Application of functional biofloc in prawn culture

Abstract

The invention discloses an application of functional biofloc in prawn culture. According to the invention, bacillus subtilis XYB4 and neutral protease are added into traditional biofloc to prepare the functional biofloc. When the functional biofloc is applied to prawn culture, the culture water quality can be purified, the content of ammonia nitrogen and nitrite nitrogen in the culture water body can be effectively reduced, so that the toxic effect of the ammonia nitrogen and the nitrite nitrogen on prawns is reduced, the growth performance and the antioxidant enzyme activity of the prawns can be improved, and the prawn breeding quality is improved. Therefore, green, environment-friendly, safe and efficient ecological prawn breeding is realized.

Description

Technical Field

[0001] The invention relates to the technical field of shrimp farming, and in particular to application of functional biofloc in shrimp farming. Background Art

[0002] my country is a major aquaculture country, and shrimp is one of the important aquaculture species in my country. Litopenaeus vannamei ) is promoted throughout the country due to its excellent characteristics such as fast growth, strong disease resistance and low feed nutrition requirements, and has quickly become one of the pillar industries of my country's aquaculture. Vannamei shrimp is one of the most important crustacean species in aquaculture, accounting for 70% of the world's total shrimp farming. However, the environmental conditions of aquaculture waters are deteriorating, and traditional aquaculture models will also cause a large amount of residual bait and feces to accumulate in the water, resulting in serious pollution of aquaculture waters, which will have a toxic effect on the growth of vannamei shrimp.

[0003] Biofloc Technology (BFT) is a sustainable aquaculture technology that can recycle nutrient waste, save resources, avoid pollution, and provide high-quality protein sources. There are two ways to cultivate bioflocs, namely in situ cultivation and ex situ cultivation. In situ cultivation is to cultivate bioflocs directly in the aquaculture pond. After calculating the appropriate carbon-nitrogen ratio based on the composition and feeding amount of the feed, select a suitable carbon source and directly put it into the water body and ensure that the dissolved oxygen and the water body are fully stirred, and bioflocs can be generated in the aquaculture pond. Bioflocs directly use ammonia nitrogen and feed residues in the water to form bacterial protein, which is reused by the aquaculture objects.

[0004] In contrast, the ex situ culture technology separates the generation of bioflocs from aquaculture activities, creates conditions for the production of bioflocs in an external reactor, collects the flocs after cultivation for feeding, or uses them as a substitute for fish meal in aquatic feed. This method is easy to implement and has strong controllability. It can also solve the problem of excessive bioflocs produced by in situ culture leading to gill filament blockage and hypoxia in cultured shrimp.

[0005] Studies have shown that the rational application of biofloc technology can significantly improve the growth performance of aquatic animals, but different aquatic animals utilize biofloc differently, and the effects of biofloc obtained by different culture methods on the growth, enzyme activity and water quality of aquaculture water of aquatic animals are also different.

[0006] Therefore, it is of great significance to provide a biofloc that promotes healthy shrimp farming and improves shrimp growth performance, enzyme activity, etc. Summary of the invention

[0007] In order to overcome the above defects and shortcomings in the prior art, the present invention provides an application of a functional biofloc in shrimp farming.

[0008] The first object of the present invention is to provide an application of biofloc in shrimp farming.

[0009] Therefore, the present invention claims the following: A biofloc is used in shrimp farming. The biofloc is obtained by 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 mixing them well, aerating the water body at the same time, culturing for 70 to 74 days, and collecting the biofloc when the ammonia nitrogen and nitrite nitrogen in the water body of the culture unit reach 0 to 0.5 mg / L. The Bacillus subtilis XYB4 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 2, 2022, with a deposit number of GDMCC NO: 62676, and is the Bacillus subtilis XYB4 in the Chinese invention patent CN116410888A; The usage ratio of the neutral protease to the disinfected seawater is (0.5-1) g:1 L.

[0010] As an practicable manner, the method for disinfecting seawater is as follows: sodium hypochlorite is added to the seawater for disinfection, and then sodium thiosulfate is added for neutralization, and disinfection is completed when there is no residual chlorine in the seawater.

[0011] The biofloc of the present invention is carried out in an ex situ culture mode, wherein the added beneficial bacteria are safe and efficient, and play a vital role in the aquaculture ecological environment system. By using beneficial microorganisms to improve the aquaculture ecological environment, the richness of the intestinal flora of the aquaculture object can be increased, thereby improving the immunity of the aquaculture object and promoting its growth.

[0012] Enzymes are biological macromolecules with special catalytic functions secreted by living cells. They 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.

