Aquatic functional feed raw material for improving the efficiency of plant protein replacing fish meal and preparation method thereof

Abstract

The present application relates to the technical field of bio-agriculture and related industries, and discloses a functional feedstuff raw material for improving the efficiency of plant protein replacing fish meal and a preparation method thereof. The functional feedstuff raw material is a mixture of a fermentation product obtained by sequentially subjecting mushroom stems, soybean meal and radix astragali residues to aerobic fermentation and anaerobic fermentation and nano-selenium. The preparation method comprises the steps of raw material preparation, enzymolysis and aerobic fermentation, anaerobic fermentation, post-treatment and the like. Based on the liver and intestinal pathological model induced by the use of plant protein to replace fish meal in the existing aquatic feed, the addition of the specially prepared functional feedstuff raw material can precisely relieve the liver and intestinal inflammatory response induced by the replacement of part of the fish meal with plant protein, and has the effects of improving the growth performance, feed utilization rate of the Trachinocephalus ovatus and improving the liver and intestinal health.

Description

Technical Field

[0001] The present invention relates to the technical field of bio - agriculture and related industries. Specifically, it relates to an aquatic functional feed raw material for improving the efficiency of replacing fish meal with plant protein and a preparation method thereof. Background Art

[0002] Trachinotus ovatus is rich in nutrients, including high - quality protein, fat - soluble vitamins and various minerals. Its meat is tender, delicious, with few spines and thick flesh, highly meeting the modern consumers' demand for aquatic products that combine health and deliciousness. In addition, the "gold" in its name and "pomfret" (homophonic with "prosper") imply "prosperity and wealth", endowing it with a cultural connotation of good luck. Therefore, it is often used as an important ingredient in wedding banquets, festivals and family dinners, forming a stable gift and catering consumption market. According to statistics, in recent years, the national aquaculture output of Trachinotus ovatus has exceeded 200,000 tons, ranking second only to Larimichthys crocea in scale and second in seawater fish aquaculture in China, with a complete industrial chain and broad market prospects.

[0003] In the aquaculture system, feed selection is crucial. As the core nutritional component of aquatic feed, protein usually accounts for 30% - 50% of the total feed mass and more than 60% of the total cost. For a long time, due to its high protein content, ideal amino acid balance, good palatability, excellent digestibility and no anti - nutritional factors, fish meal has been regarded as the golden protein standard for most aquatic animals. However, in recent years, limited by resource depletion, unstable supply, price fluctuations and the disadvantage of high carbon emissions, finding a cost - effective and sustainable fish meal replacement solution has become an urgent issue in the industry.

[0004] To achieve the sustainability goal of aquatic feed, fish meal replacement solutions are developing diversely. However, animal by - products have application limitations, and emerging protein sources are also restricted by costs and regulations. Therefore, plant protein, which combines economy and renewability, is undoubtedly the core pillar with the greatest scale advantage in constructing future diversified replacement strategies.

[0005] [[ID=ID=17]]However, plant protein has problems such as amino acid imbalance, poor palatability and containing different types of anti - nutritional factors. Replacing part of the fish meal with plant protein may induce liver and intestinal lesions in aquatic animals, and at the same time, it will also lead to growth retardation and decreased feed efficiency of aquatic animals. Therefore, developing an aquatic functional feed raw material to ensure the growth performance and body health of aquatic animals after replacing part of the fish meal with plant protein has become an urgent need in current research. Summary of the Invention

[0006] The purpose of the present invention is to provide an aquatic functional feed raw material for improving the efficiency of replacing fish meal with plant protein and a preparation method thereof, so as to solve the problems raised in the above background art.

[0007] To achieve the above objectives, on the one hand, the present invention provides an aquatic functional feed ingredient that improves the efficiency of replacing fishmeal with plant protein. The aquatic functional feed ingredient is a mixture of fermentation products obtained by aerobic fermentation and anaerobic fermentation of shiitake mushroom stems, soybean meal and astragalus residue, and nano-selenium.

[0008] Shiitake mushroom stems are rich in polysaccharides and dietary fiber, while soybean meal provides a high-quality protein matrix. Fermentation of both produces prebiotics and bioactive peptides. Astragalus residue (the solid residue obtained after extracting the main medicinal components of Astragalus through water) is specially introduced. During fermentation, its immunomodulatory components (such as Astragalus polysaccharides) are transformed by microorganisms, making them easier to absorb. This process synergizes with the fermentation products to enhance the body's antioxidant and anti-inflammatory capabilities, improving fish health at a systemic level and thus achieving liver and intestinal protection.

[0009] Preferably, the amount of the aquatic functional feed ingredient added to the aquatic feed is 1-3% by mass percentage.

[0010] Preferably, the amount of the aquatic functional feed ingredient added to the aquatic feed is 2% by mass percentage.

[0011] Preferably, the mass ratio of shiitake mushroom stems, soybean meal, and astragalus residue is (3-4):(3-4):1. This preferred ratio ensures that the fermentation product is simultaneously rich in shiitake polysaccharides, small peptides, and sufficient amounts of active ingredients from traditional Chinese medicine, achieving an ideal synergistic effect of nutrition and immune enhancement.

