阅读说明：本技术 一种具有促生长及防腐作用的枯草芽孢杆菌及其应用 (Bacillus subtilis with growth promoting and corrosion preventing effects and application thereof ) 是由 单宝龙 胡著然 王丽荣 冉新炎 陈丹丹 李圆圆 陶宁 韩广泉 齐素敏 樊梅娜 申 于 2021-06-21 设计创作，主要内容包括：本发明公开了一种具有促生长及防腐作用的枯草芽孢杆菌(Bacillus subtilis)NBL-B12005；该菌株保藏于中国典型培养物保藏中心,保藏号为：CCTCC M 2021642。本发明还提供了枯草芽孢杆菌在生物促生长剂、生物防腐剂中的应用；本发明还提供了一种生物抑菌剂,该生物抑菌剂含有上述枯草芽孢杆菌(Bacillus subtilis)NBL-B12005和/或所述枯草芽孢杆菌(Bacillus subtilis)NBL-B12005的培养物。该菌株针对引发果实腐烂的多种致腐菌、致病菌均具有良好的拮抗作用,菌悬液及发酵上清液均具有较好的抑制效果,安全性高,具有良好的抑菌、促生长、防腐的功效。(The invention discloses a Bacillus subtilis NBL-B12005 with the functions of promoting growth and preventing corrosion; the strain is preserved in China center for type culture Collection with the preservation number: CCTCC M2021642. The invention also provides the application of the bacillus subtilis in biological growth promoters and biological preservatives; the invention also provides a biological bacteriostatic agent which contains the Bacillus subtilis NBL-B12005 and/or a culture of the Bacillus subtilis NBL-B12005. The strain has good antagonistic action on various rot-causing bacteria and pathogenic bacteria which cause fruit rot, has good inhibition effect on bacterial suspension and fermentation supernatant, is high in safety, and has good effects of inhibiting bacteria, promoting growth and preventing corrosion.)

1. The Bacillus subtilis with growth promoting and preservative effects is Bacillus subtilis NBL-B12005, and has the preservation unit: china center for type culture Collection, collection address: wuhan university in Wuhan, China, the preservation number: CCTCC M2021642, date of deposit: 31/05/2021.

2. The Bacillus subtilis NBL-B12005 bacteria of claim 1 characterized as gram positive rods 0.6 x 1.0-1.4 microns in size grouped individually or in pairs to form endospores; the spores are oval with a central or sub-apical orientation; there was no swelling of the sporangia.

3. The Bacillus subtilis according to claim 1, wherein the culture medium of the Bacillus subtilis NBL-B12005 comprises 2.0-4.0% of molasses, 0% -3.0% of soybean meal/yeast extract/corn paste, and K₂HPO₄×3H₂0 0.5-1.0％，(NH₄)₂SO₄0.15-0.4%, sodium dihydrogen phosphate 0.6-1.0%, calcium carbonate 0.1-0.4%, MgSO₄×7H₂00.01-0.06%, manganese sulfate 0.02-0.12%, and water in balance, and pH 6.5-7.2.

4. The Bacillus subtilis of claim 3The Bacillus subtilis NBL-B12005 is characterized in that the culture medium of the Bacillus subtilis NBL-B12005 comprises 3.0 percent of beet molasses, 200.7 percent of K2HPO4 multiplied by 3H and (NH)₄)₂SO₄0.15%, sodium dihydrogen phosphate 0.8%, calcium carbonate 0.2%, MgSO₄×7H₂00.03 percent, manganese sulfate 0.03 percent and water in balance, and the pH value is 7.0-7.2.

5. The Bacillus subtilis according to claim 2, wherein the Bacillus subtilis NBL-B12005 is cultured at a temperature of 28 ℃ to 35 ℃ and in an inoculum size of 1% to 5%.

6. The Bacillus subtilis of claim 5, wherein the Bacillus subtilis NBL-B12005 is cultured at 30 ℃ and the inoculum size is 5%.

7. Use of the Bacillus subtilis according to any one of claims 1 to 6 wherein: bacillus subtilis NBL-B12005 is used as a biological growth promoter.

8. Use of the Bacillus subtilis according to any one of claims 1 to 6 wherein: bacillus subtilis NBL-B12005 is used as a biological preservative.

