Fish feed additive as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of aquatic feed additives, and particularly relates to a fish feed additive as well as a preparation method and application thereof. According to the invention, the fish feed additive is prepared by screening and comparing the types of the herba epimedii and a processing technology, and the optimal fish feed additive amount is optimized by screening the additive amount of the fish feed additive, so that the feed additive prepared by processing the herba epimedii medicinal material with the sheep fat oil and the feed thereof significantly improve the growth and development performance of fish; the health of the fish is not influenced. The fish feed additive inherits excellent traditional Chinese medicine processing essence, combines a modern extraction process technology, removes coarse materials and obtains essence, obtains the high-quality herba epimedii extract, strictly controls the quality control standard, realizes the effect of preparing the fish feed additive with good performance by independently using the high-quality herba epimedii extract, and has a wide prospect in the aspect of aquaculture.

Description

Technical Field

[0001] The invention belongs to the technical field of aquatic feed additives, and in particular relates to a fish feed additive and a preparation method and application thereof. Background Art

[0002] my country is the country with the largest aquatic product output and aquaculture in the world. According to the China Fisheries Statistical Yearbook, in 2022, the total output of aquatic products in China will be 69.6591 million tons, the output value of seedlings will be 84.345 billion yuan, and the total output of freshwater products will increase from 28.74 million tons in 2012 to 34.06 million tons in 2022. In 2022, the economic output value of freshwater aquaculture will account for 49% of the total aquatic economic output. Although the output of aquatic products and aquaculture have grown rapidly, they still cannot meet market demand. The per capita possession of aquatic products in China is only 47.36 kilograms, which is a huge gap. Therefore, there is an urgent need for a high-quality feed additive to improve the output and quality of aquatic animals.

[0003] Epimedium has a long history of medicinal use in my country, and was first recorded in the Shennong's Herbal Classic. The Compendium of Materia Medica states that it has the effects of "benefiting essence and qi, strengthening tendons and bones, nourishing waist and knees, and strengthening heart strength". It is used for kidney yang deficiency, impotence and spermatorrhea, tendon and bone weakness, rheumatism, pain, numbness and cramps. Epimedium contains a variety of active ingredients, mainly flavonoids, polysaccharides, volatile oils and alkaloids. Modern pharmacological studies have shown that epimedium has androgen-like and phytoestrogen-like activities, which can enhance the sexual function of animals; total flavonoids of epimedium have a regulatory effect on immune function; icariin can increase serum SOD activity and androgen levels, reduce germ cell apoptosis, improve degenerative changes in testicular tissue, and delay gonadal aging by inhibiting the expression of germ cell aging gene P16 protein; in addition, epimedium also has the effects of affecting the cardiovascular system, bone marrow and hematopoietic system functions, anti-osteoporosis, improving learning and memory, anti-radiation, and anti-tumor. Epimedium extract is listed in the "Feed Additive Variety Catalog" newly revised by the Ministry of Agriculture in July 2023. Epimedium extract has the advantages of naturalness, safety, environmental protection, and functional diversity, and can be developed and utilized as a new type of green feed additive.

[0004] The content of effective ingredients such as flavonoids in epimedium of different types or origins varies significantly. The 2020 edition of the Chinese Pharmacopoeia lists the dried leaves of Epimedium brevicomu Maxim., Epimedium sagittatum (Sieb. et Zucc.) Maxim., Epimedium pubescens Maxim. or Epimedium koreanum Nakai as epimedium medicinal materials. Liangshan Prefecture, Sichuan Province, has a unique climate and geographical environment and rich forest resources. Liangshan Prefecture makes full use of its understory resources to grow high-quality Chinese medicinal materials. Among them, the understory epimedium has been included in the second batch of national special new agricultural products in 2023. Therefore, the selection of the type of epimedium has an important impact on the quality and efficacy of epimedium.

