KR101919469B1 - Glutaraldehyde quencher comprising amino acid mixture and process for crosslinking biomaterial using the same - Google Patents

Abstract

The present invention provides a glutaraldehyde inhibitor comprising an amino acid mixture of glycine, histidine, and lysine, a method for inhibiting the activity of glutaraldehyde using the same, and a method for a crosslinking biomaterial using the same. The glutaraldehyde inhibitor, the method for inhibiting the activity of glutaraldehyde using the same, and the method for the crosslinking biomaterial using the same will contribute to improve the utilization of glutaraldehyde in various fields such as an atomic force microscope, a scanning electron microscope, a transmission electron microscope and the like.

Description

A glutaraldehyde inhibitor comprising an amino acid mixture and a method for crosslinking a biomaterial using the amino acid mixture,

The present invention relates to a glutaraldehyde inhibitor, and more particularly, to a glutaraldehyde inhibitor comprising an amino acid mixture and a method for crosslinking a biomaterial using the glutaraldehyde inhibitor.

Various types of cross-linking agents are used depending on the type of cross-linked biomaterial and the range of improvement of the desired properties. Glutaraldehyde (GA) is most widely used in a variety of fields such as histology, electron microscopy, nuclear microscopy, cell chemistry, protein chemistry, chemical sterilization, and biomedicine. In addition, GA has long been noted as an attractive crosslinking agent due to its low cost as well as commercial utility and high reactivity. GA, a linear 5-carbon dialdehyde, is a colorless, irritating oily liquid that dissolves in all proportions with water and alcohol as well as organic solvents. GA rapidly reacts with functional groups in proteins and carbohydrates near neutral pH to form thermally and chemically stable crosslinks and significantly improves elongation properties in biomaterials.

(Formaldehyde) and dialdehyde (glyoxal, 2 carbon atoms, malonalddehyde, 3 carbon atoms, succinaldehyde, 2-ethylhexyl), which have a chain length of 2 to 6 carbon atoms, as described in the prior literature on collagen crosslinking. In a reaction with 4 carbon atoms; GA, 5 carbon atoms; adipaldhede, 6 carbon atoms), a series of experiments in GA with 5 carbon atoms was maximized (I. Migneault, et al., BioTechniques, 37 (2004) 790-796, 798-802; I. Miyoshi, Acta medicinae Okayama, 17 (1963) 19-31). In addition, some chemicals, including carbodiimide, epichlorohydrin and sodium metaphosphate, have been used to cross-link biopolymers, but their crosslinking efficiency is low and their physical properties are limited. In recent years there have been attempts to increase the crosslinking efficiency and physical properties using various chemicals, but GA is still one of the most effective and notable cross-linking agents among many cross-linking agents.

GA has efficient cross-linking properties of biomaterials, but there are some limitations in that the structure of GA in aqueous solution is more controversial than other cross-linking agents. The structure of GA was reported to be present in 13 forms of various oligomers and monomers in solution (I. Migneault, et al., BioTechniques, 37 (2004) 790-796, 798-802). In addition, the products and reaction mechanisms of cross-linked biomaterials by GA were not easy to determine due to the complexity and non-uniformity of the structure, and the number of cross-links was not yet determined by chemical approach. Despite these limitations of GA, much effort is being made to improve cross-linking between GA and biomaterials.

Among the various factors affecting the crosslinking efficiency, the reaction time is an important factor in the cross-linking experiment of the biomaterial by the GA, so the reaction should be stopped and controlled within a reasonable time to effectively maintain the cross-linking and reproducibility of the biomaterial. Therefore, in order to optimize or improve cross-linking of biomaterials, a quencher capable of inactivating GA is needed. The use of such inhibitors improves the reproducibility of the resultant and enables a more quantitative assessment of the cross-linking properties of the biomaterial. However, despite the importance of these inhibitors, only a few reported as inhibitors of GA, and still satisfactory GA inhibitors have not been developed.

U.S. Patent No. 6,399,850 issued June 4, 2002.

L.H.H. Olde Damink, et al., JOURNAL OF MATERIALS SCIENCE: MATERIALS IN MEDICINE 6 (1995) 460-472. Cheung HY, et al., J Pharm Pharmacol. 1982 Apr; 34 (4): 211-4. A.F.S.A. Habeeb, et al., Archives of Biochemistry and Biophysics, Volume 126, Issue 1, July 1968, Pages 16-26 (1968.07.).

