A salt-tolerant Bacillus strain producing complex enzymes and its application in feed preparation

Abstract

This invention discloses a salt-tolerant Bacillus strain producing a complex enzyme and its application in feed preparation, belonging to the field of biotechnology. This invention provides a salt-tolerant Bacillus strain that simultaneously produces cellulase and xylanase, which can be used to produce cellulase and xylanase. The cellulase and xylanase preparations produced by this strain can efficiently degrade lignocellulose from wheat straw, corn straw, and rice straw, obtaining monosaccharides such as glucose. The strain of this invention can also be used in the preparation of ruminant feed, helping to improve feed utilization.

Description

Technical Field

[0001] This invention relates to a salt-tolerant Bacillus strain that produces complex enzymes and its application in feed preparation, belonging to the field of biotechnology. Background Technology

[0002] In the current context of "carbon neutrality," improving breeding efficiency, enhancing feed utilization, and reducing methane emissions have become crucial challenges for carbon reduction in animal husbandry. Ruminants primarily consume highly lignified roughage such as corn stalks, rice straw, and wheat straw. Even in beef cattle farming, low-quality roughage like corn stalks and rice straw accounts for no less than 50%. Ruminants rely on cellulase / xylanase secreted by rumen microorganisms to digest plant-based feeds that monogastric animals cannot utilize. However, since the cellulose and lignin components in roughage are mainly located within the cell walls, cellulase / xylanase secreted by rumen microorganisms alone cannot effectively degrade these fibers and release intracellular nutrients. Therefore, there is an urgent need to improve the utilization efficiency of cellulose in ruminant roughage. Identifying strains that improve cellulose utilization and applying them to ruminant feeding would significantly enhance the economic and social benefits of my country's animal husbandry industry. Summary of the Invention

[0003] To address the aforementioned problems in the existing technology, this invention provides a halophilic Bacillus strain that produces cellulase and xylanase. Bacillus halotolerans B757 was deposited on October 14, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No.: 62887), located at 5th Floor, Building 5, No. 100 Xianlie Middle Road, Guangzhou.

[0004] The halophilic Bacillus B757 is a Gram-positive, aerobic bacterium with short rod-shaped cells that appear pale green under a microscope; its colonies are milky white with irregular, slightly wrinkled edges. The salt-tolerant Bacillus B757 can be grown in a culture medium containing NaCl and conventional carbon and nitrogen sources (e.g., LB medium), with an optimal culture temperature of 27–38°C. o C. pH is 6.5–7.5; The salt-tolerant Bacillus B757 can tolerate the growth environment of 12% NaCl and 1.0% bile salt. When incubated for 3 hours at pH 2.0, its viable count hardly decreased, indicating that the strain can resist the adverse effects of gastric acid and high concentrations of bile salt. The salt-tolerant Bacillus B757 has a strong ability to produce cellulase and xylanase. The cellulase and xylanase described herein have the following activities: cellulase activity is 80–180 U / mL, and xylanase activity is 50–150 U / mL. Preferably, the cellulase and xylanase have the following activities: cellulase activity is 159.4 U / mL, and xylanase activity is 106.4 U / mL.

[0005] The present invention also provides a microbial inoculum containing the above-mentioned salt-resistant Bacillus B757.

[0006] In one embodiment, the microbial agent contains the halophilic Bacillus B757 and Clostridium butyricum.

[0007] In one embodiment, the ratio of halophilic Bacillus B757 to Clostridium butyricum in the microbial agent is 8 to 10:1.

[0008] In one embodiment, the microbial agent is a compound of microbial powder and excipients.

[0009] In one embodiment, the Clostridium butyricum is Clostridium butyricum (C. butyricum). Clostridium tyrobutyricum L319, accession number GDMCC No: 62289, has been published in patent application document with publication number CN115305219A.

[0010] In one embodiment, the concentration of halophilic Bacillus B757 in the microbial agent is 8 × 10⁻⁶. 10 ~8×10 12 CFU / g, Clostridium butyricum concentration was 8×10⁻⁶. 9 ~8×10 11 CFU / g.

