A Chinese herbal zeolite feed additive for increasing silk secretion

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a Chinese herbal zeolite feed additive for increasing silk secretion, wherein the composition comprises antimicrobial peptides, Chinese herbal antiviral extracts, and zeolite, wherein the antimicrobial peptides, the Chinese herbal anti-fermentation extracts, and the activated zeolite are used in combination to embody a composite mechanism of multiple actions, improve immune response, have a lasting effect, reduce the residue and use of antibiotics, and provide a new direction for new feed additives for antibacterial and viral production increase.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a Chinese herbal zeolite feed additive for improving the secretion of silkworm silk. Background Art

[0002] Silkworm suppurative disease (pus disease caused by viruses and bacteria): With long-term cultivation in the environment, silkworms will develop suppurative disease in the final silk-spinning stage, with an incidence rate of 20-30%. The frequent use of antibiotics not only causes environmental pollution, but also reduces the product quality of silk due to drug residues. In recent years, antimicrobial peptides found in many organisms have strong antibacterial activity. Compared with antibiotics, they are not easy to develop drug resistance and have less harm to organisms. They have become a hot topic in aquaculture. This study intends to use Japanese eels as the research object to extract antimicrobial peptides from the main tissues and organs of eels, providing a new way to prevent and control bacterial diseases in eels in the future. This study successfully isolated three antimicrobial peptides from epidermal mucus and spleen. Fish are an important source of antimicrobial peptides because they express all major categories of AMPs, including defensins cathelicidins, hepcidins, histone-derived peptides, etc. In recent years, the antimicrobial effect of histones and their derived peptide fragments has been widely studied, and they have been shown to have strong antimicrobial properties and have broad-spectrum activity. Existing feed CN111328914A reports that a Chinese medicine biological antiviral feed and its preparation method can give animals the advantage of preventing diseases in daily life, save time, and improve the quality of biological meat. CN111887348A provides a nano-composite antiviral feed additive and its preparation method, which can enhance the animal's own immunity and effectively kill a variety of viruses. However, they all face the problem of not being able to solve the problem of joint prevention of bacteria and viruses and increase production. Therefore, it is particularly important to carry out research on new feeds for joint synergy. Summary of the invention

[0003] The purpose of the present invention is to provide a Chinese herbal zeolite feed additive and a method for increasing the secretion of silkworm silk.

[0004] A Chinese herbal zeolite feed additive for increasing silk secretion, the composition comprising antimicrobial peptides, Chinese herbal antiviral extracts and nano zeolite supercarriers, wherein the weight proportions of the components are 30-35 parts of antimicrobial peptides, 35-45 parts of Chinese herbal antiviral fermentation extracts and 20-35 parts of nano zeolite supercarriers.

[0005] The further preparation method of the antimicrobial peptide is:

[0006] (1) Dissolve catfish mucus in distilled water at a solid-liquid ratio of 1:100 g / mL, add 0.85% w / v papain, and react at pH 7 and 37° C. for 8 h to obtain an enzymolyzate;

[0007] (2) The hydrolysate was boiled for 20 min to stop the reaction and cooled to room temperature in an ice bath. The hydrolysate was centrifuged at 2000 × g for 30 min at 6 °C and the supernatant was collected.

[0008] (3) The supernatant was filtered through a 0.22 μm filter membrane, and the peptide was purified by high-resolution gel filtration using a protein purification system. The mobile phase was 25 mmol / L PBS buffer at pH 7.0, the flow rate was 0.1 ml / min, and the detection wavelength was 250 nm. All the samples containing the target peptide were collected;

[0009] (4) The peptide liquid is freeze-dried to obtain peptide powder.

[0010] Furthermore, the target peptide has 10-15 amino acids.