The present inventors have studied glutaraldehyde inhibitors for efficiently and efficiently carrying out cross-linking of biomaterials, and have found that a mixture of glycine, histidine and lysine, which are specific amino acids, efficiently and glutaraldehyde effectively inhibits glutaraldehyde Lt; RTI ID = 0.0 > biomaterial < / RTI >

Accordingly, it is an object of the present invention to provide a glutaraldehyde inhibitor comprising a mixture of amino acids of glycine, histidine and lysine, a method for inhibiting the activity of glutaraldehyde using the same, and a crosslinking method of a biomaterial using the same.

In accordance with one aspect of the present invention, there is provided a glutaraldehyde inhibitor comprising an amino acid mixture of glycine, histidine and lysine.

In one embodiment, the amino acid mixture may be at a concentration of 25 mM or more, preferably at a concentration of 100 mM or more.

According to another aspect of the present invention there is provided a method for inhibiting the activity of glutaraldehyde comprising incubating said glutaraldehyde inhibitor with a reactant comprising glutaraldehyde.

In one embodiment, the incubation can be performed for 30 minutes or more and can be performed at a temperature of 20-40 ° C.

According to still another aspect of the present invention, there is provided a method for producing a biosurfactant, comprising: (a) treating a biomaterial with glutaraldehyde and incubating; And (b) adding the glutaraldehyde inhibitor to the reactant obtained in step (a) and incubating the mixture.

In one embodiment, the glutaraldehyde of step (a) may be used at a concentration of 0.1 wt% (w / w) or less, the incubation of step (b) 40 C < / RTI >

It has been found by the present invention that the amino acid mixture of glycine, histidine and lysine can more effectively inhibit glutaraldehyde than a single amino acid, thereby increasing the efficiency and reproducibility of cross-linking experiments with glutaraldehyde.

Therefore, the glutaraldehyde inhibitor including the amino acid mixture of glycine, histidine and lysine of the present invention, the method for inhibiting the activity of glutaraldehyde using the glutaraldehyde inhibitor, and the cross-linking method of the biomaterial using the glutaraldehyde inhibitor can be applied to various fields such as an atomic force microscope, a scanning electron microscope and a transmission electron microscope Which will contribute to improving the utilization of glutaraldehyde in various fields.

