Bacillus amyloliquefaciens xw5 and application thereof

Abstract

The application discloses a bacillus amyloliquefaciens XW5, belongs to the field of degrading bacteria screening, and the bacillus amyloliquefaciens XW5 is preserved in the China General Microbiological Culture Collection Center on May 8, 2024, has a preservation number CGMCC No.30524, and has a preservation address of No.3, Xibinxi Road, No.1 Yard, Chaoyang District, Beijing City; is classified and named as bacillus amyloliquefaciens. The strain is obtained through screening, purification and identification technologies from grape branch compost, and through research on strain characteristics, fermentation conditions and cellulase activity of the strain, it is shown that the enzyme activity of the strain is 923.20 U / g under the condition of 30 DEG C and PH = 8, and when 5% mannitol is added as a protective agent, the effective viable cell number of the bacillus amyloliquefaciens XW5 is the highest, and can reach 2.05 x 10 14 cfu / g.

Description

Technical Field

[0001] This invention relates to the field of screening and application technology of degrading bacteria, and more specifically to a Bacillus amyloliquefaciens XW5 and its application. Background Technology

[0002] Grapes are one of the main fruit trees cultivated in my country, with a vast planting area. According to data released by the International Organisation of Vine and Wine (OIV), China's grape planting area in 2020 was approximately 785,000 hectares. 2 The resulting waste grape branches will reach 9.42 million to 11.775 million tons. Composting is a common method for utilizing these branches as resources. By crushing the branches and allowing them to decompose, organic fertilizer is produced and applied as a base fertilizer. Composting is primarily a physiological and biochemical process dominated by microbial activity in the compost matrix, and it is an effective way to achieve the harmlessness, reduction, and resource utilization of grape branches. The composting process involves the transformation of organic matter by microorganisms, which can be divided into two parts: mineralization and humification. Mineralization involves breaking down large organic molecules into simpler substances; humification involves the generation of more complex humus through microbial activity. After composting, organic waste can be used as organic fertilizer in agricultural production, improving soil physical and chemical properties and promoting plant growth.

[0003] However, grapevines contain a large amount of cellulose and lignin, which presents challenges in decomposition and a long composting time. The key to degrading grapevines lies in the degradation of cellulose and lignin. Microorganisms play a crucial role in grapevine composting. Adding microbial agents during the composting of grapevines and other waste materials stimulates the production of cellulase and lignin-degrading enzymes, promoting rapid initiation and accelerating the composting process, significantly improving the quality of the organic fertilizer. Therefore, microbial strains are essential in grapevine fermentation, especially in finding a strain capable of efficiently degrading cellulose and lignin.

[0004] Therefore, how to provide a highly efficient microbial strain for degrading cellulose and apply it to branch composting is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] In view of this, the present invention provides Bacillus amyloliquefaciens XW5 and its applications.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A type of Bacillus amyloliquefaciens, XW5, was deposited on May 8, 2024, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 30524, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; and is classified as Bacillus amyloliquefaciens.

[0008] Preferably, the 16S rDNA sequence of the Bacillus amyloliquefaciens XW5 is shown in SEQ ID No. 1.

[0009] As an inventive concept with the same technical solution described above, the present invention also claims protection for the application of the Bacillus amyloliquefaciens XW5 in the degradation of cellulose.

[0010] Preferably, the application of Bacillus amyloliquefaciens XW5 in degrading cellulose in branch compost.

[0011] As an inventive concept with the same technical solution described above, the present invention also claims protection for the use of the Bacillus amyloliquefaciens XW5 in the production of cellulase.

[0012] As having the same inventive concept as the above-mentioned technical solution, this invention also claims protection for a fermentation method of Bacillus amyloliquefaciens XW5, comprising the following steps:

[0013] (3) Straw XW5 strain was streaked onto LB medium and cultured at 30°C to obtain single colonies.

[0014] (4) Inoculate single colonies into LB liquid medium and incubate at 30℃ and 180r / min for 24h to obtain culture solution;

[0015] (3) Inoculate the culture medium into sterilized solid culture medium and carry out fermentation culture in a constant temperature incubator at 30℃.

[0016] Preferably, the optimal culture medium composition for the strain in step (3) is 2% maltose, 9% trypsin, 2.5% ammonium sulfate and 2.5% potassium dihydrogen phosphate.

