Fish streptococcicosis resistant fermented feed and preparation method thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to a fish streptococcicosis-resistant fermented feed and a preparation method thereof.The bacillus subtilis subsp. Deserticola D7-HSJ9 applied to the fermented feed is obtained through self screening of an applicant, and it is detected that the strain has good salt resistance, alkali resistance and antibacterial characteristics, and the bacillus subtilis subsp. Deserticola D7-HSJ9 can be used for preparing the fish streptococcicosis-resistant fermented feed. Compared with a traditional feed processing technology, when the bacterial strain is applied to feed processing, the finished feed fermented by the technology can well retain bioactive components in the fermented feed; in the feed manufacturing process, high-temperature and high-pressure processes are omitted, energy is saved, the influence of high temperature and high pressure on probiotics is avoided, and the feed has the advantages of being large in number of viable bacteria, good in fermentation effect and the like; in addition, the product can also improve the immunity of the fish, reduce the morbidity of the fish and reduce the application of antibiotics in aquaculture, and is a green and safe fermented feed.

Description

[Technical field]

[0001] The invention relates to the technical field of microorganisms, and in particular to a fermented feed resistant to fish streptococcus disease and a preparation method 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] The conventional prevention and treatment methods for streptococcal disease are as follows: 1. Spraying chlorine preparations or quicklime throughout the pond to disinfect the water. This method often only treats the symptoms but not the root cause. As the breeding environment changes, there is still a possibility of infection with streptococcal disease after the breeding time is prolonged. Moreover, this solution cannot improve the disease resistance of fish themselves. Chlorine preparations and quicklime may also affect the growth of fish; 2. Vaccination. 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, affecting 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. Probiotics have the advantages of being pure natural, non-toxic, free of drug residues, safe and convenient, comprehensive in function and able to produce antibacterial active substances. They have become a hot topic of research at home and abroad. Using probiotics to develop new disease-resistant biological feed is the only choice to improve fish's resistance to streptococcal disease.

[0005] Fermented soft pellet feed is a new type of fermented feed. It can promote the feeding of farmed animals by processing compound feed with microbial fermentation technology. Its advantages are: fermented soft pellet feed has the complete nutritional effect of microorganisms and their metabolites and compound feed; fermented soft pellet feed contains rich primary and secondary metabolites produced by microbial fermentation, which can promote body health and improve body immunity; in addition, fermented soft pellet feed contains a large number of beneficial live bacteria, which can regulate the intestinal microecology of animals after entering the animal body and prevent pathogens from colonizing in the intestine. Therefore, it is particularly important to develop new fermented soft pellet feed with probiotic fermented compound feed to prevent streptococcal diseases in fish. [Summary of the invention]

[0006] 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 fermented soft granular feed and other products. This product can effectively improve the prevention and treatment of fish streptococcal disease and provide a green and safe biological feed for fish farming.

[0007] In order to achieve the above object, the present invention adopts the following technical scheme:

[0008] A disease-resistant fermented feed, the fermented feed contains Bacillus inaquosorum strain D7-HSJ9, whose classification name is: Bacillus inaquosorum D7-HSJ9, and whose Chinese classification name is: Bacillus subtilis desert subspecies D7-HSJ9, and whose preservation number is CCTCC NO: M20241842; the strain is preserved in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, and the preservation date is August 26, 2024.

[0009] Furthermore, the pathogenic bacteria for disease resistance inhibition is Streptococcus iniae.

[0010] Furthermore, the fermented feed also contains Saccharomyces cerevisiae and / or Lactobacillus rhamnosus; the volume ratio of the strain D7-HSJ9, Lactobacillus rhamnosus and Saccharomyces cerevisiae is 1-3:0-1:0-1.

[0011] 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.

