CN114836497A - Preparation method of maggot antibacterial peptide, composition containing maggot antibacterial peptide and preparation method of composition - Google Patents
Preparation method of maggot antibacterial peptide, composition containing maggot antibacterial peptide and preparation method of composition Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
- A61K31/722—Chitin, chitosan
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/716—Glucans
- A61K31/724—Cyclodextrins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K36/534—Mentha (mint)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/01—Hydrolysed proteins; Derivatives thereof
- A61K38/012—Hydrolysed proteins; Derivatives thereof from animals
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/40—Cyclodextrins; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention discloses a preparation method of maggot antibacterial peptide, a composition containing maggot antibacterial peptide and a preparation method thereof, wherein the preparation method of maggot antibacterial peptide comprises the following steps: step A, taking maggot eggs, feeding the maggots until the maggots begin to pupate, collecting maggot living bodies, wherein the feed for feeding at least comprises momordica grosvenori seeds; b, cleaning the maggot living bodies, cutting into pieces, adjusting the temperature to 5-15 ℃, mixing the maggot living bodies with normal saline, juicing, centrifuging, heating in a water bath, and centrifuging to obtain a maggot crude extract; and step C, taking the crude extractive solution of the maggots, adding water and glucose, mixing uniformly, sterilizing, inoculating rice mold spores for culturing, centrifuging for 3-4 days to obtain a maggot supernatant, sterilizing, centrifuging, and concentrating to obtain a maggot antibacterial peptide extracting solution. Compared with the similar products sold in the market, the maggot antibacterial peptide prepared by the application has obviously enhanced antibacterial and anti-inflammatory effects.
Description
Technical Field
The invention relates to the technical field of maggot extraction, in particular to a preparation method of maggot antibacterial peptide, a composition containing maggot antibacterial peptide and a preparation method thereof.
Background
In recent years, due to abuse of antibiotics, the drug resistance of bacteria is getting worse, so that the treatment of chronic ulcer wounds infected repeatedly in clinic is very difficult. Maggots, also known as maggots, are larvae of lucilia sericata, chrysomyia megacephala or other closely related insects of the family calliphoridae, and are attracting increasing attention due to their antibacterial and resistance-inducing properties.
At present, the commercial maggot antibacterial peptide extracting solution is basically an insect body extract obtained by drying maggots, although a certain antibacterial property is shown, the antibacterial effect is general, and the improvement space is still left.
Disclosure of Invention
In order to further improve the antibacterial effect of the maggot antibacterial peptide, the application provides a preparation method of the maggot antibacterial peptide, a composition containing the maggot antibacterial peptide and a preparation method of the composition.
A preparation method of the maggot antibacterial peptide comprises the following steps:
step A, taking maggot eggs, feeding until maggots turn yellow, and collecting maggot living bodies before pupation begins, wherein the feed contains at least momordica grosvenori seeds;
b, cleaning the maggot living bodies, shearing, adjusting the temperature to 5-15 ℃, mixing the maggot living bodies with normal saline, juicing and centrifuging, heating the supernatant in a water bath at 95-100 ℃ for 10-15min, and centrifuging again to obtain a crude maggot extract; and step C, taking the crude extract of the maggots, and mixing the crude extract with water according to a volume ratio of 1: 0.2-1, adding glucose according to the proportion that the volume of the mixed solution and the mass of the glucose are 100mL:3-5g, carrying out sterilization treatment, then inoculating rice mold spores accounting for 7-15% of the volume of the mixed solution, culturing for 3-4 days, centrifuging to obtain maggot supernatant, sterilizing, centrifuging, and concentrating to obtain the maggot antibacterial peptide extracting solution.
The method is characterized in that bran and chaff are generally used as feeds for the maggot eggs, the feeds with at least momordica grosvenori seeds are adopted for feeding the maggot eggs, the anti-inflammatory effect and the antibacterial effect of the maggot antibacterial peptide fed by the feeds with the momordica grosvenori seeds are obviously improved compared with the maggot antibacterial peptide of a common feed, and the influence of impurities in the maggot on the antibacterial and anti-inflammatory effects of the maggot antibacterial peptide can be reduced to the greatest extent by combining a special preparation method of squeezing living adult insects at low temperature, removing impurities and performing fermentation treatment, compared with the maggot extract which is usually prepared by a drying method at present, so that the antibacterial and anti-inflammatory effects of the maggot antibacterial peptide are enhanced.
