CN114381403B

CN114381403B - Bacillus bailii LOH112 and application thereof

Info

Publication number: CN114381403B
Application number: CN202210069234.6A
Authority: CN
Inventors: 葛振煌; 陆勇军; 陆正齐; 邝芷琪; 阮恒芳
Original assignee: Sun Yat Sen University
Current assignee: Sun Yat Sen University
Priority date: 2022-01-21
Filing date: 2022-01-21
Publication date: 2023-06-30
Anticipated expiration: 2042-01-21
Also published as: CN114381403A

Abstract

The invention belongs to the technical field of microorganism application, and particularly relates to bacillus bailii LOH112 and application thereof, and the invention discloses bacillus bailii LOH112 with a preservation number of CCTCC No. M20211623. The bacillus belicus has various functional characteristics: (1) Has inhibiting activity on fungi such as candida albicans and aspergillus niger; (2) Has inhibitory activity against gram-positive bacteria such as Staphylococcus aureus and Micrococcus luteus; (3) protease-producible; (4) cellulase production; (5) a strongly adherent biofilm can be formed; (6) feather degradation can be achieved. The functional characteristics enable the bacillus belicus to have various application forms, such as preventing and treating plant diseases, preparing medicines for preventing and treating fungal or bacterial diseases, preparing plant colonizers, plant biocontrol agents, biological organic fertilizers, plant growth promoters, microbial feeds, degrading feathers and the like, and have wide application prospects.

Description

Bacillus bailii LOH112 and application thereof

Technical Field

The invention belongs to the technical field of microorganism application, and particularly relates to bacillus bailii LOH112 and application thereof.

Background

Bacillus beleiensis (Bacillus velezensis) has received attention for its broad inhibitory capacity against a variety of pathogenic bacteria and fungi. In recent years, researchers successively separate various B.velezensis strains from complex environments such as soil, water, air, plant rhizosphere, animal intestinal tracts and the like, and the characteristics of wide habitat in nature are shown; meanwhile, the characteristics of high growth speed, strong stress resistance, rich metabolites, environmental friendliness and the like also provide convenience for the research and application of the novel high-performance high-temperature-resistant composite material. The research shows that the B.velezensis strains of different types have different bacteriostasis spectrums and in-vitro bacteriostasis intensities, so that the research on the bacteriostasis capacity of the B.velezensis and the separation and identification of bacteriostasis substances has higher application value.

At present, the general physiological characteristics, genetic structure and metabolite types of B.velezensis have been studied deeply, and the application potential of B.velezensis in the fields of agriculture, fermentation industry, environmental protection and the like is developed gradually. Currently, the application of b.velezensis is mainly focused on biological control of plant diseases, for example, as a common plant growth promoting rhizosphere bacteria (PGPR), b.velezensis not only competes with plant pathogens for nutrition and growth environment, but also antagonizes the growth of pathogenic bacteria by secreting secondary metabolites with bacteriostatic effect, and also stimulates plant bodies to develop induction system resistance (induced systemic resistance, ISR) to pathogens, namely, stimulates plant cells through jasmonic acid/ethylene (JA/ET) or Salicylic Acid (SA) signal transduction pathways to resist invasion of pathogenic bacteria or fungi by strengthening cell walls and synthesizing accumulated antibacterial substances. Based on the characteristics and the characteristic of drying resistance of the B.velezensis spores, the B.velezensis is prepared into dry powder biopesticide which can be stably stored for a long time, and plays an important role in preventing and controlling common agricultural disease organisms such as Erwinia amylovora (Erwinia amylovora), lawsonia solanacearum (Ralstonia solanacearum), various bacteria, fungi, nematodes and the like which cause root rot.

Another important property of velezensis is the ability to synthesize a wide variety of degrading enzymes, which has broadened its application in industry and environment. For example, the strain B. VelezensisAB has the capability of synthesizing azo reductase, so that the strain B. VelezensisAB can be applied to biological purification of azo dye polluted wastewater; it has also been found that strain b.velezensis 157 isolated from eucommia bark is capable of synthesizing higher levels of lignocellulose degrading enzymes and thus can play an important role in fermentation of agricultural byproducts such as straw, rice hulls, and the like, and production of biofuels.

Over the past decade, concerns over overuse of antibiotics have been drawn to the use of probiotics in the animal feed industry, as probiotics can improve growth performance and reduce disease risk. Currently, several bacillus species have been used as microbial additives in animal feed. The probiotic supplement for animal feed not only improves the health and production of the animal, but also diversifies and stabilizes the gastrointestinal flora of the host animal. However, little has been done about the use of b.velezensis as a probiotic in animal feed. Studies have shown that b.velezensis can affect the intestinal microbiota of animals. For example, the B.velezensis V4 strain is an effective probiotic, which promotes growth performance and controls furunculosis in aquaculture. The intestinal microbiota of mice can be improved by increasing the number of beneficial microorganisms and decreasing the number of pathogens by administering strain B.velezensis BV-7. In addition, the B.velezensisY1 strain is used as probiotics in poultry feed, and can improve the growth performance of poultry and the egg quality. It can be seen that b.velezensis has the potential to be a probiotic in the animal feed industry in addition to being a bactericide.

