CN113337423B

CN113337423B - Bacillus subtilis SCUEC7 strain and application thereof

Info

Publication number: CN113337423B
Application number: CN202110544675.2A
Authority: CN
Inventors: 王力; 李晓华; 张建国; 许琪; 寻立之; 冯喆; 王圣心; 赵思卡; 杨佳琪; 许倍滔
Original assignee: Jiangsu Shen Li Ecological Agriculture Technology Co ltd; South Central University for Nationalities
Current assignee: Jiangsu Shen Li Ecological Agriculture Technology Co ltd; South Central Minzu University
Priority date: 2021-05-19
Filing date: 2021-05-19
Publication date: 2022-07-12
Anticipated expiration: 2041-05-19
Also published as: CN113337423A

Abstract

The invention discloses a bacillus subtilis SCUEC7 strain and application thereof, wherein the bacillus subtilis SCUEC7 strain is preserved in China center for type culture Collection in 2021, 04 and 15 days, and the preservation numbers are as follows: CCTCC NO: M2021377. The SCUEC7 strain provided by the invention is separated from a mixed fermentation product of bovine gastric juice and crop straws, has an inhibiting effect on various plant pathogenic bacteria such as botrytis cinerea, pythium aphanidermatum and the like, can be used for preventing and treating crop diseases, also has an inhibiting effect on pathogenic bacteria staphylococcus aureus, and can be used in the production of foods, feeds and the like; meanwhile, the SCUEC7 strain can also promote the growth of candida utilis, and when the strain is applied to feed production, the feed fermentation can be accelerated, and the content of single-cell protein of the fermented feed can be effectively improved; the bacillus subtilis SCUEC7 strain is found to have a relaxing effect on mouse acetylcholine preshrinking tracheal rings for the first time, so that the bacillus subtilis SCUEC7 strain can be prepared into a tracheal ring relaxing medicine for treating diseases such as asthma and the like.

Description

Bacillus subtilis SCUEC7 strain and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a bacillus subtilis SCUEC7 strain and application thereof.

Background

Bacillus subtilis is one of Bacillus, widely distributed in soil and putrefactive organic matters, and easily propagated in Sucus subtilis to obtain its name. The bacterium is uniformly colored, has no capsule, flagella and can move, and gram stain is positive. The surface of the colony is rough and opaque, and the colony is white or yellowish, and is aerobic bacteria. Tryptophan can be decomposed to form indole by using protein, various sugars and starch. The bacillus subtilis can generate endogenic spores and has strong heat resistance and stress resistance. The bacillus subtilis has the advantages of high growth speed, simple nutritional requirement, easy survival, colonization and propagation, no pathogenicity, and capability of secreting various enzymes and antibiotics, so the bacillus subtilis has wide application in the fields of breeding industry, pest control, environmental remediation, medicines and the like. For example, bacillus subtilis can be metabolized to produce protease, cellulase, alpha-amylase, pectinase and other enzymes, and can be applied to enzyme production or fermentation production; the bacillus subtilis can also successfully colonize to plant rhizosphere, body surface or in vivo, and achieves the purpose of biocontrol by competition action or bacteriolysis action or generation of antibacterial substances and the like, and is used for preventing and treating plant diseases and insect pests of crops. Meanwhile, the microbial inoculum can improve the intestinal flora of animals, enhance the immunity, be used for producing animal feed, and also be used as a regulator for improving water quality and purifying water and the like.

Cellulase is a complex enzyme system capable of degrading cellulose to glucose. Among them, endoglucanases play an important role. In the hydrolysis process of cellulose, endoglucanase cuts beta-1, 4 glycosidic bonds in the cellulose to form small-fragment fibrous polysaccharide, and the small-fragment fibrous polysaccharide has wide application potential in the fields of food industry, textile industry, paper industry, biomass energy and the like. At present, part of bacillus subtilis is known to have the function of producing endoglucanase, but the problems of low enzyme production activity, slow growth and the like still exist, and the defects of production and application are not facilitated.

