CN115305212B - Bacillus subtilis and culture method and application thereof - Google Patents
Bacillus subtilis and culture method and application thereof Download PDFInfo
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Abstract
The invention provides bacillus subtilis, a preparation method and application thereof, and belongs to the technical field of microorganisms. The invention provides a bacillus subtilis G-2 with a preservation number of CGMCC No. 2177. The bacillus subtilis G-2 provided by the invention has a good lignocellulose degrading effect. The results of the examples show that the bacillus subtilis G-2 provided by the invention can grow at 10 ℃ and pH 3.5; the growth is good under the conditions of 20 ℃ and pH4.0, and the cellulase activity reaches 80U/mL; the cellulase activity reaches the highest under the conditions of 35 ℃ and pH4.0, and can reach 128U/mL.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bacillus subtilis, and a preparation method and application thereof.
Background
Lignocellulose is a main structural component in plant cell walls, and is formed by embedding lignin, cellulose and hemicellulose into each other and combining the lignin, the cellulose and the hemicellulose into a space reticular structure through covalent bonds. Because lignocellulose has large molecular weight, complex structure and stable property, the lignocellulose is extremely difficult to decompose under natural conditions. Various crop straws rich in lignocellulose, commercial crops and medical plant wastes in agricultural production in China are rich in resources and huge in yield. However, for a long time, due to the influence of factors such as life style, processing technology and poor feeding property, a large amount of crop straws and medical plant wastes are discarded or burned, and are not reasonably developed and utilized. Therefore, the development of comprehensive utilization research of lignocellulose has important practical significance for saving resources, protecting the environment, increasing the income of farmers and promoting the sustainable development of agriculture.
Common fiber degradation methods include mechanical degradation, acid degradation, alkali degradation, thermal degradation, oxidative degradation, electrochemical degradation, and biodegradation. The biodegradation process catalyzes the degradation of cellulose using cellulase produced by microorganisms, as opposed to other processes, is a mild, rapid and environmentally friendly process. The current research focus of biodegradation methods is on obtaining strains with good degradation of lignocellulose.
At present, a common bacterial strain in the biodegradation method is bacillus subtilis, and the bacillus subtilis can secrete lignocellulose in the growth process, but the existing research shows that the bacillus subtilis has low lignocellulose activity.
Disclosure of Invention
In order to solve the problems, the invention provides bacillus subtilis, and a preparation method and application thereof. The bacillus subtilis provided by the invention has good lignocellulose degrading effect.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides bacillus subtilis G-2 (Bacillus subtilis) with a preservation number of CGMCC No.21707.
The invention provides a method for culturing bacillus subtilis G-2 in the technical scheme, which comprises the following steps: inoculating the bacillus subtilis G-2 in the technical scheme to a fermentation medium to obtain bacillus subtilis G-2 bacterial liquid;
the fermentation medium comprises the following components in mass concentration: 8.0 to 13.0g/L glucose, 0.8 to 1.5g/L yeast extract, 0.4 to 0.6g/L peptone, 0.03 to 0.06g/LMgSO 4 And 4.0 to 7.0g/LCaCO 3 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the fermentation medium is 3.5-6.0.
Preferably, the conditions of the fermentation culture are as follows: the temperature is 15-40 ℃, the time is 24-72 h, and the rotating speed is 150-200 rpm.
Preferably, the bacillus subtilis G-2 is inoculated to a fermentation medium in the form of a seed solution; the inoculation amount of the seed liquid is 4-10%.
Preferably, the culture method of the seed liquid comprises the following steps: inoculating bacillus subtilis G-2 into a seed culture medium for seed culture to obtain seed liquid;
the seed culture medium comprises the following components in mass concentration: 9.0-12.0 g/L peptone, 2.0-3.0 g/L beef extract and 4.0-5.0 g/L sodium chloride; the pH of the seed culture medium is 3.5-6.0.