[0012] The present invention also includes a method for preparing the fermented feed, which comprises:

[0013] (1) Seed solution preparation: The activated D7-HSJ9 bacterial solution, the Lactobacillus rhamnosus bacterial solution and the Saccharomyces cerevisiae bacterial solution were inoculated into the D7-HSJ9 fermentation medium, the Lactobacillus rhamnosus fermentation medium or the Saccharomyces cerevisiae fermentation medium at an inoculation ratio of 2-5% by volume to obtain the corresponding seed solution. ；

[0014] (2) Mixing to prepare an initial fermentation liquid: mixing the seed liquid of step (1) with strain D7-HSJ9, Lactobacillus rhamnosus and Saccharomyces cerevisiae in a volume ratio of 1-3:0-1:0-1 to obtain an initial fermentation liquid;

[0015] (3) Preparing soft pellet feed: The initial fermentation liquid of step (2) is mixed with sterilized water at a ratio of 1:9 to obtain soft pellet fermentation liquid, and then the soft pellet fermentation liquid is added to the basic feed, mixed thoroughly, and then packaged into one-way ventilation fermentation bags, vacuumed, sealed, and fermented at 30° C. for 10-12 days to obtain the fermented feed.

[0016] Furthermore, in step (1), the pH value of D7-HSJ9 during fermentation is 6-8, the fermentation temperature is 26-35° C., and the fermentation time is 2-4 days.

[0017] Furthermore, the D7-HSJ9 fermentation medium is: 10g tryptone, 5g yeast powder, 10g sodium chloride, pH 6.8-7.2, and 1L pure water; the saccharomyces cerevisiae fermentation medium is: 20g tryptone, 10g yeast powder, 20g sodium chloride, 20g glucose, pH 6.5-7.2, and 1L pure water; the Lactobacillus rhamnosus fermentation medium is MRS broth medium.

[0018] Furthermore, the viable bacterial count of the seed liquid D7-HSJ9 in step (1) is not less than 1×10 9 CFU / ml; the number of viable Lactobacillus rhamnosus and Saccharomyces cerevisiae should not be less than 1×10 8 CFU / ml.

[0019] Furthermore, the initial fermentation broth in step (2) contains only strain D7-HSJ.

[0020] The present invention also includes application of the fermented feed in fish farming.

[0021] The present invention has the following beneficial effects:

[0022] 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 Streptococcus dolphinus. The applicant applies the strain to fermented feed processing and obtains good results. It is verified that the method of directly fermenting the finished feed with bacterial liquid in the fermentation process can better retain the biologically active components in the fermented feed. In addition, in the entire feed production process, no high temperature and high pressure process is performed, which can effectively save energy and avoid the influence of high temperature and high pressure on probiotics. The fermented feed has a large number of live bacteria (>1×10 7 CFU / g) and good fermentation effect; in addition, this product can effectively improve the immunity of fish, reduce the incidence of fish, and reduce the application of antibiotics in aquaculture. It is a green and safe fermented feed.

Brief Description of the Drawings

[0023] Figure 1This is a graph showing the growth of strain D7-HSJ9 under different salinity conditions.

[0024] Figure 2 This is a graph showing the growth of strain D7-HSJ9 under different pH conditions.

[0025] Figure 3 This is a graph showing the growth of strain D7-HSJ9 at different temperatures.

[0026] Figure 4 This is a plate image of the amylase activity test of strain D7-HSJ9.

[0027] Figure 5 This is a plate image of the cellulase activity detection of strain D7-HSJ9.

[0028] Figure 6 This is a graph showing the results of the automatic aggregation ability test of strain D7-HSJ9.

[0029] Figure 7 This is a plate image showing the antibacterial effect of strain D7-HSJ9 on Streptococcus india.

[0030] Figure 8 This is the survival curve of each group of tilapia after challenge with Streptococcus iniae. [Specific implementation method]

[0031] 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.

[0032] 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.

[0033] 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.

[0034] The yeast used in the examples is: Saccharomyces cerevisiae atcc9763, and the Lactobacillus rhamnosus is: Lactobacillus rhamnosus atcc7469 (both strains were purchased from Shanghai Beinuo Biotechnology Co., Ltd.).

[0035] Example 1

[0036] The strain D7-HSJ9 used in this example was screened by the applicant and has the following characteristics:

[0037] 1. Salt tolerance:

[0038] 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.

[0039] 2. Broader pH Survival Capability:

[0040] 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.

[0041] 3. Wide range of temperature tolerance:

[0042] 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.

[0043] 4. Functional characteristics:

[0044] (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.

[0045] (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 1 g, peptone 1 g, MgSO 4 7H 2 O 0.5g, KH 2 PO 4 1 g, agar 20 g, purified water 1 L. 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.

