A high-salt culture method for whiteleg shrimp based on biofilm

Abstract

The present invention proposes a biofilm-based high-salt culture method for whiteleg shrimp, which is characterized by comprising the following steps: adding a biological filler to a culture pond once when the whiteleg shrimp is in the flea-like larval stage to increase the salinity to 20‰-25‰; adding the biological filler to the culture pond a second time in the mysid stage to increase the salinity to 40‰-45‰; and increasing the salinity to 70‰-75‰ in the postlarval stage until the target shrimp is harvested, wherein the biological filler comprises zeolite, Bacillus natto, planktonic Clostridium and an immobilized salt solution preparation, wherein the immobilized salt solution preparation is obtained by using modified expansive soil as a carrier to adsorb a salt solution.

Description

Technical Field

[0001] The invention relates to the field of shrimp farming, and in particular to a high-salt farming method for whiteleg shrimp based on a biofilm. Background Art

[0002] White pacific shrimp (Litopenaeus vannamei), also known as Penaeus vannamei, has become a mainstream aquaculture species due to its rapid growth rate, strong adaptability, and high-density farming. However, the shrimp's open hemolymph system makes it highly susceptible to environmental factors such as temperature and salinity. Salinity fluctuations cause osmotic stress in the shrimp, which can directly affect their growth and metabolism. In severe cases, this can cause oxidative stress, inhibiting their growth and significantly reducing their survival rate. During the grow-out period, white shrimp have a weak resistance to pathogens and environmental changes, which can easily affect their physiological functions and immune defenses, reducing the shrimp's harvest. Summary of the Invention

[0003] In view of this, the purpose of the present invention is to propose a high-salt culture method for whiteleg shrimp based on biofilm, so as to improve the resistance of whiteleg shrimp to pathogenic factors and environmental changes during the rearing period and increase the harvest of shrimp.

[0004] The technical solution of the present invention is achieved as follows:

[0005] A high-salt culture method for whiteleg shrimp based on biofilm, comprising: adding biological fillers to a culture pond once when the whiteleg shrimp is in the flea-like larval stage to increase the salinity to 20‰-25‰; adding biological fillers to the culture pond a second time when the whiteleg shrimp is in the mysid stage to increase the salinity to 40‰-45‰; and increasing the salinity to 70‰-75‰ when the shrimp is in the larval stage until the target shrimp is harvested, wherein the biological fillers form a biofilm during the culture process to obtain a biofilm;

[0006] The biological filler comprises zeolite, Bacillus natto, planktonic Clostridium and an immobilized salt solution preparation. The immobilized salt solution preparation is obtained by using modified expansive soil as a carrier to adsorb salt solution.

[0007] It is further explained that the preparation method of the immobilized salt solution preparation includes: mixing the modified expansive soil with a sodium chloride solution, oscillating and adsorbing, filtering, and drying to obtain the immobilized salt solution preparation.

[0008] To further illustrate, the modified expansive soil is prepared by adding potassium permanganate solution to expansive soil, reacting in a water bath, filtering, washing, and drying.

[0009] To further illustrate, the modified expansive soil is prepared by adding 15-18M potassium permanganate solution to the expansive soil at a material-liquid weight volume ratio of 4-5g:100-140mL, reacting in a water bath at 60-65°C for 30-40min, filtering and washing until the washing liquid is colorless, and drying at 60-65°C.

[0010] It is further explained that the weight-to-volume ratio of the modified expansive soil to the sodium chloride solution is 5-6 g:30-40 mL, and the mass fraction of the sodium chloride solution is 40-50%.

[0011] It is further explained that the volume ratio of the biological filler added once to the culture pond is 0.02-0.04:1, and the volume ratio of the biological filler added twice to the culture pond is 0.02-0.04:1.

[0012] To further illustrate, the volume ratio of the biological filler added once to the culture pond is 0.4:1, and the volume ratio of the biological filler added twice to the culture pond is 0.2:1.