[0012] On the other hand, the present invention provides a method for preparing the above-mentioned functional aquatic feed ingredients that improve the efficiency of replacing fishmeal with plant protein, comprising the following steps: The dried and pulverized shiitake mushroom stems, soybean meal, and astragalus residue were combined to obtain a mixed matrix; The mixed matrix is ​​subjected to high-temperature sterilization and maturation; Add 40%-50% water by total mass to the matured mixed substrate, inoculate with compound enzyme preparation and Bacillus subtilis bacterial solution, and carry out aerobic fermentation to obtain aerobic fermentation product; A compound anaerobic bacterial agent and a quorum sensing quenching enzyme are added to the aerobic fermentation product, mixed evenly, and then anaerobic fermentation is carried out until the pH value of the fermentation system stabilizes at 4.0-4.8 to obtain the anaerobic fermentation product. The anaerobic fermentation product is dried at low temperature and then uniformly mixed with nano-selenium. Finally, it is pulverized to a particle size of ≥80 mesh to obtain the aquatic functional feed raw material.

[0013] Preferably, the compound enzyme preparation includes cellulase and protease.

[0014] Preferably, the amount of cellulase added is 500-1500 U / kg of mixed matrix, and the amount of protease added is 1000-4000 U / kg of mixed matrix.

[0015] Preferably, the compound anaerobic bacterial agent is composed of Clostridium butyricum and lactic acid bacteria at a live bacteria ratio of 1:(1-3). Butyric acid produced by Clostridium butyricum is a direct energy source for intestinal epithelial cells, effectively promoting villous repair and enhancing barrier function. Lactic acid bacteria have better colonization properties and synergistically create a healthy acidic environment. Quorum sensing quenching enzymes can specifically quench the quorum sensing signal molecules of pathogenic bacteria (such as Vibrio), inhibiting their virulence expression without killing the bacteria, thereby preventing disease and reducing the risk of intestinal infection and inflammation. The combination of these three constitutes a triple intestinal health protection mechanism of "repair (butyric acid) + stabilization (lactic acid bacteria) + defense (quenching enzymes)".

[0016] Preferably, the quorum sensing quenching enzyme is an AHL lactonease.

[0017] Preferably, the amount of nano-selenium added is 0.01-0.05% of the mass of the dried anaerobic fermentation product. Selenium is an important component of glutathione peroxidase and a key antioxidant trace element. Nano-selenium has higher bioavailability and stronger antioxidant activity. Adding this specific proportion of nano-selenium to the fermentation product can produce a synergistic effect with the antioxidants produced during fermentation (such as lentinan and astragalus polysaccharide conversion products), significantly enhancing the body's, especially the liver's, antioxidant capacity, helping to eliminate excess free radicals generated by metabolic stress, further protecting liver cells, and reducing lipid peroxidation damage.

[0018] The beneficial effects of this invention are as follows: This invention provides a targeted functional feed solution based on a model of hepatobiliary lesions induced by the replacement of fishmeal with plant protein in existing aquatic feeds. The aquatic functional feed ingredients utilize a multi-microbial synergy (aerobic bacteria consuming oxygen, anaerobic bacteria producing acid and butyric acid) and a multi-material synergy (fermentation substrate, functional bacteria, traditional Chinese medicine, nano-selenium, and enzyme quenching) mechanism to precisely alleviate liver and intestinal inflammatory responses induced by partial replacement of fishmeal with plant protein from multiple dimensions: repairing the gut (butyric acid), stabilizing the flora (lactic acid bacteria), defending against pathogens (enzyme quenching), and enhancing immunity (traditional Chinese medicine and nano-selenium). This results in improved growth performance and feed utilization in oval pomfret, as well as improved liver and intestinal health.

[0019] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0020] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings: Figure 1 HE-stained section of the intestine of the oval pomfret (5x), VH: villus height, VW: villus width, MT: muscle layer thickness; Figure 2 HE-stained section of the intestine of the oval pomfret (20x); Figure 3 Image of an oil red O-stained section of the liver of an oval pomfret (20x); Figure 4 The following is a graph showing the antioxidant indicators in the liver of the oval pomfret: CAT (catalase, A), GPX (glutathione peroxidase, B), GST (glutathione S-transferase, C), HO-1 (heme oxygenase 1, D), SOD (superoxide dismutase, E), NQO1 (quinone oxidoreductase 1, F), ROS (reactive oxygen species, G), and MDA (malondialdehyde, H). Figure 5 A graph showing the mRNA expression levels of antioxidant stress-related factors in the liver and intestines of the oval pomfret; Figure 6 A diagram showing the mRNA expression levels of GH-IGF axis genes in the liver of the oval pomfret; Figure 7 This is a diagram showing the mRNA expression levels of TOR signaling pathway genes in the liver of the oval pomfret. Detailed Implementation

[0021] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0022] It should be noted that all reagents and raw materials used in this invention are commercially available, and the reagents are of analytical grade.

