CN115851501A

CN115851501A - Bacillus, microbial preparation, culture method and application

Info

Publication number: CN115851501A
Application number: CN202211186310.8A
Authority: CN
Inventors: 赵海珍; 王安; 陆兆新; 吕凤霞; 别小妹; 孟凡强
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2022-09-27
Filing date: 2022-09-27
Publication date: 2023-03-28

Abstract

The invention discloses bacillus, a microbial preparation, a culture method and application thereof, wherein the bacillus is classified and named as LM1, the preservation number is CGMCC NO.24989, and the bacillus is preserved in China general microbiological culture Collection center in 2022, 5 months and 30 days. The bacillus of the invention has lignocellulose degrading capability, has strong capability of producing cellulase and ligninase, and can degrade lignocellulose in cereal dietary fiber through fermentation, thereby improving the content of soluble dietary fiber in the cereal dietary fiber. Therefore, the bacillus with the capability of degrading lignocellulose can be used for preparing cellulase or ligninase, fermenting grains to degrade the lignocellulose in the grains, improving the content of soluble dietary fibers and further improving the nutritional quality of the grains.

Description

Bacillus, microbial preparation, culture method and application

Technical Field

The invention relates to the technical field of microorganisms, and in particular relates to bacillus, a microbial preparation, a culture method and application.

Background

Lignocellulose consists of lignin, cellulose and hemicellulose, is an important substance for maintaining plant morphology and protecting plant tissues, and is widely present in various plant cell walls in the plant kingdom. The cellulose long chain is twisted into external hydrophobic microfibril, then lignin is combined with the destructured hemicellulose through electrostatic interaction, and the hemicellulose bridges the hydrophobic region of the cellulose microfibril to form a complex lignocellulose complex. The complex formed by the three components is difficult to degrade on one hand, so that a part of useful substances is difficult to utilize; on the other hand, some substances indirectly cause environmental pollution, such as straw (Liulin peak, he Jian, zhang Gejie, wang Mi, majin jade, screening and capability research on large fungi degraded by lignocellulose in garden waste [ J ] plateau agriculture, 2021,5 (03): 249-255).

At present, the research on lignocellulose degrading bacteria focuses on environmental management and industrial product preparation, and few researches aim at grain fermentation. The dietary fibers in cereals are classified according to water solubility into Insoluble Dietary Fibers (IDF) and water-Soluble Dietary Fibers (SDF). According to the current report, the water-soluble dietary fiber has better functional properties, such as reducing blood pressure, reducing cholesterol, regulating intestinal flora and the like. Therefore, the strain is used for fermenting the grains to degrade lignin and cellulose in the dietary fiber of the grains and increase the content of soluble dietary fiber so as to improve the functional activity of the grains (Wangbao. Highland barley dietary fiber modification and application research thereof [ D ] Anhui engineering university, 2019.).