Preferably, the seed culture conditions are: the temperature is 15-40 ℃, the time is 12-36 h, and the rotating speed is 150-200 rpm.
The invention provides application of the bacillus subtilis G-2 in lignocellulose degradation.
The invention provides a lignocellulose degrading bacterial agent, which is prepared by adopting the bacillus subtilis G-2 in the technical scheme.
Preferably, the concentration of the bacillus subtilis G-2 is 1 multiplied by 10 8 ～3×10 8 CFU/g。
The beneficial effects are that:
the invention provides bacillus subtilis G-2 with a preservation number of CGMCC No.21707. The bacillus subtilis G-2 provided by the invention has a good lignocellulose degrading effect. The results of the examples show that the bacillus subtilis G-2 provided by the invention can grow at 10 ℃ and pH 3.5; the growth is good under the conditions of 20 ℃ and pH4.0, and the cellulase activity reaches 80U/mL; the cellulase activity reaches the highest under the conditions of 35 ℃ and pH4.0, and can reach 128U/mL.
Description of biological preservation
Bacillus subtilis G-2, latin is Bacillus subtilis, and is deposited in China general microbiological culture Collection center, with deposit number: CGMCC No.21707, the preservation date 2021 is 25 days in 01 year and 25 days, and the preservation address is North Chen Liuxi Lu No.1 and No. 3 in the Chaoyang district of Beijing city.
Drawings
FIG. 1 is a graph showing the results of preliminary screening of Bacillus subtilis G-2 on Congo red plates;
FIG. 2 is a graph showing the re-screening results of Bacillus subtilis G-2 on Congo red plates;
FIG. 3 is a graph showing the results of hydrolysis circle test of Bacillus subtilis G-2 on aniline blue plate;
FIG. 4 shows the results of a B.subtilis G-2 phylogenetic tree.
Detailed Description
The invention provides bacillus subtilis G-2 with a preservation number of CGMCC No.21707. The bacillus subtilis G-2 is separated from the feces of the white star flower scarab beetles. The bacterial colony of the bacillus subtilis G-2 is irregular in appearance, milky white, opaque, small in folds and slightly provided with folds in the middle. The bacillus subtilis G-2 is dyed into purple gram, and is gram positive; spore staining showed spores; anaerobic growth-; V-P reaction+; nitrate reduction+; starch hydrolysis+; gelatin liquefaction+; by means of propionate-; d-mannitol+; d-xylose+; l-arabinose+. The bacillus subtilis G-2 can grow at 10 ℃ and pH value of 3.5; the growth is good under the conditions of 20 ℃ and pH4.0, and the cellulase activity reaches 80U/mL; the cellulase activity reaches the highest under the conditions of 35 ℃ and pH4.0, and can reach 128U/mL. The total length of the 16S rDNA gene sequence of the bacillus subtilis G-2 is 1500bp, and the specific sequence is SEQ ID No.1. By using Blast for comparison, a phylogenetic tree was constructed according to the highest similarity sequence provided by GenBank, and was identified as bacillus subtilis in combination with physiological and biochemical characteristics.
The invention provides a method for culturing bacillus subtilis G-2 in the technical scheme, which comprises the following steps: inoculating the bacillus subtilis G-2 in the technical scheme to a fermentation medium to obtain bacillus subtilis G-2 bacterial liquid;
the fermentation medium comprises the following components in mass concentration: 8.0 to 13.0g/L glucose, 0.8 to 1.5g/L yeast extract, 0.4 to 0.6g/L peptone, 0.03 to 0.06g/LMgSO 4 And 4.0 to 7.0g/LCaCO 3 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the fermentation medium is 3.5-6.0.
