Penaeus vannamei boone feed capable of rapidly generating and stabilizing biofloc and preparation method of penaeus vannamei boone feed

Abstract

The invention belongs to the technical field of aquaculture feeds, and discloses a penaeus vannamei boone feed capable of rapidly generating and stabilizing biological flocs and a preparation method of the penaeus vannamei boone feed. The feed comprises a nitrogen source, a quick-acting carbon source, a slow-release carbon source, compound microorganisms, compound vitamins, compound minerals, a carrier and a stabilizer, the quick-acting carbon source comprises at least one of molasses, glucose and brown sugar; the slow-release carbon source comprises at least one of bran, flour, corncob, peanut shell and wheat straw; the carbon-nitrogen ratio of the feed is greater than or equal to 15: 1. According to the invention, the rapid generation of the biofloc and the nutrition enrichment function are integrated into the single feed for the first time to form the low-protein dual-module carbon source feed, so that the breeding cost can be remarkably reduced. By accurately controlling the carbon-nitrogen ratio of the feed to be not less than 15: 1, the nutritional requirements of the penaeus vannamei boone and the carbon source and nitrogen source required by the growth of the biofloc are met at the same time.

Description

Technical Field

[0001] This invention belongs to the field of aquaculture feed technology, specifically relating to a feed for Litopenaeus vannamei that combines rapid biofloc formation and stability with its preparation method. Background Technology

[0002] Biological floc culture technology for Litopenaeus vannamei is a modern aquaculture technology centered on microbial technology. It promotes the reproduction of heterotrophic bacteria by regulating the carbon-to-nitrogen ratio (C / N) of the aquaculture system, thereby forming bioflocs. This achieves water purification, feed reuse, and high-efficiency aquaculture.

[0003] Bioflocs are mainly composed of bacteria, algae, protozoa, and particulate organic matter such as uneaten feed and feces. They serve as a natural food source for Litopenaeus vannamei, enabling the reuse of feed protein. Furthermore, through the assimilation by heterotrophic bacteria, the nitrification by nitrifying bacteria, and the absorption by microalgae within the flocs, they comprehensively control the concentrations of ammonia nitrogen and nitrite in the water, maintaining water quality stability. The stable formation of bioflocs is characterized by the ammonia nitrogen and nitrite concentrations in the aquaculture water remaining at low and stable levels.

[0004] In the biofloc culture model of Litopenaeus vannamei, the time it takes for bioflocs to form and stabilize significantly impacts feeding and shrimp growth. Farmers typically use external carbon sources such as molasses and brown sugar to promote biofloc formation within the culture system. However, the addition of external carbon sources often suffers from inconsistent effects and increases the operational complexity for farmers. Furthermore, the amount of external carbon source added is usually random and cannot meet the optimal carbon-nitrogen ratio required for floc growth, resulting in slow and unstable biofloc formation.

[0005] The protein content of existing commercial feeds for Litopenaeus vannamei (specific feed for Litopenaeus vannamei in small greenhouses) is usually designed to be above 43%, and the feed has a low carbon-to-protein ratio (about 9:1). Uneaten feed excretion will lead to the accumulation of ammonia nitrogen, which is not conducive to the formation of flocs.

[0006] Therefore, there is an urgent need to develop a feed that can meet the rapid growth requirements of Litopenaeus vannamei and promote the rapid formation of bioflocs in the aquaculture system, thereby achieving "multiple benefits from one feed" and reducing the overall cost of aquaculture. Summary of the Invention

[0007] This invention aims to at least solve one of the technical problems existing in the prior art. It provides a feed that achieves rapid and stable formation of bioflocs in aquaculture water through nutrient regulation and synergistic effects of microorganisms.

[0008] The first objective of this invention is to provide a low-protein dual-module carbon source feed.

[0009] The second objective of this invention is to provide the application of the feed of the first aspect of this invention in aquaculture.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention provides a low-protein dual-module carbon source feed, comprising a nitrogen source, a fast-acting carbon source, a slow-release carbon source, compound microorganisms, compound vitamins, compound minerals, a carrier, and a stabilizer. The fast-acting carbon source includes at least one of molasses, glucose, and brown sugar; The slow-release carbon source includes at least one of bran, flour, corn cob, peanut shell, and wheat straw; The carbon-to-nitrogen ratio of the feed is ≥15:1.

[0011] In some embodiments of the present invention, the carbon-to-nitrogen ratio of the feed is (15-20):1.

[0012] In some embodiments of the present invention, the nitrogen source includes at least one of fish meal, soybean meal, puffed soybeans, rapeseed meal, cottonseed meal, peanut meal, corn gluten meal, meat and bone meal, and yeast powder; preferably, it includes fish meal and soybean meal.

