Feed additive capable of resisting fish streptococcicosis and application of feed additive

Abstract

The invention relates to the technical field of microorganisms, in particular to a fish streptococcicosis-resistant feed additive and application thereof.The strain D7-HSJ9 applied to the additive is obtained by screening of an applicant, it is detected that the strain has good salt resistance, alkali resistance and antibacterial characteristics, especially has a good antibacterial effect on streptococcus, and after processing, the strain can be used for preparing a feed additive for fish streptococcicosis. The feed additive prepared by compounding the strain and traditional Chinese medicines can well inhibit the growth of streptococcus iniae and streptococcus agalactiae, the additive is used for preparing disease-resistant feed, and the additive has the remarkable advantages of being scientific in component compatibility, obvious in synergistic effect, capable of meeting the growth requirement of fish, capable of remarkably promoting the growth of the fish, capable of improving the feed utilization rate and capable of reducing the feed conversion ratio. In addition, the disease-resistant feed also has the effects of effectively improving the immunity of the fish and reducing the morbidity of the fish, and is a biological feed additive and a biological feed which are low in cost and safe in color.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, and in particular to a feed additive for resisting fish streptococcal disease and application thereof. [Background technology]

[0002] Streptococcal disease is a common bacterial disease in fish farming, posing a serious threat to marine and freshwater fish farming. Streptococcal disease has the characteristics of high mortality and strong infectiousness. The occurrence of this disease not only leads to the death of a large number of fish, but also brings huge economic losses to farmers. As the country with the largest fish production, export and consumption in the world, it is particularly important for my country to do a good job in the prevention and treatment of streptococcal disease.

[0003] In the current existing technology, the commonly used prevention and control methods for streptococcal disease are: 1. Using chlorine preparations or quicklime to spray the whole pond to disinfect the water body. This method often treats the symptoms but not the root cause. With the changes in the breeding environment, there is still a possibility of streptococcal infection after the breeding time is prolonged. Moreover, this solution cannot improve the disease resistance of tilapia itself. Chlorine preparations and quicklime may also affect the growth of fish; 2. Vaccine injection. This method is complicated to operate and cannot be widely promoted in large-scale intensive breeding. Moreover, the injection of vaccines requires surgical operation, which is generally performed by professionals. It is not easy to promote among farmers and will increase the breeding cost; 3. Feeding feed with added antibiotics. This solution will lead to the abuse of antibiotics and thus form drug-resistant bacteria. In addition, antibiotics will remain in the water and fish, causing consumers to worry about food safety.

[0004] Therefore, developing safe, healthy and effective new products has become an inevitable choice to improve the fish's ability to resist streptococcal disease. Probiotic feed additives have the advantages of being pure natural, non-toxic, free of drug residues, safe and convenient, comprehensive in function, and capable of producing antibacterial substances. They have become a hot topic of research at home and abroad. Therefore, developing new disease-resistant biological feed with probiotic fermented Chinese herbal medicine is the only choice to improve fish's resistance to streptococcal disease. [Summary of the invention]

[0005] In view of the above, it is necessary to screen strains with antibacterial activity for fish streptococcal disease, and make full use of the strains to prepare feed additives or disease-resistant feed and other products, which can effectively improve the prevention and control of streptococcal disease in fish, improve the production performance of fish, and provide a green and safe feed additive for fish farming.

[0006] In order to achieve the above object, the present invention adopts the following technical scheme:

[0007] A disease-resistant feed additive, the feed additive contains Bacillus inaquosorum strain D7-HSJ9, whose classification name is: Bacillus inaquosorum D7-HSJ9, the Chinese classification name is: Bacillus subtilis desert subspecies D7-HSJ9, and the preservation number is CCTCC NO: M20241842; the strain is preserved in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is August 26, 2024.

[0008] Furthermore, the pathogenic bacteria inhibited by the disease resistance are Streptococcus iniae and Streptococcus agalactiae.

[0009] The present invention also includes the use of the fermentation liquid of the Bacillus inaquosorum strain D7-HSJ9 in the preparation of amylase and / or cellulase.