The bacillus subtilis G-2 of the invention is preferably inoculated to the fermentation medium in the form of a seed solution. In the present invention, the method for culturing the seed liquid preferably comprises: inoculating bacillus subtilis G-2 into a seed culture medium for seed culture to obtain seed liquid. In the present invention, the seed medium preferably comprises the following components in mass concentration: 9.0-12.0 g/L peptone, 2.0-3.0 g/L beef extract and 4.0-5.0 g/L sodium chloride; further preferably, 10g/L peptone, 3.0g/L beef extract and 5.0g/L sodium chloride are included. The seed culture medium provides a proper carbon source, nitrogen source, inorganic salt and the like for the bacillus subtilis G-2, and provides good conditions for the growth of the bacillus subtilis G-2. In the present invention, the pH of the seed medium is preferably 3.6 to 6.0; further preferably 4.0 to 5.0; still more preferably 4.0. In the present invention, the temperature of the seed culture is preferably 15 to 40 ℃; further preferably 35 to 37 ℃; still more preferably 35℃or 37 ℃. In the present invention, the time for the seed culture is preferably 12 to 36 hours; still more preferably 16 to 20 hours; still more preferably 16h. In the present invention, the rotation speed of the seed fermentation is preferably 150 to 200rpm; further preferably 180 to 200rpm; still more preferably 180rpm.
After the seed liquid is obtained, the seed liquid is preferably inoculated into a fermentation medium for fermentation culture. In the invention, the inoculation amount of the seed liquid is preferably 4-10%; further preferably 6% -8%; still more preferably 6%. In the present invention, the fermentation medium preferably comprises the following components in mass concentration: 8.0 to 13.0g/L glucose, 0.8 to 1.5g/L yeast extract, 0.4 to 0.6g/L peptone, 0.03 to 0.06g/LMgSO 4 And 4.0 to 7.0g/LCaCO 3 The method comprises the steps of carrying out a first treatment on the surface of the Further preferably comprises the following components in mass concentration: 10g/L glucose, 1.5g/L yeast extract, 0.5g/L peptone, 0.05g/LMgSO 4 And 6.0g/LCaCO 3 . The fermentation medium provides a proper carbon source, nitrogen source, inorganic salt and the like for the bacillus subtilis G-2, so that the bacillus subtilis G-2 can grow optimally. In the present invention, the pH of the fermentation medium is preferably 3.5 to 6.0; further preferably 3.5 to 5.0; still more preferably 4.0. The specific pH range of the fermentation medium is the optimal growth pH range of the bacillus subtilis G-2, and the expansion growth of the bacillus subtilis G-2 is facilitated. In the present invention, the temperature of the fermentation culture is preferably 15-40 ℃; further preferably 20 to 37 ℃; still more preferably 35 ℃. In the invention, the fermentation culture time is preferably 24-72 hours; more preferably 36 to 48 hours; still more preferably 48 hours. In the present invention, the rotation speed of the seed fermentation is preferably 150 to 200rpm; further preferably 180 to 200rpm; still more preferably 180rpm. The specific fermentation conditions are favorable for fermentation, so that the bacillus subtilis G-2 has the strongest cellulase production capacity and the maximum activity of degrading lignocellulose. The method for culturing the bacillus subtilis G-2 is simple and easy to operate, and can realize large-scale production of the bacillus subtilis G-2.
The invention provides application of the bacillus subtilis G-2 in lignocellulose degradation. The bacillus subtilis G-2 has good lignocellulose degrading capability, the highest cellulase activity can reach 128U/mL, and the bacillus subtilis G-2 can be used for degrading lignocellulose and has important significance for comprehensive utilization of lignocellulose.
The invention provides a lignocellulose degrading bacterial agent, which is prepared by adopting the bacillus subtilis G-2 in the technical scheme. In the present invention, the concentration of Bacillus subtilis G-2 in the lignocellulose degrading agent is preferably 1×10 8 ～3×10 8 CFU/g; further preferably 1X 10 8 ～2×10 8 CFU/g; even more preferably 2X 10 8 CFU/g. The lignocellulose degrading bacterial agent provided by the invention has good lignocellulose degrading capability, is mild, rapid and pollution-free.