[0023] The multivitamin and mineral mixture was purchased from Shenzhen Jingji Smart Agriculture Times Co., Ltd. It consists of a vitamin premix and a mineral premix in a 1:1 mass ratio. Each kilogram of the vitamin premix contains: Vitamin A1 3.5mg, Vitamin D 30.75mg, Vitamin E 800mg, Vitamin K3 80mg, Vitamin B1 100mg, Vitamin B2 100mg, Vitamin B6 150mg, Vitamin B12 0.5mg, Nicotinic Acid 200mg, Pantothenic Acid 350mg, Biotin 0.5mg, and Vitamin C. 1050mg, inositol 1200mg, folic acid 50mg, zeolite powder 5.90g; each kilogram of mineral premix includes: glycine-iron 1.5g, glycine-copper 0.051g, glycine-manganese 0.3g, glycine-zinc 0.69g, magnesium sulfate heptahydrate 0.81g, calcium iodate 0.0051g, cobalt sulfate heptahydrate 0.0021g, sodium selenite 0.0039g, zeolite powder 6.64g.

[0024] Fishmeal was purchased from Rongcheng Haihesheng Marine Biotechnology Co., Ltd. Cottonseed protein was purchased from Xinjiang Chengrun Jinlan Biotechnology Co., Ltd. Fish lysate was purchased from Zhejiang Fengyu Marine Biological Products Co., Ltd. Corn gluten meal was purchased from Qiqihar Longjiang Fufeng Biotechnology Co., Ltd. Flour was purchased from Wudeli Group Xinghua Flour Co., Ltd. Fish oil was purchased from Guangdong Haizhiyuan Feed Co., Ltd. Soybean oil was purchased from China National Textile & Grain Oil (Zhanjiang) Industry Co., Ltd. Soybean lecithin oil was purchased from Zhanjiang Hailiangrun Trading Co., Ltd. Calcium dihydrogen phosphate was purchased from Sinochem Yunlong Co., Ltd. Natural lutein was purchased from Guangzhou Lidaer Biotechnology Co., Ltd. The preservation number for Bacillus subtilis is 1A00019, and the depositary institution is the China Marine Microbial Culture Collection Center. The preservation number for Lactobacillus plantarum is 1A07805, and the depositary institution is the China Marine Microbial Culture Collection Center. Clostridium butyricum was purchased from Wuhan Jiyesheng Chemical Co., Ltd., catalog number W01401. Example 1

[0025] Raw material preparation: The dried shiitake mushroom stems, soybean meal, and astragalus residue were crushed and passed through a 20-mesh sieve, and then mixed in a mass ratio of 3:3.5:1 to obtain 1 kg of mixed matrix; High-temperature sterilization and maturation: Sterilize and mature the mixed matrix at 80°C for 3 minutes; Enzymatic hydrolysis and aerobic fermentation: Add 450g of water to the matured mixed substrate, inoculate with cellulase (750 U / kg mixed substrate) and neutral protease (2000 U / kg mixed substrate), and simultaneously inoculate with 5% (v / w, mL / kg) Bacillus subtilis bacterial suspension (1×10⁻⁶ viable cells). 9 The aerobic fermentation product was obtained by oscillating at 200 rpm for 24 hours at 37°C with sterile air 0.6 vvm introduced simultaneously. Anaerobic fermentation: Transfer the aerobic fermentation product to an anaerobic fermenter and inoculate with 5% (v / w, mL / kg) of compound anaerobic bacteria (where the ratio of live bacteria of Clostridium butyricum to Lactobacillus plantarum is 1:2, and the total live bacteria count is 5×10⁻⁶). 9 Add CFU / mL), and add AHL lactonease (15 U / kg mixed substrate), and let stand at 37°C for anaerobic fermentation. Monitor the pH value to drop to 4.5 and stabilize, and obtain the anaerobic fermentation product; Post-processing: The anaerobic fermentation product is dried in a 48℃ hot air circulating oven until the moisture content is 8%. Then, nano selenium powder is added at a ratio of 0.03% of the fermented dry matter weight and mixed evenly in a three-dimensional mixer. Finally, it is pulverized through an 80-mesh sieve using an ultra-fine pulverizer to obtain the raw material for aquatic functional feed. Example 2

