CN100425624C - Method for simultaneous extraction of sodium heparin and antibacterial peptide of pig intestinal mucosa - Google Patents
Method for simultaneous extraction of sodium heparin and antibacterial peptide of pig intestinal mucosa Download PDFInfo
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- CN100425624C CN100425624C CNB2006100788655A CN200610078865A CN100425624C CN 100425624 C CN100425624 C CN 100425624C CN B2006100788655 A CNB2006100788655 A CN B2006100788655A CN 200610078865 A CN200610078865 A CN 200610078865A CN 100425624 C CN100425624 C CN 100425624C
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Abstract
The present invention discloses a method for synchronously extracting of sodium heparin and antibacterial peptide from pig intestinal mucosa, which comprises the following steps that a first step is to fetch a pig intestinal mucosa to add in protease formulation; after the formulation is heated to 85 to 95 DEG C and the pH value is regulated to 9.0 to 9.5, the temperature of the formulation is kept for 20 to 40 minutes; after the formulation is cooled, adsorptive resin is added to adsorb; the separating liquid is retained; the resin is reclaimed. A second step is to elute the resin reclaimed in the first step by NaCl water solution with quality percent concentration of 19 to 40%; the eluting liquid is subsided by ethanol to obtain the sodium heparin. A third step is to add the separating liquid obtained in the first step, according to the volume ratio of 1: 1 to 1: 2, into acetic acid with the volume concentration of 5 to 10% for mixing and lixiviating; the mixed liquid after lixiviated is centrifuged; upper supernatant liquid is fetched, and sediment is preserved. A fourth step is to regulate the pH value of the upper supernatant liquid obtained in the third step to 5 to 6.0; after centrifuged, the upper supernatant liquid is retained; after the upper supernatant liquid is frozen to dryness, the antibacterial peptide of the pig intestinal mucosa is obtained. The present invention simultaneously extracts the sodium heparin and the antibacterial peptide two substances from the pork butchering and processing waste-intestinal mucosa, and greatly enhances the utilization value of butchering and processing waste. The present invention has favorable economical and social benefits.
Description
Technical field
The present invention relates to the method for a kind of simultaneous extraction heparin sodium and antibacterial peptide of pig intestinal mucosa.
Background technology
Since last century end, the research of antibacterial peptide, development and use have been caused the very big interest on international biomedical boundary.
Because current microorganism is to this fact of the extensive resistance of traditional microbiotic, and the evolution course that the endogenous antibiotic peptide passed through millions of years has still kept strong antibiotic effectiveness, therefore, it is imperative to develop the new antimicrobial strategy of high-tech.And on the anti-infective new strategy of development, the natural antibiotics peptide is especially noticeable.Antibacterial peptide has wide spectrum, antimicrobial acivity efficiently, from body self, and old origin, evolving through millions of years still keeps powerful antimicrobial acivity, so may be the optimal selection of solution microorganism resistance problem.And the new revolution that may bring an antimicrobial strategy thus.
Heparin sodium is the sodium salt of heparin, be the sodium salt of the sulfuric acid glycosaminoglycan that mixes mutually of the different mucopolysaccharide of molecular weight in essence, extensively be present in Mammals Ou Li (Ehrlich) the family name mastocyte, have efficient anticoagulation, prevent blood examination formation and fall the blood effect, and can be used for treating some immunocomplex (ImmunoComplex) disease, have to be extensive use of and exploitation value.Heparin is a kind of water-soluble substances, and its molecular weight is about 3000-50000 dalton.Heparin is the natural acidic mucopolysaccharide, is linear anionic polyelectrolyte, differs, has the compounds of group that very strong negative charge chain is formed by structure, and heparin all has an effect that prolongs the blood clotting time with external in vivo, and its anticoagulation is very fast, powerful.Heparin is to thermally-stabilised, and is insensitive to general microorganism and enzyme, is white or off-white color amorphous powder.Also can make lipoprotein enzyme release and stable during the intravenous injection heparin, thereby reduce plasma triglyceride level.Pharmaceutical heparin generally can extract in the intestinal mucosa of pig or ox.
Go up the article that Wu Qi etc. has delivered a piece " the alexinic separation and purification of human neutrophil " by name in " Chinese immunology " 1993 the 9th the 2nd phase of volume, a kind of method of the human neutrophil antibacterial peptide being carried out separation and purification has wherein been proposed, to obtain the human neutrophil cytoplasmic granule, with 5% Acetic Acid Extraction, carry out Sephadex G-100 and Bio-gel P10 then and filter, obtain the antibacterial peptide of purifying.Because the intestinal tissue complex structure, foreign protein is of a great variety, thereby adopts the separation purification method of Wu Qi etc. need carry out repeatedly just reaching comparatively satisfied purification effect.
