CN113717876B

CN113717876B - Broussonetia papyrifera leaf endophytic bacterium with lignocellulose degradation function

Info

Publication number: CN113717876B
Application number: CN202110694435.0A
Authority: CN
Inventors: 何文兴; 田华; 王文波; 聂言顺; 王震; 孙超; 刘传涛; 雷伟; 张慧; 李鑫
Original assignee: Fengtang Ecological Agriculture Technology R & D Shandong Co ltd
Current assignee: Fengtang Ecological Agriculture Technology R & D Shandong Co ltd
Priority date: 2021-06-22
Filing date: 2021-06-22
Publication date: 2022-09-06
Anticipated expiration: 2041-06-22
Also published as: CN113717876A

Abstract

The invention discloses a broussonetia papyrifera leaf endophytic bacterium with a lignocellulose degradation function, which is named as BP-L-1 and classified and named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), and the preservation number is CCTCC M2021400. The broussonetia papyrifera leaf endophytic bacteria have strong capability of producing cellulase and ligninolytic enzyme, can effectively degrade lignocelluloses in the broussonetia papyrifera silage, and reduce the content of the lignocelluloses, so that the quality grade of the silage product is improved. Therefore, the broussonetia papyrifera leaf endophytic bacteria can be used for preparing cellulase and/or lignin degrading enzyme, and can be used for degrading lignocellulose, especially lignocellulose in broussonetia papyrifera; can be used for preparing silage, especially broussonetia papyrifera silage.

Description

Broussonetia papyrifera leaf endophytic bacterium with lignocellulose degradation function

Technical Field

The invention relates to a broussonetia papyrifera leaf endophytic bacterium with a lignocellulose degradation function, and belongs to the technical field of microorganisms.

Background

As is well known, the development of animal husbandry does not depart from the progress of the feed industry, and the overall development of the feed industry can affect the continuous development of animal husbandry. As a large population country, China continuously increases the demand of livestock products and the quality requirement along with the continuous improvement of the life quality of people. The current situation of 'food competition between people and livestock' is shown, the conventional feed raw materials such as corn, soybean and the like can not meet the development of animal husbandry, and broussonetia papyrifera as a high-quality unconventional high-protein feed raw material now becomes a research hotspot in the feed field.

The broussonetia papyrifera has the advantages of strong stress resistance, good adaptability, strong growth capacity and the like, is widely distributed in tropical and temperate zones of China, and grows in northeast, southwest, south China, North China and the like. The paper mulberry leaves are rich in protein, carbohydrate, amino acid and fat. However, woody plants also have a high lignocellulose content. The problems of low digestion, absorption and utilization rate, poor palatability and the like of livestock are easily caused by too high content of cellulose and lignin. Lignocellulose is subjected to catabolism of degrading bacteria to form a simple compound, so that the compound is more beneficial to absorption of livestock, and the economic value of the paper mulberry silage is improved.

Endophytes (endophytes) are fungi or bacteria that live inside the tissues and organs of healthy plants at certain or all stages, and have good affinity with plants. The endophytic bacterial strain with the lignocellulose degradation function is separated from the leaves and the roots of the broussonetia papyrifera, and the endophytic bacterial strain has important significance for reducing the content of lignocellulose in the broussonetia papyrifera leaf silage and improving the grade of silage products.

Lignocellulase producing bacteria of the species bacillus amyloliquefaciens have been studied more and more in recent years, for example:

wangkai et al (2014) screened a bacillus amyloliquefaciens FJAT-8754 with higher cellulase activity from 140 bacillus, and the initial cellulase activity of the strain is found to be 15.69 U.mL through enzyme activity detection ^-1 。

He Zhang et al (2020) performs primary screening and secondary screening of cellulase on isolated strains by a hydrolytic ring method and a CMC enzymatic saccharification method to finally obtain a strain of CMC with the enzyme activity of 31.9 U.mL ^-1 Bacillus amyloliquefaciens X10.

Liu Yu et al (2017) screened a strain of bacillus amyloliquefaciens from a corn silage sample, and after fermentation for 3 days, the enzyme activity of filter paper is determined to be 1.5U/mL, the activity of exoglucanase is determined to be 1.3U/mL, the activity of endoglucanase is determined to be 6.4U/mL, and the activity of beta-glucosidase is determined to be 2.3U/mL.