[0013] In some embodiments of the present invention, the carrier includes at least one of diatomaceous earth, activated carbon, zeolite powder and bentonite; preferably diatomaceous earth.

[0014] In some embodiments of the present invention, the stabilizer includes sodium humate and potassium fulvate.

[0015] In some embodiments of the present invention, the complex vitamins include vitamin A, vitamin D3, vitamin E, vitamin K3-MSB, vitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinamide, calcium pantothenate, folic acid, biotin, vitamin C lipoprotein, inositol, sandoquinone MAX, rice husk powder, and zeolite powder.

[0016] In some embodiments of the present invention, the composite minerals include copper sulfate, ferrous sulfate, zinc sulfate, manganese sulfate, magnesium sulfate, calcium iodate, sodium selenite, cobalt chloride, maifanite, zeolite powder, and rice husk powder.

[0017] In some embodiments of the present invention, the composite minerals include copper sulfate pentahydrate, ferrous sulfate monohydrate, zinc sulfate monohydrate, manganese sulfate monohydrate, magnesium sulfate monohydrate, calcium iodate, sodium selenite, cobalt chloride, maifanite, zeolite powder, and rice husk powder (30 mesh).

[0018] In some embodiments of the present invention, the composite microorganism is a microcapsule containing Bacillus (such as Bacillus licheniformis) and Clostridium butyricum.

[0019] In some embodiments of the present invention, the composite microorganisms are obtained by encapsulating Bacillus and Clostridium butyricum using sodium alginate and chitosan as encapsulating agents.

[0020] In some embodiments of the present invention, the composite microorganisms are prepared by a method comprising the following: Bacillus and Clostridium butyricum were mixed with sodium alginate solution to obtain sodium alginate bacterial suspension; A sodium alginate bacterial suspension was added dropwise to a calcium chloride solution and solidified to obtain gel beads. Gel beads were mixed with chitosan solution and reacted to obtain composite microorganisms.

[0021] In some embodiments of the present invention, the ratio of viable Bacillus to Clostridium butyricum is 1:(1-3), such as any one of 1:1, 1:2 or 1:3 or a range formed by any two of them.

[0022] In some embodiments of the present invention, the solvent of the sodium alginate solution is water.

[0023] In some embodiments of the present invention, the solvent of the chitosan solution is a 1%-3% aqueous acetic acid solution.

[0024] In some embodiments of the present invention, the concentration of the sodium alginate solution is 15-25 g / L, such as any value of 15, 17, 19, 20, 22, 24 or 25 g / L or a range formed by any two of them.

[0025] In some embodiments of the present invention, the concentration of Bacillus in the sodium alginate suspension is 45-55 g / L, such as any value of 45, 47, 49, 50, 52, 54 or 55 g / L or a range formed by any two of them.

[0026] In some embodiments of the present invention, the concentration of Clostridium butyricum in the sodium alginate suspension is 45-55 g / L, such as any value or a range formed by any two of 45, 47, 49, 50, 52, 54 or 55 g / L.

[0027] In some embodiments of the present invention, the mass ratio of sodium alginate to chitosan is 1:(1-3), such as any one of 1:1, 1:1.5, 1:2, 1:2.5 or 1:3 or a range formed by any two of them.

[0028] In some embodiments of the present invention, the curing time is 15-30 minutes, such as any value of 15, 17, 19, 20, 22, 24, 24 or 25 minutes or a range formed by any two of them.

[0029] In some embodiments of the invention, the reaction time is 25-40 minutes, such as any value of 25, 27, 29, 30, 32, 34, 36, 38 or 40 minutes or a range formed by any two of them.

[0030] In some embodiments of the present invention, the viable count of the Bacillus in the composite microorganism is 2-10 × 10⁻⁶. 9 cfu / g, such as 2×10 9 3×10 9 4×10 9 5×10 9 6×10 9 7×10 9 8×10 9 9×10 9 Or 10×10 9 Any value in cfu / g, or a range of values ​​formed by any two of them.

[0031] In some embodiments of the present invention, the viable count of *Clostridium butyricum* in the composite microorganism is 1-10 × 10⁻⁶. 8 cfu / g, such as 1×10 8 1.5×10 8 2×10 8 3×10 8 4×10 8 5×10 8 6×10 8 7×10 8 8×10 8 9×10 8 Or 10×10 8 Any value in cfu / g, or a range of values ​​formed by any two of them.

[0032] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises, by weight, 10-60 parts nitrogen source, 5-15 parts fast-acting carbon source, 5-35 parts slow-release carbon source, 0.1-1 parts compound microorganisms, 0.1-0.5 parts compound vitamins, 0.5-1.5 parts compound minerals, 1-5 parts carrier and 1-8 parts stabilizer.

