Function type biological floc culture method based on bacterium-enzyme synergy

Abstract

The invention discloses a method for culturing functional biofloc based on bacterium-enzyme synergy. According to the invention, bacillus subtilis XYB4 and neutral protease are added into traditional biofloc to prepare the functional biofloc. The preparation method of the functional biofloc is simple and convenient, and under the synergistic effect of the bacillus subtilis XYB4 and the neutral protease, the capacity of the functional biofloc for converting ammonia nitrogen and nitrite nitrogen can be greatly improved. Besides, the peak values of ammonia nitrogen and nitrite nitrogen in the water body are lower than the concentration of traditional biological floccules in the process of culturing the biological floccules, the influence on aquatic animals is small, and the method has a wide application prospect in aquaculture and water body purification.

Description

Technical Field

[0001] The present invention relates to the technical field of biofloc cultivation, and specifically, to a method for cultivating functional bioflocs based on the synergy of bacteria and enzymes. Background Art

[0002] Biofloc Technology (BFT) promotes the growth of heterotrophic microorganisms in the water body by adding carbon sources to the aquaculture water body to increase the carbon-nitrogen ratio (C / N) of the water body. The organic debris formed in aquaculture production is adhered together by substances including bacteria, algae, protozoa, phytoplankton, etc. to form bioflocs. Bioflocs can not only purify water quality but also serve as feed for aquatic animals, improve feed utilization rate, and further play a role in improving economic and ecological benefits.

[0003] There are two ways to cultivate bioflocs, namely in-situ cultivation and ex-situ cultivation. In-situ cultivation means directly cultivating bioflocs in the aquaculture pond. After calculating the appropriate carbon-nitrogen ratio according to the composition and feeding amount of the fed feed, selecting an appropriate carbon source and directly putting it into the water body and ensuring sufficient dissolved oxygen and water agitation, bioflocs can be generated in the aquaculture pond. In contrast, the ex-situ cultivation technology separates the generation of bioflocs from aquaculture activities, creates conditions in an external reactor to produce bioflocs, and after cultivation, collects the flocs for feeding or uses them as a substitute for fish meal in aquatic feed. This method is easy to implement, highly controllable, and can solve problems such as the blockage of the gill filaments of cultured shrimp and hypoxia caused by excessive bioflocs generated by in-situ cultivation.

[0004] Traditional biofloc cultivation techniques mostly involve adding different carbon and nitrogen sources, or on this basis, cultivating bioflocs by combining bacteria and algae, but there is little research on the cultivation of bioflocs by combining bacteria and enzymes.

[0005] During the cultivation process of bioflocs, due to the addition of substances such as carbon sources and nitrogen sources, nitrifying bacteria will carry out biochemical decomposition and transformation. During this process, ammonification, nitrification, denitrification, and assimilation are carried out respectively. Driven by these processes, the ammonia nitrogen will first increase, and then after the ammonia nitrogen decreases, the nitrite nitrogen will further accumulate, increase, and then decrease. When the concentrations of ammonia nitrogen or nitrite nitrogen with acute toxicity to aquatic animals in the water body are relatively high, there is a risk of death of aquatic animals. At the same time, the conversion ability of bioflocs themselves for ammonia nitrogen and nitrite nitrogen is also crucial.

[0006] Therefore, it is of great significance to provide a biofloc with lower peak concentrations of ammonia nitrogen and nitrite in the water body during the cultivation process and with good nitrogen conversion ability itself. Summary of the Invention

[0007] To overcome the above-mentioned defects and deficiencies in the prior art, the present invention provides a method for culturing a functional biofloc based on the synergism of bacteria and enzymes.

[0008] The first object of the present invention is to provide an application of a composition in improving the nitrogen conversion ability of bioflocs.

[0009] The second object of the present invention is to provide a method for culturing bioflocs.

[0010] Therefore, the present invention claims the following:

[0011] An application of a composition in improving the nitrogen conversion ability of bioflocs, wherein the composition comprises Bacillus subtilis XYB4 and neutral protease, and the Bacillus subtilis XYB4 was deposited at the Guangdong Microbial Culture Collection Center on August 2, 2022, with the deposit number GDMCC NO: 62676, which is the Bacillus subtilis XYB4 in Chinese Patent CN116410888A.