[0026] Raw material preparation: The dried shiitake mushroom stems, soybean meal, and astragalus residue were pulverized and passed through a 20-mesh sieve, and then mixed in a mass ratio of 3.5:4:1 to obtain 1 kg of mixed matrix; High-temperature sterilization and maturation: Sterilize and mature the mixed matrix at 80°C for 2 minutes; Enzymatic hydrolysis and aerobic fermentation: Add 400g of water to the matured mixed substrate, inoculate with cellulase (500 U / kg mixed substrate) and neutral protease (1000 U / kg mixed substrate), and simultaneously inoculate with 5% (v / w, mL / kg) Bacillus subtilis bacterial suspension (1.5 × 10⁻⁶ viable cells). 9 The aerobic fermentation product was obtained by oscillating at 200 rpm for 24 hours at 37°C with sterile air 0.5 vvm introduced simultaneously. Anaerobic fermentation: The aerobic fermentation product is transferred to an anaerobic fermenter and inoculated with 5% (v / w, mL / kg) of a compound anaerobic bacterial agent (where the ratio of viable Clostridium butyricum to Lactobacillus plantarum is 1:1, and the total viable count is 5.5 × 10⁻⁶). 9 Add CFU / mL), and add AHL lactonease (15 U / kg mixed substrate), and let stand at 37℃ for anaerobic fermentation. Monitor the pH value to drop to 4.8 and stabilize, and obtain the anaerobic fermentation product; Post-processing: The anaerobic fermentation product is dried in a 48℃ hot air circulating oven until the moisture content is 8%. Then, nano selenium powder is added at a ratio of 0.01% of the fermented dry matter weight and mixed evenly in a three-dimensional mixer. Finally, it is pulverized through a 100-mesh sieve using an ultra-fine pulverizer to obtain the raw material for aquatic functional feed. Example 3

[0027] Raw material preparation: The dried shiitake mushroom stems, soybean meal, and astragalus residue were crushed and passed through a 20-mesh sieve, and then mixed in a mass ratio of 4:3:1 to obtain 1 kg of mixed matrix; High-temperature sterilization and maturation: Sterilize and mature the mixed matrix at 80°C for 3 minutes; Enzymatic hydrolysis and aerobic fermentation: Add 500g of water to the matured mixed substrate, inoculate with cellulase (1500 U / kg mixed substrate) and neutral protease (4000 U / kg mixed substrate), and simultaneously inoculate with 5% (v / w, mL / kg) Bacillus subtilis bacterial suspension (2×10⁻⁶ viable cells). 9 The aerobic fermentation product was obtained by simultaneously introducing 0.8 vvm of sterile air at 37°C and shaking at 200 rpm for 24 h. Anaerobic fermentation: Transfer the aerobic fermentation product to an anaerobic fermenter and inoculate with 5% (v / w, mL / kg) compound anaerobic bacteria agent (where the ratio of Clostridium butyricum to Lactobacillus plantarum viable cells is 1:1, and the total viable cell count is 6×10⁻⁶). 9 Add CFU / mL), and add AHL lactonease (15 U / kg mixed substrate), and let stand at 37℃ for anaerobic fermentation. Monitor the pH value to drop to 4.0 and stabilize, and obtain the anaerobic fermentation product; Post-processing: The anaerobic fermentation product is dried in a 48℃ hot air circulating oven until the moisture content is 8%. Then, nano selenium powder is added at a ratio of 0.05% of the fermented dry matter weight and mixed evenly in a three-dimensional mixer. Finally, it is pulverized through a 100-mesh sieve using an ultra-fine pulverizer to obtain the raw material for aquatic functional feed.

[0028] Comparative Example 1 The difference compared to Example 2 is that the unfermented dry powder of shiitake mushroom stems, soybean meal, and astragalus residue is directly mixed with nano-selenium.

[0029] Comparative Example 2 Compared with Example 2, the difference is that anaerobic fermentation is not carried out, but aerobic fermentation is carried out and then directly dried and mixed with nano-selenium.

[0030] Comparative Example 3 The difference compared to Example 2 is that no AHL lactonease is added in the anaerobic fermentation step.

[0031] Comparative Example 4 The difference compared to Example 2 is that no nano-selenium powder is added in the post-processing step.

[0032] The functional feed prepared in Example 1 was added to commercially available aquatic feed for application. The specific components of the application example are shown in Table 1.

[0033] Table 1. Specific ingredients of feed in application examples

[0034] To verify the effect of aquatic functional feed ingredients on the proportion of plant protein replacing fishmeal in the feed on oval pomfret, the aquaculture effect was verified by the aquatic feeds in Application Examples 1-5. The oval pomfret was used as the research object and a 14-week aquaculture experiment was carried out. Sampling was carried out in the 14th week, and growth performance and feed efficiency were calculated at the end of the experiment.

[0035] Experimental Design This experiment selected 1500 oval pomfret with no significant difference in initial body weight and randomly divided them into 5 groups, with 3 replicates per group and 100 fish per replicate (see Table 2 for specific grouping). The experiment lasted for 14 weeks. During the rearing period, the fish were fed twice a day, in the morning and evening, and water quality indicators were continuously monitored. After each stage of the experiment, the oval pomfret were fasted for 24 hours, and the number and weight of the experimental fish in each net cage were recorded to calculate growth performance and feed efficiency.

[0036] Table 2 Experimental Grouping

[0037] In the table above, 25% fishmeal + 5% plant protein (cottonseed protein) means that, based on the feed composition of Application Example 1, an additional 5% of the total feed mass of plant protein is added to replace 5% of the total feed mass of fishmeal, that is, the percentage of fishmeal replacement is 16.67% (i.e. 5% / 30%).

