Bacillus subtilis B-1124 and application thereof in aquaculture

Abstract

The invention discloses bacillus subtilis B-1124 and application thereof in aquaculture, and belongs to the technical field of microorganisms. The bacillus subtilis B-1124 is screened from the bottom mud of the aquaculture water body, after domestication culture, the bacillus subtilis B-1124 can better adapt to the environment and can stably exist in the relatively harsh environment, and the bacillus subtilis B-1124 with the high spore yield is suitable for production, improves the survival rate of final probiotics and meanwhile has a more excellent antioxidant effect. When the bacillus subtilis B-1124 or the culture of the bacillus subtilis B-1124 is applied to the aquaculture industry, the bacillus subtilis B-1124 or the culture of the bacillus subtilis B-1124 is free of obvious toxic and side effects, the feed conversion rate of aquatic animals is increased, the intestinal oxidative stress level can be obviously improved, the generation of inflammatory factors is reduced, the ecological disorder of intestinal flora can be relieved, and the immunity of the organism can be improved; and the disease resistance of the body to streptococcus agalactiae is improved, so that the death rate of the tilapia is reduced.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Bacillus subtilis B-1124 and an application thereof in aquaculture. Background Art

[0002] Aquaculture holds an important position in the prospect of meeting the growing global demand for fish production. However, environmental degradation and disease outbreaks have caused huge economic losses to the aquaculture industry, and as the scale of intensive farming has expanded, disease outbreaks have become more frequent. Over the years, chemicals and antibiotics have been widely used in aquaculture to address these problems. However, the use of chemicals and antibiotics poses potential risks to consumers and the environment because they tend to bioaccumulate in fish tissues. In addition, the application of antibiotics and chemotherapy to control these diseases has also brought many problems, such as the spread of drug-resistant pathogens, suppression of the immune system of aquatic animals, and environmental hazards. Probiotics can be an effective alternative to antibiotics and chemicals in aquaculture and are widely used in the form of feed additives. Probiotics refer to live microorganisms that, when administered in sufficient quantities, bring health benefits to the host. Currently, probiotics in aquaculture mainly include photosynthetic bacteria, lactic acid bacteria, yeasts, and Bacillus. Among the many probiotic species discovered, Bacillus species have been shown to have better probiotic properties, which is attributed to their spore-forming ability (i.e., prolonging their duration of action). They are also able to produce antimicrobial substances that are active against many pathogens but are non-pathogenic and non-toxic. Compared with other probiotics (such as Lactobacillus), their ability to survive in different environments is more advantageous due to their good tolerance and longer duration of action. Some studies have also shown that Bacillus can regulate the antioxidant capacity of the host, relieve stress caused by pressure, prevent tissue damage, improve the disease resistance of aquatic organisms, improve feed utilization, and enhance the host immune response.

[0003] Among many probiotic species, Bacillus subtilis is one of the most widely used probiotics in aquaculture. Bacillus subtilis is widely used in livestock and poultry and aquaculture as a probiotic. Adding Bacillus subtilis to animal diets can significantly increase the number of Lactobacillus in the intestines and feces, but its mechanism has not been fully explained. In the prior art, the use of Bacillus subtilis as a feed additive in aquaculture still needs to be improved in terms of feed utilization, antioxidant capacity and enhanced disease resistance. Summary of the invention

[0004] Based on this, in order to solve the technical problem that the prior art Bacillus subtilis is used as a feed additive in aquaculture, and the feed utilization rate, antioxidant capacity and enhanced disease resistance still need to be improved, the present invention provides a Bacillus subtilis B-1124 and its application in aquaculture, and the specific technical scheme is as follows:

[0005] A Bacillus subtilis B-1124, which was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, classified and named Bacillus subtilis, and the preservation number is GDMCC NO.65327.

[0006] Furthermore, the Bacillus subtilis B-1124 is screened from the bottom mud of aquaculture water bodies.

