CN104789627B - The method that mucin is extracted from the discarded object after intestinal mucosa extraction heparin - Google Patents
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Abstract
The present invention relates to the use of animal tissue extraction mucin technology, it is especially a kind of from intestinal mucosa extract heparin after discarded object in extract mucin method.Waste plant protein leftover bits and pieces is through trypsin digestion, ethanol precipitation and ion-exchange resin purification after pig intestinal mucosa is extracted into heparin, obtain the mucin of high-purity, for its molecular weight between 200~1000kDa, protein content is 20~30wt%, 50~80wt% of total sugar content.In total reducing sugar, sialic acid content accounts for the 24~54% of total sugar weight, and GalN, GlcN, Gal and Fuc part by weight are 23~45：27~32：14~15：9~10.The present invention prepares mucin using the discarded object after intestinal mucosa extraction heparin, the comprehensive utilization ratio that intestinal mucosa extracts discarded object after heparin can be significantly improved, realize and heparin, mucin and animal feed coproduction are extracted from pig intestinal mucosa, the product obtained can be used as scientific research biochemical reagents, it is also possible to prepare humans and animals immunopotentiator and neurotrophic agent.
Description
Technical field
It the present invention relates to the use of from animal tissue extraction mucin field, it is especially a kind of to extract heparin from intestinal mucosa
The method that mucin is extracted in discarded object afterwards, contained using extraction high sialic acid in the discarded object after the processing of intestinal mucosa heparin
Measure mucin.
Background technology
Mucin (mucin) is the albumen of a kind of high glycosylation, and it is made up of albumen and oligonucleotide chain two parts, and sugar chain
Content is typically between 50%~80%.The main silk ammonia in the form of O- connections and on peptide backbone of the mucoid oligonucleotide chain
Sour (Ser) or threonine (Thr) combine, referred to as O- sugar chains.Numerous studies show, intact animal small intestinal mucosa epithelial cell table
Contain a large amount of mucins in the slime layer of face, in addition to protective epithelium cell from physically or chemically damaging, also participate in prevention
The function of the occurrence and development of a variety of diseases, such as prevent cystic fibrosis disease (Cystic Fibrosis, CF), myxoadenocarcinoma, inflammation
Disease property enteritis etc..O- sugar chain structures in mucin are complicated, and activity extensively, in small intestine cells interphase interaction, signals-modulating, is divided
Important regulating and controlling effect is played in son transport etc., directly affects the prevention grown with disease of organism.It is although various next
The structure of source mucin and the study hotspot that activity is in recent years, but due to mucin shortage of resources, separation is difficult, in the market pin
The bovine submaxillary mucin for the only scientific research sold.
China is global crude product heparin major producing country and exported country, and its raw material is more based on intestinal mucosa, and it is got a foothold
Expect albumen residue considerable amount, be taken as offal treatment more, cause the destruction of environment and the waste of resource.Examined through document
Rope, in addition to having scholar and preparing small-molecular peptides and amino acid as feed addictive using chitterlings albumen slag (Shu Xiawa,
The research of intestinal mucosa processing fent enzymolysis process, Chinese feed, 2007,6:34-36), do not given up from pig intestinal mucosa
Abandon in albumen and prepare the report of the mucin containing high sialic acid content, also without any Patents technology of disclosure.
The content of the invention
It is an object of the invention to provide the side that mucin is extracted in discarded object after a kind of processing from intestinal mucosa heparin
Method, caused small peptide and amino acid may be used as animal feed in its extraction process.Therefore, this method can realize heparin, stick
Albumen, the coproduction of animal feed three, added value of product is improved, the data that solves wastes and problem of environmental pollution.
