Composition for improving immunity of grass carp and application of composition in grass carp feed

Abstract

The invention discloses a composition for improving immunity of grass carp and application of the composition in grass carp feed, and particularly belongs to the field of functional feed. The composition for improving the immunity of the grass carp is prepared from the following components in parts by mass: 20 to 25 parts of honeysuckle flower extract, 5 to 10 parts of liquorice root extract, 2 to 5 parts of fructus forsythiae extract and 1 to 3 parts of humulus scandens extract. According to the composition for improving the immunity of the grass carp and the feed containing the composition, provided by the invention, the non-specific immunity of the grass carp can be remarkably improved, and the antiviral and anti-stress capabilities of the grass carp are enhanced; the honeysuckle extract, the liquorice extract, the fructus forsythiae extract and the scandent hop extract in the composition provided by the invention have a certain synergistic effect in the aspect of improving the immunity of the 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 one of the important freshwater farmed fish in my country. It is favored by farmers for its delicious meat, fast growth rate and strong adaptability. However, during the breeding process, grass carp is often attacked by various diseases, such as red skin disease, gill rot disease and enteritis. These diseases not only affect the growth rate and survival rate of grass carp, but also increase the breeding cost and reduce the economic benefits. Therefore, how to improve the immunity of grass carp and reduce the occurrence of diseases has become an urgent problem to be solved in the current aquaculture industry.

[0003] Traditionally, farmers have used chemical drugs and antibiotics to control diseases of grass carp, but these methods not only easily lead to drug resistance in pathogens, but also may cause potential harm to the breeding environment and human health. In recent years, with the improvement of people's awareness of food safety and environmental protection, Chinese herbal medicine has gradually been applied and promoted in the prevention and control of aquaculture diseases due to its advantages such as natural, non-toxic and non-resistant to drug resistance.

[0004] At present, there are relatively few studies on the combined application of honeysuckle extract, licorice extract, forsythia extract and Humulus tetraphylla extract in grass carp feed, and no systematic application scheme has been formed. 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 improve the immunity of grass carp and reduce the occurrence of diseases has important practical significance and application value. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide an efficient, safe and environmentally friendly composition, which is applied to grass carp feed by optimizing the ratio and preparation process 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 object, 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 method for preparing the Humulus tetraphylla extract comprises the following steps:

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

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

[0012] S1-3. Take 95v / v% ethanol aqueous solution and add it to the residue of step S1-2, reflux and extract twice, each time for 0.5h, filter, and combine the filtrate to obtain filtrate B, wherein the amount of ethanol aqueous solution added is 10 times the mass of the residue, and the reflux extraction temperature is 80°C;

[0013] S1-4. After combining the filtrates A and B, the filtrates were spray-dried to obtain a Humulus tetraphylla 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 immunity of grass carp, wherein the feed comprises the composition for improving immunity of grass carp described in 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 comprising vitamins and minerals.

[0018] In a third aspect, the present invention provides a method for preparing a feed for improving immunity of grass carp 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 are crushed by ultrafine mill;

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

[0021] S2-3. The crushed material in step S2-1 and step S2-2 is conveyed to a material modulator, and steam at 100 ° C is introduced for 10 min, and then extruded and granulated at 95 ° C using an extruded feed granulator, and the obtained feed pellets are dried at 70 ° C;

[0022] S2-4. The dried feed particles are cooled, and a mixed oil of fish oil and soybean oil is sprayed on the particles by roller spraying to obtain the feed for improving the immunity of grass carp.

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

[0024] Preferably, the feed particles 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 and triterpenoid saponins, organic acids and other ingredients, among which chlorogenic acid is one of its main active ingredients. In terms of improving immunity, honeysuckle extract works through multiple mechanisms such as promoting immune cell activity, enhancing macrophage function, promoting lymphocyte transformation, anti-inflammatory effects, antiviral activity and antibacterial effects, thereby enhancing the animal body's resistance to disease.