[0012] Preferably, the nitrogen is nitrite nitrogen.

[0013] Preferably, the nitrogen is ammonia nitrogen.

[0014] A method for culturing bioflocs, introducing disinfected seawater into a culture unit, adding a carbon source, a nitrogen source, Bacillus subtilis XYB4 and neutral protease and mixing well, while aerating the water body, culturing for 70 - 74 days, and collecting the bioflocs when the ammonia nitrogen and nitrite nitrogen in the water body of the culture unit reach 0 - 0.5 mg / L, thus obtaining.

[0015] The Bacillus subtilis XYB4 was deposited at the Guangdong Microbial Culture Collection Center on August 2, 2022, with the deposit number GDMCC NO: 62676, which is the Bacillus subtilis XYB4 in Chinese Patent CN116410888A.

[0016] The bioflocs of the present invention are cultured in an off-site manner, and the added beneficial bacteria have the characteristics of safety and high efficiency, and play a crucial role in the aquaculture ecological environment system. Using beneficial microorganisms to improve the aquaculture ecological environment can enhance the immunity of the aquaculture objects themselves and promote the growth of the aquaculture objects.

[0017] Enzymes are biological macromolecules secreted by living cells with special catalytic functions, which are widely distributed in organisms and participate in a large number of biochemical reactions in the body. The addition of enzymes can be used to enhance the catalytic digestion efficiency of aquaculture objects and increase the protein content in bioflocs.

[0018] Preferably, the dosage ratio of the disinfected seawater, carbon source, nitrogen source, Bacillus subtilis XYB4 and neutral protease is 1 L:(6-7) g:(1-3) g:(0.5-1) g.

[0019] As an implementable method, the method for disinfecting seawater is: adding sodium hypochlorite to seawater for disinfection, and then adding sodium thiosulfate for neutralization. When there is no residual chlorine in the seawater, the disinfection is completed.

[0020] As an implementable method, the carbon source is glucose.

[0021] Preferably, the nitrogen source is shrimp compound feed.

[0022] More preferably, the nitrogen source is shrimp compound feed filtered through a 500-mesh sieve.

[0023] Preferably, the effective viable count of Bacillus subtilis XYB4 is (0.86-1.63)×10 9 CFU / L.

[0024] More preferably, the effective viable count of Bacillus subtilis XYB4 is 1.2×10 9 CFU / L.

[0025] Preferably, the dissolved oxygen in the water body is maintained at 5-7 mg / L, and the salinity of the water body is maintained at 26-30.

[0026] Preferably, the temperature of the water body is maintained at 26-30 °C, and the pH of the water body is maintained at 6.5-8.5.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention discloses a method for culturing a functional biofloc based on the synergism of bacteria and enzymes. The present invention adds Bacillus subtilis XYB4 and neutral protease to a traditional biofloc to prepare a functional biofloc. The method for preparing the functional biofloc of the present invention is simple. Under the synergistic action of Bacillus subtilis XYB4 and neutral protease, the ability of the functional biofloc to convert ammonia nitrogen and nitrite nitrogen can be greatly improved. In addition, during the process of culturing the biofloc, the peak values of ammonia nitrogen and nitrite nitrogen in the water body are lower than those of the traditional biofloc, and the impact on the aquaculture animals in the water body is smaller, having broad application prospects in aquaculture and water purification. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is the change trend of ammonia nitrogen in the water body for culturing the biofloc.

[0030] Figure 2 It is the change trend of nitrite nitrogen in the water body for culturing the biofloc.

[0031] Figure 3 The change trend of nitrate nitrogen in the water body for cultivating bioflocs.

[0032] Figure 4 The ammonia nitrogen conversion ability of bioflocs obtained by different cultivation methods.

[0033] Figure 5 The nitrite nitrogen conversion ability of bioflocs obtained by different cultivation methods. Specific implementation manners

[0034] The following specific embodiments are used to further illustrate the present invention, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0035] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0036] The Bacillus subtilis XYB4 used in the examples and comparative examples was deposited on August 2, 2022 at the Guangdong Provincial Microbial Culture Collection Center, with the deposit number GDMCC NO: 62676, and the deposit address is the 5th floor of Building 59, 100th Yard, Xianlie Middle Road, Guangzhou, and it is the Bacillus subtilis XYB4 in Chinese Patent CN116410888A.

