CN103948961B - Corn protein materials, Preparation method and use - Google Patents
Corn protein materials, Preparation method and use Download PDFInfo
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- CN103948961B CN103948961B CN201410174746.4A CN201410174746A CN103948961B CN 103948961 B CN103948961 B CN 103948961B CN 201410174746 A CN201410174746 A CN 201410174746A CN 103948961 B CN103948961 B CN 103948961B
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- 235000005822 corn Nutrition 0.000 title description 30
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- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a kind of molten protein material of grain alcohol, Preparation method and use. The molten protein material of grain alcohol has higher force intensity, transparency and suitable vivo degradation speed. The molten protein material of grain alcohol of the present invention can adopt heating in water, in steam, heating and xeothermic method obtain, and the temperature controlled in heating process is 30-160 degree Celsius, and the time is 1-120 minute. The mechanical strength of the molten protein material of grain alcohol after process and transparency is improved, degradation rate and swelling ratio significantly reduce, and there is stable surface texture, do not bond. By controlling acid extraction, and then control mechanics intensity and degradation rate, and reach anti-swelling effect. The molten protein material of grain alcohol after process can be used for bio-medical purpose, and cell culture medium material and microfluidic device.
Description
Technical field
The present invention relates to the molten protein material of grain alcohol (including but not limited to zein, wheat gliadin), can pass through in water, steam heats or the method for the in type corn alcohol soluble protein of dry heat treatment obtains, to improve mechanical strength, transparency and to reduce degradation rate, especially by controlling acid extraction and then reaching the purpose of the mechanical strength, transparency and the degradation rate that control under wet condition, and the molten protein material swelling ratio of the grain alcohol after process can be reduced. The invention still further relates to the purposes of the protein material obtained by the method.
Technical background
Natural biologic material includes collagen, gelatin, sodium alginate and chitosan etc., because it is with low cost, be easily obtained, degradable and the advantage such as biocompatibility is better, it is applied to medical health field, such as cartilage substitute, implants bone material, artificial blood vessel and artificial skin etc. But, natural biologic material is because presenting the feature of mixture, and cannot match in excellence or beauty synthesising biological material in the control of degradation rate and mechanical strength. Therefore if able to search out a kind of method to control degradation rate and the mechanical strength of natural biologic material, its range of application can be greatly expanded, obtain better therapeutic effect clinically.
The natural polymer that protein is made up of several amino acids, hydrogen bond and disulfide bond that intermolecular existence is stronger interact. The mechanical property of the biomaterial based on protein is poor, need to improve the rigidity of material, thermostability and stability through methods such as ultraviolet irradiation, thermal polymerization, ferment treatment or chemical crosslinkings. Formaldehyde that chemical crosslink technique is the most frequently used or glutaraldehyde, because having toxicity, are generally avoided being used in technical field of biological material. Heat treatment is as physical method, mechanical strength and the degradation rate of protein can also be controlled by controlling heat treated temperature, and compared with the chemical method adopting cross-linking agent, it is possible to avoid the immersion of toxic compounds, improve the safety of biomaterial, reduce postprocessing working procedures.
The mechanical property of natural biologic material has larger difference under dry state and hygrometric state, and the biomaterial implanted requires also can keep good mechanical property under hygrometric state.Such as, artificial blood vessel needs to bear blood stream pressure and impact in order to avoid bursting, and nerve trachea needs to resist the pressure of externus muscle tissue in order to avoid subsiding.
The polymer with higher clarity has been widely used for micro-fluidic and cell culture system. But, polymer used such as polydimethylsiloxane and silicone rubber, cell compatibility is not highly desirable, it is necessary to improve its cell compatibility by methods such as Plasma inpouring or increase surface roughnesses. On the other hand, the polymer being raw material with oil can face exhausted problem future. Meanwhile, a large amount of uses of non-degradable material can cause environmental pollution. Based on this, research and develop naturally occurring or synthetic degradation material, significant for cell culture medium material and micro fluidic device.