[0033] In some embodiments of the invention, the low-protein dual-module carbon source feed comprises 25-35 parts by weight of fishmeal, such as any value or a range of any two of 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 parts, to provide both protein and nitrogen sources.

[0034] In some embodiments of the invention, the low-protein dual-module carbon source feed comprises 10-25 parts by weight of soybean meal, such as any value or a range of any two of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 parts, to provide both protein and nitrogen sources.

[0035] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises 5-10 parts by weight of readily available carbon source, such as any value of 5, 6, 7, 8, 9, or 10 parts, or a range formed by any combination of both. This is used to adjust the carbon-nitrogen ratio of the low-protein dual-module carbon source feed and provide energy.

[0036] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises 5-10 parts by weight of wheat bran, such as any value of 5, 6, 7, 8, 9, or 10 parts, or a range formed by any combination of both. The wheat bran acts as a slow-release carbon source, adjusting the carbon-to-nitrogen ratio of the low-protein dual-module carbon source feed.

[0037] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises 15-25 parts of flour, such as any value or a range formed by 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 parts. The flour acts as a slow-release carbon source, adjusting the carbon-to-nitrogen ratio of the low-protein dual-module carbon source feed and providing energy.

[0038] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises 0.1-0.5 parts by weight of compound microorganisms, such as any value or a range formed by any combination of 0.1, 0.2, 0.3, 0.4, or 0.5 parts. It acts as a microbial flocculant promoter, promoting microbial flocculation.

[0039] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises, by weight, 0.1-0.5 parts of compound vitamins, such as any one of 0.1, 0.2, 0.3, 0.4 or 0.5 parts or a range formed by any two of these values.

[0040] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises, by weight, 0.5-1.5 parts of compound vitamins, such as any value or a range formed by any two of 0.5, 0.7, 0.9, 1, 1.1, 1.2, 1.3, 1.4 or 1.5 parts.

[0041] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises, by weight, 1-5 parts of carrier, such as any value of 1, 2, 3, 4, or 5 parts, or a range formed by any two of these values. This serves as a microbial carrier and a microbial flocculant promoter.

[0042] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises, by weight, 1-6 parts of stabilizer, such as any value of 1, 2, 3, 4, 5, or 6 parts, or a range formed by any two of these values. It is used for floc stabilization and light-blocking algae suppression.

[0043] In some embodiments of the present invention, the low-protein dual-module carbon source feed further includes calcium dihydrogen phosphate and / or soybean phospholipid oil.

[0044] In some embodiments of the present invention, the low-protein dual-module carbon source feed comprises, by weight, 1-6 parts soybean lecithin oil, such as any value of 1, 2, 3, 4, 5, or 6 parts, or a range formed by any two of these values, for providing energy.

[0045] In some embodiments of the present invention, the low-protein dual-module carbon source feed includes 1-2 parts of calcium dihydrogen phosphate, such as any value of 1, 1.2, 1.4, 1.5, 1.6, 1.8, 1.9 or 2 parts or a range formed by any two of these values.

[0046] The low-protein dual-module carbon source feed provided by this invention adopts a dual-module carbon source design, selecting readily degradable carbon sources (molasses) and slow-release carbon sources (wheat bran, etc.) for compounding, achieving simultaneous carbon source release during feed feeding. The combination of fast-acting and slow-release carbon sources enables rapid and stable formation of bioflocs. Simultaneously, the low-protein formula design maintains a carbon-to-nitrogen ratio of ≥15:1. The low-protein formula design reduces ammonia nitrogen emissions and promotes water quality stability.

[0047] In some embodiments of the present invention, the preparation method of the low-protein dual-module carbon source feed includes the following steps: mixing the components and granulating them using a twin-screw low-temperature granulation process; in the twin-screw low-temperature granulation process, the modulation temperature is controlled below 40°C and the granulation temperature in the puffing chamber is not higher than 80°C.

[0048] Low-temperature granulation can preserve the activity of Bacillus and Clostridium butyricum.

[0049] A second aspect of the present invention provides the application of the low-protein dual-module carbon source feed of the first aspect of the present invention in aquaculture.

[0050] In some embodiments of the present invention, the aquaculture includes fish farming and shrimp farming (such as Litopenaeus vannamei).

[0051] The beneficial effects of this invention are: This invention is the first to integrate rapid biofloc formation and nutritional fortification into a single feed, creating a low-protein, dual-module carbon source feed that can significantly reduce farming costs. By precisely controlling the feed's carbon-to-nitrogen ratio to be no less than 15:1, it simultaneously meets the nutritional needs of Litopenaeus vannamei and the carbon and nitrogen sources required for biofloc growth.