9. A bacteriostatic agent comprising the compound according to any one of claims 1 to 6, wherein: the biological bacteriostatic agent contains the Bacillus subtilis NBL-B12005 and/or the Bacillus subtilis NBL-B12005.

10. A biological bacteriostatic agent according to claim 9, wherein: the effective viable count of the Bacillus subtilis NBL-B12005 is more than or equal to 20 hundred million CFU/ml.

Technical Field

The invention belongs to the technical field of microbial preservation, and particularly relates to bacillus subtilis with growth promoting and preservative effects and application thereof.

Background

At present, the preservation of fruits and vegetables in the market mainly adopts physical methods (such as refrigeration, air conditioning, ultraviolet irradiation and the like) and uses various broad-spectrum bactericides, and is mainly used after picking; however, these methods are accompanied by many problems such as high investment, high energy consumption, high pesticide residue, etc., and biological control of fruit diseases by microorganisms does not harm human health, does not cause environmental pollution, and is not easy to cause drug resistance of plants, so that the biological control technology with obvious disease prevention effect, environmental friendliness and low cost becomes a research hotspot for storage and preservation of harvested fruits and vegetables at home and abroad.

Aiming at the problems of the prior fruit and vegetable fresh-keeping technology, the microbial inoculum can be used as a substitute means for protecting agricultural products in the storage process, however, researches find that antagonistic bacteria have the defects of unstable effect, narrow preservative spectrum and the like, and the antagonistic mechanism of the antagonistic bacteria is related unilaterally through the competition of nutrition and living space or the secretion of active antibacterial substances, such as: the antagonism mechanism of yeast YT-2 is only through the competition effect with the nutrients and living space of pathogenic bacteria. Therefore, a strategy to find active biological preservatives should be to not only address the microorganisms that can colonize the damaged areas of fruit, but also to address the need for microorganisms that exhibit resistance and competition for the nutritional substrate and pathogenic bacteria under a wide range of temperature conditions.

Disclosure of Invention

Aiming at the research background, the inventor provides the bacillus subtilis with the effects of promoting growth and preserving, the bacillus subtilis plays a role in inhibiting pathogenic bacteria and putrefying bacteria through competition between nutrition and living space and secretion of active antibacterial substances, can reduce the morbidity and rotting rate of fruits, and has good application value in the aspect of fruit preservation and preservation.

In order to achieve the technical effects, the invention provides the following technical scheme:

the first purpose of the invention is to provide Bacillus subtilis with growth promoting and preservative effects, the Bacillus subtilis is separated from a peach fruit sample of a peach row in the central city of Shandong province fat city, the colony morphology of the Bacillus subtilis meets the characteristics of the Bacillus, the Bacillus subtilis has 98% similarity with 16S rDNA sequences of other known Bacillus of the same genus through genome sequencing comparison, and the accuracy of the Bacillus subtilis and the Bacillus subtilis reaches 2.201 through matrix-assisted laser desorption ionization time of flight MALDI-TOF MS mass spectrometry. According to the comprehensive morphological characteristics, the 16S rDNA gene sequence result and MALDI-TOF MS mass spectrum analysis, the strain belongs to Bacillus subtilis and is named as Bacillus subtilis NBL-B12005. The strain is preserved in China Center for Type Culture Collection (CCTCC) in 2021, 05 and 31, with the preservation address: the Wuhan university in Wuhan, China has the biological preservation number as follows: CCTCC M2021642.

Further, the Bacillus subtilis NBL-B12005 has the thallus characteristics that: gram-positive straight rod with size of 0.6 × 1.0-1.4 μm, and forming endospore by grouping individually or in pairs; the spores are oval with a central or sub-apical orientation; there was no swelling of the sporangia.

The colony characteristics are as follows: the LB plate solid culture medium forms a light beige circular colony after being cultured for 24 hours at 37 ℃, the edge is more regular, the diameter is 2-4mm, no luster exists, the center is slightly provided with concentric circular fold and bulge, and the texture is sticky.

Further, the physiological and biochemical characteristics of the Bacillus subtilis NBL-B12005 are as follows:

can hydrolyze starch and molasses, cannot utilize xylose, arabinose and mannitol, can reduce nitrate, and can grow under the conditions of 7% sodium chloride and pH5.7.

