CN102558387A - Method for extracting chitin and antibacterial peptide from fly larvae - Google Patents
Method for extracting chitin and antibacterial peptide from fly larvae Download PDFInfo
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- CN102558387A CN102558387A CN2010106029293A CN201010602929A CN102558387A CN 102558387 A CN102558387 A CN 102558387A CN 2010106029293 A CN2010106029293 A CN 2010106029293A CN 201010602929 A CN201010602929 A CN 201010602929A CN 102558387 A CN102558387 A CN 102558387A
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Abstract
The invention belongs to the field of product extraction technology and relates to a method for extracting chitin and antibacterial peptide from fly larvae. The method comprises the following steps of: firstly disinfecting and rinsing fresh fly larvae to be clean, then extruding the cleaned fly larvae to separate the fly larvae skin from visceral mass serous fluid; firstly soaking with hydrochloric acid solution, drying after washing, and then soaking with NaOH solution to remove protein; retaining residual liquid, drying after cleaning, adding the NaOH solution after crushing, carrying out water bath at the temperature of 98 DEG C, and heating for reaction; putting chitosan into acetic acid solution, stopping reaction after the chitosan is dissolved, filtering, cleaning, drying at the temperature of 50 DEG C, and obtaining the chitosan; and then adding acetic acid into the visceral mass serous fluid of the fly larvae, homogenizing, standing over the night at the temperature of 4 DEG C, filtering, adsorbing the filtrate with alginic acid, filtering, then eluting the antibacterial peptide adsorbed on the alginic acid with the hydrochloric acid solution, adjusting the pH value of the eluent to be 3.5, carrying out precipitation with NaCl, carrying out dialysis and desalting, drying and obtaining a crude antibacterial peptide product. The method has the advantages of reasonable technical route, low cost, high yield, large economic value, environmentally-friendly effect and effective utilization of the fly larvae resources for preparing natural product varieties.
Description
Technical field:
The invention belongs to the natural product extracting technique field, relate to a kind of comprehensive processing and utilization technical matters method of fly maggot, particularly a kind of from fly maggot the method for chitin extraction and antibacterial peptide.
Background technology:
The prolificacy of fly ranks first place in insect world, it is documented that a pair of fly can breed 2,660 hundred million of fly maggots in 4 months, and the fly cultivating condition is simple; It is low that envrionment conditions requires, and raw material is easy to get, and the waste material in people's animal dung, sludge of sewage treatment plants, slaughterhouse and useless watery blood all can act on the nutrition source of fly maggot; So in Similar types of materials, the aquaculture cost of fly maggot is lower, it is documented and to know; Crude protein in the dry fly maggot accounts for 55-65%, and the indispensable amino acid ratio is the high-quality protein source above non-essential amino acid; The content of chitin accounts for 30%-54.8% in the dry fly maggot, and a large amount of antimicrobial substances is arranged.At present, utilize fly maggot not see that as yet report is arranged, existing extraction process for the maturation process method that raw material adopts physics and chemical technology comprehensive processing technology to extract natural chitin and antibacterial peptide; Its principle and complicated steps; Processing condition are overcritical, and extraction cost is high, and envrionment conditions is poor; Product yield is low, and economic benefit is not obvious.So seeking to design a kind of fly maggot that utilizes is the direction of industry scientific research personnel effort for the mature technology of raw material chitin extraction, chitosan and chitooligosaccharide-and extraction antibacterial peptide.
Summary of the invention:
The objective of the invention is to overcome the shortcoming that prior art exists; Seek to design a kind of fly maggot that utilizes and be basic raw material; Technical matters method through physics and chemical technology processing chitin extraction and antibacterial peptide; This method is fully drawn the advantage of prior art, through after the comprehensive treating process economic benefit of fly maggot is multiplied.