At present, a plurality of B.velezensis strains have been isolated and identified in animals including humans and play a role in improving intestinal microecology and enhancing immunity of aquatic fish and livestock animals. However, compared with plant diseases, the current research on the ability of B.velezensis to inhibit human pathogens and effective antibacterial components is less, the physiological and biochemical mechanism of the interaction of B.velezensis with animal bodies has not been systematically elucidated, and the potential functions of the broad-spectrum antibacterial effect in the immunity and inflammation of human and animals to infectious diseases are still to be further studied.

Disclosure of Invention

To overcome the above-mentioned deficiencies of the prior art, a primary object of the present invention is to provide a Bacillus bailii LOH112.

It is a second object of the present invention to provide the use of the above Bacillus bailii LOH112.

The first object of the present invention is achieved by the following technical solutions:

bacillus belicus LOH112, wherein the Bacillus belicus LOH112 is delivered to China Center for Type Culture Collection (CCTCC) with a collection number of M20211623 at 12-13 of 2021.

The invention separates a strain LOH112 from the feces of a healthy ninety year old man in Guangdong province in China, and the 16S rDNA sequence is shown as SEQ ID NO:1, the homology with the 16S rDNA sequence of the known Bacillus beleiensis (Bacillus velezensis) was >99%, and it was confirmed that Bacillus beleiensis was the same species and different strain, and therefore, it was designated as Bacillus beleiensis (LOH 112). The strain is streaked on an LB plate for 24 hours to form white volcanic colonies with raised tops and wrinkled sides, and the strain has strong ability of producing stress-resistant dormancy of spores through spore dyeing; gram staining indicated that it was a gram positive (g+) bacterium.

The invention also provides a microbial inoculum containing the bacillus beijerinus LOH112.

Preferably, the culturing method of the bacillus belicus LOH112 comprises the following steps: and (3) streaking bacillus belicus LOH112 on an LB plate, culturing the activated strain overnight at 37 ℃, picking a single colony into an LB liquid culture medium, and culturing on a constant temperature shaking table at 37 ℃ for a period of time.

The second object of the present invention is achieved by the following technical solutions:

the application of bacillus bailii LOH112 or a microbial inoculum thereof in preventing and controlling plant fungal diseases caused by aspergillus niger.

Aspergillus niger is easy to grow and propagate in large quantity to generate enzyme heat, and flowers fall or rotting bell is caused when the Aspergillus niger infects cotton, or the Aspergillus niger is grown on succulent fruits to cause rotting or soft rot, and a large amount of black powder is generated when the Aspergillus niger infects the surfaces of onion scales; the plum rain season is also liable to cause mildew of clothes and the like. The research shows that the bacillus bailii LOH112 has a good inhibition effect on candida albicans, can be applied to preventing and controlling plant fungal diseases caused by aspergillus niger, and can be prepared into microbial fertilizers or biopesticides for preventing and controlling plant fungal diseases.

The application of bacillus bailii LOH112 or a microbial inoculum thereof in preparing medicaments for preventing and treating bacterial diseases, wherein the bacteria comprise staphylococcus aureus and micrococcus luteus.

Staphylococcus aureus is a common food-borne pathogenic microorganism that can cause a variety of diseases in humans and animals, including pseudomembranous enteritis, septicemia, sepsis, etc., and severely threatens the life safety of humans and animals. Micrococcus luteus is a conditional pathogen, and causes local tissue infection such as wound and serious infection such as endocarditis. The research shows that the bacillus bailii LOH112 has good inhibition effect on staphylococcus aureus and micrococcus luteus, and can be prepared into medicaments for treating bacterial diseases caused by staphylococcus aureus and micrococcus luteus.

The application of bacillus bailii LOH112 or a microbial inoculum thereof in preparing medicaments for preventing and treating fungal diseases, wherein the fungi are candida albicans.

Candida albicans can cause acute and chronic infections of skin, oral cavity, mucous membrane and viscera, namely candida mycosis. The research shows that the bacillus bailii LOH112 has better inhibition effect on candida albicans, and can be prepared into medicaments for treating fungal diseases caused by candida albicans.

The bacillus bailii LOH112 or the microbial inoculum thereof is applied to the preparation of plant colonizing agents, and/or plant biocontrol agents, and/or bio-organic fertilizers, and/or plant growth promoters, and/or microbial feeds.