[0037] Example 1 A cultivation method of functional bioflocs based on the synergism of bacteria and enzymes

[0038] S1. Introduce seawater into the reservoir, add sodium hypochlorite for disinfection (the recommended dosage ratio of seawater to sodium hypochlorite is 1L: 50mg, or 1m 3 : 50g), and then add sodium thiosulfate for neutralization (the recommended dosage ratio of sodium hypochlorite to sodium thiosulfate is 1g: 0.8g). After there is no residual chlorine in the seawater, obtain the seawater available for experiments, and introduce 50L of the seawater available for experiments into the biofloc cultivation barrel;

[0039] S2. Expand and activate Bacillus subtilis XYB4 to obtain a liquid bacterium agent of Bacillus subtilis XYB4, and the effective viable count of Bacillus subtilis XYB4 in the bacterium agent is 1.2×10 9 CFU / mL;

[0040] The method for expanding and activating the Bacillus subtilis XYB4 is as follows:

[0041] ① Mix 2216E liquid medium (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) with an equal proportion of ultrapure water and heat it. After full fusion, obtain medium A, and put it into a high-temperature sterilization pot for sterilization;

[0042] ② Place the sterilized medium A at about 28°C, add Bacillus subtilis XYB4 strain to the medium A, and place it in a constant temperature shaker to activate Bacillus subtilis XYB4. The constant temperature shaker is set at 36±1°C, 120rpm, and the shaker is set for 10h. After the culture is completed, use OD 600 The concentration of Bacillus subtilis XYB4 in medium A was measured to be 1.2×10 9 CFU / mL, the liquid inoculum of Bacillus subtilis XYB4 was obtained.

[0043] S3. 100 g of commercial shrimp feed powder filtered through a 500-mesh sieve, 330 g of glucose monohydrate, 50 mL of Bacillus subtilis XYB4 liquid inoculum and 25 g of neutral protease (enzyme content of 23,000 U / g) were placed in a 100-mesh filter bag, put into a biofloc culture barrel, and mixed thoroughly;

[0044] S4. Place an air stone in each biofloc culture tank to aerate the water in the culture tank and maintain the dissolved oxygen (DO) content of the water at 5-7 mg / L, the water temperature at 26-30°C, the pH at 6.5-8.5, and the salinity at 26-30. Cultivate for 72 days and test the ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the water every three days;

[0045] S5. When the ammonia nitrogen and nitrite nitrogen in the water in the culture tank are maintained at 0-0.5 mg / L, it indicates that the biofloc culture is successful.

[0046] Example 2 A method for cultivating functional biofloc based on bacterial enzyme synergy

[0047] This example is carried out with reference to Example 1, except that in step S3, the amount of neutral protease added is 50 g.

[0048] Comparative Example 1 A traditional biofloc culture method

[0049] The difference between this comparative example and Example 1 is that Bacillus subtilis XYB4 and neutral protease are not added, that is:

[0050] S1. Introduce seawater into the reservoir and add sodium hypochlorite for disinfection (the recommended dosage ratio of seawater to sodium hypochlorite is 1L:50mg, or 1m 3 : 50g), then add 1 part of sodium thiosulfate for neutralization (the recommended dosage ratio of sodium hypochlorite and sodium thiosulfate is 1g:0.8g). After there is no residual chlorine in the seawater, seawater usable for the experiment is obtained, and 50L of seawater usable for the experiment is introduced into the biofloc culture barrel;

[0051] S2. Put 100 g of shrimp commercial feed powder filtered through a 500-mesh sieve and 330 g of glucose monohydrate into a biofloc culture barrel, and mix and stir well.

[0052] S3. Place an air stone in each biofloc culture barrel, aerate the water in the culture barrel, and maintain the dissolved oxygen (DO) content of the water at 5 - 7 mg / L, the water temperature at 26 - 30 °C, the pH at 6.5 - 8.5, and the salinity at 26 - 30. Cultivate for 72 d, and detect the ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the water every three days.

