CN102321164A - Preparation method and application of insect natural antibacterial peptide products - Google Patents
Preparation method and application of insect natural antibacterial peptide products Download PDFInfo
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- CN102321164A CN102321164A CN201110136326A CN201110136326A CN102321164A CN 102321164 A CN102321164 A CN 102321164A CN 201110136326 A CN201110136326 A CN 201110136326A CN 201110136326 A CN201110136326 A CN 201110136326A CN 102321164 A CN102321164 A CN 102321164A
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Abstract
The invention discloses a preparation method and an application of insect natural antibacterial peptide products. The preparation method comprises the following steps that: 1) mealworm, housefly and dacus dorsalis hendel insects are used as raw materials; supernatant fluid is extracted through centrifugation after phosphate buffer is adopted for carrying out sufficient homogenate treatment on the raw materials; the supernatant fluid is filtered by a membrane, is primarily purified through a membrane module with the interception quantity being 30KD and is finely purified through a membrane module with the interception quantity being 10KD, then, permeate liquid is collected; the permeate liquid is chromatographed through a cation exchange column, and samples combined with the cation exchange column are collected; then, the samples combined with the cation exchange column are desalted through a membrane module with the interception quantity being 5KD, and the permeate liquid is collected; and the insect natural antibacterial peptide products can be obtained after the spray drying of the permeate liquid. The preparation method has the characteristics that that simplicity, convenience and high speed are realized, the prepared antibacterial peptide has high purity, the environment is protected, and the safety is realized. The prepared antibacterial peptide products can be widely applied to the fields of medical raw materials, food, fruit and vegetable anticorrosion and fresh keeping, feedstuff additives, skin care products and the like.
Description
Technical field
The present invention relates to a kind of preparation method and application thereof of insect natural antimicrobial peptide products.The present invention relates to the technical field of biologically active substance separation and purification.
Background technology
Antibiotic discovery such as penicillium mould has brought revolutionary leap for pathogenic bacterial infection property treatment of diseases.But the surprisingly speed that produces of pathogenic bacteria resistance and the popularity of resistance.The frequency that present anti-multiple medicines bacterium occurs surpasses the tempo of development of new antibiotic.In recent years, the microbiotic of novel structure finds that plaque is weary in clinical treatment, and the fast development of anti-medicine flora makes some infection become the difficult problem of clinical treatment.Microbiotic, Chemical Preservative add in food, feed and the makeup long-standing as the pathogenic bacteria suppressor factor.Since the 1950's, along with being widely used of microbiotic and Chemical Preservative and people improve constantly the requirement of food quality and environmental quality, their spinoff also just shows day by day, is faced with the situation that is eliminated.Be illustrated in 5,000,000 philtrums that cancer is suffered from the whole world every year according to the new portion report of announcing of AHF and National Cancer Institute, have about 50% relevantly, and microbiotic or the Chemical Preservative that suffers from about 30% in the food arranged wherein with food contamination.In order to tackle above variety of problems, people are are constantly researching and developing new type natural antibacterials and antibiotic anticorrosive additive.The antibacterial peptide that occurs in recent years is exactly one type of novel antibacterial medicine, food antiseptic antioxidant fresh and fodder additives with great development potentiality.
Antibacterial peptide is a small molecule cationic peptide class biologically active substance, extensively exists in insect, plant, animal, mikrobe and the human body.Up to the present, obtain more than 800 kind of antibacterial peptide from the organic sphere separation.Single from insect isolation identification go out more than 200 kind of antibacterial peptide.Though antibacterial peptide has resistance (antibacterium, fungi, virus, protozoon isoreactivity) widely, to normal mammalian cell nontoxicity.Antibacterial peptide does not bring out the generation of resistant mutation, most antibacterial peptides water-soluble better, to thermally-stabilised, to stronger tolerance all being arranged than heavy ion intensity, lower or higher pH.Comprehensively reported both at home and abroad, had at least the different bacterium more than 100 kinds to be killed by antibacterial peptide.Thereby antibacterial peptide can be used as medical and antibiotic alternative medicine or additive food aspect bacterinertness.The exploitation of antibacterial peptide and applied research have become the focus of area researches such as domestic and international insectology, phytology, microbiology, physiology, pharmacology.
Based on the vital role of antibacterial peptide, many scholars carry out the searching and the preparation research of novel antibacterial peptide.Seek and prepare antibacterial peptide three kinds of approach are arranged: chemosynthesis, biotechnology (recombinant technology) and the natural antibacterial peptide class of screening from biology.Chemosynthesis often receives the restriction of the nfrastructure of synthetic starting point.Also there is problems such as causing folding inaccurate protein like incorrect transcribing in biotechnology.The screening antibacterial peptide has advantages such as the source is abundant, material is natural from biology, thereby extremely antibacterial peptide research worker's favor.