[0017] As an inventive concept with the same technical solution described above, the present invention also claims protection for a microbial agent containing Bacillus amyloliquefaciens strain XW5.

[0018] Preferably, the microbial agent further includes a protectant, wherein the protectant is mannitol.

[0019] As having the same inventive concept as the above-described technical solution, the present invention also seeks protection for the use of the above-described microbial agent in the degradation of cellulose.

[0020] As can be seen from the above technical solution, compared with the prior art, the technical effects achieved by the present invention are as follows:

[0021] This invention screened a strain of *Bacillus amyloliquefaciens* XW5 from grape vine compost. Species identification was performed using 16S rRNA gene sequence analysis, and the bacterial function was determined by cellulase activity. The characteristics of this strain were further determined by screening for optimal temperature, pH, viable cell counts at different subculture times, and enzyme activity. It was found that the enzyme activity of this strain was 923.20 U / g at 30℃ and pH=8, and the viable cell count reached 1.63 × 10⁻⁶ after 14 subcultures. 10 The enzyme activity reached 534.87 U / g after 16 transfers, with a concentration of cfu / mL.

[0022] After orthogonal optimization of fermentation conditions, the cellulase activity of Bacillus amyloliquefaciens XW5 was found to be highest in a solid culture medium containing 8% maltose, 9% casein, 2.5% ammonium chloride, and 1.5% potassium dihydrogen phosphate. Furthermore, the addition of 5% mannitol as a protectant resulted in the highest effective viable count of strain XW5 in the prepared inoculum, reaching 2.05 × 10⁻⁶. 14 cfu / g. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The samples and culture media used in the examples are as follows:

[0025] Test sample

[0026] It was collected in 2021 from grape branch compost at the Wenquan Base of the Chinese Academy of Agricultural Sciences.

[0027] Test culture medium

[0028] LB medium: 10g peptone, 5g yeast extract, 10g sodium chloride, bring to a final volume of 1L, set pH to natural, and autoclave at 121℃ for 20 minutes.

[0029] Cellulose selective medium: 15g sodium carboxymethyl cellulose, 5g sodium chloride, 1g dipotassium hydrogen phosphate, 0.2g magnesium sulfate, 10g tryptone, 5g yeast extract, 20g agar, bring to a final volume of 1L, pH 7.2, autoclave at 121℃ for 20min.

[0030] Rice straw powder solid culture medium: 200g of alkali-treated rice straw powder, 800g of wheat bran, 50g of glucose, 20g of trypsin, 15g of ammonium nitrate, 10g of potassium dihydrogen phosphate, and 1500g of water. Sterilize with high-pressure steam at 121℃ for 20 minutes before use.

[0031] Example 1: Screening and Identification of Bacillus amyloliquefaciens XW5

[0032] 1. Screening of Bacillus amyloliquefaciens XW5

[0033] (1) Weigh 5g of sample into a sterile 50mL centrifuge tube, add 45mL of sterile water, and shake at 180rpm for 30min. Take 1mL of the mixture and add 9mL of sterile water to perform serial dilutions of the bacterial solution, diluting each solution to 10⁻⁶. -4 10 -5 10 -6 100 μL of the bacterial suspension was spread onto LB agar plates and incubated upside down at 30°C. After 3 days, colonies of different morphologies were picked and streaked for purification. The purification method involved picking colonies with an inoculation needle and streaking them onto four non-overlapping areas of an LB agar plate. After colonies grew on the plate, their morphology was observed. If all colonies had the same morphology, the colonies were considered purified.

[0034] (2) The purified strain was inoculated into cellulose selective medium and cultured for 3 days. It was stained with 1 mg / mL Congo red for 1 h, the stain was discarded, and the strain was destained with 1 mol / L NaCl solution. The presence or absence of a transparent zone was observed, and the length and width of the transparent zone were measured. The ratio of the transparent zone area to the colony area was calculated, and the strain with a transparent zone was preserved.