A bacillus amyloliquefaciens MU7 strain with strong cellulose and lignin degradation capacity is separated and screened from pleurotus eryngii bran by Gaosxu red et al (2018), and the laccase activity and the lignin peroxidase activity of the bacillus amyloliquefaciens MU7 strain are determined to be 0.2U/mL and 0.14U/mL respectively.

Disclosure of Invention

Aiming at the prior art, the invention provides a broussonetia papyrifera leaf endophytic bacterium with a lignocellulose degradation function, which has strong capability of producing cellulase and ligninolytic enzyme, can effectively degrade lignocellulose in broussonetia papyrifera silage, reduces the content of lignocellulose, and improves the quality grade of silage products.

The invention is realized by the following technical scheme:

a broussonetia papyrifera leaf endophytic bacterium with a lignocellulose degradation function is named as BP-L-1, is preserved in China Center for Type Culture Collection (CCTCC) at 19 months 4 and 2021, has a preservation number of CCTCC M2021400, and has a preservation address of: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province. The strain is identified to be Bacillus amyloliquefaciens (Bacillus amyloliquefaciens).

The morphological characteristics of the strain BP-L-1 are as follows: the bacterial colony is light yellow and opaque, and has rough surface, bump and irregular edge.

The physiological and biochemical characteristics of the strain BP-L-1 are as follows: the results of the starch hydrolysis experiment are positive, the results of the gelatin liquefaction experiment are negative, the results of the methyl red experiment are negative, and the results of the nitrate reduction experiment are positive.

Molecular biological identification: selecting bacterial colony, and performing liquid culture at 28 deg.C with CMC-Na liquid culture medium until bacterial liquid OD ₆₀₀ 1-1.5, 1mL of the bacterial solution was aspirated to extract genomic DNA of strain BP-L-1, 16SrDNA was subjected to genomic PCR amplification, the PCR amplification product was subjected to agarose gel electrophoresis (shown in FIG. 1), the target band was recovered, and the target band was sent to Shanghai Senno Biotech Co., Ltd for 16SrDNA gene sequencing. The sequencing result of the 16SrDNA of the strain BP-L-1 is subjected to Blast comparison in a database of NCBI, and the result shows that the homology of the strain BP-L-1 and Bacillus amyloliquefaciens is more than 99 percent (shown in figure 2).

16SrDNA sequence of the strain BP-L-1 (shown as SEQ ID NO. 1):

5’-CACCTTCGGCGGCTGGCTCCATAAAGGTTACCTCACCGACTTCGGGTGTTACAAACTCTCGTGGTGTGACGG GCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTC GAGTTGCAGACTGCGATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTGTTCTGT CCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACC GGCAGTCACCTTAGAGTGCCCAACTGAATGCTGGCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACAT CTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTAGGATTGTCA GAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTC AATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGA AACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGC TCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACAC GTGGAATTCCACTCTCCTCTTCTGCACTCAAGTTCCCCAGTTTCCAATGACCCTCCCCGGTTGAGCCGGGGGCTTTCACA TCAGACTTAAGAAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATTACCGCGGCT GCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCCGCCCTATTTGAACGGCACTTGTTCTTCCCT AACAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGAT TCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGC ATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGGTAGCCGAAGCCACC TTTTATGTCTGAACCATGCGGTTCAAACAACCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCA GGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTCGACTTGCATGTATT AGGCACGCCGCCAGCGTTCGTCCTGAGCCAGATCAAAACTCT-3’。

the paper mulberry leaf endophytic bacteria with the lignocellulose degradation function has strong capability of producing cellulase and ligninolytic enzyme, can effectively degrade lignocellulose in paper mulberry silage, reduces the content of lignocellulose, and accordingly improves the quality grade of silage products. Therefore, the broussonetia papyrifera leaf endophytic bacteria with the lignocellulose degradation function can be used for preparing cellulase and/or lignin degrading enzyme and degrading lignocellulose, especially lignocellulose in broussonetia papyrifera; can be used for preparing silage, especially broussonetia papyrifera silage.