Summary of the invention
The method that the purpose of this invention is to provide a kind of simultaneous extraction heparin sodium and antibacterial peptide of pig intestinal mucosa
The method of simultaneous extraction heparin sodium provided by the present invention and antibacterial peptide of pig intestinal mucosa comprises the steps:
1) gets pig intestinal mucosa and add protease preparation, be heated to 85-95 ℃, regulate the pH value, be incubated 20-40 minute to 9.0-9.5; After the cooling, add polymeric adsorbent and adsorb, keep parting liquid, and reclaim resin;
2) resin of step 1) recovery is the NaCl aqueous solution wash-out of 19-40% with the mass percentage concentration, and elutriant obtains heparin sodium with ethanol sedimentation;
3) parting liquid that obtains of step 1) by volume 1: it is that the acetate of 5-10% carries out stirring and leaching that 1-2 adds volumetric concentration; Mixed solution after the lixiviate is centrifugal, gets supernatant liquor, and preserves precipitation;
4) transfer step 3) gained supernatant liquor pH value to 5～6.0, centrifugal, keep supernatant liquor, freeze-drying obtains antibacterial peptide of pig intestinal mucosa.
If desired antibacterial peptide of pig intestinal mucosa is used for medicinal use, is further purified: after step 4) gained antibacterial peptide of pig intestinal mucosa is dissolved in distilled water, utilize Sephadex G-100 chromatography to remove macromole, utilize the desalination of Sephadex G-25 chromatography; The elutriant that chromatography obtains is the ultra-filtration membrane ultrafiltration of 10kD with the molecular weight cut-off earlier, and crossing molecular weight cut-off then is the ultra-filtration membrane ultrafiltration of 3kD, collects and obtains the purifying antibacterial peptide of pig intestinal mucosa that molecular weight is 3-8kD.
It is the 0.2mol/L sodium acetate solution that Sephadex G-100 chromatography removes macromolecular elutriant; The elutriant of Sephadex G-25 chromatography desalination is a distilled water.
In order to gather in the crops the active substance in the raw material more up hill and dale, active substance in the precipitation in the step 3) can also be extracted again, its method is, is 1 by volume: the ratio of 1-2 adds volumetric concentration in the centrifugation of described step 3) gained be the acetate of 5-10%, stirring and leaching; Mixed solution is centrifugal after the lixiviate, gets supernatant liquor, and merges with the supernatant liquor of step 3) gained, carries out later operation again.
In the said extracted process, the described proteolytic enzyme of step 1) can be trypsinase, and its consumption is 20g enzyme/kilogram pig intestinal mucosa; Polymeric adsorbent can be polymeric adsorbent D254, and its absorption consumption is the 5-10% of pig intestinal mucosa weight.Centrifugal all can the employing at 4 ℃ in the described leaching process carried out under the 10000rpm condition.
The result that the biologic activity of separation and purification gained antibacterial peptide of pig intestinal mucosa is studied shows that the gained antibacterial peptide of pig intestinal mucosa has the obvious suppression effect to common pathogenic bacterium such as Staphylococcus albus, streptococcus aureus, Pseudomonas aeruginosa, Salmonellas, intestinal bacteria, Aeromonas hydrophilas; Antibacterial peptide of pig intestinal mucosa also all has significant deactivation to avian infectious bronchitis virus (IBV), newcastle disease virus (NDV) and chicken infectivity bursa of Fabricius virus (IBDV).More meaningfully, antibacterial peptide of pig intestinal mucosa also can improve body non-specific immune function, specific chicken immune serum antibody horizontal and Intestinal Mucosal Immunity function, and can improve chick day weight gain, reduction material anharmonic ratio, promotes growing of chicken embryo and chick.
The present invention extracts heparin sodium and two kinds of materials of antibacterial peptide simultaneously from pork is butchered the waste intestinal mucosa of processing, improve the utility value of butchering processing waste greatly; Heparin sodium and the antibacterial peptide that obtains that extract has broad-spectrum antimicrobial, antiviral on function, do not produce resistance, and has immunoregulation effect.The inventive method separation purification method is easy, separation, extraction yield height, the raw materials used waste that uses enterprises such as pig slaughtering factory, casing source mill, the source is abundant, cost is low, realize the waste higher value application, can reduce or eliminate of the pollution of the waste of enterprises such as pig slaughtering factory, casing source mill to greatest extent, have good economic benefit and social benefit environment.