Insect is as yet not by the huge Biological resources of abundant development and use on the earth.Caste various (about 2,000,000 kinds), enormous amount is being contained very abundant biotic potential.Insect contains rich in protein and amino acid, and on average about 50%, the content of essential amino acid is very high for its crude protein content, the pattern that aminoacid profile meets or proposes near FAO.Insect body includes and also contains fat, VITAMINs, trace element, carbohydrate, antibacterial peptide, SOD, lectin, chitin and other material.Insect not only can be used as food, also has therapeuticing and health effect, i.e. integration of drinking and medicinal herbs.Housefly, tenebrio molitor and citrus fruit fly etc. are to account for important one type in the resource insect family; Use but only be used as a kind of feed protein resource for a long time, and its value in fields such as food, medicine, feed and skin care item is not carried out further investigation and abundant development and utilization.
Summary of the invention
A kind of preparation method and application thereof of insect natural antimicrobial peptide products have been the object of the present invention is to provide.The present invention has easy to be quick, the antibacterial peptide purity height of preparation and the characteristics of Environmental Safety.
In order to reach above-mentioned purpose, solution of the present invention is:
A kind of preparation method of insect natural antimicrobial peptide products comprises the steps:
1) screening of raw materials: selecting tenebrio molitor, housefly and citrus fruit fly insect for use is starting material;
2) extract and thermal treatment: after adopting phosphate buffered saline buffer that the abundant homogenate of starting material is handled through the centrifugal extraction supernatant after 10 minutes of thermal treatment in 100 ℃ of boiling water baths;
3) the tangential flow fractional separation is handled: supernatant through behind the membrane filtration successively through interception be 30KD film bag just pure, interception be that collect through liquid the consummate back of 10KD film bag;
4) cation exchange column chromatography: see through liquid through cationic exchange coloum (CM Sepharose Fast Flow post, XK 16/20) chromatography, collection combines sample with cationic exchange coloum;
5) tangential flow desalting treatment: will combine sample at last with cationic exchange coloum is 5KD film bag desalting treatment through interception, collects to see through liquid;
6) spraying drying: see through liquid and promptly obtain the insect natural antimicrobial peptide products after spray-dried.
Described step 2) adopts 4 ℃ of precoolings, 0.2M, the phosphate buffered saline buffer of pH6.0 (PB) in; And phosphate buffered saline buffer and starting material are weight ratio (0.5-1.5) ml by volume: (3-7) the mixed homogenized of g is after 4 ℃ of lixiviates centrifugal treating after 24 hours.
The described phosphate buffered saline buffer and the optimal seeking of raw and processed materials be the mixed of weight ratio 1ml:5g by volume.
Membrane filtration adopts 0.45 μ m filter membrane in the described step 3); Select for use the cross-flow ultrafiltration system that it is separated step by step, selecting interception for use is 30 KD, and tangential flow speed is 56L/H; Interception is 10 KD, tangential flow speed be the film bag of 32L/H through 2 fractional separation, concentrate 10 times at every turn.
Described film bag specification is 0.7m
2
Cation exchange column chromatography separates 2 kinds of elutriant: pH6.0 of employing, 50 mM ammonium acetate buffer solution A and pH6.0,1M ammonium acetate buffer solution B in the described step 4); Through linear gradient elution, promptly by behind 3 column volumes of 100% ammonium acetate buffer solution A-100% ammonium acetate buffer solution B wash-out, collection combines sample with cationic exchange coloum.
Adopt the cross-flow ultrafiltration system that it is carried out desalting treatment in the described step 5), selecting interception for use is 5KD, and tangential flow speed is 6L/H, collects after concentrating 10 times and sees through liquid.
Spraying drying adopts under 120 ℃ of conditions in the described step 6), and the speed of 2 mL/min is sprayed dried.
A kind of application of insect natural antimicrobial peptide products adopts the antimicrobial peptide products of method for preparing can be used as medical material, food, fruit and vegetable preserving antistaling agent, fodder additives and skin care item.
Beneficial effect of the present invention is: it is abundant, cheap that the present invention has raw material sources, easy to be quick; The antibacterial peptide purity height and the Environmental Safety of preparation; Product of the present invention is compared with the chemosynthesis antiseptic-germicide to have nontoxicly, and noresidue does not receive the restriction of the nfrastructure of synthetic starting point; Select resource insect as starting material, have higher edible safety, and it is low to have a production cost, the cycle is short, the characteristics that yield is high.The antimicrobial peptide products of preparation can be widely used in fields such as medical material, food, antiseptic preservation of fruits and vegetables and fodder additives and skin care item.