[0007] Furthermore, the screening method is as follows:

[0008] The collected aquaculture water body sludge is added to sterile physiological saline, shaken and mixed, and then kept at a temperature of 80°C to 85°C for 10min to 15min, and then cooled to below 35°C to obtain a suspension;

[0009] The suspension is inoculated into a seed culture medium, and enriched and cultured at 34° C. to 38° C. for 20 h to 24 h, and separated by line drawing, and purified to obtain a target strain with good growth and spore production;

[0010] After the target strain was subcultured for 5 times and stabilized, it was inoculated on the screening medium, and the strain with a large transparent zone was selected and identified as Bacillus subtilis B-1124.

[0011] Furthermore, the seed culture medium comprises the following components: 10 g / L to 12 g / L of peptone, 1 g / L to 5 g / L of glucose, 5 g / L to 12 g / L of yeast extract, 0.1 g / L to 0.3 g / L of magnesium sulfate and 1 g / L to 2 g / L of potassium sulfate.

[0012] Furthermore, the screening culture medium includes the following components: 1g / L~5g / L of peptone, 3g / L~7g / L of glucose, 0.01g / L~0.05g / L of calcium chloride, 0.1g / L~0.5g / L of cellulase, 0.1g / L~0.5g / L of amylase, 0.1g / L~0.5g / L of lipase, 1g / L~3g / L of antioxidants, 0.1g / L~0.3g / L of magnesium sulfate and 0.5g / L~1g / L of potassium sulfate.

[0013] Further, the cells were inoculated onto a screening medium and cultured at 35° C. to 38° C. for 24 h to 48 h.

[0014] In addition, the present application also provides an application of Bacillus subtilis B-1124, which is the application of the Bacillus subtilis B-1124 or the culture of the Bacillus subtilis B-1124 in the preparation of a bacterial agent or a feed additive.

[0015] Furthermore, the bacterial agent or the feed additive is used in aquaculture.

[0016] Furthermore, the culture of Bacillus subtilis B-1124 is inoculated into a fermentation medium with a seed liquid, and fermented for 12h to 15h at 30° C. to 38° C., a rotation speed of 150r / min to 250r / min, and a tank pressure of 0.03Mpa, and then diatomaceous earth accounting for 7% to 10% of the mass of the fermentation medium is added, and the fermentation culture is continued for 5h to 10h under the above conditions to obtain a culture of Bacillus subtilis B-1124.

[0017] Furthermore, the inoculation amount of the seed liquid is 1% to 5%.

[0018] Compared with the prior art, this application has the following beneficial effects:

[0019] 1. This application selects Bacillus subtilis B-1124 from the bottom mud of aquaculture water bodies. After domestication and cultivation, it can better adapt to the environment and can stably exist in relatively harsh environments. The high spore production ability is not only suitable for production and improves the survival rate of the final probiotics, but also can secrete amylase, protease, cellulase and lipase, and at the same time has a better antioxidant effect.

[0020] 2. The Bacillus subtilis B-1124 or the culture of Bacillus subtilis B-1124 of the present application is used in aquaculture without obvious toxic side effects, improves the feed conversion rate of aquatic animals, and can also significantly improve the level of intestinal oxidative stress, reduce the production of inflammatory factors, and alleviate the ecological disorder of intestinal flora, improve the body's immunity, and improve the body's resistance to Streptococcus agalactiae, thereby reducing the mortality rate of tilapia. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with its embodiments. It should be understood that the specific implementation methods described herein are only used to explain the present invention and do not limit the scope of protection of the present invention.

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0023] A Bacillus subtilis B-1124 in one embodiment of the present invention was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, classified and named Bacillus subtilis, and the collection number is GDMCCNO.65327.

[0024] In one embodiment, the Bacillus subtilis B-1124 is screened from the bottom mud of aquaculture water.

[0025] In one embodiment, the screening method is as follows:

[0026] The collected aquaculture water body sludge is added to sterile physiological saline, shaken and mixed, and then kept at a temperature of 80°C to 85°C for 10min to 15min, and then cooled to below 35°C to obtain a suspension;

[0027] The suspension is inoculated into a seed culture medium, and enriched and cultured at 34° C. to 38° C. for 20 h to 24 h, and separated by line drawing, and purified to obtain a target strain with good growth and spore production;

[0028] After the target strain was subcultured for 5 times and stabilized, it was inoculated on the screening medium, and the strain with a large transparent zone was selected and identified as Bacillus subtilis B-1124.