In addition, the maggot antibacterial peptide prepared by the application also has the advantages of promoting scald healing and cell repair, improving the skin resistance to stimulation caused by the external environment, reducing the skin redness and swelling degree and the like.
Preferably, the feed consists of momordica grosvenori seeds, milk powder and cane sugar according to the mass ratio of 1:0.2-0.5: 0.05-0.2.
The feed prepared by matching the momordica grosvenori seeds, the milk powder and the cane sugar according to the proportion has good antibacterial and anti-inflammatory effects on the maggot antibacterial peptide, is high in imago rate and low in death rate of maggot larvae, and is beneficial to obtaining maggot living bodies.
Preferably, in the step B, the maggot living bodies are soaked in normal saline for 4 to 5 hours at the temperature of between 0 and 4 ℃ to ensure that metabolites in the maggot living bodies are excreted completely, and then the maggot living bodies are washed by formaldehyde normal saline solution, ethanol and distilled water in sequence.
Through the low-temperature soaking treatment, the living maggot bodies are disinfected by using formaldehyde normal saline and ethanol in a combined manner, then the residual reagent on the surfaces of the living maggot bodies is washed away by using distilled water, so that the subsequent treatment steps of introducing external impurities into the living maggot bodies are reduced, the pollution to the maggot antibacterial peptides is reduced, and the exertion of the antibacterial effect of the maggot antibacterial peptides is influenced.
Preferably, in the step B, the ratio of the volume of the living maggots to the volume of the physiological saline is 1: 0.2-1 mixing.
Preferably, in the step C, the rice mold spores are cultured under the environment of 25-35 ℃ of temperature, 50-70% of humidity and 4.6-7 of pH.
A composition containing maggot antibacterial peptide is mainly prepared from the following components in percentage by mass:
10-30% of maggot antibacterial peptide extract
0.5 to 1 percent of chitosan
Herba Menthae extract 0.5-2%
61-80% of water
0 to 0.3 percent of preservative.
The mint extract, the chitosan and the maggot antibacterial peptide extracting solution are mixed according to a specific proportion, so that the synergistic antibacterial effect is obvious, and compared with the method of independently adding the maggot antibacterial peptide extracting solution, the maggot antibacterial peptide composition has the advantages that the anti-inflammatory and antibacterial effects are further enhanced, and the antibacterial and anti-inflammatory requirements under complex environments can be better met.
Preferably, the composition containing the maggot antibacterial peptide is added with the following components in percentage by mass
0.1 to 1 percent of chitin
Collagen 0.5-3%
Beta-cyclodextrin 0.5-5%.
The addition of the beta-cyclodextrin can effectively lock active ingredients of the maggot antibacterial peptide, improve the stability of active substances in the maggot antibacterial peptide and promote the synergistic cooperation of the maggot antibacterial peptide, the chitosan and the mint extract, so that the antibacterial and anti-inflammatory effects of the maggot antibacterial peptide containing composition are further improved.
A preparation process of a composition containing maggot antibacterial peptide comprises the following steps:
step 1, mixing a mint extract, chitosan and water, and uniformly stirring to obtain a mixed solution;
step 2, adding the maggot antibacterial peptide extracting solution into the mixed solution, and stirring until the maggot antibacterial peptide extracting solution is uniformly dispersed;
and 3, adding a preservative into the solution obtained in the step 2, and stirring until the preservative is uniformly dispersed to obtain the composition containing the maggot antibacterial peptide.