[0005] Studies have reported that epimedium has a good effect in livestock and poultry farming, but livestock and poultry feed additives are not completely suitable for fish feed additives. There are significant differences between the two in nutritional requirements, digestive system characteristics and types of additives. Therefore, when using epimedium as a fish feed additive, it is necessary to select appropriate processing technology and dosage according to the physiological characteristics and nutritional requirements of fish to ensure their healthy growth.

[0006] Chinese medicine processing is an important part of traditional Chinese medicine processing technology, which is of great significance to improving the efficacy and safety of Chinese medicine. In Chinese medicine processing, the safety and synergistic effect of excipients are important evaluation indicators.

[0007] Therefore, how to select the type, processing technology and dosage of Epimedium to make it suitable for fish feed additives and improve the growth and development performance of fish is a problem and challenge faced by this field. Summary of the invention

[0008] In view of the defects of the prior art, the present invention provides a fish feed additive and a preparation method thereof, aiming to improve the growth and development performance of fish.

[0009] The invention provides a fish feed additive, which consists of an epimedium extract, wherein the epimedium extract contains 3.0% or more of total flavonoids in terms of icariin, and the epimedium is epimedium pubescens.

[0010] Preferably, the epimedium is Epimedium pubescens from Liangshan Prefecture, Sichuan Province.

[0011] Preferably, the epimedium extract is obtained by processing and extracting epimedium.

[0012] Preferably, the processing methods include mutton fat roasting, ghee roasting, wine roasting, and stir-frying;

[0013] And / or, the extraction method includes ultrasonic method and heating reflux method.

[0014] Preferably, the processing method is roasting with mutton fat.

[0015] Preferably, the suet oil is Liangshan black sheep suet oil.

[0016] Preferably, the processing method is to fry the epimedium and mutton fat at 60-100° C. for 5-30 minutes until the surface turns light yellow and has an oily and shiny luster.

[0017] Preferably, in the extraction method, the extraction solvent is selected from water and 40%-85% ethanol solution; the weight volume ratio of epimedium to the extraction solvent is 1g:10-100mL; the extraction temperature is 20-40°C; the number of extractions is 1-3 times; and the extraction time for each time is 15-60min.

[0018] The present invention provides a method for preparing any one of the above-mentioned fish feed additives, which comprises the steps of processing and extracting epimedium.

[0019] The present invention provides use of any of the above-mentioned fish feed additives in preparing aquatic feed.

[0020] Preferably, the fish feed additive is added in an amount of 0.2-2 wt % of the basic aquatic feed diet.

[0021] The present invention prepares a fish feed additive by screening and comparing the types and processing techniques of epimedium, and selects the best fish feed additive by screening the addition amount of the fish feed additive, so that the feed additive and feed prepared by epimedium medicinal materials after being processed with suet oil significantly improve the growth and development performance of fish without affecting the health of fish. The present invention inherits the essence of excellent traditional processing of traditional Chinese medicine, combines modern extraction technology, removes the coarse and retains the fine, obtains high-quality epimedium extract, and strictly controls its quality control standard, so as to achieve the effect of using high-quality epimedium extract alone to prepare a fish feed additive with good performance, and has broad prospects in aquaculture.

[0022] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0023] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is the microscopic structure of the ovary of zebrafish in experimental group 1, with a magnification of 400;

[0025] Figure 2 This is the microstructure of the testis of zebrafish in experimental group 2, with a magnification of 400;

[0026] Figure 3 This is the microscopic structure of the ovary of zebrafish in experimental group 3, with a magnification of 400;

[0027] Figure 4 This is a microscopic image of the kidney of zebrafish in experimental group 4, with a magnification of 400;

[0028] Figure 5 This is the microscopic structure of the liver of zebrafish in experimental group 5, with a magnification of 400;

[0029] Figure 6 This is the microscopic structure of the testis of zebrafish in the blank control group, with a magnification of 400;

[0030] Figure 7 This is the microscopic structure of the ovary of zebrafish in the blank control group, with a magnification of 400. DETAILED DESCRIPTION

[0031] In the following examples and experimental examples, reagents and materials not particularly described are all commercially available.