[0038] 1. Growth performance and morphological indicators The average initial body weight (g), average final body weight (g) after 14 weeks of rearing, body length (cm), and total feed intake (g) of each group of oval pomfret were recorded. The weight gain rate (%) was calculated as: Weight gain rate = (final body weight - initial body weight) / initial body weight × 100%. Feed efficiency (%) was also calculated (feed efficiency = total feed intake / (final body weight - initial body weight)). Furthermore, specific growth rate (100 × (final body weight - initial body weight) / number of days) and condition factor (10) were also calculated. 0×final body weight / body length 3), feed intake rate ((total feed intake / (number of days × (final body weight - initial body weight) / 2))×100%), after dissecting each group of oval pomfret, the weight of the internal organs, liver and intestines was weighed, and the viscera-to-body ratio (viscera weight / final body weight × 100%), liver-to-body ratio (liver weight / final body weight × 100%) and intestine-to-body ratio (intestinal weight / final body weight × 100%) of each group of oval pomfret were calculated. The calculation results are shown in Table 3 below.

[0039] Table 3. Growth performance and morphological indicators of oval pomfret

[0040] Note: Different superscript letters in the same row indicate significant differences (P < 0.05).

[0041] Growth performance data show: Compared with feed group A, there were no significant differences in any indicators among oval pomfret in feed groups B and D (P>0.05); compared with feed group B, there were no significant differences in any indicators among oval pomfret in feed group C (P>0.05); compared with feed group D, the key growth performance indicators such as FBW, WGR, SGR, and FER of oval pomfret in feed group E were significantly improved, while FR was significantly decreased (P<0.05). Overall, considering the data from the five groups, the key growth performance indicators such as FBW, WGR, SGR, and FER of oval pomfret in feed group E were higher than those in the other groups, while FR was lower in all of them.

[0042] Body shape data shows: Compared with feed group A, the CF (crease concentration) of feed group B was significantly improved (P<0.05), while there were no significant differences in any morphological indicators of oval pomfret in feed group D (P>0.05). Compared with feed group B, the HSI (hyperintensity index) of oval pomfret in feed group C was significantly improved, and the RGL (ratio gravitational glutaraldehyde) was significantly decreased (P<0.05). Compared with feed group D, the VSI (vital intensity index) and HSI (hyperintensity index) of oval pomfret in feed group E were significantly improved, and the RGL (ratio gravitational glutaraldehyde) was significantly decreased (P<0.05). No significant differences were found in other morphological indicators (P>0.05). Overall, considering the data from the five groups, the VSI and HSI of oval pomfret in feed group E were higher than those in the other groups.

[0043] 2. Crude components of fish body Four fish were randomly selected from each net cage for crude component analysis, including moisture, crude protein, crude fat, and ash content. Moisture content was determined according to GB 5009.3-2016 "National Food Safety Standard - Determination of Moisture in Food", crude protein content was determined according to GB 5009.5-2016 "National Food Safety Standard - Determination of Protein in Food", crude fat content was determined according to GB / T 6434-2022 "Determination of Crude Fiber Content in Feed", and ash content was determined according to GB 5009.4-2016 "National Food Safety Standard - Determination of Ash in Food". The results are shown in Table 4.

[0044] Table 4 Crude Composition of Fish Body

[0045] Compared with feed group A, the whole fish moisture content in feed groups B and D decreased with decreasing fishmeal content. The whole fish crude fat level of oval pomfret in feed group D was significantly increased (P<0.05). Compared with feed group B, the whole fish crude fat level of oval pomfret in feed group C was significantly increased, and the whole fish moisture content was significantly decreased (P<0.05). Compared with feed group D, there was no significant difference in the whole fish crude fat content of oval pomfret in feed group E (P>0.05).

[0046] 3. Plasma nutritional metabolism indicators Six fish were randomly selected from each net cage. After anesthetizing with eugenol, blood was drawn from the caudal vein using a 1mL syringe and placed in an anticoagulant tube. The plasma was centrifuged, aliquoted, and stored at -80℃. Plasma nutritional and metabolic indicators, including total protein, triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol, were measured. All tests were performed using the corresponding reagent kits and detection methods from Nanjing Jiancheng Bioengineering Institute. Total protein was measured using the A045-2-2 kit, triglycerides using the A110-1-1 kit, total cholesterol using the A111-1-1 kit, high-density lipoprotein cholesterol using the A112-1-1 kit, and low-density lipoprotein cholesterol using the A113-1-1 kit. The test results are shown in Table 5.

[0047] Table 5 Plasma nutritional and metabolic indicators

[0048] Compared with feed group A, the triglyceride level of oval pomfret in feed group B was significantly increased, and the plasma total protein, triglycerides, total cholesterol, and high-density lipoprotein cholesterol levels of oval pomfret in feed group D were significantly increased (P<0.05). Compared with feed group B, the plasma total protein, total cholesterol, and high-density lipoprotein cholesterol levels of oval pomfret in feed group C were significantly increased (P<0.05). Compared with feed group D, the plasma total cholesterol and high-density lipoprotein cholesterol levels of oval pomfret in feed group E were significantly decreased (P<0.05). Overall, the data from all five groups showed that the plasma total protein, total cholesterol, and high-density cholesterol levels of oval pomfret in feed group C were higher than those in the other groups.