The peppermint extract, the chitosan and the water are firstly dispersed and mixed to form a mixed solution, then the maggot antibacterial peptide extracting solution is added, the dispersity of the maggot antibacterial peptide extracting solution can be effectively improved, and the maggot antibacterial peptide extracting solution is combined with all the components in the mixed solution to play a better synergistic antibacterial and anti-inflammatory effect
Preferably, the preparation of the mixed solution comprises the following steps:
step 1-1, adding chitin, collagen, mint extract and chitosan into water with the mass percentage of 20-50%, and stirring and dispersing uniformly until the chitin, the collagen, the mint extract and the chitosan are completely dissolved to obtain a premix; adding beta-cyclodextrin into the rest water, stirring and dispersing uniformly until the beta-cyclodextrin is completely dissolved to obtain a beta-cyclodextrin aqueous solution;
and step 1-2, adding the dispersed premix into a beta-cyclodextrin aqueous solution, and stirring until the dispersion is uniform to obtain a mixed solution.
By dissolving the substances step by step, the effect of dissolving and dispersing all the components is better, and the uniformly dispersed mixed solution is obtained.
In summary, the present application has the following beneficial effects:
1. the feed with at least momordica grosvenori seeds is adopted to feed the maggot eggs, a special preparation method combining living imagoes, low-temperature squeezing and biological fermentation is adopted in the maggot extraction process, the influence of impurities in the maggot antibacterial peptide on the antibacterial and anti-inflammatory effects of the maggot antibacterial peptide is reduced to the maximum extent, and the antibacterial and anti-inflammatory effects of the maggot antibacterial peptide are enhanced.
2. The composition containing the maggot antibacterial peptide is added with the mint extract, the chitosan and the maggot antibacterial peptide extracting solution in a specific ratio, has obvious synergistic antibacterial and anti-inflammatory effects, and further improves the antibacterial and anti-inflammatory effects of the composition containing the maggot antibacterial peptide.
3. By adding the beta-cyclodextrin, the chitin and the collagen with specific mass ratio, the antibacterial effect of the maggot antibacterial peptide composition can be further enhanced, and the anti-inflammatory effect of the maggot antibacterial peptide composition is further enhanced.
4, the above substances are dissolved step by step, so that the effect of dissolving and dispersing all the components is better, and the uniformly dispersed mixed solution is obtained.
Detailed Description
The following examples and comparative examples are all commercially available.
Preparation example 1
A preparation method of maggot antibacterial peptide comprises the following steps:
step A, taking maggot eggs, and mixing the momordica grosvenori, the milk powder and the cane sugar according to a mass ratio of 1: 0.2: 0.05, mixing to prepare feed, wherein tap water is drinking water, the cultivation humidity is controlled to be 70%, the pH value is 6.5, the temperature is controlled to be 20 ℃, the feed is stirred once in the morning and at the evening every day under natural illumination, the feed is raised until the maggots grow to the 3 rd larva stage and begin to yellow, and before pupation, the maggot living bodies are collected.
And step B, soaking the maggot living bodies in normal saline for 4 hours at the temperature of 4 ℃ to ensure that metabolites in the maggot living bodies are excreted completely, washing the maggot living bodies obtained in the step 1 with 3.5% formaldehyde normal saline solution and 75% ethanol which have the same volume once, finally washing the maggot living bodies with distilled water which has the same volume for three times, cutting the maggot living bodies, adjusting the temperature to be 5 ℃, and then mixing the maggot living bodies with normal saline with the mass concentration of 0.86% according to the volume ratio of 1:0.2 mixing, juicing, centrifuging at 0 deg.C and 8000r/min for 15min to remove impurities such as organelles, heating the supernatant in 100 deg.C water bath for 10min, centrifuging at 0 deg.C and 8000r/min for 15min to remove heat-labile protein impurities to obtain crude extractive solution of Chrysomyiame gacephala.