[0010] The present invention also includes a method for preparing the feed additive, which comprises: fermenting Radix Isatidis with the Bacillus inaquosorum strain D7-HSJ9 to prepare the feed additive.

[0011] Furthermore, during the fermentation of the Radix Isatidis, the mass ratio of the Radix Isatidis to water is 0.02-0.15:1; and the inoculation amount of the Bacillus inaquosorum strain D7-HSJ9 is 2-10% of the volume percentage of the substrate.

[0012] Furthermore, during fermentation of the Bacillus inaquosorum strain D7-HSJ9, the pH value is 6-8, the fermentation temperature is 26-35° C., and the fermentation time is 2-4 days.

[0013] The present invention also includes a disease-resistant feed containing a feed additive, wherein the disease-resistant feed contains 10%-15% of the feed additive in a mass percentage, and the remainder is a basic feed.

[0014] The present invention also includes a method for preparing the disease-resistant feed, which comprises: adding the feed additive in an amount of 10%-15% by mass to the basic feed, mixing the mixture evenly, and then putting the mixture into a feed machine to prepare puffed feed; after the feed is prepared, drying it at room temperature; after drying, adding vegetable oil in a ratio of 1%-3% by mass to the feed and mixing the mixture evenly to obtain the puffed feed.

[0015] Furthermore, the basic feed consists of 30-45 parts of fish meal, 10-20 parts of soybean meal, 5-15 parts of peanut meal, 4-10 parts of corn flour, 15-30 parts of high-gluten flour, 3-6 parts of yeast, 1-3 parts of squid powder / paste, 1-4 parts of fish oil, 0.1-1 parts of soybean lecithin, 1-3 parts of monocalcium phosphate, 0.3-1 parts of compound multivitamins, 0.1-0.5 parts of compound multiminerals, 0.1-0.6 parts of choline chloride and 0.1-0.5 parts of alpha starch.

[0016] The above-mentioned compound multivitamin is a mixed liquid of vitamin B1, vitamin B2, and vitamin B6; it was purchased from Guangzhou Zhengge Agriculture and Animal Husbandry Service Co., Ltd.; the compound multi-mineral is a compound of Ca, Mg, Fe, Mn, Zn, P, K, Na, and Co mineral elements; it was purchased from Shanxi Yifei Biotechnology Co., Ltd.

[0017] The present invention also includes application of the feed additive or the disease-resistant feed in fish farming.

[0018] The present invention has the following beneficial effects:

[0019] 1. The desert Bacillus subtilis subspecies D7-HSJ9 of the present invention is obtained by screening from the rhizosphere soil of mangroves by the applicant. After testing, the strain has good salt resistance, alkali resistance and antibacterial properties, especially has a good antibacterial effect on streptococci. After processing, the strain and the Chinese medicine Radix Isatidis are combined to prepare a feed additive, which can well inhibit the growth of Streptococcus dolphins and Streptococcus agalactiae, and at the same time can significantly improve the disease resistance of tilapia to Streptococcus dolphins. It is a feed additive that can inhibit streptococci.

[0020] 2. The microbial feed additive of the present invention also has the advantages of low fermentation cost, low requirements for fermentation conditions and equipment, and simple process operation.

[0021] 3. The present invention utilizes the additive to prepare disease-resistant feed. The disease-resistant feed rationally selects Chinese herbal medicine and probiotics according to the physiological characteristics of fish. The ingredients are scientifically matched and have significant synergistic effects. It can meet the growth needs of fish, significantly promote the growth of fish, improve feed utilization, and reduce feed-to-meat ratio. In addition, it can also improve the immunity of fish, reduce the incidence of fish, and reduce the use of antibiotics in aquaculture. It is a green and safe biological feed additive and biological feed.

Brief Description of the Drawings

[0022] Figure 1 This is a graph showing the growth of strain D7-HSJ9 under different salinity conditions.

[0023] Figure 2 This is a graph showing the growth of strain D7-HSJ9 under different pH conditions.

[0024] Figure 3 This is a graph showing the growth of strain D7-HSJ9 at different temperatures.

[0025] Figure 4 This is a plate image of the amylase activity test of strain D7-HSJ9.

