KR101713932B1 - Method of preparing alginate having low molecular weight using hydrogen peroxide and ultrasonic wave - Google Patents

Abstract

The present invention relates to a process for producing a low molecular weight alginate, which comprises decomposing a polymeric alginate into a low molecular weight alginate by applying hydrogen peroxide and ultrasonic waves, rapidly removing the hydrogen peroxide by using the enzyme to increase the yield of the low molecular weight alginate, And a method for producing a low molecular weight alginate.
The method for producing a low molecular weight alginate using hydrogen peroxide and ultrasonic waves according to the present invention comprises a step of dissolving a polymer alginate, a step of adding hydrogen peroxide solution to the high molecular alginate solution obtained in the dissolution step, And a post-treatment step of removing the hydrogen peroxide contained in the low molecular weight alginate solution by adding a catalase enzyme to the low molecular weight alginate solution obtained in the decomposition reaction step.

Description

[0001] The present invention relates to a method for preparing low molecular weight alginate using hydrogen peroxide and ultrasonic waves,

The present invention relates to a process for producing a low molecular weight alginate, which comprises decomposing a polymeric alginate into a low molecular weight alginate by applying hydrogen peroxide and ultrasonic waves, rapidly removing the hydrogen peroxide by using the enzyme to increase the yield of the low molecular weight alginate, And a method for producing a low molecular weight alginate.

The alginate is composed of? -L-guluronic acid [G] and? -D-mannuronic aicd [M], and an MM block, a GG block A heteropolymer block such as a homopolymer block or a MG block of polysaccharides consisting of an α-1,4 or a β-1,4 bond in turn, is used as a polysaccharide in brown algae such as seaweed or sea tangle It contains a lot.

These alginates are polymeric polysaccharides and have a wide range of properties such as gel forming ability, viscosity increasing ability, water absorbing ability, binding ability, sticking ability, lubricating ability and film forming ability depending on the molecular weight, Food, cosmetics, paper and pharmaceuticals.

In addition, recently, it has been reported that the defatting properties of alginate are emphasized, and as a dietary fiber, constipation healing, obesity suppression, anticancer action and suppression of toxicity in human body have been reported. As the functional properties of alginate as a natural fiber polysaccharide are revealed, studies are being conducted to use alginate as food, cosmetics, and medical materials.

However, conventionally used polymeric alginates have a limited dissolution time at room temperature and have a high viscosity at elevated concentration. In order to solve these drawbacks, the necessity of low molecular weight alginate has been suggested. As a biodegradable and edible film, low molecular weight alginate has been widely used as a biodegradable and edible film in that the water vapor permeability and the flexibility are increased and the low molecular alginate is added to the cholesterol and cadmium (Cd) Effect of Exhaust Emission and Glucose Concentration on Bacillus sp. Studies on the efficacy of low molecular weight alginates such as increasing storage stability by inhibiting the proliferation of sp. have been reported.

The methods of decomposing the polymer alginates used up to now include heating hydrolysis, chemical decomposition and enzymatic decomposition. It has been reported that there is a close correlation between the viscosity and the average molecular weight of the low molecular alginate produced by hydrolysis (Kim and Cho, 2000), and the chemical yields and molecular weights The decomposition method (Ikeda et al., 2000) emphasizes efficiency. However, all of these methods have difficulties in obtaining an alginate having a uniform molecular weight due to complicated processes such as decomposition time, pH, dosage of chemicals and enzymes, and are difficult to use in industrial aspects. In addition, the low-molecular-weight method of the monatin salt using the gamma ray has been actively carried out since the gamma ray generation itself can be controlled by the power source and has advantages in process control, accuracy, energy efficiency and consumer acceptability (Kang et (1999), Ikeda et al., 2000), there is a problem that the quality may vary depending on the air composition during the irradiation of gamma rays, the efficiency is low in terms of mass production, the efficiency is low in terms of high cost, have.

Korean Patent Laid-Open No. 10-2010-0005806 discloses a method for producing low-molecular alginate and low-molecular-weight alginate using hydrogen peroxide and ultrasonic waves.