And step C, taking a crude extractive solution of the maggots, and mixing the crude extractive solution with water according to a mass ratio of 1: 2, mixing to obtain a mixed solution, adding glucose according to the ratio of the volume of the mixed solution to the mass of the glucose to be 100mL:3g, sterilizing at a high temperature of 121.3 ℃ and a high pressure of 103kpa for 30min, then inoculating rice mold spores accounting for 7-15% of the volume of the mixed solution, then culturing in an environment with the temperature of 25 ℃, the humidity of 50% and the pH of 4.6, checking the growth condition of hyphae every 12h, after 4 days, centrifuging at a speed of 3000r/min for 15min at 0 ℃ to remove hyphae to obtain a maggot supernatant, sterilizing at a temperature of 121.3 ℃ and a high pressure of 103kpa for 10min to kill residual bacteria, centrifuging at a speed of 8000r/min for 15min at 0 ℃, heating in a water bath at 100 ℃ to remove most of the solvent to obtain a maggot antibacterial peptide extracting solution, and freezing and storing below 18 ℃ for later use.
Preparation example 2
A preparation method of maggot antibacterial peptide comprises the following steps:
step A, taking maggot eggs, and mixing the momordica grosvenori, the milk powder and the cane sugar according to a mass ratio of 1: 0.3: 0.1, mixing to prepare feed, wherein tap water is drinking water, the cultivation humidity is controlled to be 75%, the pH value is 7, the temperature is 25 ℃, the feed is stirred for 2 times in the morning and at the evening every day under natural illumination, the feed is raised until the maggots grow to the 3 rd larva stage and begin to yellow, and before pupation, the maggot living bodies are collected.
And step B, soaking the maggot living bodies in normal saline for 5 hours at the temperature of 4 ℃ to ensure that metabolites in the maggot living bodies are excreted completely, then washing the maggot living bodies in running water to clean large-particle substances on the surfaces, then sequentially washing the maggot living bodies with 3.2% formaldehyde normal saline solution and 75% ethanol which have the same volume as the maggot living bodies once, finally washing the maggot living bodies with distilled water which has the same volume as the normal saline solution for three times, shearing the maggot living bodies, adjusting the temperature to be 10 ℃, and then mixing the maggot living bodies with normal saline with the mass concentration of 0.86% according to the volume ratio of 1:0.5 mixing, juicing, centrifuging at 2 deg.C at 10000r/min for 12min to remove impurities such as organelles, heating the supernatant in 98 deg.C water bath for 5min, centrifuging at 2 deg.C at 10000r/min for 12min to remove heat-labile protein impurities to obtain crude extractive solution of maggot.
And step C, taking the crude extract liquid of the maggots, and mixing the crude extract liquid of the maggots with water according to a mass ratio of 1: 2, uniformly mixing, adding glucose according to the ratio of the volume of the mixed solution to the mass of the glucose to be 100mL:4g, sterilizing at 121.3 ℃ for 25min at 103kpa, inoculating rice mold spores accounting for 10% of the volume of the mixed solution, culturing under the environment of keeping the temperature at 30 ℃, the humidity at 60% and the pH value at 6, checking the growth condition of hyphae every 12h, centrifuging for 15min at the speed of 3000r/min at 0 ℃ after 3 days, removing the hyphae to obtain a maggot supernatant, sterilizing for 15min at 121.3 ℃ at 103kpa to kill residual bacteria, centrifuging for 15min at the speed of 8000r/min at 0 ℃, heating in a water bath at 100 ℃ to remove most of the solvent to obtain a maggot antibacterial peptide extracting solution, and freezing and storing below 18 ℃ for later use.
Preparation example 3
A preparation method of maggot antibacterial peptide comprises the following steps:
step A, taking maggot eggs, and mixing the momordica grosvenori, the milk powder and the cane sugar according to a mass ratio of 1: 0.5: 0.2, mixing to prepare feed, wherein tap water is drinking water, the cultivation humidity is controlled to be 80%, the pH value is 7, the temperature is 30 ℃, the feed is stirred once in the morning and at the evening every day under natural illumination, the feed is raised until the maggots grow to the 3 rd larva stage and begin to yellow, and before pupation begins, the maggot living bodies are collected.
