Mineral composite peptide as well as preparation and application thereof

Abstract

The invention belongs to the field of environmental protection technology and aquaculture, and particularly relates to a mineral composite peptide as well as preparation and application thereof. The preparation method comprises the following steps: mixing polylysine and cyclic dipeptide according to a mass ratio of (1-10): 1, dissolving in water, then adding montmorillonite, stirring to promote a polylysine cyclic dipeptide compound to be intercalated into montmorillonite mineral interlayers to form mineral composite peptides, collecting the mineral composite peptides, freeze-drying, and then mixing with an auxiliary agent to prepare the resistance-reducing feed special for aquatic products. According to the mineral composite peptide and the special feed for the aquatic animals, broad-spectrum antibacterial polylysine and cyclic dipeptide antibacterial peptide with bacteriostasis and bacterial quorum sensing inhibition functions are subjected to advantage combination, so that the protective effect of a pathogenic bacteria biological membrane on pathogenic bacteria is weakened while bacteriostasis and sterilization are realized; the feed has the function of controlling peculiar smell substances in breeding feed and a breeding system while reducing resistance and replacing resistance, and can be used as antibiotic reduction feed in the breeding industry, and the daily feeding amount is 1-10% of the body weight of bred aquatic animals.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental protection and aquaculture, and specifically relates to a mineral composite peptide and its preparation and application in the preparation of an antibiotic substitute and functional feed in aquaculture. Background Art

[0002] The widespread use of antibiotics in aquaculture has effectively controlled the outbreak of various diseases in aquatic animals and greatly promoted the development of aquaculture. However, the risk of abuse of antibiotics is becoming increasingly serious. The long-term and large-scale use of antibiotics has led to bacterial resistance and even multi-drug resistant bacteria, which has seriously threatened ecological security and human health. However, a complete ban on the use of antibiotics in aquaculture will endanger the safety of aquatic animals. Therefore, finding a suitable method to reduce or replace the use of antibiotics in aquaculture is an urgent problem to be solved.

[0003] Antimicrobial peptides are small molecule polypeptide substances encoded by genes and synthesized by ribosomes. They are generally composed of 10 to 50 amino acids and have a small molecular weight. So far, more than a thousand antimicrobial peptides have been isolated and identified from various organisms such as insects, amphibians, and mammals. These antimicrobial peptides not only have broad-spectrum antibacterial activity, but also have antifungal, antiviral and antiparasitic activities. Antimicrobial peptides have low immunogenicity and have the advantages of no residual toxicity and low resistance to drug resistance, making them a new type of antimicrobial drug with great potential. At present, the antimicrobial activity of antimicrobial peptides has attracted widespread attention, and its research and application have become a hot spot in the field of biopharmaceuticals.

[0004] Studies have found that bacteria can communicate with each other by secreting, identifying, and sensing one or more signal molecules. This phenomenon is called quorum sensing. Bacterial quorum sensing is involved in the regulation of many biological functions, including the expression of pathogenic factors, the formation of biofilms, and bacterial drug resistance. Bacterial quorum sensing inhibitors are a class of substances that can inhibit quorum sensing of a variety of microorganisms (including the formation of pathogenic biofilms).

[0005] Antimicrobial peptides can be added to feed as additives. After searching the existing technical literature, it was found that in the article published by Li Bo et al. in "Chinese Journal of Animal Husbandry and Veterinary Medicine", 2014, 41 (7): 99-103, adding cecropin antimicrobial peptide to the diet can improve the growth performance of weaned piglets, reduce feed-to-weight ratio and diarrhea rate, promote the body to produce non-specific immunity, and have no adverse effects on the growth indicators of piglets. In the article published by Xiao Fayi et al. in "Animal Health", 2019, 55 (1): 124-126, it was found that adding 1.0g / kg of antimicrobial peptide (polypeptide mycotoxin S100) to the basic feed of weaned piglets increased the average daily weight gain of piglets in the experimental group by 11.31% and reduced the diarrhea rate by 69.28% compared with the control group. However, so far, the stability of antimicrobial peptides added to feed, especially thermal stability, has not been well solved, and there has been no report on the functional combination of antimicrobial peptides with bacterial quorum sensing inhibitors and the use of such feed additives to replace antibiotics in aquaculture. Summary of the invention

[0006] The purpose of the present invention is to provide a mineral composite peptide and its preparation and application in the preparation of antibiotic substitute and functional feed in aquaculture industry in response to the current problems of abuse of antibiotics and frequent aquatic diseases.

