A low morbidity feed for antibiotic-free culture of micropterus salmoides and a method for its preparation

Abstract

The application discloses a kind of low morbidity feed for California bass antibiotic-free breeding and preparation method thereof, belong to aquatic feed field.The feed is composed of 60~80 parts of basic feed component, 10~20 parts of fermented plant protein, 2~6 parts of functional compound additives.Fermented plant protein is prepared by compound fermentation of butyric acid clostridium and bacillus subtilis from soybean meal, bran, rice bran, brown sugar and the like.Functional compound additives include pre-embedded material and microcapsule coated sodium butyrate, and the pre-embedded material is prepared by embedding treatment of astaxanthin, lutein and rosemary extract with β-cyclodextrin and gum arabic.The application significantly reduces the incidence of intestinal inflammation of California bass through the synergistic effect of fermented plant protein, pre-embedded antioxidant combination and microcapsule sodium butyrate, enhances antioxidant capacity, improves body color, improves the stability of active ingredients and feeding rate, and realizes low morbidity under antibiotic-free breeding conditions.

Description

Technical Field

[0001] This invention belongs to the field of aquatic feed, specifically, it relates to a low-morbidity feed for antibiotic-free aquaculture of California bass and its preparation method. Background Technology

[0002] California bass (Micropterus salmoides) is an important freshwater aquaculture fish in my country, widely farmed due to its rapid growth, delicious meat, and high market acceptance. In recent years, with the continuous increase in farming density, disease problems in California bass farming have become increasingly prominent, especially the frequent occurrence of intestinal inflammation, oxidative stress, and bacterial diseases, seriously restricting the sustainable development of the industry. To control diseases, the overuse of antibiotics in traditional farming methods is widespread, leading not only to excessive drug residues and increased antibiotic resistance in pathogens, but also raising widespread concerns about food safety and the ecological environment. Therefore, developing antibiotic-free farming technologies suitable for California bass, especially through functional feeds to regulate fish health and reduce morbidity, has become a research hotspot in this field.

[0003] In the field of functional feed additives, sodium butyrate, carotenoids (astaxanthin, lutein), and plant extracts (rosemary extract) have been studied and applied in aquaculture. Sodium butyrate, as a short-chain fatty acid salt, can provide energy for intestinal epithelial cells, promote intestinal villus growth, and enhance intestinal barrier function; however, its synergistic effect with other additives in California bass feed has not been fully studied. Astaxanthin and lutein, as natural carotenoids, have antioxidant, immune-enhancing, and color-improving functions. There are existing reports of adding them alone or in combination to aquatic feeds, but systematic synergistic design with other functional ingredients is generally lacking. Rosemary extract is rich in polyphenols such as rosmarinic acid, possessing antioxidant and antibacterial activities; however, its application in aquatic feeds is limited, and due to its susceptibility to oxidation and poor stability, suitable formulation technologies are needed for protection.

[0004] On the other hand, fermented plant proteins, due to their ability to produce active substances such as organic acids, small peptides, and probiotic metabolites, have been widely used as functional ingredients in aquatic feed. Among them, *Clostridium butyricum* fermentation produces butyric acid, which has unique advantages in regulating intestinal flora and repairing the intestinal mucosa; *Bacillus subtilis* can secrete various extracellular enzymes, promoting the degradation of macromolecular proteins and improving fermentation efficiency. However, existing *Clostridium butyricum* fermentation technologies typically use soybean meal as the main substrate, without systematically optimizing the fermentation substrate to suit the metabolic characteristics of the two bacteria, resulting in low butyric acid yields and limiting its functional role in feed. Furthermore, existing fermented plant proteins are mostly simply physically mixed with additives such as sodium butyrate and carotenoids, lacking a systematic design for multi-target synergistic effects.

[0005] In terms of formulation technology, astaxanthin, lutein, and rosemary extract are all sensitive to light, heat, and oxygen, and are easily degraded and inactivated during feed pelleting. Existing technologies often use β-cyclodextrin for encapsulation to improve stability; however, β-cyclodextrin alone has limited encapsulation efficiency for fat-soluble components and insufficient sustained-release properties. Developing a compound encapsulation material suitable for multiple active ingredients to improve their stability during feed processing and storage is one of the urgent technical problems to be solved in this field.

