432-2018, GB / T 6433-2006, NY / T 1459-2002 5, and GB / T20806-2022 5. Each sample was tested three times, and the average value was taken.

[0035] 3. Determination of glucosinolate content in rapeseed meal The procedure for determining the glucosinolate content in rapeseed meal before and after fermentation is as follows: 3.1 Prepare palladium chloride (PdCl2) colorimetric solution, 0.1% sodium carboxymethyl cellulose (CMC-Na) solution and 0.03 mol / L HCl solution.

[0036] 3.2 The water bath needs to be preheated before the experiment. Weigh 100 mg of crushed fermented rapeseed meal and place it in a 20 mL glass test tube. Set up 3 replicates for each group and label them. Place the glass tube in the preheated water bath and dry-heat in boiling water for 10-15 min. After that, add 10 mL of distilled water at about 90 °C to the glass tube and boil it in the water bath for another 25 min. After that, remove the test tube and cool it to room temperature. Dilute and mix it with distilled water and let it stand for 20 min. After standing, centrifuge the solution in a centrifuge at 5000 r / min for 10 min and discard the precipitate.

[0037] 3.3 Mix the reagents as follows: (1) Use a 5 mL pipette to measure 2 mL of the filtrate and place it in a 10 mL colorimetric tube. Add 4 mL of CMC-Na solution and then 2 mL of PdCl2 colorimetric solution. Mix by inverting the tube and let it stand at room temperature for 1 h. Measure its absorbance value. The value obtained is E1 after subtracting the blank control. (2) Use a 5 mL pipette to measure 2 mL of the filtrate and place it in a 10 mL colorimetric tube. Add 4 mL of CMC-Na solution and then 2 mL of 0.03 mol / L HCl solution. Mix by inverting the tube and let it stand at room temperature for 2 h. Measure the absorbance value. Subtract the blank control from the obtained value to get E2.

[0038] (3) Use distilled water to replace the filtrate as a blank control and measure the absorbance to eliminate the influence of the reagent itself.

[0039] (4) Process the obtained data according to the following formula: (i) E = E1 - E2.

[0040] (ii) Glucoside content (μmol / g) = 0.2 + 185.2E.

[0041] (iii) Degradation rate of glucosinolates (%) = (glucosinolate content before treatment - glucosinolate content after treatment) / glucosinolate content before treatment × 100%.

[0042] 4. Experimental Results (1) Results of nutritional value of fermented rapeseed meal The results are shown in the table below: After single-strain fermentation of rapeseed meal by strain CP34, the nutritional value of rapeseed meal was significantly improved, and the levels of crude protein, crude fat, neutral detergent fiber, and acid detergent fiber were all significantly increased.

[0043] Table 2 Nutritional value of rapeseed meal before and after fermentation

[0044] Example 3 The present invention further applies the strain CP34 involved in Example 1 to aquaculture, including the following process: Fifty-four selected koi carp were randomly divided into two experimental groups using a single-factor randomized controlled trial design, with three replicates in each group and nine koi carp per replicate. The control group was fed the same soybean meal diet twice daily, while the experimental groups were fed a compound fermented rapeseed meal diet instead of soybean meal. After the experiment, the final body weight (Wt) of the koi carp was measured, and their weight gain (WG, g), weight gain rate (WGR, %), and specific growth rate (SGR, % / d) were calculated using the following formulas: , , .

[0045] Subsequently, nine koi carp were randomly selected from each group and anesthetized by placing them in water containing 40 mg / L eugenol. Blood was collected from the tail veins of the koi carp, centrifuged at 8000 rpm for 15 min at 4 ℃, and the supernatant was collected to determine serum lysozyme activity.

[0046] Table 3. Growth performance and immune performance of koi.

[0047] Weight gain, weight gain rate, and specific growth rate are core indicators that directly reflect the growth performance of fish. The levels of AST and ALT in serum are key indicators for measuring the health level of the body, especially liver health. Therefore, fermented rapeseed meal can be used in special aquatic feeds. When added to the feed at a rate of 30%, compared with unfermented rapeseed meal, CP34 fermented rapeseed meal can improve growth rate, improve feed conversion efficiency, and promote liver health.

[0048] 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 them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A type of Bacillus paralicheniformis ( Bacillus paralicheniformis CP34, characterized in that, The accession number of the Bacillus paralichrysiformis CP34 is: CGMCC No. 34409.

2. A microbial agent, characterized in that, Includes Bacillus paralicheniformis CP34 or its fermentation products as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The bacterial agent is a solid bacterial agent, a liquid bacterial agent, or a microbial bacterial agent, wherein the total viable count of *Bacillus paralichrysum* CP34 in the bacterial agent is 1 × 10⁻⁶. 7-10 cfu / g.

4. The method for preparing the microbial agent according to any one of claims 2-3, characterized in that, include: All microorganisms are placed in a fermentation system for fermentation culture.

5. The preparation method according to claim 4, characterized in that, The fermentation substrate of the fermentation system is rapeseed meal, and the cultivation conditions include a temperature of 35~40℃.

6. A product characterized in that, The product includes Bacillus paralichrysogenum CP34 as described in claim 1, or the bacterial agent as described in any one of claims 2-3; the product is feed, desulfurizer, fertilizer, or plant growth promoter.

7. The use of Bacillus paralichrysogenum CP34 as described in claim 1, or the inoculum agent as described in any one of claims 2-3, in any of the following: (1) Degradation of glucosinolates; (2) Preparation of reagents for degrading glucosinolates.

8. The use of Bacillus paralichrysiformis CP34 as described in claim 1, or the inoculum as described in any one of claims 2-3, in any of the following: (1) Enhance the nutritional value of rapeseed meal; (2) Prepare a reagent for improving the nutritional value of rapeseed meal.

9. The application according to claim 8, characterized in that, The improvement of the nutritional value of rapeseed meal includes: Increase the content of crude protein, crude fat, neutral detergent fiber, and acid detergent fiber in rapeseed meal, and reduce the content of glucosinolates.

10. The use of Bacillus paralichrysiformis CP34 as described in claim 1, or the inoculum as described in any one of claims 2-3, in any of the following: (1) Improve the growth performance of fish organisms; (2) Prepare feed to improve the biological growth performance of fish; (3) Improves liver health in fish organisms; (4) Prepare feed for improving the liver health of fish organisms.

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