Corn alcohol soluble protein is a kind of degradable natural biomaterial, has good biocompatibility with Human umbilical vein endothelial cells, human liver cell and mouse fibroblast cell. [WangHJ, LinZX, LiuXM, ShengSY, WangJY.Heparin-loadedzeinmicrospherefilmandhemocompatibi lity.JControlRelease2005; 105:120-31.; SunQS, DongJ, LinZX, YangB, WangJY.Comparisonofcytocompatibilityofzeinfilmwithotherb iomaterialsanditsdegradabilityinvitro.Biopolymers2005; 78:268-74.] easily processed into type as material, such as three-dimensional rack, two-dimensional film, microsphere, tubulose, threadiness etc., the research report in bio-medical field is increasing in recent years. But, owing to corn alcohol soluble protein is mainly made up of hydrophobic amino acid, its character is had a significant impact by surrounding such as relative humidity. As water can cause it swelling, which has limited they application in some field.
Moist heat sterilization is the general sterilizing methods during cell is cultivated, and water can as the plasticizer of protein structure. Process fibroin albumen by the method for High Temperature High Pressure and can change its secondary structure, increase ��-pleated sheet structure proportion. [HuX, ShmelevK, SunL, GilES, ParkSH, CebeP, etal.Regulationofsilkmaterialstructurebytemperature-cont rolledwatervaporannealing.Biomacromolecules2011; 12:1686-96; UmIC, KweonHY, ParkYH, HudsonS.Structuralcharacteristicsandpropertiesoftheregen eratedsilkfibroinpreparedfromformicacid.IntJBiolMacromol 2001; 29:91-7; LawrenceB, OmenettoF, ChuiK, KaplanD.Processingmethodstocontrolsilkfibroinfilmbiomate rialfeatures.JMaterSci2008; 43:6967-85.] ��-pleated sheet is relevant with mechanical strength simultaneously. Nishiyama[NishiyamaY, WadaM, KugaS, MagoshiJ.Mechanicalpropertiesofsilkfibroin microcrystallinecellulosecompositefilms.JApplPolymSci200 2; 86:3425-9.] et al. find in fibroin, the appearance of ��-pleated sheet structure adds hot strength. Meanwhile, the increase of ��-pleated sheet structure reduces degradation rate. [EmmambuxMN, TaylorJR.Propertiesofheat-treatedsorghumandmaizemealandt heirprolaminproteins.JAgricFoodChem2009; 57:1045-50.]
Based on above-mentioned background document, in order to expand the range of application of corn alcohol soluble protein further, it is necessary first to itself defect when overcoming it as material: as be prone to swelling and swelling after opaque, fragility is bigger than normal.It is advantage on the one hand as its degradability of natural material, but in some occasion, for instance trypsin digestion cell during as cell culture medium material, at this moment need to stop it to degrade. In order to obtain mechanical strength, transparency and degradation rate meet such use require with corn alcohol soluble protein be base material biomaterial propose the present invention.
Summary of the invention
It is an object of the invention to provide a kind of molten protein material of grain alcohol, by water, steam or the molten protein material of the in type grain alcohol of dry heat treatment dramatically increase its mechanical strength under hygrometric state, transparency, and significantly reduce its degradation rate. Can pass through control heating-up temperature and then reach the purpose of control degradation speed and mechanical strength. It is a further object of the present invention to provide the purposes of this protein material. The molten protein material of grain alcohol can be zein, wheat gliadin.
Molten for grain alcohol protein material system is adopted zein or wheat gliadin pressed powder, it is dissolved in the ethanol water of 40��90%, it is made into 1��300mg/ml solution, this solution is coated on repeatedly on mould and forms thin film, plate or pipe, obtained zein or wheat gliadin thin film, plate or pipe directly or are immersed in pure water (in redistilled water), process 20-120 minute under the pressure condition of 60-130 DEG C and steam sterilization; Or process 1-4 week in 30-60 DEG C under saturation vapour; Or process 1-60 minute under 100-160 DEG C of dry heat condition, become zein or wheat gliadin material.
The above-mentioned processing procedure of the present invention recommends to carry out in confined space; Be generally coated on mould and form thin film, type is made in plate or management and control, and thickness is 20-1000 micron, and when relating to transparency more than 90%, usually control thickness is 20-200 micron.
First being that example is studied with zein, it was found that zein is after steam heat treated, its mechanical strength, transparency significantly improve, and its degradation rate substantially reduces. The improvement of these character can make zein material be applied to the field of tissue engineering technology such as blood vessel, nerve trachea as bio-medical material, adds that the good transparency can be also used for cell culture medium material and micro fluidic device. It has also been found that whether heat or xeothermic in water or in steam, all can improve zein mechanical strength under wet condition, transparency and its degradation rate of reduction, and by controlling heating-up temperature and the purpose of control mechanics intensity and degradation rate can be reached heat time heating time.