[0011] In one embodiment, the microbial preparation is formulated by combining single-strain powder and viable bacteria count at a viable bacteria ratio of halophilic Bacillus B757: Clostridium butyricum L319 = 10:1.

[0012] In one embodiment, the content of each microorganism in the microbial agent is as follows: halophilic Bacillus B7578 × 10 11 CFU / g, Clostridium butyricum 8×10 10 CFU / g.

[0013] In one embodiment, the excipients in the microbial agent include one or more of trehalose and chitosan oligosaccharides, and the excipients mainly play the role of dispersing single-strain powder and protecting microbial cells.

[0014] In one embodiment, the halophilic Bacillus B757 and Clostridium butyricum L319 are both obtained as dry bacterial powders through liquid fermentation, centrifugation, and spray drying processes, respectively. The dry bacterial powders are then mixed with excipients to obtain a microbial inoculant.

[0015] The present invention also provides the application of the microbial preparation in the preparation of ruminant feed.

[0016] Beneficial effects: 1. The salt-tolerant Bacillus B757 obtained by screening in this invention has strong resistance to the external environment and can completely tolerate the high osmotic pressure environment of gastric acid and intestinal bile salts in ruminants. Compared with enzyme preparations, it has stronger tolerance and more stable properties. In addition, the microbial agent has a long shelf life and is easy to transport and store.

[0017] 2. The salt-tolerant Bacillus isolated by this invention has a higher ability to produce complex enzymes such as cellulase, protease, and xylanase, and a stronger ability to degrade cellulose, which can improve the digestibility and utilization of roughage.

[0018] 3. The selected halophilic Bacillus and Clostridium butyricum strains exhibit strong complementarity, degrading cellulose through synergistic effects. Furthermore, as a novel probiotic, butyric acid, the main metabolite of Clostridium butyricum, is a crucial nutrient for the regeneration and repair of intestinal epithelial cells, and it plays a significant role in promoting animal growth. Therefore, the application of the compound microbial agent of halophilic Bacillus and Clostridium butyricum in further enhancing animal productivity while improving the digestibility and utilization of roughage is a pioneering achievement of this invention.

[0019] Preservation of biological materials Salt-resistant Bacillus ( Bacillus halotolerans B757, categorized and named Bacillus halotolerans It was deposited on October 14, 2022 at the Guangdong Provincial Center for the Preservation of Microbial Strains, with accession number GDMCCNo: 62887, located at 5th Floor, Building 5, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0020] Figure 1 The growth status of the salt-tolerant Bacillus strain B757 isolated and screened in this invention on a plate.

[0021] Figure 2 The growth curve of the salt-tolerant Bacillus strain B757 isolated and screened in this invention under high osmotic pressure and high bile salt conditions. Detailed Implementation

[0022] The microbial inoculant was produced in-house by the inventor; all other auxiliary materials were purchased from the feed market. The Clostridium butyricum used in the examples (…) Clostridium tyrobutyricum L319, accession number GDMCC No: 62289, has been published in patent application document with publication number CN115305219A.

[0023] For the determination of cellulase activity, refer to (Tan Shiyao. Study on physiological characteristics and optimization of fermentation conditions of 11 strains of Bacillus rumen-derived bacteria [D]. Dalian University of Technology, 2018.); For the determination of xylanase activity, refer to (Zhao Longmei et al. Screening, identification and characteristic analysis of xylanase-producing microorganisms [J]. Journal of Animal Ecology, 2022, 43(04):14-20.).

[0024] Cellulase activity is defined as the amount of enzyme required to hydrolyze the substrate to produce 1 μmol of glucose per minute at 25°C and pH 7.0, which is defined as 1 U.

[0025] Xylanase activity is defined as the amount of enzyme required to hydrolyze 0.01 mol xylan per minute to produce 1 μmol of reducing sugar (xylose) at 25°C and pH 7.0. One unit of enzyme activity (U) is defined as the amount of enzyme required to hydrolyze 0.01 mol xylan per minute to produce 1 μmol of reducing sugar (xylose).