[0026] Figure 5 This is a plate image of the cellulase activity detection of strain D7-HSJ9.

[0027] Figure 6 This is a plate image showing the antibacterial effect of strain D7-HSJ9 on Streptococcus india.

[0028] Figure 7 This is the survival curve of each group of tilapia after challenge with Streptococcus iniae. [Specific implementation method]

[0029] All features disclosed in this specification, or steps in all methods or processes disclosed, except mutually exclusive features and / or steps, can be combined in any manner.

[0030] Any feature disclosed in this specification (including any appended claims and abstract), unless otherwise stated, is merely an example of a series of equivalent or similar features.

[0031] The strain D7-HSJ9 (abbreviated as D7-HSJ9) used in the examples of the present application is: Bacillus inaquosorum strain D7-HSJ9, its classification name is: Bacillus inaquosorum D7-HSJ9, its Chinese classification name is: Bacillus subtilis desert subspecies D7-HSJ9, and its preservation number is CCTCC NO: M20241842; the strain is deposited in the China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is August 26, 2024.

[0032] Example 1

[0033] The strain D7-HSJ9 used in this example was screened by the applicant and has the following characteristics:

[0034] 1. Salt tolerance:

[0035] The salinity tolerance experiment of strain D7-HSJ9 in this example is as follows: LB liquid culture medium with different percentage NaCl concentrations is prepared, sterilized at 121°C for 20 min, the activated D7-HSJ9 bacterial solution is added to the LB culture medium at 2%, three parallels are set for each salinity, shaken and cultured at 28°C, and the OD of the cultured bacterial solution is measured. 600, draw a growth curve. The LB medium composition is: tryptone 10g, yeast extract 5g, sodium chloride 10g, pH 6.8-7.2, pure water 1L. The results are as follows Figure 1 As shown by Figure 1 It can be seen that strain D7-HSJ9 has a strong tolerance to salinity; and the growth rate of D7-HSJ9 is not significantly different in the salinity range of 0‰-50‰, and the growth rate is inhibited in the salinity range of 60-70‰, indicating that strain D7-HSJ9 has the physiological characteristics of high salt tolerance.

[0036] 2. Broader pH Survival Capability:

[0037] The acid-base tolerance experiment of strain D7-HSJ9 in this example is as follows: prepare liquid LB medium, adjust the medium to different pH values ​​with glacial acetic acid and NaOH, sterilize at 121°C for 20 min, inoculate D7-HSJ9 bacterial solution in the above conditioned medium at a ratio of 2%, perform 3 parallel groups for each pH value, shake culture at 28°C, and measure the OD value of the cultured bacterial solution. 600 , draw a growth curve. The results are as follows Figure 2 As shown by Figure 2 It can be seen that D7-HSJ9 grows fastest between pH 6 and 8, with no significant difference, and its growth rate is inhibited at pH 9, and its growth is significantly inhibited at pH 3-5 and pH 10. This indicates that strain D7-HSJ9 has a wide range of pH growth capabilities and can survive between pH 6 and 8.

[0038] 3. Wide range of temperature tolerance:

[0039] The temperature tolerance experiment of strain D7-HSJ9 in this example: set different temperature gradient conditions, inoculate D7-HSJ9 bacterial solution into the culture medium at a ratio of 2%, shake culture at different temperatures, and measure the OD of the culture solution 600 , draw a growth curve. The results are as follows Figure 3 As shown by Figure 3 It can be seen that the optimal growth temperature of D7-HSJ9 is 26-35°C. The growth of D7-HSJ9 at 40°C is slower than that at 30°C. The growth rate of D7-HSJ9 is inhibited at 20°C and 50°C. This shows that strain D7-HSJ9 has a strong tolerance to temperature.

[0040] 4. Functional characteristics:

[0041] (1) Amylase activity: After activating the strain D7-HSJ9, 1 μl of the bacterial solution was added to the starch medium, and Escherichia coli (without amylase activity) was used as a control. The culture was cultured for 24 hours. After the culture was completed, 15 ml of iodine solution was added to the culture medium, and the culture medium was stained for 15 minutes to observe whether there was a transparent circle. The components of the starch medium were: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 10 g of soluble starch, 20 g of agar, pH 6.8-7.2, and 1 L of pure water. The results were as follows: Figure 4 As shown, an obvious hydrolysis transparent zone can be seen, indicating that Bacillus D7-HSJ9 has strong hydrolytic amylase activity.