The Bacillus amyloliquefaciens (BP-L-1) obtained by the invention is found to have the filter paper enzyme activity of 30.56 +/-2.6 U.mL by the enzyme activity measurement ^-1 The activity of endo-beta-1, 4-glucanase is 86.67 +/-6.3 U.mL ^-1 The activity of the exo-beta-1, 4-glucanase is 75.00 +/-4.7 U.mL ^-1 The beta-glucosidase activity is 41.25 +/-2.7 U.mL ^-1 The laccase activity is 22.13 +/-1.7 U.mL ^-1 The lignin peroxidase activity was 21.50. + -. 1.2 U.mL ^-1 Is obviously superior to the strains in the prior art (the filter paper enzyme activity, the endo-beta-1, 4-glucanase activity and the exo-beta-1, 4-glucan in the invention)The enzyme activity and the beta-glucosidase activity both adopt a DNS method, the lignin peroxidase activity adopts a veratryl alcohol method, and the laccase activity adopts an ABTS method in the test process; the test method of the present invention is the same as the test method in the reference mentioned in the background art, and the data obtained by the experiment can be directly compared). Compared with other strains for producing lignocellulose degrading enzymes, the strain provided by the invention has better initial conditions for producing the cellulase strain, and can play a positive role in further research on cellulase activity optimization.

The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art. To the extent that the terms and phrases are not inconsistent with known meanings, the meaning of the present invention will prevail.

Drawings

The paper mulberry leaf endophytic bacterium BP-L-1 with lignocellulose degradation function is preserved in China Center for Type Culture Collection (CCTCC) at 19.4.2021, the preservation number is CCTCC M2021400, and the preservation addresses are as follows: eight-way Lojia mountain in Wuchang district, Wuhan, Hubei province.

FIG. 1: electrophoresis picture of 16SrDNA PCR product of strain BP-L-1.

FIG. 2: a phylogenetic tree of strain BP-L-1 based on the 16SrDNA sequence.

FIG. 3: and (3) a schematic diagram of a hydrolysis loop morphology of the candidate strain.

FIG. 4 is a schematic view of: gram-stained light microscopy of strain BP-L-1.

Detailed Description

The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.

The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

EXAMPLE 1 screening of strains

1. Bacterial strain preliminary screening

Picking fresh broussonetia papyrifera at random from hybrid broussonetia papyrifera planted in a broussonetia papyrifera planting base (located in Taian city, Shandong province), performing surface disinfection, shearing the fresh broussonetia papyrifera into pieces by using sterilized scissors, culturing the pieces on a CMC-Na solid culture medium, selecting strains with different colony morphologies, respectively inoculating the strains on the CMC-Na solid culture medium for culturing, and performing separation and purification. Inoculating the separated and purified strains on a CMC-Na solid culture medium, dyeing by using 0.5 percent Congo red after 5 days, decoloring by using 1mol/L NaCl solution, and selecting strains capable of generating transparent hydrolysis rings to be defined as candidate strains with cellulose degradation capability; inoculating the screened strain on PDA-aniline blue solid culture medium, and selecting strain capable of producing transparent hydrolysis ring on the culture medium after 5 days as candidate strain with lignin degradation capability.

According to the invention, 5 candidate strains are obtained through initial experiments, and the hydrolysis circle diameter of the candidate strain BP-L-1 is the largest by observing the hydrolysis circle of the 5 candidate strains cultured on a sodium carboxymethylcellulose (CMC-Na) culture medium and an aniline blue-PDA culture medium for three days, so that the BP-L-1 is selected as a lignocellulose degradation efficient strain, and the result is shown in figure 3.

2. Media used during the experiment:

(1) CMC-Na liquid medium: 15g of CMC-Na; 1g of yeast extract; KH (Perkin Elmer) ₂ PO ₄ ，1g；MgSO ₄ ·7H ₂ O, 0.5 g; NaCl, 0.5 g; 1000mL of distilled water; pH value, natural.

(2) CMC-Na solid medium: 14g/L agar was added to the CMC-Na liquid medium.

(3) PDA-aniline blue medium: 0.5g/L aniline blue was added to the PDA.

(4) Hertzson (Huchinson) inorganic salt broth: KH (Perkin Elmer) ₂ PO ₄ ，1.0g；NaCl，0.1g；MgSO ₄ ·7H ₂ O， 0.3g；NaNO ₃ ，2.5g；CaCl ₂ ，0.1g；FeCl ₃ 0.01 g; distilled water, 1000 mL.

(5)3, 5-dinitrosalicylic acid (DNS) reagent: 185g of potassium sodium tartrate is weighed, dissolved in 500mL of hot water, added with 6.3g of DNS and 262mL of 2M NaOH solution, added with 5g of phenol and 5g of sodium sulfite, dissolved to a constant volume of 1L, and stored in a brown reagent bottle.

