Hesperetin additive feed capable of improving intestinal health of micropterus salmoides and preparation method of hesperetin additive feed

Abstract

The invention discloses hesperetin additive feed capable of improving intestinal health of micropterus salmoides and a preparation method of the hesperetin additive feed. The feed comprises the following raw materials: 40%-50% of fish meal, 8%-12% of chicken meal, 5%-10% of cottonseed meal, 5%-8% of rapeseed meal, 6%-12% of soybean meal, 6%-10% of wheat flour, 1%-7% of fish oil, 1%-7% of soybean oil, 0.3%-1% of monocalcium phosphate, 0.1%-1% of composite vitamins, 0.1%-1% of mixed minerals, 0.1%-0.5% of choline chloride, 0.1%-1% of vitamin C, 0.1%-2% of gelatin, 1%-12% of microcrystalline cellulose and 0.1%-0.5% of hesperetin. The hesperetin is added into the feed raw materials, so that the intestinal oxidation resistance of the largemouth bass can be remarkably improved, the damage of intestinal inflammation reaction to the intestinal tract can be improved, and the intestinal health of the largemouth bass can be protected.

Description

Technical Field

[0001] The present invention relates to the field of aquatic feeds, and particularly to a hesperetin additive feed capable of improving the intestinal health of largemouth bass and a preparation method thereof. Background Art

[0002] Modern aquaculture is a rapidly developing field. The continuous growth of the global population has led to an increased demand for protein, and aquaculture has become an effective way to meet this demand. With the increasing global demand for aquatic products, the aquaculture industry has developed rapidly. As one of the most important inputs in aquaculture production, the demand for feed has also increased significantly. Largemouth bass, also known as largemouth perch or black bass, is a popular freshwater fish native to North America and belongs to carnivorous fish. Due to its delicious meat and high market demand, largemouth bass is widely cultured. According to the statistics of the China Fisheries Yearbook in 2023, the output of largemouth bass in China has reached more than 8 million tons, and it has become one of the important freshwater aquaculture varieties in China, known as the "fifth major domestic fish" and is very popular in the catering industry. The research on its nutrition and feed demand is becoming more and more urgent.

[0003] Long-term intake of high-fat feed may increase the risk of metabolic diseases in fish. The natural response of fish to infection or injury is to produce inflammation. Acute inflammation helps to resist pathogens, but chronic inflammation will have a negative impact on health. Chronic inflammation may lead to impaired immune system function, making fish more susceptible to pathogens. The fish intestine is the main place for fish to digest and absorb nutrients. A healthy intestine can effectively decompose nutrients in feed, such as proteins, fats, and carbohydrates, and improve the utilization rate of nutrients. There are a large number of immune cells and antibodies in the intestine. A healthy intestinal microbiota (intestinal flora) helps to maintain the immune balance of fish and inhibit the growth of harmful pathogens. A healthy intestine can better regulate the inflammatory response and prevent the occurrence of chronic inflammation, which helps to protect the health of fish. Selecting a suitable feed formula and additives (such as probiotics and cellulose) helps to maintain intestinal health.

[0004] At present, there has been no report on establishing a hesperetin additive feed formula capable of improving the intestinal health of largemouth bass and applying it to economic fish. Therefore, establishing a hesperetin additive feed formula is of great significance for reducing antibiotics and synthetic antibacterial drugs and improving intestinal health. Summary of the Invention

[0005] The object of the present invention is to overcome the deficiencies of the prior art and provide a hesperetin additive feed for improving the intestinal health of largemouth bass and a preparation method thereof. By using hesperetin as an additive, this feed can improve the growth performance of largemouth bass, alleviate intestinal inflammation and oxidative stress caused by high-fat feed, and effectively protect the intestinal health of largemouth bass. Moreover, the feed is complete in nutritional components and reasonable in collocation. The additives used comply with relevant regulations, are safe, green and environmentally friendly, can meet the normal growth requirements of largemouth bass, and provide theoretical guidance for the healthy breeding of largemouth bass and the development of environment-friendly feeds to make up for the deficiencies of the prior art.

[0006] The present invention provides a hesperetin additive feed for improving the intestinal health of largemouth bass, and its raw materials include, by weight percentage:

[0007] Fish meal: 40% - 50%, chicken meal: 8% - 12%, cottonseed meal: 5% - 10%, rapeseed meal: 5% - 8%, soybean meal: 6% - 12%, wheat flour: 6% - 10%, fish oil: 1% - 7%, soybean oil: 1% - 7%, calcium dihydrogen phosphate: 0.3% - 1%, compound vitamin: 0.1% - 1%, mixed minerals: 0.1% - 1%, choline chloride: 0.1% - 0.5%, vitamin C: 0.1 - 1%, gelatin: 0.1% - 2%, microcrystalline cellulose: 1% - 12%, hesperetin: 0.1% - 0.5%.

