Application of functional bio-floc in prawn culture

Abstract

The application discloses application of functional bio-floc in prawn culture. Bacillus subtilis XYB4 and neutral protease are added into traditional bio-floc to prepare the functional bio-floc. The functional bio-floc is applied to prawn culture, which not only purifies the culture water quality and effectively reduces the content of ammonia nitrogen and nitrite nitrogen in the culture water, thereby reducing the toxicity of ammonia nitrogen and nitrite nitrogen to prawns, but also improves the growth performance and antioxidant enzyme activity of prawns, so that the green, environmentally-friendly, safe and efficient ecological prawn culture is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of prawn culture, in particular, to application of a functional bio-floc in prawn culture. BACKGROUND

[0002] China is a large country of aquaculture, and prawn is one of the important aquaculture varieties in China. Litopenaeus vannamei Boone Litopenaeus vannamei is popularly cultured in all parts of China due to its fast growth, strong disease resistance, and low requirement for feed nutrition, and has rapidly become one of the pillar industries of China's aquaculture. Litopenaeus vannamei Boone is one of the most important crustacean species in aquaculture, accounting for 70% of the total amount of prawn culture in the world. However, the environmental conditions of the culture water are deteriorating, and the traditional culture mode also leads to accumulation of a large amount of residual feed and feces in the water, resulting in serious pollution of the culture water, which has a toxic effect on the growth of Litopenaeus vannamei Boone.

[0003] Bio-floc technology (BFT) is a sustainable development type of culture technology that can recycle nutrient waste, save resources, avoid pollution, and provide high-quality protein sources. There are two ways of bio-floc culture, namely, in-situ culture and ex-situ culture. In-situ culture is to culture bio-floc directly in the culture pond. After calculating the appropriate carbon-nitrogen ratio according to the composition and feeding amount of the feed, the appropriate carbon source is directly fed into the water body to ensure sufficient dissolved oxygen and water agitation, so that bio-floc can be generated in the culture pond. Bio-floc directly utilizes ammonia nitrogen and feed residues in the water to form bacterial protein, which is utilized by the cultured objects again.

[0004] In contrast, ex-situ culture technology separates the generation of bio-floc from the culture activity, creates conditions to produce bio-floc in an external reactor, and feeds the collected bio-floc after culture or uses it as a substitute for fish meal in aquatic feed. This method is easy to implement, controllable, and can solve the problems of excessive bio-floc generated by in-situ culture, such as gill filament blockage and hypoxia of cultured prawns.

[0005] Studies have shown that reasonable application of bio-floc technology can significantly improve the growth performance of aquatic animals, but different aquatic animals have different utilization of bio-floc, and the bio-floc obtained by different culture methods also has different effects on the growth, enzyme activity of aquatic animals, and water quality of the culture water.

[0006] Therefore, it is of great significance to provide a bio-floc that can promote healthy culture of prawns and improve the growth performance, enzyme activity, etc. of prawns. SUMMARY

[0007] In order to overcome the above-mentioned defects and shortcomings in the prior art, the application provides an application of a functional bio-floc in prawn culture.

[0008] A first object of the application is to provide an application of a bio-floc in prawn culture.

[0009] Therefore, the application claims the following:

[0010] An application of a bio-floc in prawn culture, the bio-floc being obtained by the following steps: introducing disinfected seawater into a culture unit, adding a carbon source, a nitrogen source, Bacillus subtilis XYB4 and neutral protease and fully mixing, at the same time, aerating the water body, culturing for 70-74 days, and collecting the bio-floc when the ammonia nitrogen and nitrite nitrogen in the water body in the culture unit reach 0-0.5 mg / L, to obtain the bio-floc.

[0011] The Bacillus subtilis XYB4 is preserved in the Guangdong Microbial Culture Collection Center on August 2, 2022, with a preservation number of GDMCC NO: 62676, and is Bacillus subtilis XYB4 in Chinese Invention Patent CN116410888A;

[0012] The neutral protease and the disinfected seawater are used in a ratio of (0.5-1) g: 1 L.

[0013] As an implementable way, the method for disinfecting seawater is to add sodium hypochlorite to the seawater for disinfection, and then add sodium thiosulfate for neutralization, so that the seawater is disinfected without residual chlorine.

[0014] The bio-floc of the application is cultured in a ectopic manner, and the added beneficial bacteria have the characteristics of safety and high efficiency, and play a crucial role in the aquatic ecological environment system. Using beneficial microorganisms to improve the ecological environment of aquaculture can improve the intestinal flora richness of the cultured objects, thereby improving the immunity and promoting the growth of the cultured objects.