[0006] Therefore, developing an antibiotic-free feed for California bass that can simultaneously achieve targeted optimization of fermented plant protein, synergistic effects of multi-component additives, and stabilization and protection of active ingredients is of great significance for promoting the development of California bass farming towards a green, efficient, and sustainable direction. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a low-morbidity feed for antibiotic-free aquaculture of California bass and its preparation method.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows: A low-morbidity feed for antibiotic-free culture of California bass is made from the following components in parts by weight: 60-80 parts of basic feed components, 10-20 parts of fermented plant protein, and 2-6 parts of functional compound additives. The fermented plant protein is obtained by drying and pulverizing a fermentation substrate after co-fermentation with Clostridium butyricum and Bacillus subtilis. The fermentation substrate contains soybean meal, wheat bran, rice bran, brown sugar, potassium dihydrogen phosphate, and magnesium sulfate, with a mass ratio of soybean meal, wheat bran, rice bran, and brown sugar of 6:2:2:1. The amounts of potassium dihydrogen phosphate and magnesium sulfate added are 0.1% and 0.05% of the total mass of soybean meal, wheat bran, rice bran, and brown sugar, respectively. The functional compound additive is composed of a pre-encapsulated material and sodium butyrate. The pre-encapsulated material is prepared by encapsulating astaxanthin, lutein, and rosemary extract with an encapsulating material containing β-cyclodextrin and gum arabic. The sodium butyrate is a microencapsulated sodium butyrate and does not participate in the encapsulation process. The mass ratio of sodium butyrate, astaxanthin, lutein, and rosemary extract is (2~4):1:(0.8~1.2):(0.1~0.3), wherein sodium butyrate is calculated as pure product; the weight of sodium butyrate in the functional compound additive is calculated as the actual weight of microencapsulated sodium butyrate. The mass ratio of β-cyclodextrin to gum arabic in the embedding material is 3:1, and the mass ratio of the total mass of the embedding material to the total mass of astaxanthin, lutein, and rosemary extract is (4~10):1.

[0009] Preferably, the conditions for the compound fermentation are as follows: the ratio of viable bacteria of Clostridium butyricum to Bacillus subtilis is 1:1, the inoculum amount is 8-12% of the total mass of the fermentation substrate after adjusting the moisture content, the fermentation temperature is 30-35℃, the fermentation time is 48-72h, and the moisture content is adjusted to 50-60% before fermentation.

[0010] Preferably, in the drying and pulverizing step, the drying temperature is ≤45℃, and the pulverized material is passed through a 60-mesh sieve.

[0011] Furthermore, the astaxanthin is derived from Haematococcus pluvialis extract; the lutein is derived from marigold extract; and the rosemary extract contains ≥10% rosmarinic acid.

[0012] The present invention also provides a method for preparing the above-mentioned functional feed, comprising the following steps: (1) Preparation of fermented plant protein: Mix soybean meal, wheat bran, rice bran and brown sugar according to the above proportion, then add potassium dihydrogen phosphate and magnesium sulfate according to the above addition amount, add water to adjust the moisture content to 50~60%, inoculate Clostridium butyricum and Bacillus subtilis with a live bacteria ratio of 1:1, and the inoculation amount is 8~12% of the total mass of the fermentation substrate after adjusting the moisture content. Ferment at 30~35℃ for 48~72h, then dry at ≤45℃, pulverize and pass through a 60 mesh sieve to obtain the product; (2) Preparation of pre-embedded material: Weigh astaxanthin, lutein and rosemary extract in a mass ratio of 1:(0.8~1.2):(0.1~0.3) and the total mass of the three is 0.35~0.9 parts by weight; then add embedding material so that the mass ratio of the total mass of the embedding material to the total mass of astaxanthin, lutein and rosemary extract is (4~10):1, and the mass ratio of β-cyclodextrin to gum arabic in the embedding material is 3:1, and perform embedding treatment to obtain pre-embedded material; (3) Weigh 60-80 parts of the basic feed components and 10-20 parts of the fermented plant protein obtained in step (1) according to the above weight parts. Mix the basic feed components and fermented plant protein and then pulverize them into ultrafine powder and pass them through an 80-100 mesh sieve. Then add sodium butyrate and the pre-embedded material obtained in step (2). The amount of sodium butyrate added is calculated based on the pure product, so that the mass ratio of sodium butyrate to astaxanthin, lutein and rosemary extract used in step (2) meets the above ratio, and the total weight parts of the pre-embedded material and sodium butyrate are 2-6 parts. Mix them evenly. (4) Granulation, drying and cooling are then performed to obtain the product.

[0013] In step (2), the embedding temperature is 50~60℃ and the time is 1~2h.

[0014] Furthermore, the sodium butyrate mentioned in step (3) is microencapsulated sodium butyrate with an effective content of 30-50%.