[0008] Furthermore, the raw materials of the hesperetin additive feed include, by weight percentage:

[0009] Fish meal: 45% - 50%, chicken meal: 10% - 12%, cottonseed meal: 5% - 7%, rapeseed meal 5% - 6%, soybean meal: 6% - 10%, wheat flour: 6% - 8%, fish oil: 2% - 7%, soybean oil: 2% - 7%, calcium dihydrogen phosphate: 0.3% - 0.5%, compound vitamin: 0.1% - 5%, mixed minerals: 0.1% - 5%, choline chloride: 0.1% - 0.3%, vitamin C: 0.1% - 0.5%, gelatin: 0.1% - 1%, microcrystalline cellulose: 1% - 10%, hesperetin: 0.125% - 0.25%.

[0010] Still further, the raw materials of the hesperetin additive feed include, by weight percentage:

[0011] Fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 2.5%, soybean oil: 2.5%, calcium dihydrogen phosphate: 0.5%, compound vitamin: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 9.175%, hesperetin: 0.125%.

[0012] Furthermore, the compound vitamin is composed of nicotinic acid, D-pantothenic acid, folic acid, D-biotin, vitamin B 12 , inositol, carnitine, vitamin A, vitamin D 3 , vitamin E, vitamin K 3 , vitamin B 1 , vitamin B 2 , vitamin B 6 and microcrystalline cellulose; and in the compound vitamin, the weight ratio of each component is: 12.5:50:5:1:0.05:0.49:75:2.25:0.75:80:10:15:15:15:4720.96.

[0013] Furthermore, the mixed minerals are composed of ferrous sulfate heptahydrate, zinc sulfate heptahydrate, manganese sulfate monohydrate, copper sulfate pentahydrate, calcium iodate hexahydrate, sodium selenite, cobalt sulfate, magnesium sulfate and microcrystalline cellulose; and in the mixed minerals, the weight ratio of each component is: 310:260:110:25:6:2:3:1200:8084.

[0014] Furthermore, the raw materials of the hesperidin additive feed include, by weight:

[0015] Fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 2.5%, soybean oil: 2.5%, calcium dihydrogen phosphate: 0.5%, compound vitamin: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 9.175%, hesperidin: 0.125%. Among them, the compound vitamin is: nicotinic acid, D-pantothenic acid, folic acid, D-biotin, vitamin B 12 , inositol, carnitine, vitamin A, vitamin D 3 , vitamin E, vitamin K 3 , vitamin B 1 , vitamin B 2 , vitamin B 6 and a composition of microcrystalline cellulose; and in the compound vitamin, the weight ratio of each component is: 12.5:50:5:1:0.05:0.49:75:2.25:0.75:80:10:15:15:15:4720.96.

[0016] The mixed minerals are ferrous sulfate heptahydrate, zinc sulfate heptahydrate, manganese sulfate monohydrate, copper sulfate pentahydrate, calcium iodate hexahydrate, sodium selenite, cobalt sulfate, magnesium sulfate and microcrystalline cellulose; and in the mixed minerals, the weight ratio of each component is: 310:260:110:25:6:2:3:1200:8084.

[0017] The present invention also provides a method for preparing the above-mentioned hesperetin additive feed that can improve the intestinal health of largemouth bass, comprising the following steps:

[0018] 1) Fish meal, chicken meal, cottonseed meal, rapeseed meal, soybean meal, and gelatin are successively pulverized and sieved.

[0019] 2) By weight percentage, it includes fish meal, chicken meal, cottonseed meal, rapeseed meal, soybean meal, wheat flour, fish oil, soybean oil, choline chloride, compound vitamin, mixed minerals, calcium dihydrogen phosphate, microcrystalline cellulose, vitamin C, gelatin, and hesperetin;

[0020] 3) After all raw material components are gradually and evenly mixed in ascending order of specific gravity, fish oil and soybean oil are added and fully mixed, and then 20 - 35% of the total weight of the above raw materials of pure water is added and fully mixed to obtain a mixed material;

[0021] 4) The mixed material after mixing in step 3) is put into an automatic granulator to make feed pellets, and then the feed pellets are sent into a dryer for drying to obtain hesperetin additive feed, which is stored in a -20°C refrigerator for later use.

[0022] Further, the compound vitamin is a composition of nicotinic acid, D-pantothenic acid, folic acid, D-biotin, vitamin B 12 , inositol, carnitine, vitamin A, vitamin D 3 , vitamin E, vitamin K 3 , vitamin B 1 , vitamin B 2 , vitamin B 6 and microcrystalline cellulose; and in the compound vitamin, the weight ratio of each component is: 12.5:50:5:1:0.05:0.49:75:2.25:0.75:80:10:15:15:15:4720.96.