For achieving the above object, the technical scheme is that：
The method that mucin is extracted in a kind of discarded object after extraction heparin from intestinal mucosa, this method include following step
Suddenly：
(1) digest：Intestinal mucosa heparin processing waste is suspended in Tris-HCl buffer solutions, is warming up to 37 DEG C,
Trypsase is added, constant temperature stirring enzymolysis, after enzymolysis terminates, boils, inactivates enzyme；
(2) alcohol precipitation：Gained enzymolysis liquid in (1) is centrifuged, ethanol is added in supernatant and is precipitated, stands 12~24h, is received
Collection precipitation, supernatant concentrated by rotary evaporation, drying, as animal feed；
(3) dialyse：Precipitation in (2) is redissolved with distilled water, is transferred in bag filter, is dialysed, removes small-molecule substance, dialysis
After end, dialyzed solution concentrated by rotary evaporation freezes, obtains mucin crude extract；
(4) purify：Mucin crude extract obtained by (3) is dissolved with distilled water, with distilled water and the sodium chloride of various concentrations
The aqueous solution is mobile phase, is isolated and purified through strong anion exchange resin, Phenol-sulphate acid method detection, merges to collect and contains mucin
Component, distilled water dialysis, is concentrated under reduced pressure, and freezes, and obtains high-purity mucin.
The described method that mucin is extracted from the discarded object after intestinal mucosa extraction heparin, this method obtain glutinous
Molecular weight of albumen distribution is 200~1000kDa, and protein content is 20~30wt%, 50~80wt% of total sugar content；Always
Sugar is by amine-galactose (GalN), Glucosamine (GlcN), galactolipin (Gal), fucose (Fuc) and sialic acid (NeuAc)
Composition, and sialic acid is the 24%~54% of total sugar weight.
It is described from intestinal mucosa extract heparin after discarded object in extract the method for mucin, except saliva in total reducing sugar
Sour outer, other monose composition includes：GalN, GlcN, Gal and Fuc, and GalN, GlcN, Gal and Fuc part by weight be 23~
45：27~32：14~15：9~10.
It is described from intestinal mucosa extract heparin after discarded object in extract the method for mucin, in step (1),
The concentration of Tris-HCl buffer solutions is 0.05~0.15mol/L, and pH is 7~9, and trypsase dosage is that discarded object feeds intake quality
1%~5%.
It is described from intestinal mucosa extract heparin after discarded object in extract the method for mucin, in step (2), made
Ethanol volumetric concentration is 90%~95%, and addition is 3~5 times of supernatant volume.
It is described from intestinal mucosa extract heparin after discarded object in extract the method for mucin, in step (3), select
Bag filter interception be 14000Da, dialysis time is 48~72h, and a water was during which changed every 3~5 hours；Or use
10000Da milipore filters carry out concentrating and desalinating in ultrafiltration instrument equipment.
It is described from intestinal mucosa extract heparin after discarded object in extract the method for mucin, it is selected in step (4)
Strong anion exchange resin is Q-Sepharose FF or Capto Q.
It is described from intestinal mucosa extract heparin after discarded object in extract the method for mucin, in step (4), stick egg
After white crude extract dissolves loading with distilled water, 2~4 column volumes are successively eluted with 0.5~1.2mol/L sodium-chloride water solution,
Eluent is collected, concentration is dialysed after desalination, is freezed, is obtained high-purity mucin.
The advantages of the present invention are：
(1) present invention obtains the height with bioactivity by initial feed extraction of intestinal mucosa heparin processing waste
Purity mucin, mucin containing high sialic acid content is extracted from intestinal mucosa leftover bits and pieces.Wherein, raw material sources are chitterlings
Mucous membrane heparin processes albumen discarded object, and aboundresources, storage level is big, securely and reliably.
(2) present invention is using intestinal mucosa heparin processing waste as initial feed, through trypsin digestion, ethanol precipitation
And strong anion exchange resin purifying can obtain the mucin of high-purity, the process route is simple, and cost is cheap, product yield phase
For discarded object charged material weight 1%~5%.