[0015] Preferably, the low-speed mixing speed in step (3) is 20~40 r / min and the time is 10~15 min.

[0016] Preferably, the granulation temperature in step (4) is 70~80℃ and the particle size is 2.5~3.5mm.

[0017] Preferably, the moisture content after drying in step (4) is ≤10%.

[0018] Compared with the prior art, the present invention has the following beneficial effects: I. This invention utilizes a co-fermentation process involving *Clostridium butyricum* and *Bacillus subtilis* to ferment a specific ratio of substrates, producing high levels of butyric acid, small peptides, and probiotic metabolites. These components effectively promote intestinal epithelial cell proliferation, repair damaged intestinal mucosa, and regulate intestinal flora balance. Simultaneously, microencapsulated sodium butyrate targets and releases butyric acid in the posterior intestinal tract, further enhancing intestinal barrier function. The synergistic effect of these two components reduces the risk of intestinal inflammation caused by high-density aquaculture at its source, thereby significantly reducing the incidence of intestinal diseases in largemouth bass without relying on antibiotics.

[0019] II. This invention combines astaxanthin, lutein, and rosemary extract in a specific ratio and uses a composite encapsulation material of β-cyclodextrin and gum arabic for encapsulation, significantly improving the stability of the active ingredients to light, heat, and oxygen, and preventing degradation and inactivation during feed pelleting. The above components synergistically exert antioxidant, free radical scavenging, macrophage activity enhancement, and body color improvement effects, effectively alleviating oxidative stress in largemouth bass, enhancing the body's resistance to bacterial diseases, and reducing overall morbidity.

[0020] Third, this invention organically integrates fermented plant protein with functional compound additives into the same feed formulation. Fermented plant protein provides basic support for intestinal repair and immune regulation, pre-encapsulated materials provide continuous antioxidant and antibacterial protection, and microencapsulated sodium butyrate provides targeted regulation of the lower intestinal tract. These three components form a three-dimensional synergistic network at different stages and targets, overcoming the limitations of single additives and the lack of systematic design in simple physical mixing. This results in a significant reduction in morbidity in antibiotic-free California bass farming conditions, while simultaneously ensuring the processing and storage stability of the feed. Detailed Implementation

[0021] The specific embodiments are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Unless otherwise specified, the raw materials and reagents used in the examples are commercially available.

[0022] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Although the invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the invention without departing from the spirit and scope of the invention, and all such modifications and substitutions should be covered within the scope of the claims of the invention. Example 1

[0023] A method for preparing a functional feed for antibiotic-free aquaculture of California bass includes the following steps: (1) Preparation of fermented plant protein: Weigh 60 kg of soybean meal, 20 kg of wheat bran, 20 kg of rice bran, and 10 kg of brown sugar. Add 0.11 kg of potassium dihydrogen phosphate and 0.055 kg of magnesium sulfate. Adjust the moisture content to 55%. Inoculate with Clostridium butyricum and Bacillus subtilis at a viable ratio of 1:1. The inoculation amount is 10% of the total mass of the fermentation substrate after adjusting the moisture content. The fermentation adopts a segmented process: first, aerobic fermentation at 35℃ for 12 h, and then anaerobic fermentation at 32℃ for 48 h. After fermentation, dry at 40℃, pulverize and pass through a 60-mesh sieve to obtain approximately 95 kg of fermented plant protein. Take 15 kg for subsequent steps and keep the rest for later use.

[0024] (2) Preparation of pre-embedded material: Weigh 0.2 kg of astaxanthin, 0.2 kg of lutein, and 0.05 kg of rosemary extract, with a total mass of 0.45 kg. Add embedding material, which includes 2.4 kg of β-cyclodextrin and 0.8 kg of gum arabic, with a total mass of 3.2 kg. Embedd at 55 °C for 1.5 h to obtain 3.65 kg of pre-embedded material.

[0025] (3) Weigh 70 kg of basic feed components and 15 kg of fermented plant protein obtained in step (1). Mix the basic feed components and fermented plant protein and then pulverize them into ultrafine powder and pass them through a 90-mesh sieve. Then add 1.5 kg of microencapsulated sodium butyrate and 3.65 kg of pre-encapsulated material obtained in step (2), wherein the effective content of microencapsulated sodium butyrate is 40% and the pure sodium butyrate mass is 0.6 kg. Mix evenly, with a low-speed mixing speed of 30 r / min and a time of 12 min. The basic feed components consist of the following ingredients: 35 kg of fish meal, 20 kg of soybean meal, 10 kg of wheat flour, 3 kg of corn gluten meal, 1 kg of fish oil, 0.5 kg of calcium dihydrogen phosphate, 0.3 kg of vitamin premix, and 0.2 kg of mineral premix.

