Composition for improving grass carp immunity and application thereof in grass carp feed

Abstract

The present invention discloses a composition for improving the immunity of grass carp and its application in grass carp feed, which specifically belongs to the field of functional feed. The composition for improving the immunity of grass carp contains the following components by mass: 20-25 parts of honeysuckle extract, 5-10 parts of licorice extract, 2-5 parts of forsythia extract and 1-3 parts of four-leaf humulus extract. The composition for improving the immunity of grass carp provided by the present invention and the feed containing the composition can significantly improve the nonspecific immunity of grass carp and enhance the antiviral and anti-stress capabilities of grass carp; the honeysuckle extract, licorice extract, forsythia extract and four-leaf humulus extract in the composition provided by the present invention have a certain synergistic effect in improving the immunity of grass carp.

Description

Technical Field

[0001] The invention belongs to the field of functional feed, and particularly relates to a composition for improving the immunity of grass carp and application of the composition in grass carp feed. Background Art

[0002] Grass carp is a key freshwater aquaculture fish species in my country, favored by farmers for its delicious meat, rapid growth rate, and strong adaptability. However, during cultivation, grass carp is often susceptible to various diseases, such as red skin disease, gill rot, and enteritis. These diseases not only affect grass carp's growth rate and survival rate, but also increase farming costs and reduce economic benefits. Therefore, how to improve grass carp's immunity and reduce the incidence of diseases has become a pressing issue in the aquaculture industry.

[0003] Traditionally, farmers have used chemical drugs and antibiotics to control grass carp diseases. However, these methods not only easily lead to the development of drug resistance in pathogens, but also pose potential risks to the aquaculture environment and human health. In recent years, with increasing awareness of food safety and environmental protection, Chinese herbal medicines have gradually been used and promoted in aquaculture disease control due to their natural properties, lack of toxic side effects, and resistance to drug resistance.

[0004] Currently, relatively little research has been conducted on the combined application of honeysuckle extract, licorice extract, forsythia extract, and Humulus tetraphylla extract in grass carp feed, and a systematic application plan has yet to be established. Therefore, developing a Chinese herbal medicine composition containing honeysuckle extract, licorice extract, forsythia extract, and Humulus tetraphylla extract and applying it to grass carp feed to enhance the carp's immunity and reduce the incidence of disease has important practical significance and application value. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide an efficient, safe and environmentally friendly composition. By optimizing the ratio and preparation process, it is applied to grass carp feed to improve the immunity and disease resistance of grass carp, promote the healthy growth of grass carp, and provide a new disease prevention and control method for the aquaculture industry.

[0006] To achieve the above objectives, the present invention discloses the following technical solutions:

[0007] In a first aspect, the present invention provides a composition for improving the immunity of grass carp, wherein the composition comprises the following components in parts by mass:

[0008]

[0009] Preferably, the preparation method of the Humulus tetraphylla extract comprises the following steps:

[0010] S1-1. Take the dried whole plant of four-leaf humulus and crush it, pass through a 60-mesh sieve to obtain four-leaf humulus powder;

[0011] S1-2. Add distilled water to the four-leaf humulus powder obtained in step S1-1, stir evenly, add the complex enzyme preparation for enzymatic hydrolysis, and filter after ultrasonication to obtain filtrate A and filter residue for standby use, wherein the mass ratio of the four-leaf humulus powder to distilled water is 1:30, the amount of the complex enzyme preparation is 6wt% of the mass of the four-leaf humulus powder, the complex enzyme preparation consists of neutral protease, β-glucanase and mannanase, the enzymatic hydrolysis time is 3h, the enzymatic hydrolysis temperature is 30°C, the ultrasonic time is 30min, and the ultrasonic frequency is 23kHz;

[0012] S1-3 take 95v / v% ethanol aqueous solution was added to the filter residue of step S1-2, reflux extraction 2 times, each 0.5h, filtered, and the combined filtrate to obtain filtrate B, wherein the amount of ethanol aqueous solution added is 10 times the mass of the filter residue, the reflux extraction temperature is 80 ℃;

[0013] S1-4. After combining filtrates A and B, the mixture was spray-dried to obtain a Humulus tetrafolia extract.