Furthermore, the Bacillus subtilis NBL-B12005 strain is proved to be non-pathogenic and non-toxic by radish seed phytotoxicity tests and veterinary toxicology studies, and can be used for microbial production.

Furthermore, the Bacillus subtilis NBL-B12005 strain is researched by a radish seed phytotoxicity test, and the result shows that the strain has no phytotoxicity and has a certain growth promoting effect on radish seedlings.

Furthermore, the Bacillus subtilis NBL-B12005 strain provided by the invention has good inhibition effect on various pathogenic bacteria causing various diseases of fruit crops, wherein the pathogenic bacteria include but are not limited to alternaria alternata, brown rot disease of peach, cladosporium cladosporioides, Gilberry disease of peach, Penicillium expansum, fusarium oxysporum and the like.

The Bacillus subtilis NBL-B12005 strain also has certain inhibition effect on pathogenic bacteria and plant pathogenic bacteria, wherein the pathogenic bacteria and the plant pathogenic bacteria comprise but are not limited to escherichia coli, ralstonia solanacearum and the like.

The bacteriostatic effect of the Bacillus subtilis NBL-B12005 strain is the combined action of the bacteria and the metabolite thereof.

Further, the culture medium of the Bacillus subtilis NBL-B12005 comprises 2.0-4.0% of molasses, 0-3.0% of soybean meal/yeast extract/corn paste and K₂HPO₄×3H₂0 0.5-1.0％，(NH₄)₂SO₄0.15-0.4%, sodium dihydrogen phosphate 0.6-1.0%, calcium carbonate 0.1-0.4%, MgSO₄×7H₂00.01-0.06%, manganese sulfate 0.02-0.12%, and water in balance, and pH 6.5-7.2.

Further, the culture medium of the Bacillus subtilis NBL-B12005 takes 1% -3% of beet molasses or cane molasses as a carbon source and 0% -4% of soybean meal as a nitrogen source.

Further, the best culture medium of the Bacillus subtilis NBL-B12005 is beet molasses 3.0%, K2HPO 4X 3H 200.7%, (NH)₄)₂SO₄0.15%, sodium dihydrogen phosphate 0.8%, calcium carbonate 0.2%, MgSO₄×7H₂00.03 percent, manganese sulfate 0.03 percent and water in balance, and the pH value is 7.0-7.2.

Further, the Bacillus subtilis NBL-B12005 is preferably cultured at 28-35 deg.C with an inoculum size of 1-5%, more preferably at 30 deg.C with an inoculum size of 5%.

The second purpose of the invention is to provide the application of the bacillus subtilis in biological growth promoters.

The third purpose of the invention is to provide the application of the bacillus subtilis in biological preservatives.

The fourth purpose of the invention is to provide a biological bacteriostatic agent which contains the Bacillus subtilis NBL-B12005 and/or the Bacillus subtilis NBL-B12005 culture.

Furthermore, the effective viable count of the Bacillus subtilis NBL-B12005 which is one of the biological bacteriostats is more than or equal to 20 hundred million CFU/ml

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

the bacillus subtilis strain provided by the invention has a good broad-spectrum antibacterial effect, has a good antagonistic effect on various spoilage bacteria and pathogenic bacteria which cause fruit rot, has a good inhibition effect on bacterial suspension and fermentation supernatant, and makes up for the defect of unstable single effect, thereby being beneficial to improving the anticorrosion effect; after fermentation optimization, higher viable count can be obtained, and the bacterial strain has high safety and good bacteriostatic, growth-promoting and anti-corrosion effects. Therefore, the strain has wider development potential in biological control of fruit preservation and freshness preservation.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.

FIG. 1 is a colony morphology of strain NBL-B12005 of the present invention;

FIG. 2 is a morphological diagram of bacterial cells of strain NBL-B12005 of the present invention;

FIG. 3 is a photograph showing the growth of 3-4 hyphae of Monilinia fructicola in the control group under the microscope in example 3;

FIG. 4 is a graph showing the effect of NBL-B12005 on the growth of hyphae of Monilinia fructicola 3-4 under a microscope in example 3;

Detailed Description

In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.