[0032] Source of Liangshan Black Sheep Suet: Buy Liangshan Black Sheep at the farmer's market in Muli County, Liangshan Prefecture, Sichuan Province, take the fat after slaughtering, remove the impurities and blood, cut the fat into small pieces, boil it in a pot, and control the temperature at 65-75℃. After the fat is completely melted, remove the residue and get Liangshan Black Sheep Suet.

[0033] The source of Epimedium pubescentis is: Puge County, Liangshan Prefecture, Sichuan Province.

[0034] Example 1 A fish feed additive and its preparation method

[0035] The fish feed additive of this embodiment is prepared by the following method:

[0036] (1) Preparation of Epimedium

[0037] Take 0.2 kg of Liangshan black sheep suet oil and heat it to melt, add 1 kg of soft hair epimedium shreds from Liangshan Prefecture, Sichuan Province, and stir-fry at 60°C until the surface turns light yellow and shiny (30 minutes), take it out and let it cool.

[0038] (2) Preparation of Epimedium Extract

[0039] The processed epimedium is subjected to ultrasonic method to prepare the epimedium extract. 10g of processed epimedium is mixed with 0.2L of 75% ethanol solution, and ultrasonicated twice at 30°C, each time for 60min. The extracted solution is concentrated and dried to obtain the extract.

[0040] The content of total flavonoids in the epimedium extract as icariin was 3.15% by UV-visible spectrophotometry.

[0041] Example 2 A fish feed additive and its preparation method

[0042] The fish feed additive was prepared according to the method of Example 1, except that:

[0043] (1) The processing temperature of Epimedium is 100℃ and the processing time is 5 minutes;

[0044] (1) The volume fraction of ethanol is 80%;

[0045] (2) The volume of ethanol is 1 L;

[0046] (3) Ultrasonic temperature is 20°C;

[0047] (4) Each extraction time is 60 min;

[0048] (5) The number of ultrasonic extractions was 1.

[0049] Example 3 A fish feed additive and its preparation method

[0050] The fish feed additive was prepared according to the method of Example 1, except that:

[0051] (1) The volume fraction of ethanol is 85%;

[0052] (2) The volume of ethanol is 0.1L;

[0053] (3) Ultrasonic temperature is 40°C;

[0054] (4) Each extraction time is 15 min;

[0055] (5) The ultrasonic extraction was performed 3 times.

[0056] Example 4 A kind of fish compound feed

[0057] The fish feed additive was prepared according to the method of Example 1, and then added to the fish feed at an addition amount of 0.2 wt% of the fish basic diet. Specifically, it is as follows:

[0058] Take 2g of epimedium extract, dissolve it in 95% ethanol, then add it to 998g of fish feed, evaporate the ethanol, and obtain the extract.

[0059] Fish feed was purchased from Santong Bioengineering (Weifang) Co., Ltd., product number 10071293121302.

[0060] Example 5 A kind of fish compound feed

[0061] The fish feed was prepared according to the method of Example 4, except that the added amount was 0.5wt% of the fish basic diet. Specifically, it was as follows:

[0062] Take 5g of epimedium extract, dissolve it in 95% ethanol, then add it to 995g of fish feed, evaporate the ethanol, and you have it.

[0063] Example 6 A kind of fish compound feed

[0064] The fish feed was prepared according to the method of Example 4, except that the added amount was 0.75wt% of the fish basic diet. Specifically, it was as follows:

[0065] Take 7.5g of epimedium extract, dissolve it in 95% ethanol, then add it to 992.5g of fish feed, evaporate the ethanol, and obtain the extract.

[0066] Example 7 A kind of fish compound feed

[0067] The fish feed was prepared according to the method of Example 4, except that the added amount was 1wt% of the basic fish diet. Specifically, it was as follows:

[0068] Take 10g of epimedium extract, dissolve it in 95% ethanol, then add it to 990g of fish feed, evaporate the ethanol, and obtain the extract.