[0035] 2. Identification of Bacillus amyloliquefaciens XW5

[0036] DNA was extracted from XW5, and its purity and integrity were determined by agarose gel electrophoresis and absorbance measurement. Then, using XW5 DNA as a template, PCR amplification was performed using primers 341F (5'-CGCCCGCCGCG CCCCGCGCCCGGCCCGCCGCCCCCGCCTACGGGAGGCAGCAG-3') and 907R (5'-CCGTCAATTCCTTTGA GTTT-3'). The target band was sent to the company for sequencing, and the obtained DNA sequence was compared with the NCBI database. The results showed that XW5 had 100% similarity to *Bacillus amyloliquefaciens*, and was identified as *Bacillus amyloliquefaciens*. The 16S rDNA sequence of this strain is shown in SEQ ID No. 1.

[0037] As shown in SEQ ID NO.1.

[0038] Example 2: Determination of cellulase activity in Bacillus amyloliquefaciens XW5

[0039] The XW5 strain was inoculated into LB liquid medium and cultured at 30℃ and 180 r / min for 24 h. Then it was transferred to rice straw powder solid medium and fermented in a constant temperature incubator at 30℃ for 7 days. After fermentation, the cellulase activity was measured, and the method was referred to "Organic Material Composting Agent" (2002).

[0040] Enzyme activity assay results showed that the cellulase activity of strain XW5 was 635.81 U / g.

[0041] Example 3: Determination of the bacterial characteristics of Bacillus amyloliquefaciens XW5

[0042] Single-factor experiments were conducted to investigate the effects of temperature, pH, and number of inoculations on cellulase production by strain XW5 and to determine the characteristics of the strain.

[0043] (1) Effect of different temperatures on cellulase activity of strain XW5

[0044] The XW5 strain cultured for 24 hours was inoculated into rice straw powder solid medium at natural pH and cultured at different temperatures of 20, 30, 40, 50, 60 and 70℃. After 7 days, its cellulase activity was measured, and the results are shown in Table 1.

[0045] Table 1 Effect of different temperatures on cellulase activity of strain XW5

[0046]

[0047] Table 1 shows that the growth and cellulase activity of the strain varied under different temperatures. Strain XW5 could produce cellulase within the range of 20–70℃, and the cellulase activity initially increased and then decreased with increasing culture temperature. At 30℃, strain XW5 exhibited a relatively high cellulase activity of 639.52 U / g. Therefore, the optimal temperature for cellulase production by strain XW5 is 30℃.

[0048] (2) Effects of different pH conditions on cellulase activity of strain XW5

[0049] The XW5 strain cultured for 24 h was inoculated into rice straw powder solid culture medium with pH values ​​of 5, 6, 7, 8 and 9, respectively, and cultured at 30℃. After 7 days, its cellulase activity was measured, and the results are shown in Table 2.

[0050] Table 2 Effects of different pH conditions on cellulase activity of strain XW5

[0051]

[0052] Table 2 shows that the growth and cellulase activity of the strain varied under different pH conditions. Strain XW5 produced cellulase within a pH range of 5–9, and the cellulase activity initially increased and then decreased with increasing pH. At pH 8, strain XW5 achieved a relatively high cellulase production capacity of 923.20 U / g. Therefore, the optimal pH for cellulase production by strain XW5 is 8.

[0053] (3) Effects of different transfection times on the viable number of XW5 strain and cellulase activity

[0054] The XW5 strain was inoculated into LB liquid medium and cultured at 180 rpm and 30℃ for 24 h. The viable cell count was then determined, and the results are shown in Table 3. Simultaneously, the culture was transferred to fresh LB medium at an inoculation density of 1 × 10⁻⁶ cells / mL. 7 The cellulase activity was recorded as cfu / mL and the number of transfers was recorded. At the same time as each transfer, the bacterial culture that had been cultured for 24 hours was inoculated into rice straw powder solid culture medium and cultured in a constant temperature incubator at 30℃ for 7 days. The cellulase activity was then measured, and the results are shown in Table 4.

[0055] Table 3. Effect of different inoculation times on the effective viable count of strain XW5

[0056]

[0057] Table 4. Effects of different transfection numbers on cellulase activity in strain XW5

[0058]

[0059]

[0060] Tables 3 and 4 show that the growth, viable cell count, and cellulase activity of the strain varied significantly with different subculture numbers. The highest viable cell count of strain XW5 (1.63 × 10⁻⁶) was observed after 14 subcultures. 10 cfu / mL; when the number of transfers was 16, the cellulase production capacity of strain XW5 reached a high level of 534.87 U / g.

