阅读说明：本技术 一种抗菌肽饲料添加剂及其制备方法 (Antibacterial peptide feed additive and preparation method thereof ) 是由 梁键钧 胡浩 王婵尤 于 2020-12-14 设计创作，主要内容包括：本发明属于饲料领域,公开了一种抗菌肽饲料添加剂及其制备方法。所述抗菌肽饲料添加剂,包括以下组分：蛙皮素、果蝇抗菌肽、大豆油、增稠剂、表面活性剂、水。所述抗菌肽饲料添加剂的制备方法,包括以下步骤：(1)将蛙皮素、果蝇抗菌肽和增稠剂加入水中得到水相；将大豆油与表面活性剂混合得到油相；(2)将步骤(1)制得的水相加入至油相中,搅拌,制得抗菌肽饲料添加剂。所述抗菌肽饲料添加剂具有很好的稳定性,且抗菌性能优良,尤其适用于作为水产养殖饲料的饲料添加剂。(The invention belongs to the field of feeds, and discloses an antibacterial peptide feed additive and a preparation method thereof. The antibacterial peptide feed additive comprises the following components: bombesin, drosophila melanogaster antibacterial peptide, soybean oil, a thickening agent, a surfactant and water. The preparation method of the antibacterial peptide feed additive comprises the following steps: (1) adding bombesin, drosophila melanogaster antibacterial peptide and thickening agent into water to obtain water phase; mixing soybean oil with a surfactant to obtain an oil phase; (2) and (2) adding the water phase prepared in the step (1) into the oil phase, and stirring to prepare the antibacterial peptide feed additive. The antibacterial peptide feed additive has good stability and excellent antibacterial performance, and is particularly suitable for being used as a feed additive of aquaculture feed.)

1. An antibacterial peptide feed additive is characterized by comprising the following components: bombesin, drosophila melanogaster antibacterial peptide, soybean oil, a thickening agent, a surfactant and water.

2. The antibacterial peptide feed additive according to claim 1, which comprises the following components in parts by weight:

3. the antibacterial peptide feed additive according to claim 1 or 2, wherein the weight ratio of bombesin to drosophila antibacterial peptide is 1: (0.2-0.4).

4. The antimicrobial peptide feed additive of claim 1 or 2, wherein the thickener is selected from at least one of pectin, xanthan gum, carrageenan, sodium carboxymethylcellulose or sodium alginate.

5. The antimicrobial peptide feed additive according to claim 1 or 2, wherein the surfactant is at least one selected from the group consisting of glycerin fatty acid ester, sucrose fatty acid ester, soybean lecithin, acetin, tartaric acid glyceride, citric acid ester, and polyglycerin fatty acid ester.

6. The method for preparing an antibacterial peptide feed additive according to any one of claims 1 to 5, comprising the steps of:

(1) adding bombesin, drosophila melanogaster antibacterial peptide and thickening agent into water to obtain water phase; mixing soybean oil with a surfactant to obtain an oil phase;

(2) and (2) adding the water phase prepared in the step (1) into the oil phase, and stirring to prepare the antibacterial peptide feed additive.

7. The method as claimed in claim 6, wherein the rotation speed of the stirring in step (2) is 500-800r/min, and the stirring time is 25-40 min.

8. A micropterus salmoides feed comprising the antimicrobial peptide feed additive of any one of claims 1 to 5.

9. The micropterus salmoides feed of claim 8, wherein the antibacterial peptide feed additive accounts for 1-2.5% of the mass of the micropterus salmoides feed.

Technical Field

The invention belongs to the field of feeds, and particularly relates to an antibacterial peptide feed additive and a preparation method thereof.

Background

Antimicrobial peptides (anti microbial peptides) are a general term for short peptides having antimicrobial activity. Most of the active peptides have the characteristics of strong basicity, thermal stability, broad-spectrum antibiosis and the like. Different antimicrobial peptides all have significant differences in peptide chain length, sequence composition and biochemical structure, but generally have two common characteristics: firstly, the antibacterial peptide belongs to cationic polypeptide; the second is that the antimicrobial peptide has an amphiphilic structure (hydrophilic and lipophilic). Scientists have discovered and isolated polypeptides with antibacterial activity from bacteria, fungi, amphibians, insects, plants, mammals and humans in sequence. The antibacterial peptide mainly has the functions of resisting bacteria, viruses, fungi, parasites, tumors and immunity.