[0007] To achieve the above object, the technical solution adopted by the present invention is: A mineral composite peptide is obtained by inserting polylysine and diketopiperazine cyclic dipeptide into modified montmorillonite, wherein the mass ratio of polylysine to diketopiperazine cyclic dipeptide is 1-10:1.

[0008] The diketopiperazine cyclic dipeptide is one or more of Cyclo(Pro-Phe), Cyclo(Pro-Tyr), Cyclo(Pro-Leu), Cyclo(Trp-Ser), Cyclo(Pro-Gly), Cyclo(Pro-His), Cyclo(Pro-Val), Cyclo(Tyr-Gly) and Cyclo(Ala-Val).

[0009] A method for preparing the mineral composite peptide comprises mixing polylysine and diketopiperazine cyclic dipeptide in a mass ratio of 1-10:1 and dissolving them in water, then adding modified montmorillonite, and stirring to promote the intercalation of polylysine and diketopiperazine cyclic dipeptide into the interlayer of montmorillonite mineral to form the mineral composite peptide.

[0010] The steps for preparing the mineral complex peptide are as follows: 1) Montmorillonite purification: Dissolve natural montmorillonite in deionized water, stir and mix, then centrifuge and collect the precipitate, dry and sieve; 2) Sodiumization of montmorillonite: The purified natural montmorillonite and Na2CO3 solid are mixed in a mass ratio of 5-10:1 and placed in deionized water, heated and stirred at 60-90°C for 3 hours, centrifuged to collect the precipitate, washed, dried, and sieved to obtain sodium montmorillonite for standby use; 3) Montmorillonite heat treatment: The sodium montmorillonite obtained in 2) is heat treated at 110-600° C. for 1-5 h, and then cooled to room temperature to obtain heat-activated montmorillonite powder; 4) Montmorillonite intercalation drug loading: The heat-activated montmorillonite powder is dispersed in an aqueous solution of 20-200 g / L polylysine and diketopiperazine cyclic dipeptide at an addition amount of 1-5% mass volume ratio (g / ml), and ultrasonic treatment is performed for 10-60 minutes until it is evenly dispersed. The suspension is then stirred for two hours to promote the insertion of polylysine and diketopiperazine cyclic dipeptide into the montmorillonite interlayer, and the complex is obtained by centrifugation, and the mineral composite peptide is obtained by washing and freeze-drying.

[0011] In the step 4), the mass ratio of polylysine to diketopiperazine cyclic dipeptide is 1-10:1.

[0012] An application of the mineral composite peptide, the mineral composite peptide is used as an antibacterial additive for aquatic animal feed.

[0013] A special feed for aquatic animals containing mineral composite peptides, wherein the mineral composite peptides are added to the feed, and the added amount thereof is 0.1-1% of the mass of the feed premix.

[0014] The feed premix comprises, by weight percentage, 30%-54% fish meal, 4%-35% soybean meal, 1%-12% shrimp meal, 5% corn meal, 1%-5% high-gluten flour, 5%-10% α-starch, 5%-7% lard, 2%-4% rapeseed oil, 2%-3% yeast powder, 1.2%-2.5% gluten powder, 0.5%-2% milk powder, 0.5%-1% egg yolk powder, 0.5%-1% complex minerals, and 0.3%-0.5% complex vitamins.

[0015] The invention discloses a preparation method for a special feed for aquatic animals containing mineral complex peptides. The mineral complex peptides are mixed with feed premix components according to the above-mentioned proportions, and after mixing, the mixture is subjected to high-temperature puffing and granulation to obtain the special feed for aquaculture.

[0016] In the high-temperature puffing and granulation steps, the moisture content of the feed is maintained at 5%-10%, the feed raw material is crushed to a particle size of 60-100 meshes, the screw speed of the puffing machine is 300-500 rpm, the puffing temperature is 120° C.-150° C., and the puffing degree is 1.8-2.5.

[0017] An application of the special feed for aquatic animals containing mineral composite peptides, wherein the feed is used as antibiotic-reduced feed in aquaculture.

[0018] The daily feeding amount of the feed is 1-10% of the body weight of the farmed aquatic animals.