[0013] It is further described that, based on weight, the biological filler comprises 30-40 parts of zeolite, 2-3 parts of Bacillus natto, 2-3 parts of planktonic Coccidioides and 40-60 parts of immobilized salt solution preparation.

[0014] It is further described that, based on weight, the biological filler comprises 34 parts of zeolite, 2 parts of Bacillus natto, 3 parts of planktonic Coccidioides and 52 parts of immobilized salt solution preparation.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention adds a biological filler prepared by zeolite, Bacillus natto, planktonic Sphaeroides and an immobilized salt solution preparation during the cultivation process of whiteleg shrimp, thereby increasing the shrimp survival rate by about 30-120%, reaching a shrimp survival rate of more than 73%, and significantly improving the survival rate in a high-salt environment with a water salinity of 75‰. After culturing for 7 weeks, the SOD enzyme activity of the whiteleg shrimp can reach more than 150 U / mg prot. The biological filler prepared by the zeolite, Bacillus natto, planktonic Sphaeroides and immobilized salt solution preparation selected by the present invention synergizes with each other to effectively enhance the tolerance of whiteleg shrimp to high salt. DETAILED DESCRIPTION

[0017] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0018] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0019] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0020] The Bacillus natto of the present invention is Bacillus natto powder purchased from Beijing Chuanxiu Technology Co., Ltd.; the Sphaerotilus natans is purchased from Shanghai Chunshi Biotechnology Co., Ltd. The Sphaerotilus natans is activated by aspirating 0.5 mL of CGY liquid culture medium and dripping it into an ampoule tube to dissolve the freeze-dried bacteria into a suspension, aspirating all the bacterial suspension, transplanting it into a CGY culture medium test tube, and culturing it at 30°C. The bacteria in the slant test tube are picked and inoculated into the CGY liquid culture medium, and cultured at 30°C and 150 r / min for 24 hours for two generations. After that, the culture solution in the logarithmic growth phase is taken, centrifuged, and the bacteria are collected and freeze-dried to obtain the Sphaerotilus natans powder.

[0021] Example 1 - Immobilized saline preparation

[0022] Preparation of modified expansive soil: 20 g of expansive soil was added to 500 mL of 15 M potassium permanganate solution (4 g:100 mL), and the mixture was reacted in a water bath at 60° C. for 30 min. The reacted expansive soil was filtered, washed until the washing liquid was colorless, and dried at 65° C. to obtain the modified expansive soil.

[0023] Preparation of immobilized salt solution preparation: 5 g of modified expansive soil was mixed with 30 mL of sodium chloride solution (5 g:30 mL), and after oscillation adsorption at 1000 r / min for 2 h, the mixture was filtered and dried at 50° C. to obtain the immobilized salt solution preparation.

[0024] Example 2 - Immobilized Saline Preparation

[0025] Preparation of modified expansive soil: 25 g of expansive soil was added to 700 mL of 18 M potassium permanganate solution (5 g: 140 mL), and the mixture was reacted in a water bath at 60° C. for 30 min. The reacted expansive soil was filtered, washed until the washing liquid was colorless, and dried at 65° C. to obtain the modified expansive soil.

[0026] Preparation of immobilized salt solution preparation: 6 g of modified expansive soil was mixed with 40 mL of sodium chloride solution (6 g:40 mL), and after oscillation adsorption at 1000 r / min for 2 h, the mixture was filtered and dried at 50° C. to obtain the immobilized salt solution preparation.

[0027] Example 3 - Biofilm Culture

[0028] The newly hatched larvae of Penaeus vannamei were reared in the laboratory for 20 days and then randomly divided into groups with a stocking density of 200,000 per cubic meter. 3The experiments were carried out in circulating water aquaculture boxes with a water volume of 50 L, a water salinity of 10‰, a water temperature of 28±1℃, a pH of 7.2±0.5, and dissolved oxygen greater than 5.5 mg / L. The water was changed regularly with the water change volume accounting for 2 / 5 of the aquaculture water. The shrimps were fed with compound feed (Yangzhou Hongda Feed Co., Ltd.) regularly with a feeding amount of 8% of the shrimp body weight. The aquaculture cycle was 73 days.