In the field of aquaculture today, the use of antibiotics in large quantities has brought about a series of adverse effects, and therefore the use of antimicrobial peptides as antibiotic substitutes is becoming a hot spot pursued in the industry. Research on replacement of antibiotics by antibacterial peptides has been developed in recent years, and although some progress has been made, the research is still in the exploration stage, and many problems to be solved are still existed in the research stage from the technical maturity, the large-scale production and the application to animal breeding. Firstly, the antibacterial peptide has various types, which have good effect when being used as a feed additive and are more economical. The second is the interaction condition between different types of antibacterial peptides. Thirdly, the content of the antibacterial peptide in animal bodies is less, natural resources are deficient, the cost of chemical synthesis is too high, and the large-scale production cannot be realized at present; it is necessary to study how to produce antibacterial peptides on a large scale from the aspect of genetic engineering techniques. Fourthly, the antibacterial peptide in the animal body is easy to be hydrolyzed by protease, is not stable enough and is difficult to reach the intestinal tract and be absorbed by the epithelial cells of the intestinal tract.

In view of the difficulties faced by the above-mentioned antibacterial peptides in the field of feed, it is desirable to provide an antibacterial peptide feed additive which is stable and has excellent antibacterial properties.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides an antibacterial peptide feed additive and a preparation method thereof, wherein the antibacterial peptide feed additive has good stability and excellent antibacterial performance, and is particularly suitable for being used as a feed additive of aquaculture feed.

An antibacterial peptide feed additive comprises the following components: bombesin, drosophila melanogaster antibacterial peptide, soybean oil, a thickening agent, a surfactant and water.

In the previous experiments, the bombesin, which is an antibacterial peptide commonly used for resisting parasites and tumors, is found to have better antibacterial and antifungal effects when being applied to the culture of aquatic animals, particularly perca salmoides. And the bombesin and the drosophila antimicrobial peptide are matched for use, so that the antibacterial effect better than that of a single antimicrobial peptide can be achieved, the disease occurrence can be greatly reduced when the Micropterus salmoides are cultured without using antibiotics, and the survival rate is improved. Meanwhile, in consideration of the situation that the stability of the antibacterial peptide is poor due to the fact that the antibacterial peptide is easily degraded by enzymes in the digestive tract, the invention carries out research on the aspect of effective coating of the antibacterial peptide, and the antibacterial peptide is coated by adopting the soybean oil, the thickening agent and the surfactant, so that the stability of the antibacterial peptide in the feed and the digestive tract of animals is improved.

Preferably, the antibacterial peptide feed additive comprises the following components in parts by weight:

more preferably, the weight ratio of bombesin to drosophila antibacterial peptide is 1: (0.2-0.4). Experiments show that when the ranpirtine and the drosophila melanogaster antibacterial peptide are in the weight ratio, a better antibacterial effect can be achieved.

Preferably, the thickening agent is selected from at least one of pectin, xanthan gum, carrageenan, sodium carboxymethylcellulose or sodium alginate.

Preferably, the surfactant is at least one selected from the group consisting of glycerin fatty acid ester, sucrose fatty acid ester, acetin, tartaric acid ester, citric acid ester and polyglycerin fatty acid ester.

In order to ensure the safety of the antibacterial peptide feed additive, the selected thickening agent and the surfactant are nontoxic edible components.

The preparation method of the antibacterial peptide feed additive comprises the following steps:

(1) adding bombesin, drosophila melanogaster antibacterial peptide and thickening agent into water to obtain water phase; mixing soybean oil with a surfactant to obtain an oil phase;

(2) and (2) adding the water phase prepared in the step (1) into the oil phase, and stirring to prepare the antibacterial peptide feed additive.

The antibacterial peptide feed additive prepared by the method is of a water-in-oil structure, and can play a certain bonding role when added into aquatic feed, so that the antibacterial peptide is not easy to dissolve in aquaculture water due to the fact that the aquatic feed is broken.

Preferably, the rotation speed of the stirring in the step (2) is 500-800r/min, and the stirring time is 25-40 min.

A micropterus salmoides feed comprises the antibacterial peptide feed additive.

Preferably, the antibacterial peptide feed additive accounts for 1-2.5% of the mass of the micropterus salmoides feed.

Compared with the prior art, the invention has the following beneficial effects:

the invention finds that the antibacterial peptide feed additive prepared by matching two antibacterial peptides, namely bombesin and drosophila melanogaster antibacterial peptide, can effectively enhance the immunity of aquatic animals, particularly micropterus salmoides, and greatly reduce the occurrence of diseases. The antibacterial peptide feed additive can be added into feed to basically replace antibiotics, so that the harm caused by abuse of antibiotics is effectively controlled.