The research shows that bacillus bailii LOH112 has the characteristic of producing protease and can be applied to the protease fields of food industry, washing industry and the like; meanwhile, the method can also be used for degrading pathogenic bacteria cell membranes in agricultural microbial fertilizers to control diseases and degrading proteins in agricultural organic fertilizers in compost to facilitate plant absorption; can also be used in microbial feed to help animals digest and absorb nutrition and improve the utilization rate of the feed by the animals. Meanwhile, bacillus bailii LOH112 also has the characteristic of producing cellulase, and can be applied to degrading the cell wall of pathogenic fungi for controlling diseases; can also be applied to the decomposition of cellulose in compost; can also be applied to feeds produced by livestock and poultry to solve the problem that monogastric animals such as pigs, chickens and the like can not utilize cellulose. In addition, bacillus bailii LOH112 also has the characteristic of forming a strong adhesive biofilm, which indicates that the bacillus bailii LOH112 has good planting capacity and can provide better rhizosphere planting effect when applied to plant antibiosis; the microbial feed can also be applied to animal feeds to facilitate the field planting of the feeds in intestinal tracts, so that the microbial feed can better exert the activities of cellulase and protease, and the digestion and utilization rate of the feeds by animals can be improved. The bacillus bailii LOH112 can be prepared into plant colonizers, plant biocontrol agents, biological organic fertilizers, plant growth promoters, microbial feeds and the like for use.

The bacillus bailii LOH112 or the bacterial agent thereof is applied to degrading feathers.

The research shows that bacillus bailii LOH112 can obviously degrade feathers, can be applied to manufacturing organic fertilizer in agriculture, and degrade pollution sources, thereby improving the environment; the method can also be applied to development of waste feathers in the feed industry, and solves the problem of insufficient protein resources in the current feed industry; and can be applied to the treatment of leather, cosmetics, medicine industry, food industry and other industries and environments.

The invention also provides a preparation method of the protease, which utilizes the protease production characteristic of bacillus bailii LOH112 to prepare the protease.

The invention also provides a preparation method of the cellulase, which utilizes the cellulase production characteristic of bacillus bailii LOH112 to prepare the cellulase.

It should be noted that the application of Bacillus bailii LOH112 is far beyond those mentioned above, and all the fields developed and utilized according to the functional characteristics of Bacillus bailii LOH112 are within the application scope of the present invention.

Compared with the prior art, the invention has the beneficial effects that:

the invention discloses bacillus beleiensis LOH112, wherein the bacillus beleiensis LOH112 is transmitted to China center for type culture collection (China center for type culture collection) for collection in the 12 th year of 2021, and the collection number is CCTCC No. M20211623. The bacillus belicus has various functional characteristics: (1) Has inhibiting activity on fungi such as candida albicans and aspergillus niger; (2) Has inhibitory activity against gram-positive bacteria such as Staphylococcus aureus and Micrococcus luteus; (3) protease-producible; (4) cellulase production; (5) a strongly adherent biofilm can be formed; (6) feather degradation can be achieved. The functional characteristics enable bacillus bailii LOH112 to have various application forms, such as preventing and treating plant diseases, preparing medicines for preventing and treating fungal or bacterial diseases, preparing plant colonizers, plant biocontrol agents, biological organic fertilizers, plant growth promoters, microbial feeds, degrading feathers and the like, and have wide application prospects.

Drawings

FIG. 1 is a morphological observation and identification of Bacillus bailii LOH112 (A is a culture curve, B is a culture plate, C is a gram stain, and D is a spore stain);

FIG. 2 is a phylogenetic tree of Bacillus bailii LOH112 and other bacilli;

FIG. 3 shows an experiment of the bacteriostasis of Bacillus bailii LOH112 on Candida albicans (left) and Aspergillus niger (right);

FIG. 4 shows an experiment of the bacteriostasis of Bacillus bailii LOH112 against Staphylococcus aureus (left) and Micrococcus luteus (right);

FIG. 5 shows the degradation of milk plates by Bacillus bailii LOH112 (left, LOH112 laboratory; right, blank);

FIG. 6 shows the degradation of cellulose plates by Bacillus bailii LOH112 (left, LOH112 laboratory; right, blank);

FIG. 7 shows the experiment of the degradation of feathers by Bacillus bailii LOH112 (left, control group of blank medium; right, experimental group with LOH112 added).

Detailed Description

The following describes the invention in more detail. The description of these embodiments is provided to assist understanding of the present invention, but is not intended to limit the present invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.

The experimental methods in the following examples, unless otherwise specified, are conventional, and the experimental materials used in the following examples, unless otherwise specified, are commercially available.

The strains referred to in the following examples include:

(1) The test strain as the object of the bacterial inhibition mechanism research is bacillus belicus (Bacillus velezensis, b. Velezensis) LOH112 strain, which is isolated from the feces of a healthy male volunteer in guangdong in China by the laboratory of the national institute of science and its intestinal microbiome research, and is frozen and preserved in a glycerin tube at-80 ℃. Typically, it is inoculated onto the surface of a plate of LB solid medium and cultured upside down in a thermostatic incubator at 37℃for 24 hours to obtain colonies, or shake-cultured in LB liquid medium in a thermostatic shaker at 37℃for 24-48 hours to obtain a fermentation broth.

