Regulation and control composition for improving metamorphosis and all-male rate of bullfrog tadpoles and preparation method and application of regulation and control composition
The high-purity luteolin complex with lecithin and lecithin is prepared by improving the extraction, separation and purification method. As a highly efficient and environmentally friendly feed additive, the problem of low metamorphism and insufficient male ratio of bullfrog tadpoles is solved. The effect of significantly increasing the perversion rate and male proportion was achieved, and the utilization of crop waste resources was promoted.
Patent Information
- Application Number
- CN202411065108.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-05
- Publication Date
- 2025-01-10
AI Technical Summary
Bullfrog tadpoles have low metamorphism and insufficient male ratio, resulting in low economic benefits of breeding. The existing use of hormones or chemical drugs leads to drug resistance and drug residues. The regulatory effects of natural active substances such as luteolin and zinc lactate have not been reported. .
By improving the extraction, separation and purification method, a composition of high-purity luteolin complex with lecithin and lecithin was prepared. Using biofermentation and ultrasonic technology, it was used as an efficient and environmentally friendly feed additive to coordinate the metamorphosis of bullfrog tadpoles. and male ratio.
It significantly increases the perversion rate and male ratio of bullfrog tadpoles, enhances the economic benefits of breeding, and utilizes crop waste resources to achieve comprehensive utilization of resources and environmental protection.
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Abstract
Description
Technical Field
[0001] The invention belongs to the field of aquaculture, and particularly relates to a regulating composition for improving the metamorphosis and all-male rate of bullfrog tadpoles, and a preparation method and application thereof. Background Art
[0002] Bullfrog is a large edible frog with tender meat, delicious taste and rich nutrition. It has been widely cultivated in China. The metamorphosis period of its tadpoles is a critical period in the breeding process, which will directly determine the success or failure of frog breeding in the later stage. However, due to the backward work of bullfrog seed conservation and breeding, inbreeding, drug abuse and other factors have led to the degeneration of parents, and the metamorphosis rate of tadpoles bred is low, which restricts the healthy development of the bullfrog seed industry chain. In addition, compared with female bullfrogs, male frogs have significant advantages such as fast growth rate, heavy hind leg weight, and high meat yield. Therefore, cultivating bullfrog tadpoles with high metamorphosis rate and male-to-female ratio has outstanding economic value and social benefits.
[0003] The metamorphosis of bullfrog tadpoles is mainly regulated by thyroid hormones, and the level of this hormone directly determines the speed and rate of metamorphosis. In addition, high aromatase activity in bullfrog seedlings promotes the conversion of androgens to estrogens, thereby reducing the proportion of male frogs. At present, hormones or chemical drugs are often used in the cultivation of bullfrog seedlings to accelerate the metamorphosis of frogs and increase the proportion of male frogs. However, the above two types of substances are prone to drug resistance and drug residues in aquatic animals during use, which restricts their widespread use in feed. In recent years, with the rise of the application of natural active substances in the fields of food and medicine, the research on plant active ingredients has received increasing attention. Luteolin is a natural flavonoid compound, and zinc lactate is an organic zinc. Both have been shown to regulate aromatase activity, thereby affecting the synthesis of sex hormones in mammals. Thyroxine is mainly composed of iodine and tyrosine. Both phosphatidylcholine complexed iodine (an organic iodine) and tyrosine can promote the synthesis of thyroid hormones in mammals. However, the physiological functions of aquatic animal tissues or organs are significantly different from those of humans and mammals. There are no reports on whether luteolin and zinc lactate can inhibit aromatase activity, increase the content of bullfrog male hormones, and increase the proportion of male frogs. At the same time, there is no research on whether lecithin-complexed iodine can promote the synthesis of bullfrog thyroid hormones and increase its metamorphosis rate. In addition, there are no reports on related products that use the different biological structure characteristics of the above four substances to scientifically match and exert synergistic effects to improve bullfrog metamorphosis and all-male rates.
[0004] Luteolin exists in large quantities in wheat bran, so using wheat bran, a waste crop, to extract luteolin and produce products containing luteolin can greatly increase the added value of wheat bran. At present, due to technical reasons and complex separation and purification processes, the amount of luteolin extracted from raw materials is low and the purity is not high, which limits the development and utilization of this natural product. Summary of the invention
[0005] In order to solve the above-mentioned deficiencies and shortcomings of the prior art, the object of the present invention is to provide a regulatory composition for improving the metamorphosis and all-male rate of bullfrog tadpoles.
[0006] Another object of the present invention is to provide a method for preparing the above-mentioned control composition for improving the metamorphosis and all-male rate of bullfrog tadpoles. The method improves the extraction amount and purity by improving the extraction, separation and purification method, extracts high-purity luteolin from bran, and synergizes luteolin, zinc lactate, phosphatidylcholine complex iodine and tyrosine to prepare a control composition for improving the metamorphosis and all-male rate of bullfrog tadpoles. As an efficient and environmentally friendly fishery feed additive, it can improve the economic benefits of bullfrog breeding.
[0007] Another object of the present invention is to provide the application of the above-mentioned regulating composition for improving the metamorphosis and all-male rate of bullfrog tadpoles.
