CN103948961A - Corn protein material as well as preparation method and application thereof - Google Patents
Corn protein material as well as preparation method and application thereof Download PDFInfo
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Landscapes
- Peptides Or Proteins (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention provides a grain alcohol-soluble protein material as well as a preparation method and application thereof. The grain alcohol-soluble protein material has relatively-high mechanical strength and transparency as well as a proper in-vivo degradation rate. The grain alcohol-soluble protein material provided by the invention can be prepared by a method comprising the steps of heating in water, heating in steam and dry heating, wherein during a heating process, the temperature is controlled to be 30 to 160 DEG C and the time is controlled to be 1-120 minutes. The mechanical strength and the transparency of the treated rain alcohol-soluble protein material are improved, the degradation rate and swelling ratio are remarkably reduced, the rain alcohol-soluble protein material also has a stable surface structure, and bonding is avoided. By controlling the heating temperature and the heating time, the mechanical strength and the degradation rate can be controlled and a swelling resisting effect is achieved. The treated grain alcohol-soluble protein material can be applied to biomedical purposes as well as cell culture substrates and micro-fluid devices.
Description
Technical field
The present invention relates to the molten protein material of grain alcohol (including but not limited to zein, wheat gliadin), can by water, the method for Steam Heating or the in type corn alcohol soluble protein of dry heat treatment obtains, to improve mechanical strength, transparency and to reduce degradation rate, particularly by controlling acid extraction and then reaching the object of controlling mechanical strength, transparency and degradation rate under wet condition, and can reduce the molten protein material swelling ratio of grain alcohol after processing.The invention still further relates to the purposes of the protein material obtaining by the method.
Technical background
Natural biologic material comprises collagen, gelatin, sodium alginate and chitosan etc., because it is with low cost, be easy to obtain, degradable and the advantage such as biocompatibility is better, be applied to medical health field, as cartilage substitute, implantable bone material, artificial blood vessel and artificial skin etc.But natural biologic material is because present the feature of mixture, synthesising biological material cannot match in excellence or beauty in the control of degradation rate and mechanical strength.If therefore can search out degradation rate and the mechanical strength that a kind of method is controlled natural biologic material, can greatly expand its range of application, obtain clinically better therapeutic effect.
The natural polymer that protein is comprised of several amino acids, the hydrogen bond that intermolecular existence is stronger and disulfide bond interact.The protein of take is poor as the mechanical property of basic biomaterial, needs to improve through methods such as ultraviolet irradiation, thermal polymerization, enzyme processing or chemical crosslinkings rigidity, thermostability and the stability of material.The formaldehyde that chemical crosslink technique is the most frequently used or glutaraldehyde, because having toxicity, generally avoid being used in technical field of biological material.Heat treatment is as physical method, by controlling heat treated temperature, also can control mechanical strength and the degradation rate of protein, and compare with adopting the chemical method of cross-linking agent, can 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 implanting requires also can keep good mechanical property under hygrometric state.For example, artificial blood vessel need to bear blood stream pressure and impact in order to avoid explosion, and nerve trachea need to be resisted the pressure of outside muscular tissue in order to avoid subside.
The polymer with higher clarity has been widely used in micro-fluidic and cell culture system.But polymer used is as polydimethylsiloxane and silicone rubber, cell compatibility is not bery desirable, need to be injected or be increased the methods such as surface roughness by plasma and improve its cell compatibility.On the other hand, take the polymer that oil is raw material 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 natural or synthetic degradation material, significant for cell culture base 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.[Wang HJ, Lin ZX, Liu XM, Sheng SY, Wang JY.Heparin-loaded zein microsphere film and hemocompatibility.J Control Release2005; 105:120-31.; Sun QS, Dong J, Lin ZX, Yang B, Wang JY.Comparison of cytocompatibility of zein film with other biomaterials and its degradability in vitro.Biopolymers2005; 78:268-74.] as material, be easy to machine-shaping, as three-dimensional rack, two-dimensional film, microsphere, tubulose, fibrous etc., in recent years increasing at the research report in bio-medical field.But because corn alcohol soluble protein is mainly comprised of hydrophobic amino acid, surrounding for example relative humidity has a significant impact its character.As water can cause its swelling, this has limited them in the application in some field.