At present, the research on lignocellulose degrading bacteria is very extensive, and more research focuses on fungi, trichoderma asperellum with high enzyme activity, which is screened from a vinasse-enriched pine needle humus sample by people such as the good lignocelluloses, the enzyme activity of carboxymethyl cellulose (CMC) reaches 9.17U/mL when fermented for 5 days, and the enzyme activity of filter paper reaches 3.50U/mL (the good lignocelluloses, the bankia, the Jinganwei, the Yangyou, the Liapium, the separation screening and the fermentation condition optimization of the cellulose degrading bacteria of white vinasse [ J ]. Chinese brewing, 2021, 40 (05): 119-123.). A strain of cladosporium is screened from the soil environment of the mountain foot mountain of the sediment. After the strain is fermented for 8 days, the enzyme activity of laccase reaches 241U/L, the enzyme activity of cellulase reaches 51U/mL, and the enzyme activity of endoglucanase reaches 2.9U/mL (research on lignocellulose degradation characteristics of Agrimonia Cladosporium sp. Bio-1 [ D ] Hunan university, 2012.). The fungi have long culture time and long fermentation period, and have certain defects. The bacillus is a gram-positive bacterium, and the bacillus has strong stress resistance and is easy to culture. Therefore, bacillus has been increasingly studied for the degradation of lignocellulose. The method is characterized in that a beleis bacillus for producing cellulase is screened from eucommia bark by an Chenlong, and before optimization, the carboxymethyl cellulase activity and the filter paper enzyme activity respectively reach 4.78U/mL and 0.73U/mL (screening and characteristic analysis of Chenlong, high-yield cellulase bacillus 157 and research on the solid fermentation and degradation functions of corn straw-soybean meal mixture [ D ] Jilin agriculture university, 2018.). Wushusha and the like screen a bacillus subtilis from a high-temperature compost sample through hydrolysis ring, straw disintegration and cellulase activity test tests, wherein the beta-glucosidase activity of the bacillus subtilis is 19.42U/mL, the CMC enzyme activity is 32.89U/mL, and the filter paper enzyme activity (FPA) is 27.38U/mL (Wushusha, lirexing, huangasia, horse jun, korea, zhang Van. High-temperature lignocellulose degrading bacteria screening and identification and composting application thereof [ J ]. Anhui agricultural science, 2021, 49 (20): 68-71 ]). Numerous studies have shown that a number of safety bacilli can be used in grain fermentation to improve the nutritional quality and functional properties of grains. Research on fermentation of red beans by using Bacillus amyloliquefaciens by Wushan et al shows that the fermentation can significantly improve the antioxidant activity and the alpha-glucosidase inhibitory activity and anticoagulant activity of the red beans (Wushan, lushuai, liujun, yanshaqing, yanqianjian and Jiangzhengqiang. Zhao etiquette uses natto bacillus fermented wheat bran to prepare dietary fiber, and finds that the water retention property, the oil retention property and various functional activities of the dietary fiber are obviously improved (Zhao etiquette. Research on preparation of the dietary fiber by natto bacillus solid-state fermented wheat bran [ D ]. Nanjing agriculture university, 2020.).

Disclosure of Invention

The invention aims to solve the defects in the prior art and provides a bacillus with a lignocellulose degradation function and a safe strain for high-yield lignocellulose. The composite material has strong capability of producing cellulase and ligninase, is safe and reliable, and can effectively degrade lignocellulose in the dietary fiber of grains, thereby improving the content of soluble dietary fiber.

In order to achieve the purpose, the invention adopts the following technical scheme:

a bacillus, the said bacillus strain is LM1, the preservation number is CGMCC NO.24989, it is preserved in China general microbiological culture Collection center in 5 months and 30 days in 2022 years, no. 3 of No. 1 of West Lu of Beijing Kogyo Beiyang district, china academy of sciences microbiology institute. Through identification, the strain is named as Bacillus velezensis according to classification.

Preferably, the Bacillus LM1 is Bacillus velezensis (Bacillus velezensis), and the nucleotide sequence of the Bacillus LM1 is shown in a sequence table SEQ ID No:1 is shown. The homology of the Bacillus LM1 and the Bacillus subtilis (Bacillus velezensis) reaches 99.93 percent. The morphological characteristics of the bacillus strain LM1 are as follows: the back of the colony is light yellow, the surface is rough, the colony has a bulge, the edge is irregular, and the colony is in a shape of a dew bead. The thallus is in the shape of a short slender rod.

The physiological and biochemical characteristics of the bacillus strain LM1 are as follows: gram stain test is positive, catalase test is positive, gelatin liquefaction test is positive, methyl red test is negative, oxidase test is positive, and starch hydrolysis test is positive.

The invention also discloses a microbial preparation which comprises the bacillus or a fermentation culture thereof.

Preferably, the fermentation culture is a fermentation broth obtained by culturing bacillus in a culture medium or a fermentation broth from which the bacteria are removed.

In any of the above embodiments, preferably, the culture medium is any one of R2A medium, LB medium, and TGY medium.