[0052] The low-protein dual-module carbon source feed provided by this invention not only meets the nutritional requirements for the growth of Litopenaeus vannamei, but also provides the necessary carrier, carbon source, nitrogen source, and probiotics for the formation and stabilization of bioflocs. The fast-acting carbon source (molasses) promotes floc formation and provides energy for microbial metabolism, indirectly improving feed utilization. The long-acting carbon source (wheat bran and flour) provides energy for the growth of Litopenaeus vannamei and continuously provides carbon for the formation of bioflocs, promoting their stability. The carrier (diatomaceous earth) provides attachment points for both flocs and microorganisms. Attached Figure Description

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The effect of different feeds on the amount of biofloc formation in small-scale ponds.

[0054] Figure 2 The effect of feeding low-protein dual-module carbon source feed on ammonia nitrogen content in small-scale ponds.

[0055] Figure 3 The effect of feeding low-protein dual-module carbon source feed on nitrite content in small-scale ponds.

[0056] Figure 4 The effect of feeding low-protein dual-module carbon source feed on the body weight of Litopenaeus vannamei in small-scale greenhouse model.

[0057] Figure 5 The study investigated the effects of feeding low-protein dual-module carbon source feed on the production performance of Litopenaeus vannamei in a small greenhouse model. In this study, A represents the statistical results of Litopenaeus vannamei yield in each treatment group, B represents the recapture rate of Litopenaeus vannamei in each treatment group, and C represents the statistical results of feed coefficient in each treatment group. Detailed Implementation

[0058] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0059] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0060] In this embodiment of the invention, the composite bacterial powder is prepared by the following method: Bacillus licheniformis and Clostridium butyricum are mixed in a 1:1 ratio of live bacteria; sodium alginate and chitosan are used as encapsulating agents, and microcapsule technology is employed to prepare microcapsules, i.e., the composite bacterial powder. The specific preparation process is as follows: (1) Solution preparation Sodium alginate bacterial suspension: Weigh 20g of sodium alginate (viscosity ≥0.02Pa·s, 20℃, Sinopharm Chemical Reagent Co., Ltd.) and dissolve it in 1L of pure water at 45℃. Stir with a magnetic stirrer until the sodium alginate is completely dissolved, forming a clear, viscous solution. Cool to room temperature. Weigh 50g of Bacillus licheniformis and 50g of Clostridium butyricum separately; first, slowly add Bacillus licheniformis to the blended gel and stir magnetically until completely dissolved; then add Clostridium butyricum and stir magnetically until completely dissolved.

[0061] Calcium chloride crosslinking solution: Weigh 30g of calcium chloride and dissolve it in pure water.

[0062] Chitosan solution: Weigh 30g of chitosan (degree of deacetylation ≥90.0%, viscosity <0.1Pa·s, Shanghai Bio-Tech Co., Ltd.) and dissolve it in 2% acetic acid aqueous solution. Stir with a magnetic stirrer until the chitosan is completely dissolved.

[0063] (2) Preparation of calcium alginate gel beads The prepared sodium alginate bacterial suspension is added dropwise to a calcium chloride solution under magnetic stirring (50 rpm). The sodium alginate bacterial suspension droplets form a gel film on the surface the moment they come into contact with the calcium chloride solution, becoming solidified gel beads. The gel beads continue to solidify in the calcium chloride solution for 20 minutes.

[0064] (3) Chitosan coating Use a sieve to remove the fixed calcium alginate gel beads, and gently rinse them with pure water to remove the calcium chloride solution from the surface. Transfer the rinsed gel beads to a chitosan solution, stir gently to ensure that each gel bead is fully in contact with the chitosan solution, and soak for 30 minutes to obtain microcapsules. Use a sieve to remove the microcapsules and gently rinse them with pure water.

[0065] (4) Storage and testing The prepared microcapsules (i.e., compound bacterial powder) are placed in a ventilated area for brief air drying until the surface of the microcapsules is no longer sticky to the touch. The microcapsules are then transferred to a refrigerator at 4°C for storage until use.

[0066] The viable counts of Bacillus licheniformis and Clostridium butyricum in the microcapsules were 2.84 × 10⁻⁶. 9 cfu / g and 1.5×10 8 cfu / g.