(3) for the present invention using intestinal mucosa heparin processing waste as initial feed, therefrom extraction has bioactivity
High-purity mucin, in addition to the preparation on heparin product is without influenceing, caused small-molecular peptides and amino acid in extraction process
It can also continue to be used as animal feed.Therefore, the present invention can reach the effect of heparin, mucin and the coproduction of animal feed three, carry
The comprehensive utilization of high chitterlings, increase added value of product, new way provided for processing such as heparin manufacturing enterprise discarded object recyclings,
Reduce environmental pollution, new economic benefit is brought for enterprise while environmental protection.
(4) present invention is using intestinal mucosa heparin processing waste as initial feed, prepared to have bioactivity
Mucin can be used as scientific research mucin biological reagent, it is also possible to prepare humans and animals immunopotentiator and nerve cell
Nutritional agents, there is the important prospect of marketing.
Brief description of the drawings
The cellulose acetate electrophoresis analysis chart of Fig. 1 mucins of the present invention.
Ten anistree degree laser light scattering instrument analysis charts of Fig. 2 mucins of the present invention.Wherein, abscissa chronomere is minute,
Ordinate is with respect to the refraction index (Δ RI) that the unit of scale is relative blank solution.
The infrared spectrum analysis figure of Fig. 3 mucins of the present invention.
The sialic acid content measure figure of Fig. 4 mucins of the present invention.
The mucin monosaccharide composition analysis figure of Fig. 5 present invention.
Embodiment
In a specific embodiment, high-purity is extracted in the heparin processing waste provided by the present invention from intestinal mucosa
The method of mucin, comprises the following steps：
(1) digest：Intestinal mucosa heparin processing waste is soaked in Tris-HCl buffer solutions, is warming up to 37 DEG C,
Trypsase is added, constant temperature stirring enzymolysis, after enzymolysis terminates, boils, inactivates enzyme；
(2) alcohol precipitation：Enzymolysis liquid obtained by (1) is centrifuged, ethanol is added in supernatant and is precipitated, stands 12~24h, is collected
Precipitation, supernatant concentrated by rotary evaporation, drying, as animal feed；
(3) dialyse：Precipitation in (2) is redissolved with distilled water, is transferred in bag filter, dialyses, removes the small-molecule substances such as desalination,
After dialysis terminates, dialyzed solution concentrated by rotary evaporation freezes, obtains mucin crude extract；
(4) purify：Mucin crude extract obtained by (3) is dissolved with distilled water, with distilled water and the sodium chloride of various concentrations
The aqueous solution is mobile phase, is isolated and purified through strong anion exchange resin, Phenol-sulphate acid method detection, merges to collect and contains mucin
Component, distilled water dialysis, is concentrated under reduced pressure, and freezes, and obtains high-purity mucin.The molecular weight of the high-purity mucin is 200
~1000kDa, protein content are 20~30wt%, 50~80wt% of total sugar content；Cellulose acetate electrophoresis detector bar
With homogeneous, gel permeation chromatography detection eluting peak is single symmetrical, and sialic acid content accounts for the 24%~54% of total sugar weight.
In step (1), it is 0.05~0.15mol/L that intestinal mucosa heparin processing waste, which is soaked in concentration, pH is 7~
In 9 Tris-HCl buffer solutions, when temperature rises to 37 DEG C, add discarded object and feed intake the trypsase of quality 1%~5%, stirring
Extraction, by boiling inactivation trypsase.In step (2), into enzymolysis supernatant the ethanol volumetric concentration that adds for 90%~
95%, addition is 3~5 times of progress alcohol precipitations of supernatant volume.In step (3), after precipitation obtained by alcohol precipitation is redissolved with distilled water,
The bag filter interception of selection is 14000Da, and dialysis time is 48~72h, and a water was during which changed every 3~5 hours.Step
(4) in, selected strong anion exchange resin is Q-Sepharose FF or Capto Q, and mucin crude extract distills
After water dissolving loading, 2~4 column volumes are successively eluted with 0.5~1.2mol/L sodium-chloride water solution, collect eluent, it is dense
After contracting dialysis desalination, freeze, obtain high-purity mucin.