[0049] 4. Organizational Structure Indicators Intestinal tissue was obtained, fixed, dehydrated, cleared, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (see...). Figure 1 and Figure 2 ) and Oil Red O staining (see Figure 3 The length, width, and muscle thickness of the villi were measured using an optical microscope, image analysis software, and microscopic image analysis system. The liver vacuolation rate and lipid droplet area were calculated. The length, width, and muscle layer thickness of the intestinal villi are shown in Table 6, the liver vacuolation rate is shown in Table 7, and the liver lipid droplet area is shown in Table 8.

[0050] Table 6. Intestinal villus length, width, and muscle layer thickness

[0051] Compared with feed group A, the intestinal villus length of oval pomfret in feed group B was significantly increased and the muscle layer thickness was significantly decreased (P<0.05). There were no significant differences in any of the intestinal data of oval pomfret in feed group D (P>0.05). Compared with feed group B, the muscle layer thickness of feed group C was significantly increased (P<0.05). Compared with feed group D, the villus length of feed group E was significantly increased (P<0.05).

[0052] Table 7 Liver vacuolation rate

[0053] Compared with feed group A, feed group B showed a significantly higher level of liver vacuolation (P<0.05). There were no significant differences in liver vacuolation levels among the other groups (P>0.05). Considering the data from all five groups of oval pomfret, the liver vacuolation rate in feed group A was lower than in the other groups.

[0054] Table 8. Liver lipid droplet area

[0055] Compared with feed group A, there were no significant differences in liver lipid droplet area between feed groups B and D (P>0.05); compared with feed group B, the liver lipid droplet area of ​​oval pomfret in feed group C was significantly lower (P<0.05); compared with feed group D, there was no significant difference in liver lipid droplet area in feed group E (P>0.05). Overall, the liver lipid droplet area of ​​oval pomfret in feed group E was lower than that in the other groups.

[0056] 5. Immune function Liver tissues from each group of oval pomfret cultured for 14 weeks were collected for antioxidant enzyme activity assays. The results are as follows: Figure 4 As shown. By Figure 4Compared with feed group A, the activity of CAT was significantly increased and the activities of GST, SOD, and NQO1 were significantly decreased in oval pomfret fed feed group B. In feed group D, the activity of GST was significantly increased and the activities of SOD and NQO1 were significantly decreased (P<0.05). Compared with feed group B, the activities of GPx, GST, HO-1, and SOD were significantly increased and the activity of CAT was significantly decreased in oval pomfret fed feed group C (P<0.05). Compared with feed group D, the activities of GST, HO-1, SOD, and NQO1 and the content of ROS were significantly increased and the content of MDA was significantly decreased in oval pomfret fed feed group E (P<0.05). There were no significant differences in other key liver antioxidant indicators (P>0.05). Based on the data of oval pomfret from the five groups, the oval pomfret in feed group C had higher GPx and HO-1 activities than the other groups, while the oval pomfret in feed group E had higher GST activity and ROS content than the other groups, and lower MDA content than the other groups.

[0057] Figure 5 This is a graph showing the mRNA expression levels of antioxidant stress-related factors in the liver (A) and intestine (B) of five groups of oval pomfret. Figure 5 The bar charts for each factor, from left to right, represent feed group A, feed group B, feed group C, feed group D, and feed group E, respectively. As shown in the figure, in the liver, compared with feed group A, feed group B showed a significantly increased mRNA expression level of nrf2 and a significantly decreased mRNA expression level of ho-1 in oval pomfret. In feed group D, the mRNA expression level of nrf2 was significantly increased, while the mRNA expression levels of mnsod and cat were significantly decreased (P<0.05). Compared with feed group B, feed group C showed a significantly increased mRNA expression level of ho-1 (P<0.05). Compared with feed group D, feed group E showed a significantly decreased mRNA expression level of cat and keap1 (P<0.05). There were no significant differences in the expression levels of other mRNAs (P>0.05). Based on the data of oval pomfret from the five groups, the mRNA expression levels of nrf2, mnsod, and ho-1 in oval pomfret from feed group C were higher than those in the other groups, while the mRNA expression levels of cat and keap1 in oval pomfret from feed group E were lower than those in the other groups.

[0058] In the gut, compared with feed group A, the mRNA expression levels of Keap1 in feed groups B and D were significantly increased, while the mRNA expression levels of nrf2, mnsod, and ho-1 were significantly decreased (P<0.05), and the mRNA expression level of ho-1 decreased with decreasing fishmeal content. Compared with feed group B, the mRNA expression levels of cat and keap1 in feed group C were significantly decreased (P<0.05). Compared with feed group D, the mRNA expression level of nrf2 in feed group E was significantly increased, while the mRNA expression levels of cat and keap1 were significantly decreased (P<0.05). There were no significant differences in the expression levels of other mRNAs (P>0.05). Overall, the data from the five groups of oval pomfret showed that the mRNA expression level of cat in feed group C was lower than in the other groups, and the mRNA expression level of keap1 in feed group E was lower than in the other groups.