The candida utilis has high protein and vitamin B content, can take urea and nitric acid as nitrogen sources, and can grow in a culture medium without adding any growth factor. It can utilize several hexoses, pentoses, urea or cheap industrial and agricultural by-products and waste materials, etc. to synthesize protein with rich nutrients, for example, it can utilize waste liquor of sulfurous acid, molasses, waste liquor of starch factory and wood hydrolysate of paper mill as carbon source, and uses urea or nitrate as nitrogen source to make growth, so that it is an excellent strain for producing unicellular protein for edible, medicinal or feed, and can greatly raise the unicellular protein content of fermented feed by promoting growth of candida utilis in the course of fermentation production of feed. At present, no report about the growth promotion of candida utilis by bacillus subtilis is found.

Asthma is a chronic airway inflammation involving various inflammatory cells, and the attack of asthma causes a large amount of acetylcholine (Ach) to be released, induces the development of calcium ion channels on the cell membrane of airway smooth muscle, and causes Ca²⁺Internal flow, tracheal contraction and tracheal constriction leading to respiratory distressIt is difficult. At present, the commonly used hormone drugs can generate drug resistance of inflammation, so that the development of new drugs capable of relaxing the trachea is of great significance. However, no relevant research report is found on the application of the bacillus subtilis in relaxing tracheal rings at present.

Disclosure of Invention

The SCUEC7 strain is separated from a mixed fermentation product of gastric juice of cattle and crop straws, and researches show that the SCUEC 3538 strain has an inhibiting effect on various pathogenic bacteria such as botrytis cinerea, pythium aphanidermatum, fusarium oxysporum, early blight of tomato, ring rot of mulberry, fusarium graminearum and the like, can be used for preventing and treating crop diseases, has an inhibiting effect on pathogenic bacteria such as staphylococcus aureus, and can be used for producing food, feed and the like; meanwhile, the SCUEC7 strain can also promote the growth of candida utilis, and when the strain is applied to feed production, the feed fermentation can be accelerated, and the single cell protein content of the fermented feed can be effectively improved; the bacillus subtilis SCUEC7 strain is found to have a relaxing effect on mice acetylcholine pre-contraction tracheal rings for the first time, so that the bacillus subtilis SCUEC7 strain can be prepared into tracheal ring relaxing medicines and is used for treating diseases such as asthma and the like.

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

one purpose of the invention is to provide a bacillus subtilis SCUEC7 strain, wherein the preservation number of the SCUEC7 strain is as follows: CCTCC NO: M2021377. The SCUEC7 strain is separated from fermentation product of mixed fermentation of cattle stomach fluid and crop straws, and has been preserved in China Center for Type Culture Collection (CCTCC) at 04 and 15 days 2021, with addresses as follows: wuhan university in Wuhan City, China.

The invention also aims to provide the application of the bacillus subtilis SCUEC7 strain in inhibiting pathogenic bacteria.

The invention also aims to provide the application of the bacillus subtilis SCUEC7 strain in preventing and controlling crop diseases.

The fourth purpose of the invention is to provide the application of the bacillus subtilis SCUEC7 strain in producing endoglucanase. The SCUEC7 strain has the advantages of high growth speed, high enzyme activity of generated endoglucanase and excellent cellulose degradation activity.

The fifth purpose of the invention is to provide the application of the bacillus subtilis SCUEC7 strain in promoting the growth of candida utilis.

The sixth purpose of the invention is to provide a microbial inoculum, which comprises: and (2) fermenting the bacillus subtilis SCUEC7 strain to obtain a zymophyte liquid, or spray drying the zymophyte liquid to obtain a dry powder microbial inoculum.