[0046] (3) Adhesion ability: The intestinal fixation ability of Bacillus D7-HSJ9 was evaluated by its automatic aggregation ability. The specific method was as follows: take the D7-HSJ9 bacterial solution cultured for 12 hours, set up 3 groups in parallel, centrifuge at 10000r / min, 4℃ for 5 minutes, collect the bacteria, wash twice with sterile PBS, resuspend with 2ml sterile double distilled water, oscillate for 30s, let stand, take the bacterial resuspension at 0, 1, 2, 3 and 24 hours, and measure its optical density at 600nm. The automatic aggregation ability (C, %) is calculated as follows: C = (1-D t / D 0 ). Among them, D 0 , D t are the optical densities at 0 and th respectively. The results are as follows Figure 6 As shown by Figure 6 It can be seen that the agglutination of Bacillus D7-HSJ9 was low in the first 3 hours, reaching 18.60%, and at 24 hours, the agglutination of Bacillus D7-HSJ9 increased significantly (P<0.05) to 89.77%, indicating that Bacillus D7-HSJ9 has good adhesion ability and can effectively colonize in the intestines of fish to form a barrier and prevent pathogens from colonizing in the intestines.

[0047] (4) 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 7 As shown in Table 1:

[0048] Table 1 Antibacterial activity of the culture supernatant of strain D7-HSJ9 against Streptococcus iniae

[0049]

[0050] Depend on Figure 7 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.

[0051] Example 2

[0052] The strain D7-HSJ9 was used to prepare fermented soft pellet feed. The specific scheme is as follows:

[0053] In this experiment, D7-HSJ9 bacterial liquid was combined with commercial feed fermented by lactic acid bacteria and yeast (Dingzu tilapia extruded feed, Jiangmen Xinjian Feed Co., Ltd.) as fermented soft pellet feed for feeding tilapia to detect the protective effect of different bacterial strains combined with fermented soft pellet feed on tilapia infected with inia Streptococcus dolphinus.

[0054] (1) Seed solution preparation: The activated D7-HSJ9 bacterial solution, Lactobacillus rhamnosus bacterial solution and Saccharomyces cerevisiae bacterial solution were inoculated at a volume percentage of 5% (the inoculation percentage of 2-5% can meet the experimental requirements) into the D7-HSJ9 fermentation medium, the Lactobacillus rhamnosus fermentation medium or the Saccharomyces cerevisiae fermentation medium to obtain the corresponding seed solution; the number of live bacteria of strain D7-HSJ9 in the seed solution was not less than 1×10 9 CFU / ml; the number of live Lactobacillus rhamnosus and Saccharomyces cerevisiae should not be less than 1×10 8 CFU / ml.

[0055] Among them, the components of D7-HSJ9 fermentation medium are: 10g tryptone, 5g yeast powder, 10g sodium chloride, pH 6.8-7.2, and 1L pure water; the fermentation conditions of D7-HSJ9 strain refer to the experimental conclusions of Example 1, and the preferred pH value is 6 (6-8), the fermentation temperature is 30°C (26-35°C), and the fermentation time is 3d (2-4d).

[0056] The fermentation medium of Saccharomyces cerevisiae is composed of 20 g tryptone, 10 g yeast powder, 20 g sodium chloride, 20 g glucose, pH 6.5-7.2, and 1 L pure water.

[0057] The Lactobacillus rhamnosus fermentation medium was MRS broth medium, which was purchased from Guangdong Huankai Microbiological Technology Co., Ltd.

[0058] (2) Mixing to prepare initial fermentation liquid: The seed liquid of step (1) is mixed in the following volume ratio to obtain initial fermentation liquid:

[0059] D7-HSJ9: Lactobacillus rhamnosus: Saccharomyces cerevisiae = 1:0:0

[0060] D7-HSJ9: Lactobacillus rhamnosus: Saccharomyces cerevisiae = 3:1:0

[0061] D7-HSJ9: Lactobacillus rhamnosus: Saccharomyces cerevisiae = 3:1:1

[0062] (3) Preparing soft pellet feed: The initial fermentation liquid of step (2) is mixed with sterilized water at a ratio of 1:9 to obtain soft pellet fermentation liquid, and then the soft pellet fermentation liquid is added to the basic feed, and the soft pellet fermentation liquid is mixed and packaged into one-way ventilation fermentation bags after the basic feed completely absorbs the soft pellet fermentation liquid. The bags are sealed after vacuuming, and fermented at 30°C for 12 days to obtain three groups of fermented soft pellet feeds (1:0:0 group, 3:1:0 group, and 3:1:1 group), that is, the corresponding soft pellet feeds are obtained; wherein the material-liquid ratio of the basic feed to the soft pellet fermentation liquid is 1:0.3-0.4.