(2) Staphylococcus aureus ATCC6538, candida albicans ATCC10231, aspergillus niger ATCC16404, and micrococcus luteus ATCC10240, were purchased from the collection of microorganisms, inc.

Table 1 strains used in experiments and culture conditions

(3) The culture medium involved is as follows:

1) LB plate: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, 20g of agar powder, distilled water to a volume of 1L, sodium hydroxide to adjust the pH to 7.2, and sterilizing at 121 ℃ for 20min under high pressure to prepare an LB plate.

2) LB liquid medium: 5g of yeast extract, 10g of tryptone, 10g of sodium chloride, distilled water to a volume of 1L, and sodium hydroxide to adjust the pH to 7.2, and sterilizing at 121 ℃ for 20 min.

3) YM plate: 5g of peptone, 10g of glucose, 3g of yeast extract, 3g of malt extract, 1L of distilled water and 20g of agar, wherein the pH is adjusted to 6.2 by citric acid, and the mixture is autoclaved at 121 ℃ for 20min and then prepared into YM plates.

4) YM liquid medium: 5g of peptone, 10g of glucose, 3g of yeast extract, 3g of malt extract and 1L of distilled water, and sterilizing at 121 ℃ for 20min under high pressure by adjusting the pH to 6.2 with citric acid.

5) PDA plate: PDA powder 40.1g, distilled water 1L,121 ℃ for 20min, after autoclave sterilization, to prepare PDA plate.

EXAMPLE 1 isolation and characterization of Bacillus bailii LOH112

Feces of a healthy male volunteer in Guangdong China was repeatedly washed 3 times with sterile water, placed in a mortar, added with an appropriate amount of sterile water, thoroughly ground into homogenate, sucked with a pipette into an appropriate amount of grinding liquid, coated on an LB plate, and cultured for 24 hours at room temperature. The colonies to be streaked and purified in the separation experiment plates were numbered with a marker and strain numbers were marked on the plates accordingly. Colonies were picked up and inoculated onto LB, and the strain was purified by plate streaking. If the strain cannot be separated by the method, colonies need to be picked from the enrichment plate, and the colonies are coated on a culture medium after being subjected to gradient dilution by an LB liquid culture medium. Then, referring to "Burjie's Manual of bacteria identification" (eighth edition), strains belonging to bacteria are first discriminated, and then the growth of colonies is observed: the growth form, whether hypha exists, whether the color is uniform and consistent, the surface and edge of a colony individual and the like are preliminarily screened to obtain a candidate strain LOH112. Finally, the obtained LOH112 strain was subjected to whole genome sequencing by Beijing Baimai Biotechnology Co.

As shown in FIGS. 1A-B, strain LOH112 was cultured in LB liquid medium for 20-24h to reach stationary phase; white volcanic colonies with raised tops and wrinkled sides were formed after streaking on LB plates for 24 hours.

The results of observation of the gram-stained and spore-stained LOH112 bacterial liquid smears under an oil microscope showed that LOH112 was a coryneform bacterium with rounded ends and slightly elongated middle, and that the bacterial cells were gram-stained purple, indicating that it was a gram-positive (G+) bacterium, as shown in FIG. 1C.

In the bacterial liquid smear dyed by spores, spores are dyed by malachite green and thalli are dyed by safranine, so that the spores and the thalli can be distinguished according to the color difference; as shown in FIG. 1D, in addition to red bacteria, a large number of green short spores were observed, which showed that LOH112 had a strong ability to produce stress-resistant dormancy, which was a spore, with a slightly shorter length and a slightly thicker diameter than the bacteria.

LOH112 strain was sequenced whole genome by beijing-baimai biotechnology limited, and 16S rDNA sequence from sequencing result was constructed with bacillus using MEGAX to develop a phylogenetic tree, which was found to have relatedness with b.velezensis FZB42 (fig. 2). BLAST alignment of 16S rDNA sequence (SEQ ID NO: 1) at NCBI Genome database revealed that it was >99% homologous to 16S rDNA sequence of known Bacillus bailii, confirming that LOH112 is a different strain of Bacillus bailii.

Finally, LOH112 strain is sent to China center for type culture collection (China center for type culture collection) for collection on 12 months 13 of 2021, and the strain is identified to be Bacillus velezensis LOH in collection name, bacillus velezensis in classification, and is collected by China center for type culture collection with collection date of 2021 and collection number of CCTCC NO: M20211623. The collection center completed the test at day 12 and 20 of 2021 to confirm the survival of the deposited microorganisms.

Velezensis is a plant growth promoting bacterium that also inhibits plant pathogens, which, based on the above characteristics, are becoming probiotics in animal feed. Tests on fish, pigs and hens have shown that b.velezensis is a safe, potential animal probiotic. Whereas potential bacillus probiotics for animals are typically isolated from the gastrointestinal tract and faeces of the animal itself, this suggests the possibility of the invention of isolating the LOH112 strain from human faeces as a probiotic. The functional properties of strain LOH112 are further investigated below.