The seventh purpose of the invention is to provide the application of the bacillus subtilis SCUEC7 strain in fermented feed. The bacillus subtilis SCUEC7 strain is applied to feed fermentation, not only can accelerate the feed fermentation, but also can greatly improve the single-cell protein content of the fermented feed by promoting the growth of candida utilis.

The invention also provides the application of the bacillus subtilis SCUEC7 strain in preparing a medicine for relaxing the tracheal rings.

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

(1) the bacillus subtilis SCUEC7 strain provided by the invention has high growth speed and high-efficiency endoglucanase production activity, and the OD of the strain after 12h culture₆₀₀3.69, the endoglucanase activity is 239.0U/mL, namely the endoglucanase has high-efficiency cellulose degradation activity and has wide application potential in the fields of food industry, textile industry, paper industry, biomass energy and the like.

(2) The bacillus subtilis SCUEC7 strain provided by the invention has an inhibiting effect on various pathogenic bacteria, can inhibit various plant pathogenic bacteria including botrytis cinerea, pythium aphanidermatum, fusarium oxysporum, early blight of tomato, ring rot of mulberry and fusarium graminearum, and can be used for disease control of crops; meanwhile, the SCUEC7 strain can also inhibit the growth of pathogenic bacteria staphylococcus aureus, so that the strain can be used in food production or feed production, the pollution of the staphylococcus aureus to food or feed is avoided, and the safety of the food or feed is ensured.

(3) The bacillus subtilis SCUEC7 provided by the invention can grow rapidly and promote the growth of candida utilis, and when the bacillus subtilis SCUEC7 is applied to the fermentation production of feeds, the fermentation of the feeds is accelerated, the growth of the candida utilis is promoted, and the content of single-cell protein in the fermented feeds is greatly improved.

(4) The invention also discovers that the fermentation liquor of the bacillus subtilis SCUEC7 strain has a relaxation effect on mouse acetylcholine preshrinking tracheal rings, and the relaxation rate reaches 44.23%, so that the bacillus subtilis SCUEC7 strain can be prepared into a medicine for relaxing the tracheal rings and is used for treating or relieving asthma and other diseases.

Drawings

FIG. 1 is a graph showing the effect of culture time on the growth and enzyme production of SCUEC7 strain in example 2 of the present invention;

FIG. 2 shows the result of detecting the growth of Staphylococcus aureus inhibited by Bacillus subtilis SCUEC7 strain in example 3 of the present invention;

FIG. 3 shows the result of the Bacillus subtilis SCUEC7 strain of the present invention in example 4 for promoting the growth of Candida utilis.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1 directed enrichment isolation and characterization of Bacillus subtilis SCUEC7 Strain

1. Targeted enrichment isolation of SCUEC7 Strain

Mixing gastric juice with crop straw in healthy cow rumen, fermenting for 48h, collecting 10g fermented product, adding 90mL CMC-Na liquid culture medium (CNB) with pH of 5.0, performing enrichment culture at 37 deg.C for 12 hr, and gradually diluting with 10 times of sterile water (10 times of gradient)^-1～10^-5) Spreading 200 μ L of the diluted solution on CMC-Na solid medium (CNA) plate, culturing at 37 deg.C for 24 hr, adding 0.2%And after dyeing with Congo red dyeing liquid for 30min, completely washing the dyeing liquid by using distilled water and 1mol/L NaCl solution in sequence, selecting bacterial colonies with larger degradation circles, carrying out scribing separation and purification for multiple times, and screening multiple strains of microorganisms with larger degradation circles. Wherein, the diameter of the transparent degradation circle of the A strain 12h culture is 22.80mm, the A strain is named as SCUEC7 strain, and the A strain has stronger cellulose degradation property.

CMC-Na liquid Medium (CNB): 10g of sodium carboxymethylcellulose, 0.5g of peptone, 0.5g of yeast extract powder, 1.5g of monopotassium phosphate, 0.3g of magnesium sulfate and 5g of sodium chloride, and adding distilled water to a constant volume of 1L and a pH value of 7.0-7.3.

