Multi-size aquatic organism bait culture method

Abstract

The invention discloses a multi-size aquatic organism bait culture method, which is characterized in that livestock and poultry waste extract liquid prepared by the culture method is used for co-culture of Rhodospiraceae bacteria and Chlorella algae, and the obtained algae is used for co-culture of zooplankton of Brachionidae and Harpacidae to finally obtain the multi-size aquatic organism bait. Compared with the harpacticoida cultured only by algae, the harpacticoida obtained by the culture method has the advantages that the EPA content is increased by 90.53%, the DHA content is increased by 13.47%, and the nutritional value is higher; a reasonable production system provides bait with multiple sizes, and the yield of chlorella algae, brachionus and harpacticoida is effectively increased; moreover, a new way for resource utilization of livestock wastes is provided, and efficient, environment-friendly and sustainable bait production is realized.

Description

Technical Field

[0001] The invention relates to aquatic biological bait, and in particular to a method for cultivating aquatic biological bait of multiple sizes. Background Art

[0002] Aquatic biological feed plays a key role in the seedling stage of aquaculture, especially in the seedling opening stage, when more than 95% of the seedlings rely on biological feed. However, there are weak links in the current biological feed production technology and application research, leading to the following major problems:

[0003] 1. Poor supply stability: Biological bait production is significantly affected by seasons and the environment. During the seedling season, insufficient supply often leads to fluctuations in production, which in turn affects the consistency of seedling quality.

[0004] 2. High cost and insufficient nutrition: Existing biological baits (such as rotifers and Artemia) have high production costs and lack of nutrients, especially ω-3 polyunsaturated fatty acids (such as EPA and DHA), which limits their effectiveness and promotion;

[0005] 3. Insufficient palatability: Rotifers are too small to meet the needs of larvae of some aquatic species; Artemia nauplii are too large to adapt to the developmental stages of different species.

[0006] As a dominant group among marine zooplankton, copepods have broad development potential and can solve the above-mentioned problems to a certain extent. Their characteristics include: strong palatability, a wide range of copepod individual sizes, which can cover the needs of a variety of aquatic larvae; high nutritional value, and balanced nutrition in terms of protein, amino acids and fatty acids; excellent digestion performance, copepods themselves contain digestive enzymes, which can make up for the underdeveloped digestive system of larvae of economic species and significantly improve the survival rate and growth rate of larvae.

[0007] Despite the significant advantages of copepods, their large-scale production still faces challenges, among which bait supply is the main bottleneck. To address this issue, new ways of waste resource utilization can be explored. The large amount of waste generated by livestock and poultry farming will cause environmental pollution if not handled properly. However, these wastes are rich in organic nutrients. Full utilization can not only reduce the cost of bait production, but also effectively alleviate the current bait supply problem in the aquatic breeding industry, and realize a more efficient, environmentally friendly and sustainable high-nutritional value bait production system. Summary of the invention

[0008] Purpose of the invention: The purpose of the present invention is to provide a method for cultivating high-nutritional-value aquatic biological baits of different types and sizes using livestock and poultry waste.

[0009] Technical solution: The multi-size aquatic biological bait cultivation method of the present invention comprises:

[0010] (1) preparing livestock and poultry waste extract;

[0011] (2) using the livestock and poultry waste extract obtained in step 1 to co-cultivate Rhodospirillaceae bacteria and Chlorellaceae algae, and obtaining Chlorellaceae algae bait after the cultivation;

[0012] (3) using the Chlorella algae bait obtained in step 2 to co-cultivate zooplankton of Brachionus rotifera and Daphnia pulex, and after the cultivation is completed, filtering and collecting with a sieve to obtain aquatic biological baits of various sizes.

[0013] Preferably, the step 1 comprises:

[0014] (11) Add 4-6 times the mass of artificial seawater with a salinity of 27-29 and a pH of 8.0-8.5 to the livestock and poultry waste and mix thoroughly;

[0015] (12) Heat and keep boiling for 25-35 minutes. Let it stand and cool, then filter out the residue and set aside.

[0016] Preferably, in step 11, the livestock and poultry waste is chicken manure.

[0017] Preferably, the step 2 comprises:

[0018] (21) 4-6 mL of the livestock and poultry waste extract obtained in step 1 is added to each liter of the culture medium, and Rhodospirillaceae bacteria and Chlorellaceae algae are inoculated, wherein the inoculation concentration of Chlorellaceae algae is 0.8-1.2×10 6 ind. / mL, the ratio of the inoculated individual amount of Rhodospirillaceae bacteria and Chlorellaceae algae was 0.1-10:1;

[0019] (22) Cultivate at 24-26°C, 1500-2500 lx, 12:12 h photoperiod for 12-16 days with shaking every 2-4 h;

[0020] (23) After the culture is completed, the mixture is centrifuged at 4000-6000 rpm for 10-20 min at 2-8 °C to obtain Chlorella algae feed.

