Bacillus for producing cellulase and application thereof

Abstract

The invention provides a bacillus for producing cellulase and application thereof, the bacillus strain XWSM3 is separated from soil of sugarcane field of Guangxi guest, the preservation number of the bacillus strain XWSM3 is GDMCC NO: 65137, the preservation date is September 14, 2024, the preservation address is Microbial Culture Collection Center in Guangzhou and Guangdong province, the preservation unit is Guangdong province Microbial Culture Collection Center, the preservation number is CGMCC NO: 65137, the preservation number is CGMCC NO: 65137, the preservation number is CGMCC NO: 65137, the preservation number is CGMCC NO: 65137, and the preservation number is CGMCC NO: 65137. The strain has relatively high enzyme production activity and good cellulase production stability, the enzyme activity of the enzyme produced by the strain reaches 16.53 U, and the yield of the prepared humic acid reaches 7.10%.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, and in particular to a cellulase-producing bacillus and application thereof. [Background technology]

[0002] Humic acid is a mixed organic molecule produced by the decomposition of animal and plant debris by microorganisms. It is widely present in water bodies such as soil, rivers, lakes, and oceans. The organizational structure of humic acid is closely related to its source. For example, the different crops grown in the soil will also affect the organizational composition of humic acid. Although the composition of humic acid is complex, its core molecule belongs to an aromatic structure and has a variety of active groups, such as phenolic hydroxyl, carboxyl, methoxy, alcohol hydroxyl, carbonyl, and some humic acids have polypeptide structures and aliphatic structures, which make humic acid show strong ion affinity as a whole. Humic acid shows strong adsorption and extremely specific metal ion chelating ability, which makes humic acid widely used in many fields. At present, humic acid is commonly used in food, medicine, ecological regulation and animal husbandry, but the most common application is humic acid to improve soil structure, fertilizer efficiency and environmental protection. Zhang Huijuan's research found that humic acid can promote wheat's ability to absorb nitrogen and phosphorus, thereby enhancing wheat's ability to absorb fertilizers and promote growth. Bu Chuangang used humic acid as raw material to prepare adsorbent, which reduced the pollution in the wastewater. Chen Zhen and others' research showed that humic acid has a significant effect in adsorbing heavy metals and plays an important role in soil pollution, prevention and restoration.

[0003] Improve soil structure, increase fertilizer efficiency and protect the environment to ensure food production quality from the source, Liu Tiandong ] et al. found that applying a certain concentration of humic acid to pepper seedlings can increase pepper yield. Zheng Zhenyong found that humic acid can promote cucumber growth and increase the nutritional content of cucumber. Zhao Jingwan

[14] et al. found that humic acid can increase the germination rate of strawberry seedlings and improve the growth environment of strawberries. Ying Yongqing et al. found that humic acid can improve the saline-alkali environment and increase rice yields. Many studies have shown that humic acid promotes the growth of crops. It also proves that humic acid has many effects on food, such as improving food quality, increasing food safety and food production. This has gradually increased the research popularity of humic acid in the food field. At the same time, these many effects also reflect the application value of humic acid in the food field and its broad market prospects.

[0004] The invention uses self-screened Bacillus as a starting strain and sugarcane bagasse as a main raw material to ferment and produce humic acid. This reaction has the advantages of being green, efficient and mild. [Summary of the invention]

[0005] In view of this, the object of the present invention is to provide a cellulase-producing Bacillus and application thereof.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] A Bacillus sp. strain XWSM3, whose deposit number is GDMCC NO: 65137, the deposit date is September 14, 2024, the deposit address is: Guangdong Microbiological Culture Collection Center, Guangzhou, China, and the deposit unit is: Guangdong Microbiological Culture Collection Center.

[0008] It is further explained that the Bacillus sp. strain XWSM3 is isolated from the soil of a sugarcane field in Laibin, Guangxi.

[0009] The present application also provides a microbial preparation comprising the Bacillus sp. strain XWSM3 as described above.

[0010] Further, the microbial preparation contains a viable count of not less than 1.0×10 6 Bacillus XWSM3 cells at 100 μg / mL.