To achieve these goals, the present invention with after sterilization of bright fly maggot process and the rinsing totally, with the pressure roller extruding, makes the fly maggot skin separate with the visceral mass slurries earlier, and the fly maggot skin is as the raw material of chitin extraction, and the visceral mass slurries are as the raw material that extracts antibacterial peptide; Its concrete technology comprises that chitooligosaccharide-purifying, insect antimicrobial peptide extract and three modules of protein powder purifying;
The chitooligosaccharide-purifying: the fly maggot to collecting screens, remove impurity after, using earlier weight percent concentration is that 1% hydrochloric acid soln soaks fly maggot skin number hour; Dry after the washes clean, use weight percent concentration to remove Deproteinization in 1-20 hour again, keep raffinate as the 4%NaOH solution soaking; Dry after the washes clean; Add the solution that the weight percentage concentration is 40%NaOH after crushed, 98 ℃ of heating of water-bath keep reaction 8 hours; Repeat to purify once, it is 1% acetic acid soln dissolving back stopped reaction that the chitosan that takes a morsel is put into weight percent concentration again, filter, washing, 50 ℃ dry chitosan; Getting chitosan, to be dissolved in weight percent concentration be 5% acetic acid soln, and adjust pH 5.5-6 adds chitin enzyme and cellulase, 50 ℃ of enzyme digestion reaction 24h; Become muddy yellow homogeneous phase non-sticky solution, filter, making the chitooligosaccharide-mean polymerisation degree in the filtrating is 10; Use ethanol sedimentation, filter, the dry chitooligosaccharide-that gets;
Insect antimicrobial peptide extracts: in fly maggot visceral mass slurries, add the concentration of acetate to 0.5mol/L, and homogenate, 4 ℃ are spent the night, and filter; Filtrating is antibacterial peptide acidic extraction thing, with Lalgine absorption, filters; Be adsorbed on the antibacterial peptide on the Lalgine with 0.2mol/L hydrochloric acid soln wash-out again, use NaOH to transfer pH value to be 3.5 elutriant after, use the NaCl precipitating; Dialysis, desalination, the dry antibacterial peptide raw product that gets;
The purifying of crude protein powder: is weight percent concentration the raffinate merging after 4%NaOH deproteinated raffinate and antibacterial peptide acidic extraction thing adsorb with Lalgine, and regulating pH value is 7,50 ℃ of vacuum concentration, and ethanol sedimentation filters, and drying makes the crude protein powder; Remaining residual acid, alkali are mixed, and transferring pH value is 7, discharging.
The present invention uses fly maggot to make the finished product as raw material through processing to be chitooligosaccharide-, antibacterial peptide and protein powder; Its chitooligosaccharide-separating and purifying technology is the 1%HCl decalcification, the 4%NaOH Deproteinization, and the 45-55%NaOH deacetylation uses complex enzyme zymohydrolysis to be chitooligosaccharide-by chitosan; Antibacterial peptide separating and purifying technology principle is: fly maggot protein is starched with acetate acidifying sex change, uses the Lalgine fractionation by adsorption, uses the hydrochloric acid wash-out, vacuum concentration, and the NaCl precipitating, the chromatography desalination gets antibacterial peptide after the drying; 4%NaOH raffinate and albumen acetate raffinate merge, and regulating PH is 7, and heating, vacuum is concentrated at 1/2 o'clock, saturated ammonium sulphate solution precipitating, and the throw out heat drying gets protein powder.
The present invention compared with prior art, its technological line is reasonable, extraction cost is low, product goes out the rate height, economic worth is big, production environment is friendly, makes the natural product kind after can effectively utilizing the processing of fly maggot resource comprehensive.
Description of drawings:
Fig. 1 is a process step flow process schematic block diagram of the present invention.
Embodiment:
Through embodiment the present invention is further described below.