[0015] Enzymes are biological macromolecules secreted by living cells with special catalytic functions, widely distributed in organisms, and involved in a large number of biochemical reactions in the body. The addition of enzymes can be used to strengthen the catalytic digestion efficiency of the cultured objects and improve the protein content in the bio-floc.

[0016] The inventors found that the bio-floc obtained by using a low dose of neutral protease can effectively improve the survival rate and CAT activity of prawns in prawn culture, and therefore preferably, the neutral protease and the disinfected seawater are used in a ratio of 0.5 g: 1 L.

[0017] Preferably, the bio-floc is used to improve the survival rate of prawns.

[0018] Preferably, the bio-floc is used to improve the antioxidant enzyme activity of the prawns.

[0019] More preferably, the antioxidant enzyme is peroxidase.

[0020] Preferably, the bio-floc is used to reduce the content of ammonia nitrogen in the water body for prawn culture.

[0021] Preferably, the bio-floc is used to reduce the content of nitrite nitrogen in the water body for prawn culture.

[0022] Preferably, the carbon source is glucose.

[0023] Preferably, the nitrogen source is prawn commercial feed.

[0024] More preferably, the effective viable count of the Bacillus subtilis XYB4 is 1.2x10 9 CFU / L.

[0025] Preferably, the Bacillus subtilis XYB4 is added once every 3 days during the cultivation of the bio-floc.

[0026] Preferably, the use amount ratio of the carbon source, the nitrogen source and the disinfected seawater is (1-3) g:(6-7) g:1 L.

[0027] More preferably, the use amount ratio of the carbon source, the nitrogen source and the disinfected seawater is 2 g:6.6 g:1 L.

[0028] As an implementable mode, the prawns are Litopenaeus vannamei, Marsupenaeus japonicus, Penaeus monodon, etc.

[0029] Compared with the prior art, the present application has the following beneficial effects:

[0030] The present application discloses an application of a functional bio-floc in prawn culture. Bacillus subtilis XYB4 and neutral protease are added to a traditional bio-floc to prepare a functional bio-floc. The functional bio-floc is applied to prawn culture, which has the following advantages:

[0031] (1) The bio-floc of the present application can purify the water quality for culture, effectively reduce the content of ammonia nitrogen and nitrite nitrogen in the water body for culture without water replacement, and the peak values of ammonia nitrogen and nitrite nitrogen in the water body during culture are lower than those in the group without the bio-floc, and the toxicity to aquatic animals is smaller.

[0032] (2) The bio-floc of the present application can maintain the water quality index of the water body for culture to be good for 1-2 months without replacing the water body for culture, which can reduce the water consumption and water replacement to some extent, so as to achieve the purpose of saving water and realize efficient and green ecological culture.

[0033] (3) The use of the bio-floc according to the present application for shrimp culture can improve the survival rate, weight gain, growth rate and antioxidant enzyme activity of the shrimp. The inventors have unexpectedly found that the bio-floc obtained by using a low dose of neutral protease can further improve the CAT activity of the shrimp, thereby increasing the survival rate of the shrimp. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The change trend of ammonia nitrogen in the water body during Litopenaeus vannamei culture.

[0035] Figure 2 The change trend of nitrite nitrogen in the water body during Litopenaeus vannamei culture.

[0036] Figure 3 The change trend of nitrate nitrogen in the water body during Litopenaeus vannamei culture.

[0037] Figure 4 The CAT enzyme activity of Litopenaeus vannamei. DETAILED DESCRIPTION

[0038] The present application will be further described below in conjunction with specific examples, but the examples do not limit the present application in any form. Unless otherwise specified, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0039] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0040] The Bacillus subtilis XYB4 used in the examples was deposited at the Guangdong Microbial Culture Collection Center on August 2, 2022, with the accession number GDMCC NO: 62676, and the address of the depositary is Building 59, No. 100, Martyrs' Road, Guangzhou, China. It is the Bacillus subtilis XYB4 in Chinese Invention Patent CN116410888A.

[0041] The composition of the shrimp commercial feed used in the examples is: crude protein ≥ 50.0%, crude fat ≥ 0%, crude fiber ≤ 5.0%, crude ash ≤ 18.0%, lysine ≥ 2.5%, total phosphorus ≥ 1.5%, and water content ≤ 10.0%.