[0023] Still further, the mixed minerals are: ferrous sulfate heptahydrate, zinc sulfate heptahydrate, manganese sulfate monohydrate, copper sulfate pentahydrate, calcium iodate hexahydrate, sodium selenite, cobalt sulfate, magnesium sulfate, and microcrystalline cellulose; and in the mixed minerals, the weight ratio of each component is: 310:260:110:25:6:2:3:1200:8084.

[0024] Still further, in step 4), the particle size of the feed pellets is 2 mm, and the drying temperature of the dryer is 60°C.

[0025] The principle of the present invention:

[0026] Flavonoids are a class of natural compounds widely present in plants and belong to polyphenolic compounds. They not only endow plants with colors but also have various biological activities beneficial to human health. Hesperetin is a flavonoid widely present in citrus fruits, especially in oranges and grapefruits, with relatively high contents. It has various biological activities and is particularly well-known for its antioxidant effect. Hesperetin can neutralize free radicals in the body and reduce oxidative stress. Free radicals are important factors leading to cell damage and aging, and scavenging them can protect the health of cells and tissues. Research shows that hesperetin can promote the activities of antioxidant enzymes in the body (such as superoxide dismutase, glutathione peroxidase, etc.). These enzymes help scavenge oxidative substances in the body and further enhance the antioxidant capacity. Hesperetin can inhibit the production of various pro-inflammatory factors, and by reducing the release of these factors, hesperetin helps alleviate the inflammatory response.

[0027] Hesperidin can enhance the integrity of intestinal epithelial cells, improve intestinal barrier function, reduce the permeability of harmful substances and pathogens. By promoting the growth of beneficial bacteria, hesperidin helps inhibit the reproduction of pathogenic bacteria, thereby improving the intestinal ecosystem. As a feed additive, hesperidin has multiple benefits such as antioxidant, immune enhancement, digestion promotion, anti-inflammatory and meat quality improvement, and can effectively improve the production performance and health level of animals. Its natural properties also make it one of the additives that have received much attention in the modern feed industry. As an additive, hesperidin can neutralize free radicals in the body and reduce oxidative stress. Free radicals are important factors leading to cell damage and aging. Removing them can protect the health of cells and tissues and has anti-inflammatory and antioxidant effects, which is particularly important for improving the overall health and immunity of animals. Hesperidin also helps improve digestive tract health, promote the balance of intestinal microorganisms, and enhance the absorption of nutrients. This is crucial for increasing the growth rate and feed conversion rate of animals. Hesperidin can affect fat metabolism and muscle growth. Adding hesperidin helps improve meat quality and flavor and increases the market value of products. So far, a large number of studies have focused on the application of hesperidin as a feed additive for mammals, and there are few studies on the effect of hesperidin as a feed additive on the intestinal health of fish, especially largemouth bass. Research shows that hesperidin can promote the activity of antioxidant enzymes in the body (such as superoxide dismutase, glutathione peroxidase, etc.). These enzymes help remove oxidative substances in the body and further enhance the antioxidant capacity. Hesperidin can inhibit the production of a variety of pro-inflammatory factors. By reducing the release of these factors, hesperidin helps reduce the inflammatory response. Research shows that hesperidin can enhance the integrity of intestinal epithelial cells, improve intestinal barrier function, reduce the permeability of harmful substances and pathogens. By promoting the growth of beneficial bacteria, hesperidin helps inhibit the reproduction of pathogenic bacteria, thereby improving the intestinal ecosystem. So far, studies have proved that hesperidin has the effect of maintaining intestinal health. Therefore, we can establish a feed formula with hesperidin as an additive and utilize its antioxidant and anti-inflammatory properties to improve the intestinal health of aquatic animals.

[0028] Therefore, we can establish a feed formula with hesperidin as an additive and utilize its antioxidant and anti-inflammatory properties to improve the intestinal health of aquatic animals.

[0029] Advantages of the present invention:

[0030] As a feed additive, hesperidin has multiple beneficial effects. Hesperidin can increase the activity of the animal's immune system and enhance the resistance to pathogenic microorganisms. This helps reduce the incidence of diseases and the use of antibiotics. Hesperidin has good antioxidant properties, can scavenge free radicals, reduce oxidative stress, thereby protecting cells and tissues and improving the overall health of animals.

[0031] Hesperetin can improve intestinal health, promote the growth of beneficial microorganisms, enhance intestinal barrier function, thereby increasing the absorption rate of nutrients and promoting animal growth. Research shows that hesperetin has anti-inflammatory effects, can reduce inflammatory responses in the intestine and other tissues, and improve the health status and production performance of animals. In meat production, the addition of hesperetin helps to improve meat quality, enhance flavor, and boost the market competitiveness of products. As a feed additive, hesperetin can improve the health and production performance of animals. This invention will provide theoretical guidance for the healthy farming of largemouth bass and the development of environmentally friendly feeds for them.