And step B, soaking the maggot living bodies in normal saline for 4 hours at the temperature of 0 ℃ to ensure that metabolites in the maggot living bodies are excreted completely, washing the maggot living bodies obtained in the step 1 in running water to clean large-particle substances on the surfaces, sequentially washing the maggot living bodies with 3.5% formaldehyde normal saline solution and 75% ethanol, finally washing the maggot living bodies with equal volume of distilled water for three times, shearing the maggot living bodies, adjusting the temperature to be 15 ℃, and then mixing the maggot living bodies with the normal saline with the mass concentration of 0.86% according to the volume ratio of 1: 1, mixing, juicing, centrifuging at 12000r/min for 15min at 4 ℃, removing impurities such as organelles and the like, heating the supernatant in water bath for 5min, centrifuging at 12000r/min for 15min at 4 ℃ again, and removing heat-unstable protein impurities to obtain the crude extract of the maggots.
And step C, taking the crude extract of the maggots, and mixing the crude extract with water according to a mass ratio of 1: 2, adding glucose according to the ratio of the volume of the mixed solution to the mass of the glucose being 100mL:5g, sterilizing at the high temperature of 121.3 ℃ and the high pressure of 103kpa for 28min, inoculating rice mold spores accounting for 15% of the volume of the mixed solution, culturing under the environment of keeping the temperature at 35 ℃, the humidity at 70% and the pH of 7, and checking the growth condition of hyphae every 12 hours after 3 days. Centrifuging at 0 deg.C at 3000r/min for 15min, and removing mycelium to obtain Chrysomyiame gacephala supernatant; sterilizing at 121.3 deg.C under high temperature and high pressure of 103kpa for 20min to kill residual bacteria, centrifuging at 0 deg.C at 8000r/min for 15min, heating in 100 deg.C water bath to remove most of solvent to obtain extract of antibacterial peptide of maggot, and freezing at-18 deg.C for storage.
Preparation of comparative example 1
A preparation method of maggot antibacterial peptide is different from the preparation example 1 in that: and (3) preparing milk powder and cane sugar into feed according to the mass ratio of 1:0.25 without adding momordica grosvenori seeds.
Example 1
A preparation method of a composition containing maggot antibacterial peptide comprises the following steps:
step 1, adding mint extract and chitosan into water with the mass percent of 20%, and stirring and dispersing uniformly at the rotating speed of 80r/min until the mint extract and the chitosan are completely dissolved to obtain a mixed solution;
step 2, adding the maggot antibacterial peptide extracting solution prepared in the preparation example 1 into the mixed solution, and stirring the mixed solution at the rotating speed of 100r/min until the mixed solution is uniformly dispersed;
and 3, adding sodium benzoate into the solution obtained in the step 2, and stirring the solution at the rotating speed of 100r/min until the solution is uniformly dispersed to obtain the composition containing the maggot antibacterial peptide.
The amounts of the above components are shown in Table 1 in g.
Example 2
A method for preparing a composition containing maggot antibacterial peptide, which is different from the composition in the example 1: the extract of the maggot antibacterial peptide of preparation example 1 was replaced with the extract of the maggot antibacterial peptide of preparation example 2 in equal amounts, the amounts of the components are detailed in table 1, and the unit is g.
Example 3
A method for preparing a composition containing maggot antibacterial peptide, which is different from the composition in the example 1: the extract of the maggot antibacterial peptide of preparation example 1 was replaced with the extract of the maggot antibacterial peptide of preparation example 3 in equal amounts, the amounts of the components are detailed in table 1, and the unit is g.
Example 4
A method for preparing a composition containing maggot antibacterial peptide, which is different from the composition in the example 1: the amounts of the components used are different, see in particular table 1, in g.
Example 5
A method for preparing a composition containing the maggot antibacterial peptide, which is different from the preparation method of the example 1 in that: adding collagen, chitin, a mint extract and chitosan into water, and stirring and dispersing uniformly at a rotating speed of 80r/min until the collagen, the chitin, the mint extract and the chitosan are completely dissolved to obtain a premix; adding beta-cyclodextrin into water, stirring and dispersing uniformly at the rotating speed of 100r/min until the beta-cyclodextrin is completely dissolved to obtain a beta-cyclodextrin aqueous solution; and adding the dispersed premix into a beta-cyclodextrin aqueous solution, and stirring at the rotating speed of 60r/min until the premix is uniformly dispersed to obtain a mixed solution.