[0042] Example 1 A method for culturing Litopenaeus vannamei using bacteria plus low enzyme functional bio-floc

[0043] 1. Culturing method of bacteria plus low enzyme functional bio-floc

[0044] In this embodiment, the culturing method of the bacteria plus low enzyme functional bio-floc includes the following steps:

[0045] S1. Introduce seawater into the reservoir, add sodium hypochlorite for disinfection (the recommended dosage ratio of seawater and sodium hypochlorite is 1 L: 50 mg, or 1 m 3 : 50 g), then add sodium thiosulfate for neutralization (the recommended dosage ratio of sodium hypochlorite and sodium thiosulfate is 1 g: 0.8 g). After there is no residual chlorine in the seawater, the seawater is obtained for use in the experiment, and 50 L of the seawater obtained for use in the experiment is introduced into the bio-floc culture barrel;

[0046] S2. The Bacillus subtilis XYB4 is expanded and activated to obtain a Bacillus subtilis XYB4 liquid inoculum, and the effective viable count of the Bacillus subtilis XYB4 in the inoculum is 1.2×10 9 CFU / mL;

[0047] The method for expanding and activating the Bacillus subtilis XYB4 is as follows:

[0048] ① The 2216E liquid medium (Qingdao Gaosi Technology Industrial Park Haibo Biotechnology Co., Ltd.) is mixed with an equal proportion of ultrapure water and heated, and after being fully mixed, a culture medium A is obtained and placed in a high-temperature sterilization pot for sterilization;

[0049] ② After the sterilization of the culture medium A is completed, the culture medium A is placed at about 28℃, and the Bacillus subtilis XYB4 strain is added to the culture medium A, and then the culture medium A is placed in a constant-temperature shaking bed for activation of the Bacillus subtilis XYB4, and the constant-temperature shaking bed is set at 36±1℃, a rotation speed of 120 rpm, and a timer of 10 h. After the culture is completed, the OD 600 of the culture medium A is measured, and when the concentration of the Bacillus subtilis XYB4 in the culture medium A is 1.2×10 9 CFU / mL, the Bacillus subtilis XYB4 liquid inoculum is obtained.

[0050] S3. 100 g of white shrimp commercial feed powder filtered through a 500-mesh screen, 330 g of glucose monohydrate, 50 mL of Bacillus subtilis XYB4 liquid inoculum, and 25 g of neutral protease (enzyme content of 23000 U / g) are placed in a 100-mesh filter bag and put into the bio-floc culture barrel for thorough mixing and stirring;

[0051] S4. One air stone is placed in each bio-floc culture barrel, the water body in the culture barrel is aerated, and the dissolved oxygen (DO) content of the water body is maintained at 5.7-7.9 mg / L, the water body temperature is 26-30℃, the pH is 7.3-8.0, the salinity is 27-30, and the culture is carried out for 72 d. During the culture, the Bacillus subtilis XYB4 liquid inoculum is added once every three days, and the addition amount is 1 mL / L. The water body ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen are detected once every three days;

[0052] S5. The water body in the culture barrel is maintained at 0-0.5 mg / L of ammonia nitrogen and nitrite nitrogen, indicating that the biological floc culture is successful.

[0053] 2. Penaeus vannamei culture

[0054] (1) Take 0.3 m 3 of plastic barrels as culture barrels, and store the experimental available seawater obtained in step S1 into each culture barrel at a ratio of biological floc: experimental available seawater = 1:100 (the volume of the biological floc is measured by an Erlenmeyer flask, and the experimental water body obtained in step 1 is allowed to stand and settle for 30 min, and then the supernatant is removed, 2.5 L of the turbid solid after settling is taken as the biological floc, and 250 L of experimental available seawater is used for Penaeus vannamei culture), and the biological floc obtained in step 1 is inoculated into the culture barrel, and 2 air stones are placed in each culture barrel to fully aerate the water body, and the dissolved oxygen content of the water body is maintained at more than 5 mg / L.

[0055] (2) Select Penaeus vannamei with a body length of 3.4±0.4 cm and a body weight of 0.5±0.2 g, and inoculate 100 Penaeus vannamei into each culture barrel. Commercial feed for Penaeus vannamei is used for feeding during the culture period, and the inoculated biological floc is activated and activated using commercial feed for Penaeus vannamei as the nitrogen source and glucose as the carbon source. After the biological floc is activated, Bacillus subtilis XYB4 liquid inoculum is added once every 3 days (the addition amount is 1 mL / L), so as to increase the content of probiotics in the culture water body and promote the nitrogen transformation rate of the water body.

[0056] During the culture period, the water temperature is maintained at 29-32℃, the total alkalinity of the water body is 150-250 mg / L (if the total alkalinity of the water body is too low, sodium bicarbonate is used for adjustment), and the deposition amount of the biological floc in the culture water body is measured regularly. If the FV 30min (30 min biological floc settlement amount) is greater than 40 mg / L, a 100-200 mesh filter screen is used for salvage to prevent the gill filaments of Penaeus vannamei from being blocked and Penaeus vannamei from dying due to oxygen deficiency caused by excessive amount of biological floc in the water body.