[0032] In summary, this technological innovation aims to increase the growth rate of largemouth bass and improve production performance. By precisely proportioning nutritional components and using more efficient raw materials, the new feed formula of this invention enables largemouth bass to gain more weight and higher production performance in a shorter time, reducing the feeding cycle, and thus enhancing the efficiency and economic benefits of aquaculture production. By establishing a new feed with hesperetin additive, this invention not only focuses on the production performance of largemouth bass, but also reduces the dependence on antibiotics and other drugs by optimizing the feed formula. It meets the overall health of aquatic animals and better protects the intestines of aquatic animals. Brief Description of the Drawings

[0033] Figure 1 Diagrams of the effects of each feed on the histological structure of the intestine, intestinal villus length, and intestinal malondialdehyde content of largemouth bass;

[0034] In the figure, A is the H&E staining diagram of the intestine,

[0035] B is the statistical chart of intestinal microvillus length. In the figure, "*" represents significant differences between different treatments (P<0.05), and "ns" represents no significant differences between different treatments (P>0.05).

[0036] C is the diagram of intestinal malondialdehyde content. In the figure, "*" represents significant differences between different treatments (P<0.05), and "ns" represents no significant differences between different treatments (P≥0.05).

[0037] Figure 2 Diagrams of the effects of each feed on the total antioxidant capacity, catalase activity, total superoxide dismutase activity, and glutathione peroxidase of largemouth bass;

[0038] In the figure, A is the diagram of total antioxidant activity in the intestine. In the figure, "*" represents significant differences between different treatments (P<0.05), and "ns" represents no significant differences between different treatments (P≥0.05).

[0039] Panel B shows the intestinal catalase activity. In the figure, "*" indicates significant differences between different treatments (P < 0.05), and "ns" indicates no significant differences between different treatments (P ≥ 0.05).

[0040] Panel C shows the intestinal total superoxide dismutase activity. In the figure, "*" indicates significant differences between different treatments (P < 0.05), and "ns" indicates no significant differences between different treatments (P ≥ 0.05).

[0041] Panel D shows the intestinal glutathione peroxidase activity. In the figure, "*" indicates significant differences between different treatments (P < 0.05), and "ns" indicates no significant differences between different treatments (P ≥ 0.05).

[0042] Figure 3 It is a diagram showing the effects of each feed on the expression of genes related to intestinal inflammatory response in largemouth bass.

[0043] In the figure, "*" indicates significant differences between different treatments (P < 0.05), and "ns" indicates no significant differences between different treatments (P ≥ 0.05). Detailed implementation methods

[0044] The present invention will be further described in detail below in conjunction with specific embodiments for those skilled in the art to understand.

[0045] Example 1

[0046] The raw materials of the hesperetin additive feed 1 that can improve the intestinal health of largemouth bass include, by weight: fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 2.5%, soybean oil: 2.5%, calcium dihydrogen phosphate: 0.5%, compound vitamin: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 9.175%, hesperetin: 0.125%, denoted as Hst1.25;

[0047] Among them, the compound vitamin is a composition of nicotinic acid, D-pantothenic acid, folic acid, D-biotin, vitamin B12, inositol, carnitine, vitamin A, vitamin D3, vitamin E, vitamin K3, vitamin B1, vitamin B2, vitamin B6 and microcrystalline cellulose; and in the compound vitamin, the weight ratio of each component is: 12.5:50:5:1:0.05:0.49:75:2.25:0.75:80:10:15:15:15:4720.96.

[0048] The mixed minerals are ferric sulfate heptahydrate, zinc sulfate heptahydrate, manganese sulfate monohydrate, copper sulfate pentahydrate, calcium iodate hexahydrate, sodium selenite, cobalt sulfate, magnesium sulfate and microcrystalline cellulose; and in the mixed minerals, the weight ratio of each component is: 310:260:110:25:6:2:3:1200:8084.

[0049] The preparation method of the hesperetin additive feed that can improve the intestinal health of Micropterus salmoides includes the following steps:

[0050] 1) Fish meal, chicken meal, rapeseed meal, cottonseed meal, soybean meal, wheat flour, choline chloride, compound vitamin, mixed minerals, calcium dihydrogen phosphate, and microcrystalline cellulose are sequentially pulverized and passed through a 40-mesh sieve;

[0051] 2) By weight percentage, it includes fish meal, chicken meal, rapeseed meal, cottonseed meal, soybean meal, wheat flour, fish oil, soybean oil, choline chloride, compound vitamin, mixed minerals, calcium dihydrogen phosphate, microcrystalline cellulose and hesperetin;

[0052] 3) After various feed ingredients are evenly mixed step by step from small to large in proportion, fish oil and soybean oil are added and fully mixed to obtain a mixed material;

[0053] 4) The mixed material after mixing in step 3) is put into an automatic granulator to make feed pellets, and then the pellet feed is sent into a dryer at a temperature of 60 °C for drying to obtain the hesperetin additive feed, which is stored in a refrigerator at -20 °C for standby. The size of the feed pellets is 2 mm, and the drying temperature of the dryer is 60 °C.