Below in conjunction with the accompanying drawings with specific embodiment to the further detailed description of technical scheme, but the present invention will
The scope of protection is asked to be not limited to the scope of embodiment statement.
Embodiment 1
Intestinal mucosa heparin processing waste 10g is weighed, is soaked in 400mL pH 8.0, concentration is 0.1mol/L's
In Tris-HCl buffer solutions, 37 DEG C are warming up to, adding trypsase, (dosage of trypsase is that discarded object feeds intake quality
3%) after, carrying out enzymolysis 4h under stirring, temperature being risen to 100 DEG C, keeps 10min, inactivate trypsase, 4500r/min is centrifuged,
Precipitation is removed, supernatant is concentrated under reduced pressure into about 100mL, adds 95% ethanol (volumetric concentration) of 4 times of volumes, stirs, 0~4 DEG C
12h is stood, precipitation is redissolved with distilled water, gone in 14000Da bag filters, and dialyse 48h, changes a water every 4h, dialysis terminates
After be concentrated under reduced pressure, freeze, obtain mucin crude extract 0.235g.Crude extract 0.1g is taken, is dissolved with 1mL distilled water, loading, through Q-
Sepharose FF (column volume 100mL) are separated, and first elute 2 column volumes with 0.5mol/L sodium-chloride water solution, then use
1.0mol/L sodium-chloride water solution elutes and collects 2 column volumes, merges eluent, concentrated freeze-dried through desalination of dialysing, and also may be used
After carrying out concentrating and desalinating using 10000Da device for ultrafiltration membrane, freeze, obtain sterling mucin PIM-1.
Embodiment 2
Intestinal mucosa heparin processing waste 20g is weighed, is soaked in 700mL pH 8.5, concentration is 0.2mol/L's
In Tris-HCl buffer solutions, 37 DEG C are warming up to, adding trypsase, (dosage of trypsase is that discarded object feeds intake quality
2%) after, carrying out enzymolysis 6h under stirring, temperature being risen to 100 DEG C, keeps 10min, inactivate trypsase, 4500r/min is centrifuged,
Precipitation is removed, supernatant is concentrated under reduced pressure into about 150mL, adds 90% ethanol (volumetric concentration) of 5 times of volumes, stirs, in 4 DEG C of refrigerators
12h is staticly settled, precipitation is redissolved with distilled water, is gone in 7000Da bag filters, and dialyse 72h, and a water, dialysis knot are changed every 4h
It is concentrated under reduced pressure, freezes after beam, obtains mucin crude extract 0.617g.Crude extract 0.2g is taken, is dissolved with 2mL distilled water, loading, warp
Capto Q (column volume 200mL) are separated, and elute 3 column volumes with 0.65mol/L sodium-chloride water solution, then use 1.3mol/L
Sodium-chloride water solution elute and collect 3 column volumes, merge eluent, dialyse desalination, it is concentrated freeze-dried, can also use
After 20000Da device for ultrafiltration membrane carries out concentrating and desalinating, freeze, obtain mucin PIM-2.
The identification and analysis of above-mentioned mucin：
1 mucin charge distribution density analysis-cellulose acetate electrophoresis
With hyaluronic acid (HA), Heparan sulfate (HS), chondroitin sulfate A (CSA), B, C (CSA, CSB, CSC) for reference substance,
With the charge density distribution of cellulose acetate electrophoresis analysis mucin, experimental result is as shown in figure 1, embodiment 1 and implementation
Two groups of mucin PIM-1 and the PIM-2 electrophoretic bands of gained of example 2 are single, and charge density distribution is between HA and HS, with a small amount of
Negative electrical charge.