And the amount of each component is different, see table 1 specifically.
Example 6
A method for preparing a composition containing maggot antibacterial peptide, which is different from the composition in the example 5 in that: the amounts of the components are different, and the specific reference is made to table 1.
TABLE 1
Comparative example 1
A method for preparing a composition containing maggot antibacterial peptide, which is different from the composition in the example 1: the maggot antibacterial peptide extract of the preparation example 1 is replaced by the maggot antibacterial peptide extract of the preparation example 1 in an equal amount.
Comparative example 2
A method for preparing a composition containing maggot antibacterial peptide, which is different from the composition in the example 1: the extract of the maggot antibacterial peptide of the preparation example 1 was replaced with water in equal amount.
Comparative example 3
A method for preparing a composition containing maggot antibacterial peptide, which is different from the composition in the example 1: the chitosan was replaced with equal amount of water.
Comparative example 4
A method for preparing a composition containing maggot antibacterial peptide, which is different from the composition in the example 1: equal amount of water was used to replace the mint extract.
Experiment 1
Irritation test
Taking the composition containing the maggot antibacterial peptide prepared in each example and proportion as a sample, according to 2.3.3.4.2 skin irritation tests of technical Specification for disinfection (2022 edition), on the day before the experiment, two sides of spinal column hairs of a rabbit are cut off, the hair removal range is about 3cm multiplied by 3cm, 0.5mL of the sample is smeared on the left-side hair removal skin with the range of 2.5cm multiplied by 2.5cm, the sample is smeared once a day and is smeared for 14 days continuously, the right-side skin is used as a control, the hair removal is carried out before smearing every day from the next day, the skin reaction is observed after one hour, the skin irritation intensity of the tested object to the skin is judged according to the skin irritation reaction scoring standard in the technical Specification for disinfection, and the skin irritation intensity grading standard is judged.
The experimental results are as follows: after the composition containing the maggot antibacterial peptide prepared by the above examples and the comparative examples is continuously applied for 1 to 14 days, no erythema or edema appears in the rabbits, and the scores are all 0 according to the reaction score standard, which indicates that the composition containing the maggot antibacterial peptide prepared by the examples and the comparative examples has no irritation to the rabbits.
Experiment 2
Bacteriostasis test
Respectively inoculating strains of Staphylococcus aureus, Escherichia coli, Candida albicans and Pseudomonas aeruginosa in nutrient broth, culturing at 37 deg.C for 24 hr, and diluting the bacterial suspension with the broth to 0.5 McLeod turbidity with McLeeb turbidimeter to reach bacterial concentration close to 1.5 × 10 8 Diluting with CFU/mL 10 times, namely injecting 100 μ L of the bacterial liquid into sterilized test tube, and injecting 10mL of culture liquid to dilute to make the concentration of bacterial liquid approach to 1.5 × 10 7 CFU/mL. Diluting the diluted concentration with sterilized cotton swabIs 10 7 100 mu L of bacterial liquid of CFU/mL staphylococcus aureus, escherichia coli, candida albicans and pseudomonas aeruginosa is uniformly coated on the surfaces of 4 nutrient agaragar. Putting 6 sterilized oxford cups (numbered: ② (r)), (100 microliter) of stock solution with corresponding types and concentrations into the holes of the oxford cups (i-example 1, i-example 5, i-comparative example 1, i-comparative example 2, i-comparative example 3, i-comparative example 4), placing the nutrient agar medium in a 37 ℃ incubator for culturing for 24 hours, and precisely measuring the diameter (mm) of a bacteriostatic ring formed by each oxford cup after the culture is finished.
The test results of experiment 2 are recorded in table 2.
TABLE 2
Different from the feed for feeding the maggot antibacterial peptide adopted in the comparative example 1, the maggot feed of the example 1 is added with the momordica grosvenori seed extract relative to the feed of the comparative example 1, other conditions are the same, and the inhibition effect of the composition containing the maggot antibacterial peptide in the example 1 on escherichia coli, candida albicans and pseudomonas aeruginosa is obviously enhanced relative to the feed of the comparative example 1 according to the data in the table 2, wherein the effect of enhancing the candida albicans is most obvious, and the feed added with the momordica grosvenori seed has obvious effect of improving the maggot antibacterial peptide to inhibit escherichia coli, candida albicans and pseudomonas aeruginosa.