CMC-Na solid Medium (CNA): 10g of sodium carboxymethylcellulose, 0.5g of peptone, 0.5g of yeast extract powder, 1.5g of monopotassium phosphate, 0.3g of magnesium sulfate, 5g of sodium chloride and 18g of agar, and adding distilled water to a constant volume of 1L and a pH value of 7.0-7.3.

2. Identification of SCUEC7 Strain

And (3) carrying out conventional gram staining on the SCUEC7 strain, wherein the result is positive, namely the gram-positive bacterium. The bacterial colony on a beef extract peptone solid medium plate is yellow white, round, irregular in edge, easy to pick up and free of pigment.

The physiological and biochemical characteristics of sceec 7 strain were identified using a conventional indole test, hydrogen sulfide test, methyl red test, citrate utilization test, voep test, propionate utilization test, citrate utilization test, nitrate reduction test, starch utilization test, and casein utilization test, and the results are shown in table 1.

TABLE 1 physiological and biochemical characteristics of SCUEC7 Strain

According to the results of Table 1, the indole test, the hydrogen sulfide test and the citrate utilization test of SCUEC7 strain were negative, and the methyl red test, the Volt-Pop test, the citrate utilization test, the starch utilization test, the propionate utilization test, the nitrate reduction test and the casein utilization test were positive.

Cloning and sequencing 16SrDNA of SCUEC7 strain, wherein the 16S rDNA sequence of the strain is shown in a sequence table SEQ ID NO: 1 is shown. Sequence alignment analysis shows that the bacillus subtilis has higher similarity with multiple strains of bacillus subtilis, and the SCUEC7 strain is identified as the bacillus subtilis by combining the morphological and physiological and biochemical characteristics of the strain.

The Bacillus subtilis SCUEC7 strain (Bacillus subtilis SCUEC7) has been deposited in China center for type culture Collection at 04/15/2021, with the addresses of: the preservation number of Wuhan university in Wuhan City of China is CCTCC NO: m2021377.

Example 2 growth and endoglucanase-producing Activity of Bacillus subtilis SCUEC7 Strain

Inoculating SCUEC7 strain seed liquid into CMC-Na liquid culture medium (CNB) with the inoculation amount of 0.5% by volume, carrying out shake culture at 37 ℃, sampling according to time, measuring the absorbance value of the strain liquid at 600nm as the growth amount of the strain, and simultaneously measuring the activity of endoglucanase generated by the strain, wherein the activity of the endoglucanase is defined and specified in international units: the amount of enzyme required to catalyze the hydrolysis of CMC-Na to produce 1. mu.g of glucose per minute was defined as one enzyme activity unit (U), and the results are shown in FIG. 1: OD of SCUEC7 strain at 0-12 h₆₀₀The enzyme activity of the endoglucanase is increased from 2.27U/mL to 239.98U/mL from 0.02 to 3.69. OD of SCUEC7 strain in 12-24 h₆₀₀The enzyme activity is between 239.98U/mL-218.10U/mL and is between 3.51-3.69. Wherein the OD of the SCUEC7 strain at the culture time of 12h₆₀₀And the activity of the endoglucanase is high, namely the SCUEC7 strain not only grows rapidly, but also generates the endoglucanase with high activity and high-efficiency cellulose degradation activity, so that the endoglucanase can be applied to the industrial production of the endoglucanase, or the fields of food industry, textile industry, paper industry, biomass energy and the like.

Example 3 pathogenic bacteria inhibitory Activity of Bacillus subtilis SCUEC7 Strain

1. Bacteriostatic activity of SCUEC7 strain on plant pathogenic bacteria

The bacteriostatic activity of the SCUEC7 strain on different plant pathogenic bacteria is researched by taking botrytis cinerea, pythium aphanidermatum, fusarium oxysporum, early blight of tomato, ring rot of mulberry and fusarium graminearum as indicator bacteria.