[0008] The purpose of the present invention is achieved through the following technical solutions:
[0009] A regulating composition for improving the metamorphosis and all-male rate of bullfrog tadpoles, comprising, by weight, 5-15 parts of lecithin complexed iodine, 5-35 parts of tyrosine, 5-25 parts of zinc lactate, 5-30 parts of luteolin and 30-50 parts of expanded perlite; the luteolin is prepared by mixing crushed peanut meal and wheat bran, uniformly mixing with sucrose, soy peptone and MgSO4·7H2O, and adding double distilled water to prepare a solid culture medium; inoculating 20-70wt% of starch buds into the solid culture medium Bacillus L-S60 and 10-60wt% of cellulase are added, the pH is adjusted to 6-7.5, and the mixture is cultured at 30-36°C. After the fermentation is finished, the mixture is dried at 50-60°C to obtain a solid fermentation product of wheat bran; petroleum ether is added to the crushed material, and ultrasonic defatting is performed to obtain a defatted fermentation product; ethanol is added to the material and allowed to stand, and ultrasonic treatment is performed at 50-60°C in a water bath, and the product is filtered and concentrated into a water liquid at 40-55°C; the product is defatted with petroleum ether and then dissolved in corresponding ethanol to determine the content; the purity of the luteolin is greater than 90%.
[0010] Preferably, the composition comprises 6-14 parts of lecithin-complexed iodine, 6-32 parts of tyrosine, 6-23 parts of zinc lactate, 7-27 parts of luteolin and 32-48 parts of expanded perlite.
[0011] More preferably, the composition comprises 7-12 parts of lecithin-complexed iodine, 8-25 parts of tyrosine, 10-20 parts of zinc lactate, 10-25 parts of luteolin and 35-45 parts of expanded perlite.
[0012] Further preferably, the composition comprises 8-10 parts of lecithin-complexed iodine, 15-20 parts of tyrosine, 14-18 parts of zinc lactate, 15-20 parts of luteolin and 37-40 parts of expanded perlite.
[0013] The preparation method of the regulating composition for improving the metamorphosis and all-male rate of bullfrog tadpoles comprises the following specific steps:
[0014] S1. The crushed peanut meal and wheat bran were mixed, mixed evenly with sucrose, soy peptone and MgSO4·7H2O, and double distilled water was added to prepare a solid medium;
[0015] S2. In a solid medium, 20 to 70 wt% of Bacillus amyloliquefaciens L-S60 and 10 to 60 wt% of cellulase were introduced, the pH was adjusted to 6 to 7.5, the mixture was cultured at 30 to 36 ° C for 3 to 5 days, and the fermentation was dried at 50 to 60 ° C to obtain a solid fermentation product of wheat bran;
[0016] S3. The solid fermentation product of wheat bran was crushed to 80 to 120 mesh and added with petroleum ether, and ultrasonically defatted twice to obtain a defatted fermentation product;
[0017] S4. After adding ethanol to the defatted fermented product and letting it stand, ultrasonic extraction was performed in a water bath at 50-60°C, and the filtrate was filtered out after cooling to 30-35°C, and concentrated into an aqueous solution at 40-55°C. After defatting with petroleum ether, the product was dissolved in the corresponding ethanol and fixed to a fixed volume to determine the content, thereby obtaining luteolin;
[0018] S5. After the lecithin-complexed iodine, tyrosine, zinc lactate, luteolin and expanded perlite are crushed separately, the mixture is passed through a 120-150 mesh sieve at a temperature of 30-40° C. and then fully mixed to obtain a regulatory composition for improving the metamorphosis and all-male rate of bullfrog tadpoles.
[0019] Preferably, the mass ratio of the peanut meal and the wheat bran in step S1 is (1.5-5):(1-5), and the mass ratio of the total mass of the peanut meal and the wheat bran, sucrose, soy peptone, MgSO4·7H2O and double distilled water is (5-7):(10-15):(10-25):(0.002-0.004):(10-15).
[0020] Preferably, the drying time in step S2 is 6 to 12 hours; the mass ratio of the solid-state fermentation product of wheat bran to petroleum ether in step S3 is 1:(10 to 25); the frequency of the ultrasound is 20 to 25 kHz, and the power of the ultrasound is 100 to 300 W.
[0021] Preferably, in step S4, the mass ratio of the defatted fermentation product to ethanol is 1:(20-50), the concentration of the ethanol is 75-95wt%, the frequency of the ultrasound is 20-40kHz, the power of the ultrasound is 100-300W, and the time of the ultrasound is 50-60min.
[0022] The application of the regulating composition for improving tadpole metamorphosis and all-male rate in bullfrog tadpole feed.
[0023] Preferably, the amount of the regulating composition added to the bullfrog tadpole feed is 0.3-1.5 wt%.
[0024] The present invention mixes bran in proportion and then adds a composite strain for fermentation, thereby obtaining luteolin with a purity higher than 90%, and using the luteolin to prepare a frog control composition. This process can greatly improve the comprehensive utilization of crop waste resources, which is of great significance for promoting farmers' income increase, environmental protection, resource conservation and sustainable development of agricultural economy. Through technical optimization and breeding experiment screening, a frog control composition is obtained based on the pharmacological synergistic relationship of luteolin, zinc lactate, lecithin complex iodine and tyrosine, which significantly improves metamorphosis and all-male rate. As an efficient and environmentally friendly aquatic feed additive, it can improve the economic benefits of bullfrog breeding.