[0026] (4) Pelletizing, drying and cooling, wherein the pelleting temperature is 75℃, the pellet size is 3.0mm, and the moisture content after drying is ≤8%, to obtain functional feed. Example 2

[0027] A method for preparing a functional feed for antibiotic-free aquaculture of California bass includes the following steps: (1) Preparation of fermented plant protein: Weigh 60 kg of soybean meal, 20 kg of wheat bran, 20 kg of rice bran, and 10 kg of brown sugar. Add 0.11 kg of potassium dihydrogen phosphate and 0.055 kg of magnesium sulfate. Adjust the moisture content to 50%. Inoculate with Clostridium butyricum and Bacillus subtilis at a viable ratio of 1:1. The inoculation amount is 8% of the total mass of the fermentation substrate after adjusting the moisture content. The fermentation adopts a segmented process: first, aerobic fermentation at 30℃ for 10 h, and then anaerobic fermentation at 30℃ for 38 h. After fermentation, dry at 35℃, pulverize and pass through a 60-mesh sieve to obtain approximately 95 kg of fermented plant protein. Take 10 kg of it for subsequent steps and keep the rest for later use.

[0028] (2) Preparation of pre-embedded material: Weigh 0.2 kg of astaxanthin, 0.16 kg of lutein, and 0.02 kg of rosemary extract, with a total mass of 0.38 kg. Add embedding material, which includes 1.14 kg of β-cyclodextrin and 0.38 kg of gum arabic, with a total mass of 1.52 kg. Embedd at 50 °C for 2 h to obtain 1.90 kg of pre-embedded material.

[0029] (3) Weigh 60 kg of basic feed components and 10 kg of fermented plant protein obtained in step (1). Mix the basic feed components and fermented plant protein and then pulverize them into ultrafine powder and pass them through an 80-mesh sieve. Then add 1.33 kg of microencapsulated sodium butyrate and 1.90 kg of pre-encapsulated material obtained in step (2), wherein the effective content of microencapsulated sodium butyrate is 30% and the pure sodium butyrate mass is 0.40 kg. Mix evenly at a low speed of 20 r / min for 15 min. The basic feed components consist of the following ingredients: 30 kg of fish meal, 17.5 kg of soybean meal, 8.5 kg of wheat flour, 2.5 kg of corn gluten meal, 0.8 kg of fish oil, 0.4 kg of calcium dihydrogen phosphate, 0.2 kg of vitamin premix, and 0.1 kg of mineral premix.

[0030] (4) Pelletizing, drying and cooling, wherein the pelleting temperature is 70℃, the pellet size is 2.5mm, and the moisture content after drying is ≤7% to obtain functional feed. Example 3

[0031] A method for preparing a functional feed for antibiotic-free aquaculture of California bass includes the following steps: (1) Preparation of fermented plant protein: Weigh 60 kg of soybean meal, 20 kg of wheat bran, 20 kg of rice bran, and 10 kg of brown sugar. Add 0.11 kg of potassium dihydrogen phosphate and 0.055 kg of magnesium sulfate. Adjust the moisture content to 60%. Inoculate with Clostridium butyricum and Bacillus subtilis at a viable ratio of 1:1. The inoculation amount is 12% of the total mass of the fermentation substrate after adjusting the moisture content. The fermentation adopts a segmented process: first, aerobic fermentation at 35℃ for 15 h, followed by anaerobic fermentation at 35℃ for 57 h. After fermentation, dry at 45℃, pulverize and pass through a 60-mesh sieve to obtain approximately 95 kg of fermented plant protein. Take 20 kg of this for subsequent steps, and keep the remainder for later use.

[0032] (2) Preparation of pre-embedded material: Weigh 0.36 kg of astaxanthin, 0.43 kg of lutein, and 0.108 kg of rosemary extract, with a total mass of 0.898 kg. Add embedding material, which includes 2.7 kg of β-cyclodextrin and 0.9 kg of gum arabic, with a total mass of 3.6 kg. Incubate at 60 °C for 1 h to obtain 4.50 kg of pre-embedded material.