[0053] S4. When the ammonia nitrogen and nitrite nitrogen in the water in the culture barrel are maintained at 0 - 0.5 mg / L, it indicates that the biofloc culture is successful.

[0054] Comparative Example 2 A method for culturing single-enzyme bioflocs

[0055] This comparative example is different from Example 1 in that Bacillus subtilis XYB4 is not added, that is:

[0056] S1. Introduce seawater into a reservoir, add sodium hypochlorite for disinfection (the recommended dosage ratio of seawater to sodium hypochlorite is 1 L: 50 mg, or 1 m 3 : 50 g), and then add sodium thiosulfate for neutralization (the recommended dosage ratio of sodium hypochlorite to sodium thiosulfate is 1 g: 0.8 g). After there is no residual chlorine in the seawater, obtain the seawater available for experiments, and introduce 50 L of the seawater available for experiments into a biofloc culture barrel;

[0057] S2. Put 100 g of shrimp commercial feed powder filtered through a 500-mesh sieve, 330 g of glucose monohydrate, and 25 g of neutral protease (enzyme content is 23000 U / g) into a biofloc culture barrel, and mix and stir well.

[0058] S3. Place an air stone in each biofloc culture barrel, aerate the water in the culture barrel, and maintain the dissolved oxygen (DO) content of the water at 5 - 7 mg / L, the water temperature at 26 - 30 °C, the pH at 6.5 - 8.5, and the salinity at 26 - 30. Cultivate for 72 d, and detect the ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the water every three days.

[0059] S4. When the ammonia nitrogen and nitrite nitrogen in the water in the culture barrel are maintained at 0 - 0.5 mg / L, it indicates that the biofloc culture is successful.

[0060] Comparative Example 3 A method for culturing single-bacteria bioflocs

[0061] This comparative example is different from Example 1 in that neutral protease is not added, that is:

[0062] S1. Introduce seawater into the reservoir and add sodium hypochlorite for disinfection (the recommended dosage ratio of seawater to sodium hypochlorite is 1L:50mg, or 1m 3 : 50g), then add sodium thiosulfate for neutralization (the recommended dosage ratio of sodium hypochlorite and sodium thiosulfate is 1g:0.8g). After there is no residual chlorine in the seawater, seawater usable for the experiment is obtained, and 50L of seawater usable for the experiment is introduced into the biofloc culture barrel;

[0063] S2. The Bacillus subtilis XYB4 was expanded and activated to obtain a liquid inoculum of Bacillus subtilis XYB4. The effective viable cell count of Bacillus subtilis XYB4 in the inoculum was 1.2×10 9 CFU / mL;

[0064] The method for expanding and activating the Bacillus subtilis XYB4 is as follows:

[0065] ① Mix 2216E liquid culture medium (Qingdao Hi-Tech Industrial Park Haibo Biotechnology Co., Ltd.) with ultrapure water in equal proportions and heat them. After fully blending, obtain culture medium A, and place it in a high-temperature sterilizer for sterilization;

[0066] ② Place the sterilized medium A at about 28°C, add Bacillus subtilis XYB4 strain to the medium A, and place it in a constant temperature shaker to activate Bacillus subtilis XYB4. The constant temperature shaker is set at 36±1°C, 120rpm, and the shaker is set for 10h. After the culture is completed, use OD 600 The concentration of Bacillus subtilis XYB4 in medium A was measured to be 1.2×10 9 CFU / mL, the liquid inoculum of Bacillus subtilis XYB4 was obtained.

[0067] S3. 100 g of commercial shrimp feed powder filtered through a 500-mesh sieve, 330 g of glucose monohydrate and 50 mL of Bacillus subtilis XYB4 liquid inoculant were placed in a biofloc culture barrel and mixed thoroughly;

[0068] S4. Place an air stone in each biofloc culture tank to aerate the water in the culture tank and maintain the dissolved oxygen (DO) content of the water at 5-7 mg / L, the water temperature at 26-30°C, the pH at 6.5-8.5, and the salinity at 26-30. Cultivate for 72 days and test the ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the water every three days;

[0069] S5. When the ammonia nitrogen and nitrite nitrogen in the water in the culture tank are maintained at 0-0.5 mg / L, it indicates that the biofloc culture is successful.