[0025] Compared with the prior art, the present invention has the following beneficial effects:
[0026] 1. The control composition of the present invention uses luteolin, zinc lactate, iodine lecithin complex and tyrosine as raw materials, and makes full use of the different characteristics of their biological structures to scientifically match them in order to exert synergistic effects. At the same time, it makes full use of crop waste resources, produces luteolin with the help of biological fermentation and other technologies, and adds it to the control composition.
[0027] 2. The present invention promotes the healthy development of the bullfrog seed industry chain and improves the economic benefits of breeders by improving the metamorphosis and all-male rate of bullfrog seedlings.
[0028] 3. The present invention makes full use of the different biological structures of raw materials to scientifically match and exert synergistic effects. Combining biological fermentation and ultrasonic purification technology greatly increases the comprehensive utilization of crop waste resources. The control composition of the present invention can increase the synthesis of thyroid hormones in bullfrog tadpoles, promote their metamorphosis, and significantly increase the ratio of male frogs, and is used in the field of bullfrog seed breeding. DETAILED DESCRIPTION
[0029] The content of the present invention is further described below in conjunction with specific examples, but it should not be construed as limiting the present invention. If not specifically indicated, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.
[0030] Example 1
[0031] 1. Grind 5 parts of peanut meal and wheat bran in a mass ratio of 4.5:2, mix them thoroughly with 10 parts of sucrose, 15 parts of soy peptone and 0.002 parts of MgSO4·7H2O, and add 10 parts of double distilled water to prepare a solid culture medium.
[0032] 2. After the solid culture medium is sterilized, a mixture of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 3:5 is added, the total inoculation amount is 30wt%, the bottling amount is 40vol%, the pH is adjusted to 6.0, and the culture is carried out at 32°C for 3 days. After the fermentation is completed, it is dried at 60°C for 12h to obtain a solid-state fermentation product of wheat bran.
[0033] 3. The solid fermentation product of wheat bran was crushed to 80 mesh, petroleum ether was added at a material-liquid mass ratio of 1:10, and defatted twice using ultrasonic 20kHz, 100W power to obtain defatted fermentation product.
[0034] 4. After defatting the fermented product, 100 mL of 95 wt% ethanol was added at a feed-liquid mass ratio of 1:50, and then allowed to stand. The product was extracted in a 50°C water bath at 40 kHz, 300 W power ultrasound for 60 min. After cooling to room temperature, the filtrate was filtered out and concentrated into an aqueous solution at 55°C. After defatting with petroleum ether, the product was dissolved in the corresponding ethanol and the content was determined to obtain luteolin.
[0035] 5. 5 parts of lecithin complexed iodine, 5 parts of tyrosine, 10 parts of zinc lactate, 30 parts of luteolin and 50 parts of expanded perlite were crushed at 40°C and passed through a 120-mesh sieve respectively, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0036] Example 2
[0037] 1. Grind 6 parts of peanut meal and wheat bran in a mass ratio of 4:2, mix them thoroughly with 10 parts of sucrose, 10 parts of soy peptone and 0.004 parts of MgSO4·7H2O, and add 10 parts of double distilled water to prepare a solid culture medium.
[0038] 2. After the solid culture medium is sterilized, a mixed solution of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 4:5 is inoculated, the total inoculation amount is 20wt%, the bottling amount is 40vol%, the pH is adjusted to 6.0, and the culture is carried out at 32°C for 3 days. After the fermentation is completed, it is dried at 50°C for 6h to obtain a solid-state fermentation product of wheat bran.
[0039] 3. The solid fermentation product of wheat bran was crushed to 80 mesh, petroleum ether was added at a material-liquid mass ratio of 1:10, and ultrasonic 25kHz, 100W power was used for defatting twice to obtain defatted fermentation product.
[0040] 4. After defatting the fermented product, 100 mL of 95 wt% ethanol was added at a feed-liquid mass ratio of 1:30, and then allowed to stand. The product was extracted in a 55°C water bath at 20 kHz, 300 W power ultrasound for 50 min. After cooling to room temperature, the filtrate was filtered out and concentrated into an aqueous solution at 50°C. After defatting with petroleum ether, the product was dissolved in the corresponding ethanol and the content was determined to obtain luteolin.
[0041] 5. 10 parts of lecithin complexed iodine, 5 parts of tyrosine, 25 parts of zinc lactate, 10 parts of luteolin and 50 parts of expanded perlite were crushed separately at 30°C and passed through a 120-mesh sieve, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0042] Example 3
[0043] 1. 5.5 parts of peanut meal and wheat bran in a mass ratio of 4:5 were crushed, mixed with 14 parts of sucrose, 20 parts of soy peptone and 0.004 parts of MgSO4·7H2O, and 15 parts of double distilled water were added to prepare a solid culture medium.