[0019] The advantages of the present invention are: 1) The present invention adds antimicrobial peptides to minerals through intercalation to form mineral composite peptides, which can effectively avoid the technical difficulties of loss and inactivation of antimicrobial peptides in the preparation process, especially in the heating and puffing process. It can also effectively avoid the poor stability and easy diffusion and loss of antimicrobial peptides when used in aquaculture environments, thus overcoming many industrial problems.

[0020] 2) The present invention combines broad-spectrum antibacterial polylysine and cyclic dipeptide antibacterial peptides with antibacterial and bacterial quorum sensing inhibition functions to form a composite peptide, which can complement each other's advantages, while inhibiting and killing bacteria, weakening the protective effect of pathogen biofilm on pathogens, and further strengthening the killing effect of pathogens. And related additives are used as raw materials to prepare reduced-volume preparations to reduce the use of antibiotics in the aquaculture industry.

[0021] 3) The mineral composite peptides and the doped feeds of the present invention not only reduce antibiotics but also have the function of controlling odor substances. The addition of mineral composite peptides strengthens the adsorption and removal of odor substances in aquaculture feed and aquaculture systems. At the same time, the presence of mineral composite peptides is conducive to the full use of heating and puffing in the feed preparation process, which can further strengthen the removal of odor substances in aquaculture feed and reduce the accumulation of odor substances in aquaculture systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The infrared spectrum of the mineral composite peptide provided by the embodiment of the present invention includes polylysine cyclodipeptide complex (EPL), natural montmorillonite (MMT-Ca), sodium-modified montmorillonite (MMT-Na), thermally modified montmorillonite (mMMT) and montmorillonite mineral composite peptide (EPL-mMMT).

[0023] Figure 2 A diagram showing the bactericidal effect of the mineral composite peptide provided in an embodiment of the present invention; wherein, a diagram showing the antibacterial effect of the mineral composite peptide on Escherichia coli (a, b, c) and Staphylococcus aureus (d, e, f); the treatments from left to right are: blank control; montmorillonite; montmorillonite mineral composite peptide.

[0024] Figure 3 Transmission electron micrographs of the products provided in the examples of the present invention; wherein, a, b, c: natural montmorillonite; d, e, f: sodium-based montmorillonite; g, h, i: thermally modified montmorillonite; j, k, l: mineral composite peptides. DETAILED DESCRIPTION

[0025] The present invention is further described below by way of examples, however the present invention is not limited to the following examples.

[0026] The natural montmorillonite in the following examples was commercially available.

[0027] In the following embodiments, the composite minerals are composite mineral powders prepared from defatted rice bran as a matrix, with a ratio of 12 g / kg calcium, 8 g / kg phosphorus, 30 mg / kg iron, 1 mg / kg copper, 0.01 mg / kg cobalt, 10 mg / kg manganese, 20 mg / kg zinc, 0.1 mg / kg iodine, and 0.01 mg / kg selenium; the composite vitamins are commercially available multidimensional preparations (purchased from Xinxiang Huaxu Trading Co., Ltd.), with a ratio of vitamin A (2000-13000000 IU / kg), vitamin B1 (≥350 mg / kg), vitamin D3 (3500-10000000 IU / kg), and vitamin E (≥350 IU / kg); in the following embodiments, the composite vitamins are vitamin A (100000 IU / kg), vitamin B1 (400 mg / kg), vitamin D3 (100000 IU / kg), and vitamin E (400 IU / kg).

[0028] The complex vitamins in the following embodiments are vitamin A (100,000 IU / kg), vitamin B1 (400 mg / kg), vitamin D3 (100,000 IU / kg), and vitamin E (400 IU / kg). Example 1

[0029] Preparation of special feed for aquatic animals containing mineral complex peptides: 1) Montmorillonite purification: Dissolve natural montmorillonite in deionized water at a ratio of 40 g / L, stir and mix, then centrifuge at 500 rpm for 8 min, discard the supernatant, collect the purified montmorillonite at the bottom, place it in an oven, dry it at 40°C, grind it through a 200-mesh sieve, and seal it for later use.