The bacillus subtilis SCUEC7 strain is taken and cultured on CNB culture medium for 12h at 37 ℃ to obtain culture solution, then the culture solution is centrifuged for 10min at 8000r/min, 5 mu L of culture supernatant is taken to carry out bacteriostasis experiment, the diameter of the bacteriostasis zone is measured, and the conventional bacillus subtilis ATCC6633 strain is taken as a control, and the measurement result is shown in the table 2: the diameters of the bacteriostatic rings of the supernatant of the bacillus subtilis SCUEC7 strain on botrytis cinerea, pythium aphanidermatum, fusarium oxysporum, early blight of tomato, ring rot of mulberry and fusarium graminearum are respectively as follows: 27.3, 25.2, 12.3, 17.7, 22.2 and 22.0 mm. The conventional bacillus subtilis ATCC6633 strain only has an inhibitory effect on botrytis cinerea, the diameter of an inhibition zone is 9.5mm, and the conventional bacillus subtilis ATCC6633 strain does not have an inhibitory effect on other plant pathogenic bacteria. Compared with the conventional bacillus subtilis, the bacillus subtilis SCUEC7 strain obtained by screening has obvious inhibition effect on botrytis cinerea, pythium aphanidermatum, fusarium oxysporum, early blight of tomato, ring rot of mulberry and fusarium graminearum, and can be widely applied to the control of various crop diseases.

TABLE 2 bacteriostatic effect of culture supernatants of SCUEC7 strain and ATCC6633 strain

Note: the outer diameter of the oxford cup is 8mm

2. Bacteriostatic activity of SCUEC7 strain on staphylococcus aureus

Staphylococcus aureus (s. aureus) is a common food-borne pathogenic microorganism, which causes food poisoning.

The diameter of the inhibition zone of the 12h culture of the SCUEC7 strain is 15.5 +/-0.9 mm by taking the bacillus subtilis ATCC6633 strain as a control, and the supernatant of the bacillus subtilis ATCC6633 strain has no inhibition effect on staphylococcus aureus. Namely, compared with the conventional bacillus subtilis, the bacillus subtilis SCUEC7 strain has obvious inhibiting effect on pathogenic staphylococcus aureus.

The inhibitory effect of 5 μ L of sceec 7 strain 12h culture supernatant, sceec 7 strain 12h culture bacterial suspension, sceec 7 strain 24h culture supernatant and sceec 7 strain 24h culture bacterial suspension on staphylococcus aureus is shown in fig. 2, where a is 24h culture bacterial suspension, B is 24h culture supernatant, C is 12h culture bacterial suspension and D is 12h culture supernatant. The results show that the inhibition zone diameters of the supernatant and the bacterial suspension of the SCUEC7 strain 24h culture for inhibiting staphylococcus aureus are 11.1mm and 14.2mm respectively, and the inhibition zone diameters of the supernatant and the bacterial suspension of the SCUEC7 strain 12h culture for inhibiting staphylococcus aureus are 11.5mm and 15.5mm respectively. The supernatant and the bacterial suspension of the bacillus subtilis SCUEC7 bacterial strain culture have obvious inhibiting effect on the growth of staphylococcus aureus, so that the bacillus subtilis SCUEC7 bacterial strain culture can be applied to food production or feed production, the safety of food or feed is ensured, and the harm of the staphylococcus aureus to human bodies and animals is avoided.