[0069] Example 8 A kind of fish compound feed

[0070] The fish feed was prepared according to the method of Example 4, except that the added amount was 2wt% of the fish basic diet. Specifically, it was as follows:

[0071] Take 20g of epimedium extract, dissolve it in 95% ethanol, then add it to 980g of fish feed, evaporate the ethanol, and obtain the extract.

[0072] Example 9 A kind of fish compound feed

[0073] The fish feed was prepared according to the method of Example 7, except that the suet oil was commercially available from Nantan Comprehensive Farmers' Market in Xichang City, Liangshan Prefecture.

[0074] Comparative Example 1 A fish feed

[0075] The fish feed was prepared according to the method of Example 7, except that:

[0076] (1) Epimedium is not processed;

[0077] (2) Epimedium Herba Epimedium Herba Epimedium Herba Epimedium Herba Epimedium Herba Epimedium Herba Epimedium was purchased from Yizhou Pharmacy in Xichang City, Liangshan Prefecture.

[0078] Comparative Example 2: A fish feed

[0079] The fish feed was prepared according to the method of Example 7, except that:

[0080] (1) The suet oil is commercially available suet oil;

[0081] (2) Epimedium: Commercially available Epimedium medicinal materials were used.

[0082] Comparative Example 3: A fish feed

[0083] The fish feed was prepared according to the method of Example 7, except that the epimedium was commercially available.

[0084] The technical solution of the present invention is further illustrated by experiments below.

[0085] Experimental Example 1 Optimal amount of fish feed additive

[0086] 1. Experimental Methods

[0087] (1) Fish breeding

[0088] The AB strain zebrafish were bred in natural pairs and kept in 28℃ aquaculture water. The light switch was artificially controlled, the day-night ratio was 14:10, and the pH of the aquaculture water was controlled at 6.5-8.5.

[0089] (2) Experimental groups

[0090] The zebrafish were randomly divided into 6 groups, namely, a blank control group, and experimental groups 1 to 5. The blank control group was fed only with a basic diet; the experimental group 1 was fed with the fish compound feed of Example 4; the experimental group 2 was fed with the fish compound feed of Example 5; the experimental group 3 was fed with the fish compound feed of Example 6; the experimental group 4 was fed with the fish compound feed of Example 7; and the experimental group 5 was fed with the fish compound feed of Example 8. There were 30 fish in each group, with a male-female ratio of 1:1.

[0091] (3) Performance testing

[0092] The experimental culture period of zebrafish was 4 weeks. After 4 weeks of culture, the body length, body weight, testis weight and ovary weight of zebrafish were tested.

[0093] 2. Experimental Results

[0094] The preferred results of the addition amount are shown in Table 1: Compared with the blank control group, the length, weight, testis weight and ovary weight of zebrafish in the experimental group were significantly increased, indicating that the fish feed additive of the present invention can improve the growth performance and reproductive organ development of zebrafish.

[0095] The data of experimental groups 1-5 show that as the amount of fish feed additive added increases within the range of 0.2-1wt%, the body length, body weight, testis weight and ovary weight of zebrafish all increase; when the amount added is greater than 1wt%, the body length, body weight, testis weight and ovary weight of zebrafish no longer increase significantly, indicating that the preferred amount of fish feed additive added is 1wt%.

[0096] Table 1 Zebrafish growth and development parameters

[0097]

[0098]

[0099] Note: Compared with the blank control group, *p<0.05, **p<0.01, ***p<0.001.

[0100] The above results show that the fish feed additive of the present invention can improve the growth performance and reproductive organ development of zebrafish. The preferred addition amount of the fish feed additive is 1wt% of the basic fish diet.

[0101] Experimental Example 2 Optimization of Epimedium species and processing technology

[0102] 1. Experimental Methods

[0103] (1) Fish breeding

[0104] The AB strain zebrafish were bred in natural pairs and kept in 28℃ aquaculture water. The light switch was artificially controlled, the day-night ratio was 14:10, and the pH of the aquaculture water was controlled at 6.5-8.5.