[0030] 2) Sodiumization of montmorillonite: The purified natural montmorillonite and Na2CO3 solid were mixed in a mass ratio of 5:1 and placed in deionized water (the concentration of the purified natural montmorillonite and Na2CO3 solid in deionized water was 5%), heated and stirred at 60°C for 3h, centrifuged at 4000rpm for 6min to remove the supernatant, and the montmorillonite at the bottom of the centrifuge tube was collected. The montmorillonite was then washed twice with deionized water to obtain sodium-based montmorillonite. The obtained sodium-based montmorillonite was placed in an oven for drying at 40°C, ground through a 200-mesh sieve, and sealed for storage for later use.

[0031] 3) Montmorillonite heat treatment: The sodium-montmorillonite obtained above was put into a crucible, and the temperature was raised to 110° C. in a muffle furnace and maintained for 5 hours. When the muffle furnace dropped to a certain temperature of 50° C., the sample was taken out and cooled to room temperature to obtain heat-activated montmorillonite powder.

[0032] 4) Montmorillonite intercalation drug loading: Heat-activated montmorillonite powder was dispersed in an aqueous solution of 20 g / L polylysine and diketopiperazine cyclic dipeptide at a 1% addition amount (wherein, 1 g of heat-activated montmorillonite powder was dispersed in 100 ml of an aqueous solution of 20 g / L polylysine and diketopiperazine cyclic dipeptide; polylysine and diketopiperazine cyclic dipeptide in the aqueous solution were mixed at a mass ratio of 1:1; wherein the diketopiperazine cyclic dipeptide was a mixture of Cyclo (Pro-Phe), Cyclo (Pro-Tyr), Cyclo (Pro-His), and Cyclo (Pro-Val) at a ratio of 1:1:1), ultrasonically treated for 60 minutes until uniformly dispersed, and then the suspension was stirred for two hours to promote the insertion of polylysine and diketopiperazine cyclic dipeptide into the montmorillonite interlayer, and the complex was separated by centrifugation at 4000 rpm for 6 minutes, and washed with deionized water. The obtained product was freeze-dried and ground through a 200-mesh sieve to obtain a mineral composite peptide (see Figure 1 ).

[0033] Depend on Figure 1 By comparing the infrared spectrum of montmorillonite, it can be found that the montmorillonite mineral complex peptide has a peak at 3239 cm -1 , 2936cm -1 , 2853cm -1 , 1668cm -1 and 1568cm -1 Five characteristic peaks were added. Referring to the characteristic peak positions and peak shapes of polylysine cyclodipeptide, it can be determined that the newly added characteristic peaks of montmorillonite mineral composite peptide are the symmetric stretching vibration peaks of EPL imino group (NH2), the antisymmetric and symmetric stretching vibration peaks of methylene group (-CH2-), amide I band and amide II band. This result can confirm that the polylysine cyclodipeptide complex was successfully intercalated into the interlayer of montmorillonite.

[0034] The antibacterial experiment was carried out using the mineral complex peptide obtained above, specifically: Escherichia coli or Staphylococcus aureus was cultured in LB liquid medium at 37°C and 170 rpm for 6 h. 8 CFU / mL of bacterial suspension was diluted 10 times with sterile water until a concentration of 10 3 CFU / mL bacterial suspension. Weigh 1.0g of the mineral complex peptide prepared above into a conical flask, and add 1mL of 103 CFU / mL bacterial suspension and 19mL PBS buffer. The blank control group did not add any sample, but only added 1mL of 10 3 CFU / mL bacterial suspension and 19mL PBS buffer. All conical bottles were placed in a shaker at 150rpm and 37℃ for 6h. After the incubation, 100μL of the mixture was transferred to LB nutrient agar medium, evenly spread, and placed in a 37℃ incubator for 24h to observe the growth of colonies on the surface of the medium.

[0035] It can be seen from the blank control group culture medium plate ( Figure 2 A and Figure 2 In (d), both E. coli and Staphylococcus aureus grew all over the culture medium plate. Figure 2 Medium c and Figure 2 It can be observed in Figure f that the addition of mineral complex peptides prevented the two strains from growing, proving that mineral complex peptides have a strong inhibitory ability against a variety of pathogens.

[0036] 5) The mineral complex peptide is added to the feed premix at a mass ratio of 0.1%, and after mixing, it is subjected to high-temperature puffing and granulation to obtain a special feed for aquaculture.