Example 1 screening and identification of strains

(1) In vitro bacteriostatic test of the strains

Separating bacterial strains from 8 samples such as peach fruits, peach leaves, soil, fruit stalks and the like collected from central peach rows in Shandong province fat cities, weighing 1g of the samples, adding the samples into 100mL of sterile water, shaking up in a thermostat at 37 ℃, carrying out gradient dilution on sample liquid, coating the diluent liquid in a flat plate containing an NA solid culture medium, and culturing overnight at 37 ℃. The strains were isolated from the plates on which colonies grew and purified, and transferred to NA slant medium for storage.

100 strains of bacteria that can sporulate were isolated from the samples. Three saprophytic bacteria (alternaria alternate, brown rot of peach and cladosporium cladosporioides) separated and purified from rotten peach fruits are taken as test objects, a perforation method is used for researching the bacteriostatic activity of the fermentation liquor of the separated and purified strains, and the strains with obvious bacteriostatic action are selected. And primarily screening several strains with different degrees of bacteriostatic pathogenic bacteria expansion according to the diameter of the bacteriostatic zone. The bacteriostatic activity of the isolated and purified strains is shown in table 1:

TABLE 1 antifungal Activity of the strains

(2) Fruit in vivo bacteriostasis test

The colonies with obvious bacteriostasis are shaken in an LB liquid culture medium for 24 hours at the temperature of 30 ℃ for 200r/min, and then the peach in-vivo experiment is carried out. Soaking peach fruits in a sodium hypochlorite solution with the practical concentration of 2% for sterilization for 2-3min, then washing away residual sodium hypochlorite by using sterile normal saline, and placing the peach fruits in a sterile operating table for natural drying for later use. A hole with the diameter of 5mm and the depth of about 3mm is artificially punched on the equator of the fruit by using a sterile puncher. mu.L of antagonist suspension (1X 10) was added to each well using a pipette gun⁸CFU/ml), standing in a sterile workbench for 2h, and then making the sterile suspension flow in and around the wound of the fruit grain. Then 20. mu.L of a pathogen spore suspension (1X 10) was added to the wound with a pipette⁵CFU/ml) and placing for 2 hours at room temperature, sealing with a preservative film after no flowing bacterial suspension is left at the wound and around the fruit, placing the fruit in a plastic basin, placing the fruit in an incubator at the temperature of 28 ℃/93% RH for constant-temperature moisture-preserving culture, observing and recording the rotting condition of the fruit after 5 days, measuring the lesion diameter of the wound, counting the morbidity, and adding sterile water (control 2) and a corresponding pathogen spore suspension (control 1) into a control group. 10 peach fruits were selected for each treatment, 3 replicates and the experiment was repeated 2 times.

The formula for calculating the incidence is as follows: incidence (%) is the number of fruits treated per the number of fruits treated × 100.

According to the incidence of the fruit and the spreading condition of the lesion after wound inoculation, the strain with antagonism is selected.

In this example, 2 strains were screened at a concentration of 1X 10⁹More than CFU/ml can completely inhibit the Monilinia fructicola strains, wherein the NBL-B12005 strain has the best control effect. And performing species identification on the plants according to the characteristics of the plants.

(3) Identification of strains

The purified B8 strain was characterized under a microscope as shown in fig. 2: gram-positive bacteria in the form of bacteria, bacilli, 0.6X 1.0-1.4 microns in size, arranged singly, in pairs or in short chains, form endospores. The spores are oval with a central or sub-apical orientation. There was no swelling of the sporangia.

Colony characteristics, as shown in fig. 1: the LB plate solid culture medium forms a light beige circular colony after being cultured for 24 hours at 37 ℃, the edge is more regular, the diameter is 2-4mm, no luster exists, the center is slightly provided with concentric circular fold and bulge, and the texture is sticky.