[0021] Preferably, the culture medium in step 21 is F / 2 culture medium; the Rhodospirillaceae bacteria is Rhodopseudomonas palustris, and the Chlorellaceae algae is Chlorella vulgaris.

[0022] Preferably, step 3 comprises:

[0023] (31) In artificial seawater with salinity of 27-29 and pH 8.0-8.5, the inoculation concentration was 1×10 4 -1×10 7ind. / mL of Chlorella family algae, Brachionus rotifer zooplankton at a concentration of 40-4000ind. / L, and Daphnia family zooplankton at a concentration of 35-45ind. / L;

[0024] (32) Cultivate for 12-16 days at 24-26°C, 1500-2500 lx of light, and 12:12 h photoperiod, with 50% water replacement per day, replacing with artificial seawater containing Chlorella algae;

[0025] (33) After the cultivation is completed, the bait is collected by sieve filtration to obtain aquatic biological baits of various sizes.

[0026] Preferably, in step 31, the zooplankton of the Brachionus rotifer family is Brachionus plicatilis; the zooplankton of the Daphnia pulex family is Daphnia japonica; the inoculated individual numbers of the zooplankton of the Brachionus rotifer family and Daphnia pulex family are the same; in step 33, the aperture of the sieve is 62-66 μm.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The Daphnia pulex cultured with chicken manure extract-Rhodospirillaceae bacteria-Chlorellaceae algae-Brachius rotifer has higher EPA and DHA contents. Compared with traditional cultured Daphnia pulex, the EPA content is increased by 90.53%, the DHA content is increased by 13.47%, and the nutritional value is higher; 2. The reasonable production system effectively increases the yield of Chlorellaceae algae, Brachionus rotifer and Daphnia pulex while providing baits of multiple sizes; 3. It provides a new way to utilize livestock and poultry waste resources, and realizes efficient, environmentally friendly and sustainable production of baits with high nutritional value. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a statistical graph of chlorophyll concentration of Chlorella vulgaris under different concentrations of Rhodopseudomonas palustris;

[0029] Figure 2 The population growth rate diagram of Brachionus plicatilis and Daphnia japonica with different initial proportions;

[0030] Figure 3 This is a graph showing the population growth rate of Daphnia pulex and Brachionus plicatilis at different seawater Chlorella concentrations. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below.

[0032] Example 1: Chicken manure extract for co-cultivation of Rhodopseudomonas palustris and Chlorella vulgaris

[0033] 1. Place 200g chicken manure in 1L water, heat and keep boiling for 30min, let it stand and cool, filter out the residue and set aside;

[0034] 2. Add 5 mL of chicken manure extract per liter of F / 2 medium and inoculate 1×10 6 ind. / mL concentration of seawater Chlorella, and then inoculated with 0 and 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 ind. / mL concentration of Rhodopseudomonas palustris,

[0035] Among them, F / 2 culture medium includes: NaNO3 (25g / L); H2PO4·H2O (5g / L); Na2SiO3·9H2O (30g / L); FeCl3·6H2O (3.15g / L); Na2EDTA·2H2O (4.36g / L); CuSO4·5H2O (0.0098g / L); Na2MoO4·2H2O (0.0063g / L); ZnSO4·7H2O (0.022g / L); CoCl·6H2O (0.01g / L); MnCl2·4H2O (0.18g / L); Vitamin B 12 (0.0005g / L); Biotin(0.0005g / L);

[0036] 3. Cultivate at 25℃, 2000lx light, 12:12h photoperiod, shake every 3h, and compare the biomass of marine Chlorella by chlorophyll concentration after 14d.

[0037] At the same time, F / 2 medium without chicken manure extract was set up and 1×10 6 Ind. / mL concentration of seawater Chlorella was cultured as a control.

[0038] The results are as follows Figure 1 As shown in the figure, compared with the control group (without chicken manure extract and Rhodopseudomonas palustris), the addition of chicken manure extract can effectively increase the biomass of marine Chlorella (expressed by chlorophyll content). In addition, the addition of photosynthetic bacteria at a concentration of 1×10 6 ind. / mL, the biomass of seawater Chlorella was the highest, that is, the optimal culture ratio of Rhodospirillaceae bacteria to Chlorella algae was 1:1.