[0059] Figure 6 This is a graph showing the mRNA expression levels of the GH-IGF axis genes in the livers of five groups of oval pomfret. Figure 7 This is a diagram showing the mRNA expression levels of TOR signaling pathway genes in the livers of five groups of oval pomfret. Figure 6 and Figure 7 The bar chart corresponding to each gene, from left to right, represents feed group A, feed group B, feed group C, feed group D, and feed group E, respectively. Figure 6 and Figure 7 It was found that, compared with group A, the expression level of irs-1 mRNA in oval pomfret was significantly increased and the expression level of igf-1 mRNA was significantly decreased in group B, while the expression level of irs-1 mRNA was significantly increased in group D (P<0.05). Compared with group B, the expression levels of igf-1 and irs-1 mRNA were significantly increased in group C (P<0.05). Compared with group D, the expression level of gh mRNA was significantly increased in group E (P<0.05). There were no significant differences in the expression levels of other mRNAs (P>0.05). Based on the data from the five groups of oval pomfret, the expression levels of igf-1 and irs-1 mRNA were higher in group C than in the other groups, and the expression level of gh mRNA was higher in group E than in the other groups.

[0060] In the liver, compared with feed group A, the mRNA expression levels of akt-1, s6, and 4ebp-1 in feed groups B and D decreased with decreasing fishmeal content. The mTOR mRNA expression level in *Sinibrama ovatus* was significantly decreased in feed group B (P<0.05). Compared with feed group B, the mTOR mRNA expression level in feed group C was significantly increased (P<0.05). Compared with feed group D, the mRNA expression levels of s6 and 4ebp-1 were significantly increased in feed group E (P<0.05). There were no significant differences in the expression levels of other mRNAs (P>0.05). Overall, the data from the five groups of *Sinibrama ovatus* showed that the mTOR and s6 mRNA expression levels in feed group C were higher than in the other groups.

[0061] As can be seen from the above, the aquatic functional feed ingredients in the aquatic feed of the present invention have a positive impact on improving the growth performance, nutritional composition and metabolism, tissue structure and immune performance of oval pomfret by replacing fishmeal in their feed, mainly as follows: (1) The addition of aquatic functional feed ingredients to aquatic feed has a significant promoting effect on the growth performance and feed efficiency of oval pomfret. Compared with other groups, the key growth performance indicators such as FBW, WGR, SGR and FER of oval pomfret in feed group E are higher than those in other groups, while FR is lower than that in other groups; (2) The crude fat level of oval pomfret in feed group C was significantly increased and its level was higher than that of other groups; (3) The addition of aquatic functional feed ingredients to the feed has a promoting effect on the nutrient intake and absorption of oval pomfret. Compared with other groups, the levels of various plasma nutrient metabolism indicators of oval pomfret in feed group C were significantly increased, such as total plasma protein, total cholesterol, high-density lipoprotein cholesterol and low-density lipoprotein cholesterol; (4) The addition of aquatic functional feed ingredients to the feed has a significant effect on protecting the intestinal structure. Compared with other groups, the intestinal villus length, villus width and muscle layer thickness of oval pomfret in feed group C were significantly increased; (5) The addition of aquatic functional feed ingredients to the feed has a positive effect on the liver health of oval pomfret. Compared with other groups, the liver vacuolation level and liver fat content of oval pomfret in feed group E were significantly reduced and remained at a low level compared with other groups. (6) The addition of aquatic functional feed ingredients to the feed significantly improved the liver's antioxidant stress level and immune response. Compared with other groups, feed group C had better antioxidant capacity and the oval pomfret were in a healthier state; (7) The addition of aquatic functional feed ingredients to the feed can activate the GH-IGF axis and TOR signaling pathway, which can promote the growth and immune performance of oval pomfret.

[0062] In conclusion, functional aquatic feed ingredients, by replacing fishmeal with plant protein in the feed, significantly improved the growth performance, nutritional metabolism, and immune function of oval pomfret, demonstrating a significant promoting effect on their growth and development. All indicators showed that both feed group C and feed group E significantly improved the growth and immune performance of oval pomfret.

[0063] Application Example 6 The aquatic functional feed ingredients prepared in Example 2 were used to replace the aquatic functional feed ingredients in Application Example 3.

[0064] Application Example 7 The aquatic functional feed ingredients prepared in Comparative Example 1 were used to replace the aquatic functional feed ingredients in Application Example 3.

[0065] Application Example 8 The aquatic functional feed ingredients prepared in Comparative Example 2 were used to replace the aquatic functional feed ingredients in Application Example 3.

[0066] Application Example 9 The aquatic functional feed ingredients prepared in Comparative Example 3 were used to replace the aquatic functional feed ingredients in Application Example 3.

[0067] Application Example 10 The aquatic functional feed ingredients prepared in Comparative Example 4 were used to replace the aquatic functional feed ingredients in Application Example 3.