Description of drawings
Fig. 1 is preparation technology's schema of the present invention.
Embodiment
Come further explain the present invention below in conjunction with Fig. 1 and specific embodiment, but embodiment of the present invention is not limited thereto.
Embodiment 1
At first take by weighing fruit bat, housefly and citrus fruit fly resource insect; Sterile purified water cleans the back seasoning, adds phosphate buffered saline buffer (PB, the 0.2M of 4 ℃ of precoolings in the volume weight ratio of 1ml: 5g; PH6.0); (described phosphate buffered saline buffer and raw-material blending ratio can adopt (0.5-1.5) ml: (3-7) arbitrary value in the volume weight ratio of g) after the abundant homogenized, centrifugal behind 4 ℃ of refrigerator lixiviate 24 h (4 ℃, centrifugal 30 minutes of 10000 * g); Precipitate lixiviate repeatedly for several times, merge supernatant.Supernatant was put in 100 ℃ of boiling water baths thermal treatment 10 minutes, then in centrifugal 30 minutes (4 ℃, 10000 * g), collect supernatant.Supernatant is behind 0.45 μ m membrane filtration; Select for use the cross-flow ultrafiltration system that it is separated step by step; Selecting interception for use is that (all concentrate 10 times, film bag specification is 0.7m through 2 tangential flow fractional separation for the film bag (tangential flow speed is 32L/H) of 30 KD film bags (tangential flow speed is 56L/H), 10 KD
2)); Collect the liquid that sees through after 10 KD film bag tangential flows are separated; Last cationic exchange coloum (CM Sepharose Fast Flow post, XK 16/20) carries out chromatographic separation, adopts 2 kinds of elutriant: pH6.0 in the chromatography process; 50 mM ammonium acetate buffer solution A and pH6.0,1M ammonium acetate buffer solution B; Through linear gradient elution, promptly by behind 3 column volumes of 100% ammonium acetate buffer solution A-100% ammonium acetate buffer solution B wash-out, collection combines sample with cationic exchange coloum.Final sample carries out desalination through 5KD film bag (tangential flow speed is 6L/H), collects after concentrating 10 times and sees through liquid, and the speed of 2 mL/min is sprayed driedly under 120 ℃ of conditions, collects spray powder, promptly gets the insect natural antimicrobial peptide products.The method of present embodiment is easy fast, and the antibacterial peptide purity of preparation is high.The antimicrobial peptide products of preparation can be widely used in medical material---antibacterials, food, antiseptic preservation of fruits and vegetables and fodder additives and skin-care product additive.
Claims (9)
1. the preparation method of an insect natural antimicrobial peptide products is characterized in that comprising the steps:
1) screening of raw materials: selecting tenebrio molitor, housefly and citrus fruit fly insect for use is starting material;
2) extract and thermal treatment: after adopting phosphate buffered saline buffer that the abundant homogenate of starting material is handled through the centrifugal extraction supernatant after 10 minutes of thermal treatment in 100 ℃ of boiling water baths;
3) the tangential flow fractional separation is handled: supernatant through behind the membrane filtration successively through interception be 30KD film bag just pure, interception be that collect through liquid the consummate back of 10KD film bag;
4) cation exchange column chromatography: see through liquid through cation exchange column chromatography, collection combines sample with cationic exchange coloum;
5) tangential flow desalting treatment: will combine sample at last with cationic exchange coloum is 5KD film bag desalting treatment through interception, collects to see through liquid;
6) spraying drying: see through liquid and promptly obtain the insect natural antimicrobial peptide products after spray-dried.
2. the preparation method of a kind of insect natural antimicrobial peptide products as claimed in claim 1 is characterized in that: adopt 4 ℃ of precoolings, 0.2M, the phosphate buffered saline buffer of pH6.0 described step 2); And phosphate buffered saline buffer and starting material are weight ratio (0.5-1.5) ml by volume: (3-7) the mixed homogenized of g is after 4 ℃ of lixiviates centrifugal treating after 24 hours.
3. the preparation method of a kind of insect natural antimicrobial peptide products as claimed in claim 2 is characterized in that: described phosphate buffered saline buffer and starting material are the mixed of weight ratio 1ml:5g by volume.