[0011] The present application also provides a method for producing cellulase, and the specific method is: under sterile operation, a sterilized inoculation ring is inoculated with Bacillus XWSM3 into a fermentation medium (pH = 6.5) that has been sterilized at high temperature, and the fermentation culture is carried out at 31°C and 180r / min in a shaking incubator for 24 hours. Then, the fermentation liquid is inoculated into a new fermentation medium according to the inoculation amount of 10.0%, and the culture is carried out at a constant temperature of 31°C in a shaking incubator for 96 hours. The supernatant is obtained by centrifugation at 10000r / min and 15min, which is the crude cellulase enzyme solution.

[0012] The present application also provides an application of the above-mentioned Bacillus sp. XWSM3 in the production of humic acid products.

[0013] The present application also provides a method for preparing the humic acid as described above, the method comprising the following steps:

[0014] The preserved Bacillus XWSM3 was inoculated on the slant medium and cultured in a constant temperature incubator at 31°C for 2 days to obtain activated strains. The strains were washed with sterile water to obtain a concentration of 1.0×10 6 The suspension of Bacillus XWSM3 with a concentration of 100 μg / mL was inoculated into the fermentation medium optimized in a shake flask, and cultured in a constant temperature incubator at 31°C for 96 hours to obtain the crude humic acid.

[0015] In the present invention, it is further described that the slant culture medium is CMC culture medium; the formula of the CMC culture medium is: 2g CMC-Na, 1g yeast powder, 2g peptone, 1g sodium chloride, 4g agar, 0.04g magnesium sulfate, 0.2g potassium dihydrogen phosphate, 200mL water, pH 6.5.

[0016] In the present invention, it is further described that the fermentation medium formula after the shake flask optimization is: 5g bagasse, 20g 12°Bx molasses, 4g peptone, 1g ammonium sulfate, and the molasses pH is adjusted to 6.0 with 1mol / L sodium hydroxide solution.

[0017] The present application also discloses the application of the above-mentioned Bacillus sp. XWSM3 in the fields of industry and agriculture.

[0018] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0019] The bacillus sp. XWSM3 of the present invention is a strain isolated and screened from the soil of a sugarcane field in Laibin, Guangxi. The study found that the bacterium is a cellulase-producing bacillus sp. XWSM3. In order to study the enzyme production ability of the bacterium, a preliminary study of single factor and orthogonal experiments was conducted. The applicant determined that the optimal conditions for the fermentation and enzyme production of bacillus sp. XWSM3 are as follows: fermentation time 96h, inoculation amount 10%, fermentation temperature 31°C, maltose 7g, soybean powder 5g, CMC-Na 1g, dipotassium hydrogen phosphate 0.1g, magnesium sulfate 0.1g, sodium chloride 0.1g, 100mL distilled water, initial pH 6.5, and the enzyme activity of the enzyme produced by the bacterium is up to 16.53U / mL. It can be seen that the bacterium has high enzyme production activity and good stability.

[0020] The applicant has determined through research that the optimal conditions for Bacillus sp. XWSM3 fermentation to produce humic acid are: fermentation time 96h, fermentation temperature 31°C, inoculation amount 10%, 5g bagasse, 20g 12°Bx molasses, 4g peptone, 1g ammonium sulfate, initial pH 7.0, and the humic acid content produced by the bacteria reaches 7.10%.

Brief Description of the Drawings

[0021] Figure 1 This is a morphological diagram of the Bacillus XWSM3 strain on a plate according to an embodiment of the present application;

[0022] Figure 2 This is a graph showing the effect of different carbon sources on the enzyme production capacity of Bacillus XWSM3;

[0023] Figure 3This is a graph showing the effect of different carbon source concentrations on the enzyme production capacity of Bacillus XWSM3;

[0024] Figure 4 This is a diagram showing the effect of different nitrogen sources on the enzyme production capacity of Bacillus XWSM3;

[0025] Figure 5 This is a graph showing the effect of different nitrogen source concentrations on the enzyme production capacity of Bacillus XWSM3;

[0026] Figure 6 This is a graph showing the effect of different pH on the enzyme production capacity of Bacillus XWSM3;

[0027] Figure 7 This is the effect of different fermentation times on the humic acid production capacity of Bacillus XWSM3;

[0028] Figure 8 This is the effect of different pH on the humic acid production capacity of Bacillus XWSM3;

[0029] Fig. 9 This is the effect of different inoculation amounts of bacteria on the humic acid production capacity of Bacillus XWSM3;