[0028] Licorice extract contains polysaccharides, glycosides, alkaloids, organic acids, volatile oils and flavonoids, which play an important role in improving the immunity of grass carp. The polysaccharides and glycosides in licorice extract can activate the non-specific enzyme activity of grass carp and the phagocytic activity of immune cells, thereby improving the non-specific immunity of grass carp. At the same time, licorice extract can effectively enhance the phagocytic activity of phagocytes in the reticuloendothelial system, promote antibody production, accelerate antigen-antibody reaction and lymphocyte transformation.

[0029] The main components of Forsythia extract include phenylethanol 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, and reduce the invasion of pathogens, thereby indirectly enhancing immunity.

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

[0031] The present invention study found that honeysuckle extract, licorice extract, forsythia extract and humulus four-leaf 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 antiviral and antibacterial effects.

[0032] Beneficial effects of the present invention:

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

[0034] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. However, the following embodiments are only preferred embodiments of the present invention, not all. For the sake of clarity, all features of the actual embodiments are not 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 of the Rubiaceae family. In a specific embodiment, the whole plant of Galium bungei Steud. is dried for extraction. The Galium bungei Steud. is purchased from Guangzhou Qingping Chinese 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 was 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 crush 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 humus powder obtained in step S1-1, stir evenly, add the composite enzyme preparation for enzymolysis, filter after ultrasonication to obtain filtrate A and filter residue for standby use, wherein the mass ratio of the four-leaf humus powder to distilled water is 1:30, the amount of the composite enzyme preparation is 6wt% of the mass of the four-leaf humus powder, and the composite enzyme preparation is composed of neutral protease, β-glucanase and mannanase in a mass ratio of 1:2:2, the enzymolysis time is 3h, the enzymolysis temperature is 30°C, the ultrasonic time is 30min, and the ultrasonic frequency is 23kHz;

[0048] S1-3. Take 95v / v% ethanol aqueous solution and add it to the residue of step S1-2, reflux and extract twice, each time for 0.5h, filter, and combine the filtrate to obtain filtrate B, wherein the amount of ethanol aqueous solution added is 10 times the mass of the residue, and the reflux extraction temperature is 80°C;

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

[0050] Preparation of the composition:

[0051] The raw materials in Table 1 are accurately weighed according to their mass fractions, and placed in a high-speed mixer 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 Composition⑤ Composition ⑥ Composition ⑦ Composition⑧ 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 addition.

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

[0056] S2-1. Accurately weigh the mass fractions of raw materials in Table 2 for standby use;

[0057] S2-2. The soybean meal, fish meal, bran, cottonseed meal, rapeseed meal, rice bran, starch are crushed by an ultrafine grinder, the composition is added to the premix and mixed, and then placed in a grinder to grind so that all ingredients can pass through a 100-mesh screen;

[0058] S2-3. All the crushed materials were conveyed to the material modulator, and steam at 100 ° C was introduced for 10 min, and then extruded and granulated at 95 ° C using an extruded feed pelletizer, and the obtained feed pellets were dried at 70 ° C 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 by drum spraying 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 moisture and contamination.

[0061] Table 2 Mass fraction of feed raw materials

[0062]

[0063]

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

[0065] Performance Test:

[0066] Test of serum immune indexes of grass carp

[0067] 1 Experimental design

[0068] The experiment selected 135 healthy grass carps weighing 100±5g each, and divided them into 9 treatments according to the single-factor experimental design, with 3 replicates in each treatment and 5 grass carps in each replicate. Each group of grass carps was fed with the above-prepared feed for improving grass carp immunity (see Table 3 for specific grouping) for 8 weeks. After the experiment, blood of grass carps was collected from each replicate to test blood immune indicators. The experimental grouping is 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 raised in 27 canvas pools (3×3×1.5m), with 5 grass carps in each canvas pool. The grass carps were fed twice a day (9:00 and 16:00), and the daily feeding amount was about 3.5% of the grass carp's body weight. The feeding amount was adjusted at any time according to the grass carp's body weight and weather conditions, but the feeding amount for each group was ensured to be consistent. Before morning feeding every day, the sewage pipe should be promptly discharged of residual bait and feces. The water temperature was controlled at about 26℃, the pH value was about 7.5, and the dissolved oxygen content was greater than 5.5mg / L.