Example 4 promoting action of Bacillus subtilis SCUEC7 Strain on growth of Candida utilis

By taking candida utilis as an indicator, respectively adding 5 mu L of a supernatant (D) of a 12h culture of a bacillus subtilis SCUEC7 strain, a suspension (C) of a 12h culture of a bacillus subtilis SCUEC7 strain, a supernatant (B) of a 24h culture of a bacillus subtilis SCUEC7 strain and a suspension (A) of a 24h culture of a bacillus subtilis SCUEC7 strain into an Oxford cup (the outer diameter is 8mm) for experiment, and observing the growth condition of candida utilis on a plate after culturing for 16h at 28 ℃, wherein the results are shown in figure 3: the bacillus subtilis SCUEC7 strain can promote the growth of the candida utilis, wherein the diameters of a 24-hour culture supernatant and a bacterial suspension for promoting the growth circle of the candida utilis are respectively 19.8mm and 21.2mm, and the diameters of a 12-hour culture supernatant and a bacterial suspension for promoting the growth circle of the candida utilis are respectively 20.2mm and 21.5mm, so that the bacillus subtilis SCUEC7 strain culture supernatant and the bacterial suspension have promotion effects on the growth of the candida utilis.

50mL of potato dextrose liquid medium was inoculated with Candida utilisAdding different volumes of culture supernatant of Bacillus subtilis SCUEC7 strain cultured for 12h and CNB liquid culture medium to make total volume of the culture solution be 2500 μ L, culturing at 28 deg.C for 12h, and determining OD of the culture solution₆₀₀The value was recorded as the growth amount of Candida utilis, and as a result, as shown in Table 3, the growth of Candida utilis in the supernatant of the culture to which Bacillus subtilis SCUEC7 strain was added was remarkably accelerated as compared with the supernatant of the culture to which SCUEC7 strain was not added. 250 mu L of the supernatant of the culture of the bacillus subtilis SCUEC7 strain is added into 50mL of beef extract peptone liquid culture medium, the growth speed of the candida utilis is 2.3 times that of the candida utilis which is not added, and 2500 mu L of the supernatant is added, the growth speed of the candida utilis is 2.9 times that of the candida utilis which is not added, so that the bacillus subtilis SCUEC7 strain has the characteristic of promoting the growth of the candida utilis, and the bacillus subtilis SCUEC7 strain can be applied to the fermentation of the feed with the candida utilis to increase the content of single-cell protein in the feed and improve the quality of the fermented feed.

TABLE 3 Effect of culture supernatant of SCUEC7 Strain on the growth of Candida utilis

Example 5 application of Bacillus subtilis SCUEC7 Strain in feed fermentation

1. Activation of Bacillus subtilis SCUEC7 Strain

Activating the bacillus subtilis SCUEC7 strain stored on the slant, inoculating on CMC-Na solid culture medium (CNA), and culturing at constant temperature of 30 deg.C for 12 h; selecting strains on the activated culture medium, inoculating into CMC-Na liquid culture medium (CNB), culturing at 30 deg.C for 24 hr to obtain culture solution. Or further spray drying to obtain a dry powder microbial inoculum, wherein the optimized spray drying process conditions are as follows: the corn starch is used as a carrier for spray drying, the air inlet temperature is 160 ℃, the material concentration is 15 percent, the feeding speed is 20mL/min, and the viable count of the bacillus subtilis SCUEC 7-containing strain is 1 multiplied by 10¹¹cfu/g～1×10¹²cfu/g。

2. Bacillus subtilis SCUEC7 strain for fermenting feed

(1) The application method of the bacillus subtilis SCUEC7 strain in the fermented straw feed comprises the following steps: crushing straws, adding corresponding auxiliary materials, placing the crushed straws into a straw feed fermentation machine, sterilizing the straws for 2 hours at 75-80 ℃, inoculating SCUEC7 strain liquid or powder (namely, 1 kilogram of dry powder microbial inoculum or 1 liter of SCUEC7 strain culture solution is added into one ton of straws) with the inoculation amount of one thousandth, inoculating a proper amount of Candida utilis yeast with the water content of about 50 percent, and sealing and packaging the straws by a sealing machine. And ATCC6633 strain is used as a control group instead of SCUEC7 strain, and only Candida utilis is inoculated without adding Bacillus subtilis as a blank group.