[0105] (2) Experimental groups

[0106] The zebrafish were randomly divided into 4 groups, namely experimental group 4, experimental group 6, experimental group 7, experimental group 8, experimental group 9 and blank control group. Experimental group 4 was fed with the fish compound feed of Example 7, experimental group 6 was fed with the fish compound feed of Comparative Example 1, experimental group 7 was fed with the fish compound feed of Comparative Example 2, experimental group 8 was fed with the fish compound feed of Example 9, and experimental group 9 was fed with the fish compound feed of Comparative Example 3. The blank control group was fed with only the basic diet. There were 20 fish in each group, with a male-female ratio of 1:1.

[0107] (3) Performance testing

[0108] The experimental culture period of zebrafish was 4 weeks. After 4 weeks of culture, the body length, body weight, testis weight and ovary weight of zebrafish were tested.

[0109] 2. Experimental Results

[0110] The results of experimental groups 6-9 are shown in Tables 2-5. The growth rate results of each group are shown in Table 6.

[0111] Table 2 Growth and development parameters of zebrafish in experimental group 6

[0112]

[0113]

[0114] Note: Compared with experimental group 4 and experimental group 6, *** p<0.001.

[0115] As shown in Table 2, compared with experimental group 6, the body length, body weight, testis weight, and ovary weight of zebrafish in experimental group 4 were significantly increased. The preparation process and addition amount of experimental group 6 were the same as those of experimental group 4, but commercially available Epimedium medicinal materials were used and were not processed. The comparative study results show that the feed additive made from the selected Epimedium pubescens medicinal materials produced in Liangshan Prefecture, Sichuan Province and processed with Liangshan black sheep suet oil can better improve the growth and development performance of zebrafish.

[0116] Table 3 Growth and development parameters of zebrafish in experimental group 7

[0117]

[0118] Note: Compared with experimental group 4 and experimental group 7, * p<0.05, ** p<0.01, *** p<0.001.

[0119] As shown in Table 3, compared with experimental group 7, the body length, body weight, testis weight, and ovary weight of zebrafish in experimental group 4 were significantly increased. The preparation process and addition amount of experimental group 7 were the same as those of experimental group 4, but the commercially available Epimedium medicinal material was used and processed with commercially available suet oil. The comparative study results show that the feed additive made from the selected pubescent Epimedium medicinal material produced in Liangshan Prefecture, Sichuan Province and processed with Liangshan black sheep suet oil can better improve the growth and development performance of zebrafish.

[0120] Table 4 Growth and development parameters of zebrafish in experimental group 8

[0121]

[0122] Note: Compared with experimental group 4 and experimental group 8, * p<0.05.

[0123] As shown in Table 4, compared with experimental group 8, the body length, body weight, testis weight, and ovary weight of zebrafish in experimental group 4 were significantly increased. The preparation process and addition amount of experimental group 8 were the same as those of experimental group 4, but commercially available suet oil was used for processing. The comparative study results show that the processing technology of selected black sheep suet oil plays an important role in improving the growth and development performance of zebrafish.

[0124] Table 5 Growth and development parameters of zebrafish in experimental group 9

[0125]

[0126] Note: Compared with experimental group 4 and experimental group 9, * p<0.05, ** p<0.01.

[0127] As shown in Table 5, compared with experimental group 9, the body length, body weight, testis weight, and ovary weight of zebrafish in experimental group 4 were significantly increased. The preparation process and addition amount of experimental group 9 were the same as those of experimental group 4, but commercially available Epimedium medicinal materials were used. The comparative study results show that the feed prepared with selected Epimedium pubescens medicinal materials produced in Liangshan Prefecture, Sichuan Province can better improve the growth and development performance of zebrafish; the selection of Epimedium pubescens medicinal materials plays an important role.

[0128] As shown in Table 6, compared with the blank control group, the body length, body weight, testis weight and ovary weight of zebrafish in experimental group 4 increased significantly, among which the body length increased by 14.8%, the body weight increased by 88.7%, the testis weight increased by 140%, and the ovary weight increased by 90.4%. Compared with experimental groups 7, 8, and 9, the growth and development indicators of experimental group 4 also increased significantly, indicating that the feed additive made of selected Epimedium pubescens medicinal materials and processed with Liangshan black sheep suet oil can significantly improve the growth performance and reproductive organ development of zebrafish.