[0030] Fig.10 This is a graph showing the effect of different fermentation temperatures on the humic acid production capacity of Bacillus XWSM3. [Specific implementation method]

[0031] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0032] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0033] 1. Screening of bacterial strains

[0034] Soil samples were taken from the sugarcane fields in Laibin, Guangxi. CMC medium was used to form a screening medium to screen the cellulase-producing strains. The process was as follows: strain screening: 1 g of soil was added to 9 mL of sterile saline to make 10 -1 The sample solution of concentration was diluted to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 Concentration, set aside for use. Take the last three dilutions, separate the strains on the CMC medium plate by spreading method, separate by streaking on the plate, purify repeatedly, and obtain the pure culture of the isolated strain. Store the plate in a 4℃ refrigerator.

[0035] CMC medium is specifically CMC agar slant solid medium, and its formula is: 2g CMC-Na, 1g yeast powder, 2g peptone, 1g sodium chloride, 4g agar, 0.04g magnesium sulfate, 0.2g potassium dihydrogen phosphate, 200mL water, pH 6.5

[0036] 2. Identification of strains and optimization of fermentation conditions

[0037] 1. Identification of bacterial species

[0038] (1) Observation of colony morphology: The slant bacteria were inoculated into a plate containing CMC medium and cultured at 37°C for 48 h. The colony morphology was observed.

[0039] (2) 16S rDNA Identification of the Strain: The purified strain was sent to Guangdong Microbial Culture Collection Center for identification. Nucleotide BLAST analysis showed that the 16S rDNA sequence homology with the Bacillus strain was 100%, and it was preliminarily determined that the XWSM3 strain was Bacillus.

[0040] The preservation information of the screened Bacillus XWSM3 is as follows: the name is Bacillus sp. XWSM3, its classification name is Bacillus sp. XWSM3, the preservation number is GDMCC NO: 65137, the preservation date is September 14, 2024, the preservation address is: 5th Floor, Experimental Building, No. 100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province, and the preservation unit is: Guangdong Provincial Microbiological Culture Collection Center.

[0041] The morphological characteristics of Bacillus sp. XWSM3 on a plate are as follows: Figure 1 As shown, the colonies of this strain are white and sticky with a moist and smooth surface.

[0042] 2. Study on optimization of enzyme production conditions of Bacillus sp. XWSM3

[0043] The basic formula of CMC medium is: CMC medium: 2g CMC-Na, 1g yeast powder, 2g peptone, 1g sodium chloride, 4g agar, 0.04g magnesium sulfate, 0.2g potassium dihydrogen phosphate, 200mL water, pH 6.5

[0044] (1) Optimization of carbon source and its concentration

[0045] Bacillus sp. XWSM3 was inoculated with CMC medium, and the carbon source variables were sucrose, starch, CMC-Na, glucose, and maltose. The liquid medium was prepared and placed on a shaker at 37°C, with a speed of 180 r / min, and fermented for 96 hours, and centrifuged at 10000 r / min for 10 minutes. On this basis, the effect of maltose concentration (3, 5, 7, 9, 11%) on the enzyme production of the strain was further explored, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 1 and Figure 2 , 3 .

[0046] Table 1 Effect of different carbon sources on enzyme production of Bacillus XWSM3

[0047]

[0048] (2) Optimization of nitrogen source

[0049] Bacillus sp. XWSM3 was inoculated with CMC medium, and beef extract, peptone, yeast powder, ammonium sulfate, and soybean powder were selected as the only nitrogen source in the fermentation medium. The liquid medium was prepared and placed on a shaker at 37°C, 180r / min, fermented for 96h, and centrifuged at 10000r / min for 10min. On this basis, the effect of soybean powder concentration (1, 3, 5, 7, 9%) on the enzyme production of the strain was further explored, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 2 and Figure 4 , Figure 5 .

[0050] Table 2 Effect of different nitrogen sources on enzyme production capacity of Bacillus XWSM3

[0051]

[0052] (3) Effect of different pH values ​​on the enzyme production capacity of Bacillus sp. XWSM3

[0053] Bacillus sp. XWSM3 was inoculated with CMC medium, and the initial pH of the medium was adjusted to 5, 5.5, 6, 6.5, and 7, and the supernatant was taken to measure the enzyme activity. The liquid medium was prepared and placed on a shaker at 37°C, with a speed of 180 r / min, and the fermentation culture was cultured for 96 hours. The fermentation liquid was taken out and centrifuged at 10000 r / min for 10 minutes, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 3 and Figure 6 .