[0063] The soft pellet feed prepared by the above method was subjected to live bacteria count and antibacterial property verification, and the specific scheme is as follows:

[0064] (1) Statistics of viable bacteria:

[0065] The initial live bacterial count of the soft pellet feed in step (3) before fermentation begins and the final live bacterial count after 12 days of fermentation (fermentation for 10-12 days can meet the experimental requirements) are counted. The specific method is: take 0.1 g of the feed at the beginning / end of fermentation and place it in a 1.5 ml sterile centrifuge tube, add 0.9 ml of sterile water to the centrifuge tube, crush the feed to make it evenly dissolved in the water, place it on a shaker (180 rpm, 28°C) and shake and mix for 30 minutes, take 100 μl of the mixed solution for gradient dilution, and count the number of live bacteria per gram of fermented soft pellet feed.

[0066] Table 2 shows the statistics of the number of live bacteria (CFU / g) in the fermented soft pellet feed at the beginning and end

[0067]

[0068] As can be seen from Table 2, the number of live bacteria in the three groups of fermented soft pellet feeds at the beginning and end was maintained at 1×10 7 CFU / g or above; indicating that the fermented soft pellet feed has more beneficial microorganisms.

[0069] (2) Antibacterial verification:

[0070] Take three groups of soft pellet feeds prepared after 12 days of fermentation, dry and grind them, weigh 30g each, and use excess ethyl acetate (solid-liquid ratio = 1:3-4) for organic extraction. The extraction time is 2 days. After the extraction, filter paper is used for solid-liquid separation; the organic phase is concentrated by rotary evaporation to obtain a concentrated solution, and the concentrated solution is fixed to obtain a concentrated standard solution; the extracted concentrated solution obtained under the same conditions of ordinary commercial feed is used as the control group. The two groups of extracts were used for antibacterial experiments, with the indicator bacteria being Streptococcus iniae, ethyl acetate as a blank control, and 50μg / ml chloramphenicol as a positive control. The antibacterial experiment was carried out by the filter paper method to detect the antibacterial activity of the three groups of fermented soft pellet feeds against Streptococcus iniae. The specific operation of the filter paper method antibacterial experiment is as follows: take 100 μl of activated Streptococcus indioides, apply it on the LB solid plate, stick the sterilized filter paper on the LB solid plate coated with Streptococcus indioides, add the sample to be tested to the filter paper (5 μl on each filter paper), invert it in a 28°C incubator, culture for 12 hours, and measure the antibacterial diameter. The results are shown in Table 3:

[0071] Table 3 shows the antibacterial activity of fermented soft pellet feed against Streptococcus india

[0072]

[0073] It can be seen from Table 3 that after the three groups of fermented soft pellet feeds were extracted, the active substance concentrates obtained had antibacterial activity against Streptococcus dolphins, and the antibacterial effect of the (1:0:0) group was higher than the antibacterial activity of 50 μg / ml chloramphenicol against Streptococcus dolphins, indicating that the fermented soft pellet feeds contained active substances that had antibacterial effects against Streptococcus dolphins.

[0074] Example 3

[0075] The soft pellet feed prepared in Example 2 was used to feed fish in a breeding experiment. The specific method is as follows:

[0076] The above three groups of fermented soft pellet feed were used to feed tilapia. The specific breeding experiment was as follows: the experimental fish was Nile tilapia, with an initial body weight of 5.24±0.26g, and three parallel groups were set up, with 60 fish in each group. The breeding experiment lasted for 28 days. After the experiment, each group of tilapia was challenged with inia streptococcus to detect the disease resistance of each group of tilapia. Among them, the experimental group was fermented soft pellet feed including 1:0:0 group, 3:1:1 group and 3:1:0 group, and the control group was ordinary commercial feed.