[0011] Furthermore, the amino acid sequence of the identified peptide is chromatographically separated by a nanoliter flow rate chromatography system, the sample enters the chromatographic analysis column, and the peptide segments are separated and then subjected to mass spectrometry analysis and sequencing by a mass spectrometer. The chromatographic conditions of the chromatographic analysis column are as follows: within 50 minutes, the flow rate is 300 nL / min; the gradient of the mobile phase A liquid is 0-3 minutes, 95-90%; 3-4.5 minutes, 90-95%; 4.5-28 minutes, 72-75%; 28-40 minutes, 60%-8 0%, the gradient of mobile phase B was 0-5min, 3%; 3-5.5min, 3-10%; 4.5-28min, 8-35%; 28-35min, 32-45%; 31-44min, 44-60%; 34-45min, 99%, and the peptides obtained from de novo sequencing analysis were identified and matched with reference to the protein database. All identified peptides were compared through the NCBL website, and sequences with no corresponding function reported and with a homology of less than 70% were selected: SEQ ID 1: DKDAPAVWLWIKDVNIL, SEQ ID 2: IKVVDWDVDVGWLWD, and SEQ ID 3: WLWDWPKASDVDKK.

[0012] Furthermore, the Chinese herbal antiviral extract is prepared by: adding distilled water and 60% [v / v] ethanol to Fucus vesiculosus, and extracting the extract in two batches using an ultrasonic extractor for 3 hours; decompressing and concentrating the extract after removing the solid, adding a composite enzyme in which a hydrolase and a flavor enzyme are mixed in a ratio of 1:1, and adding 1% to 5%, heating at 95°C for 20 minutes using a centrifuge Combi-514R, and separating the enzyme at 3,000rpm and 4°C for 20 minutes, collecting only the supernatant, filtering the supernatant through a 0.22μm filter membrane, and purifying the peptide by high-resolution gel filtration using a protein purification system, using a mobile phase of 25mmol / L PBS buffer and eluting at pH7.0, a flow rate of 0.1ml / min, and a detection wavelength of 250nm, to collect all the peptides containing the target peptide;

[0013] Furthermore, the target peptide has 10-15 amino acids.

[0014] Furthermore, the amino acid sequence of the identified refined peptide is chromatographically separated by a chromatography system with a nanoliter flow rate, the sample enters the chromatographic analysis column, and after the peptide segments are separated, mass spectrometry analysis is performed using a mass spectrometer, sequencing is performed, and the peptides obtained by de novo sequencing analysis are identified and matched with reference to a protein database, all identified peptides are compared through the NCBL website, and sequences SEQ ID 4: AWVIKDSWDKDAPKLL, SEQ ID 5: IKVGWLVDWDKASDN, and SEQ ID 6: WLIDNWVDPDDKK that have not reported corresponding functions and have a homology of less than 70% are selected.

[0015] Further: the preparation method of the nano zeolite supercarrier is as follows: add 30g of silica to commercial zeolite and ball mill. Ball mill at 2500r / min for 3 hours in a ball mill, sieve out the powder in the tank with a sieve, heat it to 650℃ at a rate of 5.5℃ / min and maintain this temperature for oxidative calcination for 4 hours, and then crush it through a 100-mesh sieve.

[0016] The technical effects achieved by the present invention are:

[0017] The use of this product can reduce the incidence of bacteria and viruses, the overall mortality rate, and promote silk production; the use of adding small molecule peptides and activated zeolite can significantly reduce the incidence and mortality rate compared with the embodiment without adding them. The activated zeolite can immobilize small molecule peptides. Due to its excellent ion exchange effect, the porous physical adsorption effect can extend the period of efficacy; the additives have no chemical residues, are environmentally friendly, and have a simple preparation process. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 , antibacterial and antiviral combined test;

[0019] Figure 2, feed feeding comparison test;

[0020] Figure 3 , immune response test results. DETAILED DESCRIPTION

[0021] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the accompanying drawings.

[0022] Embodiment 1

[0023] A Chinese herbal zeolite feed additive for increasing silk secretion, the composition comprising antimicrobial peptides, Chinese herbal antiviral extracts and nano zeolite supercarriers, wherein the weight proportion of the components is 30 g of antimicrobial peptides, 35 g of Chinese herbal antiviral extracts and 20 g of nano zeolite supercarriers.

[0024] The further preparation method of the antimicrobial peptide is:

[0025] (1) Dissolve catfish mucus in distilled water at a solid-liquid ratio of 1:100 g / mL, add 0.85% w / v papain, and react at pH 7 and 37° C. for 8 h to obtain an enzymolyzate;

[0026] (2) The hydrolysate was boiled for 20 min to stop the reaction and cooled to room temperature in an ice bath. The hydrolysate was centrifuged at 2000 × g for 30 min at 6 °C and the supernatant was collected.