[0013] The inventors found that the bioflocs cultivated with low doses of neutral protease can effectively improve the survival rate and CAT activity of shrimp in shrimp farming. Therefore, preferably, the dosage ratio of the neutral protease to the disinfected seawater is 0.5 g: 1 L.

[0014] Preferably, the biofloc is used to improve the survival rate of shrimps.

[0015] Preferably, the biofloc is used to increase the antioxidant enzyme activity of shrimp.

[0016] More preferably, the antioxidant enzyme is peroxidase.

[0017] Preferably, the biofloc is used to reduce the content of ammonia nitrogen in water for shrimp farming.

[0018] Preferably, the biofloc is used to reduce the content of nitrite nitrogen in water for shrimp farming.

[0019] Preferably, the carbon source is glucose.

[0020] Preferably, the nitrogen source is commercial shrimp feed.

[0021] More preferably, the effective viable count of the Bacillus subtilis XYB4 is 1.2×10 9 CFU / L.

[0022] Preferably, during the cultivation of the biofloc, Bacillus subtilis XYB4 is added once every 3 days.

[0023] Preferably, the usage ratio of the carbon source, nitrogen source and sterilized seawater is (1-3) g: (6-7) g: 1 L.

[0024] More preferably, the usage ratio of the carbon source, nitrogen source and sterilized seawater is 2 g:6.6 g:1 L.

[0025] As an prawn that can be implemented, the prawn is Penaeus vannamei, Penaeus japonicus, Penaeus monodon, etc.

[0026] Compared with the prior art, the present invention has the following beneficial effects: The present invention discloses an application of a functional biofloc in shrimp farming. The present invention adds Bacillus subtilis XYB4 and neutral protease to a traditional biofloc to prepare a functional biofloc. The application of the functional biofloc in shrimp farming has the following advantages: (1) The bioflocs of the present invention can purify the aquaculture water quality and effectively reduce the content of ammonia nitrogen and nitrite nitrogen in the aquaculture water without changing the water. The peak values ​​of ammonia nitrogen and nitrite nitrogen in the water during the aquaculture period are lower than those in the group without adding bioflocs, and the toxic effect on aquatic animals is smaller.

[0027] (2) The biofloc of the present invention can maintain good water quality indicators of the aquaculture water for 1 to 2 months without replacing the aquaculture water, and can reduce the amount of aquaculture water and water replacement to a certain extent, thereby achieving the purpose of saving water and realizing efficient green ecological aquaculture.

[0028] (3) The use of the bioflocs of the present invention for shrimp farming can improve the survival rate, weight gain, growth rate and antioxidant enzyme activity of shrimp. The inventors unexpectedly found that the bioflocs cultivated with a low dose of neutral protease can further improve the CAT activity of shrimp, thereby increasing the survival rate of shrimp. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the changing trend of ammonia nitrogen in the aquaculture water during the culture of Penaeus vannamei.

[0030] Figure 2 This is the changing trend of nitrite nitrogen in the aquaculture water during the culture of Penaeus vannamei.

[0031] Figure 3 This is the changing trend of nitrate nitrogen in the aquaculture water during the culture of Penaeus vannamei.

[0032] Figure 4 is the CAT enzyme activity of Penaeus vannamei. DETAILED DESCRIPTION

[0033] The present invention is further described below with reference to specific examples, but the examples 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 art.

[0034] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0035] The Bacillus subtilis XYB4 used in the embodiment was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 2, 2022, with a deposit number of GDMCC NO: 62676, and the deposit address is 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou. It is the Bacillus subtilis XYB4 in the Chinese invention patent CN116410888A.

[0036] The commercial shrimp feed used in the examples has the following components: crude protein ≥50.0%, crude fat ≥0%, crude fiber ≤5.0%, crude ash ≤18.0%, lysine ≥2.5%, total phosphorus ≥1.5%, and moisture ≤10.0%.

[0037] Example 1 A method for cultivating Litopenaeus vannamei using bacteria plus low-enzyme functional biofloc 1. Cultivation method of bacteria plus low enzyme functional biofloc In this embodiment, the method for culturing the bacteria-plus-low-enzyme functional biofloc includes the following steps: S1. Introduce seawater into the reservoir and 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), 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, seawater usable for the experiment is obtained, and 50 L of seawater usable for the experiment is introduced into the biofloc culture barrel; S2. 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; The method for expanding and activating the Bacillus subtilis XYB4 is as follows: 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; 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, 120 rpm, and the shaker is set for 10 h. 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.