Accompanying drawing explanation
Table 1 is the maximum mechanical strength (n=3) that the axial tension of corn protein materials before and after processing, radial drawing and tractive stretch.
Figure 1A is the pancreatin degradation curve of the corn protein materials after processing and untreated corn protein materials, and B is the profile photo after 2,4,6,8 days enzymolysis. The degradation rate of vertical coordinate Degradationoftubes and pipe; Abscissa is degradation time.
Fig. 2 is the swelling and transparency results of the corn protein materials after processing and untreated corn protein materials. A and a is treated corn protein materials sample, and B and b is untreated zein sample, and A and B shoots under drying regime, and a and b is swelling rear shooting.
Fig. 3 is the zein thin film of the spectrophotometer detection transmitance to light, and A1 and A2 is the testing result under dry state, and B1 and B2 is the testing result (n=3) under hygrometric state.Vertical coordinate Transmittance and light transmittance; Abscissa is Wavelength (wavelength). Untreated represents untreated samples; Sample after Treated representative process.
Fig. 4 is the difference of the swelling ratio of the corn protein materials (Treated) after processing and untreated corn protein materials (Untreated). Vertical coordinate Swellingratio and swelling ratio; Abscissa is swelling time.
Fig. 5 is NIH3T3 cell (left side) and proliferative conditions (the 5th day) on corn protein materials surface (right side) after processing on culture plate.
Detailed description of the invention
In order to be better understood from the present invention, the present invention is expanded on further following by embodiment and performance test. Embodiment one
The aqueous solution of material 1,90% ethanol; 2, zein
Step:
Weigh certain mass zein pressed powder, it is dissolved in the ethanol water of 90%, it is made into 100mg/ml solution, this solution is coated on repeatedly on plate (pipe) shape mould, finally give the zein film (pipe) of wall thickness 50-200 micron. Finally obtained zein film (pipe) is immersed in secondary water, steam heating (121 DEG C, 105kPa, 20 minutes).
Embodiment two
The aqueous solution of material 1,75% ethanol; 2, zein
Step:
Weigh certain mass zein pressed powder, it is dissolved in the ethanol water of 75%, it is made into 300mg/ml solution, this solution is coated on repeatedly on plate (pipe) shape mould, finally give the zein film (pipe) of wall thickness 800 microns. Finally will process (30 DEG C, 4 weeks) under obtained zein film (pipe) saturation vapour.
Embodiment three
The aqueous solution of material 1,40% ethanol; 2, zein
Step:
Weigh certain mass zein pressed powder, it is dissolved in the ethanol water kind of 40%, it is made into 1mg/ml solution, this solution is coated on repeatedly on plate (pipe) shape mould, finally give the zein film (pipe) of wall thickness 500 microns. Finally obtained zein film (pipe) is immersed in secondary water, heats (90 DEG C, 120 minutes).
Embodiment four
The aqueous solution of material 1,60% ethanol; 2, zein
Step:
Weigh certain mass zein pressed powder, it is dissolved in the ethanol water of 60%, it is made into 40mg/ml solution, this solution is coated on repeatedly on plate (pipe) shape mould, finally give the zein film (pipe) of wall thickness 20 microns. Finally by obtained zein film (pipe) heating (160 DEG C, 60 minutes).
Performance test one
The mechanical strength of the corn protein materials that embodiment one obtains has been studied. Processing method, for being soaked in secondary water, is placed in 121 DEG C, and in the steam sterilization pan of 105kPa, the process time is 20 minutes. The difference of the mechanical strength of comparison process and untreated sample, tractive after detecting radial drawing, axial tension respectively and getting lines crossed. From the data of table 1 it can be seen that the mechanical strength of sample after processing is significantly higher than untreated sample.
The maximum mechanical strength (n=3) that table 1 axial tension, radial drawing and tractive stretch
Performance test two
Zein pancreatin degradation rate at different heating temperature has been studied. The difference of the pancreatin degradation rate processing sample and untreated samples that comparing embodiment three obtains. It can be seen in fig. 1 that treated corn protein materials sample, pancreatin degradation rate is remarkably decreased, and the untreated zein sample after 4,6,8 days enzymolysis can lose original shape (1B).
Performance test three
The corn protein materials that embodiment one is obtained carries out transparency experiment, the corn protein materials of comparison process and untreated zein sample transparency difference under dry state and hygrometric state.