(6) Enzyme-producing fermentation medium: 5g of CMC-Na; NaNO ₃ ，3g；K ₂ HPO ₄ ，1g；KCl，0.5g；MgSO ₄ ， 0.5g；FeSO ₄ 0.01 g; 1000mL of distilled water; the pH value is 5.5-6.0.

(7)0.2mol/L acetate buffer at pH 4.8:

solution A: adding distilled water to dilute the glacial acetic acid to 1000 mL;

and B, liquid B: weighing 27.22g of sodium acetate, and dissolving in 1000mL of distilled water;

sucking 40mL of the solution A and 60mL of the solution B and mixing uniformly.

3. Identification of strains

The strain BP-L-1 obtained by primary screening is identified by microscopic examination, colony appearance observation and 16SrDNA gene sequence. The strain BP-L-1 is selected and cultured under the environment of 28 ℃, a gram staining experiment is carried out after 5 days of culture, the morphological characteristics of the bacteria are observed under an oil lens, the bacteria are observed to be rod-shaped, the color of the thallus is purple, and therefore the strain BP-L-1 is gram positive bacteria, as shown in figure 4.

According to the manual of identifying common bacteria systems and the manual of identifying Bergey bacteria, the physiological and biochemical characteristic experiments of the strain BP-L-1 are carried out, and the method comprises the following experiments: the results of the starch hydrolysis test are positive, the gelatin liquefaction test is negative, the methyl red test is positive, the nitrate reduction test is positive, and the results are shown in table 2.

TABLE 2 physiological and biochemical characteristics of the strains

4. Determination of cellulase secretion ability of strain BP-L-1

The enzyme activity unit is defined as that 1 enzyme activity unit (U) is required for 1ml of crude enzyme solution to catalyze a substrate to produce 1mg of glucose within 1min at a pH of 5.0 and 40 ℃, and the enzyme activity is expressed as the amount of the enzyme activity unit (U) contained in each ml of fermentation broth.

And (3) determining the cellulase production activity of the strain BP-L-1 by using a DNS method, and respectively determining the filter paper enzyme activity, the endo-beta-1, 4-glucanase activity, the exo-beta-1, 4-glucanase activity and the beta-glucosidase activity.

(1) Preparation of crude enzyme solution

Inoculating the strain BP-L-1 into 15mL of fermentation medium, and carrying out shake culture for 2-3 h at 28 ℃ and at 200 rpm/min; centrifuging at 4 deg.C and 10000rpm/min for 10min to obtain supernatant, which is crude enzyme solution.

(2) Drawing of glucose standard curve

Preparing 1.0mg/mL glucose solution as standard glucose mother liquor, respectively sucking 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL glucose solution in a 25mL graduated test tube, diluting to 1mL with distilled water, adding 3mL DNS developer, boiling water bath for 10min, cooling to room temperature, diluting to 25mL with distilled water, and mixing. And (3) adjusting zero by using a blank tube, measuring an absorbance value at 540nm, and drawing a standard curve by using the glucose concentration as an abscissa and the absorbance value as an ordinate.

(3) Filter paper enzyme activity assay

Cutting Xinhua No. one filter paper into small strips of 1cm multiplied by 3cm (about 50mg), putting the small strips into a 25mL graduated test tube, adding 3mL of acetic acid-sodium acetate buffer solution and 1mL of enzyme solution, uniformly mixing, completely soaking the filter paper strips in the mixed solution, carrying out water bath at 50 ℃ for 1h, then adding 3mL of DNS developer, carrying out boiling water bath for 10min, placing the mixed solution in cold water to cool to room temperature, using distilled water to fix the volume to 25mL, and measuring the absorbance value at the wavelength of 540nm by using an ultraviolet spectrophotometer.

(4) Determination of endo-beta-1, 4-glucanase activity

Adding 3mL of acetic acid-sodium acetate buffer solution containing 0.5% sodium carboxymethylcellulose into a 25mL test tube, adding 1mL of enzyme solution, placing the test tube into a 50 ℃ water bath kettle, carrying out water bath for 0.5h, adding 3mL of DNS reagent, carrying out boiling water bath for 10min, cooling to room temperature, adding 18mL of distilled water, and measuring the absorbance value at the wavelength of 540nm by using an ultraviolet spectrophotometer.