[0044] 2. After the solid culture medium is sterilized, a mixture of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 5:3 is added, the total inoculation amount is 30wt%, the bottling amount is 40vol%, the pH is adjusted to 7.0, and the culture is carried out at 32°C for 3 days. After the fermentation is completed, it is dried at 50°C for 10h to obtain a solid-state fermentation product of wheat bran.
[0045] 3. The solid fermentation product of wheat bran was crushed to 100 mesh, petroleum ether was added at a material-liquid mass ratio of 1:15, and ultrasonic 20kHz, 100W power was used for defatting twice to obtain defatted fermentation product.
[0046] 4. After defatting, add 100 mL of 75 wt% ethanol at a feed-liquid mass ratio of 1:35 to the fermented product and let it stand. Then use 20 kHz, 300 W power ultrasound in a 50 ° C water bath and extract it in a 55 ° C water bath for 60 min. After cooling to room temperature, filter out the filtrate, concentrate it into an aqueous solution at 40 ° C, defatted with petroleum ether, and then dissolved in the corresponding ethanol to determine the content to obtain luteolin.
[0047] 5. 15 parts of lecithin complexed iodine, 5 parts of tyrosine, 20 parts of zinc lactate, 15 parts of luteolin and 45 parts of expanded perlite were crushed separately at 30°C and passed through a 120-mesh sieve, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0048] Example 4
[0049] 1. Grind 7 parts of peanut meal and wheat bran in a mass ratio of 3:1, mix them thoroughly with 15 parts of sucrose, 25 parts of soy peptone and 0.004 parts of MgSO4·7H2O, and add 10 parts of double distilled water to prepare a solid culture medium.
[0050] 2. After sterilizing the solid culture medium, inoculate a mixture of Bacillus amyloliquefaciens L-S60 and cellulase at a mass ratio of 4.5:6, with a total inoculation amount of 20wt%, a bottling amount of 50vol%, adjust the pH to 7.0, and culture at 36°C for 3 days. After fermentation, dry at 50°C for 12h to obtain a solid fermentation product of wheat bran.
[0051] 3. The solid fermentation product of wheat bran was crushed to 90 mesh, petroleum ether was added at a material-liquid mass ratio of 1:25, and ultrasonic 20kHz, 300W power was used for defatting twice to obtain defatted fermentation product.
[0052] 4. Add 100 mL of 75 wt% ethanol to the defatted fermentation product at a feed-liquid mass ratio of 1:25, let it stand, and then extract it in a 55°C water bath at 20 kHz, 300 W power ultrasound for 60 min. After cooling to room temperature, filter out the filtrate, concentrate it into an aqueous solution at 45°C, defatted it with petroleum ether, and then dissolved it with the corresponding ethanol to determine the content, thereby obtaining luteolin.
[0053] 5. 5 parts of lecithin complexed iodine, 35 parts of tyrosine, 5 parts of zinc lactate, 5 parts of luteolin and 50 parts of expanded perlite were crushed separately at 30°C and passed through a 130-mesh sieve, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0054] Example 5
[0055] 1. Grind 6.5 parts of peanut meal and wheat bran in a mass ratio of 5:1, mix them thoroughly with 10 parts of sucrose, 20 parts of soy peptone and 0.004 parts of MgSO4·7H2O, and add 15 parts of double distilled water to prepare a solid culture medium.
[0056] 2. After the solid culture medium is sterilized, a mixed solution of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 6:5 is added, the inoculation amount is 20wt%, the bottling amount is 50vol%, the pH is adjusted to 7.5, and the culture is carried out at 36°C for 3 days. After the fermentation is completed, it is dried at 55°C for 12h to obtain a solid-state fermentation product of wheat bran.
[0057] 3. The solid fermentation product of wheat bran was crushed to 90 mesh, petroleum ether was added at a material-liquid mass ratio of 1:20, and ultrasonic 20kHz, 300W power was used for defatting twice to obtain defatted fermentation product.
[0058] 4. After defatting the fermented product, 100 mL of 75 wt% ethanol was added at a feed-liquid mass ratio of 1:20, and the product was allowed to stand. Then, the product was extracted in a water bath at 60°C at 20 kHz and 300 W power for 50 min. After cooling to room temperature, the filtrate was filtered out and concentrated into an aqueous solution at 40°C. After defatting with petroleum ether, the product was dissolved in the corresponding ethanol and the content was determined to obtain luteolin.
[0059] 5. 5 parts of lecithin complexed iodine, 30 parts of tyrosine, 10 parts of zinc lactate, 15 parts of luteolin and 40 parts of expanded perlite were crushed separately at 30°C and passed through a 130-mesh sieve, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0060] Example 6
[0061] 1. Grind 5 parts of peanut meal and wheat bran in a mass ratio of 5:3, mix them thoroughly with 15 parts of sucrose, 20 parts of soy peptone and 0.004 parts of MgSO4·7H2O, and add 10 parts of double distilled water to prepare a solid culture medium.
[0062] 2. After the solid culture medium is sterilized, a mixture of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 2:1 is added, the inoculation amount is 40wt%, the bottling amount is 40vol%, the pH is adjusted to 7.0, and the culture is carried out at 36°C for 3 days. After the fermentation is completed, it is dried at 50°C for 12h to obtain a solid-state fermentation product of wheat bran.