[0026] Example 1: Isolation and Identification of Haloxytolerant Bacillus B757 Sample Collection: Selecting the optimal sampling time for microbial isolation and considering local climate conditions, samples were collected from a radiation-contaminated area in April. After removing the topsoil (5 cm) at each sampling point, soil samples (5-30 cm deep, thoroughly mixed) were placed in sterile sampling tubes and then placed in a lead-lined container at -4°C. o Store in refrigerator (C) for later use.

[0027] Strain isolation: Take 1 g of the above soil sample, add it to 99 ml of sterile physiological saline, mix well, and then add 1 ml of the liquid to a liquid culture medium with CMC (carboxymethyl cellulose) as the sole carbon source. Incubate at 30°C. o C. After culturing at 200 rpm for 24 h, 1 ml of the liquid was added to a liquid culture medium with CMC as the sole carbon source, and incubated at 30°C. o C. Continue shaking culture at 200 rpm for 24 h, repeating this enrichment culture three times. Finally, after serial dilution of the culture medium, spread it on plates using CMC as the sole carbon source, and incubate at 30°C. o The culture was carried out at C for 48 h. Based on the size of the clear zone produced by the colonies on the plate culture medium, 50 strains that could effectively utilize CMC were screened. The larger the clear zone, the stronger the ability of the strain to produce cellulase.

[0028] Next, the isolated single colonies were inoculated onto LB medium supplemented with 2% AZCL-wheat arabinoxylan for screening, and the colonies were cultured at 30°C. o Incubate at C for 48 h to test for xylanase activity in the colonies. Based on the blue hydrolysis zones produced on LB plates, strains with large hydrolysis zones are numbered and transferred to slant agar plates as target strains for later use.

[0029] The cellulase and xylanase activities of six selected Bacillus strains were measured. Single colonies of the strains were picked, inoculated into LB medium, and cultured at 30℃ for 24 h. The enzyme activities in the fermentation broth were then detected. The results are shown in Table 1. One strain with the highest cellulase and xylanase production capacity was obtained, preserved in glycerol, and designated as B757. 16S rDNA identification confirmed it to be a halophilic Bacillus. Bacillus halotolerans As can be seen, the halophilic Bacillus B757 screened in this invention has a strong ability to produce cellulase and xylanase, and can be made into bacterial powder for use in the research and development of various feed products.

[0030] Table 1. Results of enzyme activity assay for the target strain

[0031] Example 2: Validation of the tolerance performance of halophilic Bacillus B757 Seed culture medium (g / L): glucose 10, K2HPO4 4.8, KH2PO4 3.5, (NH4)2SO4 2, MgCl2 0.16, CaCl2 0.02, Na2MoO4·2H2O 0.0024, FeCl3 0.0018, MnCl2·2H2O 0.0015, NaCl 10, initial pH 7.0.

[0032] (1) Growth performance under 12% NaCl conditions The seed culture of halophilic Bacillus B757 was transferred to a seed culture medium containing 12% NaCl at an inoculation rate of 2%. Samples were taken at regular intervals to measure the OD. 600 The absorbance at each location was used to plot cell growth curves, with sampling time on the x-axis and absorbance value on the y-axis, as shown below. Figure 2 As shown, the bacterial concentration OD of the strain under 12% NaCl conditions 600 It can reach 2.2.

[0033] (2) Growth performance under 1.0% bile salt conditions The seed culture of halophilic Bacillus B757 was transferred to a seed culture medium containing 1.0% bile salts at an inoculation rate of 2%. Samples were taken at regular intervals to measure the OD. 600 The absorbance at each sampling point was used to plot cell growth curves with sampling time on the x-axis and absorbance value on the y-axis. For example... Figure 2 As shown, the maximum bacterial concentration (OD) of the strain under 1.0% bile salt conditions was... 600 It reached 1.52.