Moist heat sterilization is the general sterilizing methods in cell culture, and water can be used as the plasticizer of protein structure.By the method for High Temperature High Pressure, process fibroin albumen and can change its secondary structure, increase β-pleated sheet structure proportion.[Hu X, Shmelev K, Sun L, Gil ES, Park SH, Cebe P, et al.Regulation of silk material structure by temperature-controlled water vapor annealing.Biomacromolecules 2011; 12:1686-96; Um IC, Kweon HY, Park YH, Hudson S.Structural characteristics and properties of the regenerated silk fibroin prepared from formic acid.Int J Biol Macromol2001; 29:91-7; Lawrence B, Omenetto F, Chui K, Kaplan D.Processing methods to control silk fibroin film biomaterial features.JMater Sci 2008; 43:6967-85.] β-pleated sheet is relevant with mechanical strength simultaneously.Nishiyama[Nishiyama Y, Wada M, Kuga S, Magoshi J.Mechanical properties of silk fibroin – microcrystalline cellulose composite films.J Appl Polym Sci2002; 86:3425-9.] etc. people find that, in fibroin, the appearance of β-pleated sheet structure has increased hot strength.Meanwhile, the increase of β-pleated sheet structure has reduced degradation rate.【Emmambux?MN,Taylor?JR.Properties?of?heat-treated?sorghum?and?maize?meal?and?their?prolamin?proteins.J?Agric?Food?Chem2009；57:1045-50.】
Based on above-mentioned background document, in order further to expand the range of application of corn alcohol soluble protein, the defect of itself while first needing to overcome it as material: as be easy to opaque after swelling and swelling, fragility is bigger than normal etc.As its degradability of natural material, be advantage on the one hand, but in some occasion, for example, during as cell culture base material, will use trypsin digestion cell, at this moment need to stop its degraded.For the biomaterial that corn alcohol soluble protein is base material of take that obtains that mechanical strength, transparency and degradation rate meet that such use requires has proposed the present invention.
Summary of the invention
The object of the present invention is to provide the molten protein material of a kind of grain alcohol, by water, steam or the molten protein material of the in type grain alcohol of dry heat treatment significantly increase its mechanical strength, transparency under hygrometric state, and significantly reduce its degradation rate.Can be by controlling heating-up temperature and then reaching the object of control degradation speed and mechanical strength.Another object of the present invention is to provide the purposes of this protein material.The molten protein material of grain alcohol can be zein, wheat gliadin.
The molten protein material of grain alcohol system is adopted to zein or wheat gliadin pressed powder, be dissolved in 40～90% ethanol water, be made into 1～300mg/ml solution, this solution is repeatedly coated on and on mould, forms thin film, plate or pipe, by resulting zein or wheat gliadin thin film, plate or pipe directly or be immersed in (in redistilled water) in pure water, under the pressure condition of 60-130 ℃ and steam sterilization, process 20-120 minute; Or in 30-60 ℃, process 1-4 week under saturation vapour; Or process 1-60 minute under 100-160 ℃ of dry heat condition, become zein or wheat gliadin material.
Above-mentioned processing procedure of the present invention is recommended in confined space carries out; Conventionally be coated on and on mould, form thin film, plate or management and control and make type, thickness is 20-1000 micron, and when relating to transparency and being greater than 90%, usually controlling thickness is 20-200 micron.