[0014] Further preferably, the complex enzyme preparation consists of neutral protease, β-glucanase and mannanase in a mass ratio of 1:2:2.

[0015] In a second aspect, the present invention provides a feed for improving the immunity of grass carp, wherein the feed comprises the composition for improving the immunity of grass carp according to the first aspect.

[0016] Preferably, the feed further comprises soybean meal, fish meal, bran, cottonseed meal, rapeseed meal, rice bran, starch, fish oil, soybean oil and premix.

[0017] Further preferably, the premix is a mixture containing vitamins and minerals.

[0018] In a third aspect, the present invention provides a method for preparing the feed for improving grass carp immunity according to the second aspect, the preparation method comprising the following steps:

[0019] S2-1. The soybean meal, fish meal, bran, cottonseed meal, rapeseed meal, rice bran, starch were ultrafinely pulverized;

[0020] S2-2. The composition for improving grass carp immunity is added to the premix and mixed, and then placed in a grinder;

[0021] S2-3. The crushed material in step S2-1 and step S2-2 was transported to the material modulator, steam was introduced at 100 ° C for 10 min, and then extruded and granulated at 95 ° C using an extruded feed pelletizer, and the resulting feed pellets were dried at 70 ° C;

[0022] S2-4. The dried feed pellets are cooled and sprayed with a mixture of fish oil and soybean oil using a drum spraying method to obtain the feed that improves grass carp immunity.

[0023] Preferably, the powders obtained in steps S2-1 and S2-2 can both pass through a 100-mesh sieve.

[0024] Preferably, the feed pellets are dried to a moisture content of ≤8%.

[0025] In a fourth aspect, the present invention provides use of the composition for improving the immunity of grass carp described in the first aspect in preparing a feed having the effect of improving the immunity of grass carp.

[0026] In the present invention:

[0027] Honeysuckle extract is rich in flavonoids, iridoids, triterpenes, triterpenoid saponins, and organic acids, with chlorogenic acid being one of its main active ingredients. In terms of boosting immunity, honeysuckle extract works through multiple mechanisms, including promoting immune cell activity, enhancing macrophage function, promoting lymphocyte transformation, and exhibiting anti-inflammatory, antiviral, and antibacterial effects, thereby enhancing the body's resistance to disease.

[0028] Licorice extract contains polysaccharides, glycosides, alkaloids, organic acids, volatile oils, and flavonoids, all of which play a significant role in boosting grass carp's immunity. The polysaccharides and glycosides in licorice extract can activate the body's nonspecific enzymes and the phagocytic activity of immune cells, thereby enhancing the carp's nonspecific immunity. Licorice extract also effectively enhances the phagocytic activity of phagocytes in the reticuloendothelial system, promotes antibody production, accelerates antigen-antibody reactions, and promotes lymphocyte transformation.

[0029] The main components of Forsythia extract include phenylethanoid glycosides, lignans, phenolic acids, flavonoids, terpenes and volatile oils, natural alcohols and their glycosides. Forsythia extract has immunomodulatory activity and can enhance the body's nonspecific immune function. It can activate macrophages and natural killer cells, and improve their ability to phagocytize and kill viral infections. It has anti-inflammatory and antibacterial effects, can reduce inflammatory responses, reduce the invasion of pathogens, and thus indirectly enhance immunity.

[0030] The main components of the extract of Humulus four-leaf include flavonoids, alkaloids, phenolic acid compounds, polysaccharides, etc. The polysaccharides and flavonoids in the extract of Humulus four-leaf can effectively enhance the nonspecific immune function of grass carp.

[0031] The present study found that honeysuckle extract, licorice extract, forsythia extract and humulus quadrangularis extract have a synergistic effect in improving the immunity of grass carp. They jointly improve the immunity and disease resistance of grass carp by enhancing nonspecific immune function, activating immune cells, improving antioxidant capacity, improving intestinal health, and having antiviral and antibacterial effects.