20 anistree degree laser light scattering instrument measure mucin molecular weight
Using High Performance Gel Permeation chromatogram (HPGPC) and ten anistree degree laser light scattering instrument and Composition distribution combinations, measure
PIM-1 and PIM-2 molecular weight, as a result such as Fig. 2.Wherein PIM-1 molecular weight is 368~910kDa, and PIM-2 molecular weight is
209~858kDa, and laser, with showing that it is overlapping that difference signal rushes, peak type is single symmetrical, and purity is higher.
The infrared spectrum analysis of 3 mucins
Mucin infrared spectrum analysis prepared by the present invention is as shown in figure 3, by taking PIM-2 as an example, 3366cm-1Locate as sugared ring
Upper hydroxyl O-H stretching vibrations, 1652cm-1And 1544cm-1Amido link and sugared (HexNAc) acetyl of aminoglucose respectively in albumen
C=O and N-H flexible and change angular oscillation, 1232cm in base-1Locate the S=O stretching vibrations for sulfate, and the absorption peak strength
It is smaller, show that sulfate radical content is few in mucin.
The monose composition measuring of 4 mucins
Mucin PIM-1 and PIM-2 prepared in the embodiment of the present invention are subjected to acid degradation, using anti-phase C18 chromatograms
Post-monose species and ratio contained by high performance liquid chromatography centering is measured, as a result such as monose in Fig. 4, PIM-1 and PIM-2
In composition in addition to sialic acid, mainly it is made up of GalN, GlcN, Gal and Fuc, its part by weight is respectively 44.6:27.9:
14.4:10.0 with 23.8:31.5:14.4:9.2.
Protein content and the sialic acid content measure of 5 mucins
Protein content in mucin is determined using Folin- phenol method, using acid degradation-DMB derivatives-high performance liquid chromatography
Sialic acid content in mucin is determined, spectrum analysis are shown in Fig. 5.As a result show, the present invention prepared by mucin in PIM-1 with
PIM-2 protein contents are respectively 21wt%, 27wt%.In mucin prepared by the present invention, PIM-1 and PIM-2 total reducing sugar contain
Amount respectively 50wt% and 80wt%, PIM-1 and PIM-2 Total silicic acid accounts for the 24% and 54% of total sugar weight respectively.
O- sugar chain structures parse in 6 mucins
In order to further investigate the mucin fine structure in chitterlings source, using reproducibility β-null method in mucin
O- sugar chains discharged to obtain various oligosaccharides alcohol, after exclusion chromatography isolates and purifies, using electrospray ionization tandem matter
Spectrum is analyzed, and 25 kinds of oligosaccharide structures of gained the results are shown in Table 1.The mucin that is obtained of the present invention for sugar chain type with Core2 and
Based on Core3, the substitution of part of sulfuric acid root occurs in GlcNAc C-6 positions, and surface antigen type is mainly Blood group H
Type, belong to the mucin of high sialylation.
In summary, the mucin that the present invention extracts by raw material of intestinal mucosa heparin processing waste is high saliva
Mucin is acidified, because purity is higher, source is sufficient, can be developed into scientific research mucin biochemical reagents, and humans and animals are exempted from
Epidemic disease reinforcing agent and neurotrophic agent, there is market potential application value.
Above example is merely to illustrate technical scheme, rather than is limited；Although with reference to foregoing reality
Example is applied the present invention is described in detail, for the person of ordinary skill of the art, still can be to foregoing implementation
Technical scheme described in example is modified, or carries out equivalent substitution to which part technical characteristic；And these are changed or replaced
Change, the essence of appropriate technical solution is departed from the spirit and scope of claimed technical solution of the invention.