Example 1
Isolation and identification of Bacillus subtilis G-2
The bacillus subtilis G-2 is separated from the feces of the white star flower scarab beetles. The invention adopts a cross marking method to perform primary screening on a Congo red plate. The preparation method of Congo red culture medium comprises the following steps: sodium carboxymethylcellulose 4.0g, mgSO 4 ·7H 2 O 0.5g，KH 2 PO 3 1.0 g，(NH 4 ) 2 SO 4 2.0g, agar 20.0g, congo red 0.2g, steamingDistilled water was dissolved to 1000mL. Congo red is added into a culture medium, can be combined with macromolecular polysaccharide, and cellulose is macromolecular polysaccharide, so that the addition of Congo red on the cellulose-containing plate can be colored, and when bacteria grow to generate cellulase, cellulose in the culture medium is decomposed into micromolecular sugar, and transparent rings appear. FIG. 1 is a graph showing the results of preliminary screening of Bacillus subtilis G-2 on Congo red plates (cross-hatching).
And (3) re-screening by adopting Congo red plates and aniline blue plates. The preparation method of the aniline blue culture medium comprises the following steps: yeast powder 10.0g, glucose 20.0g, aniline blue 0.1g, agar 20.0g, distilled water to 1000mL. The aniline blue plate is similar to Congo red plate in principle, and the aniline blue plate is also used for screening lignin degrading enzyme strain.
FIG. 2 is a graph showing the results of a hydrolysis circle test of Bacillus subtilis G-2 in Congo red plates (plate diffusion method); FIG. 3 is a graph showing the results of hydrolysis circle test of Bacillus subtilis G-2 on aniline blue plate (plate diffusion method). The transparent circle diameter measurement results of the bacillus subtilis G-2 are shown in Table 1.
TABLE 1 transparent circle diameter measurement results of Bacillus subtilis G-2
The bacillus subtilis G-2 strain provided by the invention grows on an NA culture medium, wherein the preparation method of the NA culture medium comprises the following steps: 10.0g of peptone, 3.0g of beef extract, 5.0g of sodium chloride, 15.0-20.0 g of agar, and distilled water to 1000mL, and regulating pH to 7.2-7.4. The obtained colony has irregular appearance, milky white color, opaqueness, smaller folds and slightly raised folds in the middle; gram staining is purple, is a gram positive bacterium; spore staining showed spores; anaerobic growth-; V-P reaction+; nitrate reduction+; starch hydrolysis+; gelatin liquefaction+; by means of propionate-; d-mannitol+; d-xylose+; l-arabinose+.
The genome extraction kit is used for extracting genome DNA of the strain, and the PCR technology is used for determining that the total length of a 16SrDNA gene sequence is 1500bp, the specific sequence is SEQ ID No.1, and the specific sequence is as follows:
ACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGTCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTACCGCCCTATTCGAACGGTACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTTGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTATGTTTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACGCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTT。
by using Blast for comparison, a phylogenetic tree was constructed according to the highest similarity sequence provided by GenBank, and was identified as bacillus subtilis in combination with physiological and biochemical characteristics, and the result of the phylogenetic tree is shown in fig. 4.
Example 2
Effect of culture temperature on Bacillus subtilis G-2 culture
Inoculating bacillus subtilis G-2 into a seed culture medium, and shake culturing for 16 hours at 180rpm by a shaking table at 37 ℃ to prepare seed liquid. Wherein, the seed culture medium consists of the following components in mass concentration: 10g/L peptone, 3.0g/L beef extract and 5.0g/L sodium chloride, and the pH is 7.2-7.4.