[0070] Example 4: Trends of changes in ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in water

[0071] I. Experimental Methods

[0072] During the cultivation of bioflocs, the initial nitrogen content in the water body was not high. Due to the addition of substances such as carbon sources and nitrogen sources, nitrifying bacteria would carry out biochemical decomposition and transformation. During this process, ammonification, nitrification, denitrification, and assimilation were carried out respectively. Driven by these processes, the ammonia nitrogen would increase first, and then after the ammonia nitrogen decreased, the nitrite nitrogen would further accumulate, increase, and then decrease. When the concentration of ammonia nitrogen or nitrite nitrogen, which is acutely toxic to aquatic animals, is relatively high, there is a risk of death for aquatic animals.

[0073] During the cultivation of the functional bioflocs based on the synergism of bacteria and enzymes (Example 1) and the traditional bioflocs (Comparative Example 1), the ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the water body were detected every three days to observe the changing trends of ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the water body.

[0074] II. Experimental Results

[0075] The changing trend of ammonia nitrogen in the water body during the cultivation of the functional bioflocs based on the synergism of bacteria and enzymes (Example 1) and the traditional bioflocs (Comparative Example 1) is shown in Table 1 and Figure 1 as follows. It can be seen from Table 1 that during the cultivation of the bioflocs in Example 1 and Comparative Example 1, the ammonia nitrogen concentration reached the peak on the 18th day. However, the ammonia nitrogen peak of the bioflocs in Example 1 was 15.1% lower than that in Comparative Example 1, indicating that the bioflocs obtained by cultivation in Example 1 had a lower ammonia nitrogen peak and less impact on aquatic animals.

[0076] The changing trend of ammonia nitrogen in the bioflocs obtained by cultivation in Example 2 was not significantly different from that in the bioflocs in Example 1.

[0077] Table 1 Changing trend of ammonia nitrogen in the water body (x±s)

[0078]

[0079] Note: Compared with the traditional bioflocs group at the same time point, * P<0.005, ** P<0.001.

[0080] The changing trend of nitrite nitrogen in the water body during the cultivation of the functional bioflocs based on the synergism of bacteria and enzymes (Example 1) and the traditional bioflocs (Comparative Example 1) is shown in Table 2 and Figure 2 as follows. It can be seen from Table 2 that during the cultivation of the bioflocs in Example 1 and Comparative Example 1, the nitrite nitrogen concentration reached the peak on the 42nd day. However, the nitrite nitrogen peak of the bioflocs in Example 1 was 11.3% lower than that in Comparative Example 1, indicating that the bioflocs obtained by cultivation in Example 1 had a lower nitrite nitrogen peak and less impact on aquatic animals.

[0081] The change trend of nitrite nitrogen in the bioflocs cultured in Example 2 was not much different from that of the bioflocs in Example 1.

[0082] Table 2 Change trend of nitrite nitrogen in water body (x±s)

[0083]

[0084] Note: Compared with the traditional biofloc group at the same time point, * P<0.005, ** P<0.001.

[0085] During the cultivation process of the functional bioflocs based on the cooperation of bacteria and enzymes (Example 1) and the traditional bioflocs (Comparative Example 1), the change trend of nitrate nitrogen in the water body is shown in Table 3 and Figure 3 as follows. It can be seen from Table 3 that during the cultivation process of the bioflocs in Example 1 and Comparative Example 1, the nitrate nitrogen began to increase continuously after 30 days and gradually stabilized after 66 days.

[0086] The change trend of nitrate nitrogen in the bioflocs cultured in Example 2 was not much different from that of the bioflocs in Example 1.

[0087] Ammonia nitrogen and nitrite nitrogen are toxic to aquatic organisms to a certain extent. One of the main functions of bioflocs is to convert ammonia nitrogen and nitrite nitrogen into relatively non-toxic nitrate nitrogen. Combining the trend changes of ammonia nitrogen and nitrite nitrogen concentrations, it can be seen that the bioflocs of the present invention were successfully cultured.

[0088] Table 3 Change trend of nitrate nitrogen in water body (x±s)

[0089]

[0090] Note: Compared with the traditional biofloc group at the same time point, * P<0.005, ** P<0.001.