The ESI-CID-MS/MS parsings of O- sugar chains in the mucin of table 1
Claims (6)
1. the method for mucin is extracted in the discarded object after a kind of extraction heparin from intestinal mucosa, it is characterised in that this method
Comprise the following steps：
(1) digest：Intestinal mucosa heparin processing waste is suspended in Tris-HCl buffer solutions, is warming up to 37 DEG C, is added
Trypsase, constant temperature stirring enzymolysis, after enzymolysis terminates, boils, inactivates enzyme；
(2) alcohol precipitation：Gained enzymolysis liquid in (1) is centrifuged, ethanol is added in supernatant and is precipitated, stands 12~24h, it is heavy to collect
Form sediment, supernatant concentrated by rotary evaporation, drying, as animal feed；
(3) dialyse：Precipitation in (2) is redissolved with distilled water, is transferred in bag filter, is dialysed, removes small-molecule substance, dialysis terminates
Afterwards, by dialyzed solution concentrated by rotary evaporation, freeze, obtain mucin crude extract；
(4) purify：Mucin crude extract obtained by (3) is dissolved with distilled water, with distilled water and the aqueous sodium chloride of various concentrations
Liquid is mobile phase, is isolated and purified through strong anion exchange resin, Phenol-sulphate acid method detection, merges and collects the group containing mucin
Point, distilled water dialysis, it is concentrated under reduced pressure, freezes, obtain high-purity mucin；
The mucin range of molecular weight distributions that this method obtains is 200~1000kDa, and protein content is 20~30wt%, always
50~80wt% of sugared content；Total reducing sugar is by amine-galactose (GalN), Glucosamine (GlcN), galactolipin (Gal), fucose
(Fuc) formed with sialic acid (NeuAc), and sialic acid is the 24%~54% of total sugar weight；
In total reducing sugar in addition to sialic acid, other monose composition includes：GalN, GlcN, Gal and Fuc, and GalN, GlcN, Gal and
Fuc part by weight is 23~45：27~32：14~15：9~10.
2. the method for mucin is extracted in the discarded object after the extraction heparin according to claim 1 from intestinal mucosa, its
It is characterised by, in step (1), the concentration of Tris-HCl buffer solutions is 0.05~0.15mol/L, and pH is 7~9, and trypsase is used
Measure and feed intake the 1%~5% of quality for discarded object.
3. the method for mucin is extracted in the discarded object after the extraction heparin according to claim 1 from intestinal mucosa, its
It is characterised by, in step (2), used ethanol volumetric concentration is 90%~95%, and addition is the 3~5 of supernatant volume
Times.
4. the method for mucin is extracted in the discarded object after the extraction heparin according to claim 1 from intestinal mucosa, its
It is characterised by, in step (3), the bag filter interception of selection is 14000Da, and dialysis time is 48~72h, during which every 3~5
Hour changes a water；Or concentrating and desalinating is carried out in ultrafiltration instrument equipment using 10000Da milipore filters.
5. the method for mucin is extracted in the discarded object after the extraction heparin according to claim 1 from intestinal mucosa, its
It is characterised by, in step (4), selected strong anion exchange resin is Q-Sepharose FF or Capto Q.
6. the method for mucin is extracted in the discarded object after the extraction heparin according to claim 1 from intestinal mucosa, its
It is characterised by, in step (4), after mucin crude extract dissolves loading with distilled water, with 0.5~1.2mol/L aqueous sodium chloride
Liquid successively elutes 2~4 column volumes, collects eluent, and concentration is dialysed after desalination, is freezed, is obtained high-purity mucin.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102604915A (en) * | 2012-03-27 | 2012-07-25 | 华中农业大学 | Method for jointly extracting a variety of proteins from egg white |
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2015
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604915A (en) * | 2012-03-27 | 2012-07-25 | 华中农业大学 | Method for jointly extracting a variety of proteins from egg white |
Non-Patent Citations (3)
| Title |
|---|
| 猪小肠黏膜加工下脚料酶解工艺的研究;舒夏娃等;《中国饲料》;20070320(第6期);摘要部分和第34页左栏第1段 * |
| 酶法提取肠渣饲用蛋白质工艺的研究;孔凡伟等;《粮食与饲料工业》;20110715(第7期);摘要部分 * |
| 鸡卵类粘蛋白的分离与纯化;境界121212;《百度文库》;20130414;正文部分 * |
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