[0057] The culture time of Penaeus vannamei is 21 days, and the ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen contents in the culture water body are measured once every 3 days during the culture period. After the culture is completed, the growth performance and antioxidant enzyme activity of Penaeus vannamei are counted.

[0058] Example 2: A method for culturing Penaeus vannamei using a bacterial enzyme functional biological floc

[0059] This example is carried out according to Example 1, and the difference is that in step S3, the addition amount of neutral protease is 50 g.

[0060] Comparative Example 1

[0061] 1. Take 0.3 m 3 of plastic barrels as culture barrels, and store the experimental available seawater obtained in the above step S1 into each culture barrel. Place 2 air stones in each culture barrel to fully aerate the water body, and maintain the dissolved oxygen content of the water body to be greater than 5 mg / L.

[0062] 2. Select Litopenaeus vannamei with a body length of 3.4±0.4 cm and a body weight of 0.5±0.2 g, and put them into the culture barrels. Put 100 Litopenaeus vannamei into each culture barrel, and feed the Litopenaeus vannamei with commercial feed during the culture period.

[0063] During the culture period, maintain the water temperature of the culture water body to be 29-32℃, and the total alkalinity of the water body to be 150-250 mg / L (if the total alkalinity of the water body is too low, use sodium bicarbonate to adjust).

[0064] The Litopenaeus vannamei culture time is 21 days, during which the ammonia nitrogen, nitrite nitrogen and nitrate nitrogen contents in the culture water body are measured every 3 days. After the culture ends, the growth performance and antioxidant enzyme activity of the Litopenaeus vannamei are counted.

[0065] Test Example 1 Change Trend of Ammonia Nitrogen, Nitrite Nitrogen and Nitrate Nitrogen in Culture Water Body

[0066] I. Experimental Method

[0067] During the culture period of the Litopenaeus vannamei, the ammonia nitrogen, nitrite nitrogen and nitrate nitrogen contents in the culture water body are measured every 3 days, and the change trends of the ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the culture water bodies of Example 1, Example 2 and Comparative Example 1 are observed.

[0068] II. Experimental Method

[0069] 1. Change Trend of Ammonia Nitrogen and Nitrite Nitrogen

[0070] The change trend of the ammonia nitrogen in the culture water bodies of Example 1, Example 2 and Comparative Example 1 is shown in Figure 1 . As can be seen from Figure 1 , the culture water body of Comparative Example 1 appeared peak values on the 6th day and the 15th day, respectively, and gradually stabilized after 15 days, with the ammonia nitrogen content maintained at about 3 mg / L.

[0071] While the peak values of the culture water bodies of Example 1 and Example 2 appeared on the 6th day, which was significantly lower than the ammonia nitrogen peak value of Comparative Example 1, and the ammonia nitrogen peak values of Example 1 and Example 2 gradually decreased after the 6th day, eventually decreasing to 0-0.5 mg / L, indicating that the bio-floc in Example 1 and Example 2 can effectively reduce the ammonia nitrogen content in the culture water body.

[0072] The change trend of nitrite nitrogen in the aquaculture water bodies of Example 1, Example 2 and Comparative Example 1 is shown in Figure 2 As shown in Figure 2 It can be seen that the concentration of nitrite nitrogen in the aquaculture water body of Comparative Example 1 gradually increased after the 9th day, and the content of nitrite nitrogen reached about 20 mg / L at the 21st day.

[0073] While the peak value of nitrite nitrogen concentration in the aquaculture water bodies of Example 1 and Example 2 was only about 5 mg / L, and gradually decreased after the 15th day and the 12th day respectively, and finally reduced to 0-0.5 mg / L, indicating that the bio-floc in Example 1 and Example 2 can effectively reduce the content of nitrite nitrogen in the aquaculture water body.

[0074] When the concentration of ammonia nitrogen or nitrite nitrogen, which is acutely toxic to aquatic animals, is high, the aquatic animals are at risk of death. Figure 1 And Figure 2 The results show that the peak values of ammonia nitrogen and nitrite nitrogen in the aquaculture water bodies are lower when using the methods of Example 1 and Example 2 to breed Litopenaeus vannamei, and the influence on Litopenaeus vannamei is smaller. Therefore, adding bio-floc to the aquaculture water body can effectively improve the ecological environment and purify the water quality of shrimp culture. Moreover, the present application carries out the breeding of Litopenaeus vannamei without water change, and to some extent, the present application can also reduce the amount of water used for breeding, so as to achieve the purpose of saving water and realizing efficient and green ecological breeding.

