Application of perilla protein in preparation of feed for improving immunity of Basa fish

Abstract

The invention provides an application of perilla protein in preparation of a feed for improving the immunity of Basa fish. According to the application, the perilla protein subjected to enzymolysis by papain or trypsin or the immune peptide in the perilla protein can effectively improve the immunity of the basa fish and resist infection of parasites such as trichodina and the like.

Description

Field of the Invention

[0001] The present application belongs to the field of feed and protein. Specifically, the present application provides the application of perilla protein in the preparation of feed for improving the immunity of basa fish. Background Art

[0002] Basa fish belongs to the order Siluriformes and is native to the Mekong River in Vietnam. The fish has a large yield, delicious meat and no intramuscular spines. It is an excellent freshwater edible fish. Basa fish is the largest export variety of Vietnamese aquatic products and is also farmed in many southern provinces of my country. Basa fish has low requirements for water quality and oxygen, and has a strong resistance to bacterial infection. In actual farming, the most serious harm to it is the parasites of the gills or skin, such as wheelworms and ringworms.

[0003] Perilla is a herbaceous plant of the genus Perilla in the family Lamiaceae, mainly distributed in East Asia and South Asia. Perilla has a long history of medicinal and edible use, and can be used to prepare oils and spices. In traditional Chinese medicine, it is believed to have the effects of dispelling cold, promoting qi and relieving fullness, and calming the fetus. Modern medical research has found that perilla and many of its ingredients have antibacterial and anti-inflammatory, anti-tumor, and analgesic effects.

[0004] The most common use of perilla is oil extraction. The perilla meal left after oil extraction contains a large amount of perilla protein, which is currently mainly used as feed and fertilizer, or even abandoned. Based on the known medicinal uses of perilla, perilla protein can be deeply processed and its functions explored to develop feed or medicinal ingredients. Summary of the invention

[0005] On the one hand, the present application provides the use of perilla protein in preparing feed for improving the immunity of Basa fish.

[0006] On the other hand, the present application provides the use of perilla protein in the preparation of a drug for improving the immunity of Basa fish.

[0007] On the other hand, the present application provides the use of Perilla protein in the preparation of a drug for preventing parasitic infection.

[0008] Furthermore, the parasite is a wheelworm.

[0009] Furthermore, the perilla protein is perilla protein enzymatically hydrolyzed by papain or trypsin.

[0010] Furthermore, the preparation method of the perilla protein hydrolyzed by papain or trypsin is: using water as a solvent to prepare an enzymatic hydrolysis composition containing 100 mg / mL perilla protein, 500 U / mL papain or trypsin, and pH 7-7.5; enzymatically hydrolyzing the enzymatic hydrolysis composition at 45-50° C. for 4-5 hours.

[0011] Furthermore, the preparation method of the perilla protein is: crush the defatted perilla meal and add it into water with a mass 20 times that of the defatted perilla meal, mix; adjust the pH to 10; treat in a 60-70°C water bath for 1 hour; centrifuge to obtain the supernatant; adjust the pH of the supernatant to 4.5; centrifuge to obtain the precipitate; wash the precipitate with water and then freeze-dry to obtain the perilla protein.

[0012] Furthermore, the perilla protein is a perilla protein immune peptide with a sequence of SEQ ID NO.1 or SEQ ID NO.2.

[0013] Furthermore, the feed contains compound feed.

[0014] The compound feed can be various commercially available compound feeds or homemade compound feeds, and can be in the form of powder, granules, etc.

[0015] Furthermore, the medicine contains excipients acceptable in veterinary medicine.

[0016] Those skilled in the art can determine the specific types of acceptable excipients in the veterinary drugs based on veterinary medicine books and relevant regulatory provisions for veterinary drugs and poultry products, including but not limited to fillers, dispersants, adhesives, lubricants, flavoring agents, coating agents, solvents, co-solvents, pH adjusters, osmotic pressure regulators, etc.

[0017] The present application also provides a feed for pangasius, which contains the above-mentioned perilla protein.

[0018] The present application also provides a drug for improving the immunity of pangasius and resisting parasitic infection, wherein the drug comprises the above-mentioned perilla protein.