[0077] After the breeding experiment, 30 fish from each group were taken for the infestation experiment with Streptococcus indioides to detect the effect of fermented soft pellet feed on the survival rate of tilapia. The specific method was as follows: the pathogenic bacteria Streptococcus indioides was activated three times, and the Streptococcus indioides that was activated for the third time (1×10 8CFU / ml) for the challenge test; the experiment was divided into five groups: blank group (control group fish injected with PBS), negative control group (control group fish injected with Streptococcus dolphinus), (1:0:0) group, (3:1:1) group and (3:1:0) group. The negative control group and the three fermented soft pellet feed groups were injected with 50μl / fish of Streptococcus dolphinus, 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 8 As shown. Figure 8 It can be seen that within 7 days after the challenge, the three groups of fermented soft pellets all showed resistance to S. iniae. The survival rates of tilapia in each fermented soft pellet feed group were 60% in the (1:0:0) group, 56.67% in the (3:1:1) group, and 40% in the (3:1:0) group. Compared with the control group, the immune protection rates were increased by 42.86%, 38.10%, and 14.29%, respectively. This shows that fermented soft pellet feed can improve the disease resistance of tilapia to S. iniae, and D7-HSJ9 fermentation alone has the best effect on tilapia against S. iniae.

[0078] 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, and also has good adhesion ability, can colonize in the fish intestine, and has a good antibacterial effect on streptococci. After processing, the soft granular feed prepared by using the strain can significantly improve the disease resistance of tilapia to dolphin streptococci, and is a green and safe feed for preventing and treating fish streptococcus.

[0079] 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 fermented feed, characterized in that: The fermented feed contains Bacillus inaquosorum strain D7-HSJ9, and its preservation number is CCTCC NO: M20241842.

2. The fermented feed according to claim 1, characterized in that: The pathogenic bacteria for disease resistance inhibition is Streptococcus iniae.

3. The fermented feed according to claim 1, characterized in that: The fermented feed also contains Saccharomyces cerevisiae and / or Lactobacillus rhamnosus; the volume ratio of the strain D7-HSJ9, Lactobacillus rhamnosus and Saccharomyces cerevisiae is 1-3:0-1:0-1.

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

5. A method for preparing the fermented feed according to claim 1, characterized in that: The method is: (1) Seed solution preparation: The activated D7-HSJ9 bacterial solution, the Lactobacillus rhamnosus bacterial solution and the Saccharomyces cerevisiae bacterial solution are inoculated into the D7-HSJ9 fermentation medium, the Lactobacillus rhamnosus fermentation medium or the Saccharomyces cerevisiae fermentation medium at an inoculation ratio of 2-5% by volume to obtain the corresponding seed solution; (2) Mixing to prepare an initial fermentation liquid: mixing the seed liquid of step (1) with strain D7-HSJ9, Lactobacillus rhamnosus and Saccharomyces cerevisiae in a volume ratio of 1-3:0-1:0-1 to obtain an initial fermentation liquid; (3) Preparing soft pellet feed: The initial fermentation liquid of step (2) is mixed with sterilized water at a ratio of 1:9 to obtain soft pellet fermentation liquid, and then the soft pellet fermentation liquid is added to the basic feed, mixed thoroughly, and then packaged into one-way ventilation fermentation bags, vacuumed, sealed, and fermented at 30° C. for 10-12 days to obtain the fermented feed.

6. The method according to claim 5, characterized in that The pH value of D7-HSJ9 during fermentation in step (1) is 6-8, the fermentation temperature is 26-35° C., and the fermentation time is 2-4 days.

7. The method according to claim 5, characterized in that The D7-HSJ9 fermentation medium is: 10g of tryptone, 5g of yeast powder, 10g of sodium chloride, pH 6.8-7.2, and 1L of pure water; the saccharomyces cerevisiae fermentation medium is: 20g of tryptone, 10g of yeast powder, 20g of sodium chloride, 20g of glucose, pH 6.5-7.2, and 1L of pure water; the Lactobacillus rhamnosus fermentation medium is an MRS broth medium.

8. The method according to claim 5, characterized in that The viable bacterial count of the seed liquid D7-HSJ9 in step (1) is not less than 1×10 9 CFU / ml; the number of viable Lactobacillus rhamnosus and Saccharomyces cerevisiae should not be less than 1×10 8 CFU / ml.

9. The method according to claim 5, characterized in that The initial fermentation broth in step (2) contains only strain D7-HSJ.

10. Use of the fermented feed according to any one of claims 1 to 3 or the fermented feed prepared by the method according to claims 5 to 9 in fish farming.

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