And selecting a single colony, inoculating the single colony in an LB liquid culture medium, performing shake culture at 37 ℃ and 180rpm for 24 hours, and extracting the genomic DNA of the strain from 1-5ml of bacterial liquid by using a bacterial genomic DNA extraction kit. The 16S rRNA universal primers 27f (5'-agagtttgatcctggctcag-3') and 1492r (5'-ggttaccttgttacgactt-3') amplified the genomic DNA of the strain and sent to Shanghai Piracetam bioengineering GmbH for sequencing. The BLAST software compares and analyzes the strain and the following strains of the same sex can reach more than 99.50 percent: bacillus subtilis strain 168 (99.65%), Bacillus subtilis strain DSM 10 (99.50%), and Bacillus subtilis strain JCM 1465 (99.50%).

Through matrix-assisted laser desorption ionization time of flight MALDI-TOF MS mass spectrometry, the accuracy of the strain and Bacillus subtilis reaches 2.201.

According to the comprehensive morphological characteristics, the 16S rDNA gene sequence result and MALDI-TOF MS mass spectrum analysis, the strain belongs to Bacillus subtilis (Bacillus subtilis) and is named as Bacillus subtilis NBL-B12005; and provides a gene sequence of the Bacillus subtilis NBL-B12005, and the length is 1413 bp.

Example 2 phytotoxicity and growth promotion test

The bacterial phytotoxicity detection is carried out by taking radish seeds as a model test object, the influence of the strain on the growth and development of plants is researched, and the phytotoxicity of the strain is evaluated. The seeds were soaked in 2% concentration of single bacterial liquid, 5% concentration of single bacterial liquid and distilled water (control group) for 10 min. Then washing with distilled water, uniformly placing in a culture dish paved with wet filter paper, and placing in a constant temperature incubator with the temperature set to 24 ℃. The germination rate of the seeds is recorded after 3-4 days, and the seedling lengths of the experimental group and the control group are recorded after 7 days. The phytotoxicity is judged by observing the germination rate of the seeds and the growth condition of the seedlings. The results are shown in Table 2.

TABLE 2 influence of bacterial liquid on germination percentage of radish seeds and growth of seedlings

As can be seen from Table 2, there was no significant difference in the germination rates between the radish seeds treated with the 2% and 5% bacterial solutions and the control seeds treated with water. The length of the 7-day seedlings exceeded the control group by 8-16%. From the data obtained, it can be concluded that the studied bacteria not only have no phytotoxicity, but also have a growth promoting effect on radish seedlings,

and veterinary toxicology research is carried out, and the result shows that the bacillus subtilis NBL-B12005 is nonpathogenic and nontoxic and can be used for microbial production.

Example 3 antifungal Activity Spectrum of Bacillus subtilis

On the basis of laboratory research, improved Maynell is selected and cultured for 2 days at 30 ℃ under the condition that 3% of cane molasses and 2% of soybean meal are respectively used as a carbon source and a nitrogen source, and the number of viable bacteria and the number of spores respectively reach 3.3 multiplied by 10⁹CFU/ml and 2.9X 10⁹Spores/ml.

The inhibition effect of Bacillus subtilis on various plant pathogenic fungi and bacteria causing diseases of fruit crops is measured by adopting a plate perforation antagonism method. The lower layer culture medium is 2% PDA culture medium, 1ml of pathogenic bacteria and 5ml of 1.2% PDA culture medium are mixed uniformly to form the upper layer culture medium after solidification, a hole puncher punches the hole (9cm) after solidification, 100 microliters of bacterial liquid is added, the mixture is kept still for 2h in a refrigerator, and the mixture is cultured upright for 4d at 30 ℃. The bacteriostasis spectrum of the bacteria to be detected is detected by measuring the diameter of the bacteriostasis zone, and the results are shown in tables 3 and 4.

TABLE 3 antifungal Activity Profile 1 of Bacillus subtilis NBL-B12005

TABLE 4 antibacterial Activity Profile 2 of Bacillus subtilis NBL-B12005

The results showed that Bacillus subtilis NBL-B12005 had good inhibitory effects on various pathogenic bacteria causing various diseases of fruit crops (Alternaria alternata Alternaria sp., Monilinia persica 3-4Monilinia fructicola, Cladosporium Cladosporium sp., Gilbertella persica Persicaria, Monilinia persica 2Monilinia fructicola, Fusarium oxysporum Fusarium avenaceum, Neurospora Alternaria Alternata, Cladosporium cladosporioides Cladosporoides, Penicillium expansum Expanum, Neurospora Alternaria Alternata), not only formed a growth inhibition zone with a zone diameter of 20.8-29.0mm, but also swelled and stopped the development of air bubbles on pathogenic fungi under the action of the pathogens.