[0042] (2) Cellulose activity: After the strain D7-HSJ9 was activated and cultured, it was added dropwise to CMC-Na medium and cultured for 24 h. After the colonies grew out, it was stained with 0.1% Congo red solution for 15 min and the dye was discarded. Then, an appropriate amount of 1 mol / L NaCl solution was added for decolorization for 30 min. PBS was used as a negative control and cellulase solution (200 U / ml) was used as a positive control to observe the presence of transparent circles. CMC-Na medium culture medium components: sodium carboxymethyl cellulose (CMC-Na) 15 g, NH 4 NO 3 1g, peptone 1g, MgSO 4 7H 2 O0.5g, KH 2 PO 4 1g, agar 20g, purified water 1L. The results are as follows Figure 5 As shown, obvious transparent circles can be seen, indicating that Bacillus D7-HSJ9 has the ability to produce cellulase and has a certain ability to decompose cellulose.

[0043] (3) Antibacterial effect on Streptococcus dolphinus: Using Streptococcus dolphinus as the indicator bacteria, the Oxford cup method was used to detect the antibacterial experiment of the test strain D7-HSJ9. The specific operation is: first pour a layer of LB solid culture medium in the plate, and after solidification, take 500μl of the activated bacterial solution and add it to the plate, and then pour the unsolidified LB solid culture medium to mix the indicator bacterial solution; use sterile tweezers to clamp the sterilized Oxford cup and place it on the unsolidified culture medium to punch holes. After the culture medium solidifies, use sterile tweezers to pull out the Oxford cup, and use a sterile inoculation loop to pick out the agar block in the hole. Put 100μl of strain D7-HSJ9 bacterial solution into the Oxford cup, and use the same amount of chloramphenicol with a concentration of 50μg / ml as the positive control, and sterile PBS as the negative control. The culture medium with the added sample is placed upright and cultured in a 28℃ incubator for 12 to 16 hours, and the size of the inhibition zone is observed. The results are as follows Figure 6 As shown in Table 1:

[0044] Table 1 Antibacterial activity of the culture supernatant of strain D7-HSJ9 against Streptococcus iniae

[0045]

[0046] Depend on Figure 6 As can be seen from Table 1, strain D7-HSJ9 has a good antibacterial effect on S. iniae, and the antibacterial effect of strain D7-HSJ9 bacterial solution is equivalent to that of 50 μg / ml chloramphenicol.

[0047] Example 2

[0048] The strain D7-HSJ9 was used to prepare feed additives. The specific scheme is as follows:

[0049] The product is prepared by fermenting the Chinese herbal medicine Radix Isatidis (produced in Bozhou, Anhui, Jiangsu and Hebei) with the strain D7-HSJ9; the specific method is as follows: drying and crushing the Radix Isatidis and then passing it through an 80-mesh sieve, weighing the Radix Isatidis powder and dissolving it in water to prepare a mixed solution of Chinese herbal medicine: water = 0.02:1, 0.10:1, and 0.15:1, sterilizing at 121°C for 20 minutes for use; inoculating the activated strain D7-HSJ9 bacterial solution into the mixed solution at a volume percentage of 10% (an inoculation ratio of 2-10% can meet experimental requirements) for fermentation, the fermentation conditions are 30°C, 180r / min, and the fermentation time is 3 days (a fermentation time of 2-4 days can meet experimental requirements), and after the fermentation is completed, it becomes a microbial feed additive.