The method comprises: adding 40 to 60 parts by weight of hydrogen peroxide in an amount of 10 (w / v) to 40 (w / v) per 1 part by weight of alginate to dissolve the mixture; and treating the mixture with ultrasonic waves .

There is a problem in that the hydrogen peroxide added to decompose the polymer alginate can not be effectively removed.

Korean Patent Publication No. 10-2010-0005806: Low-molecular alginate and low-molecular-weight alginate using hydrogen peroxide and ultrasonic wave

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in order to solve the above problems, and it is an object of the present invention to provide a method for preparing a polymeric alginate by decomposing a polymeric alginate into a low molecular weight alginate by adding hydrogen peroxide and ultrasonic waves, The present invention also provides a method for producing a low molecular weight alginate,

In order to accomplish the above object, the present invention provides a method for producing a low molecular weight alginate using hydrogen peroxide and ultrasonic waves, comprising: dissolving a polymer alginate; A decomposition reaction step of adding hydrogen peroxide solution to the polymer alginate solution obtained in the dissolution step and then decomposing the polymer alginate into a low molecular weight alginate by irradiation with ultrasonic waves; And a post-treatment step of adding catalase enzyme to the low molecular weight alginate solution obtained in the decomposition reaction step to remove hydrogen peroxide contained in the low molecular weight alginate solution.

The decomposition reaction step is characterized in that the ultrasonic wave is irradiated for 16 to 22 hours while maintaining the temperature of the polymer alginate solution at 50 to 80 캜.

The post-treatment step is characterized in that the catalase enzyme concentration in the low molecular weight alginate solution is 1 to 100 ppm.

The low molecular weight alginate has a weight average molecular weight of 4082 to 14108 Da, and Escherichia coli , Staphylococcus aureus , and Pseudomonas aeruginosa .

The present invention can decompose a polymeric alginate into a low molecular weight alginate having a molecular weight of 4,082 Da to 14,108 Da using hydrogen peroxide and ultrasonic waves, thereby increasing the yield of the low molecular weight alginate and improving the antimicrobial activity.

In addition, the present invention can efficiently produce low molecular weight alginate by decomposing into low molecular alginate and then rapidly removing hydrogen peroxide using catalase enzyme.

FIG. 1 is a schematic view showing a dissolution part for dissolving a polymer alginate, a decomposition part for adding ultrasonic waves after adding hydrogen peroxide solution to a polymer alginate solution, and a post-processing part for removing hydrogen peroxide by adding an enzyme to a low molecular weight alginate solution Figure 2 is a front view schematically illustrating a modularized device.
FIG. 2 is a graph showing the concentration of hydrogen peroxide remaining and the concentration of consumed hydrogen peroxide when the low molecular weight is progressed according to the decomposition reaction temperature.
FIG. 3 is a graph of MALS-GPC (Multi Angle Light Scattering Gel Permeation Chromatography) of alginate according to decomposition reaction time at the time of low molecular weight progress according to decomposition reaction temperature.
FIG. 4 is a graph showing the concentration of residual hydrogen peroxide with time at the time of enzyme treatment by varying the concentration of the catalase enzyme.
FIG. 5 shows the FT-IR results of the low molecular alginate according to the decomposition reaction temperature.
FIG. 6 shows the H ¹ -NMR results of the low molecular weight alginate according to the decomposition reaction temperature.
FIG. 7 is a photograph of an antibacterial test result of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at a concentration of a polymer alginate having a weight average molecular weight of 401,271 Da at a concentration of 1.00%.
8A is a photograph of an antibacterial test result for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at a concentration of a low molecular weight alginate having a weight average molecular weight of 14,108 Da at 1.00%.
FIG. 8b is a photograph of the result of antibacterial test against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at a concentration of low molecular weight alginate having a weight average molecular weight of 14,108 Da of 0.50%.
FIG. 8c is a photograph of an antibacterial test result of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at 0.10% of low molecular weight alginate having a weight average molecular weight of 14,108 Da.
9.9a is a photograph of an antibacterial test result of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa in 1.00% of low molecular weight alginate having a weight average molecular weight of 4,082 Da.
FIG. 9.b is a photograph of an antibacterial test result against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at 0.10% of low molecular weight alginate having a weight average molecular weight of 4,082 Da.
Fig. 9.c is a photograph of an antimicrobial test result of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa at 0.05% of low molecular weight alginate having a weight average molecular weight of 4,082 Da.