[0054] Example 2

[0055] The preparation method of the hesperetin additive feed 2 that can improve the intestinal health of Micropterus salmoides is basically the same as that of the hesperetin additive feed 1 in Example 1, the difference is that:

[0056] The raw materials of the hesperetin additive feed 2 that can improve the intestinal health of Micropterus salmoides include by weight: fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 2.5%, soybean oil: 2.5%, calcium dihydrogen phosphate: 0.5%, compound vitamin: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 9.05%, hesperetin: 0.25%, denoted as Hst2.5.

[0057] Example 3

[0058] The preparation method of the hesperetin additive feed 3 that can improve the intestinal health of Micropterus salmoides is basically the same as that of the hesperetin additive feed 1 in Example 1, the difference is that:

[0059] The raw materials of the hesperidin additive feed 3 that can improve the intestinal health of Micropterus salmoides include, by weight: fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 6.5%, soybean oil: 6.5%, calcium dihydrogen phosphate: 0.5%, compound vitamin: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 1.175%, hesperidin: 0.125%, denoted as HFD + Hst1.25.

[0060] Example 4

[0061] The preparation method of the hesperidin additive feed 4 that can improve the intestinal health of Micropterus salmoides is basically the same as that of the hesperidin additive feed 1 in Example 1, except that:

[0062] The raw materials of the hesperidin additive feed 4 that can improve the intestinal health of Micropterus salmoides include, by weight: fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 6.5%, soybean oil: 6.5%, calcium dihydrogen phosphate: 0.5%, compound vitamin: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 1.05%, hesperidin: 0.25%, denoted as HFD + Hst2.5.

[0063] Control 1

[0064] The raw materials of the feed include, by weight: fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 2.5%, soybean oil: 2.5%, calcium dihydrogen phosphate: 0.5%, compound vitamin: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 9.3%, denoted as Control.

[0065] Control 2 (high fat)

[0066] The raw materials of the hesperidin additive feed 3 that can improve the intestinal health of Micropterus salmoides include, by weight: fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 6.5%, soybean oil: 6.5%, calcium dihydrogen phosphate: 0.5%, compound vitamin: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 1.3%, denoted as HFD.

[0067] The preparation methods of the feeds in the above Controls 1 - 2 are as follows:

[0068] 1) Crush the other raw materials except fish oil and soybean oil in sequence and pass them through a 40-mesh sieve, accurately weigh them according to the formula, mix the various feed ingredients step by step according to their specific gravity, add soybean oil and fish oil and mix them well, then add 40% distilled water (based on the weight percentage of the total mixture) and mix them well to obtain a premix:

[0069] 2) The mixed material obtained in step 1) is put into an automatic granulator to make feed particles with a size of 2 mm, and then the granular feed is put into a dryer and dried at 60° C. to obtain a finished feed product, which is stored at -20° C. for later use.

[0070] In the above feed, fish meal contains 67.1% crude protein.

[0071] Complex vitamins (calculated per kg of feed): niacin 12.5 mg, D-pantothenic acid 50 mg, folic acid 510 mg, D-biotin 1 mg, vitamin B12 0.05 mg, inositol 0.49 mg, carnitine 75 mg, vitamin A 2.25 mg, vitamin D3 0.75 mg, vitamin E 80 mg, vitamin K3 10 mg, vitamin B1 15 mg, vitamin B2 15 mg, vitamin B6 15 mg, microcrystalline cellulose 4720.96 mg.

[0072] The mixed minerals (calculated per kg of feed) include: 310 mg of iron sulfate heptahydrate, 260 mg of zinc sulfate heptahydrate, 110 mg of manganese sulfate monohydrate, 25 mg of copper sulfate pentahydrate, 6 mg of calcium iodate hexahydrate, 2 mg of sodium selenite, 3 mg of cobalt sulfate, 1200 mg of magnesium sulfate and 8084 mg of microcrystalline cellulose.

[0073] Based on the feeds prepared in the above embodiments and comparative examples, six kinds of feeds prepared in embodiments 1-4 and comparative examples 1 and 2 (high-fat) (the six kinds of feeds use fish meal, soybean meal, rapeseed meal, cottonseed meal, and chicken meal as proteins, and soybean oil and fish oil as fat sources) were used to explore their effects on the growth performance and intestinal health of largemouth bass, in order to evaluate the feasibility of applying the hesperidin additive feed of the present invention to largemouth bass.