First with zein, be that example is studied, found that, zein is after Steam Heating is processed, and its mechanical strength, transparency significantly improve, and its degradation rate obviously reduces.The improvement of these character can make zein material as bio-medical material, be applied to the field of tissue engineering technology such as blood vessel, nerve trachea, adds that the good transparency can also be for cell culture base material and micro fluidic device.No matter the present invention also finds it is heating or xeothermic in water or in steam, all can improve zein its degradation rate of mechanical strength, transparency and reduction under wet condition, and can be by controlling heating-up temperature and reaching the object of control mechanics intensity and degradation rate heat time heating time.
Accompanying drawing explanation
Table 1 is the maximum mechanical strength (n=3) that axial tension, radial drawing and the tractive of the zein material before and after processing stretches.
Figure 1A is zein material after processing and the pancreatin degradation curve of untreated zein material, and B is through 2,4, the profile photo after 6,8 days enzymolysis.Vertical coordinate Degradation of tubes is the degradation rate of pipe; Abscissa is degradation time.
Fig. 2 is zein material after processing and swelling and the transparency results of untreated zein material.A and a are treated zein material samples, and B and b are untreated zein samples, and A and B take under drying regime, and a and b take after swelling.
Fig. 3 is the transmitance of the zein thin film that detects of spectrophotometer to light, and A1 and A2 are the testing result under dry state, and B1 and B2 are the testing result (n=3) under hygrometric state.Vertical coordinate Transmittance is light transmittance; Abscissa is Wavelength (wavelength).Untreated represents untreated samples; Rear sample is processed in Treated representative.
Fig. 4 is the difference of the swelling ratio of zein material (Treated) after processing and untreated zein material (Untreated).Vertical coordinate Swelling ratio is swelling ratio; Abscissa is swelling time.
Fig. 5 be NIH3T3 cell on culture plate (left side) and process after the propagation situation (the 5th day) of zein material surface (right side).
The specific embodiment
For a better understanding of the present invention, will further set forth the present invention by embodiment and performance test below.Embodiment mono-
The aqueous solution of material 1,90% ethanol; 2, zein
Step:
Take certain mass zein pressed powder, be dissolved in 90% ethanol water, be made into 100mg/ml solution, this solution is coated on plate (pipe) shape mould repeatedly, finally obtain the zein film (pipe) of wall thickness 50-200 micron.Finally resulting zein film (pipe) is immersed in intermediate water to Steam Heating (121 ℃, 105kPa, 20 minutes).
Embodiment bis-
The aqueous solution of material 1,75% ethanol; 2, zein
Step:
Take certain mass zein pressed powder, be dissolved in 75% ethanol water, be made into 300mg/ml solution, this solution is coated on plate (pipe) shape mould repeatedly, finally obtain the zein film (pipe) of 800 microns of wall thickness.Finally will under resulting zein film (pipe) saturation vapour, process (30 ℃, 4 weeks).
Embodiment tri-
The aqueous solution of material 1,40% ethanol; 2, zein
Step:
Take certain mass zein pressed powder, be dissolved in 40% ethanol water kind, be made into 1mg/ml solution, this solution is coated on plate (pipe) shape mould repeatedly, finally obtain the zein film (pipe) of 500 microns of wall thickness.Finally resulting zein film (pipe) is immersed in intermediate water to heating (90 ℃, 120 minutes).
Embodiment tetra-
The aqueous solution of material 1,60% ethanol; 2, zein
Step:
Take certain mass zein pressed powder, be dissolved in 60% ethanol water, be made into 40mg/ml solution, this solution is coated on plate (pipe) shape mould repeatedly, finally obtain the zein film (pipe) of 20 microns of wall thickness.Finally by resulting zein film (pipe) heating (160 ℃, 60 minutes).