[0032] Beneficial effects of the present invention:

[0033] The composition for improving the immunity of grass carp provided by the present invention comprises honeysuckle extract, licorice extract, forsythia extract and humulus quadrangularis extract, which have a certain synergistic effect in improving the immunity of grass carp. The composition and feed containing the composition can significantly improve the nonspecific immunity of grass carp and enhance the antiviral and anti-stress capabilities of grass carp. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to specific embodiments. However, the following embodiments are merely preferred embodiments of the present invention and are not exhaustive. For the sake of clarity, not all features of the actual embodiments are described.

[0035] Based on the embodiments in the implementation manner, other embodiments obtained by those skilled in the art without making any creative work shall all fall within the protection scope of the present invention.

[0036] In the present invention:

[0037] Honeysuckle extract: purchased from Shaanxi Xinyanghe Biotechnology Co., Ltd., specification 10:1;

[0038] Licorice extract: purchased from Sichuan Xiaoye Bencao Biotechnology Co., Ltd., specification 10:1;

[0039] Forsythia suspensa extract: purchased from Sichuan Xiaoye Bencao Biotechnology Co., Ltd., specification 10:1;

[0040] Galium bungei Steud. is a plant of the genus Galium in the Rubiaceae family. In a specific embodiment, the whole plant of Galium bungei Steud. is dried and extracted. Galium bungei Steud. is purchased from Guangzhou Qingping Chinese Herbal Medicine Market.

[0041] Neutral protease: enzyme activity of 50 U / g, purchased from Yunnan Boshiao Biotechnology Co., Ltd.;

[0042] β-Glucanase: Enzyme activity was 100 U / g, purchased from Yunnan Boshiao Biotechnology Co., Ltd.

[0043] Mannanase: enzyme activity of 120 U / g, purchased from Yunnan Boshiao Biotechnology Co., Ltd.

[0044] Other raw materials are commercially available.

[0045] Preparation of Humulus tetraphylla extract:

[0046] S1-1. Take the dried four-leaf Humulus whole plant and grind it in a high-speed mill, pass through a 60-mesh sieve, and obtain four-leaf Humulus powder;

[0047] S1-2. Add distilled water to the four-leaf humulus powder obtained in step S1-1, stir evenly, add the complex enzyme preparation for enzymatic hydrolysis, filter after ultrasonication to obtain filtrate A and filter residue for standby use, wherein the mass ratio of the four-leaf humulus powder to distilled water is 1:30, the amount of the complex enzyme preparation is 6wt% of the mass of the four-leaf humulus powder, and the complex enzyme preparation is composed of neutral protease, β-glucanase and mannanase in a mass ratio of 1:2:2, the enzymatic hydrolysis time is 3h, the enzymatic hydrolysis temperature is 30°C, the ultrasonic time is 30min, and the ultrasonic frequency is 23kHz;

[0048] S1-3 take 95v / v% ethanol aqueous solution was added to the filter residue of step S1-2, reflux extraction 2 times, each 0.5h, filtered, and the combined filtrate to obtain filtrate B, wherein the amount of ethanol aqueous solution added is 10 times the mass of the filter residue, the reflux extraction temperature is 80 ℃;

[0049] S1-4. After combining filtrates A and B, the mixture was spray-dried to obtain a Humulus tetrafolia extract.

[0050] Preparation of the composition:

[0051] The raw materials in Table 1 were accurately weighed in parts by mass, placed in a high-speed blender for compounding and mixing to obtain a composition for improving grass carp immunity.

[0052] Table 1 Mass fraction of raw materials of the composition

[0053] Raw materials\group Composition ① Composition ② Composition ③ Composition ④ Composition ⑤ Composition ⑥ Composition ⑦ Composition ⑧ Honeysuckle extract 20 23 25 23 23 23 7 23 Licorice Extract 5 7 10 7 7 7 23 3.5 Forsythia suspensa extract 2 3.5 5 3.5 - - 3.5 7 Humulus tetraphylla extract 1 2 3 - 2 - 2 2

[0054] Note: “-” in the table means no additive.