(5) Exo-beta-1, 4-glucanase activity assay

Adding 3mL of acetic acid-sodium acetate buffer solution containing 0.5% microcrystalline cellulose into a 25mL test tube, adding 1mL of enzyme solution, carrying out water bath at 50 ℃ for 2h, adding 3mL of DNS reagent, carrying out boiling water bath for 10min, cooling with ice water, diluting to 25mL with distilled water, and measuring the absorbance value at the wavelength of 540nm by using an ultraviolet spectrophotometer.

(6) Beta-glucosidase activity assay

Taking 3mL of acetic acid-sodium acetate buffer solution containing 1% salicylic acid, adding 1mL of enzyme solution, carrying out water bath at 50 ℃ for 30min, adding 3mL of DNS reagent, carrying out boiling water bath for 10min, cooling with ice water, and measuring the absorbance value at the wavelength of 540nm by using an ultraviolet spectrophotometer.

The measurement results are shown in Table 3.

TABLE 3 Activity of the strains BP-L-1 with four cellulases

5. Determination of lignin degrading enzyme secretion capability of strain BP-L-1

According to the fact that two enzymes of lignin can act on different substrates, different substrates are selected, and the change of absorbance of the substrates is measured by an ultraviolet spectrophotometer under different wavelengths, so that the two enzyme activities are calculated.

(1) Preparation of crude enzyme solution

Inoculating the strain into 100mL of liquid fermentation medium, performing constant temperature shaking culture at 28 ℃ and 220rpm for 4-5 days, centrifuging an appropriate amount in a centrifuge tube at 4 ℃ and 10000rpm for 10 minutes, and obtaining a supernatant, namely a crude enzyme solution.

(2) Laccase (Lac) enzyme activity determination

Determined by the rate at which the enzyme oxidizes ABTS at 420 nm. At 25 ℃, the total reaction system is 3mL, 2mL is 0.5mmol/L ABTS, 1mL enzyme solution is added, and the change of the absorbance value with time within 0-3min is measured by ultraviolet spectrophotometry at 420 nm. The amount of enzyme required to convert 1. mu. mol of ABTS per minute was defined as 1 activity unit (U) and the extinction coefficient ε was 36000 mol ^-1 ·L·cm ^-1 。

(3) Lignin peroxidase (Lip) enzyme activity assay

The reaction was carried out at 37 ℃ in a total reaction system of 3mL, containing 1.85mL of 200mmol/L sodium tartrate buffer (pH 3) and 0.1mL of 24mmol/L veratryl alcohol and 1.0mL of the enzyme solution, and after preheating to 37 ℃, 0.05mL of 20mmol/L H was added ₂ O ₂ The reaction was started and the absorbance increase was measured before and after 3min at 310nm with an ultraviolet spectrophotometer. Defining the enzyme quantity for oxidizing 1 mu mol veratryl alcohol in 1min as one enzyme activity unit (U), and the extinction coefficient epsilon is 9300mol ^-1 ·L·cm ^-1 。

Through determination, the laccase activity of the strain BP-L-1 is 22.13U/mL, and the lignin peroxidase activity is 21.5U/mL, as shown in Table 4.

TABLE 4 two lignin-degrading enzyme activities of the strains BP-L-1

6. Determination of capability of strain BP-L-1 in degrading lignocellulose in paper mulberry leaves

Picking broussonetia papyrifera leaves from a broussonetia papyrifera planting base, cleaning the broussonetia papyrifera leaves by using distilled water, placing the broussonetia papyrifera leaves in an oven at 80 ℃ for drying until the weight is constant, and dividing the three groups into an initial group, a control group and a treatment group, wherein each group is used for performing three parallel experiments. The initial group was directly tested for cellulose, hemicellulose and lignin content without any treatment. Each conical flask of the control group and the strain BP-L-1 group contains 2.50g of broussonetia papyrifera leaves and 100mL of inorganic salt nutrient solution, and the control group does not contain the strain solution and is added with 10mL of distilled water; 10mL of BP-L-1 bacterial solution was added to the treated group.

(1) Determination of cellulose: hydrolyzing the acid washed fiber with 10mL of 72% sulfuric acid for 3h, adding 45mL of distilled water, standing overnight, filtering with a sand core funnel the next day until the filtrate becomes neutral, placing in a 60 ℃ oven to dry to constant weight (m3), placing in a dryer at room temperature, and weighing.

(2) And (3) determination of lignin: the remaining residue was burned in a muffle furnace at 550 ℃ for 2.5 hours, taken out when the temperature was reduced to about 200 ℃, placed in a desiccator at room temperature, and weighed (m 4).