[0033] (3) Weigh 80 kg of basic feed components and 20 kg of fermented plant protein obtained in step (1). Mix the basic feed components and fermented plant protein and then pulverize them into ultrafine powder and pass them through a 100-mesh sieve. Then add 1.44 kg of microencapsulated sodium butyrate and 4.50 kg of pre-encapsulated material obtained in step (2), wherein the effective content of microencapsulated sodium butyrate is 50% and the pure sodium butyrate mass is 0.72 kg. Mix evenly at a low speed of 40 r / min for 10 min. The basic feed components consist of the following ingredients: 40 kg of fish meal, 23 kg of soybean meal, 11.5 kg of wheat flour, 3.5 kg of corn gluten meal, 1 kg of fish oil, 0.5 kg of calcium dihydrogen phosphate, 0.3 kg of vitamin premix, and 0.2 kg of mineral premix.

[0034] (4) Pelletizing, drying and cooling, wherein the pelleting temperature is 80℃, the pellet size is 3.5mm, and the moisture content after drying is ≤10% to obtain functional feed.

[0035] Comparative Example 1 Objective: To verify the irreplaceable nature of plant protein fermented by the combined fermentation of Clostridium butyricum and Bacillus subtilis.

[0036] Difference from Example 1: Fermented plant protein was not used; instead, an equal amount of ordinary soybean meal was used. It lacks active metabolites such as small peptides and butyric acid produced by Clostridium butyricum / Bacillus subtilis fermentation.

[0037] Preparation method: (1) Weigh 70 kg of basic feed components and 15 kg of ordinary soybean meal, mix them, and then pulverize them into ultrafine powder and pass them through a 90-mesh sieve.

[0038] (2) Preparation of pre-embedded material: Same as step (2) in Example 1, weigh 0.2 kg of astaxanthin, 0.2 kg of lutein and 0.05 kg of rosemary extract, add 2.4 kg of β-cyclodextrin and 0.8 kg of gum arabic, and embed at 55°C for 1.5 h to obtain 3.65 kg of pre-embedded material.

[0039] (3) Add 1.5 kg of microencapsulated sodium butyrate (effective content 40%, pure product 0.6 kg) and 3.65 kg of pre-encapsulated material, mix evenly, mix at low speed 30 r / min for 12 min.

[0040] (4) Pelletizing, drying and cooling: Pelletizing temperature 75℃, particle size 3.0mm, moisture content ≤8% after drying to obtain feed.

[0041] Comparative Example 2 Objective: To verify the key role of pre-encapsulation treatment in the stability of active ingredients.

[0042] Difference from Example 1: Astaxanthin, lutein, and rosemary extract were not encapsulated with β-cyclodextrin / gum arabic.

[0043] Preparation method: (1) Preparation of fermented plant protein: Same as step (1) in Example 1, to obtain 15 kg of fermented plant protein.

[0044] (2) Weigh out 0.2 kg of unencapsulated astaxanthin, 0.2 kg of lutein and 0.05 kg of rosemary extract, mix them with fermented plant protein and 70 kg of basic feed components, and then pulverize them through a 90-mesh sieve.

[0045] (3) Add 1.5 kg of microencapsulated sodium butyrate (40% effective content, 0.6 kg pure product), mix evenly, mix at a low speed of 30 r / min for 12 min.

[0046] (4) Granulation, drying and cooling: Same as step (4) in Example 1.

[0047] Comparative Example 3 Objective: To verify the advantages of microencapsulated sodium butyrate for intestinal targeted release.

[0048] Difference from Example 1: Ordinary sodium butyrate was used, without microencapsulation.

[0049] Preparation method: (1) Preparation of fermented plant protein: Same as step (1) in Example 1, to obtain 15 kg of fermented plant protein.

[0050] (2) Preparation of pre-embedded material: Same as step (2) in Example 1, to obtain 3.65 kg of pre-embedded material.

[0051] (3) Weigh 70 kg of basic feed components and 15 kg of fermented plant protein, mix them, and then pulverize them through a 90-mesh sieve. Then add 0.6 kg of ordinary sodium butyrate (non-microencapsulated) (pure product, the same amount as the pure product in Example 1) and 3.65 kg of pre-embedded material, and mix them evenly.

[0052] (4) Granulation, drying and cooling: Same as step (4) in Example 1.

[0053] I. Incidence of intestinal inflammation (%) Detection method: 1. Aquaculture experiment: Each group was fed with California bass (initial weight about 50g, 30 fish per group, 3 replicates) for 60 consecutive days.

[0054] 2. Sampling: After the experiment, each fish was dissected and its complete intestine (from foregut to hindgut) was removed and rinsed with physiological saline.

[0055] 3. Pathological observation: Intestinal tissue was fixed with 10% formalin, embedded in paraffin, sectioned, stained with hematoxylin and eosin (HE), and observed under an optical microscope.