The invention also discloses a screening method of the bacillus, which comprises the following steps:

step (1), strain enrichment and preliminary screening: weighing a soil sample, pouring the soil sample into a conical flask filled with sterile water, oscillating, sucking a supernatant, and adding the supernatant into a lignin enrichment medium and a cellulose enrichment medium; carrying out gradient dilution on the enrichment culture solution, coating the dilution solution in an LB solid culture medium added with nystatin, putting the LB solid culture medium into a constant-temperature incubator for culture, and screening single colonies with different forms visible to naked eyes; selecting single colonies with different forms, streaking on aniline blue solid plates and sodium carboxymethyl cellulose plates, selecting strains with obvious effects, inoculating the strains into a filter paper culture medium, and observing the disintegration condition of the filter paper;

step (2), re-screening of strains: respectively streaking the preliminarily screened strains, selecting a single colony, inoculating the single colony to a liquid LB culture medium, culturing to obtain a seed solution, absorbing the seed solution, inoculating the seed solution to a ligninase production culture medium and a cellulase production culture medium, culturing, and centrifuging the culture solution to obtain a supernatant.

In any of the above embodiments, preferably, in the step (1), the lignin-rich culture is performed at 30 ℃ and 200rpm for 3d, and the cellulose-rich culture is performed at 30 ℃ and 200rpm until the filter paper strips are completely dissolved.

In any of the above schemes, preferably, the cellulose-rich medium in step (1) comprises 1-3g/L of cellulose, 1-3g/L of peptone, 3-6g/L of sodium chloride, 0-0.5g/L of calcium chloride, 0.5-1g/L of potassium dihydrogen phosphate, 1-3g/L of ammonium sulfate, 0.5-1g/L of magnesium sulfate, and 1L of water, wherein 3-5 filter paper strips are arranged in the cellulose-rich medium.

In any of the above schemes, preferably, the lignin-rich medium in step (1) comprises 16-20g/L of alkaline lignin, 1-3g/L of ammonium sulfate, 0.5-1g/L of magnesium sulfate, 0.5-1g/L of potassium dihydrogen phosphate, 3-6g/L of sodium chloride, 1-3g/L of peptone and 1L of water.

In any of the above schemes, preferably, the LB solid medium in the step (1) comprises 5g/L yeast extract, 10g/L tryptone, 10g/L sodium chloride, 15g/L agar and 1L water.

In any of the above schemes, preferably, the sodium carboxymethyl cellulose solid culture medium in the step (1) comprises 1-3g/L of sodium carboxymethyl cellulose, 1-3g/L of ammonium sulfate, 0.5-2g/L of dipotassium hydrogen phosphate, 0.5-1g/L of magnesium sulfate, 0.5-2g/L of sodium chloride and 1L of water.

In any of the above schemes, preferably, the aniline blue solid culture medium in the step (1) comprises 5g/L of yeast extract, 10g/L of tryptone, 10g/L of sodium chloride, 15g/L of agar, 0.1-0.5g/L of aniline blue and 1L of water.

In any of the above schemes, preferably, the ligninase production medium in the step (2) comprises 10-20g/L of lignin, 0.5-1g/L of magnesium sulfate, 1-3g/L of potassium dihydrogen phosphate, 2-6g/L of ammonium sulfate, 10-20g/L of peptone, 3-5g/L of yeast extract powder, 0.5-1g/L of sodium chloride and 1L of water.

In any of the above schemes, preferably, the cellulase production medium in step (2) comprises 10-20g/L sodium carboxymethylcellulose, 0.5-1g/L magnesium sulfate, 1-3g/L potassium dihydrogen phosphate, 2-6g/L ammonium sulfate, 10-20g/L peptone, 3-5g/L yeast extract powder and 1L water.

In any of the above schemes, preferably, the liquid LB medium in step (2) comprises yeast extract 5g/L, tryptone 10g/L, sodium chloride 10g/L, and water 1L.

The invention also discloses a method for fermenting highland barley in the liquid state by the bacillus strain, which comprises the steps of marking the strain, selecting a single colony, inoculating the single colony in a liquid LB culture medium, and culturing; sterilizing the highland barley culture medium prepared according to different material-water ratios, and then inoculating the seed liquid into the highland barley culture medium for fermentation for different days.

The invention also discloses the identification method of the bacillus, which comprises the steps of sucking 3mL of bacterial liquid cultured by the culture method, extracting the genome DNA of the strain LM1, carrying out genome PCR amplification on 16S rDNA, and carrying out agarose gel electrophoresis on PCR amplification products; sequencing the 16S rDNA gene of the PCR amplification product; the sequencing result of the strain LM1 is subjected to Blast comparison in a NCBI database, and the result shows that the homology of the strain LM1 and Bacillus velezensis reaches 99.93 percent.