[0073] 3. Immune index detection

[0074] On the 56th day of the experiment, before the morning feeding of grass carp, about 10 mL of blood was collected from each grass carp, and the serum was collected by centrifugation. Lysozyme, superoxide dismutase, malondialdehyde, alkaline phosphatase and catalase in the serum were mainly detected. The detection method was referred to the instructions of the kit, and the kit was 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, and the LSD method was used to analyze the 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 were expressed as the mean ± standard deviation of each data.

[0077] 5 Results and analysis

[0078] The effects of feed on grass carp immunity on the immune indicators 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 among the groups (p < 0.05), and data marked with the same lowercase letters indicate no significant differences among the groups (p > 0.05).

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

[0083] It can be seen from Example 2 that when the Humulus tetraphylla extract and the Forsythia suspensa extract are respectively missing, the performance of the overall formula is greatly affected, which proves that the honeysuckle extract, licorice extract, Forsythia suspensa extract and Humulus tetraphylla extract in the composition provided by the present invention have a certain synergistic effect in improving the immunity of grass carp.

[0084] It can be seen from Example 2 that when the mass ratio of honeysuckle extract, licorice extract, forsythia 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 description is only a preferred embodiment of the present invention and is 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 can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope 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; Licorice extract 5-10 parts; 2-5 parts of Forsythia suspensa extract; 1-3 parts of Humulus tetraphylla extract.

2. The composition for improving the immunity of grass carp according to claim 1, characterized in that: The preparation method of the Humulus tetraphylla extract comprises the following steps: S1-1. Take the dried whole plant of four-leaf humus and crush it, pass it through a 60-mesh sieve, and obtain four-leaf humus powder; S1-2. Add distilled water to the four-leaf humus powder obtained in step S1-1, stir evenly, add the composite enzyme preparation for enzymolysis, filter after ultrasonication to obtain filtrate A and filter residue for standby use, wherein the mass ratio of the four-leaf humus powder to distilled water is 1:30, the amount of the composite enzyme preparation is 6wt% of the mass of the four-leaf humus powder, the composite enzyme preparation is composed of neutral protease, β-glucanase and mannanase, the enzymolysis time is 3h, the enzymolysis temperature is 30°C, the ultrasonic time is 30min, and the ultrasonic frequency is 23kHz; S1-3. Take 95v / v% ethanol aqueous solution and add it to the residue of step S1-2, reflux and extract twice, each time for 0.5h, filter, and combine the filtrate to obtain filtrate B, wherein the amount of ethanol aqueous solution added is 10 times the mass of the residue, and the reflux extraction temperature is 80°C; S1-4. After combining the filtrates A and B, the filtrates were spray-dried to obtain a Humulus tetraphylla extract.

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

2.

4. A feed for improving immunity of grass carp, characterized in that: The feed contains the composition for improving the immunity of grass carp according to any one of claims 1 to 3.

5. The feed for improving immunity of grass carp according to claim 4, 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.

6. The method for preparing the feed for improving grass carp immunity according to claim 5, 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 are crushed by ultrafine mill; S2-2. The composition for improving grass carp immunity is added to the premix and mixed, and then crushed in a grinder; S2-3. The crushed material in step S2-1 and step S2-2 is conveyed to a material modulator, and steam at 100 ° C is introduced for 10 min, and then extruded and granulated at 95 ° C using an extruded feed granulator, and the obtained feed pellets are dried at 70 ° C; S2-4. The dried feed particles are cooled, and a mixed oil of fish oil and soybean oil is sprayed on the particles by roller spraying to obtain the feed for improving the immunity of grass carp.

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

8. The preparation method according to claim 6, characterized in that: The feed particles are dried to a moisture content of ≤8%.

9. Use of the composition for improving the immunity of grass carp according to any one of claims 1 to 3 in preparing grass carp feed.

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