[0055] Preparation of feed to improve grass carp immunity:

[0056] S2-1 according to Table 2 raw materials by mass parts accurately weighed standby;

[0057] S2-2 soybean meal, fish meal, bran, cottonseed meal, rapeseed meal, rice bran, starch after ultrafine grinding mill, the composition was added to the premix mixed, placed in a grinder pulverized so that all ingredients can pass through a 100 mesh sieve;

[0058] S2-3 all the crushed material was transported to the material modulator, passed through 100 ℃ steam modulation 10min, and then at 95 ℃ using an extruded feed pelletizer for extrusion granulation, the resulting feed pellets were dried at 70 ℃ to a moisture content of ≤8%;

[0059] S2-4. The dried feed pellets were cooled and sprayed with a mixture of fish oil and soybean oil using a drum spraying method to obtain feed that improves grass carp immunity;

[0060] S2-5. The obtained grass carp immunity-enhancing feed is transported to a packaging warehouse for packaging to prevent the feed from getting damp and contaminated.

[0061] Table 2 Mass fraction of feed raw materials

[0062]

[0063]

[0064] Note: The premix is herbivorous fish premix 1211, purchased from Guangzhou Nutriera Biotechnology Co., Ltd.

[0065] Performance testing:

[0066] Grass carp serum immune index test

[0067] 1 Experimental design

[0068] The study involved 135 healthy grass carp, each weighing 100 ± 5 g. A single-factor experimental design was used, with each group equally divided into nine treatments, three replicates per treatment, and five grass carp per replicate. Each group of grass carp was fed the immune-enhancing diet described above (see Table 3 for specific groupings) for eight weeks. After the study, blood samples were collected from each replicate for immune markers. The experimental groupings are shown in Table 3.

[0069] Table 3 Experimental groups

[0070] Group deal with control group Comparative Example 6 Preparation of Feed Example 1 Example 1 Preparation of feed Example 2 Example 2 Preparation of feed Example 3 Example 3 Preparation of feed Comparative Example 1 Comparative Example 1 Preparation of feed Comparative Example 2 Comparative Example 2 Preparation of feed Comparative Example 3 Comparative Example 3 Preparation of feed Comparative Example 4 Comparative Example 4 Preparation of Feed Comparative Example 5 Comparative Example 5 Preparation of feed

[0071] 2. Breeding management

[0072] The experimental fish were housed in 27 canvas ponds (3 × 3 × 1.5 m), with five grass carp per pond. The grass carp were fed twice daily (at 9:00 AM and 4:00 PM), with the daily feed amount representing approximately 3.5% of their body weight. Feeding amounts were adjusted based on the fish's weight and weather conditions, ensuring consistency across all groups. Before each morning feeding, any remaining bait and feces were promptly removed from the drainage pipes. The water temperature was maintained at approximately 26°C, the pH at approximately 7.5, and the dissolved oxygen content at greater than 5.5 mg / L.

[0073] 3. Immune index detection

[0074] On day 56 of the experiment, before the morning feeding, approximately 10 mL of blood was collected from each grass carp and centrifuged to collect serum. Serum levels of lysozyme, superoxide dismutase, malondialdehyde, alkaline phosphatase, and catalase were assayed according to the kit instructions. The kits were purchased from Shanghai ELISA Biotechnology Co., Ltd.

[0075] 4 Data Analysis

[0076] SPSS19 was used to analyze the significance of differences in the serum immune indicators of grass carp in each group. The LSD method was used to analyze multiple comparisons between groups. P>0.05 indicated that the difference was not significant, and p<0.05 indicated that the difference was significant. The results are expressed as mean ± standard deviation of each data.

[0077] 5 Results and Analysis

[0078] The effects of feeds that improve grass carp immunity on the immune indicators of grass carp are shown in Table 4.

[0079] Table 4 Effects of feeds that improve grass carp immunity on grass carp immune indicators

[0080]

[0081] Note: Data in the same column marked with different lowercase letters indicate significant differences between groups (p < 0.05), and data marked with the same lowercase letters indicate no significant differences between groups (p > 0.05).

[0082] Compared with the control group, Examples 1-3 showed that the immune indicators of lysozyme, alkaline phosphatase, superoxide dismutase, and catalase in Examples 1-3 were significantly higher than those in the control group. That is to say, the composition of honeysuckle extract, licorice extract, forsythia extract and humulus quadrangularis extract provided by the present invention can significantly improve the nonspecific immunity of grass carp and enhance the antiviral and anti-stress capabilities of grass carp.