[0029] The experiment was divided into 5 groups, namely, biofilm group, preparation group, salinity group, microbial group and conventional group. The experiments were as follows:

[0030] Biofilm group: When the newly hatched larvae of Penaeus vannamei are in the flea-like larvae stage, biological fillers are added to the breeding pond (v / v=0.04:1), and the water salinity is increased to 20‰. The biological fillers form biofilms during the breeding process and obtain biofilms; when they are in the mysid stage (the flea-like larvae undergo 9 molts), biological fillers are added to the breeding pond again (v / v=0.02:1), and after 5 days of breeding, the water salinity is adjusted to 40‰; when entering the larval stage, the water salinity can be directly adjusted to 75‰ and the adult shrimp are harvested.

[0031] The zeolite was crushed, and 340 g of zeolite powder, 20 g of Bacillus natto, 30 g of planktonic Clostridium perfringens and 520 g of the immobilized saline solution preparation of Example 1 were mixed and ball-milled for 30 minutes to obtain a biofiller.

[0032] Preparation group: When the newly hatched larvae of Penaeus vannamei are in the flea-like larvae stage, the immobilized salt solution preparation of Example 1 is added to the culture pond (v / v=0.04:1), and the water salinity is increased to 20‰; when the larvae are cultured to the mysid stage (the flea-like larvae undergo 9 molts), the immobilized salt solution preparation of Example 1 is again added to the culture pond (v / v=0.02:1), and after culturing for 5 days, the water salinity is adjusted to 40‰; when entering the larval stage, the water salinity can be directly adjusted to 75‰, and the shrimp are harvested.

[0033] Salinity group: When the newly hatched larvae of white shrimp are in the flea stage, the water salinity is increased to 20‰; when they are in the mysid stage (the flea larvae undergo 9 molts), the water salinity is adjusted to 40‰; when they enter the larval stage, the water salinity can be directly adjusted to 75‰ and the shrimp are harvested.

[0034] Microbiome: When the newly hatched larvae of Penaeus vannamei were in the flea-like stage, Bacillus natto and planktonic Clostridium were added to the culture pond (v / v = 0.04:1, m 纳豆菌 :m 浮游球衣菌 =2:3), and at the same time, the water salinity was increased to 20‰; when the shrimp were in the mysid stage (the flea-like larvae underwent 9 molts), Natto bacteria and planktonic Clostridium were added to the culture pond again (v / v = 0.02:1, m纳豆菌 :m 浮游球衣菌 =2:3), after 5 days of culture, adjust the water salinity to 40‰; when entering the shrimp larvae stage, the water salinity can be directly adjusted to 75‰ and the shrimp can be harvested.

[0035] Conventional group: The salinity of the water body was gradually adjusted by 0.2-0.4 per day until the salinity of the water body reached 75‰, and then the shrimps were harvested.

[0036] The hepatopancreas of shrimp was dissected, and the superoxide dismutase (SOD) activity was measured using a kit (Beijing Solebaugh Technology Co., Ltd.). The results are shown in Table 1.

[0037] Table 1 Changes in survival rate and SOD enzyme activity of shrimp in different culture cycles

[0038]

[0039] After 10 weeks of cultivation, the survival rate of shrimp in the biofilm group was the highest, reaching 73.6%. Compared with the conventional group, the survival rate of the biofilm group increased by about 57%, indicating that adding biological fillers prepared with zeolite, Bacillus natto, planktonic Coccidioides and immobilized salt solution preparations during shrimp cultivation can improve the survival rate of shrimp; compared with the preparation group and the microbial group, the survival rate of the biofilm group increased by about 118% and 33% respectively, indicating that zeolite, Bacillus natto, planktonic Coccidioides and immobilized salt solution preparations synergistically improved the survival rate of shrimp, especially the preparation group. Directly adding the immobilized salt solution preparation into the breeding pond caused water pollution and affected the living environment of the shrimp.