[0067] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0068] Example 1 A low-protein dual-module carbon source feed, by weight, is made from the following raw materials: 30 parts fish meal (crude protein ≥ 65%), 15 parts soybean meal (protein > 46%), 10 parts molasses, 5 parts wheat bran, 3 parts soybean lecithin oil, 20 parts flour, 1.5 parts calcium dihydrogen phosphate, 0.2 parts compound vitamin premix, 1 part compound mineral premix, 0.2 parts compound Bacillus subtilis, 4 parts sodium humate, and 3 parts diatomaceous earth; Each kilogram of vitamin premix (hereinafter the same) contains: Vitamin A 8g, Vitamin D3 3.5g, Vitamin E (50%) 150g, Vitamin K3-MSB 21g, Vitamin B1 16g, Vitamin B2-80% 16.5 parts, Vitamin B6 13.5g, 1% Vitamin B12 5g, Nicotinamide 20g, Calcium Pantothenate 15g, Folic Acid 2.5g, Biotin-2% 12.5g, Vitamin C Lipid 300g, Inositol 108g, Santoquinone MAX 1.5g, 80-mesh Rice Husk Powder 207g, and Zeolite Powder 100g.

[0069] Each kilogram of mineral premix (the same below) contains: 40g copper sulfate pentahydrate, 180g ferrous sulfate monohydrate, 93g zinc sulfate monohydrate, 23g manganese sulfate monohydrate, 500g magnesium sulfate monohydrate, 10g calcium iodate-5%, 3g sodium selenite-5%, 8g cobalt chloride-5%, 100g maifanite, 100g zeolite powder, and 112g 30-mesh rice husk powder.

[0070] The preparation method of the above-mentioned low-protein dual-module carbon source feed includes the following steps: Fish meal, soybean meal, molasses, wheat bran, flour, calcium dihydrogen phosphate, compound vitamin premix, compound mineral premix, compound bacterial powder, sodium humate, and diatomaceous earth are mixed according to the proportions in the formula, pulverized, and then passed through an 80-mesh sieve. Molasses is first passed through an 80-mesh sieve and then mixed with the powdered raw materials. After all the raw materials are mixed, they are processed into 1.1mm diameter pellets using a twin-screw extruder (Yanggong TSE65S type twin-screw wet extruder) employing a twin-screw low-temperature pelleting process (the pelleting temperature is controlled to not exceed 60℃ by water cooling).

[0071] The carbon-to-nitrogen ratio of the protein dual-module carbon source feed in this embodiment is 15.6:1.

[0072] Example 2 A low-protein dual-module carbon source feed, by weight, is made from the following raw materials: 25 parts fish meal (crude protein ≥ 65%), 25 parts soybean meal (protein > 46%), 3 parts soybean lecithin oil, 5 parts molasses, 10 parts wheat bran, 20 parts flour, 1.5 parts calcium dihydrogen phosphate, 0.2 parts compound vitamin premix, 1 part compound mineral premix, 0.1 parts compound bacterial powder, 4 parts sodium humate, and 3 parts diatomaceous earth.

[0073] The preparation method of the above-mentioned low-protein dual-module carbon source feed for Litopenaeus vannamei is the same as in Example 1.

[0074] The carbon-to-nitrogen ratio of the protein dual-module carbon source feed in this embodiment is 16.2:1.

[0075] Comparative Example 1 A conventional feed for Litopenaeus vannamei contains the following ingredients by weight: 25 parts fishmeal (crude protein ≥65%), 35 parts soybean meal, 20 parts wheat flour, 1.5 parts calcium dihydrogen phosphate, 0.2 parts compound vitamin premix, and 1 part compound mineral premix.

[0076] The preparation method of the above-mentioned feed for Litopenaeus vannamei is the same as in Example 1.

[0077] In this embodiment, the carbon-to-nitrogen ratio of the feed for Litopenaeus vannamei is 13.5:1.

[0078] Effect Example 1. Winter greenhouse farming test of Litopenaeus vannamei. The experiment was conducted at the Haiweilai Aquaculture Service Company base (Guanghai Town, Taishan City, Guangdong Province). The base had 30 aquaculture sheds, each measuring 10m × 40m with a water depth of 80cm, and each shed stocked with 60,000 Litopenaeus vannamei shrimp. Before the experiment, the sheds were divided into three groups: a control group, Example 1 group, and Example 2 group, with three sheds in each group. The control group, Example 1 group, and Example 2 group were fed the feed of Control Group 1 and the low-protein dual-module carbon source feed of Examples 1 and 2, respectively. After the start of the experiment, the daily feeding amount was calculated as 3% of the shrimp mass in the sheds, and the daily feeding was divided into four feedings at 7:00, 11:00, 15:00, and 19:00.

[0079] After the start of culture, the biofloc content was measured every 3 days, and water samples were collected and immediately taken back to the laboratory to test for ammonia nitrogen and nitrite content. Every 10 days, the size of the Litopenaeus vannamei was measured. After 90 days of culture, the yield, recapture rate, and feed conversion ratio of Litopenaeus vannamei were calculated. Ten Litopenaeus vannamei were taken from each shed, and their hepatopancreas, intestines, and stomach were dissected on ice. After dissection, they were immediately placed in liquid nitrogen and brought back to the laboratory and refrigerated at -80°C for later use.