[0054] Table 3 Effect of different fermentation medium pH on enzyme production capacity of Bacillus XWSM3

[0055] project pH(5) pH(5.5) pH(6) pH(6.5) pH(7) Enzyme activity (U / mL) 10.38 11.72 12.94 14.66 12.78

[0056] (4) Orthogonal optimization of enzyme production conditions

[0057] Based on the results of the single factor test, the carbon source (A, 5%, 7%, 9% 3 levels), nitrogen source (B, 3%, 5%, 7% 3 levels), pH (C, 6, 6.5, 7.0 3 levels) and blank group were selected to design a 3-factor 3-level orthogonal test, using the L9 (34) type orthogonal design. The orthogonal test results are shown in Table 4.

[0058] Table 4 Results of orthogonal test for optimization of enzyme production conditions of strains

[0059]

[0060]

[0061] (5) Verification test

[0062] According to the orthogonal experimental results, the verification test was carried out, the rotation speed was 180r / min, the fermentation culture was carried out for 96h, the fermentation liquid was taken out and centrifuged at 10000r / min for 10min, and the supernatant was taken to measure the enzyme activity. The results are shown in Table 5.

[0063] Table 5 Test verification results

[0064] project Test 1 Test 2 Test 3 average value Enzyme activity (U / mL) 16.23 16.99 16.37 16.53

[0065] Through Tables 1 to 5 and Figures 2 to 6 The enzyme activity data detected by single factor optimization and orthogonal experiment can determine that the optimal conditions for enzyme production by Bacillus sp. XWSM3 are: fermentation time 96h, inoculation size 10%, fermentation temperature 31℃, maltose 7g, soybean powder 5g, CMC-Na 1g, potassium dihydrogen phosphate 0.1g, magnesium sulfate 0.1g, sodium chloride 0.1g, 100mL distilled water, initial pH 6.5, and the maximum enzyme activity of the bacteria is 16.53U / mL.

[0066] 3. Application of Bacillus sp. XWSM3: Humic acid production

[0067] Study on the optimization of fermentation conditions for humic acid production using Bacillus sp. XWSM3

[0068] Bagasse solid fermentation medium: 5 g bagasse, 20 g 12° Bx molasses, 4 g peptone, 1 g ammonium sulfate, pH 6.0.

[0069] (1) Effects of different fermentation times on the humic acid production capacity of Bacillus XWSM3

[0070] Bacillus sp. XWSM3 was inoculated into bagasse solid fermentation medium, and the humic acid content was measured after fermentation for 72, 84, 96, 108, and 120 hours at a temperature of 31°C and a pH of 6.0. The results are shown in Tables 6 and Figure 7 .

[0071] Table 6 Effects of different fermentation times on the humic acid production capacity of Bacillus XWSM3

[0072]

[0073] (2) Effects of different pH values ​​on the humic acid production capacity of Bacillus XWSM3

[0074] Bacillus sp. XWSM3 was inoculated into bagasse solid fermentation medium, and the pH was adjusted to 5, 6, 7, 8, and 9, respectively. The humic acid content was measured after fermentation at 31°C for 96 hours. The results are shown in Table 7 and Figure 8 .

[0075] Table 7 Effect of different pH values ​​on the humic acid production capacity of Bacillus XWSM3

[0076]

[0077] (3) Effect of different inoculation amounts on the humic acid production capacity of Bacillus XWSM3

[0078] Bacillus sp. XWSM3 was inoculated with bagasse solid fermentation medium, and the inoculation amount was adjusted to 6, 8, 10, 12, and 14% respectively. The humic acid content was measured after fermentation for 96 hours at a temperature of 31°C and a pH of 7. The results are shown in Table 8 and Fig. 9 .

[0079] Table 8 Effect of different inoculation amounts on the humic acid production capacity of Bacillus XWSM3

[0080]

[0081] (4) Effects of different fermentation temperatures on the humic acid production capacity of Bacillus XWSM3

[0082] Bacillus sp. XWSM3 was inoculated with bagasse solid fermentation medium at pH 7.0 and 10% inoculation amount. The humic acid content was measured by sampling at 25°C, 28°C, 31°C, 34°C and 37°C for 96h. The results are shown in Table 9 and Fig.10 .