[0074] Table 1 Feed formula and nutritional composition of comparative examples 1-2 and examples 1-4

[0075]

[0076] Analysis of the experimental effects of feed breeding prepared in the above examples and comparative examples

[0077] The experimental largemouth bass juveniles were from the same batch of artificially cultivated fry provided by Zhongxiang Tianchi Fisheries Co., Ltd. Before the experiment, the largemouth bass were temporarily raised with commercial feed for 2 weeks to adapt to the aquaculture environment. Before the start of the formal experiment, the largemouth bass were fasted for 24 h. Largemouth bass with uniform size (about 6.62 g) and healthy fish bodies were randomly assigned to 18 culture barrels (divided into 6 experimental groups, with 3 replicates in each group). The volume of the aquaculture water in the barrels was 300 L, and the aquaculture water was tap water with aeration and chlorine removal. There were 25 fish in each culture barrel. Feed was fed twice a day, at 8:00 am and 4:00 pm on time to satiation. The experiment lasted for 10 weeks. During the whole aquaculture experiment, continuous aeration for 24 h, natural light, water temperature of 25.00 - 26.00 °C, pH of 7.5 - 7.99, ammonia nitrogen of 0.02 ± 0.025 mg / L, dissolved oxygen of 6.0 - 6.4 mg / L, and nitrite of 0.002 ± 0.001 mg / L were maintained.

[0078] At the end of the 10 - week aquaculture experiment, the largemouth bass were fasted for 24 hours before sampling. Then, they were anesthetized with MS - 222 (100 mg / L water), the total weight of the fish in each barrel was weighed and the number of fish was recorded to calculate the growth rate and survival rate of the largemouth bass. Then, the weight of each individual largemouth bass was weighed in turn and the body length was measured to calculate the condition factor. The visceral mass and intestine of the largemouth bass were separated and weighed to calculate the viscerosomatic index and intestine - somatic index. The collected intestinal tissue samples were fixed with 4% formalin buffer for histological (H&E section) detection of the largemouth bass intestine, and H&E sections and intestinal health - related indicators were analyzed. The analysis of each index used mature methods in domestic and foreign studies. All values were expressed as mean ± standard error. One - way ANOVA and independent T - test analysis were used for each feed, and Duncan's multiple comparison method was used to compare the differences between different treatment groups. P < 0.05 indicated a significant difference between different treatment groups.

[0079] Table 2. Effects of each feed on the growth performance, feed efficiency and morphological parameters of largemouth bass

[0080]

[0081]

[0082] Note: The data in the table are mean ± standard error (n = 3); among the data in the same row, those without the same lowercase letter annotation indicate significant differences from each other (P < 0.05).

[0083] From the growth indices, feed utilization rates, and morphological parameters in Table 2, it can be seen that the hesperetin additive feed affects the final average weight, weight gain rate, specific growth rate, feed coefficient, and feed intake of largemouth bass (P<0.05). Compared with the negative control group, adding 1.25 - 2.5 g / kg of hesperetin to the diet in Examples 1 and 2 decreased the final average weight, weight gain rate, specific growth rate, and feed intake of largemouth bass. However, compared with the positive control group (high-fat), adding 1.25 - 2.5 g / kg of hesperetin to the diet in Examples 3 and 4 increased the final average weight, weight gain rate, and feed intake of largemouth bass, showing a dose-dependent effect. Compared with the control group (high-fat), adding 1.25 - 2.5 g / kg of hesperetin to the diet in Examples 3 and 4 decreased the feed coefficient, showing a dose-dependent effect. Compared with the control group, adding 1.25 - 2.5 g / kg of hesperetin to the diet in Examples 1 and 2 had no significant effect on the viscerosomatic index, intestine-somatic index, and condition factor of largemouth bass (p>0.05). Compared with the control group (high-fat), adding 1.25 - 2.5 g / kg of hesperetin to the diet in Examples 3 and 4 had no significant effect on the viscerosomatic index, intestine-somatic index, and condition factor of largemouth bass. This indicates that adding a certain amount of hesperetin to the diet can improve the growth performance of largemouth bass.

[0084] From Figure 1 it can be seen that adding hesperetin to the feed affects the intestinal health of largemouth bass. From the results of intestinal histology, it can be concluded that in Examples 1 and 2, compared with the control group, the intestinal villus length was significantly increased, and the malondialdehyde content in the intestine was decreased, showing a dose-dependent effect. Compared with the control group (high-fat), adding 1.25 - 2.5 g / kg of hesperetin to the diet in Examples 3 and 4 significantly increased the intestinal microvillus length of largemouth bass and decreased the malondialdehyde content in the intestine, improving the intestinal health status.