[0050] (1) Determine the antibacterial properties of the additives. The specific test method is as follows:

[0051] The above three kinds of Radix Isatidis from different origins (Chinese herbal medicine: water = 0.02: 1, 0.10: 1, 0.15: 1) were fermented with strain D7-HSJ9, and then centrifuged at 4000rpm / min. The obtained supernatant was subjected to antibacterial test by Oxford cup method, and the indicator bacteria were Streptococcus iniae and Streptococcus agalactiae. The control of the experiment was Radix Isatidis liquid without D7-HSJ9 bacteria (Chinese herbal medicine: water = 0.15: 1), PBS blank control solution, chloramphenicol positive control solution, and D7-HSJ9 bacterial culture supernatant. The results are shown in Table 2.

[0052] Table 2 Antibacterial effect of fermentation of Chinese herbal medicine Radix Isatidis by strain D7-HSJ9

[0053]

[0054] As shown in Table 2, the product of Isatis indigotica fermented by strain D7-HSJ9 has a good antibacterial effect on Streptococcus dolphins and Streptococcus agalactiae, and is significantly stronger than the bacterial culture supernatant of D7-HSJ9 that has not been fermented by traditional Chinese medicine. There is no significant difference in the antibacterial activity of Isatis indigotica fermented by D7-HSJ9 from three origins.

[0055] (2) Determination of MIC of additives against Streptococcus dolphinus and Streptococcus agalactiae, the specific method is as follows:

[0056] The same amount (initial volume) of the bacterial fermentation Chinese medicine feed additives with different Chinese herbal medicine addition ratios was taken, and extracted with an organic solvent ethyl acetate (additive: organic solvent = 1:2). The control group was a D7-HSJ9 single bacterial fermentation broth under the same conditions. After extraction, the liquid was separated with a separatory funnel, and the organic phase was concentrated by rotary evaporation to obtain a concentrate with different additives. The concentrate was evaporated from the organic solvent in a fume hood to obtain dry matter. The dry matter was resuspended in DMSO to the initial volume of the bacterial fermentation Chinese medicine feed additive as an active substance extract. The minimum inhibitory concentration of Streptococcus dolphins and Streptococcus agalactiae was detected by the two-fold dilution method, and the dilution multiple of the active substance extract was used as an indicator for evaluating the MIC of different bacterial fermentation Chinese medicine feed additives. The results are shown in Table 3:

[0057] Table 3 The highest dilution multiples of the inhibition of Streptococcus india and Streptococcus agalactiae by Chinese herbal feed additives in different proportions of bacterial fermentation

[0058]

[0059] It can be seen from Table 3 that the bacterial fermentation Chinese medicine feed additives with different Chinese herbal medicine addition ratios have higher inhibitory activity against Streptococcus dolphinus than the control group, among which the dilution multiples of 10% addition and 15% addition are 16 times, which is significantly higher than the antibacterial activity of the control group; similar effects were achieved in the experiment on Streptococcus agalactiae, indicating that the bacterial fermentation Chinese medicine feed additive of the present invention can significantly improve the antibacterial effect against Streptococcus.

[0060] Example 3

[0061] The feed prepared by the feed additive of Example 2 was used to feed fish in a breeding experiment, and the specific method was as follows:

[0062] In this experiment, D7-HSJ09 fermented Radix Isatidis was used as a feed additive to prepare extruded feed for feeding tilapia, and the protective effect of the feed additive on tilapia infected with Streptococcus iniae was detected.

[0063] The feed preparation steps are as follows: prepare fermented isatis root with "Chinese herbal medicine: water = 0.02:1, 0.10:1, 0.15:1", fully mix the various components of the feed raw materials, and after mixing, evenly mix the above-mentioned D7-HSJ09 fermented isatis root liquid with an addition amount of 10-15% by mass (wherein the feed group adding "Chinese herbal medicine: water = 0.02:1" fermented Chinese medicine is named "2% addition group", the feed group adding "Chinese herbal medicine: water = 0.10:1" fermented Chinese medicine is named "10% addition group", the feed group adding "Chinese herbal medicine: water = 0.15:1" fermented Chinese medicine is named "15% addition group", and the control group does not add fermented Chinese medicine), put the mixed feed raw materials into a feed machine (Hebei Norda Machinery) to make puffed feed, dry the feed at room temperature after it is made, add vegetable oil at a mass percentage of 1%-3% and mix evenly with the feed after drying, which is the disease-resistant feed used in the experiment.