Meanwhile, the research results of the project show that the soybean oil is adopted, and the proper amount of the thickening agent and the surfactant are added, so that the antibacterial peptide can be well wrapped, the degradation of digestive enzymes in an animal body to the antibacterial peptide is reduced, and the antibacterial peptide can fully exert the effect. Therefore, the related research of the department on the packaging technology of the antibacterial peptide provides a certain reference for the application of the antibacterial peptide in the field of aquatic feeds.

Detailed Description

In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples are given for illustration. It should be noted that the following examples are not intended to limit the scope of the claimed invention.

The starting materials, reagents or apparatuses used in the following examples are conventionally commercially available or can be obtained by conventionally known methods, unless otherwise specified.

Example 1

The embodiment provides an antibacterial peptide feed additive which comprises the following components in parts by weight: 2 parts of bombesin, 1 part of drosophila antibacterial peptide, 3 parts of soybean oil, 2 parts of thickening agent (sodium carboxymethylcellulose), 6 parts of surfactant (glycerin fatty acid ester) and 8 parts of water.

The preparation method of the antibacterial peptide feed additive in the embodiment specifically comprises the following steps:

(1) adding bombesin, drosophila melanogaster antibacterial peptide and thickening agent into water to obtain water phase; mixing soybean oil with a surfactant to obtain an oil phase;

(2) and (2) adding the water phase prepared in the step (1) into the oil phase, and stirring at the speed of 800r/min for 25min to prepare the antibacterial peptide feed additive.

Example 2

The embodiment provides an antibacterial peptide feed additive which comprises the following components in parts by weight: 3 parts of bombesin, 0.5 part of drosophila antibacterial peptide, 2 parts of soybean oil, 2 parts of thickener (sodium alginate), 6 parts of surfactant (citrate) and 10 parts of water.

The preparation method of the antibacterial peptide feed additive in the embodiment specifically comprises the following steps:

(1) adding bombesin, drosophila melanogaster antibacterial peptide and thickening agent into water to obtain water phase; mixing soybean oil with a surfactant to obtain an oil phase;

(2) and (2) adding the water phase prepared in the step (1) into the oil phase, and stirring at the speed of 500r/min for 40min to prepare the antibacterial peptide feed additive.

Example 3

The embodiment provides an antibacterial peptide feed additive which comprises the following components in parts by weight: 4 parts of bombesin, 1 part of drosophila antibacterial peptide, 4 parts of soybean oil, 3 parts of a thickening agent (carrageenan), 6 parts of a surfactant (tartaric acid glyceride) and 14 parts of water.

The preparation method of the antibacterial peptide feed additive in the embodiment specifically comprises the following steps:

(1) adding bombesin, drosophila melanogaster antibacterial peptide and thickening agent into water to obtain water phase; mixing soybean oil with a surfactant to obtain an oil phase;

(2) and (2) adding the water phase prepared in the step (1) into the oil phase, and stirring at the speed of 600r/min for 30min to prepare the antibacterial peptide feed additive.

Comparative example 1

The comparative example provides an antibacterial peptide feed additive which comprises the following components in parts by weight: 4 parts of bombesin and 1 part of drosophila melanogaster antibacterial peptide.

The preparation method of the antibacterial peptide feed additive in the comparative example specifically comprises the following steps: mixing bombesin and drosophila melanogaster antibacterial peptide to obtain antibacterial peptide feed additive.

Comparative example 2

The comparative example provides an antibacterial peptide feed additive which comprises the following components in parts by weight: 5 parts of bombesin, 4 parts of soybean oil, 3 parts of a thickening agent (carrageenan), 6 parts of a surfactant (tartaric acid glyceride) and 14 parts of water.

The preparation method of the antibacterial peptide feed additive in the comparative example specifically comprises the following steps:

(1) adding bombesin and thickener into water to obtain water phase; mixing soybean oil with a surfactant to obtain an oil phase;

(2) and (2) adding the water phase prepared in the step (1) into the oil phase, and stirring at the speed of 600r/min for 30min to prepare the antibacterial peptide feed additive.

Comparative example 3

The comparative example provides an antibacterial peptide feed additive which comprises the following components in parts by weight: 5 parts of drosophila antibacterial peptide, 4 parts of soybean oil, 3 parts of thickening agent (carrageenan), 6 parts of surfactant (tartaric acid glyceride) and 14 parts of water.

The preparation method of the antibacterial peptide feed additive in the comparative example specifically comprises the following steps:

(1) adding the drosophila melanogaster antibacterial peptide and the thickening agent into water to obtain a water phase; mixing soybean oil with a surfactant to obtain an oil phase;

(2) and (2) adding the water phase prepared in the step (1) into the oil phase, and stirring at the speed of 600r/min for 30min to prepare the antibacterial peptide feed additive.