[0019] Perilla is a food raw material, and the safety of its enzymatic hydrolysis products after being hydrolyzed by food enzymes is relatively guaranteed. The enzymatic hydrolysates and immune peptides of the present application provide a new type of feed additive and drug option for Basa fish farming. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is the mass spectrum of the immune peptide ZS-6;

[0021] Figure 2 is the mass spectrum of the immune peptide ZS-7;

[0022] Figure 3 This study investigates the effects of feeding pangasius with immune peptides and enzymatic hydrolysates on the nonspecific immunity (complement C3, IgM) of the fish. DETAILED DESCRIPTION

[0023] Example 1 Preparation of Perilla Protein Hydrolysate

[0024] Perilla protein was produced using defatted perilla meal from a certain oil production enterprise as raw material. The defatted perilla meal was crushed and added into water with a mass of 20 times that of the defatted perilla meal, mixed; the pH was adjusted to 10; the mixture was treated in a 60-70℃ water bath for 1 hour; the supernatant was centrifuged; the pH of the supernatant was adjusted to 4.5; the precipitate was centrifuged; the precipitate was washed with water and freeze-dried to obtain perilla protein.

[0025] The perilla protein obtained in Example 1 was enzymatically hydrolyzed using papain, bromelain, ficin, and trypsin (Xiasheng, food grade). The enzymatic hydrolyzate was further concentrated and freeze-dried, and the specific conditions were as follows:

[0026] Table 1 Enzymatic hydrolysis conditions

[0027]

[0028] Example 2 Basa fish breeding experiment

[0029] Experimental location and conditions:

[0030] The experimental site was located in a farm in the suburbs of Nanning City, Guangxi Zhuang Autonomous Region. The experimental time was from June to July. The breeding pond was a fully disinfected concrete pool, and the breeding water was groundwater that had been dried and aerated.

[0031] Experimental fish:

[0032] Basa fish raised in indoor breeding glass ponds were used as experimental subjects to ensure that there was basically no interference from parasites; Basa fish with obvious wheel worm infection on the gills and skin that were manually identified from seriously infected fish ponds were selected as diseased fish; the specifications of the Basa fish used were all 1kg±100g, which was close to the commercial specifications.

[0033] Feeding plan:

[0034] 85 healthy basa fish were stocked in each 20 square meter breeding pond at a density lower than the conventional breeding density, using the commercially available Yihai brand basa fish compound feed (added with 0.1% by weight of perilla protein hydrolysate), and following the conventional method of feeding (daily feeding amount 2%), management and regulation conditions, but without the use of antiparasitic drugs such as copper sulfate and avermectin. See Table 2 for details:

[0035] Table 2 Experimental conditions of each culture pond

[0036]

[0037] After 60 days of breeding, the number of deaths of basa fish in each pond was counted, and the infection status of basa fish was manually inspected one by one (manual inspection of the gills and skin for more than 3 white spots infected by parasites, falling scales, and bleeding was considered infected).

[0038] The results showed that due to sufficient disinfection before the experiment and low stocking density, all the basa fish in the control pond 1 survived and had no obvious symptoms of parasitic infection. Therefore, the infection in other fish ponds can basically be considered to be caused by wheelworms; 32 basa fish died in the control pond 2 where infected fish were introduced, and the infection rate of the surviving fish reached 81.1%; the situation in ponds 4 and 5 was similar to that in pond 2; the situation in ponds 3 and 6 was significantly better, with only 11 and 8 deaths respectively, and the disease rate of the remaining fish was less than 15%.

[0039] Table 3 Culture experiment results

[0040]

[0041]

[0042] Therefore, the use of papain hydrolysate and trypsin hydrolysate of perilla protein in feed can increase the immunity of Basa fish and improve its disease resistance to parasites.

[0043] From the subsequent practical use, it can be seen that papain hydrolysate and trypsin hydrolysate of perilla protein also have good inhibitory effects on Dactylopidae.

[0044] Example 3 Identification of Immunity Peptides

[0045] In order to clarify the effect and obtain products with better feeding and medicinal value, the applicant tried to identify functional peptides in papain hydrolysates and trypsin hydrolysates of perilla protein.