[0039] Example 2: Co-cultivation of marine Chlorella vulgaris with Brachionus plicatilis and Daphnia japonica

[0040] 1. According to Example 1, the chicken manure extract was used to co-culture Rhodopseudomonas palustris and Chlorella vulgaris. After the culture was completed, the mixture was centrifuged at 4°C and 5000 rpm for 15 min to obtain Chlorella vulgaris bait, which was then stored at 4°C.

[0041] 2. Inoculate 1×106 ind. / mL concentration of the seawater Chlorella cultured in step 1, and 40 ind. / mL of Daphnia pulex, and then inoculated with 40, 400, and 4000 ind. / mL of Brachionus plicatilis rotifer, respectively.

[0042] Among them, artificial seawater includes: NaCl (24.54g / L); KBr (0.1g / L); KCl (0.7g / L); H3BO3 (0.003g / L); Na2SO4 (4.09g / L); NaHCO3 (0.185g / L); NaF (0.003g / L); CaCl2·2H2O (1.54g / L); MgCl2·6H2O (11.1g / L); SrCl2·6H2O (0.017g / L), with a salinity of 28 and a pH of 8.3; Daphnia japanensis was provided by Jiangsu Key Laboratory of Aquatic Biological Feed. The Brachionus plicatilis rotifers used in the experiment were filtered out with a mesh with a pore size of 150μm, placed in a 500mL glass beaker, and picked into the experimental culture system according to the experimental density. The sex ratio of the picked Japanese tiger-spotted Daphnia pulex was 1; the Brachionus plicatilis rotifers were collected from the ponds in the river crab nursery area in Sheyang, Jiangsu Province. The Brachionus plicatilis rotifers used in the experiment were filtered out with a mesh with a pore size of 112μm, placed in a 100mL glass beaker, and picked into the experimental culture system according to the experimental density;

[0043] 3. Cultivate at 25℃, 2000lx light, 12:12h photoperiod. After 14 days, filter with 77μm pore size silk sieve, and count the population growth rate of Brachionus plicatilis and Daphnia japonica.

[0044] At the same time, artificial seawater was inoculated with 1×10 6 Ind. / mL concentration of Chlorella vulgaris and 40ind. / mL of Daphnia japanensis were cultured as controls.

[0045] The results are as follows Figure 2 As shown in the figure, the initial individual size of Chlorella vulgaris-Taenia japonica-Brachionus plicatilis was 1×10 5 :4:4, the population growth rate of Japanese tiger daphnia-Brachius plicatilis was the best. In addition, compared with the traditional method of culturing Daphnia with only Chlorella (control group), the co-cultivation method of Brachius rotifer and Daphnia can better promote the population growth of Daphnia. With the same Chlorella input, co-cultivation can obtain a greater biological bait yield.

[0046] Example 3: Optimizing the co-culture of Brachionus plicatilis and Daphnia japonica by adjusting the concentration of Chlorella vulgaris in seawater

[0047] 1. According to Example 1, the chicken manure extract was used to co-culture Rhodopseudomonas palustris and Chlorella vulgaris. After the culture was completed, the mixture was centrifuged at 4°C and 5000 rpm for 15 min to obtain Chlorella vulgaris bait, which was then stored at 4°C.

[0048] 2. Inoculate 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 ind. / mL concentration of the seawater Chlorella cultured in step 1, as well as 40 ind. / mL of Daphnia pulex and 40 ind. / mL of Brachionus plicatilis.

[0049] Among them, artificial seawater includes: NaCl (24.54g / L); KBr (0.1g / L); KCl (0.7g / L); H3BO3 (0.003g / L); Na2SO4 (4.09g / L); NaHCO3 (0.185g / L); NaF (0.003g / L); CaCl2·2H2O (1.54g / L); MgCl2·6H2O (11.1g / L); SrCl2·6H2O (0.017g / L), with a salinity of 28 and a pH of 8.3; Daphnia japanensis was provided by Jiangsu Key Laboratory of Aquatic Biological Feed. The Brachionus plicatilis rotifers used in the experiment were filtered out with a mesh with a pore size of 150μm, placed in a 500mL glass beaker, and picked into the experimental culture system according to the experimental density. The sex ratio of the picked Japanese tiger-spotted Daphnia pulex was 1; the Brachionus plicatilis rotifers were collected from the ponds in the river crab nursery area in Sheyang, Jiangsu Province. The Brachionus plicatilis rotifers used in the experiment were filtered out with a mesh with a pore size of 112μm, placed in a 100mL glass beaker, and picked into the experimental culture system according to the experimental density;