[0029] In one embodiment, the seed culture medium comprises the following components: 10 g / L to 12 g / L peptone, 1 g / L to 5 g / L glucose, 5 g / L to 12 g / L yeast extract, 0.1 g / L to 0.3 g / L magnesium sulfate and 1 g / L to 2 g / L potassium sulfate.

[0030] In one embodiment, the screening medium comprises the following components: 1g / L~5g / L peptone, 3g / L~7g / L glucose, 0.01g / L~0.05g / L calcium chloride, 0.1g / L~0.5g / L cellulase, 0.1g / L~0.5g / L amylase, 0.1g / L~0.5g / L lipase, 1g / L~3g / L antioxidant, 0.1g / L~0.3g / L magnesium sulfate and 0.5g / L~1g / L potassium sulfate.

[0031] In one embodiment, the cells are inoculated onto a screening medium and cultured at 35° C. to 38° C. for 24 h to 48 h.

[0032] In one embodiment, the antioxidant is eugenol.

[0033] In addition, the present application also provides an application of Bacillus subtilis B-1124, which is the application of the Bacillus subtilis B-1124 or the culture of the Bacillus subtilis B-1124 in the preparation of a bacterial agent or a feed additive.

[0034] In one embodiment, the bacterial agent or the feed additive is used in aquaculture.

[0035] In one embodiment, the culture of Bacillus subtilis B-1124 is inoculated into a fermentation medium with a seed liquid, and fermented for 12h to 15h at 30°C to 38°C, a rotation speed of 150r / min to 250r / min, and a tank pressure of 0.03Mpa. After that, diatomaceous earth accounting for 7% to 10% of the mass of the fermentation medium is added, and the fermentation culture is continued for 5h to 10h under the above conditions to obtain a culture of Bacillus subtilis B-1124.

[0036] In one embodiment, the inoculation amount of the seed solution is 1% to 5%.

[0037] In one embodiment, the fermentation medium comprises the following ingredients: 0.2 g / L to 0.5 g / L sucrose, 1 g / L to 2 g / L potassium hydrogen phosphate, 1 g / L to 3 g / L yeast extract, 0.03 g / L to 0.08 g / L magnesium sulfate, 0.1 g / L to 0.3 g / L manganese sulfate, 15 g / L to 20 g / L soybean cake powder and 0.01 g / L to 0.02 g / L sodium chloride.

[0038] The Bacillus subtilis B-1124 obtained by the above scheme has strong adaptability and is applied in aquaculture to improve the body's feed conversion rate and significantly improve the body's immunity, thereby reducing the risk of disease.

[0039] The embodiments of the present invention will be described in detail below with reference to specific examples.

[0040] Embodiment 1:

[0041] Screening of Bacillus subtilis B-1124:

[0042] The collected aquaculture water sediment is added to sterile physiological saline, shaken and mixed, and then kept at 85°C for 10 minutes, and then cooled to below 35°C to obtain a suspension;

[0043] The suspension was inoculated into a seed culture medium (the seed culture medium includes the following components: 10 g / L peptone, 3 g / L glucose, 6 g / L yeast extract, 0.3 g / L magnesium sulfate and 2 g / L potassium sulfate), enriched and cultured at 37° C. for 24 h, and separated by line drawing to purify the target strain with good growth and spore production;

[0044] After the target strain was subcultured for 5 times and stabilized, it was inoculated into a screening medium (the screening medium included the following ingredients: 5 g / L peptone, 3 g / L glucose, 0.01 g / L calcium chloride, 0.1 g / L cellulase, 0.1 g / L amylase, 0.1 g / L lipase, 3 g / L eugenol, 0.3 g / L magnesium sulfate and 0.5 g / L potassium sulfate), cultured at 36°C for 24 hours, and strains with large transparent zones were screened and identified as Bacillus subtilis B-1124.