16S rDNA sequence(1545bp，SEQ ID NO：1)：

AAAGGAGGTGATCCAGCCGCACCTTCCGATACGGCTACCTTGTTACGACTTCACCC CAATCATCTGTCCCACCTTCGGCGGCTGGCTCCTAAAAGGTTACCTCACCGACTTCGGGT GTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAAGTAGGGAACTTATTCACCGC GGCATGCTGATCCGCGATTACTAGCGATTCCAGCTTCACGCAGTCGAGTTGCAGACTGC GATCCGAACTGAGAACAGATTTGTGGGATTGGCTTAACCTCGCGGTTTCGCTGCCCTTTG TTCTGTCCATTGTAGCACGTGTGTAGCCCAGTTCATAAGGGGCATGATGATTTGACGTCA TCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCACCTTAGAGTGCCCAACTGAATGCTG GCAACTAAGATCAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGA GCTGACGACAACCATGCACCACCTGTCACTCTGCCCCCGAAGGGGACGTCCTATCTCTA GGATTGTCAGAGGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCA CATGCTCCACCGCTTGTGCATTTCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTC CCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACA CTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCAC GCTTTCGCTCCTCAGCGTCAGTTACAGACCAGAGAGTCGCCTTCGCCACTGGTGTTCCT CCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAA GTTCCCCAGTTTCCAATGACCCTCCCGGTTGAGCCGGGGGCTTTCACATCAGACTTAAG AAACCGCCTGCGAGCCCTTTACGCCCAATAATTCCGGACAACGCTTGCCACCTACGTATT ACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCC GTCCTATTTGAACGGCACTTGTTCTTCCCTAACAACAGAGCTTTACGATCCGAAAACCTT CATCACTCACGCGGCGTTGCTCCGTCAGACTTTCGTCCATTGCGGAAGATTCCCTACTGC TGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAG GTCGGCTACGCATCGTCGCCTTGGTGAGCCGTTACCTCACCAACTAGCTAATGCGCCGC GGGTCCATCTGTAAGTGGTAGCCGAAGCCACCTTTTACGTCTGAACCATGCGGTTCAAA CAAGCATCCGGTATTAGCCCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACC CACGTGTTACTCACCCGTCCGCCGCTAACATCAGGGAGCAAGCTCCCATCTGTCCGCTC GACTTGCATGTATTAGGCACGCCGCCAGCGTTCGTCCTGAGCCAGGATCAAACTCTCCG ATAA。

EXAMPLE 2 bacteriostatic action of Bacillus bailii LOH112

1. Inhibition of fungi candida albicans and aspergillus niger

(1) Culture of strains

1) Bacillus beleiensis LOH112 culture: culturing strain preserved at-80deg.C on LB plate, culturing overnight in 37 deg.C constant temperature incubator, collecting single colony in LB liquid culture medium, and culturing on 37 deg.C constant temperature shaking table for 24 hr to obtain bacterial liquid. The concentration of the bacterial liquid is measured by an ultraviolet spectrophotometer, and the bacterial liquid is diluted to 0.01Abs for standby by an LB liquid culture medium.

2) Culturing candida albicans: culturing strain preserved at-80deg.C on YM plate at 30deg.C overnight, collecting single colony in YM liquid culture medium, and culturing at 30deg.C for 24 hr to obtain bacterial liquid. The concentration of the bacterial liquid was determined by an ultraviolet spectrophotometer and diluted to 0.01Abs with YM liquid medium for use.

3) Culturing Aspergillus niger: culturing strain stored at-80deg.C on PDA plate, culturing in constant temperature incubator at 30deg.C for 2-5d, eluting spores on plate with sterile water,and (5) resuspension to obtain aspergillus niger liquid. Aspergillus niger spore concentration was determined using a hemocytometer and diluted to 10 with sterile water ⁶ And (3) keeping the mixture in a constant volume (mL).

(2) Test method

100 mu L of candida albicans liquid with the concentration of 0.01Abs or 10 mu L of candida albicans liquid is dripped on the surface of an LB plate ⁶ Per mL of Aspergillus niger spore suspension, and uniformly coating; then a round sterile filter paper sheet with the diameter of 5mm is stuck to the center of the culture medium; 5uL of bacillus besii LOH112 bacterial liquid with the concentration of 0.01Abs is dripped on a filter paper sheet; the candida albicans flat plate is placed in a 30 ℃ incubator to be cultivated for 24 hours, and the aspergillus niger flat plate is placed in the 30 ℃ incubator to be cultivated for 2-3 days.

The results showed that LOH112 formed a distinct zone of inhibition for candida albicans and aspergillus niger compared to the control with the addition of the blank medium (fig. 3), indicating that it has an inhibitory effect on the growth of both fungi.