[0038] S3. Put 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) into a 100-mesh filter bag, put it into the biofloc culture barrel, and mix and stir thoroughly; 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 5.7-7.9 mg / L, the water temperature at 26-30°C, the pH at 7.3-8.0, and the salinity at 27-30. Cultivate for 72 days, during which Bacillus subtilis XYB4 liquid inoculant is added every 3 days at a dosage of 1 mL / L. Detect ammonia nitrogen, nitrite nitrogen, and nitrate nitrogen in the water every three days. 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.

[0039] 2. Farming of Penaeus vannamei (1) Take 0.3 m 3plastic barrels as breeding barrels, and the experimentally available seawater obtained in the above step S1 is stored in each breeding barrel, with the ratio of biofloc: experimentally available seawater = 1:100 (the volume of biofloc is measured using an Inhofe conical flask, and the experimental water successfully cultured in step 1 is taken, and allowed to stand and settle for 30 min, and then the supernatant is removed, and 2.5 L of turbid solids after sedimentation are taken to obtain biofloc, and then 250 L of experimentally available seawater is taken for the cultivation of Penaeus vannamei), the biofloc obtained by the culture in step 1 is put into the breeding barrels, and 2 air stones are placed in each breeding barrel to fully aerate the water to maintain the dissolved oxygen content of the water greater than 5 mg / L.

[0040] (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 put them into culture barrels. Put 100 Penaeus vannamei into each culture barrel. During the culture period, feed the shrimp with commercial feed, and use the commercial feed as the nitrogen source and monohydrate glucose as the carbon source to activate the biofloc inoculated in step (1). After the biofloc is activated, add Bacillus subtilis XYB4 liquid inoculant (addition amount is 1 mL / L) every 3 days to increase the probiotic content of the culture water and promote the nitrogen conversion rate of the water.

[0041] During the breeding period, the water temperature was maintained at 29-32°C, the total alkalinity of the water was maintained at 150-250 mg / L (if the total alkalinity of the water is too low, sodium bicarbonate was used to adjust it), and the deposition of biofloc in the breeding water was measured regularly. 30min When (the amount of biofloc sedimentation within 30 minutes) is greater than 40 mg / L, a 100-200 mesh filter is used to salvage it to prevent the gill filaments of Penaeus vannamei from being blocked and the shrimp from dying due to lack of oxygen, which is caused by excessive amount of biofloc in the water.

[0042] The shrimp farming period was 21 days, during which the ammonia nitrogen, nitrite nitrogen and nitrate nitrogen contents in the farming water were measured every 3 days. After the farming period, the growth performance and antioxidant enzyme activity of the vannamei shrimp were counted.

[0043] Example 2 A method for cultivating Litopenaeus vannamei using bacteria-enzyme functional biofloc This example is carried out with reference to Example 1, except that in step S3, the amount of neutral protease added is 50 g.

[0044] Comparative Example 1 1. Take 0.3 m 3 Plastic barrels are used as breeding barrels, and the experimental seawater obtained in the above step S1 is stored in each breeding barrel. Two air stones are placed in each breeding barrel to fully aerate the water body to maintain the dissolved oxygen content of the water body greater than 5 mg / L.

[0045] 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 put them into culture barrels. Put 100 Penaeus vannamei into each culture barrel and feed them with commercial shrimp feed during the culture period.

[0046] During the breeding period, maintain the breeding water temperature at 29-32°C and the total alkalinity of the water at 150-250 mg / L (if the total alkalinity of the water is too low, use sodium bicarbonate to adjust it).

[0047] The shrimp farming period is 21 days, during which the ammonia nitrogen, nitrite nitrogen and nitrate nitrogen contents in the farming water are measured every 3 days. After the farming period, the growth performance and antioxidant enzyme activity of Penaeus vannamei are counted.

[0048] Test Example 1: Changes in ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in aquaculture water 1. Experimental Methods During the shrimp farming period, the ammonia nitrogen, nitrite nitrogen and nitrate nitrogen contents in the farming water were measured every 3 days, and the changing trends of ammonia nitrogen, nitrite nitrogen and nitrate nitrogen in the farming water of Example 1, Example 2 and Comparative Example 1 were observed.

[0049] 2. Experimental Methods 1. Changing trends of ammonia nitrogen and nitrite nitrogen The variation trend of ammonia nitrogen in the aquaculture water of Example 1, Example 2 and Comparative Example 1 is as follows: Figure 1 As shown. Figure 1 It can be seen that the aquaculture water of Comparative Example 1 reached peak values ​​on the 6th and 15th days, respectively, and gradually stabilized after 15 days, with the ammonia nitrogen content maintained at about 3 mg / L.