The invention also discloses applications of the bacillus in degradation of lignocellulose, preparation of cellulase and ligninase and application of fermented grains in improvement of soluble dietary fiber content.

The invention has the following beneficial effects:

(1) The bacillus of the invention has lignocellulose degradation capability, has strong capability of producing cellulase and ligninase, and can degrade lignocellulose in the grain dietary fiber through fermentation, thereby improving the content of soluble dietary fiber in the grain dietary fiber. Therefore, the bacillus with the capability of degrading lignocellulose can be used for preparing cellulase or ligninase, can be used for fermenting grains to degrade the lignocellulose in the grains, improves the content of soluble dietary fiber and further improves the nutritional quality of the grains.

(2) According to the Bacillus belgii (LM 1) obtained by the invention, the enzyme activity determination result shows that the activity of endo-cellulase is 37.16U/mL, the activity of filter paper enzyme is 22.16U/mL, the activity of laccase is 31.68U/L, the activity of lignin peroxidase is 290.8U/L, and the activity of manganese peroxidase is 27.78U/L (in the invention, the activity determination of endo-cellulase and the activity determination of filter paper enzyme adopt a DNS method, the activity determination of laccase adopts an ABTS method, the activity determination of lignin peroxidase adopts a veratryl alcohol method, and the activity determination of manganese peroxidase adopts a manganese peroxidase methodMnSO ₄ Method). The invention achieves certain enzyme activity under the screening condition, and can effectively degrade lignocellulose in the highland barley powder.

Drawings

FIG. 1 shows the fading of solid aniline blue plates of different strains;

FIG. 2 is a liquid aniline blue fading blank set;

FIG. 3 shows the fading of liquid aniline blue of strain LM 1;

FIG. 4 shows the transparent circle of strain LM 1;

FIG. 5 is a diagram of the enzyme activity of candidate strains;

FIG. 6 is an electrophoresis diagram of 16 SrDNAPR products of Bacillus strain LM 1;

FIG. 7 is a phylogenetic tree of Bacillus strain LM1 based on the 16SrDNA sequence;

FIG. 8 is a diagram showing the colony and thallus morphology of Bacillus strain LM 1;

FIG. 9 is a chart showing the results of hemolysis experiments of Bacillus strain LM1 and control group.

Detailed Description

The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. The various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art. The materials used in the tests and the test methods are described generally or specifically. 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 unless otherwise specified, and are conventional experimental methods, detection methods and the like in the prior art unless otherwise specified.

Example 1

Screening of strains

1. Bacterial strain enrichment and preliminary screening

Weighing 10g of soil sample (collected 5-10cm below phoenix tree of Nanjing agriculture university) and pouring into a conical flask containing 90mL of sterile water,after 30 ℃ and 30min shaking at 200rpm, 5mL of the supernatant was aspirated and added to the lignin and cellulose rich medium. Culturing lignin at 30 deg.C and 200rpm for 3d, and culturing cellulose at 30 deg.C and 200rpm until the filter paper strips are completely dissolved. The enrichment culture solution is diluted to 10 degrees in a gradient way ^-6 100 mu L of the diluent is taken and coated in LB solid culture medium added with nystatin, and the mixture is put into a constant temperature incubator at 30 ℃ for 24h. In total, 20 single colonies with different morphologies visible to the naked eye were screened.

Selecting single bacterial colonies with different forms, streaking on aniline blue solid plate, selecting 6 strains with obvious fading effect, inoculating to aniline blue liquid culture medium at 30 deg.C and 200rpm, culturing for 3d, and culturing at OD ₅₉₀ And (5) measuring the absorbance change value, and quantitatively detecting the fading rate. The fading of aniline blue by different strains is shown in table 1 and fig. 1-3. Researches show that the 6 strains have lignin degradation capability, wherein the fading rate of liquid aniline blue of LM1 and LM5 reaches over 90 percent, which shows that the strains have good lignin degradation capability.