[0045] The screened Bacillus subtilis B-1124 has stable colony growth, irregular edges, positive Gram staining, rod-shaped under a microscope, submesospores, non-inflated sporangia, and can secrete amylase, protease, cellulase and lipase. The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, and was classified and named Bacillus subtilis, with a preservation number of GDMCC NO.65327.

[0046] Embodiment 2:

[0047] Culture of Bacillus subtilis B-1124:

[0048] The seed liquid was inoculated into a fermentation medium (the fermentation medium included the following components: 0.3 g / L sucrose, 2 g / L dipotassium hydrogen phosphate, 3 g / L yeast extract, 0.03 g / L magnesium sulfate, 0.1 g / L manganese sulfate, 20 g / L bean cake powder and 0.01 g / L sodium chloride) at an inoculum amount of 4%. After fermentation for 12 hours at 35° C., a rotation speed of 150 r / min and a tank pressure of 0.03 MPa, diatomaceous earth accounting for 7% of the mass of the fermentation medium was added, and the fermentation culture was continued for 10 hours under the above conditions to obtain a culture of Bacillus subtilis B-1124.

[0049] Embodiment 3:

[0050] Antibacterial test of Bacillus subtilis B-1124:

[0051] The Bacillus subtilis B-1124 screened in the example was cultured at 37° C. for 24 h, diluted with sterile saline, and then spread on agar plates. The antibacterial species tested were: Streptococcus agalactiae, Escherichia coli, and Staphylococcus aureus. The diameter of the antibacterial zone was tested, and the antibacterial effect was calculated. The results are shown in Table 1 below.

[0052] Table 1: In vitro antibacterial results

[0053] Test bacteria Streptococcus agalactiae Escherichia coli Staphylococcus aureus Antibacterial rate% 98.5％ 99.7％ 99.9％

[0054] From the data analysis of Table 1, it can be seen that the Bacillus subtilis B-1124 screened in the present application has an excellent antibacterial effect.

[0055] Embodiment 4:

[0056] The culture of Bacillus subtilis B-1124 in Example 2 was concentrated, and the feed rate was controlled under the conditions of an inlet air temperature of 115°C and an outlet air temperature of 75°C, the bacterial powder was 300 mesh, and the number of viable bacteria in the bacterial powder was 10 10 CFU / g.

[0057] The bacterial powder is added to the basic fish feed, mixed and granulated, and the mass ratio of the bacterial powder to the basic fish feed is 1:99.

[0058] The sample after mixing the plasmid was prepared according to 1g / 99mL sterile water to obtain a sample suspension, and the suspension sample was placed in an 85°C water bath for 15 minutes, and then the sample suspension was diluted 10 times with sterile water, and the appropriate dilution gradient was selected to be spread on the LB nutrient agar medium, and placed in a 37°C incubator for inversion and culture for 24 hours. After visible colonies grew on the plate, the number of viable bacteria in the sample was calculated. Three groups of replicates were set, and the results are shown in Table 2.

[0059] Table 2: Number of viable cells

[0060] Suspension sample 1 Suspension sample 2 Suspension sample 3 <![CDATA[2.4×10 7 ]]> <![CDATA[2.5×10 7 ]]> <![CDATA[2.3×10 7 ]]>

[0061] It should be noted that the fish basic feed is composed of the following ingredients: 35% fish meal, 12% bone meal, 18% soybean meal, 17% protein powder, 5% chicken meal, 3% rapeseed cake, 5% soybean oil, 1% vitamin premix, 1% mineral premix, 0.5% choline chloride, and 2.5% lecithin.

[0062] From the data analysis in Table 2, it can be seen that during the processing of the fish feed in this application, the number of live bacteria remains at 2.3×10 7 The above, and excellent stability in fish feed, can not only ensure its use effect, but also ensure its shelf life.

[0063] Application Example 1:

[0064] The feed sample after mixing the plasmid in Example 4 was subjected to a feeding test. The feeding object was large yellow croaker. The fish were fed once in the morning and evening, and the number of large yellow croakers fed was the same, 100 in each group. The quality of the feed sample fed each time was the same. A control group was set up, and the control group was fed with the same quality of the above-mentioned fish basic feed.