(2) Detecting the quality of the fermented feed: during the fermentation period of the feed, the number of bacillus subtilis and the number of candida utilis in the feed (Table 4), the number of the nutrient components and the number of microorganisms in the fermented feed (Table 6) are respectively measured by sampling at 0d, 1d, 5d and 10d

TABLE 4 variation of Bacillus subtilis amounts in straw feed at different fermentation times

As can be seen from Table 4, compared with the control group, the number of the Bacillus subtilis in the fermented feed added with the SCUEC7 strain is 10.98log CFU/g when the fermented feed is fermented for 10 days, which is obviously higher than 9.16log CFU/g of the control group, and the growth speed of the Bacillus subtilis SCUEC7 strain in the fermented feed is high, so that the fermentation of the feed can be accelerated, the fermentation time is shortened, and the production cost of the feed is effectively reduced.

TABLE 5 growth of Candida utilis by SCUEC7 strain in fermented feed

As can be seen from Table 5, compared with the blank group without the addition of the SCUEC7 strain, the number of Candida utilis added in the control group with the addition of the Bacillus subtilis ATCC6633 is remarkably reduced and is only 6.97 +/-0.12 log CFU/g, while the number of Candida utilis in the fermented feed with the addition of the SCUEC7 strain is remarkably increased and is as high as 9.09log CFU/g, namely after the SCUEC7 strain is added in the feed, the growth rate of the Candida utilis in the feed is remarkably increased, so that the quality of the fermented feed can be greatly improved.

TABLE 6 fermented feed nutrient composition and microbiological assay results

As can be seen from Table 6, compared with the control group, the content of crude protein in the fermented feed added with the Bacillus subtilis SCUEC7 strain is increased by 37.5%, and the content of crude fiber is reduced by more than 42.4%, namely, in the process of feed fermentation, the content of crude protein in the feed is obviously increased and the content of crude fiber is reduced by adding the SCUEC7 strain.

Example 6 the diastolic Effect of Bacillus subtilis SCUEC7 Strain on the Pre-contracted tracheal Ring of Acetylcholine in mice

1. Pretreatment: 15 SPF-grade BALB/c mice 8-10 weeks old are purchased from the center of experimental animals in Hubei province. The mice are sacrificed by intravenous injection of pentobarbital sodium at 150mg/kg, the trachea is cut off, the attached tissues are removed, and the trachea ring with the length of 3-4 mm is cut. The trachea ring is sleeved into a triangular hook of the tension transducer, the upper end of the hook is connected with the tension transducer, the lower end of the hook is fixed on the hook above the oxygen outlet, and the hook is soaked in a PSS perfusion tank with the temperature of 37 ℃ for oxygen introduction. The tracheal ring preload was set at 0.3 g. The high calcium solution (PSS) is replaced once every 15min, and the solution is replaced for 4 times in total, so that the pre-balance is completed. Using 100 mu mol/L acetylcholine (Ach) to perform pre-stimulation on tracheal rings, eluting the acetylcholine (Ach) by PSS after the tension reaches a plateau, and finishing 1-time pre-stimulation when the tracheal ring tension reaches a baseline. The official experiment was started 3 times after pre-stimulation.

2. The experimental process comprises the following steps: after the tracheal rings are balanced and pre-stimulated, 100 mu mol/L acetylcholine (Ach) is added into a perfusion groove to stimulate the tracheal rings to enable the tracheal rings to shrink to the maximum value, the tracheal rings enter a plateau period, and the muscle tension value displayed by a tension transducer is read. Then 100. mu.L, 200. mu.L, 300. mu.L of culture supernatant and bacterial suspension of Bacillus subtilis SCUEC7 strain cultured for 12h were added, respectively, for relaxing tracheal rings. After the relaxation effect had stabilized, the muscle tone values were read and the relaxation ratio was determined, and the statistics of the muscle tone values and the relaxation ratio are shown in table 7.