[0027] (3) The supernatant was filtered through a 0.22 μm filter membrane, and the peptide was purified by high-resolution gel filtration using a protein purification system. The mobile phase was 25 mmol / L PBS buffer at pH 7.0, the flow rate was 0.1 ml / min, and the detection wavelength was 250 nm. All the samples containing the target peptide were collected;

[0028] (4) The peptide liquid is freeze-dried to obtain peptide powder.

[0029] The amino acid sequence of the identified peptide is chromatographically separated by a chromatographic system with a nanoliter flow rate. The sample enters the chromatographic analysis column. After the peptide segments are separated, mass spectrometry analysis and sequencing are performed by a mass spectrometer. The chromatographic conditions of the chromatographic analysis column are as follows: within 50 minutes, the flow rate is 300nL / min; the gradient of the mobile phase A liquid is 0-3min, 95-90%; 3-4.5min, 90-95%; 4.5-28min, 72-75%; 28-40min, 60%-80% , the gradient of mobile phase B was 0-5min, 3%; 3-5.5min, 3-10%; 4.5-28min, 8-35%; 28-35min, 32-45%; 31-44min, 44-60%; 34-45min, 99%. The peptides obtained from de novo sequencing analysis were identified and matched with reference to the protein database, and all identified peptides were compared through the NCBL website, and sequences with no corresponding function reported and less than 70% homology were selected: SEQ ID 1: DKDAPAVWLWIKDVNIL, SEQ ID2: IKVVDWDVDVGWLWD, SEQ ID 3: WLWDWPKASDVDKK.

[0030] The preparation method of the Chinese herbal antiviral extract is as follows: adding distilled water and 60% [v / v] ethanol to Fucus vesiculosus, extracting the extract in two batches using an ultrasonic extractor for 3 hours; decompressing and concentrating the extract after removing the solid, adding a composite enzyme in which a hydrolase and a flavor enzyme are mixed in a ratio of 1:1, and adding 1%, heating at 95°C for 20 minutes using a centrifuge Combi-514R, and separating the enzyme at 3,000 rpm and 4°C for 20 minutes, collecting only the supernatant, filtering the supernatant through a 0.22 μm filter membrane, and purifying the peptide by high-resolution gel filtration using a protein purification system, using a mobile phase of 25 mmol / L PBS buffer and eluting at pH 7.0, a flow rate of 0.1 ml / min, and a detection wavelength of 250 nm, to collect all the peptides containing the target peptide;

[0031] The amino acid sequence of the identified refined peptide is chromatographically separated by a chromatography system with a nanoliter flow rate, the sample enters the chromatographic analysis column, and the peptide segment is separated and then subjected to mass spectrometry analysis and sequencing by a mass spectrometer. The peptides obtained by de novo sequencing analysis are identified and matched with reference to a protein database, all identified peptides are compared through the NCBL website, and sequences SEQ ID 4: AWVIKDSWDKDAPKLL, SEQ ID 5: IKVGWLVDWDKASDN, and SEQID 6: WLIDNWVDPDDKK that have not reported corresponding functions and have a homology of less than 70% are selected.

[0032] The nano zeolite supercarrier is prepared by adding 30g of silica to commercial zeolite and ball milling. The ball mill is milled at a speed of 2500r / min for 3 hours, the powder in the jar is sieved out with a screen, the temperature is raised to 650°C at a speed of 5.5°C / min and the temperature is maintained for oxidative calcination for 4 hours, and then crushed to pass through a 100-mesh sieve.

[0033] Embodiment 2

[0034] Only the Fucus peptide extract in Example 1 is removed, and the other preparation processes are the same to prepare the product.

[0035] Embodiment 3

[0036] Only the small molecule peptide extract in Example 1 is removed, and the other preparation processes are the same to prepare the product.

[0037] Embodiment 4

[0038] Only the activated zeolite component in Example 1 is removed, and the other preparation processes are the same to obtain the product.

[0039] Embodiment 5

[0040] Toxicological and immune response tests of catfish small molecule peptides and Fucus vesiculosus extracts. Toxicity assessment

[0041] Macrophage density = 1×10 6 / mL, 300 μL per well was inoculated in a 96-well plate, and cultured in a 38°C, 5% CO2 incubator for 2D, and different concentrations of the small molecule polypeptide SEQ NO.1-6 described in Example VI were added. The blank control group and the positive control group were respectively added with culture medium and LPS (100 ng / mL) and continued to culture for 24h; the 96-well plate was taken out, the old culture medium was discarded, and the cells were cultured in a 37°C, 5% CO2 incubator for 90min, and then the absorbance at 450nm was measured with an enzyme marker. The results are shown in Table 1. Within the concentration range of 5-200 μg / mL, there was no toxicity to macrophages.

