KR20140048362A - Radiation biomolecules bacterial cellulose nanofibers and method of manufacture - Google Patents

Abstract

The present invention relates to microbial cellulose nanofibers using radiation irradiation and a method for manufacturing the same, includes the steps of: freeze-drying cellulose gel by microorganism metabolites of citrus pulp; irradiating the freeze-dried cellulose gel by radiation and preparing graft cellulose fibers for grafting (meth)acrylic acid; manufacturing biomolecular nanofibers for polymerizing the graft cellulose fibers and extracellular matrix proteins; and freeze-drying the biomolecular nonofibers. According to the present invention, the microbial cellulose nanofibers can be used as recycling resources of by-product and also be used for developing a biomimetic scaffold for tissue engineering, thereby obtaining enormous economical effects and exhibiting high technological value. [Reference numerals] (AA) Bacterial cellulose nanofibers; (BB,CC,DD) Protein/Peptide

Description

TECHNICAL FIELD [0001] The present invention relates to a biodegradable cellulose nanofiber,

The present invention relates to a microbial cellulose nano fiber using a radiation irradiation technique and a manufacturing method thereof.

Tissue engineering is a new technology that can be effectively applied to the treatment of injuries and malfunctions of tissues and organs caused by the increase in external leisure activities and the entry into the age of aging. Organs and tissues based on various engineering methods.

The human body system consists of a very complex system based on cells. Cells maintain their life phenomena by interaction with three - dimensional microstructures called proteins, saccharides and extracellular matrix composed of these complexes. Through specific binding to the extracellular matrix, the cells settle in a three-dimensional space, maintain the intrinsic shape of the cell, and express the cell-specific trait.

Cellulose is a polysaccharide composed of β-1,4-glucose as a main component of cell walls of higher plants widely found in nature. Plant cellulose is composed of heteropoly-saccharide mixed with other polysaccharides such as pectin, hemicelluloses, and lignin, resulting in low crystallinity and poor mechanical strength and adsorption. On the other hand, unlike vegetable cellulose, cellulose produced by bacteria is a pure cellulose aggregate containing no polysaccharide such as pectin, hemicelluloses and lignin, and is a microfibril having a thickness of about 0.1 μm. Is a three-dimensional network structure formed by hydrogen bonding. Therefore, it has high crystallinity and excellent physical properties such as mechanical strength, adsorptivity, water retentivity, suspension stability, and binding property and is widely used as a new material in foods, cosmetics and pharmaceuticals industry.

Citrus is the largest fruit product in Korea, which is produced in Jejudo with annual production of 50 ~ 600 thousand tons. Most citrus juice is used as a raw material, but some citrus juice is stored in citrus juice concentrate at the previous stage. It is produced in the most limited season and has many difficulties in preservation processing. In addition, the treatment of citrus peels and by-products is costly, and the need for the development of new materials using citrus peels is emerging. Korean Patent Laid-Open No. 10-2004-0069587 (Patent Document 1) for the purpose of recycling citrus peel has disclosed a technique for producing dietary fiber, flavonoid, feed and the like by recycling citrus peel.

As one of the methods of using citrus peel, there have been attempts to produce microbial cellulose and the like from citrus peel for use in biomaterials. However, the microbial cellulose has a disadvantage of low cell adhesiveness and proliferation efficiency because there is no cell attachment ligand necessary for effectively regenerating the tissue.

Korean Patent Publication No. 10-2004-0069587

It is an object of the present invention to solve the above problem, and it is an object of the present invention to provide a method for modifying a microbial cellulose produced from citrus peel into biomaterials.

Specifically, it is an object of the present invention to provide a support for tissue engineering which enables the synthesis of microbial cellulose with an extracellular matrix by modifying the microbial cellulose using a radiation irradiation technique.

The present invention to achieve the above object

Preparing a cellulosic gel by lyophilization by microbial metabolism of citrus peel;

Preparing grafted cellulose fiber by irradiating the freeze-dried cellulosic gel with (meth) acrylic acid by irradiation;

Preparing a biomolecule nanofiber that polymerizes the extracellular matrix protein on the grafted cellulose fiber; And

Lyophilizing the biomolecule nanofibers;

The present invention also provides a method for producing microbial cellulose nanofibers.

The present invention also provides microbial cellulose nanofibers prepared by the above-mentioned method.