[0064] The basic feed raw material components mentioned above are: 32 parts of fish meal, 15 parts of soybean meal, 10 parts of peanut meal, 6 parts of corn flour, 20 parts of high-gluten flour, 5 parts of yeast, 2 parts of squid meal / paste, 2 parts of fish oil, 0.5 parts of soybean lecithin, 2 parts of monocalcium phosphate, 0.7 parts of compound multivitamin, 0.2 parts of compound multimineral, 0.5 parts of choline chloride and 0.3 parts of alpha starch. Each feed raw material is dried and crushed and then passed through an 80-mesh sieve for use. (Among them, the general ratio of basic feed raw materials in breeding applications is as follows: 30-45 parts of fish meal, 10-20 parts of soybean meal, 5-15 parts of peanut meal, 4-10 parts of corn flour, 15-30 parts of high-gluten flour, 3-6 parts of yeast, 1-3 parts of squid powder / paste, 1-4 parts of fish oil, 0.1-1 parts of soybean lecithin, 1-3 parts of monocalcium phosphate, 0.3-1 parts of compound multivitamins, 0.1-0.5 parts of compound multi-minerals, 0.1-0.6 parts of choline chloride and 0.1-0.5 parts of alpha starch, which can meet the production needs).

[0065] The above-mentioned compound multivitamin is a mixed liquid of vitamin B1, vitamin B2, and vitamin B6; it was purchased from Guangzhou Zhengge Agriculture and Animal Husbandry Service Co., Ltd.; the compound multi-mineral is a compound of Ca, Mg, Fe, Mn, Zn, P, K, Na, and Co mineral elements; it was purchased from Shanxi Yifei Biotechnology Co., Ltd.

[0066] The above feed was used to feed tilapia. The specific breeding experiment was as follows: the experimental fish was Nile tilapia, with an initial body weight of 14.628±1.61g. Three parallel groups were set up, with 60 fish in each group. The breeding experiment lasted for 60 days. After the experiment, the growth performance indicators of each group of tilapia were counted, and each group of tilapia was challenged with inia streptococcus to detect the disease resistance effect of each group of tilapia. The experimental group was fed with fermented Chinese medicine feed, and the control group was fed with ordinary feed without addition.

[0067] The above feed was fed to tilapia for 60 days, and the growth performance indicators of the fish were counted at the end of the day. The specific statistical indicators are as follows:

[0068] Initial average weight = initial weight of experimental fish / number of experimental fish

[0069] Final average weight = final weight of experimental fish / number of experimental fish

[0070] Weight gain rate WGR = [(W 末 -W 初 ) / W 初 ]×100%

[0071] Specific growth rate SGR = (lnW 末 -lnW 初 ) / D×100%

[0072] Where W 末 is the final body weight, W 初 is the initial body weight and D is the experimental period of 60 days.

[0073] Feed coefficient % = [feed amount / (final body weight - initial body weight) × 100%]; where the final body weight and initial body weight are both the average body weight of each group. The results are shown in Table 4:

[0074] Table 4 Effects of adding fermented Chinese medicine feed on the growth performance of tilapia

[0075]

[0076] Note: * indicates significant difference compared with the control group (P<0.05).

[0077] As can be seen from Table 4, the final average weight, weight gain rate and specific growth rate of the group with 15% fermented Chinese medicine feed were significantly higher than those of the other groups and the control group, and the feed coefficient was significantly lower than that of the control group. This shows that adding fermented Chinese medicine feed to conventional feed can significantly improve the growth performance of tilapia and improve feed utilization.