[0042] Table 1. Toxicological evaluation

[0043]

[0044]

[0045] Effects of small molecule peptides on the expression of genes related to immune factor assessment:

[0046] Macrophage density = 2×10 6 / mL, inoculated in 96-well plates, cultured in a CO2 incubator for 2D, and added with small molecule peptide amino acid sequences of different concentrations. (25, 50, 100, 200μg / mL), the control group and the positive control group were added with culture medium and LPS (100ng / mL), and the cell supernatant was collected after 24h of culture to determine the production of related immune cytokines. The results are shown in Table 2. Figure 1 As shown, within the concentration range of 5-10 μg / mL, Figure 3 The immune factors shown responded successfully and reconstructed effectively, and CD45 factor could be released. The induction effect of catfish small molecule peptide was better than that of Fucus vesiculosus extract, and the combination of the two could enhance the effect.

[0047] Table 2. Immune cell CD45 factor response test

[0048]

[0049] Embodiment 6

[0050] Antibacterial and antiviral tests of small molecule peptides and Fucus vesiculosus extract.

[0051] Table 3. Antibacterial and viral tests

[0052]

[0053] Figure 1 As shown, the inhibition rates of catfish small molecule peptides against Escherichia coli, Staphylococcus aureus and Candida albicans were all greater than 95%; the inactivation rate of Fucus vesiculosus extract against influenza A virus and coronavirus was as high as 99%;

[0054] Embodiment 7

[0055] 400 silkworms of 30 days old were selected and divided into a control group and a test group according to the principle of random allocation, with 50 silkworms in each group. The control group was fed with ordinary feed, and the test group was fed with the feed of the present invention. During the test, the feed was quantitatively fed, the temperature in the house was about 20 degrees Celsius, and regular disinfection was carried out according to routine management. Feeding was carried out 3 times a day, and attenuated viruses and bacteria were inoculated after 30 days. A control test (CK was a commercially available conventional feed) was set up. After 30 days, the silk increase rate, mortality rate, and morbidity rate were recorded every day in the test interval.

[0056] Table 4. Feeding test of adult silkworms.

[0057]

[0058] Test statistics Figure 2As shown, the use of this product can reduce the morbidity and mortality rates; adding small molecule peptides and activated zeolites can significantly reduce the morbidity and mortality rates compared with the embodiments without adding them. The activated humic acid zeolite can immobilize the small molecule peptide extract, and the porous physical adsorption effect can improve the intestines and prolong the period of efficacy.

[0059] The above is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. The structures, devices and operating methods not specifically described and explained in the present invention shall be implemented according to the conventional means in the art unless otherwise specified and limited.

Claims

1. A Chinese herbal zeolite feed additive for increasing silk secretion, the additive comprising antimicrobial peptides, Chinese herbal antiviral fermentation extracts and nano zeolite supercarriers, wherein the weight ratio of each component of the additive is 30-35 parts of antimicrobial peptides, 35-45 parts of Chinese herbal antiviral fermentation extracts and 20-35 parts of nano zeolite supercarriers; The antimicrobial peptides are SEQ ID 1: DKDAPAVWLWIKDVNIL, SEQ ID 2: IKVVDWDVDVGWLWD, SEQ ID 3: WLWDWPKASDVDKK; The amino acids of the Chinese herbal antiviral fermentation extract are: SEQ ID 4: AWVIKDSWDKDAPKLL, SEQ ID 5: IKVGWLVDWDKASDN, SEQ ID 6: WLIDNWVDPDDKK; The preparation method of the nano zeolite supercarrier is as follows: commercially available zeolite is added with silicon dioxide in a ratio of 10:1 and ball-milled at a speed of 2500 r / min in a ball mill for 3 hours, the powder in the jar is sieved out with a sieve, the temperature is raised to 750° C. at a speed of 6.5° C. / min and the temperature is maintained for oxidation calcination for 5 hours, and then crushed to pass through a 100-mesh sieve.

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