Example 4 Effect of different treatment solutions of Bacillus subtilis NBL-B12005 on the growth of pathogenic bacteria

The experimental method comprises the following steps: inoculating a bacillus subtilis strain into an LB culture medium, and carrying out constant-temperature shaking culture for 12-24h at the temperature of 30 ℃ and under the condition of 200r/min to obtain a bacillus fermentation liquid; centrifuging the Bacillus fermentation liquid at 12000r/min at 4 deg.C for 10min, and filtering the supernatant with 0.22 μm filter membrane to obtain Bacillus fermentation supernatant; the cells were suspended in the same volume of sterile physiological saline to obtain a cell suspension. And (3) determining the bacteriostatic effect of different treatment solutions of the bacillus subtilis on peach fruit pathogenic bacteria by adopting an in-vitro plate punching method. The results are shown in Table 5.

TABLE 5 bacteriostatic effect of different treating solutions on peach rot-causing bacteria

Different treatment liquids	Fermentation liquor	Bacterial suspension	Fermentation supernatant
				Diameter mm of bacteriostatic zone	26.0±1.0	17.5±1.1	23.5±1.2

The results show that the three different treatment liquids have certain inhibition effects on peach fruit pathogenic bacteria, and the fermentation liquid and the fermentation supernatant have obvious inhibition effects. The antibacterial effect of the fermentation liquor is stronger. This indicates that the antagonism of the strains of the invention is the combined action of the bacteria and their metabolites; the antagonistic mechanism of the strains according to the invention may be the competition of nutrition with the living space and the secretion of active antibacterial substances.

EXAMPLE 5 Medium and culture Condition optimization

The influence of different carbon source and nitrogen source culture media on the viable count and the bacteriostatic activity of bacillus subtilis (b.subtilis) NBL-B12005 was studied in a laboratory using a triangular flask in a shaker.

The specific method comprises the following steps: taking the cultured inclined plane, carrying out streak separation on an LB (Langmuir Blodgett) plate, culturing for 20-24h, then selecting a single colony to inoculate in an LB liquid culture medium under an aseptic condition, culturing for 18-24h at 30 ℃ under a shaking condition of 180rpm to obtain a seed solution, inoculating the seed solution in different culture media according to the inoculation amount of 2% (v/v), carrying out shaking culture for 48h at 180rpm under the temperature of 30 ℃, and detecting the viable count and the bacteriostatic activity. The results are shown in Table 6.

TABLE 6 influence of different carbon and nitrogen source media on the viable count and bacteriostatic activity of Bacillus subtilis NBL-B12005

Culture medium	Viable count CFU/mL	Zone of inhibition diameter, mm, for M.fructicola 3-4
			1	2.0×109	20
2	2.6×109	23
			3	3.0×109	28
4	2.3×109	23
			5	2.6×109	24
6	2.2×109	22
			7	2.6×109	25
8	2.7×109	27

Note that molasses and various additives of carbon and nitrogen sources were used in all media: 1-beet molasses (1%), 2-beet molasses (2%), 3-beet molasses (3%), 4-beet molasses (3%) + yeast extract (0.25%), 5-beet molasses (3%) + corn paste (0.25%), 6-cane molasses (1%), 7-cane molasses (3%), 8-cane molasses (5%).

The result shows that the viable count and the bacteriostatic activity of the bacillus subtilis NBL-B12005 achieve the best effect in the No. 3 culture medium. The organic nitrogen source (yeast extract and corn extract) supplemented in the culture medium reduces the biomass accumulation level and the bacteriostatic activity of the strain to a certain extent. According to the comprehensive analysis of viable count and bacteriostatic activity, the fermentation medium of the bacillus subtilis NBL-B12005 is finally obtained as follows: beet molasses 3.0%, K₂HPO₄×3H₂0 0.7％，(NH₄)₂SO₄0.15%, sodium dihydrogen phosphate 0.8%, calcium carbonate 0.2%, MgSO₄×7H₂00.03 percent and 0.03 percent of manganese sulfate; the pH value is 7.0-7.2. The above-described optimized medium was used to perform culture temperature and inoculum size optimization experiments in a laboratory environment. The results are shown in tables 7 and 8.