The microbial cellulose nanofiber according to the present invention can be used as a biomaterial by producing microbial cellulose, specifically microbial fermentation cellulosic gel, from citrus peel, thereby solving the problem of recycling citrus peel as a by-product of waste, and also having an environmentally friendly and economical effect You can expect.

This is because it is not harmful to the human body and nature as well as recycling resources of used byproducts by introducing a substance having excellent biocompatibility by using radiation technology without using chemicals necessary for the manufacture of conventional tissue engineering scaffolds And biomimetic scaffolds for tissue engineering can be expected to have enormous economic effects.

The present invention also relates to a method for producing a microbial fermentation cellulosic gel, which is capable of synthesizing a microbial fermentation cellulosic gel, which is a natural material, with an extracellular matrix by using a radiation technique, thereby improving tissue and cell affinity Can be provided.

These techniques are superior to conventional biomaterials in terms of biocompatibility. Specifically, since the support for tissue engineering according to the present invention exhibits excellent ability in cell adsorption, proliferation and adsorption of cells, the cell affinity of the support can be enhanced It is expected to be useful as a technology.

FIG. 1 is a schematic view showing the stepwise bonding structure of microbial cellulose nanofibers according to the present invention.
2 is a scanning electron microscope photograph of (a) Example 2 and (b) Example 3. Fig.
FIG. 3 shows the result of UV-VIS spectrometer measurement of (a) Example 1 (BC) and Example 2 (AAc-BC) after staining with toluidine blue (TBO) and (b) .
FIG. 4 is a graph showing the results of gelatin-AAc-BCM gelatin-AAC-BC polymerized with (a) Example 1 (BC), (b) Example 2 (AAc- BC) were observed by fluorescence microscopy.
FIG. 5 is a graph showing changes in the surface properties of ATR-AAC-BC obtained from Example 1 (BC), Example 2 (AAc-BC) and Example 3 (Gelatin- FTIR graph.

The present invention relates to a method for preparing a grafted cellulose fiber, comprising the steps of preparing cellulose gel by microbial metabolism of citrus peel by lyophilization, preparing grafted cellulose fiber by irradiating the lyophilized cellulosic gel with radiation to (meth) acrylic acid, A step of preparing biomolecule nanofibers for polymerizing extracellular matrix proteins on the fibers; And lyophilizing the biomolecule nanofibers; The present invention also provides a method for producing microbial cellulose nanofibers.

In the present invention, the extracellular matrix protein may include a three-dimensional nanostructure composed of one or two or more selected from gelatin, collagen, fibrin, laminin, fibronectin and elastin.

In the present invention, the extracellular matrix protein may further comprise a polypeptide.

In the present invention, the freeze-drying may be performed at -80 to -50 ° C for 3 to 5 days.

The step of preparing the biomolecule nanofibers includes the step of polymerizing 1-ethyl-3- (3-methylmethoxypropyl) carbodiimide and N-hydroxysuccinimide.

The graft may be prepared by immersing the graft in a (meth) acrylic acid solution and then irradiating 5 to 100 kGy of gamma rays.

The present invention also provides a method for producing microbial cellulose nanofibers, which further comprises removing unreacted (meth) acrylic acid present in the grafted cellulose fibers

The microbial cellulose nanofibers produced by the above methods are included in the scope of the present invention.

Also included within the scope of the present invention are tissue engineering supports comprising the microbial cellulose nanofibers.

Hereinafter, the present invention will be described in more detail.

Biodegradability in tissue engineering accounts for a considerable proportion. If non-degradable polymers are used, the support that remains in the tissue after complete regeneration of the lesion is recognized as an external substance and the inflammation reaction may continue to occur. Must accompany. In addition, if biodegradation does not occur, the newly formed tissue is difficult to regenerate into the support and can not be an effective fusion with surrounding tissues. Biodegradable polymers are polymers that are degraded in the body by hydrolysis or degradation enzymes. Depending on the method of obtaining the polymer, the biodegradable polymer can be largely divided into a natural polymer and a synthetic polymer. Natural polymers include proteins such as gelatin, collagen, fibrin and elastin, and polysaccharide series such as alginic acid and hyaluronic acid, and synthetic polymers include polymers and copolymers of polyester and the like.

The inventors of the present invention have discovered that biocompatible nanofibers can be provided by applying cellulosic gels based on microbial metabolism of citrus peel instead of natural polymers and synthetic polymers conventionally used as biodegradable polymers, thereby completing the present invention.

In the present invention, acrylic acid is graft-polymerized on microbial cellulose nanofibers by using a radiation technique, and microbial cellulose nanofibers can be provided by binding proteins or peptides having excellent cell affinity.