Embodiment
Embodiment 1, simultaneous extraction heparin sodium and antibacterial peptide of pig intestinal mucosa
Fs: the extraction of heparin sodium
Step 1 is got the byproduct pig intestinal mucosa of casing processing, adds trypsin inhibitor and stirs, and its consumption is 20 gram enzyme/kilogram pig intestinal mucosas, is heated to 90 ℃, regulates pH value 9.0-9.5, constant temperature 20-40 minute with sodium hydroxide.
Step 2 after the cooling, adds the polymeric adsorbent D254 that cleans up and adsorbs, and resin demand is 5% of an intestinal mucosa weight.
Step 3, liquid after the absorption and resin isolation, liquid is used to extract antibacterial peptide, reclaims resin, with 19-30% (weight) NaCl aqueous solution wash-out;
Step 4, elutriant precipitates with straight alcohol, the heparin sodium crude that collecting precipitation goes out, kept dry.Through identifying that the content of heparin sodium can reach more than 65% in this product.
Subordinate phase: the extraction of antibacterial peptide
Step 5 is got above-mentioned steps three and is isolated liquid, adds 5% acetate, stirring and leaching 24h by 1: 1 (v/v);
Step 6, in 4 ℃, the centrifugal 30min of 10000rpm gets supernatant with mixture.
Step 7, with five lixiviates once more set by step of the precipitation after centrifugal, carry out according to step 6 more centrifugal, keep supernatant liquor after, continue next step;
Step 8 merges supernatant liquor twice, transfers its pH value to 5～6.0, and is centrifugal, removes precipitation, keeps supernatant liquor, obtains the thick product of antibacterial peptide of pig intestinal mucosa (but freeze-drying preservation).
If desired antibacterial peptide of pig intestinal mucosa is used for medicinal use, is further purified:
Lyophilized products is dissolved in distilled water, utilize Sephadex G-100 chromatography to remove macromole (with 0.2mol/L sodium acetate wash-out) and utilize Sephadex G-25 chromatography desalination (using the distilled water wash-out), the elutriant that chromatography obtains is the ultra-filtration membrane ultrafiltration of 10kD with the molecular weight cut-off earlier, crossing molecular weight cut-off then is the ultra-filtration membrane ultrafiltration of 3kD, collects the pure product of antibacterial peptide that molecular weight is 3-8kD that obtain.
With the products obtained therefrom ultraviolet detection, measure the absorption value of OD260 nanometers, if absorption value is arranged, prove albumen at the 260nm place, can carry out bacteriostatic experiment.
The functional experiment of embodiment 2, antibacterial peptide of pig intestinal mucosa
1, bacteriostatic test
Adopt micro-agarose disperse test method(s) to detect the effect of antibacterial peptide of pig intestinal mucosa to bacteriums such as Staphylococcus albus, streptococcus aureus, Pseudomonas aeruginosa, Salmonellas, the strain of intestinal bacteria natural separation, all grand Aeromonas hydrophilas, the result shows that antibacterial peptide of pig intestinal mucosa all has in various degree inhibition and killing action to these bacteriums, sterilizing rate does not wait by 59.5%～98.5%, has disclosed antibacterial peptide of pig intestinal mucosa and has had very strong anti-microbial effect and wider antimicrobial spectrum.
2, antiviral experiment
Antiviral experimental result shows that antibacterial peptide of pig intestinal mucosa can make the hemagglutinative titer of IBV in the chick embryo allantoic liquid reduce to 1.0log2 (antibacterial peptide of pig intestinal mucosa is handled the virus group) by 7.3log2 (virus group), suppresses the caused chicken embryo of IBV pathology; Can also make the chicken infected sickness rate of NDV reduce to 40% (ABPs handles NDV and attacks the poison group) by 100% (NDV attacks the poison group), corresponding mortality ratio reduces to 13.3% by 60%.Make the chicken infected sickness rate of IBDV reduce to 33.3% (ABPs handles IBDV and attacks the poison group) by 100% (IBDV attacks the poison group), corresponding mortality ratio reduces to 20% by 80%.Disclose antibacterial peptide of pig intestinal mucosa IBV, NDV and IBDV are had certain inhibition and killing action.