TABLE 1 fading rates of liquid aniline blue for different strains

Simultaneously, a single bacterial colony is spotted on a sodium carboxymethylcellulose flat plate, after the single bacterial colony is cultured for 3d at 30 ℃, congo red dyeing is carried out, 1mol/L sodium chloride solution is decolorized, the size of a transparent ring is observed, the diameter of the bacterial colony and the diameter of the transparent ring are measured, a bacterial strain with obvious effect is selected and inoculated in a filter paper culture medium, and the filter paper disintegration effect is observed. The congo red transparent circle sizes of the different strains are shown in table 2. Research shows that 6 strains of bacteria all generate obvious transparent rings, and the ratio of the LM1 transparent rings to the diameter of bacterial colonies is higher (5.66), which indicates that the cellulose degradation capability is better. The results of the strain LMI clearing circle are shown in FIG. 4. The filter paper disintegration effect of different strains is shown in table 3, and all strains have the capability of disintegrating the filter paper, wherein the disintegration effect of LM1 is the best, and further shows that the cellulose degradation capability of LM1 is the strongest.

TABLE 2 Congo red transparent circle size of different strains

TABLE 3 disintegration of filter paper for different strains

Note: "+" indicates the degree of filter damage, and more "+" indicates greater damage.

2. Bacterial strain rescreening

Respectively scribing and selecting single colonies of the preliminarily screened strains, inoculating the single colonies into a liquid LB culture medium, culturing for 24 hours at 30 ℃ and 200rpm to obtain seed liquid, absorbing 2mL of the seed liquid, inoculating the seed liquid into a ligninase production culture medium and a cellulase production culture medium, culturing for 3 days at 30 ℃ and 200rpm, respectively taking 2mL of the culture liquid at 8000r/min, centrifuging for 10 minutes to obtain supernatant, and taking the supernatant to measure the enzymatic activity of laccase, lignin peroxidase, manganese peroxidase, endo-cellulase and filter paper (the result is shown in figure 5). Researches show that the degradation capacities of lignin and cellulose are integrated, the activities of various corresponding enzymes produced by the strain LM1 are higher, and the activities of laccase, lignin peroxidase, manganese peroxidase, endo-cellulase and filter paper enzyme respectively reach 31.68U/L,290.8U/L,27.78U/L,37.16U/mL and 22.16U/mL.

The LM1 strain with better effect is obtained by primary screening and secondary screening. Therefore, LM1 is selected as a lignocellulose-degrading high-efficiency strain.

3. Culture medium used in experimental process

LB solid medium: 5g/L of yeast extract, 10g/L of tryptone, 10g/L of sodium chloride, 15g/L of agar and 1L of water.

LB liquid medium: 5g/L of yeast extract, 10g/L of tryptone, 10g/L of sodium chloride and 1L of water.

Cellulose enrichment medium: 1-3g/L of cellulose, 1-3g/L of peptone, 3-6g/L of sodium chloride, 0-0.5g/L of calcium chloride, 0.5-1g/L of monopotassium phosphate, 1-3g/L of ammonium sulfate, 0.5-1g/L of magnesium sulfate, 1L of water and 3-5 filter paper strips.

Lignin enrichment medium: 16-20g/L of alkaline lignin, 1-3g/L of ammonium sulfate, 0.5-1g/L of magnesium sulfate, 0.5-1g/L of monopotassium phosphate, 3-6g/L of sodium chloride, 1-3g/L of peptone and 1L of water.

Sodium carboxymethyl cellulose solid medium: 1-3g/L of sodium carboxymethylcellulose, 1-3g/L of ammonium sulfate, 0.5-2g/L of dipotassium hydrogen phosphate, 0.5-1g/L of magnesium sulfate, 0.5-2g/L of sodium chloride and 1L of water.

Aniline blue solid medium: 5g/L of yeast extract, 10g/L of tryptone, 10g/L of sodium chloride, 15g/L of agar, 0.1-0.5g/L of aniline blue and 1L of water.

Aniline blue liquid medium: 5g/L of yeast extract, 10g/L of tryptone, 10g/L of sodium chloride, 0.1-0.5g/L of aniline blue and 1L of water.