[0085] From Figure 2 the results of antioxidant enzymes and total antioxidant capacity, compared with the control group, adding 1.25 - 2.5 g / kg of hesperetin to the diet in Examples 1 and 2 significantly increased the activities of catalase, total antioxidant enzyme, glutathione peroxidase, and superoxide dismutase in the intestine of largemouth bass. Compared with the control group (high-fat), adding 1.25 - 2.5 g / kg of hesperetin to the diet in Examples 3 and 4 significantly increased the activities of catalase, total antioxidant enzyme, glutathione peroxidase, and superoxide dismutase in the intestine of largemouth bass. This indicates that adding a certain amount of hesperetin to the feed can increase the intestinal antioxidant capacity of largemouth bass and improve the intestinal health status of largemouth bass.

[0086] From Figure 3From the results of the gene expression related to intestinal inflammatory factors, it can be concluded that compared with the comparative example, the diets supplemented with 1.25 - 2.5 g / kg of hesperidin in Example 1 and Example 2 significantly reduced the mRNA expression levels of the pro-inflammatory factors tnf-α, il-1β, il-6, and il-8 in the intestine of largemouth bass, and significantly increased the mRNA expression levels of the anti-inflammatory factors tgf-β and il-10 in the intestine of largemouth bass. Compared with the comparative example (high-fat), the diets supplemented with 1.25 - 2.5 g / kg of hesperidin in Example 3 and Example 4 significantly reduced the mRNA expression levels of the pro-inflammatory factors tnf-α, il-1β, il-6, and il-8 in the intestine of largemouth bass, and significantly increased the mRNA expression levels of the anti-inflammatory factors tgf-β and il-10 in the intestine of largemouth bass. Generally speaking, these data indicate that adding a certain amount of hesperidin to the feed will increase the expression of anti-inflammatory factors and reduce the expression of pro-inflammatory factors in the intestine of largemouth bass.

[0087] To sum up, in this experiment, compared with the comparative example (high-fat), adding hesperidin can, to a certain extent, alleviate the negative effects brought by high fat, enhance the growth performance of largemouth bass, and 2.5 g / kg can better alleviate the negative effects brought by high fat. Compared with the comparative example, adding 1.25 g / kg and 2.5 g / kg of hesperidin can, to a certain extent, enhance the intestinal antioxidant capacity of largemouth bass and better protect the fish intestine, and the effect of 2.5 g / kg is more obvious. Combining all the examples and comparative examples, it can be concluded that adding 2.5 g / kg of hesperidin has a better effect. On the one hand, this invention will provide a theoretical basis for the healthy farming of largemouth bass and its feed development. It can provide a certain theoretical basis for improving production efficiency, reducing breeding costs, and reducing the use of drugs such as antibiotics. It promotes the development of the aquaculture industry towards the direction of sustainability, high efficiency, and innovation.

[0088] Other parts not described in detail are all prior arts. Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A hesperidin additive feed for improving the intestinal health of largemouth bass, characterized in that: Its raw materials include, by weight: fish meal: 40%-50%, chicken meal: 8%-12%, cottonseed meal: 5%-10%, rapeseed meal: 5%-8%, soybean meal: 6%-12%, wheat flour: 6%-10%, fish oil: 1%-7%, soybean oil: 1%-7%, monocalcium phosphate: 0.3%-1%, complex vitamins: 0.1%-1%, mixed minerals: 0.1%-1%, choline chloride: 0.1%-0.5%, vitamin C: 0.1%-1%, gelatin: 0.1%-2%, microcrystalline cellulose: 1%-12%, hesperidin: 0.1%-0.5%.

2. The hesperidin additive feed capable of improving the intestinal health of largemouth bass according to claim 1, characterized in that: Its raw materials include by weight percentage: Fish meal: 45%-50%, chicken meal: 10%-12%, cottonseed meal: 5%-7%, rapeseed meal 5%-6%, soybean meal: 6%-10%, wheat flour: 6%-8%, fish oil: 2%-7%, soybean oil: 2%-7%, monocalcium phosphate: 0.3%-0.5%, multivitamins: 0.1%-0.5%, mixed minerals: 0.1%-0.5%, choline chloride: 0.1%-0.3%, vitamin C: 0.1%-0.5%, gelatin: 0.1%-1%, microcrystalline cellulose: 1%-10%, hesperetin: 0.125%-0.25%.

3. The hesperidin additive feed capable of improving the intestinal health of largemouth bass according to claim 1 or 2, characterized in that: The raw materials of the hesperetin additive feed include, by weight percentage: Fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 2.5%, soybean oil: 2.5%, monocalcium phosphate: 0.5%, multivitamins: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 9.175%, hesperetin: 0.125%.