FIG. 1 shows the effect of various amino acids as an inhibitor against GA. GA inhibition with increasing amino acid concentration was measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and increased as the concentration of lysine (K) (A), glycine (G) (B), histidine (H) (C) and arginine (R) GA [lane 1, low range molecular weight (MW) marker; Lane 2, 2 mg / ml of 1-Cys TkPrx as a control; Lane 3, 1-Cys TkPrx and 0.1% GA (v / v) at 2 mg / ml; Lane 4-8, 1-Cys TkPrx at 2 mg / ml, 0.1% GA (v / v) and different concentrations of amino acids (5-100 mM). All experiments were performed at room temperature (RT) for 1 minute.
Figure 2 shows the GA cross-linking effect of an amino acid mixture, comparing (A) the GA inhibitory effect with the same concentration of single amino acids (G, H and K) and their mixtures (KG, KH, GH and GHK) [Lanes 1 and 2, low range MW marker as control and 1-Cys TkPrx at 2 mg / ml; Lane 3, 1-Cys TkPrx and 0.1% GA (v / v) at 2 mg / ml; Lanes 4-6, 1-Cys TkPrx at 2 mg / ml, 0.1% GA (v / v) and different amino acids (K, G and H); Lane 7-9, 2 mg / ml 1-Cys TkPrx, 0.1% GA (v / v) and two different amino acid mixtures (KG, KH and GH); Lanes 10, 2 mg / ml 1-Cys TkPrx, 0.1% GA (v / v) and 3 different amino acid mixtures (GHK) [Lanes 1-3, same experimental conditions as (A); Lane 4, 2 mg / ml 1-Cys TkPrx, 0.1% GA (v / v) and 100 mM GHK; Lanes 5-6, 1-Cys TkPrx at 2 mg / ml, 0.1% GA (v / v) and different concentrations of H (10 mM and 100 mM, respectively). All experiments were performed at room temperature for 1 minute.
FIG. 3 shows the optimization of GHK for GA inhibition, which is the optimization result of the GA inhibition effect by the change of reaction time (A) and the change of concentration of GHK (B) [(A) lanes 1 and 2, low range MW Marker and 2 mg / ml of 1-Cys TkPrx; Lane 3, 1-Cys TkPrx and 0.1% GA (v / v) at 2 mg / ml; Lane 4-10, 0.1% GA and 100 mM GHK were mixed and incubated for various times (1 to 1440 minutes) and reacted with 1-Cys TkPrx for 1 minute. (B) ; Lane 4-10, 0.1% GA and various concentrations of GHK (10-200 mM), incubated for 30 min and reacted with 1-Cys TkPrx] (C) GHK inhibition response to high concentration GA [Lanes 1-2, (A); Lane 3, 1 mg / ml 1-Cys TkPrx and 2.5% GA (v / v); Lane 4-10, 2.5% GA and different concentrations of GHK (with stepwise concentration increase), incubated for 30 minutes and reacted for 1 minute with 1-Cys TkPrx. (D) GHK for different concentrations of GA Inhibition reaction (lanes 1-3, (C); Lanes 4-10, 100 mM GHK and different concentrations of GA (with stepwise concentration reduction) were mixed and incubated for 30 minutes and reacted with 1-Cys TkPrx for 1 minute.
Figure 4 shows the inhibitory effect of GHK on GA cross-linking under EM. (A) Negative stained EM image of 1-Cys TkPrx. (B) 1-Cys TkPrx protein was mixed with 0.1% GA and incubated for 1 min (A and B) showed hollow (hollow) similar spherical structures in both control and GA cross-linked groups. (C) The structure of 1-Cys TkPrx was degraded by dithiothreitol (DTT), a reducing agent. (D) 0.1% GA mixed with 100 mM GHK for 30 min at RT and a mixture of GA and GHK were reacted with 1-Cys TkPrx for 1 min.

The present invention provides glutaraldehyde inhibitors comprising an amino acid mixture of glycine, histidine and lysine. The amino acid may be the structure of L-glycine, L-histidine and L-lysine, respectively.

In one embodiment, the amino acid mixture may be at a concentration of 25 mM or more, preferably at a concentration of 100 mM or more.

The present invention also provides a method for inhibiting the activity of glutaraldehyde, which comprises incubating the glutaraldehyde inhibitor together with a reactant containing glutaraldehyde.

In one embodiment, the incubation may be performed for at least 30 minutes, preferably at least 40 minutes.

In one embodiment, the incubation can be performed at a temperature of 20-40 째 C, preferably 20-30 째 C, and most preferably 25 째 C.

Also, the present invention provides a method for producing a microorganism, comprising the steps of: (a) treating a biomaterial with glutaraldehyde and incubating; And (b) incubating the reactant obtained in step (a) by adding the glutaraldehyde inhibitor and incubating the biotin.

In one embodiment, the glutaraldehyde of step (a) may be used at a concentration of 0.1% by weight or less, preferably 0.5% by weight, more preferably 2.5% by weight.

In one embodiment, the incubation of step (b) may be performed for at least 30 minutes, preferably at least 40 minutes.

In one embodiment, the incubation of step (b) can be performed at a temperature of 20-40 째 C, preferably 20-30 째 C, and most preferably 25 째 C.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not limited thereto.