(3) Determination of acid-washed fibers: accurately weighing about 1g of sample (m), placing the sample in a straight-tube beaker, adding 100mL of acidic detergent, a few drops of decalin and 0.5g of anhydrous sodium sulfite, covering the beaker on a condensing device on an electric furnace, boiling within 5-10 min, continuously keeping slight boiling for 60min, taking off the straight-tube beaker after the boiling is finished, pouring the solution in the beaker into a glass crucible (m1) with known weight arranged on a filter flask for filtering, completely transferring the residue in the beaker into the glass crucible, washing the glass crucible and the residue with boiling water, and washing until the filtrate is neutral. Wash twice with 20mL of acetone and suction filter. After placing the glass crucible in an oven at 105 ℃ for 2h, cooling in a desiccator for 30min and weighing until the weight is constant (m 2).

(4) Determination of neutral detergent fiber: about 1g of sample (m5) was accurately weighed into a straight beaker, and 100mL of neutral detergent and several drops of decalin and 0.5g of anhydrous sodium sulfite were added. The beaker is sleeved with a condensing device and placed on an electric furnace, and boiled within 5-10 min and kept for micro-boiling for 60 min. After boiling, the straight beaker was removed, the solution in the beaker was poured into a glass crucible (m6) of known weight mounted on a filter flask for filtration, the residue in the beaker was removed completely, and the glass crucible and the residue were washed with boiling water until the filtrate was neutral. Washed twice with 20ml of acetone and filtered with suction. After placing the glass crucible (m7) in an oven at 105 ℃ for 2h, cooling in a drier for 30min and weighing until constant weight.

(5) Content calculation

Acid wash fiber calculation formula:

cellulose calculation formula:

the lignin calculation formula is as follows:

neutral detergent fiber calculation formula:

hemicellulose calculation formula: x% ═ X ₄ -X ₁

The measurement results are shown in Table 5.

TABLE 5 degradation of lignocellulose by the strain BP-L-1

As can be seen from Table 4, after 20 days of fermentation, the strain BP-L-1 has a significant effect on the degradation rate of cellulose, hemicellulose and lignin in the broussonetia papyrifera silage (P is less than 0.05); compared with a control group, the treatment of adding the strain BP-L-1 improves the degradation rate of cellulose by plus 26.42 percent, the degradation rate of hemicellulose by plus 39.78 percent and the degradation rate of lignin by plus 17.54 percent, and has obvious promotion effect on the degradation of lignocellulose.

The strain BP-L-1(Bacillus sp.) of the invention is a strain of endophyte of plants, wherein the strain is a bacterium existing in healthy tissues and organs of broussonetia papyrifera leaves; through morphological identification and molecular identification, the bacillus is determined to be a bacillus strain and has the natural characteristic of being capable of carrying out microbial fermentation under aerobic and anaerobic conditions. Under the culture condition of 28 ℃, the strain BP-L-1(Bacillus sp.) can secrete cellulase and ligninase at the same time, and the enzyme production activity is high. Moreover, lignocellulose in the paper mulberry silage can be effectively degraded, so that an excellent strain resource is provided for overcoming the limitation of high lignocellulose content in the paper mulberry silage, and a resource basis and a technical support are provided for the subsequent research and development of silage fermentation microbial agents.

The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims. All publications, patents, and patent applications cited in this specification are herein incorporated by reference as if each such publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

Reference to the literature

Wangkai, Lanjiang forest, Liubo, etc. Bacillus amyloliquefaciens FJAT-8754 for producing cellulase and amylase, and optimizing the fermentation conditions [ J ] Fujian agricultural science report 2014,29(4):357 and 363.

The screening and identification of Bacillus amyloliquefaciens high-yield cellulase strains [ J ]. Fujian agricultural science, 2020,35(7): 781-.

Liu Yu, Li Yang, Yi jade Yi, etc. separation and identification of a bacillus amyloliquefaciens producing cellulase and preliminary study on suitable conditions of enzymatic reaction thereof J. livestock ecology newspaper, 2017, (4) 58-62.

The separation and identification of the feed microorganism and the effect on the fermentation of pleurotus eryngii bran [ J ] microbiology report, 2018,58(12): 2110-2122.