The present invention relates to a method for producing a cellulose gel, comprising the steps of: preparing cellulose gel by microbial metabolism of citrus peel; A step of preparing grafted cellulose fibers by irradiating the cellulose gel with radiation to graft (meth) acrylic acid; And a biomolecule nanofiber to polymerize the extracellular matrix protein on the grafted cellulose fiber; The present invention also provides a method for producing microbial cellulose nanofibers.

First, a step of preparing cellulose gel by microbial metabolism of citrus peel by lyophilization will be described in detail.

The preparation of the cellulose gel can be carried out by subjecting the citrus peel fermentation broth to a fermentation step.

In the present invention, citrus peel is obtained from citrus, and "citrus fruit" is citrus fruit cultivated in southern Europe and the like, grape fruit as a mutant species of a cultivar cultivated in the United States of America and the like, It is grown in India, Italy and so on. Sour orange is cultivated all over the world such as India, Kumquat or Yuzu cultivated in China or Korea, Halla bong, Jean oriental, Tangerine, Citrus, , Citron, chrysanthemum, mandarin, sardine, persimmon, tangerine and the like, all of which are called "citrus peel".

The citrus peel fermentation broth is preferably prepared at about 40 to 70 ° C for 3 to 12 hours after inoculation with sugar chain enzymes such as Pectines 100 L, Viscozyme L, AMG 300 L and Celluclast 1.5 L. At this time, the sugar content is not particularly limited, but it is preferably within the range of 1 to 30 brix. The amount of sugar inoculated with the enzyme is not limited, but when the efficiency is considered, it is preferable to inoculate 0.01 to 1% (w / w) of the citrus peel.

The strain can be applied to various strains within a range in which the cellulose gel is produced, and most preferably, Gluconacetobacter hanseni TL-2C can be used.

At this time, the seedlings are inoculated in the fermentation broth at 10% (v / v), and the inoculation amount is in the range of ¹ x ^{10 3} to 1 x 10 ⁷ cells / ml but is not necessarily limited thereto.

Political fermentation is carried out under aerobic conditions and takes 7 to 30 days at 20 ° C to 40 ° C, but is not limited thereto.

The cellulosic gel thus prepared may be used by the Jeju Rural Development Administration.

The cellulosic gel is prepared by lyophilization and lyophilization is performed at -80 to -50 ° C for 3 to 5 days. When the wet cellulosic gel is dried at room temperature, the thickness and porosity of the cellulosic gel decrease as the water is blown from the gel. Therefore, when the freeze-drying method is used, the water in the cellulose gel is crystallized into ice at low temperature, and when the pressure is lowered, the water is sublimated and the thickness of the original cellulose is maintained. Among these methods, since the initial wet cellulosic gel can adjust the crystal size of water according to the freezing temperature, a gel having various pore sizes can be produced. For example, when cooled slowly at -20 ° C, ice crystals increase, and at 80 ° C, water suddenly freezes and crystals decrease. Therefore, it is possible to control the porosity of the gel by controlling the crystallization rate according to the cooling temperature of water.

Next, the production step of the grafted cellulose fiber in which (meth) acrylic acid is grafted by irradiating the cellulose gel with radiation will be described in detail.

The cellulosic gel according to the present invention has low cell adhesiveness and proliferation efficiency due to the absence of cell attachment ligand necessary for effectively regenerating the tissue. The present invention introduces radiation technology to solve this problem. The principle of the technique using radiation is that radicals are generated in the monomers by polymerization and polymerized to form polymers, or cross-linking of polymer chains or grafting of monomers occurs due to the radicals of the polymer chains. Since these radiation techniques do not use crosslinkers or initiators which are harmful to human body compared to chemical methods, there is no need for separate purification process after the reaction, and the sterilization process, which is an essential step when used in the medical field, can also be replaced by irradiation.

The cellulosic gel is preferably cut into a size of 3 to 10 mm in diameter.

Irradiation means gamma ray. Specifically, gamma rays of ⁶⁰ Co source (ACEL type C-1882, Korea Atomic Energy Instiute) can be irradiated at 5 ~ 100 kGy.

At this time, the (meth) acrylic acid can be specifically acrylic acid solution, more specifically, a 5 wt% acrylic acid solution of 0.1 M NaOH salt may be used, but it is not limited thereto. For example, 1 to 10 wt% of an acrylic acid solution can be used.