4. The hesperidin additive feed capable of improving the intestinal health of largemouth bass according to any one of claims 1 to 3, characterized in that: The complex vitamin is composed of niacin, D-pantothenic acid, folic acid, D-biotin, vitamin B12, inositol, carnitine, vitamin A, vitamin D3, vitamin E, vitamin K3, vitamin B1, vitamin B2, vitamin B6 and microcrystalline cellulose; and the weight ratio of each component in the complex vitamin is: 25:100:10:2:0.1:0.98:150:4.3:1.25:160:20:30:30:30:9436.

37.

5. The hesperidin additive feed capable of improving the intestinal health of largemouth bass according to any one of claims 1 to 3, characterized in that: The mixed mineral is composed of ferric sulfate heptahydrate, zinc sulfate heptahydrate, manganese sulfate monohydrate, copper sulfate pentahydrate, calcium iodate hexahydrate, sodium selenite, cobalt sulfate, magnesium sulfate and microcrystalline cellulose; and the weight ratio of each component in the mixed mineral is: 299:221:62.9:12:2.3:0.7:1.4:1000:8400.

7.

6. The hesperidin additive feed capable of improving the intestinal health of largemouth bass according to claim 3, characterized in that: The raw materials of the hesperidin additive feed include, by weight: fish meal: 45%, chicken meal: 10%, cottonseed meal: 5%, rapeseed meal: 6%, soybean meal: 8%, wheat flour: 8%, fish oil: 2.5%, soybean oil: 2.5%, calcium dihydrogen phosphate: 0.5%, complex vitamins: 1%, mixed minerals: 1%, choline chloride: 0.1%, vitamin C: 0.1%, gelatin: 1%, microcrystalline cellulose: 9.175%, hesperidin: 0.125%, wherein the complex vitamins are: niacin, D-pantothenic acid, folic acid, D-biotin, vitamin B 12 , inositol, carnitine, vitamin A, vitamin D3, vitamin E, vitamin K3, vitamin B1, vitamin B2, vitamin B6 and microcrystalline cellulose; and in the complex vitamins, the weight ratio of each component is: 12.5:50:5:1:0.05:0.49:75:2.25:0.75:80:10:15:15:15:4720.

96. The mixed minerals include iron sulfate heptahydrate, zinc sulfate heptahydrate, manganese sulfate monohydrate, copper sulfate pentahydrate, calcium iodate hexahydrate, sodium selenite, cobalt sulfate, magnesium sulfate and microcrystalline cellulose; and the weight ratio of each component in the mixed minerals is: 310:260:110:25:6:2:3:1200:8084.

7. A method for preparing a hesperidin additive feed capable of improving the intestinal health of largemouth bass according to any one of claims 1 to 3, comprising the following steps: 1) Grind and sieve fish meal, chicken meal, cottonseed meal, rapeseed meal, soybean meal and gelatin in sequence; 2) fish meal, chicken meal, cottonseed meal, rapeseed meal, soybean meal, wheat flour, fish oil, soybean oil, choline chloride, multivitamins, mixed minerals, monocalcium phosphate, microcrystalline cellulose, vitamin C, gelatin and hesperidin by weight percentage; 3) After various raw material components are mixed step by step according to their specific gravity, fish oil and soybean oil are added and mixed thoroughly, and then purified water accounting for 20-35% of the total weight of the above raw materials is added and mixed thoroughly to obtain a mixture; 4) The mixed material after step 3) is put into an automatic granulator to make feed pellets, and then the feed pellets are sent to a dryer for drying to obtain hesperidin additive feed, which is stored in a -20°C refrigerator for standby use.

8. The method for preparing the hesperidin additive feed capable of improving the growth and health of largemouth bass according to claim 7, characterized in that: The compound vitamins are: niacin, D-pantothenic acid, folic acid, D-biotin, vitamin B 12 , inositol, carnitine, vitamin A, vitamin D3, vitamin E, vitamin K3, vitamin B1, vitamin B2, vitamin B6 and microcrystalline cellulose; And in the multivitamin, the weight ratio of each component is: 12.5:50:5:1:0.05:0.49: 75：2.25：0.75：80：10： 15： 15： 15：4720.96。 9. The method for preparing the hesperidin additive feed capable of improving the intestinal health of largemouth bass according to claim 7, characterized in that: The mixed minerals are: iron sulfate heptahydrate, zinc sulfate heptahydrate, manganese sulfate monohydrate, copper sulfate pentahydrate, calcium iodate hexahydrate, sodium selenite, cobalt sulfate, magnesium sulfate and microcrystalline cellulose; and in the mixed minerals, the weight ratio of each component is: 310:260:110:25:6:2:3:1200:8084.

10. The method for preparing the hesperidin additive feed capable of improving the intestinal health of largemouth bass according to claim 7, characterized in that: In the step 4), the particle size of the feed particles is 2 mm, and the drying temperature of the dryer is 60°C.

Images