<Examples>

1. Materials and Methods

1.1. Cloning and protein expression

The full-length 1-Cys TkPrx (tk0537) gene was prepared as described in the prior art [S. Lee, B. Jia, J. Liu, BP Pham, JM Kwak, YH Xuan, GW Cheong, PloS one, 10 (2015) e0125325. E. coli BL21 (DE3) PlysS (Stratagene, USA) cells used for protein expression (production) were cultured in Luria-Bertani (LB) medium containing 50 μg · ml ^-1 of kanamycin at 37 ° C., with stirring to ₆₀₀ = 1) were incubated (at 180 rpm), the protein expression was induced with 1 mM isopropyl thio -β-D- galacto-pyrano side (isopropyl-β-D-thiogalactopyranoside , IPTG, Sigma-Aldrich) And incubation was continued for an additional 4 hours. Cells were pelleted by centrifugation at 4,424 g for 10 min. The pellet was washed with a buffer containing 25 mM Tris-HCl (pH 8.0) and 100 mM NaCl and precipitated again by centrifugation under the same conditions. After washing, the pellet was resuspended in lysis buffer containing 25 mM Tris-HCl, 100 mM NaCl and 5 mM imidazole (pH 8.0) and incubated at 40% amplitude for an effective time of 6 minutes (2 seconds on, 2 seconds off) Lt; / RTI &gt; and homogenized. The homogenate was incubated at 70 ° C for 30 minutes to remove the thermodynamically unstable protein such as E. coli protein. The supernatant was separated by centrifugation at 34,957 g for 20 minutes, and then equilibrated with the lytic buffer. Column (Bio-Rad, Hercules, Calif.). Recombinant 1-Cys TkPrx was eluted using an imidazole concentration gradient (20-250 mM). After separation, the buffer solution was exchanged with 1-Cys TkPrx in HEPES buffer for cross-linking experiments.

1.2 GA Processing

1-Cys TkPrx (2 mg / ml) was treated with GA (0.1% or 2.5%) at room temperature (25 ° C) for 1 minute or for various times. GA solution was prepared using 2.5% stock of water from an ampoule containing 25% GA (Agar Scientific, UK).

1.3 SDS-PAGE

12.5% linear gradient gel and Bio-Rad 'mini-slab' gel system were used. 20 [mu] l of protein solution (2 mg / ml concentration) was mixed with 5 [mu] l of denaturation buffer and then loaded onto the gel. The molecular weight markers ^{(Bio-Rad, ProSieve ® QuadColor} ™ Protein Markers) to evaluate the molecular weight of the protein was used. The gel was stained in a dye [Coomassie brilliant blue R-250 (ELPIS BIOTECH, PowerStain (TM) Brilliant Blue Staining Solution, Korea) for 2 hours. The dyed gel was decolorized in 40% methanol, 10% acetic acid, 50% tertiary distilled water solution. The image of the discolored gel was recorded using a scanning machine (EPSON, HP Officejet Pro K8600).

1.4 Electron microscopy and single particle analysis

Purified 1-Cys TkPrx was diluted to a final concentration of 100 nM with 25 mM Tris-Cl buffer (pH 8.0) containing 300 mM NaCl, 1 mM DTT and 0.5 mM EDTA. 5 μl of the protein solution was used for negative (negative) staining using a carbon-coated copper grid (1% uranyl acetate) glow-discharged (Harric Plasma, Ithaca, NY) (Kim et al., Journal of molecular cell biology, 4 (2012)], which is incorporated herein by reference in its entirety. 258-261; S. Lee, et al., Biochemical and biophysical research communications, 469 (2016) 1028-1033). The prepared grid was examined using a transmission electron microscope (Philips Tecnai T10 transmission electron microscope, FEI, USA) operating at 100 kV. Images were recorded at a magnification of 34,000 (0.32 nm / pixel) on a camera (2K x 2K Gatan US1000 CCD camera).

2. Results

2.1. GA inhibition of aqueous solutions of amino acids

Chemical cross-linking by GA is a well-known reaction, but the inhibition of GA is still unclear. To determine if amino acids could inhibit the cross-linking of GA, various concentrations of several amino acids were mixed and incubated with 0.1% GA in 2-mg / ml 1-Cys TkPrx solution for 30 minutes (see Figure 1). The level of GA inhibition by lysine was determined by measuring the mobility of 1-Cys TkPrx on 12.5% SDS-PAGE (see Figure 1A). Lysine decreased the cross-linking of 1-Cys TkPrx by GA in a concentration-dependent manner. From the lysine concentration of 25 mM, the cross-linking pattern showed a decreasing tendency and decreased more than 70% at about 100 mM concentration. In particular, the oligomeric form showed a reduction of more than 90% (see Figure 1A). Next, GA inhibition by other amino acids such as glycine, histidine and arginine was measured. Glycine and histidine more effectively reduced cross-linking of GA and 1-Cys TkPrx than lysine (see FIGS. 1B and 1C). In the case of glycine, cross-linking was reduced from 10 mM and almost all of the highly oligomeric forms were inhibited at 50 mM (see FIG. 1B). Histidine showed stronger inhibitory effect on crosslinking than glycine. In particular, at 100 mM, not only did the highly oligomeric form disappear completely, but also the dimeric form was also reduced by 90% (see FIG. 1C). Unlike other amino acids, arginine showed no inhibitory effect (see FIG. 1D).