[0078] After the breeding experiment, 30 fish from each group were taken for the S. indica challenge experiment to detect the effect of adding fermented Chinese medicine feed on the survival rate of tilapia. The specific method was as follows: the pathogenic bacteria S. indica was activated three times, and the S. indica that was activated for the third time (1×10 8CFU / ml) for the challenge test; the experiment was divided into three groups: blank group (control group fish injected with PBS), negative control group (control group fish injected with Streptococcus dolphin), 2% added group (fermented Chinese medicine feed + injection of Streptococcus dolphin), 10% added group (fermented Chinese medicine feed + injection of Streptococcus dolphin), 15% added group (fermented Chinese medicine feed + injection of Streptococcus dolphin); the negative control group and the group added with fermented Chinese medicine feed were injected with 50ul of Streptococcus dolphin per fish, and the blank group was injected with the same amount of sterile PBS. After the challenge, the survival of each group of fish within 7 days was counted. The results are shown in Figure 7 As shown, from Figure 7 It can be seen that after the attack, the survival rate of the 15% added group (66.7%) and the survival rate of the 10% added group (60%) were significantly higher than those of the negative control group (6.67%). The protective effect of the fermented Chinese medicine group was increased by 64.29% compared with the negative control group, which indicates that fermented Chinese medicine can significantly enhance the disease resistance of tilapia to Streptococcus indolii.

[0079] In summary, the Bacillus subtilis desert subspecies D7-HSJ9 of the present invention is obtained by screening by the applicant. The strain has good salt resistance, alkali resistance and antibacterial properties, especially has a good antibacterial effect on Streptococcus iniae. After processing, the strain and the Chinese medicine Radix Isatidis are combined to prepare a feed additive, which can well inhibit the growth of Streptococcus iniae, and at the same time can significantly improve the disease resistance of tilapia to Streptococcus iniae. It is a green and safe feed additive that can inhibit Streptococcus iniae.

[0080] The above-described embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several modifications 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 present invention shall be subject to the attached claims.

Claims

1. A disease-resistant feed additive, characterized in that: The feed additive contains Bacillus inaquosorum strain D7-HSJ9, and its preservation number is CCTCC NO: M20241842.

2. The feed additive according to claim 1, characterized in that The pathogenic bacteria inhibited by the disease resistance are Streptococcus iniae and Streptococcus agalactiae.

3. Use of the fermentation broth of Bacillus inaquosorum strain D7-HSJ9 as claimed in claim 1 in the preparation of amylase and / or cellulase.

4. A method for preparing the feed additive as claimed in claim 1, characterized in that: The method comprises the following steps: fermenting Radix Isatidis with the Bacillus inaquosorum strain D7-HSJ9 to prepare the product.

5. The method according to claim 4, characterized in that During the fermentation of the isatis root, the mass ratio of the isatis root to water is 0.02-0.15:1; and the inoculation amount of the Bacillus inaquosorum strain D7-HSJ9 is 2-10% of the volume percentage of the substrate.

6. The method according to claim 5, characterized in that The pH value of the Bacillus subtilis desert subspecies (Bacillusinaquosorum) strain D7-HSJ9 during fermentation is 6-8, the fermentation temperature is 26-35° C., and the fermentation time is 2-4 days.

7. A disease-resistant feed comprising the feed additive according to any one of claims 1 or 2, characterized in that: The disease-resistant feed contains 10%-15% by mass of feed additives, and the remainder is basic feed.

8. A method for preparing the disease-resistant feed as claimed in claim 7, characterized in that: The method comprises the following steps: adding the feed additive in an amount of 10% to 15% by mass to the basic feed, mixing the mixture evenly, and then putting the mixture into a feed machine to prepare expanded feed; air-drying the prepared feed at room temperature; and adding vegetable oil in a proportion of 1% to 3% by mass to the feed and mixing the mixture evenly to obtain the expanded feed.

9. The disease-resistant feed according to claim 7 or the method according to claim 8, characterized in that: The basic feed consists of 30-45 parts of fish meal, 10-20 parts of soybean meal, 5-15 parts of peanut meal, 4-10 parts of corn meal, 15-30 parts of high-gluten flour, 3-6 parts of yeast, 1-3 parts of squid meal / paste, 1-4 parts of fish oil, 0.1-1 parts of soybean lecithin, 1-3 parts of monocalcium phosphate, 0.3-1 parts of compound multivitamins, 0.1-0.5 parts of compound multiminerals, 0.1-0.6 parts of choline chloride and 0.1-0.5 parts of alpha starch.

10. Use of the feed additive according to any one of claims 1 or 2 or the disease-resistant feed according to claim 7 in fish farming.

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