Candida albicans can cause acute and chronic infections of skin, oral cavity, mucous membrane and viscera, namely candida mycosis. Aspergillus niger is easy to grow and propagate in large quantity to generate enzyme heat, and flowers fall or rotting bell is caused when the Aspergillus niger infects cotton, or the Aspergillus niger is grown on succulent fruits to cause rotting or soft rot, and a large amount of black powder is generated when the Aspergillus niger infects the surfaces of onion scales; the plum rain season is also liable to cause mildew of clothes and the like. Therefore, bacillus bailii LOH112 has better inhibition effect on human pathogenic fungi such as candida albicans and plant pathogenic fungi such as aspergillus niger and has better application prospect in the fields of medicine and agriculture.

2. Inhibition of the gram-positive bacteria staphylococcus aureus and micrococcus luteus

(1) Culture of strains

1) Staphylococcus aureus culture: culturing strain preserved at-80deg.C on LB plate with streak, standing in 37 deg.C constant temperature incubator overnight, culturing until colony grows on the plate, selecting single colony into LB liquid medium, and culturing at 37 deg.C constant temperature for 24 hr to obtain bacterial liquid. The concentration of the bacterial liquid is measured by an ultraviolet spectrophotometer, and the bacterial liquid is diluted to 0.01Abs for standby by an LB liquid culture medium.

2) Culturing micrococcus luteus: culturing strain preserved at-80deg.C on LB plate with streak, standing in 37 deg.C constant temperature incubator overnight, culturing until colony grows on the plate, selecting single colony into LB liquid medium, and culturing at 37 deg.C constant temperature for 24 hr to obtain bacterial liquid. The concentration of the bacterial liquid is measured by an ultraviolet spectrophotometer, and the bacterial liquid is diluted to 0.01Abs for standby by an LB liquid culture medium.

(2) Test method

Dripping 100 mu L of staphylococcus aureus bacterial liquid with the concentration of 0.01Abs or micrococcus luteus bacterial liquid with the concentration of 0.01Abs on the surface of the flat plate, and uniformly coating; then a round sterile filter paper sheet with the diameter of 5mm is stuck to the center of the culture medium; 5uL of bacillus besii LOH112 bacterial liquid with the concentration of 0.01Abs is dripped on a filter paper sheet; the plates were placed in an incubator at 37℃and incubated 24h.

The results showed that LOH112 formed a distinct zone of inhibition against staphylococcus aureus and micrococcus luteus (fig. 4) compared to the control with the blank medium drop, indicating that it has inhibitory effect on the growth of both fungi.

Staphylococcus aureus is a common food-borne pathogenic microorganism that can cause a variety of diseases in humans and animals, including pseudomembranous enteritis, septicemia, sepsis, etc., and severely threatens the life safety of humans and animals. Micrococcus luteus is a conditional pathogen, and causes local tissue infection such as wound and serious infection such as endocarditis. Therefore, bacillus bailii LOH112 has good inhibition effect on gram-positive bacteria such as staphylococcus aureus, micrococcus luteus and the like, and has good application prospect in the field of bacterial infection diseases.

EXAMPLE 3 protease-producing Properties of Bacillus bailii LOH112

Identification and measurement of the ability of Bacillus belicus LOH112 to secrete protease hydrolysis protein was performed on the basis of an agar well diffusion experiment: after the skim milk flat plate is configured, a round sterile filter paper sheet with the diameter of 5mm is stuck to the center of the flat plate; 2uL of bacillus beijerinus LOH112 bacteria solution with the concentration of 0.01Abs is dripped on a filter paper sheet, 2uL of blank culture medium is dripped on a control group, and the flat plate is placed in a 37 ℃ incubator for 1d of culture; the diameter of the degradation circle of the protein on the plate was observed. The skim milk plate medium used was: 5g/L beef extract, 10g/L peptone, 1g/L LNaCl,16g/L agar, pH 7.2.

The results show that compared with a control of dripping a blank culture medium, LOH112 can remarkably degrade proteins to form an obvious degradation ring (figure 5), which shows that bacillus bailii LOH112 can produce protease and can be used for extracting the protease and applied to the protease fields such as food industry or washing industry; meanwhile, the method can also be used for degrading pathogenic bacteria cell membranes in agricultural microbial fertilizers to control diseases and degrading proteins in agricultural organic fertilizers in compost to facilitate plant absorption; can also be used in microbial feed to help animals digest and absorb nutrition and improve the utilization rate of the feed by the animals.