It is also preferable that the cellulose gel is immersed in the acrylic acid solution and irradiated with the radiation. Therefore, the content ratio of the acrylic acid solution and the cellulose gel is suitably in the range capable of immersing the cellulose gel, specifically, it is preferably 50 to 99% by weight of the acrylic acid solution and 1 to 50% by weight of the cellulosic gel.

The grafted cellulose fiber grafted with (meth) acrylic acid may further include a step of removing unreacted (meth) acrylic acid. Specifically, the grafted cellulose fiber may be washed with tertiary distilled water for 50 to 100 hours (Meth) acrylic acid can be removed.

Next, the production steps of the biomolecule nanofibers for polymerizing the extracellular matrix protein on the grafted cellulose fiber will be described in detail.

The grafted cellulose fibers can polymerize extracellular matrix proteins using an EDC / NHS reaction. (EDC) and N-hydroxysuccinimide (NHS). More specifically, the step of polymerizing z- (N (N, N-dimethylaminopropyl) carbodiimide -Morpholino) ethylsulfonic acid (MES) buffer solution in which EDC and NHS are dissolved, immersing the grafted cellulose fiber in a mixture, dissolving the extracellular matrix protein in the dissolution solution, and stirring. At this time, the mixing ratio of EDC and NHS mixed solution may be selected from 1: 9 to 9: 1.

The extracellular matrix protein comprises a three-dimensional nanostructure consisting of one or more selected from gelatin, collagen, fibrin, laminin, fibronectin and elastin.

The polypeptide may also be used as an extracellular matrix protein.

The biomolecule nanofibers in which the extracellular matrix protein is polymerized may further include a step of removing unreacted extracellular matrix proteins, and specifically, z- (N-morpholino) ethylsulfonic acid and phosphate buffered saline And washing can be performed for 20 hours to 30 hours.

Finally, the step of freeze-drying the nanofibers will be described in detail.

In the present invention, when the cellulosic nanofibers are lyophilized, the degree of crosslinking of the cellulose gel fibers is increased, and tensile strength, elasticity, and restoring force are increased. As a result, a superior support for tissue engineering has been provided.

It is preferable that the freeze-drying is performed at -80 to -50 캜 for 3 to 5 days.

The following examples are intended to illustrate the present invention and should not be construed as limiting the scope of the invention.

≪ Example 1 > Freeze-drying of cellulosic gel by microbial metabolism of citrus peel

Cellulose gel (Jeju Rural Development Administration), which was prepared by fermenting citrus peel as a raw material at 30 ° C for 14 days by using Glucoacetobacter hansen TL-2C, was incubated at -78 ° C for 24 hours and at -50 ° C for 48 hours with a freeze dryer Lyophilized and prepared.

Example 2: Preparation of grafted cellulose fiber

The lyophilized cellulose gel was cut into a size of 8 mm and immersed in a weight ratio of 1: 1 of the cellulosic gel and a 5 wt% acrylic acid solution of 0.01 M NaOH salt.

(ACEL type C-1882, Korea Atomic Energy Institute) was irradiated to the immersed cellulosic gel at a dose rate of 10 kGy / hr at 25 kGy. After the irradiation, grafted cellulose fibers were prepared by washing with distilled water for 72 hours in order to remove unreacted acrylic acid.

Example 3 Production of Biomolecule Nanofibers

The grafted cellulose fibers were immersed in a solution of EDC and NHS in a volume ratio of 1: 1 in 0.1 M z- (N-morpholino) ethylsulfonic acid (MES) buffer solution (pH 5.07, 5 mg / (2 mg / mL), and the mixture was stirred for 12 hours. Biomolecule nanofibers were prepared by washing for 24 hours in a 1: 1 mixture of z- (N-morpholino) ethylsulfonic acid and phosphate buffered saline to remove unreacted gelatin.

Example 4 Freeze-drying Biomolecule Nanofibers

The washed biomolecule nanofibers were freeze-dried at -78 ° C for 72 hours using a freeze dryer.

The prepared microbial cellulose nanofibers were analyzed by various methods and their properties were evaluated.

1. Scanning electron microscope (SEM) measurement

The effect of acrylic acid and gelatin bound to the surface of the cellulose gel in the biomolecule nanofibers was examined using an electron microscope (SEM, JSM-6400, JEOL, Japan) at 10 kv, 10 to 12 mm distance . The samples were measured by gold coating for 70 seconds using a sputter coater.