[0061] Example 4: Optimization of fermentation conditions for Bacillus amyloliquefaciens XW5

[0062] Fermentation conditions were optimized using rice straw powder solid culture medium. Single-factor and orthogonal experiments were conducted to investigate the effects of carbon source, nitrogen source (slow-release nitrogen source and fast-release nitrogen source) and inorganic salts on cellulase production by strain XW5 during culture, and the optimal fermentation conditions were determined.

[0063] 1. Single-factor experiments were conducted to determine the types of carbon sources, nitrogen sources (slow-acting and fast-acting nitrogen sources), and inorganic salts.

[0064] (1) Carbon source of fermentation medium

[0065] Eight treatments were set up with carbon sources, namely fructose, glucose, mannitol, sucrose, lactose, maltose, starch, and molasses, to replace glucose in the rice straw powder solid culture medium. The culture medium was then prepared according to the specified ratio and sterilized at 121℃ for 20 min. After the culture medium cooled, 1 mL of XW5 bacterial suspension activated for 18 h was inoculated and cultured at 30℃. Cellulase activity was measured after 7 days, and the results are shown in Table 5.

[0066] Table 5 Effects of different carbon sources on cellulase activity of strain XW5

[0067]

[0068] As shown in Table 5, the addition of different carbon sources to the culture medium will affect the growth of the strain. When the carbon source is maltose, the cellulase activity of strain XW5 is the highest, reaching 934.76 U / g. Therefore, maltose is selected as the optimal carbon source for culturing strain XW5.

[0069] (2) Delayed-acting nitrogen source and readily available nitrogen source in fermentation culture medium

[0070] Nine treatments were set up using slow-release nitrogen sources, including casein, yeast powder, trypsin, corn flour, corn steep liquor, casein, soybean peptone, peanut meal powder, and soybean flour, to replace peptone in rice straw powder solid culture medium. The corresponding culture media were then prepared and sterilized at 121℃ for 20 min. After the culture medium cooled, 1 mL of XW5 bacterial suspension activated for 18 h was inoculated and cultured at 30℃. Cellulase activity was measured after 7 days, and the results are shown in Table 6.

[0071] Five treatments were set up with available nitrogen sources: ammonium chloride, ammonium sulfate, urea, ammonium nitrate, and diammonium hydrogen phosphate, to replace ammonium nitrate in the rice straw powder solid culture medium. The corresponding culture media were then prepared, sterilized and cooled, and 1 mL of XW5 bacterial suspension activated for 18 h was inoculated and cultured at 30 °C. Cellulase activity was measured after 7 days, and the results are shown in Table 7.

[0072] Table 6. Effects of different delayed-release nitrogen sources on cellulase activity in strain XW5

[0073]

[0074] Table 7 Effects of different readily available nitrogen sources on cellulase activity in strain XW5

[0075]

[0076] As shown in Tables 6 and 7, when trypsin was used as the delayed nitrogen source, the cellulase activity of strain XW5 was the highest, reaching 535.53 U / g, significantly higher than other delayed nitrogen sources. When ammonium sulfate was used as the readily available nitrogen source, the cellulase activity of strain XW5 was the highest, reaching 1171.51 U / g, significantly higher than other readily available nitrogen sources. Therefore, trypsin and ammonium sulfate were selected as the optimal delayed and readily available nitrogen sources for culturing strain XW5.

[0077] (3) Inorganic salts in fermentation medium

[0078] Ten inorganic salts were used to replace potassium dihydrogen phosphate in rice straw powder solid culture medium. These salts were then used to prepare the corresponding culture media. After sterilization and cooling, 1 mL of XW5 bacterial suspension activated for 18 h was inoculated and cultured at 30 °C. Cellulase activity was measured after 7 days. The results are shown in Table 8.

[0079] Table 8 Effects of different inorganic salts on cellulase activity of strain XW5

[0080]

[0081] As shown in Table 8, the cellulase activity of strain XW5 was the highest when potassium dihydrogen phosphate was used as the inorganic salt, reaching 602.48 U / g. Therefore, potassium dihydrogen phosphate was selected as the most suitable inorganic salt for culturing strain XW5.