The example 1 is added with the maggot antibacterial peptide, the chitosan and the mint extract respectively compared with the comparative examples 2-4, the effect of the composition containing the maggot antibacterial peptide in the example 1 on inhibiting staphylococcus aureus, escherichia coli, candida albicans and pseudomonas aeruginosa is obviously improved compared with the comparative examples 2-4, and the combination of the maggot antibacterial peptide extract, the chitosan and the mint extract is proved to generate obvious synergistic antibacterial effect.
Experiment 3
Mouse ear swelling experiment (anti-inflammatory experiment)
According to GQT-ZY-DL-002, the guide book of mouse ear swelling test, the compositions containing the maggot antimicrobial peptides prepared in example 1, example 5 and comparative examples 1-5 were used as samples to perform a mouse ear swelling test.
The test method is as follows:
1. the SPF-grade KM mice after qualified quarantine are randomly grouped into 10 groups, namely a test object group and a model control group of examples 1 and 5 and comparative examples 1-4.
2. After the mice are classified into groups for quarantine, the left ear is not treated to be a background value, the inner side and the outer side of the right ear are coated with 30u L xylene solution, after 30min, the model control group is not treated, the two sides of the right ear of the mice in the test object group are coated with 100mg of corresponding samples, after the samples are coated for 1h, cervical vertebra is whitened to kill the mice, the test objects are wiped off, the two ears are cut off, and the ear pieces (the same parts of the left ear and the right ear) with the diameter of 8mm are weighed and recorded. The weight difference between the left ear and the right ear of the same animal is the swelling degree.
Swelling degree-right ear weight-left ear weight
The swelling inhibition rate is (average swelling degree of model control group-average swelling degree of test substance group)/average swelling degree of model control group x 100%.
3. Statistical analysis, experimental data expressed as mean soil standard deviation (x days S) and independent sample T-test was performed using SPSS software.
And (3) test results: compared with the model control group, the ear swelling degree of the mice in the test object group is obviously reduced, the difference has statistical significance (P is less than 0.05), and the inhibition effect of the ear swelling of the mice is shown in table 3.
TABLE 3
| Swelling degree of ear (mg) | Inhibition ratio of ear swelling (%) | |
| Model control group | 23.41±2.86 | - |
| Example 1 | 13.97±1.45 | 40.32 |
| Example 5 | 12.03±0.64 | 48.61 |
| Comparative example 1 | 18.16±1.41 | 22.43 |
| Comparative example 2 | 23.34±2.57 | 0.30 |
| Comparative example 3 | 13.98±2.78 | 40.28 |
| Comparative example 4 | 14.08±1.26 | 39.85 |
As can be seen from comparison of the data of example 1 and comparative example 1 in table 3, the anti-inflammatory effect of the feed supplemented with momordica grosvenori seed-fed maggots of example 1 was enhanced relative to comparative example 1.
Compared with the example 1, when the extractive solution of the maggot antibacterial peptide is not added in the comparative example 2, the maggot antibacterial peptide composition has no obvious anti-inflammatory effect, so that the addition of the maggot antibacterial peptide extractive solution plays a key role in improving the anti-inflammatory effect of the maggot antibacterial peptide composition, and the comparison of the results of the example 1 and the comparative examples 3-4 shows that the anti-inflammatory effect of the maggot antibacterial peptide composition is improved to a certain extent after the maggot antibacterial peptide extractive solution, the chitosan and the mint extracts are added simultaneously.
The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.