TABLE 7 relaxation of acetylcholine (Ach) preshrinked tracheal rings by culture supernatants and suspensions of SCUEC7 Strain

As can be seen from Table 7, the culture supernatant and the bacterial suspension of Bacillus subtilis SCUEC7 strain of the present invention both had a certain vasodilation effect on tracheal rings, and the effect of the bacterial suspension was superior to that of the culture supernatant, wherein 300. mu.L of the bacterial suspension of Bacillus subtilis SCUEC7 strain cultured for 12 hours had the best vasodilation effect on tracheal rings, and the vasodilation ratio reached 44.23%. Therefore, the bacterial suspension or culture supernatant of the bacterium can be prepared into a medicine for dilating the tracheal ring, and is used for treating or relieving diseases such as asthma and the like.

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 changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Sequence listing

<110> Zhongnan national university

<120> bacillus subtilis SCUEC7 strain and application thereof

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gattactagc gattccagct tcacgcagtc gagttgcaga ctgcgatccg aactgagaac 180

agatttgtgg gattggctta acctcgcggt ttcgctgccc tttgttctgt ccattgtagc 240

acgtgtgtag cccaggtcat aaggggcatg atgatttgac gtcatcccca ccttcctccg 300

gtttgtcacc ggcagtcacc ttagagtgcc caactgaatg ctggcaacta agatcaaggg 360

ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga caaccatgca 420

ccacctgtca ctctgccccc gaaggggacg tcctatctct aggattgtca gaggatgtca 480

agacctggta aggttcttcg cgttgcttcg aattaaacca catgctccac cgcttgtgcg 540

ggcccccgtc aattcctttg agtttcagtc ttgcgaccgt actccccagg cggagtgctt 600

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cggcgtggac taccagggta tctaatcctg ttcgctcccc acgctttcgc tcctcagcgt 720

cagttacaga ccagagagtc gccttcgcca ctggtgttcc tccacatctc tacgcatttc 780

accgctacac gtggaattcc actctcctct tctgcactca agttccccag tttccaatga 840

ccctccccgg ttgagccggg ggctttcaca tcagacttaa gaaaccgcct gcgagccctt 900

tacgcccaat aattccggac aacgcttgcc acctacgtat taccgcggct gctggcacgt 960

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tccgtcagac tttcgtccat tgcggaagat tccctactgc tgcctcccgt aggagtctgg 1140

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gctaacatca gggagcaagc tcccatctgt ccgctcgact tgc 1423

Claims

1. A bacillus subtilis SCUEC7 strain, wherein the preservation number of the SCUEC7 strain is as follows: CCTCC NO: M2021377.

2. The use of the bacillus subtilis sceec 7 strain of claim 1 for inhibiting pathogenic bacteria.

3. The use according to claim 2, wherein said pathogenic bacteria comprise: botrytis cinerea, pythium aphanidermatum, Fusarium oxysporum, early blight of tomato, ring rot of mulberry, Fusarium graminearum and Staphylococcus aureus.

4. The use of the Bacillus subtilis SCUEC7 strain of claim 1 for the control of crop diseases.

5. Use of the bacillus subtilis SCUEC7 strain of claim 1 for the production of an endoglucanase.

6. The use of the Bacillus subtilis SCUEC7 strain of claim 1 for promoting the growth of Candida utilis.

7. A microbial inoculum, comprising: the bacillus subtilis SCUEC7 strain as defined in claim 1 is fermented to obtain a zymocyte solution, or the zymocyte solution is spray-dried to obtain a dry powder microbial inoculum.

8. The use of the Bacillus subtilis SCUEC7 strain of claim 1 in fermented feed.

9. The use of claim 8, wherein the dry powder inoculum and/or the zymogen inoculum of the bacillus subtilis SCUEC7 strain is inoculated into fermented straw feed, and then inoculated with candida utilis.

10. The use of the bacillus subtilis SCUEC7 strain of claim 1 for the preparation of a diastolic tracheal ring medicament.