[0083] Compared with Comparative Examples 1-3, Example 2 shows that when the four-leaf humulus extract and the forsythia extract are respectively missing, the performance of the overall formula is greatly affected, which proves that the honeysuckle extract, licorice extract, forsythia extract and four-leaf humulus extract in the composition provided by the present invention have a certain synergistic effect in improving the immunity of grass carp.

[0084] Comparison of Example 2 with Comparative Examples 4 and 5 shows that when the mass ratio of honeysuckle extract, licorice extract, forsythia suspensa extract and humulus quadrangularis extract in the composition provided by the present invention is not within the specific ratio range provided by the present invention, the performance of the overall formula will be affected.

[0085] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A composition for improving the immunity of grass carp, characterized in that: The composition comprises the following components in parts by mass: 20-25 parts of honeysuckle extract; 5-10 parts of licorice extract; 2-5 parts of Forsythia suspensa extract; 1-3 parts of Humulus tetraphylla extract; The preparation method of the Humulus tetraphylla extract comprises the following steps: S1-1. Take the dried whole plant of four-leaf humulus and crush it, pass through a 60-mesh sieve to obtain four-leaf humulus powder; S1-2. Add distilled water to the four-leaf humulus powder obtained in step S1-1, stir evenly, add the complex enzyme preparation for enzymatic hydrolysis, and filter after ultrasonication to obtain filtrate A and filter residue for standby use, wherein the mass ratio of the four-leaf humulus powder to distilled water is 1:30, the amount of the complex enzyme preparation is 6wt% of the mass of the four-leaf humulus powder, and the complex enzyme preparation consists of neutral protease, β-glucanase and mannanase. The enzymatic hydrolysis time is 3h, the enzymatic hydrolysis temperature is 30°C, the ultrasonic time is 30min, and the ultrasonic frequency is 23kHz; S1-3 take 95v / v% ethanol aqueous solution was added to the filter residue of step S1-2, reflux extraction 2 times, each 0.5h, filtered, and the combined filtrate to obtain filtrate B, wherein the amount of ethanol aqueous solution added is 10 times the mass of the filter residue, the reflux extraction temperature is 80 ℃; S1-4. After combining filtrates A and B, the mixture was spray-dried to obtain a Humulus tetrafolia extract.

2. The composition for improving the immunity of grass carp according to claim 1, characterized in that: The complex enzyme preparation consists of neutral protease, beta-glucanase and mannanase in a mass ratio of 1:2:

2.

3. A feed for improving grass carp immunity, characterized in that: The feed contains the composition for improving the immunity of grass carp according to claim 1 or 2.

4. The feed for improving grass carp immunity according to claim 3, characterized in that: The feed also contains soybean meal, fish meal, bran, cottonseed meal, rapeseed meal, rice bran, starch, fish oil, soybean oil and premix.

5. The method for preparing the feed for improving grass carp immunity according to claim 4, characterized in that: The preparation method comprises the following steps: S2-1. The soybean meal, fish meal, bran, cottonseed meal, rapeseed meal, rice bran, starch were ultrafinely pulverized; S2-2. The composition for improving grass carp immunity is added to the premix and mixed, and then placed in a grinder; S2-3. The crushed material in step S2-1 and step S2-2 was transported to the material modulator, steam was introduced at 100 ° C for 10 min, and then extruded and granulated at 95 ° C using an extruded feed pelletizer, and the resulting feed pellets were dried at 70 ° C; S2-4. The dried feed pellets are cooled and sprayed with a mixture of fish oil and soybean oil using a drum spraying method to obtain the feed that improves grass carp immunity.

6. The preparation method according to claim 5, characterized in that The powders obtained in steps S2-1 and S2-2 can both pass through a 100-mesh sieve.

7. The preparation method according to claim 5, characterized in that The feed pellets are dried to a moisture content of ≤8%.

8. Use of the composition for improving grass carp immunity according to claim 1 or 2 in preparing grass carp feed.

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