[0083] Table 9 Effect of different fermentation temperatures on the humic acid production capacity of Bacillus XWSM3

[0084]

[0085] (5) Orthogonal optimization of humic acid production conditions

[0086] Based on the results of the single factor test, the inoculum size (A, 5%, 7%, 9% 3 levels), fermentation temperature (B, 3%, 5%, 7% 3 levels), pH (C, 6, 6.5, 7.0 3 levels) and blank group were selected to design a 3-factor 3-level orthogonal test, using the L9 (34) type orthogonal design. The orthogonal test results are shown in Table 10.

[0087] Table 10 Results of orthogonal test for optimization of enzyme production conditions of strains

[0088]

[0089] (6) Verification test

[0090] A verification test was carried out based on the orthogonal experimental results, and the results are shown in Table 11.

[0091] Table 11 Test verification results

[0092] project Test 1 Test 2 Test 3 average value Enzyme activity (U / mL) 6.93 7.29 7.07 7.10

[0093] Through Tables 6 to 11 and Figures 7 to 10 The humic acid content data detected by single factor optimization and orthogonal test can determine the optimal conditions for Bacillus sp. XWSM3 fermentation to produce humic acid: fermentation time 96h, inoculation amount 10%, fermentation temperature 31℃, 5g bagasse, 20g 12°Bx molasses, 4g peptone, 1g ammonium sulfate, initial pH 7.0, and humic acid content as high as 7.10%.

[0094] The humic acid content was determined according to the national standard NYT1971-2010.

[0095] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A Bacillus sp. strain XWSM3, whose deposit number is GDMCC NO: 65137, deposit date: September 14, 2024, deposit address: Guangdong Microbiological Culture Collection Center, Guangzhou, China, deposit unit: Guangdong Microbiological Culture Collection Center.

2. The Bacillus sp. strain XWSM3 according to claim 1, characterized in that The strain was isolated from the soil of a sugarcane field in Laibin, Guangxi.

3. A microbial preparation comprising the Bacillus sp. strain XWSM3 according to claim 1.

4. The microbial preparation according to claim 3, characterized in that The number of viable bacteria in the microbial preparation is not less than 1.0×10 6 Bacillus XWSM3 cells at 100 μg / mL.

5. A method for producing cellulase, characterized in that: The specific method is as follows: under aseptic operation, a sterile inoculation ring is used to inoculate Bacillus XWSM3 into a high-temperature sterilized fermentation medium (pH = 6.5), and after constant temperature fermentation and cultivation at 31°C and 180 r / min in a shaking incubator for 24 hours, the fermentation liquid is inoculated into a new fermentation medium at a constant temperature of 31°C and cultured at a shaking incubator for 96 hours according to an inoculation amount of 10.0%, and the supernatant is obtained by centrifugation at 10000 r / min and 15 min, which is the crude cellulase enzyme solution.

6. Use of the Bacillus sp. XWSM3 as claimed in claim 1 in producing humic acid products.

7. A method for preparing humic acid as claimed in claim 6, characterized in that: The method comprises the following steps: The preserved Bacillus XWSM3 was inoculated on the slant medium and cultured in a constant temperature incubator at 31°C for 2 days to obtain activated strains. The strains were washed with sterile water to obtain a concentration of 1.0×10 6 The suspension of Bacillus sp. XWSM3 with a concentration of 100 μg / mL was inoculated into the fermentation medium optimized in a shake flask, and cultured in a constant temperature incubator at 31°C for 96 hours to obtain the crude humic acid.

8. The method according to claim 7, characterized in that The slant culture medium is CMC culture medium; the formula of the CMC culture medium is: 2g CMC-Na, 1g yeast powder, 2g peptone, 1g sodium chloride, 4g agar, 0.04g magnesium sulfate, 0.2g potassium dihydrogen phosphate, 200mL water, pH 6.

5.

9. The method according to claim 7, characterized in that The fermentation medium formula after the shake flask optimization is: 5g bagasse, 20g 12°Bx molasses, 4g peptone, 1g ammonium sulfate, and the molasses pH is adjusted to 6.0 with 1mol / L sodium hydroxide solution.

10. Use of the Bacillus sp. XWSM3 according to claim 1 in industrial and agricultural fields.

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