Fig. 2 is the swelling and transparency results of the corn protein materials after processing and untreated corn protein materials. A and a is treated corn protein materials sample, and B and b is untreated zein sample, and A and B shoots under drying regime, and a and b is swelling rear shooting. Photo is to scribble the coverslip photographs of protein films, and the coverslip of coating covers on school badge. Coating layer thickness is 50 ��m. It can be seen that the corn protein materials sample after processing, through swelling, under hygrometric state, remain in that higher transparency.
Fig. 3 is the zein thin film of the spectrophotometer detection transmitance to light, and A1 and A2 is the testing result under dry state, and B1 and B2 is the testing result (n=3) under hygrometric state. The thickness of A1 and B1 is 50 microns, and the thickness of A2 and B2 is 100 microns. Data result and the photographic result of Fig. 2 in figure are completely the same. Still higher transparency (> 90%, 450nm-800nm can be kept) under corn protein materials sample hygrometric state after proof process.
Performance test four
Carry out studying (n=3) to the swelling ratio of the corn protein materials that embodiment two obtains. From fig. 4, it can be seen that the swelling ratio of the corn protein materials after processing is substantially less than untreated zein sample, and swelling ratio maintains low-level.
Performance test five
The corn protein materials that embodiment four is obtained is cultivated as cell and the effect of micro-fluidic base material has been studied. Fig. 5 shows the cell proliferative conditions of optical microphotograph Microscopic observation: compared with 48 well culture plates, propagation on NIH3T3 cell corn protein materials thin film after treatment is without significant difference, and due to its higher transparency, it is possible to directly by optical microscope observation of cell form. Illustrate that corn protein materials meets the requirements as cell culture medium material and micro fluidic device.
Claims (6)
1. the molten protein material of grain alcohol, it is characterized in that adopting the molten protein solid powder of grain alcohol, it is dissolved in the ethanol water of 40��90%, it is made into 1��300mg/ml solution, this solution is coated on repeatedly on mould and forms thin film, plate or pipe, by the obtained molten protein films of grain alcohol, plate or pipe by heating in water, steam heats or xeothermic method processes, by the obtained molten protein films of grain alcohol, plate or pipe, directly or be immersed in pure water steam heating, process 20-120 minute under the pressure condition of 60-130 DEG C and steam sterilization; Or process 1-4 week in 30-60 DEG C under saturation vapour; Or process 1-60 minute under 100-160 DEG C of dry heat condition; The molten protein material of described grain alcohol is zein, wheat gliadin.
2. the molten protein material of grain alcohol as claimed in claim 1, is characterized in that described pure water is redistilled water.
3. the molten protein material of grain alcohol as claimed in claim 1, is characterized in that the thickness of described thin film, plate or pipe is 20-1000 micron.
4. the molten protein material of grain alcohol as claimed in claim 1, is characterized in that described heat-treatment process all carries out in confined space.
5. the molten protein material of grain alcohol as claimed in claim 1 is for micro fluidic device or cell culture medium material.
6. the molten protein material of grain alcohol as described in right 1 is for the bio-medical material of Biodegradable nerve conduit or degradable artificial blood vessel.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1555892A (en) * | 2004-01-08 | 2004-12-22 | 上海交通大学 | Preparation method of plant origion alcohrl soluble protein three dimentional support |
| CN101757692A (en) * | 2010-03-11 | 2010-06-30 | 上海交通大学 | Cereals alcohol-soluble protein conduit and preparation method thereof |
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| CN1476825A (en) * | 2003-07-04 | 2004-02-25 | 中国科学院上海有机化学研究所 | Grain prolamin microsphere and preparation method |
| EP2007219A1 (en) * | 2006-03-13 | 2008-12-31 | NATURIN GmbH & CO. | Biodegradable protein based thermoset compositions, preparation methods and applications thereof |
| CN102516778A (en) * | 2011-11-29 | 2012-06-27 | 上海交通大学 | Cereal protein-based micro-carrier for large-scale culture of cells, and preparation method and application of micro-carrier |
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| CN1555892A (en) * | 2004-01-08 | 2004-12-22 | 上海交通大学 | Preparation method of plant origion alcohrl soluble protein three dimentional support |
| CN101757692A (en) * | 2010-03-11 | 2010-06-30 | 上海交通大学 | Cereals alcohol-soluble protein conduit and preparation method thereof |
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