2.2. Effect of amino acid combination on cross-linking with GA

In the result (FIG. 1A), it was confirmed that each amino acid (G, H and K) showed a GA inhibitory effect in a concentration-dependent manner. KH (lysine + histidine), GH (glycine + histidine) and GHK (glycine + histidine + lysine), which are mixtures of equal amounts of 25 mM individual amino acids, ] Was prepared, mixed with 0.1% GA, incubated for 30 minutes, and then added to 1-Cys TkPrx solution (see FIG. 2A). As a result of comparing GA inhibition efficiency by mixing two amino acids, the mixture of KH and GH effectively inhibited the cross-linking reaction of 1-Cys TkPrx and GA over KG. These results are consistent with the results of single amino acid inhibition experiments (see Figures 1 and 2A). The GA inhibitory effect of the three amino acids of the GHK mixture was also confirmed. At 25 mM concentration, there was no significant difference between single amino acid (H), two amino acid mixtures (KH and GH) and three amino acid mixtures (GHK). Previous experiments have shown that GA inhibition is maximized when 100 mM of amino acid is used, so the inhibitory effects of 100 mM His and GHK are compared. 100 mM GHK mixture was prepared by mixing equal amounts of 100 mM of individual amino acids (glycine (G), histidine (H), lysine (K)). As shown in Fig. 1C, histidine (H) exhibited a dimer-type band at 100 mM, but did not show band migration other than monomer in GHK (see Fig. 2B). These results indicate that the use of amino acids containing GA inhibitory ability in the form of a mixture is more effective in inhibiting GA. After this experiment, GHK was used as a GA inhibitor.

2.3. Optimized for use as a GA inhibitor Incubation  Time and GHK  density

When using cross-linked biomaterials by GA for atomic force microscopy and scanning or transmission electron microscopy experiments, be aware of the signal noise and the formation of artifacts linked to the background. Some artifacts can be reduced using low-concentration GA or low-temperature electron microscopy, but the potential of GA is not fully realized due to the presence of artifacts. To maximize the cross-linking effect of GA, an optimized inhibition reaction is required to reduce signal noise and background. First, to determine the optimum reaction time of GHK as a GA inhibitor, the concentrations of GHK and GA were fixed at 100 mM and 0.1% (w / w), respectively, and crosslinking experiments were performed (see FIG. 3A). The 100 mM GHK mixture significantly reduced the reactivity of the aldehyde group of GA. Nearly all dimer - type band shifts disappeared in response time after about 30 minutes. In the next experiment, it was confirmed that the GA aldehyde group was completely inhibited at the GHK concentration of 30 mM or more when the reaction time and the GA concentration were fixed (see FIG. 3B). According to the results of FIGS. 3A and 3B, it can be seen that when 0.1% GA is used, more than 30 mM GHK should be incubated for at least 30 minutes to completely inhibit GA. In protein-protein response experiments using low concentrations of GA (less than 0.1%), the above results can be used to increase the utility of GA.

GA is known to play an important role in immunocytochemistry and histology experiments and is used at various concentrations of 0.5% to 2.5% or 6% for the production of biological samples (plants, brain, cells and bacteria). In the present invention, the inhibitory ability of GHK in fixed 2.5% GA in protein solution was investigated. Low concentrations of GHK and high concentrations of GA caused protein aggregates and artifacts in the stacking gel (see Figure 3C). Figure 3C shows that the GA inhibitory potency increases as the GHK concentration increases when the GA concentration is at a fixed 2.5%. The oligomer and dimer bands began to decrease with increasing GHK concentration and the dimer band did not appear at about 100 mM GHK concentration. It should be noted that although the oligomeric form is not present, artificial products remain in the lowest part of the gel due to the excess of GA (see bottom of FIG. 3C). In order to find the optimum GA concentration that does not form the artifact, the experiment was performed using various concentrations of GA after fixing the time and concentration (see FIG. 3D). Artifacts formed at the bottom of the gel disappeared at a concentration of about 0.25% GA. These results indicate that the balance of GHK and GA concentrations should be considered to minimize unnecessary cross-linking and artifact formation.