Product effectiveness testing

The data show that the protease activity in intestinal tracts of the micropterus salmoides is highest, and the optimal pH value of the intestinal protease is about 8. Therefore, the alkaline protease is selected to replace intestinal protease of the Micropterus salmoides to carry out in-vitro proteolysis test, and the hydrolysis Degree (DH) refers to the percentage of peptide bonds hydrolyzed in protein to the total peptide bonds. Wherein the concentration of the alkaline protease is 3%, the hydrolysis temperature is 50 ℃, the hydrolysis time is 6 hours, and the pH value is controlled to be 8-8.5. The degree of proteolysis of the antimicrobial peptide feed additives of examples 1-3 and comparative example 3 was determined by the OPA (o-phthalaldehyde) method, which is based on the principle that o-phthalaldehyde forms a yellow compound with the free α -amino group in the presence of β -mercaptoethanol, which has a characteristic absorption at 340nm and its OD value can be detected by a spectrophotometer, after hydrolysis was complete, and the specific data for the degree of hydrolysis are shown in table 1:

TABLE 1 hydrolysis degree of antimicrobial peptide feed additives

Example 1

Example 2

Example 3

Comparative example 1

Comparative example 2

Comparative example 3

Degree of hydrolysis

13.4％

12.7％

12.2％

32.5％

15.6％

14.3％

As can be seen from Table 1, the hydrolysis degree of the antibacterial peptide feed additive in the comparative example 1 is far greater than that of the antibacterial peptide feed additives in the examples 1-3 and the comparative examples 2-3, which shows that the antibacterial peptide is effectively wrapped by adding the soybean oil, the thickening agent and the surfactant on the basis of the antibacterial peptide, the hydrolysis of protease on the antibacterial peptide can be reduced, and the stability of the antibacterial peptide is improved.

The antibacterial peptide feed additives prepared in the examples 1 to 3 and the comparative examples 1 to 3 are respectively added into basic weever feed formulas (25% of bean cake, 10% of chicken meal, 25% of fish meal, 15% of bran, 15% of flour, 6% of fish oil, 2% of compound vitamin, 1% of inorganic salt and 1% of feed adhesive), so that 1.5% of the antibacterial peptide feed additive is obtained.

The prepared micropterus salmoides feed is used for carrying out a growth culture test on micropterus salmoides, a circulating water culture system is used for carrying out the growth culture test, 600 commercially available micropterus salmoides with basically the same growth condition are randomly divided into 6 groups, and each group of 100 fish is subjected to the growth culture test under the environment of the same temperature and pH value and under the condition of not using any antibiotic for 60 days; the feeding method and the culture condition are both conventional methods and conditions.

After the culture test is finished, the survival rate of the micropterus salmoides, the incidence rate of diseases of liver, gallbladder and intestinal tract and the activity of superoxide dismutase (SOD) in the liver are measured, and the test results are shown in Table 2:

TABLE 2 survival rate, disease incidence and liver superoxide dismutase (SOD) activity of Micropterus salmoides

From table 2 it can be derived: the survival rate, the disease incidence rate and the trends shown by three indexes of superoxide dismutase and the like are basically consistent. Namely, the effect of the examples 1 to 3 is obviously stronger than that of the comparative examples 1 to 3; example 3 is superior to examples 1-2 in effect; comparative example 2 is superior to comparative example 3 in effect, and comparative example 3 is superior to comparative example 1 in effect.

Although all of examples 1 to 3 employ the drosophila melanogaster antibacterial peptide and bombesin in combination, the weight ratio of bombesin to drosophila melanogaster antibacterial peptide in example 3 is a more preferable ratio than that in examples 1 to 2, and thus better antibacterial and immunity-enhancing effects can be achieved.

As can be seen by comparing comparative example 2 with comparative example 3, although the parts by weight of the antimicrobial peptides used in both were kept the same (5 parts each), the effect obtained in comparative example 2 was better than that obtained in comparative example 3, indicating that the application of bombesin to the cultivation of Micropterus salmoides exhibited better antibacterial and antifungal effects than the drosophila antimicrobial peptides. Comparison of comparative examples 2-3 with examples 1-3 shows that the use of drosophila antimicrobial peptides in combination with bombesin provides significantly better results than the use of drosophila antimicrobial peptides alone.

Through the analysis of the comparative example 1, the bombesin and the drosophila antimicrobial peptide of the comparative example 1 are not coated, so that the stability is poor, the hydrolysis degree of the bombesin and the drosophila antimicrobial peptide after being ingested by the micropterus salmoides is higher, the antibacterial performance is poor, and the immunity of the fed micropterus salmoides cannot be effectively enhanced.