Present embodiment with the pressure roller extruding, makes the fly maggot skin separate with the visceral mass slurry earlier with after sterilization of bright fly maggot process and the rinsing totally, and the fly maggot skin is as the raw material of chitin extraction, and the visceral mass slurries are as the raw material of extraction antibacterial peptide;
(1) purifying of low chitosan: the fly maggot skin contains a large amount of chitins, protein, fat, mineral substance etc., and in order to produce a desired effect, at first using weight percent concentration is that 1% hydrochloric acid soln soaks fly maggot skin number hour; After the washes clean, the oven dry back uses weight percent concentration to remove Deproteinization in 1-20 hour as the 4%NaOH solution soaking, after the washes clean; Oven dry; Add the solution that the weight percentage concentration is 40%NaOH after crushed, 98 ℃ of heating of water-bath keep reaction 8 hours; Repeat to purify once, it is 1% acetic acid soln dissolving back stopped reaction that the chitosan that takes a morsel is put into weight percent concentration again, filter, washing, 50 ℃ dry chitosan; Getting chitosan, to be dissolved in weight percent concentration be 5% acetic acid soln adjust pH 5.5-6, adds chitin enzyme and cellulase, 50 ℃ of enzyme digestion reaction 24h; Become muddy yellow homogeneous phase non-sticky solution, filter, making the chitosan oligomer mean polymerisation degree in the filtrating is 10; Use ethanol sedimentation, filter, the dry chitosan oligomer that gets;
(2) insect antimicrobial peptide extracts: in fly maggot visceral mass slurries, add the concentration of acetate to 0.5mol/L, and homogenate, 4 ℃ are spent the night, and filter; Filtrating is antibacterial peptide acidic extraction thing, with Lalgine absorption, filters; With 0.2mol/L hydrochloric acid soln wash-out, use NaOH to transfer pH value to be 3.5 the elutriant after, use the NaCl precipitating; Dialysis, desalination, the dry antibacterial peptide raw product that gets;
(3) purifying of crude protein powder: is weight percent concentration the raffinate merging after Deproteinated raffinate of 4%NaOH and antibacterial peptide acidic extraction thing adsorb with Lalgine, and regulating pH value is 7,50 ℃ of vacuum concentration, and ethanol sedimentation filters, and drying makes the crude protein powder; Remaining residual acid, alkali are mixed, and transferring pH value is 7, discharging.
Present embodiment uses fly maggot to make the finished product as raw material through processing to be chitooligosaccharide-, antibacterial peptide and protein powder; Its chitooligosaccharide-separating and purifying technology principle is: the 1%HCl decalcification, and the 4%NaOH Deproteinization, the 45-55%NaOH deacetylation uses complex enzyme zymohydrolysis to be chitooligosaccharide-by chitosan; Antibacterial peptide separating and purifying technology principle is that fly maggot protein is starched with acetate acidifying sex change, uses the Lalgine fractionation by adsorption, uses the hydrochloric acid wash-out, vacuum concentration, and the NaCl precipitating, the chromatography desalination gets antibacterial peptide after the drying; 4%NaOH raffinate and albumen acetate raffinate merge, and regulating PH is 7, and heating, vacuum is concentrated at 1/2 o'clock, saturated ammonium sulphate solution precipitating, and the throw out heat drying gets protein powder.
Embodiment 1: get ight soil and cultivate fly maggot 100g, use 1/5000KMnO
4After the solution rinsing 10 minutes, the dregs of beans of input 1/100 slurry was cultivated 1 day, made fly maggot emptying liquid manure; With the clear water washing once, get bright fly maggot 91g again, the pressure roller extruding; Get fly maggot skin 35g and fly maggot visceral mass slurry 56g, fly maggot micromicro chitin extraction and chitosan, fly maggot visceral mass slurry can extract antibacterial peptide.
Embodiment 2:
(1) chitin preparation: 35g fly maggot skin adds the 250ml three-necked flask, adds 50ml 1% hydrochloric acid soln, and heating is 4 hours in 38-40 ℃ of water-bath, filters; Washing is extremely neutral for several times, 50 ℃ of dryings, and exsiccant fly maggot skin adds 4%NaOH50ml; Heating is 5 hours in the 96-98 water-bath, filters, and washing is for several times to neutral; Dry below 50 ℃, get chitin 4.9g, yield 14%.
(2) Preparation of Chitosan: chitin 4.9g is soaked among the NaOH of 100ml45%-55%; Sustained reaction is newly prepared the NaOH of 100ml45%-55% after 8 hours again in water-bath 98-99 ℃, in water-bath 98-99 ℃, continues to heat 8-16 hour; When treating that product dissolves fully in 1%-2% acetate; Reaction finishes, and washing is to the neutral 3.4g chitosan that gets, and yield is 70% of a chitin.
(3) preparation of chitooligosaccharide-: get fly maggot chitosan 3.4g and be dissolved in the 100ml acetate, regulate pH value, add chitin enzyme and cellulase 4g to 5.5-6; 50-55 ℃ of enzyme digestion reaction 10 hours, boiled 10 minutes at 100 ℃ again, use ethanol sedimentation then; The centrifuging and taking throw out with the acetone dehydration, takes off ester again; Get throw out, the dry chitooligosaccharide-white powder 2.04 that gets, yield is 60%.