[0056] 4. Judgment criteria: Normal: Intestinal villi are intact, mucosal epithelial cells are neatly arranged, and there is no inflammatory cell infiltration.

[0057] Mild inflammation: slight shortening of intestinal villi, with infiltration of a small number of lymphocytes and macrophages.

[0058] Moderate inflammation: Intestinal villi are significantly shortened and fused, the submucosa is widened, and a large number of inflammatory cells infiltrate.

[0059] Severe inflammation: loss of intestinal villi, mucosal ulceration, bleeding or necrosis.

[0060] 5. Calculation formula: Intestinal inflammation incidence (%) = (Number of fish tails with mild, moderate or severe inflammation / Total number of fish in the experiment) × 100%.

[0061] II. Serum superoxide dismutase (SOD) activity (U / mL) Detection method: 1. Blood collection: After the aquaculture experiment, blood was collected from the tail vein of each fish, left to stand at room temperature for 30 minutes, centrifuged at 3000 r / min for 10 minutes, and the upper serum was collected and stored at -80℃.

[0062] 2. Reagent kit assay: The xanthine oxidase method (WST-1 method) SOD assay kit (Nanjing Jiancheng Bioengineering Institute, catalog number A001-3) was used.

[0063] 3. Operating steps: Prepare the reagents according to the kit instructions, and set up test wells, control wells, and blank wells.

[0064] Add 10 μL of sample, 20 μL of enzyme working solution, and 200 μL of substrate application solution to each well, and mix well.

[0065] Incubate at 37℃ for 20 min, and measure the absorbance (OD value) at a wavelength of 450 nm.

[0066] 4. Calculation: SOD activity (U / mL) = (Control OD - Measured OD) / Control OD × Dilution factor × Standard concentration (calculated according to the kit formula). Measure each sample three times and take the average.

[0067] III. Carotenoid deposition (mg / kg, calculated as astaxanthin + lutein) Detection method (using back skin and muscle tissue as samples): 1. Sampling: Take about 5g of a mixture of skin and muscle above the lateral line on the back of each fish and homogenize it.

[0068] 2. Extraction: Add 10 mL of acetone-petroleum ether (1:1, v / v) mixed solvent, sonicate for 15 min, centrifuge at 4000 r / min for 10 min, and collect the supernatant. Repeat the extraction until colorless, and combine the supernatants.

[0069] 3. Saponification: Add 2 mL of 10% KOH-methanol solution to the supernatant and saponify for 30 min in the dark.

[0070] 4. Determination: Dilute to 25 mL with petroleum ether and measure the absorbance (characteristic absorption peak of astaxanthin) at a wavelength of 474 nm. At the same time, prepare a standard curve using astaxanthin standards.

[0071] 5. Calculation formula: Carotenoid content (mg / kg) = (C × V × n) / m, where C is the concentration (mg / L) obtained from the standard curve, V is the fixed volume (L), n is the dilution factor, and m is the sample mass (kg).

[0072] IV. Retention rate of pre-embedded materials (%) Definition: The total active ingredient retention rate of astaxanthin, lutein, and rosemary extract in pre-encapsulated materials after granulation (high temperature 70-80℃) and subsequent drying and cooling.

[0073] Detection method: 1. Sampling before granulation: Take the pre-embedded material prepared in step (2) and determine the contents of astaxanthin, lutein and rosmarinic acid according to the method of the example / comparative example (determine separately), and calculate the total active ingredient content (mg / g).

[0074] Astaxanthin / lutein: High performance liquid chromatography (HPLC), C18 column, mobile phase methanol-water (95:5), flow rate 1.0 mL / min, detection wavelength 474 nm.

[0075] Rosmarinic acid: HPLC, mobile phase acetonitrile-0.1% phosphoric acid water (25:75), detection wavelength 330 nm.

[0076] 2. Sampling after pelleting: Take the final finished feed and extract and determine the content of the three active ingredients using the same method (converted to the theoretical content of pre-encapsulated substances in the feed).

[0077] 3. Calculation formula: Retention rate (%) = (Total amount of the three active ingredients in the feed after pelleting / Total amount of the three active ingredients in the pre-coated material before pelleting) × 100%. Comparative Example 2, since it was not encapsulated, directly measured the retention rate of the unencapsulated components in the feed.

[0078] V. Sodium butyrate retention rate (%) Definition: The effective retention ratio of sodium butyrate in microencapsulated sodium butyrate (or ordinary sodium butyrate) after granulation, drying, and cooling.

[0079] Detection method: 1. Sampling before granulation: Take the powder after mixing in step (3) (after adding sodium butyrate and before granulation) and determine the sodium butyrate content.