TABLE 7 influence of temperature on the viable count and bacteriostatic activity of Bacillus subtilis NBL-B12005 strain

TABLE 8 Effect of inoculum size on viable count of Bacillus subtilis NBL-B12005 Strain

As is apparent from the experimental data in tables 7 and 8, the optimum temperature of Bacillus subtilis NBL-B12005 is 30 ℃; to achieve the highest viable count, 3% -5% of the inoculum size should be used.

Example 7 in vivo fruit preservation Effect test

In order to examine the preservative effect of the strain on the harvested fruits, in this example, peaches and pears are respectively taken as representatives of the fruits, and healthy and nondestructive fruits with consistent sizes are selected for in vivo experiments on the fruits.

(1) The test method comprises the following steps: soaking and sterilizing the fruits for 2-3min by using a sodium hypochlorite solution with the practical concentration of 2%, then washing away residual sodium hypochlorite by using sterile normal saline, and naturally airing the fruits in a sterile operating table for later use. A hole with the diameter of 5mm and the depth of about 3mm is artificially punched on the equator of the fruit by using a sterile puncher. And adding 20 mu L of liquid microbial inoculum into each hole by using a pipette gun, standing in an aseptic workbench for 2h, and allowing no flowing bacterial suspension to flow in and around the wound of the fruit grain. Then 20. mu.L of a pathogen spore suspension (1X 10) was added to the wound with a pipette⁵CFU/ml), standing at room temperature for 2h, sealing with plastic wrap after no flowing bacterial suspension is left around the fruit wound, placing in plastic basin, culturing at constant temperature and humidity in incubator at 28 deg.C/93% RH, and 5d laterThe decay of the fruit was observed and recorded, the lesion diameter was measured and the incidence was counted, and sterile water (control 2) and the corresponding pathogen spore suspension (control 1) were added to the control group. 10 fruits were selected for each treatment, 3 replicates and the experiment was repeated 2 times.

The calculation formula is as follows:

incidence (%) is the number of fruits treated per the number of fruits treated × 100.

Degree of inhibition (%) of pathogen spread (lesion diameter of control 1-lesion diameter of treated group)/lesion diameter of control 1 ] × 100

(2) Inhibiting effect of different treating liquids on peach brown rot

In this example, a bacterial suspension, fermentation broth, fermentation supernatant were obtained according to the method of example 4. The results are shown in Table 9.

TABLE 9 inhibitory Effect of different treatment solutions on peach brown rot

Group of	Incidence of disease%	Diameter of lesion, mm
			Control 1	100	68.5
Bacterial suspension	20	15.2
			Fermentation liquor	0	0
Fermentation supernatant	10	10.8

The results show that the bacterial suspension, the fermentation liquor and the supernatant have certain inhibition effect on peach brown rot (M.fructicola 3-4), wherein the fermentation liquor has the best effect, and the supernatant and the bacterial suspension are used secondly. This example demonstrates again that the strain of the invention is a combination of bacterial cells and their metabolites to control the development of post-harvest rot in fruits.

(3) Antiseptic effect of fermentation liquid with different concentrations in peach fruit body

In this example, the respective concentrations were set to 1X 10⁶、1×10⁷、1×10⁸、1×10⁹CFU/ml of Bacillus subtilis NBL-B12005 fermentation broth, T4, T3, T2 and T1 groups, respectively, the results are shown in Table 10.

TABLE 10 antiseptic effect of different concentrations of bacteria on peach fruits

The results show that the preservative effect of the 4 treatment groups on peaches on peach brown rot M.fructicola 3-4 and Alternaria alternata sp can reach more than 60 percent, wherein the concentration is 1 multiplied by 10⁹The CFU/ml fermentation liquor can completely inhibit the growth of pathogenic bacteria after peach harvest, reduces the occurrence of microbial diseases after peach harvest, and has obvious antagonistic action. Second 1X 10⁸The spoilage rate of the CFU/ml fermentation liquor group is only 10%, and the inhibition rates of the CFU/ml fermentation liquor group on two fruit diseases are 86.0% and 77.1%, respectively.