[0050] 3. Cultivate at 25°C, 2000 lx light, 12:12h photoperiod, discard 50% of artificial seawater every 24h and replace with 1×10 4 , 1×10 5 , 1×10 6 , 1×10 7 The artificial seawater of the Chlorella vulgaris cultured in step 1 was filtered with a 77 μm pore size silk screen after 14 days at a concentration of ind. / mL, and the population growth rates of Brachionus plicatilis and Daphnia japonica were counted;

[0051] The results are as follows Figure 3 As shown in the figure, the population growth rate of Daphnia pulex was not affected by the concentration of Chlorella, but the population growth rate of Brachionus rotifer increased with the increase of Chlorella concentration. Therefore, the concentration of Chlorella should be controlled at 1×10 7 ind. / mL or less to prevent the Brachionus rotifer population from growing too fast.

[0052] Example 4: Analysis of nutritional components of the chicken manure extract-Rhodospirillaceae bacteria-Chlorellaceae algae-Brachia rotifer-Daphnia granatum system

[0053] 1. According to Example 1, the chicken manure extract was used to co-culture Rhodopseudomonas palustris and Chlorella vulgaris. After the culture was completed, the mixture was centrifuged at 4°C and 5000 rpm for 15 min to obtain Chlorella vulgaris bait, which was then stored at 4°C.

[0054] 2. Inoculate 1×10 6 ind. / mL concentration of Chlorella vulgaris cultured in step 1, 40 ind. / L of Daphnia pulex and 40 ind. / L of Brachionus plicatilis,

[0055] Among them, artificial seawater includes: NaCl (24.54g / L); KBr (0.1g / L); KCl (0.7g / L); H3BO3 (0.003g / L); Na2SO4 (4.09g / L); NaHCO3 (0.185g / L); NaF (0.003g / L); CaCl2·2H2O (1.54g / L); MgCl2·6H2O (11.1g / L); SrCl2·6H2O (0.017g / L), with a salinity of 28 and a pH of 8.3; Daphnia japanensis was provided by Jiangsu Key Laboratory of Aquatic Biological Feed. The Brachionus plicatilis rotifers used in the experiment were filtered out with a mesh with a pore size of 150μm, placed in a 500mL glass beaker, and picked into the experimental culture system according to the experimental density. The sex ratio of the picked Japanese tiger-spotted Daphnia pulex was 1; the Brachionus plicatilis rotifers were collected from the ponds in the river crab nursery area in Sheyang, Jiangsu Province. The Brachionus plicatilis rotifers used in the experiment were filtered out with a mesh with a pore size of 112μm, placed in a 100mL glass beaker, and picked into the experimental culture system according to the experimental density;

[0056] 3. Cultivate at 25°C, 2000lx light, 12:12h photoperiod, and filter with 64μm pore size silk screen after 14 days to obtain multi-size aquatic biological bait;

[0057] 4. The adult Japanese tiger-spotted Daphnia was further filtered using a 550 μm pore size silk sieve to detect the fatty acid composition in its body.

[0058] At the same time, artificial seawater was inoculated with 1×10 6 Ind. / mL concentration of Chlorella vulgaris and 40ind. / mL of Daphnia japonica were cultured under the same conditions as controls.

[0059] The results are shown in Table 1. The Daphnia pulex obtained by the co-culture system of chicken manure extract-Rhodospirillaceae bacteria-Chlorellaceae algae-Brachionus rotifer-Daphnia pulex has higher EPA and DHA contents. Compared with the traditional culture of Daphnia pulex, the EPA content is increased by 90.53%, the DHA content is increased by 13.47%, and the nutritional value is higher.

[0060] Table 1 Comparison of fatty acid composition of Daphnia japanensis cultured by different methods (percentage of total fatty acids)