[0063] 3. The solid fermentation product of wheat bran was crushed to 100 mesh, petroleum ether was added at a material-liquid mass ratio of 1:20, and ultrasonic 20kHz, 300W power was used for defatting twice to obtain defatted fermentation product.
[0064] 4. Add 100 mL of 75% ethanol to the defatted fermentation product at a feed-liquid mass ratio of 1:25, let it stand, and then extract it in a 55°C water bath at 20 kHz, 100 W power ultrasound for 60 min. After cooling to room temperature, filter out the filtrate, concentrate it into an aqueous solution at 45°C, defatted it with petroleum ether, and then dissolved it with the corresponding ethanol to determine the content, so as to obtain luteolin.
[0065] 5. 5 parts of lecithin complexed iodine, 10 parts of tyrosine, 25 parts of zinc lactate, 10 parts of luteolin and 50 parts of expanded perlite were crushed separately at 45°C and passed through a 140-mesh sieve, and then fully mixed. During this process, the mixture must be uniform and stable to obtain a control composition.
[0066] Example 7
[0067] 1. Grind 6 parts of peanut meal and wheat bran in a mass ratio of 5:4, mix them thoroughly with 15 parts of sucrose, 25 parts of soy peptone and 0.002 parts of MgSO4·7H2O, and add 10 parts of double distilled water to prepare a solid culture medium.
[0068] 2. After the solid culture medium is sterilized, a mixed solution of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 7:5 is added, the inoculation amount is 40wt%, the bottling amount is 50vol%, the pH is adjusted to 7, and the culture is carried out at 36°C for 3 days. After the fermentation is completed, it is dried at 55°C for 12h to obtain a solid-state fermentation product of wheat bran.
[0069] 3. The solid fermentation product of wheat bran was crushed to 100 mesh, petroleum ether was added at a material-liquid mass ratio of 1:20, and ultrasonic 20kHz, 300W power was used for defatting twice to obtain defatted fermentation product.
[0070] 4. After defatting, add 100 mL of 75 wt% ethanol at a feed-liquid mass ratio of 1:20 to the fermented product, let it stand, and then extract it in a 55°C water bath at 20 kHz, 200 W power ultrasound for 80 min. After cooling to room temperature, filter out the filtrate, concentrate it into an aqueous solution at 40°C, defatted it with petroleum ether, and then dissolved it with the corresponding ethanol to determine the content, thereby obtaining luteolin.
[0071] 5. 15 parts of lecithin complexed iodine, 15 parts of tyrosine, 15 parts of zinc lactate, 5 parts of luteolin and 50 parts of expanded perlite were crushed separately at 35°C and passed through a 140-mesh sieve, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0072] Example 8
[0073] 1. Grind 5 parts of peanut meal and wheat bran in a mass ratio of 5:2, mix them thoroughly with 12 parts of sucrose, 20 parts of soy peptone and 0.002 parts of MgSO4·7H2O, and add 15 parts of double distilled water to prepare a solid culture medium.
[0074] 2. After the solid culture medium is sterilized, a mixture of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 5:1 is added, the inoculation amount is 40wt%, the bottling amount is 40vol%, the pH is adjusted to 7.5, and the culture is carried out at 36°C for 3 days. After the fermentation is completed, it is dried at 50°C for 12h to obtain a solid-state fermentation product of wheat bran.
[0075] 3. The solid fermentation product of wheat bran was crushed to 100 mesh, petroleum ether was added at a material-liquid mass ratio of 1:25, and ultrasonic 25kHz, 300W power was used for defatting twice to obtain defatted fermentation product.
[0076] 4. After defatting, add 100 mL of 75 wt% ethanol at a feed-liquid mass ratio of 1:25 to the fermented product, let it stand, and then extract it in a water bath at 55°C for 60 min at 20 kHz and 300 W power ultrasound. After cooling to room temperature, filter out the filtrate, concentrate it into an aqueous solution at 55°C, defatted it with petroleum ether, and then dissolved it with the corresponding ethanol to determine the content, thereby obtaining luteolin.
[0077] 5. 5 parts of lecithin complexed iodine, 10 parts of tyrosine, 20 parts of zinc lactate, 20 parts of luteolin and 45 parts of expanded perlite were crushed separately at 40°C and passed through a 150-mesh sieve, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0078] Example 9
[0079] 1. Grind 7 parts of peanut meal and wheat bran in a mass ratio of 3:2, mix them thoroughly with 10 parts of sucrose, 15 parts of soy peptone and 0.004 parts of MgSO4·7H2O, and add 10 parts of double distilled water to prepare a solid culture medium.
[0080] 2. After the solid culture medium is sterilized, a mixture of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 6:1 is added, the inoculation amount is 30wt%, the bottling amount is 40vol%, the pH is adjusted to 7.5, and the culture is carried out at 36°C for 3 days. After the fermentation is completed, it is dried at 55°C for 8h to obtain a solid-state fermentation product of wheat bran.
[0081] 3. The solid fermentation product of wheat bran was crushed to 100 mesh, petroleum ether was added at a solid-liquid ratio of 1:20, and ultrasonic 25kHz, 200W power was used for defatting twice to obtain defatted fermentation product.