Claims (9)
1. A preparation method of maggot antibacterial peptide is characterized by comprising the following steps: the method comprises the following steps:
step A, taking maggot eggs, feeding until maggots turn yellow, and collecting maggot living bodies before pupation begins, wherein the feed contains at least momordica grosvenori seeds;
step B, cleaning the maggot living bodies, shearing the maggot living bodies, adjusting the temperature to be 5-15 ℃, mixing the maggot living bodies with normal saline, juicing and centrifuging the mixture, heating the supernatant in water bath at 95-100 ℃ for 10-15min, and centrifuging the supernatant again to obtain a maggot crude extract;
and step C, taking a crude extractive solution of the maggots, and mixing the crude extractive solution with water according to a volume ratio of 1: 0.2-1, obtaining a mixed solution, adding glucose according to the proportion that the volume of the mixed solution and the mass of the glucose are 100mL:3-5g, carrying out sterilization treatment, then inoculating rice mold spores accounting for 7-15% of the volume of the mixed solution, culturing for 3-4 days, centrifuging to obtain a maggot supernatant, sterilizing, centrifuging, and concentrating to obtain the maggot antibacterial peptide extracting solution.
2. The method for preparing maggot antibacterial peptide according to claim 1, characterized in that: the feed consists of momordica grosvenori seeds, milk powder and cane sugar according to the mass ratio of 1:0.2-0.5: 0.05-0.2.
3. The method for preparing maggot antibacterial peptide according to claim 1, characterized in that: and in the step B, the maggot living bodies are washed by formaldehyde normal saline solution and ethanol in sequence and then by distilled water.
4. The method for preparing maggot antibacterial peptide according to claim 1, characterized in that: in the step B, the volume ratio of the maggot living bodies to the physiological saline is 1: 0.2-1 mixing.
5. The method for preparing maggot antibacterial peptide according to claim 1, characterized in that: in the step C, the rice mold spores are cultured under the environment with the temperature of 25-35 ℃, the humidity of 50-70% and the pH value of 4.6-7.
6. A composition containing maggot antibacterial peptide is characterized in that: the paint is mainly prepared from the following components in percentage by mass:
the extract of the maggot antibacterial peptide of any one of claims 1-5 10-30%
0.3 to 1 percent of chitosan
Mint extract 0-2%
20 to 80 percent of water
0 to 0.3 percent of preservative.
7. The maggot antibacterial peptide composition according to claim 6, wherein the composition comprises the following components: the composition containing the maggot antibacterial peptide is also added with the following components in percentage by mass
0.1 to 1 percent of chitin
Collagen 0.5-3%
Beta-cyclodextrin 0.5-5%.
8. A process for preparing a maggot antibacterial peptide composition according to any one of claims 6-7, which comprises the following steps: the method comprises the following steps:
step 1, mixing a mint extract, chitosan and water, and uniformly stirring to obtain a mixed solution;
step 2, adding the maggot antibacterial peptide extracting solution into the mixed solution, and stirring until the maggot antibacterial peptide extracting solution is uniformly dispersed;
and 3, adding a preservative into the solution obtained in the step 2, and stirring until the preservative is uniformly dispersed to obtain the composition containing the maggot antibacterial peptide.
9. The maggot antimicrobial peptide-containing composition according to claim 8, wherein the composition comprises: the preparation of the mixed solution comprises the following steps:
step 1-1, adding chitin, collagen, mint extract and chitosan into water with the mass percentage of 20-50%, and stirring and dispersing uniformly until the chitin, the collagen, the mint extract and the chitosan are completely dissolved to obtain a premix; adding beta-cyclodextrin into the rest water, stirring and dispersing uniformly until the beta-cyclodextrin is completely dissolved to obtain a beta-cyclodextrin aqueous solution;
and step 1-2, adding the dispersed premix into a beta-cyclodextrin aqueous solution, and stirring until the dispersion is uniform to obtain a mixed solution.
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| CN113599498A (en) * | 2021-08-13 | 2021-11-05 | 陈日和 | Composite antibacterial peptide and preparation method thereof, and bee venom conditioning cream capable of rapidly relieving pain and preparation method thereof |
| CN113599498B (en) * | 2021-08-13 | 2023-09-26 | 广东日和堂医药科技有限公司 | Composite antibacterial peptide and preparation method thereof, and bee venom conditioning cream capable of quickly relieving pain and preparation method thereof |
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