3, intramuscular injection experiment
Adopt the method for intramuscular injection, give chicken, each 10 μ g dosage injection chitling road antibacterial peptide according to 1 time weekly; The physiological saline of control group injection same dose.The result shows the average daily gain of experimental group chick, and (15.54 ± 0.20g) are significantly higher than control group (11.10 ± 0.19g) (p＜0.05); Experimental group material anharmonic ratio (2.27 ± 0.04) significantly is lower than control group (2.60 ± 0.05) (p＜0.05).This shows that antibacterial peptide of pig intestinal mucosa can improve the speed of growth and the feed conversion rate of chick, improve the growth indexes of chicken, promote chick growth to grow.Simultaneously, find also under study for action that antibacterial peptide of pig intestinal mucosa can promote the chicken growth of the embryo to grow, and significantly improves the intestinal villus length and the V/C value of each section intestinal tube.
Claims (5)
1, the method for a kind of simultaneous extraction heparin sodium and antibacterial peptide of pig intestinal mucosa comprises the steps:
1) gets pig intestinal mucosa and add trypsinase, be heated to 85-95 ℃, regulate the pH value, be incubated 20-40 minute to 9.0-9.5; After the cooling, add polymeric adsorbent and adsorb, keep parting liquid, and reclaim resin;
2) resin of step 1) recovery is the NaCl aqueous solution wash-out of 19-40% with the mass percentage concentration, and elutriant obtains heparin sodium with ethanol sedimentation;
3) parting liquid that obtains of step 1) by volume 1: it is that the acetate of 5-10% carries out stirring and leaching that 1-2 adds volumetric concentration; Mixed solution after the lixiviate is centrifugal, gets supernatant liquor, and preserves precipitation;
4) transfer step 3) gained supernatant liquor pH value to 5～6.0, centrifugal, keep supernatant liquor, freeze-drying obtains antibacterial peptide of pig intestinal mucosa;
5) after the gained antibacterial peptide of pig intestinal mucosa is dissolved in distilled water, utilize Sephadex G-100 chromatography to remove macromole, utilize the desalination of Sephadex G-25 chromatography; The elutriant that chromatography obtains is the ultra-filtration membrane ultrafiltration of 10kD with the molecular weight cut-off earlier, and crossing molecular weight cut-off then is the ultra-filtration membrane ultrafiltration of 3kD, collects and obtains the purifying antibacterial peptide of pig intestinal mucosa that molecular weight is 3-8kD.
2, method according to claim 1 is characterized in that: it is the 0.2mol/L sodium acetate solution that Sephadex G-100 chromatography removes macromolecular elutriant; The elutriant of Sephadex G-25 chromatography desalination is a distilled water.
3, method according to claim 1 is characterized in that: be 1 by volume: the ratio of 1-2 adds volumetric concentration in the centrifugation of described step 3) gained be the acetate of 5-10%, stirring and leaching; Mixed solution is centrifugal after the lixiviate, gets supernatant liquor, and merges with the supernatant liquor of step 3) gained.
4, according to claim 1 or 2 or 3 described methods, it is characterized in that: the described tryptic consumption of step 1) is 20g enzyme/kilogram pig intestinal mucosa; Described polymeric adsorbent is polymeric adsorbent D254, and its absorption consumption is the 5-10% of pig intestinal mucosa weight.
5, according to claim 1 or 2 or 3 described methods, it is characterized in that: centrifugal in the described leaching process all is at 4 ℃, carries out under the 10000rpm condition.
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Cited By (1)
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|---|---|---|---|---|
| CN101601437B (en) * | 2009-06-22 | 2012-12-12 | 陈道才 | Chicken intestinal peptide and production process thereof |
Families Citing this family (5)
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| CN101209082B (en) * | 2007-12-20 | 2011-08-31 | 武汉工业学院 | Preparation of functional feed protein DPS |
| CN101773522B (en) * | 2010-03-03 | 2011-12-21 | 中国农业科学院饲料研究所 | Animal intestinal mucosa extract and preparation method as well as application thereof |
| CN104119458A (en) * | 2014-07-07 | 2014-10-29 | 广元市海天实业有限责任公司 | Process using intestinal casing waste liquid as raw material for production of sodium heparin and intestinal membrane protein |
| CN112280774B (en) * | 2019-07-27 | 2023-04-07 | 烟台东诚药业集团股份有限公司 | Method for combined preparation of DNA sodium salt and antibacterial peptide |
| CN114230688B (en) * | 2021-12-24 | 2022-12-13 | 武汉瑞法医疗器械有限公司 | Simple and efficient heparin sodium recovery method for blood purification |
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| CN101601437B (en) * | 2009-06-22 | 2012-12-12 | 陈道才 | Chicken intestinal peptide and production process thereof |
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