Inoculating the seed solution into a fermentation medium with an inoculum size of 6.0%, wherein the fermentation medium consists of the following components in mass concentration: 10g/L glucose, 1.5g/L yeast extract, 0.5g/L peptone, 0.05g/LMgSO 4 And 6.0g/LCaCO 3 The pH is 7.2-7.4. The fermentation temperature was adjusted to 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃ and 40 ℃ each 2 times of treatment, and after 48 hours of cultivation, the growth of the bacteria was observed and the cellulase activity of the supernatant of the fermentation broth centrifugation was measured. The results of the measurements are shown in Table 2, where "-" does not grow, "w" grows weakly, "+" grows, "++" grows better and "++ +" grows better.
TABLE 2 growth Properties of the strains of the invention under different temperature conditions
As is clear from the results shown in Table 2, the strain of the present invention grew well under the conditions of 25 to 40℃and was able to grow at a low temperature of 15 to 20℃and had low temperature tolerance. At the temperature of 10-35 ℃, the cellulase activity produced by the bacillus subtilis G-2 is gradually increased along with the increase of the temperature, the cellulase activity reaches the highest value at the temperature of 35 ℃ and is about 128.0U/mL, and then the cellulase activity shows a decreasing trend. The bacillus subtilis G-2 still has the enzyme activity of more than 80U/mL at 20 ℃.
Example 3
Effect of pH of fermentation Medium on Bacillus subtilis G-2 culture
The seed solution of Bacillus subtilis G-2 was cultured in the same manner as in example 2.
The seed solution was inoculated into fermentation media having pH values of 3.0, 3.5, 4.0, 4.5, 5.0, 5.5 and 6.0 in an inoculum size of 6.0%, wherein the composition and concentration of the fermentation media were the same as those of example 2. Each treatment was repeated 2 times, incubated at 37℃for 48 hours, the growth of the bacteria was observed, and the cellulase activity of the supernatant of the fermentation broth centrifugation was measured. The results of the measurements are shown in Table 3, wherein "-" does not grow, "w" grows weakly, "+" grows, "++" grows better and "++ +" grows better.
TABLE 3 growth Properties of the strains of the invention under different temperature conditions
As is clear from the results shown in Table 3, the strain of the present invention can grow at a pH of 3.5 to 6.0, but grows weakly at a pH of 3.5, and grows preferably at a pH of 4.0 to 4.5.
The bacillus subtilis G-2 provided by the invention has good lignocellulose degrading effect and can grow under the conditions of 10 ℃ and pH 3.5; the growth is good under the conditions of 20 ℃ and pH4.0, and the cellulase activity reaches 80U/mL; the cellulase activity reaches the highest under the conditions of 35 ℃ and pH4.0, and can reach 128U/mL.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, one may obtain other embodiments from this embodiment without inventiveness, which are all within the scope of this invention.
Sequence listing
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<120> a strain of bacillus subtilis, and culture method and application thereof
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gtcgagttgc agactgcgat ccgaactgag aacagatttg tgggattggc ttaacctcgc 180
ggtttcgctg ccctttgttc tgtccattgt agcacgtgtg tagcccaggt cataaggggc 240
atgatgattt gacgtcatcc ccaccttcct ccggtttgtc accggcagtc accttagagt 300
gcccaactga atgctggcaa ctaagatcaa gggttgcgct cgttgcggga cttaacccaa 360
catctcacga cacgagctga cgacaaccat gcaccacctg tcactctgcc cccgaagggg 420
acgtcctatc tctaggattg tcagaggatg tcaagacctg gtaaggttct tcgcgttgct 480