[0082] 2. Orthogonal experiments to determine the nutrient content in the culture medium.

[0083] Based on orthogonal experimental design, the maltose, trypsin, ammonium sulfate and potassium dihydrogen phosphate contents in the solid culture medium of strain XW5 were optimized. Three gradients were set for each substance, as shown in Table 9. Strain XW5 was inoculated into the corresponding contents of the culture medium and cultured for 7 days. Its cellulase activity was measured and the results are shown in Table 10.

[0084] Table 9 Nutrient gradient settings

[0085]

[0086] Table 10 Results of the orthogonal experiment on nutrient content

[0087]

[0088]

[0089] The orthogonal experimental results of nutrient content of strain XW5 are shown in Table 7. When the solid-state fermentation conditions of strain XW5 were 2% maltose, 9% trypsin, 2.5% ammonium sulfate and 2.5% potassium dihydrogen phosphate, the cellulase activity of XW5 was the highest, reaching 666.96 U / g, which was significantly higher than other treatment groups.

[0090] Example 5: Effects of different protectants on XW5 inoculant

[0091] The XW5 strain was inoculated into rice straw powder solid culture medium and cultured in a constant temperature incubator at 30℃ for 7 days. Casein, mannitol, humic acid, trehalose and chitosan were selected as protectants and added to the solid culture medium containing XW5 bacteria at a concentration of 5%. The medium was then dried in a 30℃ oven until the moisture content was 5%. After that, it was crushed and placed in a sealed bag for storage at room temperature in the dark. After 12 months, the number of effective viable bacteria, the number of miscellaneous bacteria and the cellulase activity were determined according to the standard of "Agricultural Microbial Agents" (2006). The results are shown in Table 11.

[0092] Table 11 Effects of different protectants on XW5 bacterial inoculum

[0093]

[0094] As shown in Table 11, in terms of effective viable bacteria count, strain XW5 had the highest effective viable bacteria count, reaching 2.05 × 10⁻⁶, when 5% mannitol was used as a protectant. 14 The CFU / g values ​​were significantly higher than those of other protectants. Furthermore, when mannitol was used as the protectant, the cellulase activity of the prepared bacterial agent was higher at 457.84 U / g, also higher than that of other protectants. Therefore, this invention uses 5% mannitol as the protectant, which not only increases the effective viable cell count of the bacterial agent, facilitating the preservation of strain XW5, but also maintains the cellulase activity of the strain.

[0095] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0096] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A type of Bacillus amyloliquefaciens XW5, characterized in that, The *Bacillus amyloliquefaciens* XW5 was deposited on May 8, 2024, at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 30524, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; and classified as *Bacillus amyloliquefaciens*. Bacillus amyloliquefaciens ; The 16S rDNA sequence of the Bacillus amyloliquefaciens XW5 is shown in SEQ ID No.

1.

2. The application of Bacillus amyloliquefaciens XW5 according to claim 1 in the degradation of cellulose.

3. The application of Bacillus amyloliquefaciens XW5 according to claim 1 in the production of cellulase.

4. A fermentation method for Bacillus amyloliquefaciens XW5 according to claim 1, characterized in that, Includes the following steps: (1) Straw XW5 strain was streaked onto LB medium and cultured at 30°C to obtain single colonies. (2) Inoculate single colonies into LB liquid medium and incubate at 30℃ and 180r / min for 24h to obtain culture solution; (3) Inoculate the culture medium into sterilized solid culture medium and carry out fermentation culture in a constant temperature incubator at 30℃.

5. The fermentation method of Bacillus amyloliquefaciens XW5 according to claim 4, characterized in that, The optimal culture medium composition for the strain in step (3) is 2% maltose, 9% trypsin, 2.5% ammonium sulfate and 2.5% potassium dihydrogen phosphate.

6. A microbial inoculant, characterized in that, The bacterial agent includes Bacillus amyloliquefaciens XW5 as described in claim 1.

7. A microbial inoculant according to claim 6, characterized in that, It also includes a protective agent, namely mannitol.

8. The application of the microbial agent according to any one of claims 6-7 in the degradation treatment of cellulose.

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