[0037] The premix ingredients, calculated by mass percentage, include 46% fish meal, 16% soybean meal, 5.5% shrimp meal, 5% corn meal, 3% high-gluten flour, 8% α-starch, 6% lard, 3% rapeseed oil, 2.5% yeast powder, 2% gluten powder, 1% milk powder, 0.8% egg yolk powder, 0.8% complex minerals, and 0.4% complex vitamins.

[0038] 6) The prepared mineral complex peptides and their special feeds for aquatic animals were used as antibiotic-reduced feeds for factory farming of freshwater fish such as eels, salmon, sea bass, and mandarin fish; the daily feeding amount of each aquatic animal feed was 1% of the body weight of the farmed aquatic animals. At the same time, the feed premix of the special feed was used as a control and compared with the above dosage. The use of antibiotics in the two groups during the farming period was zero. The mortality rates of the control groups of eels, salmon, sea bass, and mandarin fish were 25%, 28%, 21%, and 24%, respectively. The mortality rates of the groups fed with mineral complex peptide special feed were 7%, 9%, 5%, and 6%, respectively. No odorous substances were detected in the group fed with mineral complex peptide special feed. Example 2

[0039] Preparation of special feed for aquatic animals containing mineral complex peptides: 1) Montmorillonite purification: Dissolve natural montmorillonite in deionized water at a ratio of 50 g / L, stir and mix, then centrifuge at 560 rpm for 5 min, discard the supernatant, collect the purified montmorillonite at the bottom, place it in an oven, dry it at 50°C, grind it through a 200-mesh sieve, and seal it for later use.

[0040] 2) Sodiumization of montmorillonite: The purified natural montmorillonite and Na2CO3 solid were mixed in a mass ratio of 10:1 and placed in deionized water (the concentration of the purified natural montmorillonite and Na2CO3 solid in deionized water was 5%), heated and stirred at 80°C for 3h, centrifuged at 4200rpm for 5min to remove the supernatant, and the montmorillonite at the bottom of the centrifuge tube was collected. The montmorillonite was then washed twice with deionized water to obtain sodium-based montmorillonite. The obtained sodium-based montmorillonite was placed in an oven for drying at 50°C, ground through a 200-mesh sieve, and sealed for storage for later use.

[0041] 3) Montmorillonite heat treatment: The sodium-montmorillonite prepared as above was put into a crucible, heated to 300° C. in a muffle furnace and maintained for 3 hours. When the temperature of the muffle furnace dropped to 50° C., the sample was taken out and cooled to room temperature to obtain heat-activated montmorillonite powder.

[0042] 4) Montmorillonite intercalation drug loading: The heat-activated montmorillonite powder was dispersed in a 100 g / L aqueous solution of polylysine and diketopiperazine cyclic dipeptide at a 2% addition amount (polylysine and diketopiperazine cyclic dipeptide were mixed in a mass ratio of 5:1 in the aqueous solution; wherein the diketopiperazine cyclic dipeptide was a mixture of Cyclo (Tyr-Gly), Cyclo (Ala-Val), and Cyclo (Pro-Leu) at a mass ratio of 1:1:1), and ultrasonically treated for 30 minutes until uniformly dispersed, and then the suspension was stirred for two hours to promote the insertion of polylysine and diketopiperazine cyclic dipeptide into the montmorillonite interlayer, and the complex was separated by centrifugation at 4200 rpm for 8 min, and washed with deionized water. The obtained product was freeze-dried and ground through a 200-mesh sieve to obtain a mineral composite peptide (see Figure 3 ).

[0043] The morphology of montmorillonite treated by different modification methods was observed by transmission electron microscopy. As can be seen from the figure below, calcium-based montmorillonite exhibits a stacked layered structure on a 200nm scale ( Figure 3 a), and all have irregular lamellar structures with interlayer spacing less than 1 nm ( Figure 3 Middle b, Figure 3 c). After sodiumization and heat treatment, the montmorillonite structure is still retained, and the interlayer spacing of sodium-based montmorillonite increases after calcination at 450 °C. After polylysine and cyclodipeptide intercalation, the interlayer spacing of montmorillonite increases significantly (see Figure 3). Due to ion exchange, the interlayer spacing of montmorillonite increases due to the large molecular volume of long-chain polylysine and cyclic dipeptide. This confirms that the mineral composite peptide was successfully prepared.

[0044] 5) The mineral complex peptide is added to the feed premix at a mass ratio of 0.5%, and after mixing, it is subjected to high-temperature puffing and granulation to obtain a special feed for aquaculture.