[0075] 2. Change trend of nitrate nitrogen

[0076] As shown in Figure 3 The results show that the concentration of nitrate nitrogen in the aquaculture water bodies of Example 1, Example 2 and Comparative Example 1 continues to increase. Ammonia nitrogen and nitrite nitrogen have a certain toxicity to aquatic animals, and one of the main functions of bio-floc is to convert ammonia nitrogen and nitrite nitrogen into relatively non-toxic nitrate nitrogen. Figure 3 The results show that the yield of nitrate nitrogen in the examples is higher than that in the comparative example after adding bio-floc, indicating that bio-floc can effectively promote the conversion of ammonia nitrogen and nitrite nitrogen in the aquaculture water body.

[0077] Test Example 2: Effect of bio-floc on the growth performance and antioxidant enzyme activity of Litopenaeus vannamei

[0078] I. Experimental method

[0079] After the end of the breeding, the Litopenaeus vannamei was weighed, and the body length, body weight, survival rate, weight gain rate and specific growth rate were calculated:

[0080] Weight gain rate = (final body weight - initial body weight) / initial body weight x 100%;

[0081] Specific growth rate = (ln final body weight - ln initial body weight) / breeding period × 100%;

[0082] At the same time, the peroxidase (CAT) activity in the hepatopancreas of Litopenaeus vannamei was determined using a kit.

[0083] 2. Experimental Results

[0084] Growth performance results are shown in Table 1. The results demonstrate that the growth performance of the shrimp in the Examples significantly increased compared to Comparative Example 1. Notably, the survival rate of shrimp cultured using the low-enzyme functionalized biofloc of Example 1 was 6.59% higher than that of the medium-enzyme functionalized biofloc of Example 2.

[0085] Table 1 Growth performance of Litopenaeus vannamei

[0086]

[0087] Note: The results are expressed as "mean ± standard deviation". Different letters indicate significant differences. P <0.05.

[0088] The results of antioxidant enzyme activity are shown in Table 2 and Figure 4 The results showed that compared with Comparative Example 1, the antioxidant enzyme activities of the shrimp in the examples were significantly increased. Notably, the CAT activity of the shrimp in the culture of the shrimp using the low-enzyme functional biofloc of Example 1 increased by 30.8% compared with that in Example 2.

[0089] Table 2 Antioxidant enzyme activities of Litopenaeus vannamei

[0090]

[0091] Note: The results are expressed as "mean ± standard deviation".

[0092] The above data demonstrate that an increase in neutral protease levels in bioflocs leads to a decrease in CAT levels in Litopenaeus vannamei, resulting in a decrease in survival rate. This further demonstrates that aquatic animals utilize biofloc differently. Therefore, when using bioflocs for aquaculture, it is necessary to cultivate appropriate bioflocs for different aquaculture species in order to maximize the benefits of biofloc technology in aquaculture.

[0093] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. An application of biofloc in shrimp farming, characterized in that: The bioflocs are obtained by culturing the following steps: introducing disinfected seawater into a culture unit, adding a carbon source, a nitrogen source, Bacillus subtilis XYB4 and a neutral protease and fully mixing, aerating the water, culturing for 70 to 74 days, and collecting the bioflocs when the ammonia nitrogen and nitrite nitrogen in the water of the culture unit reach 0 to 0.5 mg / L; The Bacillus subtilis XYB4 was deposited in Guangdong Provincial Microbiological Culture Collection Center on August 2, 2022, with the deposit number GDMCC NO: 62676; The usage ratio of the neutral protease to the disinfected seawater is (0.5-1) g:1 L.

2. The use according to claim 1, characterized in that The usage ratio of the neutral protease to the disinfected seawater is 0.5 g:1 L.

3. The use according to claim 1 or 2, characterized in that The biofloc is used for improving the survival rate of shrimp.

4. The use according to claim 1 or 2, characterized in that The biofloc is used for improving the antioxidant enzyme activity of shrimp.

5. The use according to claim 4, characterized in that The antioxidant enzyme is peroxidase.

6. The use according to claim 1 or 2, characterized in that The biofloc is used to reduce the ammonia nitrogen content in water for shrimp farming.

7. The use according to claim 1 or 2, characterized in that The biofloc is used to reduce the content of nitrite nitrogen in water for shrimp farming.

8. The use according to claim 1 or 2, characterized in that The carbon source is glucose.

9. The use according to claim 1 or 2, characterized in that: The nitrogen source is commercial shrimp feed.

10. The use according to claim 1, characterized in that During the cultivation of the biofloc, Bacillus subtilis XYB4 was added every 3 days.

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