SEM photographs of the grafted cellulose fibers of Example 2 are shown in FIG. 2 (a), and SEM photographs of the gelatin-polymerized biomolecule nanofibers of Example 3 are shown in FIG. 2 (b).

2. Toluidine blue O (Toluidine blue O) staining and quantification

Also, TBO was used to determine the carboxyl group of acrylic acid in the cellulose gel. The measurement method is as follows.

In a 24-well plate, 500 μl of TBO solution (0.01 M HCl, 60 mg NaCl, 12 mg TBO) and grafted cellulose fiber were added and stirred for 6 hours, followed by washing with distilled water until TBO was no longer dissolved . The solution was then dissolved in a solution of 0.1 M NaOH and ethanol in a volume ratio of 1: 4 (v / v), and then measured at 630 nm using a UV-VIS spectrometer (PowerWave XS, Biotek, USA). 3 (a), the cellulose gel (BC) of Example 1 was not dyed while the grafted nanofibers (AAc-BC) of Example 2, in which acrylic acid was grafted, Color was confirmed. Fig. 3- (b) shows the results of quantifying the amount of TBO stained on the graft nanofibers. BC was measured to be 0.0195 + 0.1384 and AAc-BC was measured to be 6.3827 + 0.7710 (ug / mg). As a result, it was confirmed that the TBO reaction occurred due to the binding of the carboxyl group.

3. Fluorescein staining

To determine whether the gelatin was polymerized well, the following measurements were made using fluorescamine.

350 μl of 0.2 M borate buffer (pH 9) and 1 μl of fluorescein solution (1 mg) were added to each of cellulose gel (BC), graft nanofiber (AAc-BC) and gelatin-polymerized biomolecule nanofibers / mL acetone) were mixed together, and the mixture was stirred at a high speed for 1 minute using a voltex, and then observed under a fluorescence microscope. As shown in FIG. 4, green fluorescence was not observed in BC and AAc-BC, but gelatin was bound to gelatin-AAc-BC because green fluorescence appeared.

4. ATR-FTIR measurement

In order to confirm the chemical characteristics of the surfaces of cellulose gel (BC), graft nanofibers (AAc-BC) and gelatin-biomolecule nanofibers (G-AAc-BC), attenuated total reflectance-Fourier transform infrared spectroscopy -FTIR) (Bruker TEMSOR 37, Bruker AXS. Inc., Germany). The spectra ranged from 500 to 4000 cm ^-1 at 4 cm ^{- 1} resolution and 64 scans.

5 is a graph of ATR-FTIR in which surface characteristics of (a) BC, (b) AAc-BC and (c) Gelatin-AAc-BC are analyzed. The peak of the carboxyl group of AAc-BC was found at 1720 cm ^-1 . G-AAc-BC combines the gelatin has been found a new peak at 1650 cm ^-1, 1550cm ^-1. This peak shows that amide (Ⅰ) and (Ⅱ) in gelatin bind gelatin to AAc-BC.

Claims (9)

Preparing a cellulosic gel by lyophilization by microbial metabolism of citrus peel;
Preparing grafted cellulose fiber by irradiating the freeze-dried cellulosic gel with (meth) acrylic acid by irradiation;
Preparing a biomolecule nanofiber that polymerizes the extracellular matrix protein on the grafted cellulose fiber; And
Lyophilizing the biomolecule nanofibers;
≪ / RTI > The method of claim 1, wherein
Wherein the extracellular matrix protein comprises a three-dimensional nanostructure composed of one or more selected from gelatin, collagen, fibrin, laminin, fibronectin and elastin. The method of claim 1, wherein
Wherein said extracellular matrix protein further comprises a polypeptide; and a method for producing microbial cellulose nanofibers The method of claim 1,
Wherein the freeze-drying is performed for 3 to 5 days at -80 to -50 ° C. The method of claim 1, wherein
Wherein the step of preparing the biomolecule nanofibers comprises polymerizing 1-ethyl-3- (3-methylmethoxypropyl) carbodiimide and N-hydroxysuccinimide. The method of claim 1,
Wherein the graft is immersed in a (meth) acrylic acid solution and irradiated with 5 to 100 kGy of gamma rays. The method of claim 1, wherein
And removing unreacted (meth) acrylic acid existing in the grafted cellulose fiber. A microbial cellulose nanofiber produced by the method of any one of claims 1 to 7. The method of claim 8,
A support for tissue engineering comprising the microbial cellulose nanofibers.

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