[0091] Nitrogen conversion ability of the bioflocs in Example 5

[0092] I. Experimental method

[0093] After the bioflocs were successfully cultured (i.e., the concentrations of ammonia nitrogen and nitrite nitrogen in the bioflocs were less than 0.5 mg / L, recorded as 0h), ammonium chloride (AR, Shanghai Guoyao) and sodium nitrite (AR, Shanghai Guoyao) were used to artificially increase the ammonia nitrogen concentration in the biofloc water of each group (Example 1 and Comparative Examples 1 to 3) to about 20 mg / L and the nitrite nitrogen concentration to about 5 mg / L. Aeration was continued for 24h. During this period, the concentrations of ammonia nitrogen and nitrite nitrogen were detected every 4h, and the measured data were the nitrogen conversion capacity.

[0094] 2. Experimental Results

[0095] The ammonia nitrogen conversion capacity of the bioflocs in Example 1 and Comparative Examples 1 to 3 within 24 hours is shown in Tables 4 and Figure 4 As shown in Table 4, the ammonia nitrogen conversion rate of the biofloc prepared in Example 1 is much higher than that of the traditional biofloc, and is 22.6% higher than that of the biofloc in Comparative Example 1, indicating that the biofloc in Example 1 has excellent ability in converting ammonia nitrogen.

[0096] Table 4 Ammonia nitrogen conversion capacity (unit: mg / L, x±s)

[0097]

[0098] Note: Compared with the traditional biofloc group, the expressions with different letters are significant, P<0.05.

[0099] The nitrite conversion capacity of the bioflocs in Example 1 and Comparative Examples 1 to 3 within 24 hours is shown in Tables 5 and Figure 5 As shown in Table 5, under the synergistic effect of Bacillus subtilis XYB4 and neutral protease, the nitrite nitrogen conversion rate of the biofloc prepared in Example 1 was increased by 80.2% compared with the biofloc in Comparative Example 1, indicating that the biofloc in Example 1 has excellent ability in converting nitrite nitrogen.

[0100] It has been determined that the biofloc of Example 2 also has excellent ability in converting ammonia nitrogen and nitrite nitrogen.

[0101] Table 5 Nitrite nitrogen conversion capacity (unit: mg / L, x±s)

[0102]

[0103]

[0104] Note: Compared with the traditional biofloc group, the expressions with different letters are significant, P<0.05.

[0105] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a composition for improving the nitrogen conversion capacity of biological flocs, characterized in that: The composition comprises Bacillus subtilis XYB4 and neutral protease, and the Bacillus subtilis XYB4 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 2, 2022, with a collection number of GDMCC NO: 62676.

2. The use according to claim 1, characterized in that: The nitrogen is nitrite nitrogen.

3. The use according to claim 1, characterized in that: The nitrogen is ammonia nitrogen.

4. A method for cultivating biofloc, characterized in that: The sterilized seawater is introduced into the culture unit, and a carbon source, a nitrogen source, Bacillus subtilis XYB4 and a neutral protease are added and mixed thoroughly, and the water body is aerated at the same time. The culture is carried out for 70 to 74 days, and when the ammonia nitrogen and nitrite nitrogen in the water body of the culture unit reach 0 to 0.5 mg / L, the biofloc is collected to obtain; The Bacillus subtilis XYB4 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 2, 2022, with the deposit number GDMCC NO: 62676.

5. The culture method according to claim 4, characterized in that The usage ratio of the disinfected seawater, carbon source, nitrogen source and neutral protease is 1L: (6-7)g: (1-3)g: (0.5-1)g.

6. The culture method according to claim 4, characterized in that: The carbon source is glucose.

7. The culture method according to claim 4, characterized in that: The nitrogen source is a shrimp feed.

8. The culture method according to claim 4, characterized in that: The effective viable bacterial count of the Bacillus subtilis XYB4 is (0.86-1.63)×10 9 CFU / L.

9. The culture method according to claim 4, characterized in that: The dissolved oxygen of the water body is maintained at 5-7 mg / L, and the salinity of the water body is maintained at 26-30.

10. The culture method according to claim 4, characterized in that: The temperature of the water body is maintained at 26-30° C., and the pH of the water body is maintained at 6.5-8.5.

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