Embodiment 3: gets 56g fly maggot visceral mass slurry and adds the concentration of acetate to 0.5mol/L, and homogenate, 4 ℃ are spent the night, centrifugal; Filter, get 200ml fly maggot antibacterial peptide acid extraction liquid, behind the adding Lalgine, stirred 1 hour; Antibacterial peptide is fully adsorbed by Lalgine, and 4 ℃ leave standstill, and spend the night, and use the B suction filtration; Collect the Lalgine that has adsorbed antibacterial peptide,, receive elutriant, after use solid sodium-acetate adjusting pH value is 3.5 with 0.2mol/L hydrochloric acid wash-out; NaCl with 320g/L saltouts, after spending the night, through dialysis tubing dialysis, desalination, the dry 0.6g antibacterial peptide bullion that gets.
Embodiment 4:4%NaOH chitin extraction raffinate and the two merging of antibacterial peptide acetic acid extraction liquid raffinate, regulating pH value is 7, and heating, vacuum is concentrated into 1/2, uses the saturated ammonium sulphate precipitating, and the deposition oven dry is crude protein dried bean noodles powder 13g.
The related solution of present embodiment is weight percent concentration except that indicating.
Claims (1)
1. the method for chitin extraction and antibacterial peptide from a fly maggot; After it is characterized in that elder generation is with sterilization of bright fly maggot process and rinsing totally,, the fly maggot skin is separated with the visceral mass slurries with the pressure roller extruding; The fly maggot skin is as the raw material of chitin extraction, and the visceral mass slurries are as the raw material that extracts antibacterial peptide; Concrete technology comprises that chitooligosaccharide-purifying, insect antimicrobial peptide extract and three modules of protein powder purifying;
The chitooligosaccharide-purifying: the fly maggot to collecting screens, remove impurity after, using earlier weight percent concentration is that 1% hydrochloric acid soln soaks fly maggot skin number hour; Dry after the washes clean, use weight percent concentration to remove Deproteinization in 1-20 hour again, keep raffinate as the 4%NaOH solution soaking; Dry after the washes clean; Add the solution that the weight percentage concentration is 40%NaOH after crushed, 98 ℃ of heating of water-bath keep reaction 8 hours; Repeat to purify once, it is 1% acetic acid soln dissolving back stopped reaction that the chitosan that takes a morsel is put into weight percent concentration again, filter, washing, 50 ℃ dry chitosan; Getting chitosan, to be dissolved in weight percent concentration be 5% acetic acid soln, and adjust pH 5.5-6 adds chitin enzyme and cellulase, 50 ℃ of enzyme digestion reaction 24h; Become muddy yellow homogeneous phase non-sticky solution, filter, making the chitooligosaccharide-mean polymerisation degree in the filtrating is 10; Use ethanol sedimentation, filter, the dry chitooligosaccharide-that gets;
Insect antimicrobial peptide extracts: in fly maggot visceral mass slurries, add the concentration of acetate to 0.5mol/L, and homogenate, 4 ℃ are spent the night, and filter; Filtrating is antibacterial peptide acidic extraction thing, with Lalgine absorption, filters; Be adsorbed on the antibacterial peptide on the Lalgine with 0.2mol/L hydrochloric acid soln wash-out again, use NaOH to transfer pH value to be 3.5 elutriant after, use the NaCl precipitating; Dialysis, desalination, the dry antibacterial peptide raw product that gets;
Crude protein powder purifying: is weight percent concentration the raffinate merging after 4%NaOH deproteinated raffinate and antibacterial peptide acidic extraction thing adsorb with Lalgine, and regulating pH value is 7,50 ℃ of vacuum concentration, and ethanol sedimentation filters, and drying makes the crude protein powder; Remaining residual acid, alkali are mixed, and transferring pH value is 7, discharging.