[0082] 4. Add 100 mL of 75 wt% ethanol to the defatted fermentation product at a solid-liquid ratio of 1:25, let it stand, and then extract it in a 50°C water bath at 20 kHz, 300 W power ultrasound for 50 min. After cooling to room temperature, filter out the filtrate, concentrate it into an aqueous solution at 50°C, defatted it with petroleum ether, and then dissolved it with the corresponding ethanol to determine the content, thereby obtaining luteolin.
[0083] 5. 15 parts of lecithin complexed iodine, 10 parts of tyrosine, 12 parts of zinc lactate, 13 parts of luteolin and 50 parts of expanded perlite were crushed separately at 50°C and passed through a 150-mesh sieve, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0084] Example 10
[0085] 1. Grind 7 parts of peanut meal and wheat bran in a mass ratio of 1.5:2, mix them well with 15 parts of sucrose, 15 parts of soy peptone and 0.004 parts of MgSO4·7H2O, and add 15 parts of double distilled water to prepare a solid culture medium.
[0086] 2. After the solid culture medium is sterilized, a mixture of Bacillus amyloliquefaciens L-S60 and cellulase in a mass ratio of 5:2 is added, the inoculation amount is 30wt%, the bottling amount is 40vol%, the pH is adjusted to 7.2, and the culture is carried out at 36°C for 3 days. After the fermentation is completed, it is dried at 50°C for 8h to obtain a solid-state fermentation product of wheat bran.
[0087] 3. The solid fermentation product of wheat bran was crushed to 100 mesh, petroleum ether was added at a solid-liquid ratio of 1:25, and ultrasonic 25kHz, 300W power was used for defatting twice to obtain defatted fermentation product.
[0088] 4. After defatting the fermented product, 100 mL of 80 wt% ethanol was added at a solid-liquid ratio of 1:25, and the product was allowed to stand. Then, the product was extracted in a 50°C water bath at 20 kHz and 300 W power ultrasound for 60 min. After cooling to room temperature, the filtrate was filtered out and concentrated into an aqueous solution at 55°C. After defatting with petroleum ether, the product was dissolved in the corresponding ethanol and the content was determined to obtain luteolin.
[0089] 5. 12 parts of lecithin complexed iodine, 10 parts of tyrosine, 13 parts of zinc lactate, 15 parts of luteolin and 40 parts of expanded perlite were crushed separately at 35°C and passed through a 150-mesh sieve, and then fully mixed. During this process, the mixture must be ensured to be uniform and stable to obtain a control composition.
[0090] Application Example 1
[0091] Bullfrog tadpoles (about 0.02g) breeding test and result analysis: This breeding test was carried out at the Zhongshan Innovation Base of South China Agricultural University. The bullfrog tadpoles used were purchased from Guanxing Agricultural Technology Co., Ltd. 360 tadpoles of neat specifications were selected and randomly placed in 12 breeding tanks (30L of water in each tank), 30 tadpoles in each breeding tank. 1wt% of lecithin complex iodine, 1wt% of tyrosine and 1wt% of the regulating composition of Example 1 were added to the basic feed (20wt% fish meal, 20wt% soybean meal, 17wt% rapeseed meal, 19wt% cottonseed meal, 2wt% fish oil, 2wt% soybean oil, 17wt% dextrin and 2wt% dicalcium phosphate) to prepare (1) lecithin complex iodine feed; (2) tyrosine feed; (3) regulating composition feed, the feed formula components are shown in Table 1. The tadpoles were cultured for 12 weeks, during which the feeding and growth conditions of the tadpoles were observed. After the breeding, blood and tissue samples were collected for subsequent index analysis, and the results are shown in Table 2.
[0092] The weight gain rate, hind limb budding rate, limb budding rate, tail elimination rate, froglet rate, and serum thyroid hormone content of the tadpoles fed with the feed containing the control composition were significantly higher than those of the lecithin complexed iodine group and the tyrosine group. The above results show that the feed containing the control composition prepared in Example 1 can improve the growth performance of tadpoles, increase the thyroid hormone content of the body, and promote their metamorphosis.
[0093] Table 1 Test feed formula
[0094]
[0095] Table 2 Effects of different feeds on tadpole growth, metamorphosis and blood biochemical indicators
[0096]
[0097] Note: The same lowercase letters in the same row indicate no significant difference
[0098] Application Example 2
[0099] Bullfrog tadpoles (about 0.04g) breeding test and result analysis: This breeding test was carried out at the Zhongshan Innovation Base of South China Agricultural University. The bullfrog tadpoles used were purchased from Guanxing Agricultural Technology Co., Ltd. 360 tadpoles of neat specifications were selected and randomly placed in 12 breeding tanks (30L of water in each tank), with 30 tadpoles in each breeding tank. 1wt% of the regulating composition of Example 3, 1wt% of the regulating composition of Example 6 and 1wt% of the regulating composition of Example 10 were added to the basic feed (20wt% fish meal, 20wt% soybean meal, 17wt% rapeseed meal, 19wt% cottonseed meal, 2wt% fish oil, 2wt% soybean oil, 17wt% dextrin and 2wt% dicalcium phosphate) to prepare (1) regulating composition 3 feed group; (2) regulating composition 6 feed group; (3) regulating composition 10 feed group. The tadpoles were cultured for 12 weeks, during which the feeding and growth of the tadpoles were observed. After the breeding, blood and tissue samples were collected for subsequent index analysis. The results are shown in Table 3.