2.4 Under an electron microscope On GHK  Visualization of inhibition by

The interaction of the native 1-Cys TkPrx protein with GA was evaluated by electron microscopy (see FIG. 4). The portion of the negative stained protein showed a spherical structure with a hollow star inside. 4A and 4B, it can be seen that the 0.1% GA treated protein has a clearer structure through cross-linking with GA without structural changes such as unwanted aggregation. In addition, 1-Cys TkPrx reduced by dithiothreitol (DTT) was used to investigate the inhibitory ability of GHK. Figure 4C shows 1-Cys TkPrx treated with 5 mM DTT for 12 hours. The cyclic oligomer structure was separated into small pieces by treatment with a reducing agent such as DTT. The mixture of GA and GHK was incubated at RT for 30 min, then applied to the reduced form of 1-Cys TkPrx and stained with 1% uranyl acetate to confirm the structural change. It was confirmed that the cross-linking reaction did not proceed by GHK, and these results were consistent with FIG. From the SDS-PAGE and EM image results, it can be seen that GHK can be used as an inhibitor to inhibit GA cross-linking.

3. Discussion

GHK  Optimization of GA and its artifacts In control It is important

As can be seen in Figure 3, optimization of the concentration and incubation time used is an important factor controlling the GA response. (See lane 6-8 of FIG. 3C), or by only a monomeric form (see FIG. 3C) by controlling the GHK concentration while keeping the incubation time and the amount of GA used in a fixed state Lanes 9 and 10), and a high oligomeric complex (see lanes 4 and 5 in Fig. 3C). However, it is interesting to note that artifacts are still present at the bottom of the SDS-PAGE gel. These artifacts can be described as by-products produced by excess GA and residual amino acids. Therefore, it is necessary to control the amount of GA used in consideration of the generation of artifacts in the crosslinking experiment by GA. As can be seen in FIG. 3, when the GA concentration was lowered while maintaining the GHK level constant, the artifacts began to disappear and disappeared completely at 0.1% and 0.05% GA concentrations. Therefore, in order to obtain the best results, it is important to optimize the concentration of GA as well as the GHK concentration used in the protein solution.

4. Summary and Conclusion

Glutaraldehyde is a chemical crosslinking agent widely used as a crosslinking agent or tissue anchor in protein chemistry and electron microscopy. In applications for protein sample processing, unstable interactions between biological molecules, often separated into discrete particles, are cross-linked to a stable structure. Thus, glutaraldehyde can be applied to stabilize biocompatible materials, including intermolecular interactions of proteins, proteins and proteins in cells, cell organelle membrane lipids, oligomers. Although the cross-linking reaction using GA is a well-known reaction, the inhibitory effect on the GA used as a cross-linking agent is still unclear. In the present invention, the specific inhibition reaction with GA was investigated by using various amino acids as candidates of GA inhibitor in order to eliminate the formation of cross-linking reaction of GA and to prevent nonspecific coagulation in long-term storage of biological samples. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was used to investigate inhibitory candidates such as L-arginine, L-lysine, L-glycine and L-histidine. The mixtures and methods of inhibition of the present invention are applicable to a wide range of sample treatments including protein-protein interactions for microscopic observation, cell fixation and purification of polymeric conjugates.

Claims (10)

Glutaraldehyde inhibitor comprising a mixture of amino acids of glycine, histidine and lysine at a concentration of 100 mM or more.
delete delete A method for inhibiting the activity of glutaraldehyde, comprising the step of incubating the glutaraldehyde inhibitor of claim 1 with a reactant comprising glutaraldehyde.
5. The method of claim 4, wherein the incubation is performed for at least 30 minutes.
5. The method of claim 4, wherein the incubation is performed at a temperature of 20-40 &lt; 0 &gt; C.
(a) treating the biomaterial with glutaraldehyde and incubating; And
(b) adding the glutaraldehyde inhibitor of claim 1 to the reaction product obtained in step (a) and incubating
Of the biomaterial.
The method according to claim 7, wherein the glutaraldehyde of step (a) is used in a concentration of 0.1% by weight or less.
8. The method according to claim 7, wherein the incubation of step (b) is performed for at least 30 minutes.
8. The method according to claim 7, wherein the incubation of step (b) is carried out at a temperature of 20-40 占 폚.

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