[0080] Detection method: Biofloc content: Take 1L of water sample in an Inhofe conical tube, let it stand for 5min, and record the content of sediment as the biofloc content; Ammonia nitrogen detection: (1) Preparation of standard curve: Use a pipette to pipette 0.5, 1.0, 3.0, 5.0, 7.0 and 10.0 mL of ammonia nitrogen standard solution respectively, and dilute with water to 50 mL. Add 1.0 mL of potassium sodium tartrate solution, mix well, then add 1.0 mL of Nessler solution, mix well, let stand for 10 min, and measure the absorbance at a wavelength of 420 nm using a 20 mL cuvette with pure water as a control. (2) Sample determination: Take 5 mL of the filtered water sample and add it to a 20 mL colorimetric tube, dilute with water to the mark, add 1.0 mL of potassium sodium tartrate solution, mix well, then add 1.0 mL of Nessler solution, mix well, let stand for 10 min, and measure the absorbance at a wavelength of 420 nm using a 20 mL cuvette with pure water as a control.

[0081] Nitrite detection: (1) Preparation of standard curve: Use a pipette to take 0.5, 1.0, 3.0, 5.0, 7.0 and 10.0 mL of nitrite standard solution respectively, and dilute with water to the mark. Add 1.0 mL of colorimetric reagent, stopper tightly, mix well, let stand for 20 min, and measure the absorbance at a wavelength of 540 nm, with pure water as the control. (2) Sample determination: Take 5 mL of the filtered water sample and add it to a 50 mL colorimetric tube, dilute with water to the mark, add 1.0 mL of colorimetric reagent, let stand for 20 min, and measure the absorbance at 540 nm, with pure water as the control.

[0082] Protease activity assay: The Folin-phenol method was used. 1 mL of 1% casein solution and 5 mL of 0.025 mol / L phosphate buffer were added to a test tube and incubated at 37°C for 10 min. 1 mL of sample (digestive tract tissue homogenate) was then quickly added, and the mixture was incubated at 37°C for 20 min. The reaction was terminated by adding 3 mL of 10% trichloroacetic acid using a pipette. 1 mL of the filtrate was added to 5 mL of 0.55 mol / L sodium carbonate solution and 0.5 mL of Folin reagent. The mixture was incubated in a water bath (37°C) for 15 min, and the color was measured at 680 nm. For the control tube, the stop solution was added first, followed by the enzyme solution. One unit of protease activity was defined as the production of 1 μg of tyrosine per minute.

[0083] Amylase activity assay: The 3,5-dinitrosalicylic acid method was used. 1 mL of a 1% soluble starch solution was preheated in a 37°C water bath for 10 min. 0.2 mL of sample (digestive tract homogenate) was added, and the mixture was reacted in a 37°C water bath for 5 min. Immediately after reaction, 1 mL of 3,5-dinitrosalicylic acid colorimetric reagent was added, and the mixture was placed in a boiling water bath for 5 min. After cooling under running water, the solution was diluted 20 times, and the colorimetric value was measured at 540 nm. In the control tube, the colorimetric reagent was added first, followed by the sample. One unit of amylase activity is defined as the amount of starch that can be dissolved to produce 1 μmol of maltose per minute at pH 7.5.

[0084] Aquaculture results: (1) Effects of low-protein dual-module carbon source feed on water quality in small-scale ponds for Litopenaeus vannamei in winter Feeding with the low-protein dual-module carbon source diets of Examples 1 and 2 resulted in increased biofloc formation starting on day 45 of culture, reaching its maximum on days 57 and 60, respectively. In the comparative group, biofloc formation began on day 54 of culture, reaching its maximum on day 72. During the culture of Litopenaeus vannamei, the maximum biofloc formation in the comparative group was 16.75 mL / L, in Example 1 it was 21.55 mL / L, and in Example 2 it was 22.1 mL / L. Figure 1 ).

[0085] During the aquaculture trial, the nutritional composition of bioflocs collected on days 50 and 90 of aquaculture was analyzed. Crude protein was determined using the Kjeldahl method with a Kjeldahl nitrogen analyzer (Kjeltec-8400, FOSS Tecator, Haganas, Sweden). Crude fat was determined using the Soxhlet extraction principle with a Soxhlet extractor (Soxtec-2055, FOSS Tecator, Haganas, Sweden). The results are shown in Table 1. As can be seen from Table 1, there was no significant difference in the nutritional composition of the bioflocs when different low-protein dual-module carbon source diets were fed. Specifically, on day 50 of aquaculture, there was no significant difference in protein and fat content among the bioflocs in the examples and comparative examples; on day 90 of aquaculture, there was no significant difference in protein and fat content among the bioflocs in the treatment groups, with the highest protein content in the bioflocs of comparative example group 1 and the lowest protein content in the bioflocs of example group 1.