[0080] Sample preparation: Take 1g of powder, add 10mL of distilled water, extract by sonication for 10min, centrifuge at 4000r / min, and take the supernatant.

[0081] Determination: Gas chromatography (GC) or ion chromatography is used. GC method recommended: The sample is acidified with phosphoric acid, butyric acid is extracted with diethyl ether, methylated, and then injected. Quantification is performed using an FID detector and external standard method.

[0082] 2. Sampling after pelleting: Take the final finished feed and determine the sodium butyrate content using the same method.

[0083] 3. Calculation formula: Retention rate (%) = (Sodium butyrate content in feed after pelleting / Sodium butyrate content in powder before pelleting) × 100%.

[0084] VI. Food intake rate (%) Detection method: 1. Aquaculture management: Feed twice a day, at 9:00 am and 4:00 pm, with each feeding amount being about 3-5% of the total weight of the fish. Collect any uneaten feed 30 minutes after feeding.

[0085] 2. Record data: Feeding amount (g): Weigh the feed before each feeding.

[0086] Residual bait (g): Siphon out the residual bait after 30 minutes, dry it at 65℃ until constant weight, and then weigh it.

[0087] Total feeding amount = sum of feeding amounts at each feeding.

[0088] Total feed intake = Total feed amount - Total uneaten feed amount.

[0089] 3. Calculation formula: Feed intake rate (%) = (Total feed intake / Total feed amount) × 100%. Each group of experiments was repeated 3 times, and the average value was taken.

[0090] The test data is shown in the table below: Analysis of the table data yields the following: Each embodiment significantly outperformed the three comparative examples in key indicators such as gut health, antioxidant capacity, body color improvement, active ingredient retention rate, and palatability. The average incidence of intestinal inflammation in Examples 1-3 was 6.7%, a 62% reduction compared to the comparative example's average of 17.7%; the average serum superoxide dismutase (SOD) level was 307 U / mL, a 53% increase compared to the comparative example's average of 200 U / mL; the average carotenoid deposition was 42.7 mg / kg, an 88% increase compared to the comparative example's average of 22.7 mg / kg; and the average food intake rate was 97%, a 5% increase compared to the comparative example's average of 92%. Example 1, using intermediate parameters, showed the best performance: intestinal inflammation below 5%, SOD reaching 320 U / mL, carotenoid deposition at 45 mg / kg, active ingredient retention rate exceeding 90%, and food intake rate at 98%. Although Examples 2 and 3, using endpoint values, showed slightly lower results, they were still significantly better than all comparative examples, demonstrating the significant technological advancement of this patented technology.

[0091] Three comparative examples verified the irreplaceable nature of the three core features: fermented plant protein, pre-encapsulation treatment, and microencapsulated sodium butyrate. Comparative Example 1, using ordinary soybean meal instead of fermented plant protein, showed a high incidence of intestinal inflammation (25%), significantly higher than in the other examples, demonstrating the indispensability of fermentation metabolites for gut health. Comparative Example 2, using unencapsulated astaxanthin, lutein, and rosemary extracts, showed only 15 mg / kg of carotenoid deposition and less than 60% retention of pre-encapsulated material, proving that encapsulation treatment is crucial for ensuring the stability of active ingredients and improving body color. Comparative Example 3, using ordinary sodium butyrate instead of microencapsulated sodium butyrate, showed an intake rate of only 85% and a sodium butyrate retention rate of less than 70%, demonstrating that microencapsulation technology effectively improves palatability and sodium butyrate bioavailability.

[0092] In summary, the combination of fermented plant protein, pre-encapsulation treatment, and microencapsulation of sodium butyrate produces a significant synergistic effect, rather than a simple additive effect. It forms a three-dimensional synergistic network at different stages of action, namely intestinal repair, antioxidant defense, and targeted release, achieving excellent results that cannot be expected by those skilled in the art through a single feature, fully demonstrating the inventiveness and practical value of this invention.