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| CN2010106029293A CN102558387A (en) | 2010-12-13 | 2010-12-13 | Method for extracting chitin and antibacterial peptide from fly larvae |
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Cited By (20)
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| CN102755258A (en) * | 2012-07-20 | 2012-10-31 | 梁小毛 | Skin cleaning healthcare solution and preparation method thereof |
| CN102755259A (en) * | 2012-07-20 | 2012-10-31 | 梁小毛 | Mouth healthcare cleaning solution and preparation method thereof |
| CN102755260A (en) * | 2012-07-20 | 2012-10-31 | 姚春宝 | Female pudendum cleaning health-care fluid and preparation method thereof |
| CN103908668A (en) * | 2014-04-03 | 2014-07-09 | 浙江大学 | Bots antibacterial peptide adjuvant and preparation method thereof, and vaccine formulation containing adjuvant and use thereof |
| CN105524158A (en) * | 2016-01-18 | 2016-04-27 | 任辉 | Fly maggot antibacterial peptide, encoding gene, carrier, engineering bacteria and application thereof |
| FR3031115A1 (en) * | 2014-12-31 | 2016-07-01 | Ynsect | PRODUCTION OF CHITINES FROM INSECTS BY ENZYMATIC HYDROLYSIS COMPRISING A COMBINATION OF PRELIMINARY STEPS |
| FR3031113A1 (en) * | 2014-12-31 | 2016-07-01 | Ynsect | PROCESS FOR PRODUCTION OF PRODUCT (S) OF INTEREST FROM INSECT BY ENZYMATIC HYDROLYSIS |
| CN105949273A (en) * | 2016-04-25 | 2016-09-21 | 张福义 | Preparation method for original antibacterial peptide serous fluid from housefly larvae |
| CN107257632A (en) * | 2014-12-31 | 2017-10-17 | 昆虫公司 | composition containing chitin and digestible protein |
| FR3060947A1 (en) * | 2016-12-28 | 2018-06-29 | Ynsect | METHOD OF TREATING INSECTS COMPRISING THE SEPARATION OF CUTICLES FROM THE MOLLE PART OF INSECTS THEN SEPARATING THE MOLLE PART INTO THREE FRACTIONS |
| FR3060946A1 (en) * | 2016-12-28 | 2018-06-29 | Ynsect | METHOD OF TREATING INSECTS COMPRISING THE SEPARATION OF CUTICLES FROM THE MOLLE PART OF INSECTS USING A BAND SEPARATOR |
| CN108220361A (en) * | 2018-03-15 | 2018-06-29 | 佛山汇沐化学科技有限公司 | A kind of extracting method of julid chitosan and its application |
| WO2018122353A1 (en) * | 2016-12-28 | 2018-07-05 | Ynsect | Method for treating insects, in which the cuticles are separated from the soft part of the insects using a belt separator |
| WO2018122352A1 (en) * | 2016-12-28 | 2018-07-05 | Ynsect | Method for treating insects, in which the cuticles are separated from the soft part of the insects, and the soft part is then separated into three fractions |
| CN110279720A (en) * | 2019-07-31 | 2019-09-27 | 浙江佰科堂生物科技股份有限公司 | A kind of maggot processed product and its application in adjusting intestinal flora |
| CN113080315A (en) * | 2021-05-08 | 2021-07-09 | 湖南自然创造生物科技有限公司 | Preparation method and application of insect-based protein powder product |
| RU2759518C1 (en) * | 2021-05-12 | 2021-11-15 | Федеральное государственное учреждение «Федеральный исследовательский центр «Фундаментальные основы биотехнологии» Российской академии наук» | Composition for producing minced meat |
| RU2761649C1 (en) * | 2021-05-13 | 2021-12-13 | Федеральное государственное учреждение «Федеральный исследовательский центр «Фундаментальные основы биотехнологии» Российской академии наук» | Composition for the preparation of filling for waffles |
| CN113907189A (en) * | 2021-11-18 | 2022-01-11 | 江门鑫肽生物蛋白有限公司 | Application of fly maggot breeding waste and method for extracting fly maggot metabolite |
| RU2790403C2 (en) * | 2016-12-28 | 2023-02-17 | Инсект | Method for treatment of insects, including separation of cuticle from soft part of insects, using belt separator, with subsequent separation of soft part into three fractions |
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| JP2018502203A (en) * | 2014-12-31 | 2018-01-25 | インセクト | Enzymatic hydrolysis of chitin, hydrolysates, and insects to produce one or more desired products |
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| CN110099572A (en) * | 2016-12-28 | 2019-08-06 | 昆虫公司 | Method including the processing insect for using belt separator to separate the cuticula of insect with soft section |
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Application publication date: 20120711 |