4. the preparation method of a kind of insect natural antimicrobial peptide products as claimed in claim 1 is characterized in that: membrane filtration adopts 0.45 μ m filter membrane in the described step 3); Select for use the cross-flow ultrafiltration system that it is separated step by step, selecting interception for use is 30 KD, and tangential flow speed is 56L/H; Interception is 10 KD, tangential flow speed be the film bag of 32L/H through 2 fractional separation, concentrate 10 times at every turn.
5. the preparation method of a kind of insect natural antimicrobial peptide products as claimed in claim 4 is characterized in that: described film bag specification is 0.7m
2
6. the preparation method of a kind of insect natural antimicrobial peptide products as claimed in claim 1; It is characterized in that: cation exchange column chromatography separates 2 kinds of elutriant: pH6.0 of employing in the described step 4); 50 mM ammonium acetate buffer solution A and pH6.0,1M ammonium acetate buffer solution B; Through linear gradient elution, promptly by behind 3 column volumes of 100% ammonium acetate buffer solution A-100% ammonium acetate buffer solution B wash-out, collection combines sample with cationic exchange coloum.
7. the preparation method of a kind of insect natural antimicrobial peptide products as claimed in claim 1; It is characterized in that: adopt the cross-flow ultrafiltration system that it is carried out desalting treatment in the described step 5); Selecting interception for use is 5KD, and tangential flow speed is 6L/H, collects after concentrating 10 times and sees through liquid.
8. the preparation method of a kind of insect natural antimicrobial peptide products as claimed in claim 1 is characterized in that: spraying drying adopts under 120 ℃ of conditions in the described step 6), and the speed of 2 mL/min is sprayed dried.
9. the application of an insect natural antimicrobial peptide products as claimed in claim 1 is characterized in that: adopt the antimicrobial peptide products of method for preparing can be used as medical material, food, fruit and vegetable preserving antistaling agent, fodder additives and skin care item.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104336324A (en) * | 2013-07-31 | 2015-02-11 | 天津鑫泽众生物技术有限公司 | Probiotic antibacterial peptide and its preparation method |
| CN104356215A (en) * | 2014-10-08 | 2015-02-18 | 广州格拉姆生物科技有限公司 | Antibacterial peptide of bactrocera dorsalis hendel and derivatives and application of antibacterial peptide |
| CN104472552A (en) * | 2014-11-21 | 2015-04-01 | 沈阳化工研究院有限公司 | Tenebrio molitor extract as well as separation method and application thereof |
| CN104740599A (en) * | 2015-03-03 | 2015-07-01 | 昆明理工大学 | Compound housefly antimicrobial peptide preparation |
| CN107156547A (en) * | 2017-07-03 | 2017-09-15 | 广州禾立田生物科技有限公司 | A kind of feed of artificial breeding natural enemy ladybug |
| CN107903300A (en) * | 2017-11-16 | 2018-04-13 | 罗乌支 | A kind of method that native peptides are extracted from fly |
| CN110272474A (en) * | 2019-05-15 | 2019-09-24 | 中国农业大学 | Barley worm natural antibacterial albumen and the preparation method and application thereof |
| CN114097711A (en) * | 2021-11-23 | 2022-03-01 | 华南理工大学 | Method for inducing yellow mealworms to produce antibacterial peptide, preparation method and application of yellow mealworms antibacterial peptide |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104336324A (en) * | 2013-07-31 | 2015-02-11 | 天津鑫泽众生物技术有限公司 | Probiotic antibacterial peptide and its preparation method |
| CN104356215A (en) * | 2014-10-08 | 2015-02-18 | 广州格拉姆生物科技有限公司 | Antibacterial peptide of bactrocera dorsalis hendel and derivatives and application of antibacterial peptide |
| CN104472552A (en) * | 2014-11-21 | 2015-04-01 | 沈阳化工研究院有限公司 | Tenebrio molitor extract as well as separation method and application thereof |
| CN104740599A (en) * | 2015-03-03 | 2015-07-01 | 昆明理工大学 | Compound housefly antimicrobial peptide preparation |
| CN107156547A (en) * | 2017-07-03 | 2017-09-15 | 广州禾立田生物科技有限公司 | A kind of feed of artificial breeding natural enemy ladybug |
| CN107903300A (en) * | 2017-11-16 | 2018-04-13 | 罗乌支 | A kind of method that native peptides are extracted from fly |
| CN110272474A (en) * | 2019-05-15 | 2019-09-24 | 中国农业大学 | Barley worm natural antibacterial albumen and the preparation method and application thereof |
| CN114097711A (en) * | 2021-11-23 | 2022-03-01 | 华南理工大学 | Method for inducing yellow mealworms to produce antibacterial peptide, preparation method and application of yellow mealworms antibacterial peptide |
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Application publication date: 20120118 |