Filter paper disintegration medium: 20-30 filter paper strips, 0.1-0.5g/L magnesium sulfate, 3-6g/L ammonium sulfate, 0.5-2g/L dipotassium hydrogen phosphate, 3-6g/L sodium chloride, 10-20g/L peptone, 10-20g/L yeast extract powder and 1L water.

A cellulase production culture medium: 10-20g/L sodium carboxymethylcellulose, 0.5-1g/L magnesium sulfate, 1-3g/L potassium dihydrogen phosphate, 2-6g/L ammonium sulfate, 10-20g/L peptone, 3-5g/L yeast extract powder and 1L water.

Ligninase-producing culture medium: 10-20g/L of lignin, 0.5-1g/L of magnesium sulfate, 1-3g/L of monopotassium phosphate, 2-6g/L of ammonium sulfate, 10-20g/L of peptone, 3-5g/L of yeast extract powder, 0.5-1g/L of sodium chloride and 1L of water.

4. Method for measuring various enzyme activities

(1) And (3) enzyme activity determination of laccase (Lac): total reaction 3.15mL: after mixing 100mmol/L malonic acid-sodium malonate buffer (pH = 4.5) and 0.6mmol/LABTS in equal amounts, 3mL of the mixture was taken, 150. Mu.L of the supernatant was added at 30 ℃ to start the reaction, and the change in absorbance at 420nm for 1min was measured. The amount of enzyme catalyzing 1. Mu. Mol of a substrate within 1min is defined as one enzyme activity unit (U).

(2) And (3) measuring the activity of lignin peroxidase (LiP): total reaction 3mL:200mmol/L sodium tartrate buffer (pH = 3.0) 1.5mL,40mmol/L veratrum300 mu L of alcoholic solution, 150 mu L of supernatant and 1.02mL of deionized water. Adding 20mmol/LH at 30 DEG C ₂ O ₂ The reaction was started at 30. Mu.L, and the change in absorbance at 310nm for 1min was measured. The amount of enzyme that oxidizes 1. Mu. Mol of resveratrol within 1min is defined as one enzyme activity unit (U).

(3) Manganese peroxidase (Mnp) enzyme activity determination: total reaction 3mL:100mmol/L malonic acid-sodium malonate buffer (pH = 4.5) 1.5mL,10mmol/LMnSO ₄ Solution 300. Mu.L, supernatant 150. Mu.L, deionized water 1.02mL. Adding 10mmol/LH at 30 DEG C ₂ O ₂ The reaction was started at 30. Mu.L, and the change in absorbance at 270nm for 1min was measured. The amount of enzyme that oxidizes 1. Mu. Mol MnSO4 within 1min is defined as one enzyme activity unit (U).

(4) And (3) measuring the enzyme activity of carboxymethyl cellulase (CMCase): the measurement was performed by the DNS method. Adding 100 μ L of 1% CMC-Na citric acid buffer solution and 50 μ L of crude enzyme solution into a test tube, mixing the two solutions, carrying out water bath at 50 ℃ for 30min, adding 200 μ L of the solution, boiling in boiling water bath for 5min, cooling with ice water, adding 1mL of distilled water, mixing, adding 200 μ L of the solution into an ELISA plate, and measuring the absorbance at 540 nm. Hydrolysis of sodium carboxymethylcellulose to produce 1. Mu.g of reducing sugars within 1min is defined as 1 enzyme activity unit (U/mL).

(5) Filter paper enzyme activity (FPA) assay: the measurement was performed by the DNS method. Adding 1 × 3cm filter paper strip, 200 μ L citric acid buffer solution and 100 μ L crude enzyme solution into a test tube, mixing the three solutions, water-bathing at 50 deg.C for 30min, adding 400 μ L DNS solution, boiling in boiling water bath for 5min, cooling with ice water, adding 2mL distilled water, mixing, adding 200 μ L into an ELISA plate, and measuring absorbance at 540 nm. Hydrolysis of the filter paper to yield 1. Mu.g of reducing sugars within 1min was defined as 1 enzyme activity unit (U/mL).