[0100] The weight gain rate, hind limb sprouting rate, limb sprouting rate, tail elimination rate, froglet rate, and serum thyroid hormone content of the tadpoles fed the feed containing the regulating composition 10 were significantly higher than those of the tadpoles in the regulating composition 3 feed group and the regulating composition 6 feed group. The above results show that the feed containing the regulating composition prepared in Example 10 is more effective in promoting the growth and metamorphosis of tadpoles.
[0101] Table 3 Effects of different feeds on tadpole growth, metamorphosis and blood biochemical indicators
[0102]
[0103] Note: The same lowercase letters in the same row indicate no significant difference
[0104] Application Example 3
[0105] Bullfrog tadpoles (about 0.03g) breeding experiment and result analysis: This breeding experiment was carried out at the Zhongshan Innovation Base of South China Agricultural University. The bullfrog tadpoles used were purchased from Guanxing Agricultural Technology Co., Ltd. 360 tadpoles of neat specifications were selected and randomly placed in 12 breeding tanks (30L of water in each tank), with 30 tadpoles in each breeding tank. 1wt% of zinc lactate, 1wt% of luteolin and 1wt% of the regulating composition of Example 2 were added to the basic feed (20wt% fish meal, 20wt% soybean meal, 17wt% rapeseed meal, 19wt% cottonseed meal, 2wt% fish oil, 2wt% soybean oil, 17wt% dextrin and 2wt% dicalcium phosphate) to prepare (1) zinc lactate feed group; (2) luteolin feed group; (3) regulating composition feed group. The tadpoles were cultured for 12 weeks, during which the feeding and growth of the tadpoles were observed. After the breeding, blood and tissue samples were collected for subsequent index analysis. The specific results are shown in Table 4. The weight gain rate, froglet rate, serum thyroid hormone content and male frog rate of tadpoles fed with the feed containing the regulating composition were significantly higher than those of the tadpoles in the zinc lactate feed group and the luteolin feed group. The above results show that the feed containing the regulating composition prepared in Example 2 can improve the growth performance of tadpoles, increase the thyroid hormone content of the body, and promote their metamorphosis. At the same time, the regulating composition 2 feed group also increases the rate of male frogs.
[0106] Table 4 Effects of different feeds on tadpole growth, metamorphosis, sex ratio and blood biochemical indicators
[0107]
[0108] Note: The same lowercase letters in the same row indicate no significant difference.
[0109] Application Example 4
[0110] Bullfrog tadpoles (about 0.08g) breeding test and result analysis: This breeding test was carried out at the Zhongshan Innovation Base of South China Agricultural University. The bullfrog tadpoles used were purchased from Guanxing Agricultural Technology Co., Ltd. 360 tadpoles of neat specifications were randomly placed in 12 breeding tanks (30L of water in each tank), 30 tadpoles in each breeding tank. 1wt% of the regulating composition of Example 4, 1wt% of the regulating composition of Example 8 and 1wt% of the regulating composition of Example 10 were added to the basic feed (20wt% fish meal, 20wt% soybean meal, 17wt% rapeseed meal, 19wt% cottonseed meal, 2wt% fish oil, 2wt% soybean oil, 17wt% dextrin and 2wt% dicalcium phosphate) to prepare (1) regulating composition 4 feed group; (2) regulating composition 8 feed group; (3) regulating composition 10 feed group. The tadpoles were cultured for 12 weeks, during which the feeding and growth of the tadpoles were observed. After the breeding, blood and tissue samples were collected for subsequent index analysis. The specific results are shown in Table 5. The weight gain rate, froglet rate, serum thyroid hormone content and male frog rate of tadpoles fed with the feed containing the regulating composition 10 were significantly higher than those of the tadpoles fed with the feed containing the regulating composition 4 and 8. The results show that the feed containing the regulating composition prepared in Example 10 is more effective in promoting the growth and metamorphosis of tadpoles and increasing the male ratio.
[0111] Table 5 Effects of different feeds on tadpole growth, metamorphosis, sex ratio and blood biochemical indicators
[0112]
[0113] Note: The same lowercase letters in the same row indicate no significant difference
[0114] The present invention combines the growth and physical and chemical index results of relevant breeding experiments to confirm that the regulatory composition can significantly promote the growth and metamorphosis of tadpoles and increase the male ratio.