[0086] Table 1. Effects of feeding low-protein dual-module carbon source feed on biofloc formation in small-scale ponds.

[0087] The ammonia nitrogen content in the aquaculture water was measured during the aquaculture period, and the results are as follows: Figure 2As shown, the ammonia nitrogen level in the culture water gradually increased during days 30-40 of culture. On day 42, the ammonia nitrogen content in the comparative group pond reached its maximum (7.05 mg / kg), while in Example 1 group it reached its maximum (3.00 mg / kg) on ​​day 45, and in Example 2 group it reached its maximum (5.9 mg / kg) on ​​day 45. The highest ammonia nitrogen content in the comparative group pond was significantly higher than that in Example 1 group. On day 63 of culture, the ammonia nitrogen in the comparative group decreased to a safe level (1.0 mg / kg), while in Example 1 group it decreased to a safe level on day 51, and in Example 2 group it decreased to a safe level on day 54. After day 70 of culture, the ammonia nitrogen content in the culture pond gradually decreased and remained relatively stable.

[0088] The results were obtained by measuring the nitrite content in the aquaculture water during the aquaculture period. Figure 3 As shown, around day 33 of culture, the nitrite level in the culture water gradually increased; on day 51, the nitrite content in the control group pond reached its maximum (10.90 mg / kg), the nitrite content in Example 1 group pond reached its maximum (5.2 mg / kg) on ​​day 51, and the nitrite content in Example 2 group pond reached its maximum (7.9 mg / kg) on ​​day 48. The nitrite content in Examples 1 and 2 groups returned to a safe level (1.0 mg / kg) on ​​day 60, while the nitrite content in the control group pond only decreased to a safe level on day 81.

[0089] (2) Effects of low-protein dual-module carbon source diet on the growth performance of Litopenaeus vannamei in small greenhouse model Shrimp from each treatment group were sampled, and their weights were measured. The results are as follows: Figure 4 As shown. Starting from day 80 of culture, significant differences in the size of the Pacific white shrimp in the Example 1 group and the Comparative group appeared, with the sample size from largest to smallest being Example 1 group > Example 2 group > Comparative group. On day 80 of culture, the Pacific white shrimp in Example 1 group were significantly larger than those in the Comparative group ( p The shrimp size in Example 1 group was ≤0.05, showing no significant difference from the control group; the shrimp size in Example 2 group was larger than that in the control group, but there was no significant difference. On day 90 of culture, the shrimp size in Example 1 group was significantly larger than that in the control group (≤0.05), showing no significant difference from the control group. p (≤0.05), with no significant difference from Example 2; the size of the whiteleg shrimp in Example 2 was larger than that in the comparative example, but there was no significant difference.

[0090] The yield of Litopenaeus vannamei in each treatment group was further analyzed, and the results are as follows: Figure 5 As shown. By Figure 5 As shown in section A, the yield of greenhouse-raised Litopenaeus vannamei fed with the low-protein dual-module carbon source feed of Examples 1 and 2 was significantly higher than that of Control Example 1, and the yield of Example 1 group was significantly higher than that of Example 2 group.p ≤0.05). By Figure 5 As shown in Figure B, the recapture rate of Litopenaeus vannamei in the comparative group was significantly lower than that in Example 1 and Example 2. p (≤0.05), there was no significant difference between Example 1 group and Example 2 group. Figure 5 As can be seen from C, the feed conversion ratio of the comparative group was significantly higher than that of the Example 1 group and the Example 2 group ( p (≤0.05), there was no significant difference between Example 1 group and Example 2 group.

[0091] (3) Effects of low-protein dual-module carbon source feed on digestive enzyme activity in Litopenaeus vannamei in small-scale greenhouse model After the aquaculture trial, the protease activities of the hepatopancreas and stomach of Litopenaeus vannamei in each treatment group were measured, and the results are shown in Table 2. As can be seen from the data in Table 2, the low-protein dual-carbon-source modular diet had no significant effect on the protease activities of the hepatopancreas and stomach of Litopenaeus vannamei, but significantly affected the intestinal protease activities. The intestinal protease activity of the Example 1 group was significantly higher than that of the comparative group, while the intestinal protease activity of the Example 2 group was not significantly different from that of the comparative group and the Example 1 group.

[0092] Table 2. Effects of different experimental diets on the activity of digestive tract proteases in Litopenaeus vannamei.

[0093] After the aquaculture trial, the amylase activities of the hepatopancreas and intestines of Litopenaeus vannamei in each treatment group were measured, and the results are shown in Table 3. Table 3 shows that the low-protein dual-carbon source modular diet had no significant effect on the amylase activity of the hepatopancreas of Litopenaeus vannamei, but significantly affected the intestinal amylase activity. The intestinal amylase activity of the Example 1 group was significantly higher than that of the control group (…). p (≤0.05), the intestinal amylase activity in the Example 2 group was higher than that in the control group, but there was no significant difference.