[0061] Fatty cids (%) Traditional culture of Daphnia Daphnia pulex P-value C12:0 0.05±0.01 0.06±0.02 0.69 C13:0 0.05±0.01 0.07±0.02 0.21 C14:0 0.63±0.02 4.55±0.63 0.01 C15:0 0.52±0.03 0.78±0.04 0.00 C16:0 20.94±0.13 20.42±0.43 0.16 C16:1 2.41±0.16 13.66±0.60 0.00 C17:0 1.06±0.03 2.83±0.08 0.00 C18:0 6.31±0.10 8.76±0.30 0.00 C18:1n9c 12.75±0.38 9.92±0.46 0.00 C18:2n6c 12.37±0.45 3.42±0.47 0.00 C20:0 2.77±0.09 0.64±0.02 0.00 C18:3n3 9.03±0.21 0.44±0.06 0.00 C20:1 2.25±0.53 0.80±0.17 0.03 C21:0 0.14±0.01 0.17±0.02 0.06 C20:2 0.60±0.01 0.26±0.06 0.01 C22:0 2.77±0.09 1.46±0.07 0.00 C20:3n3 2.10±0.08 0.17±0.02 0.00 C22:1n9 0.98±0.01 0.40±0.11 0.01 C20:4n6 0.76±0.03 3.06±0.26 0.00 C23:0 0.63±0.02 1.16±0.06 0.00 C20:5n3(EPA) 3.38±0.03 6.44±0.92 0.03 C24:0 2.34±0.08 3.26±0.33 0.03 C24:1 0.14±0.01 0.25±0.08 0.12 C22:6n3(DHA) 15.00±0.15 17.02±0.84 0.01

Claims

1. A method for cultivating multi-size aquatic biological bait, characterized in that: include: (1) preparing livestock and poultry waste extract; (2) using livestock and poultry waste extract to co-cultivate Rhodospirillaceae bacteria and Chlorellaceae algae, and obtaining Chlorellaceae algae bait after the cultivation; (3) Using Chlorella algae bait to co-cultivate Brachionus rotifers and Daphnia pulex zooplankton, after the cultivation is completed, the bait is filtered and collected through a sieve to obtain aquatic biological baits of various sizes.

2. The method for cultivating multi-size aquatic biological bait according to claim 1, characterized in that: The step 1 comprises: (11) Add 4-6 times the mass of artificial seawater with a salinity of 27-29 and a pH of 8.0-8.5 to the livestock and poultry waste and mix thoroughly; (12) Heat and keep boiling for 25-35 minutes. Let it stand and cool, then filter out the residue and set aside.

3. The method for cultivating multi-size aquatic biological bait according to claim 2, characterized in that: In step 11, the livestock and poultry waste is chicken manure.

4. The method for cultivating multi-size aquatic biological bait according to claim 1, characterized in that: The step 2 comprises: (21) 4-6 mL of the livestock and poultry waste extract obtained in step 1 is added to each liter of the culture medium, and Rhodospirillaceae bacteria and Chlorellaceae algae are inoculated, wherein the inoculation concentration of Chlorellaceae algae is 0.8-1.2×10 6 ind. / mL, the ratio of the inoculated individual amount of Rhodospirillaceae bacteria and Chlorellaceae algae was 0.1-10:1; (22) Cultivate at 24-26°C, 1500-2500 lx, 12:12 h photoperiod for 12-16 days with shaking every 2-4 h; (23) After the culture is completed, the mixture is centrifuged at 4000-6000 rpm for 10-20 min at 2-8 °C to obtain Chlorella algae feed.

5. The method for cultivating multi-size aquatic biological bait according to claim 4, characterized in that: The culture medium in step 21 is F / 2 culture medium.

6. The method for cultivating multi-size aquatic biological bait according to claim 4, characterized in that: In step 21, the bacteria of the Rhodospirillaceae family is Rhodopseudomonas palustris, and the algae of the Chlorella family is Chlorella vulgaris.

7. The method for cultivating multi-size aquatic biological bait according to claim 1, characterized in that: The step 3 comprises: (31) In artificial seawater with salinity of 27-29 and pH 8.0-8.5, the inoculation concentration was 1×10 4 -1×10 7 ind. / mL of Chlorella family algae, Brachionus rotifer zooplankton at a concentration of 40-4000ind. / L, and Daphnia family zooplankton at a concentration of 35-45ind. / L; (32) The culture was carried out at a temperature of 24-26 °C, a light intensity of 1500-2500 lx, and a photoperiod of 12:12 h for 12-16 days, with a 50% water change every day, replacing it with artificial seawater containing Chlorella algae. (33) After the cultivation is completed, the bait is collected by sieve filtration to obtain aquatic biological baits of various sizes.

8. The method for cultivating multi-size aquatic biological bait according to claim 7, characterized in that: In the step 31, the zooplankton of the Brachionus rotifer family is Brachionus plicatilis; and the zooplankton of the Daphnia family is Daphnia japonica.

9. The method for cultivating multi-size aquatic biological bait according to claim 7, characterized in that: In the step 31, the inoculated zooplankton of Brachionidae and Daphniadae are the same in number.

10. The method for cultivating multi-size aquatic biological bait according to claim 7, characterized in that: In the step 33, the mesh aperture is 62-66 μm.

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