[0115] 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. A control composition for improving the metamorphosis and all-male rate of bullfrog tadpoles, characterized in that: The regulating composition comprises 5 to 15 parts of lecithin complexed iodine, 5 to 35 parts of tyrosine, 5 to 25 parts of zinc lactate, 5 to 30 parts of luteolin and 30 to 50 parts of expanded perlite in parts by weight; the luteolin is prepared by mixing crushed peanut meal and wheat bran, mixing evenly with sucrose, soy peptone and MgSO4·7H2O, and then adding double distilled water to prepare a solid culture medium; 20 to 70 wt% of Bacillus amyloliquefaciens L-S6 is inoculated into the solid culture medium; The invention discloses a method for preparing a solid-state fermentation product of wheat bran by mixing 0 and 10 to 60 wt % of cellulase, adjusting the pH to 6 to 7.5, culturing at 30 to 36° C., drying after fermentation, and obtaining a solid-state fermentation product of wheat bran; then adding petroleum ether to the crushed material, performing ultrasonic defatting to obtain a defatted fermentation product, adding ethanol to the material, allowing the material to stand, performing ultrasonic defatting at 50 to 60° C. in a water bath, filtering the material, concentrating the material into a water solution at 40 to 55° C., defatting the material with petroleum ether, and then dissolving the material with corresponding ethanol to determine the content; the purity of the luteolin is greater than 90%.
2. The control composition for improving the metamorphosis and all-male rate of bullfrog tadpoles according to claim 1, characterized in that: The composition comprises 6-14 parts of lecithin complexed iodine, 6-32 parts of tyrosine, 6-23 parts of zinc lactate, 7-27 parts of luteolin and 32-48 parts of expanded pearlite.
3. The control composition for improving the metamorphosis and all-male rate of bullfrog tadpoles according to claim 2, characterized in that: The composition comprises 7-12 parts of lecithin complexed iodine, 8-25 parts of tyrosine, 10-20 parts of zinc lactate, 10-25 parts of luteolin and 35-45 parts of expanded pearlite.
4. The control composition for improving the metamorphosis and all-male rate of bullfrog tadpoles according to claim 3, characterized in that: The composition comprises 8-10 parts of lecithin complexed iodine, 15-20 parts of tyrosine, 14-18 parts of zinc lactate, 15-20 parts of luteolin and 37-40 parts of expanded pearlite.
5. The method for preparing the regulating composition for improving the metamorphosis and all-male rate of bullfrog tadpoles according to any one of claims 1 to 4, characterized in that: The specific steps include: S1. The crushed peanut meal and wheat bran were mixed, mixed evenly with sucrose, soy peptone and MgSO4·7H2O, and double distilled water was added to prepare a solid medium; S2. In a solid medium, 20 to 70 wt% of Bacillus amyloliquefaciens L-S60 and 10 to 60 wt% of cellulase were added, the pH was adjusted to 6.0 to 7.5, the temperature was raised at 30 to 36 ° C, and the fermentation was dried at 50 to 60 ° C to obtain a solid fermentation product of wheat bran; S3. The solid fermentation product of wheat bran was crushed to 80 to 120 mesh and added with petroleum ether, and ultrasonically defatted twice to obtain a defatted fermentation product; S4. After adding ethanol to the defatted fermented product and letting it stand, ultrasonic extraction was performed in a water bath at 50-60°C, and the filtrate was filtered out after cooling to 30-35°C, and concentrated into an aqueous solution at 40-55°C. After defatting with petroleum ether, the product was dissolved in the corresponding ethanol and fixed to a fixed volume to determine the content, thereby obtaining luteolin; S5. After the lecithin-complexed iodine, tyrosine, zinc lactate, luteolin and expanded perlite are crushed separately, the mixture is passed through a 120-150 mesh sieve at a temperature of 30-40° C. and then fully mixed to obtain a regulatory composition for improving the metamorphosis and all-male rate of bullfrog tadpoles.
6. The method for preparing the regulating composition for improving the metamorphosis and all-male rate of bullfrog tadpoles according to claim 5, characterized in that: The mass ratio of the peanut meal and the wheat bran in step S1 is (1.5-5):(1-5), and the mass ratio of the total mass of the peanut meal and the wheat bran, sucrose, soy peptone, MgSO4·7H2O and double distilled water is (5-7):(10-15):(10-25):(0.002-0.004):(10-15).
7. The method for preparing the regulating composition for improving the metamorphosis and all-male rate of bullfrog tadpoles according to claim 5, characterized in that: The drying time in step S2 is 6 to 12 hours, and the culturing time is 3 to 5 days; the mass ratio of the solid-state fermentation product of wheat bran to petroleum ether in step S3 is 1:(10 to 25); the frequency of the ultrasound is 20 to 25 kHz, and the power of the ultrasound is 100 to 300 W.
8. The method for preparing the regulating composition for improving the metamorphosis and all-male rate of bullfrog tadpoles according to claim 5, characterized in that: In step S4, the mass ratio of the defatted fermentation product to ethanol is 1:(20-50), the concentration of the ethanol is 75-95wt%, the frequency of the ultrasound is 20-40kHz, the power of the ultrasound is 100-300W, and the time of the ultrasound is 50-60min.
9. Use of the regulating composition for improving tadpole metamorphosis and all-male rate according to any one of claims 1 to 4 in bullfrog tadpole feed.
10. Use of the control composition for improving the metamorphosis and all-male rate of bullfrog tadpoles according to claim 9 in bullfrog tadpole feed, characterized in that: The amount of the regulating composition added to the bullfrog tadpole feed is 0.3-1.5 wt%.