Sequence listing

<110> Shangtang ecological agriculture science and technology research & development (Shandong) Co Ltd

<120> a broussonetia papyrifera leaf endophytic bacterium with lignocellulose degradation function

<141> 2021-06-22

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1474

<212> DNA

<213> Bacillus amyloliquefaciens

<400> 1

caccttcggc ggctggctcc ataaaggtta cctcaccgac ttcgggtgtt acaaactctc 60

gtggtgtgac gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc atgctgatcc 120

gcgattacta gcgattccag cttcacgcag tcgagttgca gactgcgatc cgaactgaga 180

acagatttgt gggattggct taacctcgcg gtttcgctgc cctttgttct gtccattgta 240

gcacgtgtgt agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc 300

cggtttgtca ccggcagtca ccttagagtg cccaactgaa tgctggcaac taagatcaag 360

ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg 420

caccacctgt cactctgccc ccgaagggga cgtcctatct ctaggattgt cagaggatgt 480

caagacctgg taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg 540

cgggcccccg tcaattcctt tgagtttcag tcttgcgacc gtactcccca ggcggagtgc 600

ttaatgcgtt agctgcagca ctaaggggcg gaaaccccct aacacttagc actcatcgtt 660

tacggcgtgg actaccaggg tatctaatcc tgttcgctcc ccacgctttc gctcctcagc 720

gtcagttaca gaccagagag tcgccttcgc cactggtgtt cctccacatc tctacgcatt 780

tcaccgctac acgtggaatt ccactctcct cttctgcact caagttcccc agtttccaat 840

gaccctcccc ggttgagccg ggggctttca catcagactt aagaaaccgc ctgcgagccc 900

tttacgccca ataattccgg acaacgcttg ccacctacgt attaccgcgg ctgctggcac 960

gtagttagcc gtggctttct ggttaggtac cgtcaaggtg ccgccctatt tgaacggcac 1020

ttgttcttcc ctaacaacag agctttacga tccgaaaacc ttcatcactc acgcggcgtt 1080

gctccgtcag actttcgtcc attgcggaag attccctact gctgcctccc gtaggagtct 1140

gggccgtgtc tcagtcccag tgtggccgat caccctctca ggtcggctac gcatcgtcgc 1200

cttggtgagc cgttacctca ccaactagct aatgcgccgc gggtccatct gtaagtggta 1260

gccgaagcca ccttttatgt ctgaaccatg cggttcaaac aaccatccgg tattagcccc 1320

ggtttcccgg agttatccca gtcttacagg caggttaccc acgtgttact cacccgtccg 1380

ccgctaacat cagggagcaa gctcccatct gtccgctcga cttgcatgta ttaggcacgc 1440

cgccagcgtt cgtcctgagc cagatcaaaa ctct 1474

Claims

1. A paper mulberry leaf endophytic bacterium with lignocellulose degradation function is named as BP-L-1, classified and named as Bacillus amyloliquefaciens (Bacillus amyloliquefaciens), is preserved in China center for type culture Collection in 2021 in 4 months and 19 days, has a preservation number of CCTCC NO: M2021400, and has a preservation address of: eight-path Lojia mountain in Wuchang region of Wuhan city, Hubei province.

2. A broussonetia papyrifera leaf endophytic bacterium having a lignocellulose degrading function according to claim 1, wherein: the morphological characteristics of the strain BP-L-1 are as follows: the bacterial colony is light yellow and opaque, has rough surface, bulges and irregular edge.

3. The broussonetia papyrifera endophytic bacteria with lignocellulose degrading function according to claim 1, wherein: the physiological and biochemical characteristics of the strain BP-L-1 are as follows: the results of the starch hydrolysis experiment are positive, the results of the gelatin liquefaction experiment are negative, the results of the methyl red experiment are negative, and the results of the nitrate reduction experiment are positive.

4. Use of the broussonetia papyrifera leaf endophytic bacterium with a lignocellulose degrading function according to any one of claims 1 to 3 in preparation of cellulase and/or lignin degrading enzyme.

5. Use of the paper mulberry leaf endophytic bacterium having a lignocellulose degradation function according to any one of claims 1 to 3 in degrading lignocellulose.

6. Use according to claim 5, characterized in that: the lignocellulose is lignocellulose in a paper mulberry.

7. Use of the paper mulberry leaf endophytic bacterium with lignocellulose degradation function in any one of claims 1-3 for preparing silage.

8. Use according to claim 7, characterized in that: the silage is prepared from paper mulberry as a raw material.