[0034] (3) Tolerance performance under pH 2.0 conditions Salt-tolerant Bacillus B757 was inoculated into seed culture medium and cultured to mid-log phase. 1 mL of fermentation broth was taken, centrifuged, and the supernatant was discarded, retaining the bacterial cells. The cells were resuspended in pH 2.0 (HCl-adjusted) medium, shaken well, and treated for 3 h. The culture was then diluted and spread onto plates, with three replicates per sample. The plates were incubated at 30°C for 24 h, and the survival rate under the treatment conditions was calculated (survival rate = number of viable bacteria after incubation / number of viable bacteria before incubation). The experiment showed that after 3 h of incubation at pH 2.0, the survival rate still reached 97.38%, with almost no effect.

[0035] Example 3: Preparation of microbial inoculum containing halophilic Bacillus B757 (1) Preparation of Clostridium butyricum inoculum The seed culture medium used was commercially available Reinforced Clostridium Medium (RCM). (Qingdao Haibo Biotechnology Co., Ltd., Product No.: HB0316) The isolated single colonies were inoculated into RCM medium and cultured at 30°C for 12 h to obtain seed culture.

[0036] The fermentation medium was a modified version of Clostridium Growth Medium (CGM) (see Jiang Ling. Research on the Production of Butyric Acid from Inexpensive Biomass by Fermentation of Clostridium butyricum Immobilized in Fiber Beds [D]. South China University of Technology, 2010.). Specifically: Waste shrimp and crab shells from kitchen waste were collected, washed, dried, ground into powder, and passed through a 100-mesh sieve. The obtained shrimp and crab shell powder was used directly as a nitrogen source, added to the liquid culture medium at a concentration of 25 g / L. This medium (1 L) also contained 5 g NaCl, 1.5 g K2HPO4, 0.6 g MgSO4·7H2O, 0.03 g FeSO4·7H2O, and 20 g glucose.

[0037] Batch fermentation of Clostridium butyricum L319 was carried out in a 50 L fully automated mechanically stirred fermenter, with 25 L of fermentation medium added. Before fermentation, the fermenter was heated to 121 °C. o Sterilize at 115°C for 20 min, then add fermentation medium and sterilize at 115°C. o Sterilize at C for 20 min. After cooling, purge the tank with sterile nitrogen to achieve an anaerobic environment. Then, inoculate the seed culture into the fermenter at a 5% inoculum rate. Set the stirring speed and temperature to 100 rpm and 30 rpm, respectively. o C, and the pH of the fermentation broth was controlled at 6.0 by adding 8 M NaOH.

[0038] When the bacterial cells reach the mid-to-late logarithmic growth stage (approximately 12 hours), the fermentation broth is aliquoted into centrifuge cups, sealed, and centrifuged at 5000 rpm for 4 seconds. o Centrifuge at 1°C for 15 min, discard the supernatant. Collect the centrifuged sample and prepare bacterial powder by spray drying. Spray drying operating conditions: inlet air temperature 200°C. o C, outlet air temperature 80 o C. 4 × 10⁴ units of live bacteria 9 CFU / g or higher.

[0039] (2) Preparation of halophilic Bacillus B757 inoculum Fermentation medium composition (liquid, g / L): glucose 30, peptone 20, yeast extract 15, potassium dihydrogen phosphate 1.5, magnesium sulfate 1.0, initial pH 7.0.

[0040] Salt-tolerant Bacillus B757 was inoculated into seed culture medium and incubated at 30°C. o C, aerated culture for 18 h to obtain liquid seed; then, the liquid seed was inoculated into a 50 L fermenter containing sterilized fermentation medium at a volume fraction of 10%, and incubated at 30°C. o Bacterial cultures were obtained by culturing at 4°C, 200 r / min, and an aeration rate of 0.8 vvm for 40 h (logarithmic growth phase); the bacterial cultures were then collected at 4°C. o C. Centrifuge at 5000 r / min for 15 min, discarding the supernatant. Collect the centrifuged sample and prepare bacterial powder by spray drying. Spray drying operating conditions are: inlet air temperature 200... o C, outlet air temperature 80 o C. 8 × 10⁸ units of live bacteria 10 CFU / g or higher.