[0065] Wherein, weight gain rate % = (final average weight - initial average weight) × 100 / initial average weight;

[0066] Survival rate % = final number of fish × 100 / initial number of fish;

[0067] The enzyme activities of superoxide dismutase (SOD) and catalase (CAT) in the liver and lysozyme (LZM) in the serum were determined by ELISA. The results are shown in Table 3.

[0068] Table 3: Feeding effect

[0069]

[0070] As can be seen from Table 3, compared with the control group without bacterial powder added, the weight gain rate and survival rate of the large yellow croaker in the application example are significantly higher than those of the control group, and the growth index of the large yellow croaker in the application example 1 group is also better than that of the control group, indicating that the addition of Bacillus subtilis B-1124 can enhance the antioxidant capacity, significantly improve the growth performance, survival rate and immune function of large yellow croaker, increase the yield per unit area and the total amount, thereby increasing the income of aquaculture and improving the efficiency.

[0071] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0072] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A Bacillus subtilis B-1124, characterized in that The strain was deposited in the Guangdong Provincial Microbiological Culture Collection Center on October 23, 2024, classified and named Bacillus subtilis, and the collection number is GDMCC NO.65327.

2. The Bacillus subtilis B-1124 according to claim 1, characterized in that The Bacillus subtilis B-1124 is screened from the bottom mud of aquaculture water bodies.

3. The Bacillus subtilis B-1124 according to claim 1, characterized in that The screening method is as follows: The collected aquaculture water body sludge is added to sterile physiological saline, shaken and mixed, and then kept at a temperature of 80°C to 85°C for 10min to 15min, and then cooled to below 35°C to obtain a suspension; The suspension is inoculated into a seed culture medium, and enriched and cultured at 34° C. to 38° C. for 20 h to 24 h, and separated by line drawing, and purified to obtain a target strain with good growth and spore production; After the target strain was subcultured for 5 times and stabilized, it was inoculated on the screening medium, and the strain with a large transparent zone was selected and identified as Bacillus subtilis B-1124.

4. The Bacillus subtilis B-1124 according to claim 3, characterized in that The seed culture medium comprises the following components: 10 g / L to 12 g / L of peptone, 1 g / L to 5 g / L of glucose, 5 g / L to 12 g / L of yeast extract, 0.1 g / L to 0.3 g / L of magnesium sulfate and 1 g / L to 2 g / L of potassium sulfate.

5. The Bacillus subtilis B-1124 according to claim 3, characterized in that The screening culture medium comprises the following components: 1g / L-5g / L of peptone, 3g / L-7g / L of glucose, 0.01g / L-0.05g / L of calcium chloride, 0.1g / L-0.5g / L of cellulase, 0.1g / L-0.5g / L of amylase, 0.1g / L-0.5g / L of lipase, 1g / L-3g / L of antioxidants, 0.1g / L-0.3g / L of magnesium sulfate and 0.5g / L-1g / L of potassium sulfate.

6. The Bacillus subtilis B-1124 according to claim 5, characterized in that Inoculate onto screening medium and culture at 35°C to 38°C for 24h to 48h.

7. An application of Bacillus subtilis B-1124, characterized in that: The application is the application of the Bacillus subtilis B-1124 described in claim 1 or the culture of the Bacillus subtilis B-1124 described in claim 1 in the preparation of a bacterial agent or a feed additive.

8. The use according to claim 7, characterized in that: The bacterial agent or the feed additive is used in aquaculture.

9. The use according to claim 7, characterized in that: The culture of Bacillus subtilis B-1124 is inoculated with seed liquid into a fermentation medium, and fermented for 12h to 15h at 30° C. to 38° C., a rotation speed of 150r / min to 250r / min, and a tank pressure of 0.03Mpa, then diatomaceous earth accounting for 7% to 10% of the mass of the fermentation medium is added, and the fermentation culture is continued for 5h to 10h under the above conditions to obtain the culture of Bacillus subtilis B-1124.

10. The use according to claim 9, characterized in that: The inoculation amount of the seed liquid is 1% to 5%.

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