EXAMPLE 4 cellulase-producing Properties of Bacillus bailii LOH112

Identification and measurement of the cellulose degrading ability of Bacillus bailii LOH112 were performed on the basis of an agar well diffusion method: after the cellulose flat plate is configured, a round sterile filter paper sheet with the diameter of 5mm is stuck to the center of the flat plate; 2uL of bacillus beljavensis LOH112 bacterial solution with the concentration of 0.01Abs is dripped on a filter paper sheet, 2uL of blank culture medium is dripped on a control group, the flat plate is reversely placed in a 37 ℃ incubator for 1d, the flat plate is dyed for 10min by using 1mg/mL congo red dye, and the undyed dye is washed twice by using deionized water; the diameter of the degradation circle of cellulose on the plate was observed. The cellulose plate medium used was: 1.5g/L K ₂ HPO ₄ ,1.5g/L(NH ₄ ) ₂ SO ₄ ,0.3g/L MgSO ₄ ·7H ₂ O,0.2g/L CaCl·2H ₂ O,5g/LNaCl, 0.3g/L urea, 10g/L CMC-Na,0.3g/L peptone, 16g/L agar, pH 7.2.

The results show that compared with a control of dropwise adding a blank culture medium, LOH112 can remarkably degrade cellulose to form an obvious degradation ring (figure 6), which shows that bacillus bailii LOH112 can produce cellulase and can be used for extracting the cellulase, and has wide application prospects in both food industry and environmental industry; can be applied to degrading the cell wall of pathogenic fungi for controlling diseases; can also be applied to the decomposition of cellulose in compost; can also be applied to feeds produced by livestock and poultry to solve the problem that monogastric animals such as pigs, chickens and the like can not utilize cellulose.

EXAMPLE 5 Properties of Bacillus bailii LOH112 to form a strongly adherent biofilm

The biofilm formation capacity was quantified using crystal violet staining. The method comprises the following steps: overnight cultured Bacillus bailii LOH112 was diluted to 1X 10 ⁶ CFU/mL. Subsequently, 200. Mu.L of the diluted bacterial liquid was transferred to a 96-well plate, 6 multiple wells were made, and cultured at 37℃for 24 hours, and a blank medium was used as a control. After incubation, the medium was discarded, the cells were rinsed 3 times with PBS to remove impurities and planktonic bacteria. Next, 200. Mu.L of methanol was added to each well to fix the biofilm, and after 15 minutes, the methanol was discarded, and after drying, 200. Mu.L of 0.1% crystal violet was added. After 5min of crystal violet staining, excess dye solution was washed away, and 200 μl of 33% glacial acetic acid was added to each well. Finally, the microplate reader was oscillated at high speed for 20min and the optical density was measured at 595nm wavelength.

Depending on the critical ODc value (ODc equals the mean of the blank wells plus 3 times the standard deviation), biofilms can be divided into several types: OD.ltoreq. ODc indicates no adhesion (-), ODc < OD.ltoreq.2 ODc indicates weak adhesion (+), 2ODc < OD.ltoreq.4 ODc is medium adhesion (++), OD >4ODc is strong attachment force (+++). Each experiment was repeated 3 times and averaged.

The results show that bacillus beleiensis LOH112 can form a strong adhesive biofilm (table 2), which shows that bacillus beleiensis LOH112 has good colonization capability and can provide better rhizosphere colonization effect when applied to plant antibiosis; the microbial feed can also be applied to animal feeds to facilitate the field planting of the feeds in intestinal tracts, so that the microbial feed can better exert the activities of cellulase and protease, and the digestion and utilization rate of the feeds by animals can be improved.

TABLE 2 biofilm assay results

EXAMPLE 6 degradation of feathers by Bacillus bailii LOH112

(1) Preparation of feather culture medium: the feather is mainly used as a feed, and the feather powder is obtained by washing chicken feather and then primarily crushing the chicken feather into filaments by a crusher. When in preparation, 20g of feather powder, 5g of sodium chloride, 0.4g of monopotassium phosphate and 0.1g of magnesium sulfate are added, distilled water is used for constant volume to 1L, and finally, the mixture is subjected to high-pressure sterilization at 121 ℃ for 20 min.

(2) Test method

The cultured Bacillus bailii bacterial solution was centrifuged at 5000 Xg for 5min at 4℃and the supernatant was discarded, washed twice with sterile water, and the pellet was resuspended in sterile water to concentrate the bacterial cells to 10.0Abs. Wherein, 300uL of bacterial liquid is taken from the experimental group and added into a chicken feather culture medium of 2.70mL, and finally 3.0mL of chicken feather culture medium contains bacillus bailii of 1.0 Abs; the blank group was prepared by adding 300uL of sterile water to 2.70mL of chicken feather medium, and finally 3.0mL of chicken feather medium. The experimental group and the blank group were subjected to shaking culture at a constant temperature of 220rpm for 24 hours at 37℃to observe the degradation of feathers.

The results show that compared with a control group of a blank culture medium, LOH112 can remarkably degrade feathers, which indicates that bacillus bailii LOH112 can produce keratinase, can be applied to agriculture to manufacture organic fertilizer and degrade pollution sources, thereby improving the environment; the method can also be applied to development of waste feathers in the feed industry, and solves the problem of insufficient protein resources in the current feed industry; and can be applied to the treatment of leather, cosmetics, medicine industry, food industry and other industries and environments.