[0093] It should be understood that the above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It should not be considered that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A low-morbidity feed for antibiotic-free aquaculture of California bass, characterized in that, It is made from the following components in parts by weight: 60-80 parts of basic feed components, 10-20 parts of fermented plant protein, and 2-6 parts of functional compound additives; The fermented plant protein is obtained by drying and pulverizing a fermentation substrate after co-fermentation with Clostridium butyricum and Bacillus subtilis. The fermentation substrate contains soybean meal, wheat bran, rice bran, brown sugar, potassium dihydrogen phosphate, and magnesium sulfate, with a mass ratio of soybean meal, wheat bran, rice bran, and brown sugar of 6:2:2:

1. The amounts of potassium dihydrogen phosphate and magnesium sulfate added are 0.1% and 0.05% of the total mass of soybean meal, wheat bran, rice bran, and brown sugar, respectively. The functional compound additive is composed of a pre-encapsulated material and sodium butyrate. The pre-encapsulated material is prepared by encapsulating astaxanthin, lutein, and rosemary extract with an encapsulating material containing β-cyclodextrin and gum arabic. The sodium butyrate is a microencapsulated sodium butyrate and does not participate in the encapsulation process. The mass ratio of sodium butyrate, astaxanthin, lutein, and rosemary extract is (2~4):1:(0.8~1.2):(0.1~0.3), wherein sodium butyrate is calculated as pure product; the weight of sodium butyrate in the functional compound additive is calculated as the actual weight of microencapsulated sodium butyrate. The mass ratio of β-cyclodextrin to gum arabic in the embedding material is 3:1, and the mass ratio of the total mass of the embedding material to the total mass of astaxanthin, lutein, and rosemary extract is (4~10):

1.

2. The low-morbidity feed according to claim 1, characterized in that, The conditions for the compound fermentation are as follows: the ratio of viable bacteria of Clostridium butyricum to Bacillus subtilis is 1:1, the inoculum amount is 8-12% of the total mass of the fermentation substrate after adjusting the moisture content, the fermentation temperature is 30-35℃, the fermentation time is 48-72h, and the moisture content is adjusted to 50-60% before fermentation.

3. The low-morbidity feed according to claim 1, characterized in that: In the drying and pulverizing step, the drying temperature is ≤45℃, and the pulverized product is passed through a 60-mesh sieve.

4. The low-morbidity feed according to claim 1, characterized in that: The astaxanthin is derived from Haematococcus pluvialis extract; the lutein is derived from marigold extract; and the rosemary extract contains ≥10% rosmarinic acid.

5. A method for preparing low-morbidity feed as described in any one of claims 1 to 4, characterized in that, Includes the following steps: (1) Preparation of fermented plant protein: Mix soybean meal, wheat bran, rice bran and brown sugar in the proportions described in claim 1, add potassium dihydrogen phosphate and magnesium sulfate in the amounts described in claim 1, add water to adjust the moisture content to 50-60%, inoculate with Clostridium butyricum and Bacillus subtilis with a live bacteria ratio of 1:1, and the inoculation amount is 8-12% of the total mass of the fermentation substrate after adjusting the moisture content. Ferment at 30-35℃ for 48-72 hours, then dry at ≤45℃, pulverize and pass through a 60-mesh sieve to obtain the product; (2) Preparation of pre-embedded material: Weigh astaxanthin, lutein and rosemary extract in a mass ratio of 1:(0.8~1.2):(0.1~0.3) and the total mass of the three is 0.35~0.9 parts by weight; then add embedding material so that the mass ratio of the total mass of the embedding material to the total mass of astaxanthin, lutein and rosemary extract is (4~10):1, and the mass ratio of β-cyclodextrin to gum arabic in the embedding material is 3:1, and perform embedding treatment to obtain pre-embedded material; (3) Weigh 60-80 parts of the basic feed components and 10-20 parts of the fermented plant protein obtained in step (1) according to the weight parts of claim 1, and mix the basic feed components and fermented plant protein and then pulverize them into ultrafine powder and pass them through an 80-100 mesh sieve; then add sodium butyrate and the pre-embedded material obtained in step (2). The amount of sodium butyrate added is calculated based on the pure product, so that the mass ratio of sodium butyrate to astaxanthin, lutein and rosemary extract used in step (2) meets the ratio of claim 1, and the total weight parts of the pre-embedded material and sodium butyrate are 2-6 parts, and mix them evenly. (4) Granulation, drying and cooling are then performed to obtain the product.

6. The preparation method according to claim 5, characterized in that: In step (2), the embedding temperature is 50~60℃ and the time is 1~2h.

7. The preparation method according to claim 5, characterized in that: The low-speed mixing speed in step (3) is 20~40 r / min, and the time is 10~15 min.

8. The preparation method according to claim 5, characterized in that: In step (4), the granulation temperature is 70~80℃ and the particle size is 2.5~3.5mm.

9. The preparation method according to claim 5, characterized in that: The moisture content after drying in step (4) is ≤10%.

10. The preparation method according to claim 5, characterized in that: The sodium butyrate mentioned in step (3) is microencapsulated sodium butyrate with an effective content of 30-50%.

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