Performance test one
The mechanical strength of the zein material that embodiment mono-is obtained is studied.Processing method is for being soaked in intermediate water, and is placed in 121 ℃, and in the steam sterilization pan of 105kPa, the processing time is 20 minutes.Relatively process and the difference of the mechanical strength of untreated sample, detect respectively radial drawing, axial tension and get lines crossed after tractive.From the data of table 1, can find out, the mechanical strength of the 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
Pancreatin degradation rate to zein at different heating temperature is studied.The difference of the processing sample that comparing embodiment three obtains and the pancreatin degradation rate of untreated samples.From Figure 1A, can find out, treated zein material sample, pancreatin degradation rate significantly declines, and through 4,6, the untreated zein sample after 8 days enzymolysis can lose original shape (1B).
Performance test three
The zein material that embodiment mono-is obtained carries out transparency experiment, zein material and the transparency difference of untreated zein sample under dry state and hygrometric state relatively processed.
Fig. 2 is zein material after processing and swelling and the transparency results of untreated zein material.A and a are treated zein material samples, and B and b are untreated zein samples, and A and B take under drying regime, and a and b take after swelling.Photo is to scribble the coverslip photographs of albumen thin film, and the coverslip of coating covers on school badge.Coating layer thickness is 50 μ m.As can be seen from the figure, the zein material sample after processing, through swelling, has still kept higher transparency under hygrometric state.
Fig. 3 is the transmitance of the zein thin film that detects of spectrophotometer to light, and A1 and A2 are the testing result under dry state, and B1 and B2 are 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.The photo result of data result in figure and Fig. 2 is in full accord.Under zein material sample hygrometric state after proof is processed, still can keep higher transparency (>90%, 450nm-800nm).
Performance test four
The swelling ratio of the zein material that embodiment bis-is obtained is studied (n=3).As can be seen from Figure 4, the swelling ratio of the zein material after processing is significantly lower than untreated zein sample, and swelling ratio maintains low-level.
Performance test five
The zein material that embodiment tetra-is obtained is studied as the effect of cell culture and micro-fluidic base material.Fig. 5 has shown the cell proliferation situation of optical microphotograph Microscopic observation: compare with 48 well culture plates, propagation on the zein material film of NIH3T3 cell after processing is without significant difference, and due to its higher transparency, can directly use optics microscope observing cell form.Illustrate that zein material meets the requirements as cell culture base material and micro fluidic device.
Claims (8)
1. the molten protein material of grain alcohol, it is characterized in that adopting the molten protein solid powder of grain alcohol, be dissolved in 40～90% ethanol water, be made into 1～300mg/ml solution, this solution is repeatedly coated on mould and forms thin film, plate or pipe, resulting corn alcohol soluble protein thin film, plate or pipe are processed by heating in water, Steam Heating or xeothermic method.
2. the molten protein material of grain alcohol as claimed in claim 1, is characterized in that the described molten protein material of grain alcohol is zein, wheat gliadin.
3. the preparation method of the molten protein material of grain alcohol as claimed in claim 1, it is characterized in that resulting corn alcohol soluble protein thin film, plate or pipe, directly or be immersed in Steam Heating in pure water, under the pressure condition of 60-130 ℃ and steam sterilization, process 20-120 minute; Or in 30-60 ℃, process 1-4 week under saturation vapour; Or process 1-60 minute under 100-160 ℃ of dry heat condition, become zein or wheat gliadin material.
4. method as claimed in claim 3, is characterized in that described pure water is redistilled water.
5. method as claimed in claim 3, is characterized in that the thickness of described thin film, plate or pipe is 20-1000 micron.
6. method as claimed in claim 3, is characterized in that described heat-treatment process all carries out in confined space.
7. the molten protein material of grain alcohol as claimed in claim 1 is for food and bio-medical material, cell culture base material and micro fluidic device.
8. the molten protein material purposes of grain alcohol as claimed in claim 1, is characterized in that described bio-medical material is Biodegradable nerve conduit, degradable artificial blood vessel.
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| CN112062994A (en) * | 2020-09-09 | 2020-12-11 | 苏州大学 | Egg white bioplastic, preparation method and application thereof |
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