tcgaattaaa ccacatgctc caccgcttgt gcgggccccc gtcaattcct ttgagtttca 540
gtcttgcgac cgtactcccc aggcggagtg cttaatgcgt tagctgcagc actaaggggc 600
ggaaaccccc taacacttag cactcatcgt ttacggcgtg gactaccagg gtatctaatc 660
ctgttcgctc cccacgcttt cgctcctcag cgtcagttac agaccagaga gtcgccttcg 720
ccactggtgt tcctccacat ctctacgcat ttcaccgcta cacgtggaat tccactctcc 780
tcttctgcac tcaagttccc cagtttccaa tgaccctccc cggttgagcc gggggctttc 840
acatcagact taagaaaccg cctgcgagcc ctttacgccc aataattccg gacaacgctt 900
gccacctacg tattaccgcg gctgctggca cgtagttagc cgtggctttc tggttaggta 960
ccgtcaaggt accgccctat tcgaacggta cttgttcttc cctaacaaca gagctttacg 1020
atccgaaaac cttcatcact cacgcggcgt tgctccgtca gactttcgtc cattgcggaa 1080
gattccctac tgctgcctcc cgtaggagtc tgggccgtgt ctcagtccca gtgtggccga 1140
tcaccctctc aggtcggcta cgcatcgttg ccttggtgag ccgttacctc accaactagc 1200
taatgcgccg cgggtccatc tgtaagtggt agccgaagcc accttttatg tttgaaccat 1260
gcggttcaaa caaccatccg gtattagccc cggtttcccg gagttatccc agtcttacag 1320
gcaggttacc cacgtgttac tcacgcgtcc gccgctaaca tcagggagca agctcccatc 1380
tgtccgctcg actt 1394
Claims (6)
1. The application of bacillus subtilis G-2 bacterial liquid in lignocellulose degradation is characterized in that the preservation number of the bacillus subtilis G-2 is CGMCC No. 2107;
the preparation method of the bacillus subtilis G-2 bacterial liquid comprises the following steps: inoculating the bacillus subtilis G-2 to a fermentation medium to obtain bacillus subtilis G-2 bacterial liquid;
the fermentation medium comprises the following components in mass concentration: 8.0 to 13.0g/L glucose, 0.8 to 1.5g/L yeast extract, 0.4 to 0.6g/L peptone, 0.03 to 0.06g/LMgSO 4 And 4.0 to 7.0g/LCaCO 3 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the fermentation medium is 6.0;
the conditions of the fermentation culture are as follows: the temperature is 37 ℃, the time is 48 hours, and the rotating speed is 150-200 rpm.
2. The use according to claim 1, characterized in that the bacillus subtilis G-2 is inoculated to the fermentation medium in the form of a seed liquid; the inoculation amount of the seed liquid is 4-10%.
3. The use according to claim 2, wherein the seed liquid is cultivated by the following method: inoculating bacillus subtilis G-2 into a seed culture medium for seed culture to obtain seed liquid;
the seed culture medium comprises the following components in mass concentration: 9.0-12.0 g/L peptone, 2.0-3.0 g/L beef extract and 4.0-5.0 g/L sodium chloride; the pH of the seed culture medium is 3.5-6.0.
4. The use according to claim 3, wherein the seed culture conditions are: the temperature is 15-40 ℃, the time is 12-36 h, and the rotating speed is 150-200 rpm.
5. A lignocellulose degrading bacterial agent, characterized in that the bacterial agent is prepared by using the bacillus subtilis G-2 of claim 1;
the preparation method comprises the steps of inoculating the bacillus subtilis G-2 to a fermentation medium to obtain bacillus subtilis G-2 bacterial liquid;
the fermentation medium comprises the following components in mass concentration: 8.0 to 13.0g/L glucose, 0.8 to 1.5g/L yeast extract, 0.4 to 0.6g/L peptone, 0.03 to 0.06g/LMgSO 4 And 4.0 to 7.0g/LCaCO 3 The method comprises the steps of carrying out a first treatment on the surface of the The pH of the fermentation medium is 6.0;
the conditions of the fermentation culture are as follows: the temperature is 37 ℃, the time is 48 hours, and the rotating speed is 150-200 rpm.
6. The lignocellulose degrading bacterial agent of claim 5, wherein the concentration of bacillus subtilis G-2 in the lignocellulose degrading bacterial agent is 1 x 10 8 ～3×10 8 CFU/g。
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