[0129] Table 6 Effects of Epimedium and its processing technology on the growth and development performance of zebrafish

[0130]

[0131]

[0132] Note: The growth rates of each parameter are obtained by comparing with the data of the blank control group in Experimental Example 1.

[0133] The above results show that the feed additive prepared with Epimedium improves the growth and development performance of zebrafish, especially the development of reproductive organs; the processing technology has a certain influence on improving the efficacy of Epimedium; the feed additive made by carefully selecting Epimedium pubescens and processing it with Liangshan black sheep fat can significantly improve the growth and development performance of zebrafish.

[0134] Experimental Example 3 Biosafety of Fish Formula Feed

[0135] 1. Experimental Methods

[0136] Zebrafish were raised according to the method of Experimental Example 1 and cultured in groups. After 4 weeks of culture, the zebrafish in each group were dissected and the ovary, testis, liver and kidney tissues were taken for pathological examination.

[0137] 2. Experimental Results

[0138] Pathological test results such as Figure 1-7 As shown: no obvious pathological changes were found in the ovaries, testes, livers and kidneys of the zebrafish in the experimental groups 1-5; no obvious pathological changes were found in the testes and ovaries of the blank control group. This shows that the fish feed additive and the fish compound feed of the present invention have good biosafety and will not affect the health of zebrafish.

[0139] Through the above embodiments and experimental examples, it can be seen that the present invention screens and compares the amount of fish feed additives, the types of epimedium medicinal materials, and the types of suet oil used for processing. The results show that the feed additive prepared from epimedium improves the performance of zebrafish, especially the degree of development of reproductive organs. The feed additive made from the epimedium medicinal material processed with Liangshan black sheep suet oil can significantly improve the growth and development performance of zebrafish without affecting the health of the fish. The present invention inherits the essence of excellent traditional processing of traditional Chinese medicine, combines modern extraction technology, removes the coarse and retains the fine, obtains high-quality epimedium extract, and strictly controls its quality control standards, so as to achieve the effect of using high-quality epimedium extract alone to make a fish feed additive with good performance, and has broad prospects in aquaculture.

Claims

1. A fish feed additive, characterized in that: The invention is composed of epimedium extract, wherein the epimedium extract contains total flavonoids calculated as icariin ≥ 3.0%, and the epimedium is epimedium pubescens.

2. The fish feed additive according to claim 1, characterized in that: The epimedium extract is prepared by processing and extracting epimedium.

3. The fish feed additive according to claim 2, characterized in that: The processing methods include mutton fat roasting, ghee processing, wine processing, and stir-frying; And / or, the extraction method includes ultrasonic method and heating reflux method.

4. The fish feed additive according to claim 3, characterized in that: The processing method is roasting with mutton fat.

5. The fish feed additive according to claim 4, characterized in that: The suet oil is Liangshan black sheep suet oil.

6. The fish feed additive according to claim 4, characterized in that: The processing method comprises the following steps: frying epimedium and mutton fat at a temperature of 60-100° C. until the surface becomes light yellow and glossy.

7. The fish feed additive according to claim 3, characterized in that: In the extraction method, the extraction solvent is selected from water and 40%-85% ethanol solution; the weight volume ratio of epimedium to the extraction solvent is 1g:10-100mL; the extraction temperature is 20-40°C; the extraction times are 1-3 times; and the extraction time for each time is 15-60min.

8. The method for preparing the fish feed additive according to any one of claims 1 to 7, characterized in that: The herb is prepared through processing and extraction.

9. Use of the fish feed additive according to any one of claims 1 to 7 in preparing aquatic feed.

10. The use according to claim 9, characterized in that: The amount of fish feed additive added is 0.2-2wt% of the basic diet of aquatic feed.

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