[0045] The premix ingredients, by mass percentage, include 54% fish meal, 4% soybean meal, 12% shrimp meal, 5% corn meal, 1% high-gluten flour, 5% α-starch, 7% lard, 2% rapeseed oil, 3% yeast powder, 2.5% gluten powder, 2% milk powder, 1% egg yolk powder, 1% complex minerals, and 0.5% complex vitamins.

[0046] 6) The prepared mineral complex peptides and their special feeds for aquatic animals were used as antibiotic-reduced feeds for aquaculture of marine fish such as grouper and grouper bream respectively; the daily feeding amount of each aquatic animal feed was 5% of the body weight of the aquatic animal. At the same time, the feed premix of the special feed was used as a control and compared with the above dosage. The use of antibiotics in the two groups during the breeding period was zero. The mortality rates of grouper and grouper bream in the control group were 27% and 30%, respectively, and the mortality rates of grouper and grouper bream in the mineral complex peptide breeding group were 6% and 8%, respectively. No odorous substances were detected in the group fed with the mineral complex peptide special feed. Example 3

[0047] Preparation of special feed for aquatic animals containing mineral complex peptides: 1) Montmorillonite purification: Dissolve natural montmorillonite in deionized water at a ratio of 60 g / L, stir and mix, then centrifuge at 550 rpm for 5 min, discard the supernatant, collect the purified montmorillonite at the bottom, place it in an oven, dry it at 80°C, grind it through a 200-mesh sieve, and seal it for later use.

[0048] 2) Sodiumization of montmorillonite: The purified natural montmorillonite and Na2CO3 solid were mixed in a mass ratio of 10:1 and placed in deionized water (the concentration of the purified natural montmorillonite and Na2CO3 solid in deionized water was 10%), heated and stirred at 90°C for 3h, centrifuged at 4200rpm for 8min to remove the supernatant, and the montmorillonite at the bottom of the centrifuge tube was collected. The montmorillonite was then washed twice with deionized water to obtain sodium-based montmorillonite. The obtained sodium-based montmorillonite was placed in an oven for drying at 60°C, ground through a 200-mesh sieve, and sealed for storage for later use.

[0049] 3) Montmorillonite heat treatment: The sodium-montmorillonite prepared as above was put into a crucible, heated to 600° C. in a muffle furnace and maintained for 1 hour. When the temperature of the muffle furnace dropped to 60° C., the sample was taken out and cooled to room temperature to obtain heat-activated montmorillonite powder.

[0050] 4) Montmorillonite intercalation drug loading: The heat-activated montmorillonite powder was dispersed in a 200 g / L aqueous solution of polylysine and diketopiperazine cyclic dipeptide at a 5% addition amount (polylysine and diketopiperazine cyclic dipeptide were mixed in a mass ratio of 10:1 in the aqueous solution; wherein the diketopiperazine cyclic dipeptide was a mixture of Cyclo (Trp-Ser) and Cyclo (Pro-Gly) at a mass ratio of 1:1), ultrasonically treated for 60 minutes until uniformly dispersed, and then the suspension was stirred for two hours to promote the insertion of polylysine and diketopiperazine cyclic dipeptide into the montmorillonite interlayer, and the complex was obtained by centrifugation at 4000 rpm for 10 minutes, and washed with deionized water. The obtained product was freeze-dried and ground through a 200-mesh sieve to obtain a mineral composite peptide.

[0051] 5) The mineral complex peptide is added to the feed premix at a mass ratio of 1%, and after mixing, it is subjected to high-temperature puffing and granulation to obtain a special feed for aquaculture.

[0052] The premix ingredients, by mass percentage, include 30% fish meal, 35% soybean meal, 1% shrimp meal, 5% corn meal, 5% high-gluten flour, 10% α-starch, 5% lard, 4% rapeseed oil, 2% yeast powder, 1.2% gluten powder, 0.5% milk powder, 0.5% egg yolk powder, 0.5% complex minerals, and 0.3% complex vitamins.