[0050] The peak values ​​of the aquaculture water in Examples 1 and 2 appeared on the 6th day, which was significantly lower than the ammonia nitrogen peak value in Comparative Example 1. Moreover, after the 6th day, the ammonia nitrogen peak values ​​in Examples 1 and 2 gradually decreased, and finally decreased to 0-0.5 mg / L, indicating that the biofloc in Examples 1 and 2 can effectively reduce the ammonia nitrogen content in the aquaculture water.

[0051] The variation trend of nitrite nitrogen in the aquaculture water of Example 1, Example 2 and Comparative Example 1 is as follows: Figure 2 As shown. Figure 2 It can be seen that the nitrite nitrogen concentration in the aquaculture water of Comparative Example 1 gradually increased after the 9th day, and the nitrite nitrogen content reached about 20 mg / L on the 21st day.

[0052] The peak nitrite nitrogen concentration in the aquaculture water 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 decreased to 0-0.5 mg / L, indicating that the biofloc in Example 1 and Example 2 can effectively reduce the nitrite nitrogen content in the aquaculture water.

[0053] When the concentration of ammonia nitrogen or nitrite nitrogen, which are acutely toxic to aquatic animals, is high, aquatic animals are at risk of death. Figure 1 and Figure 2 The results show that the ammonia nitrogen and nitrite nitrogen peaks of the culture water body are lower when the methods of Example 1 and Example 2 are used to culture Litopenaeus vannamei, and the impact on Litopenaeus vannamei is smaller. Therefore, adding biofloc to the culture water body can effectively improve the ecological environment of shrimp culture and purify the water quality. In addition, the present invention cultured Litopenaeus vannamei without changing the water. To a certain extent, the present invention can also reduce the amount of water used for culture, thereby achieving the purpose of saving water and realizing efficient green ecological culture.

[0054] 2. Changing trends of nitrate nitrogen Depend on Figure 3 The results show that the concentration of nitrate nitrogen in the aquaculture water of Example 1, Example 2 and Comparative Example 1 continued to increase. Ammonia nitrogen and nitrite nitrogen are toxic to aquatic animals, and one of the main functions of biofloc is to convert ammonia nitrogen and nitrite nitrogen into relatively non-toxic nitrate nitrogen. Figure 3 The results show that after adding biofloc, the yield of nitrate nitrogen in the example is higher than that in the control example, indicating that biofloc can effectively promote the conversion of ammonia nitrogen and nitrite nitrogen in aquaculture water.

[0055] Test Example 2 Effects of biofloc on growth performance and antioxidant enzyme activity of Litopenaeus vannamei 1. Experimental Methods After the culture was completed, the shrimp were weighed, and the body length, body weight, survival rate, weight gain rate and specific growth rate were calculated: Weight gain rate = (final body weight - initial body weight) / initial body weight × 100%; Specific growth rate = (ln final body mass - ln initial body mass) / breeding cycle × 100%; At the same time, the peroxidase (CAT) activity in the hepatopancreas of Penaeus vannamei was determined using a kit.

[0056] 2. Experimental Results The growth performance results are shown in Table 1. The results show that the growth performance of the shrimp in the examples is significantly increased compared to that in comparative example 1. It is worth noting that the survival rate of the shrimp in the culture of the low enzyme functional biofloc of example 1 is 6.59% higher than that of the medium enzyme functional biofloc of example 2.

[0057] Table 1 Growth performance of Penaeus vannamei

[0058] Note: The results are expressed as "mean ± standard deviation". Different letters indicate significant differences. P <0.05.

[0059] The results of antioxidant enzyme activities are shown in Table 2 and Figure 4 The results show that the antioxidant enzyme activities of the shrimp in the examples are significantly increased compared with those in comparative example 1. It is worth noting that the CAT activity of the shrimp increased by 30.8% compared with that in example 2 when the low-enzyme functional biofloc of example 1 was used to culture the shrimp.

[0060] Table 2 Antioxidant enzyme activities of Litopenaeus vannamei

[0061] Note: The results are expressed as “mean ± standard deviation”.

[0062] From the above data, it can be seen that the increase in the neutral protease content in the bioflocs leads to a decrease in the CAT content of the vannamei shrimp, which leads to a decrease in the survival rate, further indicating that aquatic animals use bioflocs differently. Therefore, in the process of using bioflocs to breed aquatic animals, it is necessary to cultivate suitable bioflocs for different breeding objects in order to maximize the benefits of biofloc technology in aquaculture.

[0063] 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 equivalent replacement methods and are included in the protection scope 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 body, culturing for 70 to 74 days, and collecting the bioflocs when the ammonia nitrogen and nitrite nitrogen in the water body of the culture unit reach 0 to 0.5 mg / L; The Bacillus subtilis XYB4 was deposited in Guangdong 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 shrimps.

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 content of ammonia nitrogen 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 is added once every 3 days.

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