[0040] After 7 weeks of culture, the SOD enzyme activity of the biofilm group had reached 156.82U / mg prot. Shrimp have an open hemolymph system and are extremely susceptible to environmental factors such as temperature and salinity. Under high-salt stress, shrimp will cause oxidative stress response in the biological body, and the body will produce a large amount of SOD enzyme to dismutate reactive oxygen species to alleviate the oxidative stress response. The SOD enzyme activity of the biofilm group was the highest, indicating that adding biological fillers prepared with zeolite, natto bacteria, planktonic Clostridium and immobilized salt solution preparations during shrimp culture can enhance the tolerance of whiteleg shrimp to high salt.

[0041] When the biofilm group entered the larval stage, the water salinity was adjusted to 70‰. After 10 weeks of cultivation, the survival rate of the whiteleg shrimp was 78.2%. At the same time, the biofilm group adjusted the raw material dosage of the biological filler (400g of zeolite powder, 30g of natto bacteria, 20g of planktonic Clostridium and 600g of the immobilized salt solution preparation of Example 1). After 10 weeks of cultivation, the survival rate of the whiteleg shrimp was 71.8%.

[0042] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A high-salt culture method for whiteleg shrimp based on biofilm, characterized in that: This includes adding biological fillers to the culture pond once when the white shrimp are in the flea-like larval stage to increase the salinity to 20‰-25‰; adding biological fillers to the culture pond twice during the mysid stage to increase the salinity to 40‰-45‰; and increasing the salinity to 70‰-75‰ during the postlarval stage until the target shrimp are harvested. The biological fillers will form biofilms during the culture process to obtain biofilms. The biological filler comprises zeolite, Bacillus natto, planktonic Clostridium and an immobilized salt solution preparation, wherein the immobilized salt solution preparation is obtained by using modified expansive soil as a carrier to adsorb salt solution; The preparation method of the immobilized salt solution preparation comprises: mixing the modified expansive soil with a sodium chloride solution, oscillating and adsorbing, filtering, and drying to obtain the immobilized salt solution preparation; The modified expansive soil is prepared by adding potassium permanganate solution into expansive soil, reacting in a water bath, filtering, washing, and drying.

2. A high-salt culture method for whiteleg shrimp based on biofilm according to claim 1, characterized in that: The modified expansive soil is prepared by adding 15-18M potassium permanganate solution to the expansive soil at a material-liquid weight volume ratio of 4-5g:100-140mL, reacting in a water bath at 60-65°C for 30-40min, filtering and washing until the washing solution is colorless, and drying at 60-65°C.

3. A high-salt culture method for whiteleg shrimp based on biofilm according to claim 1, characterized in that: The weight-to-volume ratio of the modified expansive soil to the sodium chloride solution is 5-6 g:30-40 mL, and the mass fraction of the sodium chloride solution is 40-50%.

4. A high-salt culture method for whiteleg shrimp based on biofilm according to claim 1, characterized in that: The volume ratio of the biological filler added once to the culture pond is 0.02-0.04:1, and the volume ratio of the biological filler added twice to the culture pond is 0.02-0.04:

1.

5. A high-salt culture method for whiteleg shrimp based on biofilm according to claim 4, characterized in that: The volume ratio of the biological filler added once to the culture pond is 0.04:1, and the volume ratio of the biological filler added twice to the culture pond is 0.02:

1.

6. A high-salt culture method for whiteleg shrimp based on biofilm according to claim 1, characterized in that: In parts by weight, the biological filler comprises 30-40 parts of zeolite, 2-3 parts of Bacillus natto, 2-3 parts of planktonic Coccidioides and 40-60 parts of immobilized salt solution preparation.

7. A biofilm-based high-salt culture method for whiteleg shrimp according to claim 1, characterized in that: In parts by weight, the biological filler comprises 34 parts of zeolite, 2 parts of Bacillus natto, 3 parts of planktonic Coccidioides and 52 parts of immobilized salt solution preparation.

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