[0046] The hydrolysate was centrifuged using a 3kDa ultrafiltration tube and desalted by column. The LC-MS / MS method was used for separation and identification: the liquid phase was Shimadzu LC-20AD (self-assembled C18 column 75μm×25cm; mobile phase 1: 2% ACN, 0.2% formic acid, mobile phase 2: 98% CAN, 0.2% formic acid; column flow rate was 300nL / min, column temperature was 40°C), directly connected to a mass spectrometer (Thermo Fisher MALDI-TOP, primary scanning range: 200-1800m / z, AGC target: 3e 6 , resolution: 70000, maximum injection time: 50ms):

[0047] The mass spectrometry results were analyzed using MaxQuant 1.5.3.30 and identified using Mascot 2.3.02. The Uniprot database was analyzed and two immunoreactive peptides were obtained from the papain hydrolysate of perilla protein and the trypsin hydrolysate of perilla protein.

[0048] Due to the predicted toxicity and poor synthesis difficulty, two active peptides were removed from the papain hydrolysate. The two active peptides from the perilla protein trypsin hydrolysate were commissioned to NovoPro (Shanghai Newpro) for solid phase resin (Fmoc) synthesis. The synthetic mass spectrum is shown in Figure 1 and Figure 2 shown.

[0049] SEQ ID NO.1: IVAWSP;

[0050] SEQ ID NO. 2: FGLVQPG.

[0051] Example 4: Verification of the Effect of Immune Peptides

[0052] Four experimental pools were set up, and 10 sub-adult basa fish weighing 500 g ± 100 g were stocked in each indoor breeding glass pool (2 m × 1.5 m × 0.7 m). Commercially available Yihai brand basa fish compound feed (without other ingredients, 0.005% by weight of two synthetic peptides, and 0.1% by weight of perilla protein trypsin hydrolysate) was used, and the fish were fed (daily feeding amount 2%), managed, and regulated according to the conventional adult fish method.

[0053] Table 2 Experimental conditions of each culture pond

[0054]

[0055] After 30 days of feeding, none of the four groups of fish died. After killing the fish, the intestinal samples of each group of fish were taken (surface samples are greatly affected by the environment, so intestinal tissue with a large number of epithelial cells and representing the immune ability of the digestive system was used), mixed, and 10 times the mass of PBS (pH 7.0) was added to break the homogenate and centrifuged to obtain the supernatant. The protein concentration was detected using Tian Enze's BAC protein quantification kit, and the IgM and complement 3 detection kit (turbidimetric method) of Klamar was used to detect the content of IgM and complement 3 (average of 3 samples).

[0056] The results are as follows Figure 3 The results show that adding one twentieth of the weight of the enzymatic hydrolysate ZS-6 and ZS-7 to the feed can achieve a more significant effect of enhancing the nonspecific immunity of fish than the enzymatic hydrolysate. After designing a suitable genetic engineering bacteria for mass expression, it is expected to be used as a feed or drug ingredient.

Claims

1. Application of perilla protein in the preparation of feed for improving the immunity of Basa fish.

2. Application of perilla protein in the preparation of drugs for improving the immunity of Basa fish.

3. Application of perilla protein in the preparation of drugs for preventing parasitic infections.

4. The use according to claim 3, wherein the parasite is Trichodina.

5. The use according to any one of claims 1 to 4, wherein the perilla protein is perilla protein enzymatically hydrolyzed by papain or trypsin.

6. The use according to claim 5, wherein the preparation method of the perilla protein hydrolyzed by papain or trypsin is: using water as a solvent to prepare an enzymatic hydrolysis composition containing 100 mg / mL perilla protein, 500 U / mL papain or trypsin, and pH 7-7.5; and enzymatically hydrolyzing the enzymatic hydrolysis composition at 45-50°C for 4-5 hours.

7. The use according to claim 5 or 6, wherein the preparation method of perilla protein is: crush the defatted perilla meal and add it into water with a mass 20 times that of the defatted perilla meal, mix; adjust the pH to 10; treat in a 60-70°C water bath for 1 hour; centrifuge to obtain the supernatant; adjust the pH of the supernatant to 4.5; centrifuge to obtain the precipitate; wash the precipitate with water and then freeze-dry to obtain the perilla protein.

8. The use according to any one of claims 1 to 4, wherein the perilla protein is a perilla protein immune peptide with a sequence of SEQ ID NO.1 or SEQ ID NO.

2.

9. The use according to any one of claims 1, 5-8, wherein the feed comprises compound feed.

10. The use according to any one of claims 2 to 8, wherein the medicine contains excipients acceptable in veterinary drugs.

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