[0041] (3) Preparation of compound microbial inoculants The Clostridium butyricum inoculum prepared in step (1) and the halophilic Bacillus B757 were compounded according to the ratio of live bacteria B757 to Clostridium butyricum L319 = 10:1. The compounded microorganisms were then mixed with excipients (trehalose or chitosan oligosaccharide) to obtain the microbial inoculum, wherein the volume ratio of bacteria to excipients was 1:2.

[0042] Example 4: Effect of microbial inoculants containing halophilic Bacillus B757 on roughage utilization rate The microbial inoculant obtained in Example 3 was mixed with wheat, rice, and corn straw that had been crushed and passed through a 200-mesh sieve (i.e., 9 g / 100 g straw was added), and water was added to ensure full absorption. The blank control was not treated with inoculant (the excipients from the microbial inoculant prepared in Example 3 were used instead). Three replicates were set for each substrate, and the mixtures were stored and fermented at room temperature (25°C) for 60 days. The residue was then transferred to a pre-weighed 200-mesh nylon bag. The crude fiber content was determined according to the crude fiber determination method for nylon bags described by Hu Yanping et al. (Wu Qiujue et al. Experimental study on the determination of crude fiber in feed using nylon bags [J]. Feed Industry, 2005(01):55-56.), and the crude fiber degradation rate was calculated.

[0043] As shown in Table 2, the microbial agent of Example 3 achieved a crude fiber hydrolysis rate of 25.4% for rice straw and 20.3% for wheat straw.

[0044] Table 2. Effects of microbial inoculants on the degradation rate of crude fiber in feed.

[0045] Comparative Example 1: The specific implementation method is the same as in Example 4, except that the inoculant is replaced with an inoculant containing only halophilic Bacillus B757 prepared according to the method in Example 3. The amount of inoculant added is 9 g / 100 g rice straw. The results show that the crude fiber hydrolysis rate is only 21.4%.

[0046] Comparative Example 2: The specific implementation method is the same as in Example 4, except that the inoculant is replaced with an inoculant containing only Clostridium butyricum L319 prepared according to the method in Example 3. The amount of inoculant added is 9 g / 100 g rice straw. The results show that the crude fiber hydrolysis rate is only 19.8%.

[0047] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A halophilic Bacillus strain producing cellulase and xylanase ( Bacillus halotolerans B757 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on October 14, 2022, with accession number GDMCC No: 62887.

2. A microbial agent containing the halophilic Bacillus B757 as described in claim 1.

3. The microbial agent according to claim 2, characterized in that, The microbial agent contains the salt-resistant Bacillus B757 and Clostridium butyricum as described in claim 1. Clostridium tyrobutyricum L319, the Clostridium butyricum ( Clostridium tyrobutyricum L319 has the accession number GDMCC No: 62289.

4. The microbial agent according to claim 3, characterized in that, The ratio of the concentrations of halophilic Bacillus B757 and Clostridium butyricum L319 in the microbial agent is (8-10):

1.

5. The microbial inoculant according to any one of claims 2 to 4, characterized in that, The microbial agent is a compound of microbial powder and excipients.

6. The microbial agent according to claim 3 or 4, characterized in that, The concentration of halophilic Bacillus B757 in the microbial agent was 8 × 10⁻⁶. 10 ~8×10 12 The concentration of Clostridium butyricum L319 was 8 × 10⁻⁶ CFU / g. 9 ~8×10 11 CFU / g.

7. The use of the salt-tolerant Bacillus B757 of claim 1 in the production of cellulase and xylanase.

8. The application of the salt-tolerant Bacillus B757 of claim 1 or the microbial agent of any one of claims 2 to 6 in the preparation of ruminant feed.

9. The application according to claim 8, characterized in that, The application includes the degradation of coarse fibers from wheat straw, rice straw, or corn straw.

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