(4) In vivo antiseptic effect of crown pear

In this implementationIn the examples, the concentration was 1X 10⁸CFU/ml and 1X 10⁷NBL-B12005 fermentation broth at CFU/ml and suspensions (1X 10) of Bacillus subtilis AFB041 and AFB096 preferred in the invention patent 201410190713.9⁷CFU/ml) compared the preservative effect of gill's peach in the Huangguan pear body. Sterile water (control 2) and the corresponding pathogen spore suspension (control 1) were added to the control group. As shown in table 11.

TABLE 11 preservative Effect on Huangguan Pear fruits

The results showed that the concentration was 1X 10⁸The incidence of CFU/ml in the hay-treated group was 0, at the same concentration (1X 10)⁷CFU/ml), the morbidity rate of the strain treatment group is only 15%, and the morbidity rate and lesion diameter are both obviously lower than those of the bacillus subtilis AFB041 and AFB096 bacterial suspensions in the invention patent 201410190713.9. Therefore, the bacterial strain fermentation liquid has extremely obvious preservative effect in fruits. Not only can reduce the occurrence of fruit putrefaction, but also can inhibit the growth of putrefactive bacteria.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Shandong blue Biotech Ltd

<120> bacillus subtilis with growth promoting and antiseptic effects and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1413

<212> DNA

<213> 2 Ambystoma laterale x Ambystoma jeffersonianum

<400> 1

ggcggctggc tcctaaaggt tacctcaccg acttcgggtg ttacaaactc tcgtggtgtg 60

acgggcggtg tgtacaaggc ccgggaacgt attcaccgcg gcatgctgat ccgcgattac 120

tagcgattcc agcttcacgc agtcgagttg cagactgcga tccgaactga gaacagattt 180

gtgggattgg cttaacctcg cggtttcgct gccctttgtt ctgtccattg tagcacgtgt 240

gtagcccagg tcataagggg catgatgatt tgacgtcatc cccaccttcc tccggtttgt 300

caccggcagt caccttagag tgcccaactg aatgctggca actaagatca agggttgcgc 360

tcgttgcggg acttaaccca acatctcacg acacgagctg acgacaacca tgcaccacct 420

gtcactctgc ccccgaaggg gacgtcctat ctctaggatt gtcagaggat gtcaagacct 480

ggtaaggttc ttcgcgttgc ttcgaattaa accacatgct ccaccgcttg tgcgggcccc 540

cgtcaattcc tttgagtttc agtcttgcga ccgtactccc caggcggagt gcttaatgcg 600

ttagctgcag cactaagggg cggaaacccc ctaacactta gcactcatcg tttacggcgt 660

ggactaccag ggtatctaat cctgttcgct ccccacgctt tcgctcctca gcgtcagtta 720

cagaccagag agtcgccttc gccactggtg ttcctccaca tctctacgca tttcaccgct 780

acacgtggaa ttccactctc ctcttctgca ctcaagttcc ccagtttcca atgaccctcc 840

ccggttgagc cgggggcttt cacatcagac ttaagaaacc gcctgcgagc cctttacgcc 900

caataattcc ggacaacgct tgccacctac gtattaccgc ggctgctggc acgtagttag 960

ccgtggcttt ctggttaggt accgtcaagg tgccgcccta tttgaacggc acttgttctt 1020

ccctaacaac agagctttac gatccgaaaa ccttcatcac tcacgcggcg ttgctccgtc 1080

agactttcgt ccattgcgga agattcccta ctgctgcctc ccgtaggagt ctgggccgtg 1140

tctcagtccc agtgtggccg atcaccctct caggtcggct acgcatcgtc gccttggtga 1200

gccgttacct caccaactag ctaatgcgcc gcgggtccat ctgtaagtgg tagccgaagc 1260

caccttttat gtctgaacca tgcggttcag acaaccatcc ggtattagcc ccggtttccc 1320

ggagttatcc cagtcttaca ggcaggttac ccacgtgtta ctcacccgtc cgccgctaac 1380

atcagggagc aagctcccat ctgtccgctc gac 1413