[0053] 6) The prepared mineral complex peptides and their special feeds for aquatic animals were used as antibiotic-reduced feeds for shrimp, crab, and crucian carp farming respectively; the daily feeding amount of each aquatic animal feed was 1% of the body weight of the farmed aquatic animal. At the same time, the feed premix of the special feed was used as a control and compared with the above dosage. The use of antibiotics during the breeding period of the two groups was zero. The mortality rates of shrimp, crab, and crucian carp in the control group were 19%, 17%, and 12%, respectively, and the mortality rates of shrimp, crab, and crucian carp in the mineral complex peptide group were 6%, 5%, and 0%, respectively. No odorous substances were detected in the group fed with the mineral complex peptide special feed.

Claims

1. A mineral composite peptide, characterized in that: The mineral composite peptide is obtained by inserting polylysine and diketopiperazine cyclic dipeptide into modified montmorillonite, wherein the mass ratio of polylysine to diketopiperazine cyclic dipeptide is 1-10:

1.

2. The mineral composite peptide according to claim 1, characterized in that: The diketopiperazine cyclic dipeptide is one or more of Cyclo(Pro-Phe), Cyclo(Pro-Tyr), Cyclo(Pro-Leu), Cyclo(Trp-Ser), Cyclo(Pro-Gly), Cyclo(Pro-His), Cyclo(Pro-Val), Cyclo(Tyr-Gly) and Cyclo(Ala-Val).

3. A method for preparing the mineral composite peptide according to claim 1, characterized in that: Polylysine and diketopiperazine cyclic dipeptide are mixed and dissolved in water at a mass ratio of 1-10:1, and then the modified montmorillonite is added and stirred to promote the intercalation of polylysine and diketopiperazine cyclic dipeptide into the interlayer of montmorillonite mineral to form a mineral composite peptide.

4. The method for preparing the mineral composite peptide according to claim 3, characterized in that: The steps for preparing the mineral complex peptide are as follows: 1) Montmorillonite purification: Dissolve natural montmorillonite in deionized water, stir and mix, then centrifuge and collect the precipitate, dry and sieve; 2) Sodiumization of montmorillonite: The purified natural montmorillonite and Na2CO3 solid are mixed in a mass ratio of 5-10:1 and placed in deionized water, heated and stirred at 60-90°C for 3 hours, centrifuged to collect the precipitate, washed, dried, and sieved to obtain sodium montmorillonite for standby use; 3) Montmorillonite heat treatment: The sodium-montmorillonite obtained in 2) is heat-treated at 110-600° C. for 1-5 h, and then cooled to room temperature to obtain heat-activated montmorillonite powder; 4) Montmorillonite intercalation drug loading: The heat-activated montmorillonite powder is dispersed in an aqueous solution of 20-200 g / L polylysine and diketopiperazine cyclic dipeptide at a mass volume ratio of 1-5%, and ultrasonic treatment is performed for 10-60 minutes until uniform dispersion. The suspension is then stirred for two hours to promote the insertion of polylysine and diketopiperazine cyclic dipeptide into the montmorillonite interlayer. The complex is obtained by centrifugation, and the mineral composite peptide is obtained by washing and freeze-drying.

5. The method for preparing the mineral composite peptide according to claim 4, characterized in that: In the step 4), the mass ratio of polylysine to diketopiperazine cyclic dipeptide is 1-10:

1.

6. An application of the mineral composite peptide according to claim 1, characterized in that: The mineral composite peptide is used as an antibacterial additive for aquatic animal feed.

7. A special feed for aquatic animals containing mineral complex peptides, characterized in that: The mineral complex peptide according to claim 1 is added to the feed, and its addition amount is 0.1-1% of the mass of the feed premix.

8. The special feed for aquatic animals containing mineral complex peptides according to claim 7, characterized in that: The feed premix comprises, by weight percentage, 30%-54% fish meal, 4%-35% soybean meal, 1%-12% shrimp meal, 5% corn meal, 1%-5% high-gluten flour, 5%-10% α-starch, 5%-7% lard, 2%-4% rapeseed oil, 2%-3% yeast powder, 1.2%-2.5% gluten powder, 0.5%-2% milk powder, 0.5%-1% egg yolk powder, 0.5%-1% complex minerals, and 0.3%-0.5% complex vitamins.

9. An application of the special feed for aquatic animals containing mineral composite peptides according to claim 7, characterized in that: The feed is used as antibiotic-reduced feed in aquaculture.

10. The use of the special feed for aquatic animals containing mineral complex peptides according to claim 9, characterized in that: The daily feeding amount of the feed is 1-10% of the body weight of the farmed aquatic animals.

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