The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. It will be apparent to those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, and yet fall within the scope of the invention.

Sequence listing

<110> university of Zhongshan

<120> Bacillus bailii LOH112 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1545

<212> DNA

<213> 16S rDNA sequence(Bacillus velezensis)

<400> 1

aaaggaggtg atccagccgc accttccgat acggctacct tgttacgact tcaccccaat 60

catctgtccc accttcggcg gctggctcct aaaaggttac ctcaccgact tcgggtgtta 120

caaactctcg tggtgtgacg ggcggtgtgt acaaagtagg gaacttattc accgcggcat 180

gctgatccgc gattactagc gattccagct tcacgcagtc gagttgcaga ctgcgatccg 240

aactgagaac agatttgtgg gattggctta acctcgcggt ttcgctgccc tttgttctgt 300

ccattgtagc acgtgtgtag cccagttcat aaggggcatg atgatttgac gtcatcccca 360

ccttcctccg gtttgtcacc ggcagtcacc ttagagtgcc caactgaatg ctggcaacta 420

agatcaaggg ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga 480

caaccatgca ccacctgtca ctctgccccc gaaggggacg tcctatctct aggattgtca 540

gaggatgtca agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac 600

cgcttgtgca tttcgtcaat tcctttgagt ttcagtcttg cgaccgtact ccccaggcgg 660

agtgcttaat gcgttagctg cagcactaag gggcggaaac cccctaacac ttagcactca 720

tcgtttacgg cgtggactac cagggtatct aatcctgttc gctccccacg ctttcgctcc 780

tcagcgtcag ttacagacca gagagtcgcc ttcgccactg gtgttcctcc acatctctac 840

gcatttcacc gctacacgtg gaattccact ctcctcttct gcactcaagt tccccagttt 900

ccaatgaccc tcccggttga gccgggggct ttcacatcag acttaagaaa ccgcctgcga 960

gccctttacg cccaataatt ccggacaacg cttgccacct acgtattacc gcggctgctg 1020

gcacgtagtt agccgtggct ttctggttag gtaccgtcaa ggtgccgtcc tatttgaacg 1080

gcacttgttc ttccctaaca acagagcttt acgatccgaa aaccttcatc actcacgcgg 1140

cgttgctccg tcagactttc gtccattgcg gaagattccc tactgctgcc tcccgtagga 1200

gtctgggccg tgtctcagtc ccagtgtggc cgatcaccct ctcaggtcgg ctacgcatcg 1260

tcgccttggt gagccgttac ctcaccaact agctaatgcg ccgcgggtcc atctgtaagt 1320

ggtagccgaa gccacctttt acgtctgaac catgcggttc aaacaagcat ccggtattag 1380

ccccggtttc ccggagttat cccagtctta caggcaggtt acccacgtgt tactcacccg 1440

tccgccgcta acatcaggga gcaagctccc atctgtccgc tcgacttgca tgtattaggc 1500

acgccgccag cgttcgtcct gagccaggat caaactctcc gataa 1545

Claims

1. The bacillus beleiensis LOH112 is characterized in that the bacillus beleiensis LOH112 is sent to China center for type culture collection (China center for type culture collection) for collection (CCTCC No. M20211623) at the 12 th month 13 of 2021.

2. A microbial preparation comprising the Bacillus belicus LOH112 as claimed in claim 1.

3. The microbial inoculum of claim 2, wherein the bacillus beijerinus LOH112 is cultured by the following method: and (3) streaking bacillus belicus LOH112 on an LB plate, culturing the activated strain overnight at 37 ℃, picking a single colony into an LB liquid culture medium, and culturing on a constant temperature shaking table at 37 ℃ for a period of time.

4. Use of bacillus belgium LOH112 according to claim 1 or of the microbial inoculum according to claim 2 for controlling plant fungal diseases caused by aspergillus niger.

5. The use of bacillus belgium LOH112 according to claim 1 or a microbial agent according to claim 2 for the preparation of a medicament for the prevention and treatment of bacterial diseases, wherein the bacteria are staphylococcus aureus and micrococcus luteus.

6. The use of bacillus belgium LOH112 according to claim 1 or a microbial inoculum according to claim 2 for the preparation of a medicament for the control of fungal diseases, characterized in that the fungus is candida albicans.

7. Use of bacillus belicus LOH112 according to claim 1 or a microbial inoculum according to claim 2 for the preparation of plant biocontrol agents and/or bio-organic fertilizers.

8. Use of bacillus belgium LOH112 according to claim 1 or a microbial agent according to claim 2 for degrading feathers.

9. A method for producing a protease, characterized in that the protease is produced by utilizing the protease-producing property of Bacillus bailii LOH112 according to claim 1.

10. A method for producing cellulase, characterized in that the cellulase is produced by utilizing the cellulase-producing property of Bacillus bailii LOH112 according to claim 1.