5. Identification of strains

The LM1 strain obtained by primary screening and secondary screening is subjected to microscopic examination, colony appearance observation and 16SrDNA gene sequence identification. An electrophoresis chart of a 16SrDNAPCR product of the strain LM1 is shown in figure 6, which shows that the length of the strain LM1 is about 1500bp approximately, and a phylogenetic tree of the strain LM1 based on a 16SrDNA sequence is shown in figure 7, and the affinity of the strain LM1 and the Bacillus belgii is found to be extremely high and reaches 99.93%.

The strain is picked, a single colony is streaked, the morphology is observed, the back of the colony is light yellow, the surface is rough, the colony has a bump, the edge is irregular, and the colony is in a shape of a dew bead. After culturing at 30 ℃ and 200rpm for 20 hours, gram staining was performed, and morphological characteristics of the cells were observed under an oil lens, and the LM1 strain was gram-positive because the cells were short and thin rod-shaped and purple in color, as shown in FIG. 8.

The physiological and biochemical characteristic experiments of the strain LM1 are carried out according to Bergey's Manual of systematic identification, which comprises the following experiments: according to the results in table 4, the catalase test, the gelatin liquefaction test, the oxidase test and the starch hydrolysis test of the strain LM1 were all positive, and the methyl red test was negative.

TABLE 4 physiological and biochemical characteristics of the strain LM1

Note: "+" indicates positive reaction, and "-" indicates negative reaction.

6. Safety test of strains

(1) Antibiotic sensitivity experiments: 100 mu L of bacterial liquid is sucked and evenly coated on an LB solid culture medium, 8 drugs (tetracycline, penicillin, erythromycin, chloramphenicol, gentamicin, vancomycin, norfloxacin and cefoxitin) are selected according to clinical common antibiotics, drug sensitive paper sheets of the drugs are closely attached to the culture medium, the culture medium is placed in a constant temperature incubator at 37 ℃ for 24 hours to record the diameter of an inhibition zone, and the drug sensitivity judgment is carried out according to the antibacterial drug sensitivity test standard, and the results are shown in Table 5. The strain LM1 is sensitive to 8 antibiotics shown in Table 5 and is a safe strain.

TABLE 5 Strain LM1 drug sensitivity test results

(2) Hemolysis test: the single colony of the selected strain was streaked on a blood plate and cultured in a 37 ℃ incubator for 24 hours to see whether a lysoloop was produced, and the results are shown in FIG. 9. The strain LM1 produced no hemolytic rings on the blood plates, indicating that the strain is safe.

Example 2 fermentation of highland barley with Strain

1. Content change of soluble dietary fiber and insoluble dietary fiber after highland barley fermentation by strain LM1

(1) Liquid fermentation of the strain highland barley: the strain is streaked, and a single colony is selected and inoculated in a liquid LB culture medium for 30 ℃, and is cultured to logarithmic phase under the condition of 200rpm, so that the seed liquid is obtained. Inoculating the seed liquid into sterilized highland barley culture medium with different material-liquid ratios according to the inoculation amount of 3% for fermentation for 2-5d.

(2) Extracting dietary fiber and measuring the content: the fermented highland barley culture medium is firstly put into boiling water bath for 10min to inactivate enzyme, and then is cooled and centrifuged at 8000rpm and 10min to respectively obtain supernatant and precipitate.

SDF extraction and content determination: the supernatant was transferred to a beaker, added with 4 volumes of 95% ethanol, and precipitated with ethanol overnight. Centrifuging the obtained liquid at 8000rpm for 10min, washing the obtained precipitate for three times by using 95% ethanol, drying to obtain the soluble dietary fiber, and weighing.

(3) Taking 10g of raw material highland barley whole grain, slightly modifying an extraction method based on GB.5009, adding water according to a material-liquid ratio (g/mL) of 1: 10, stirring, adding 250 mu L of high-temperature alpha-amylase (2 ten thousand U/mL) in a water bath kettle at 95 ℃ for oscillation for 35min, adding 4mg of alkaline protease (200U/mg) in the water bath kettle at 60 ℃ for oscillation for 30min, adjusting the pH value of a reaction system to 4.5 +/-0.02 after the reaction is finished, and adding 20 mu L of saccharifying enzyme (10 ten thousand U/mL) in the reaction system in the water bath kettle at 60 ℃ for oscillation for 30min. After cooling to room temperature, the mixture was centrifuged.