[0094] Table 3. Effects of different experimental diets on the activity of amylase in the digestive tract of Litopenaeus vannamei.

[0095] In summary, the low-protein dual-module carbon source feed provided in Examples 1-2 of this invention has the following advantages in the farming of Litopenaeus vannamei: Advantages in water quality regulation: The low-protein dual-module carbon source feed has a significant effect on regulating the water quality of small-scale ponds. In Examples 1 and 2, the biofloc formation time of the low-protein dual-module carbon source feed was 9-15 days earlier than that of Control Example 1, with maximum formation amounts reaching 21.55 mL / L and 22.1 mL / L respectively, significantly higher than the 16.75 mL / L of the Control Example group. Regarding ammonia nitrogen control, the maximum ammonia nitrogen value in Example 1 (3.00 mg / kg) was significantly lower than that in the Control Example group (7.05 mg / kg), and the recovery time to safe levels was 9 days earlier. The control of nitrite content also showed significant advantages; the maximum value in Example 1 was 40%-52% lower than that in the Control Example group, and the recovery time to safe levels was 24 days earlier.

[0096] Performance Enhancement Advantages: When fed the low-protein dual-module carbon source feed of Example 1, the yield of Litopenaeus vannamei was the highest, significantly better than the other two groups; the recapture rate was significantly higher than the comparative group ( p ≤0.05); the feed conversion ratio was significantly lower than that of the control group ( p ≤0.05).

[0097] Low-protein dual-module carbon source feed optimizes shrimp digestion, effectively improves the farming environment, and significantly enhances production performance. Among them, the low-protein dual-module carbon source feed of Example 1 showed the best performance in all key indicators and has the best application value.

[0098] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A feed comprising a nitrogen source, a readily available carbon source, a slow-release carbon source, compound microorganisms, compound vitamins, compound minerals, a carrier, and a stabilizer; The fast-acting carbon source includes at least one of molasses, glucose, and brown sugar; The slow-release carbon source includes at least one of bran, flour, corn cob, peanut shell and wheat straw; The feed has a carbon-to-nitrogen ratio of ≥15:

1.

2. The feed according to claim 1, characterized in that, The nitrogen source includes at least one of fish meal, soybean meal, puffed soybeans, rapeseed meal, cottonseed meal, peanut meal, corn gluten meal, meat and bone meal, and yeast powder.

3. The feed according to claim 1, characterized in that, The composite microorganism is a microcapsule containing Bacillus and Clostridium butyricum.

4. The feed according to claim 3, characterized in that, The composite microorganisms are obtained by encapsulating Bacillus and Clostridium butyricum using sodium alginate and chitosan as encapsulating agents.

5. The feed according to claim 4, characterized in that, The composite microorganisms were prepared by the following methods: Bacillus and Clostridium butyricum were mixed with sodium alginate solution to obtain sodium alginate bacterial suspension; A sodium alginate bacterial suspension was added dropwise to a calcium chloride solution and solidified to obtain gel beads. Gel beads were mixed with chitosan solution and reacted to obtain composite microorganisms.

6. The feed according to claim 5, characterized in that, The curing time is 15-30 minutes; and / or the reaction time is 25-40 minutes.

7. The feed according to any one of claims 1-5, characterized in that, The carrier includes at least one of diatomaceous earth, activated carbon, zeolite powder, and bentonite; and / or, the stabilizer includes sodium humate and potassium humate.

8. The feed according to any one of claims 1-5, characterized in that, The complex vitamins include vitamin A, vitamin D3, vitamin E, vitamin K3, vitamin B1, vitamin B2, vitamin B6, vitamin B12, nicotinamide, calcium pantothenate, folic acid, biotin, vitamin C ester, inositol, sardine MAX, rice husk powder, and zeolite powder; and / or, the complex minerals include copper sulfate, ferrous sulfate, zinc sulfate, manganese sulfate, magnesium sulfate, calcium iodate, sodium selenite, cobalt chloride, maifanite, zeolite powder, and rice husk powder.

9. The feed according to any one of claims 1-5, characterized in that, By weight, the feed comprises 10-60 parts nitrogen source, 5-15 parts readily available carbon source, 5-35 parts slow-release carbon source, 0.1-1 parts compound microorganisms, 0.1-0.5 parts compound vitamins, 0.5-1.5 parts compound minerals, 1-5 parts carrier and 1-8 parts stabilizer.

10. The use of the feed according to any one of claims 1-9 in aquaculture.

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