[0068] The feeds used in Application Examples 6-10 were tested for their aquaculture effects using the same methods as above. After the aquaculture experiment, the experimental fish were infected with a specific pathogen (such as Aeromonas hydrophila) and the number of deaths was recorded. The formula was: Survival rate after challenge (%) = (Number of fish surviving after challenge / Total number of fish in the challenge experiment) × 100%. All test results are shown in Table 9.

[0069] Table 9 Results of Aquaculture Effect Testing

[0070] Results analysis: Application Example 7: The growth performance and feed efficiency were the lowest among all groups, with the highest liver lipid droplet area and the lowest survival rate after viral challenge. This indicates that untreated raw materials, due to the failure to degrade anti-nutritional factors, not only severely inhibited growth and feed utilization but also directly led to severe liver damage and extremely poor disease resistance. This, in turn, confirms that fermentation is an absolute prerequisite for producing any beneficial effects, and that simple physical mixing is ineffective.

[0071] Application Example 8 (Aerobic Fermentation Only): Its growth performance was not significantly different from Application Example 6, demonstrating that aerobic fermentation effectively improved nutrient availability. However, its liver lipid droplet area was still significantly higher than that of Application Example 6, and the improvement in survival rate after challenge was limited (based on Application Example 7). This indicates that aerobic fermentation alone cannot produce enough key metabolites (such as butyrate) to effectively alleviate the metabolic burden on the liver and systematically enhance disease resistance, highlighting the functional specificity and necessity of the subsequent anaerobic fermentation step.

[0072] Application Example 9 (without quenching enzyme): It showed no significant differences from Application Example 6 in growth, most plasma and intestinal structural parameters. However, its survival rate after viral challenge was significantly lower than that of Application Example 6. This indicates that while the absence of quorum sensing quenching enzyme (AHL lactonease) does not affect basal nutritional efficacy, it significantly weakens the actual disease resistance conferred by fermentation products on animals against specific pathogens. This demonstrates that quorum sensing quenching enzyme is not a dispensable component, but a key component for the specific goals of "healthy farming" and "disease prevention."

[0073] Application Example 10 (without nano-selenium): Its basic growth and structural indicators were not significantly different from those of Application Example 6, but its survival rate after viral challenge was still slightly lower than that of Application Example 6. This indicates that the absence of nano-selenium limits the ultimate upper limit of the body's immune defense and protection capabilities under stress conditions (such as pathogen infection). Therefore, the combination of nano-selenium is a key link to achieve the best disease resistance and stress resistance effects. The preferred 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 specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention. Furthermore, it should be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0074] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for preparing a functional feed ingredient for oval pomfret that can alleviate liver and intestinal inflammatory responses induced by partial replacement of fishmeal with plant protein, characterized in that, Includes the following steps: The dried and pulverized shiitake mushroom stems, soybean meal, and astragalus residue were compounded together. The astragalus residue was the solid residue obtained after water extraction of astragalus. The mass ratio of the shiitake mushroom stems, soybean meal, and astragalus residue was (3-4):(3-4):1, to obtain a mixed matrix. The mixed matrix is ​​subjected to high-temperature sterilization and maturation; Add 40%-50% water by total mass to the matured mixed substrate, inoculate with a compound enzyme preparation and Bacillus subtilis bacterial solution, wherein the compound enzyme preparation includes cellulase and protease, and perform aerobic fermentation to obtain aerobic fermentation product. A compound anaerobic bacterial agent and a quorum sensing quenching enzyme are added to the aerobic fermentation product. The compound anaerobic bacterial agent is composed of Clostridium butyricum and lactic acid bacteria in a live bacteria ratio of 1:(1-3). The quorum sensing quenching enzyme is AHL lactonease. After mixing evenly, anaerobic fermentation is carried out until the pH value of the fermentation system stabilizes at 4.0-4.8 to obtain the anaerobic fermentation product. The anaerobic fermentation product is dried at low temperature and then uniformly mixed with nano-selenium. The amount of nano-selenium added is 0.01-0.05% of the mass of the dried anaerobic fermentation product. Finally, it is pulverized to a particle size ≥80 mesh to obtain the oval pomfret functional feed raw material.

2. The method for preparing the oval pomfret functional feed ingredient according to claim 1, which can alleviate liver and intestinal inflammatory responses induced by partial replacement of fishmeal with plant protein, is characterized in that, The amount of cellulase added is 500-1500 U / kg of mixed matrix, and the amount of protease added is 1000-4000 U / kg of mixed matrix.

3. The oval pomfret functional feed ingredient prepared by the method of claim 1 or 2, which can alleviate the inflammatory response of the liver and intestine induced by the replacement of part of the fish meal with plant protein.

4. The oval pomfret functional feed ingredient according to claim 3, which can alleviate liver and intestinal inflammatory responses induced by partial replacement of fishmeal with plant protein, is characterized in that... The amount of the oval pomfret functional feed ingredient added to the oval pomfret feed is 1-3% by mass percentage.

5. The oval pomfret functional feed ingredient according to claim 4, which can alleviate liver and intestinal inflammatory responses induced by partial replacement of fishmeal with plant protein, is characterized in that... The amount of the oval pomfret functional feed ingredient added to the oval pomfret feed is 2% by mass.

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