SDF extraction and content determination: adding 4 times volume of 95% ethanol into the supernatant, precipitating with ethanol overnight, centrifuging the obtained liquid with a centrifuge at 8000rpm for 10min, washing the obtained precipitate with 95% ethanol for three times, drying to obtain soluble dietary fiber, weighing, and measuring the results shown in Table 6. The content of the fermented highland barley SDF is almost higher than that of the unfermented highland barley under different feed-water ratios and fermentation time, and reaches the highest value (7.76 g/100 g) when the fermentation time is 3d at the feed-water ratio of 1: 10, which is 1.44 times of the content of the unfermented highland barley SDF.

TABLE 6 SDF content before and after fermentation of highland barley (g/100 g)

The strain LM1 is separated from soil, identified as Bacillus velezensis (Bacillus velezensis) through morphology, physiology, biochemistry and molecular biology, and can perform microbial fermentation under a facultative anaerobic condition. The cellulase and the ligninase can be produced by culturing at the temperature of 30 ℃, and the enzyme production activity is higher. But also can be used for grain fermentation, degrading lignocellulose in dietary fiber and improving the content of soluble dietary fiber. The strain resource is provided for the subsequent grain fermentation, and a foundation is provided for the research and development of a subsequent fermentation microbial agent.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical scope of the present invention and the equivalent alternatives or modifications according to the technical solution and the inventive concept of the present invention within the technical scope of the present invention.

Claims

1. A Bacillus, comprising: the bacillus is classified and named as LM1, the preservation number is CGMCC NO.24989, and the bacillus is preserved in China general microbiological culture Collection center in 2022, 5 months and 30 days.

2. The bacillus of claim 1, wherein: the Bacillus LM1 is Bacillus velezensis (Bacillus velezensis), and the nucleotide sequence of the Bacillus LM1 is shown in a sequence table SEQ ID No:1, the homology of the Bacillus LM1 and the Bacillus velezensis (Bacillus velezensis) reaches 99.93 percent.

3. A microbial preparation comprising a culture medium and the Bacillus of claim 1 or 2 or a fermentation culture thereof.

4. The microbial preparation of claim 3, wherein the fermentation culture is a fermentation broth obtained by culturing the Bacillus sp in a medium or a thallus-removed fermentation broth.

5. The microbial preparation of claim 4, wherein the medium is any one of R2A medium, LB medium and TGY medium.

6. The method for culturing Bacillus according to claim 1 or 2, comprising the steps of:

step (2), re-screening of strains: respectively marking and selecting single colonies of the preliminarily screened strains, inoculating the single colonies into a liquid LB culture medium, culturing to obtain a seed solution, absorbing the seed solution, inoculating the seed solution into a ligninase production culture medium and a cellulase production culture medium, culturing, and centrifuging the culture solution to obtain a supernatant.

7. The method for culturing Bacillus according to claim 6, wherein the LB solid medium in step (1) comprises yeast extract 5g/L, tryptone 10g/L, naCl 10g/L, agar 15g/L, and water 1L.

8. The method for identifying Bacillus according to claim 1 or 2,

sucking the bacterial liquid cultured by the culture method of claim 6, extracting the genome DNA of the strain LM1, performing genome PCR amplification on 16S rDNA, and performing agarose gel electrophoresis on PCR amplification products; sequencing the 16S rDNA gene of the PCR amplification product; the sequencing result of the strain LM1 is subjected to Blast alignment in a database of NCBI.

9. The method for liquid fermentation of highland barley by bacillus strain as claimed in claim 1 or 2, wherein the strain is streaked and a single colony is selected and inoculated in the liquid LB medium for culture; the single colony is selected by streaking the strain and inoculated in a liquid LB culture medium for culture to logarithmic phase to obtain a seed solution; inoculating the seed liquid into a highland barley culture medium of a sterilization liquid, fermenting in a shaking table, and extracting the soluble dietary fiber after fermenting for a certain time.

10. Use of the bacillus according to claim 1 or 2 for degradation of lignocellulose, for the preparation of cellulases and ligninases, for fermenting cereals for increasing the content of soluble dietary fibers.