Hereinafter, a method for producing a low molecular weight alginate using hydrogen peroxide and ultrasonic waves according to a preferred embodiment of the present invention will be described in detail.

The method for producing a low molecular weight alginate of the present invention comprises a dissolution step of dissolving a polymer alginate, a step of dissolving the polymer alginate into a low molecular weight alginate by irradiating ultrasonic waves after adding hydrogen peroxide solution to the polymer alginate solution obtained in the dissolution step, And a post-treatment step of removing the hydrogen peroxide contained in the low molecular weight alginate solution by adding a catalase enzyme to the low molecular weight alginate solution obtained in the decomposition reaction step.

1. Dissolution step

First, the polymer alginate is dissolved in the dissolution step.

Polymer alginate is a type of polysaccharide contained in the cell wall or cell epilepsy of brown algae and is a linear unbranched chemical polymer containing α-L-glucuronate and β-D-maleuronate residues. In the present invention, the polymer alginate means a weight average molecular weight of 400,000 Da or more.

Polymeric alginates useful for lower molecular weight in the present invention include monovalent salts of alginic acid salts such as sodium and potassium alginate, silver alginate as well as the esterified form of alginate, for example, propylene glycol alginate . All such esterified forms are included within the definition of the alginate salt used in the present invention. Further examples of alginates which may be used in the present invention include magnesium alginate and 3-ethanolamine alginate. These alginates may be used singly or in combination of two or more.

The polymeric alginate can be extracted from commercially available products or from brown algae such as seaweed or sea tangle using known methods.

The prepared polymer alginate is dissolved in distilled water to obtain a polymer alginate solution. The concentration of the polymeric alginate in the polymeric alginate solution may be 1 to 5% (mass percentage, hereinafter, concentration% means mass percentage).

2. Decomposition reaction step

Hydrogen peroxide water is added to the polymer alginate solution obtained in the above-mentioned dissolution step, and the polymer alginate is decomposed into low molecular weight alginate by irradiating ultrasonic waves.

100 parts of polymeric alginate solution, 10 to 50 parts of hydrogen peroxide solution may be added to the skin. When the hydrogen peroxide solution is added to the polymer alginate solution, the concentration of hydrogen peroxide in the polymer alginate solution is preferably 1%.

Hydrogen peroxide water is added to the polymer alginate solution, and the polymer alginate is decomposed into low molecular weight alginate by irradiation with ultrasonic waves while stirring.

In the decomposition reaction step, ultrasonic waves can be irradiated for 16 to 22 hours while the temperature of the polymeric alginate solution is maintained at 50 to 80 캜. 48 to 60 kHz can be used as an ultrasonic wave.

If the decomposition reaction temperature is less than 50 캜, the molecular motion of the alginate molecule is limited, and the structural change due to the ultrasonic wave is not smoothly performed. If the temperature exceeds 100 캜, the risk of explosion by OH radicals generated by hydrogen peroxide increases, .

As described above, the present invention can increase the efficiency of decomposition into hydroxy radical using hydrogen peroxide, increase the amount of radicals generated by ultrasonic irradiation, and decompose the polymer alginate in a simple and quick manner by the generated radicals.

3. Post-processing step

Catalase enzyme is added to the low molecular weight alginate solution to remove the hydrogen peroxide in the low molecular weight alginate solution obtained in the decomposition reaction step.

The catalase enzyme concentration in the low molecular weight alginate solution may be from 1 to 100 ppm for efficient removal of hydrogen peroxide. For example, it is preferable to add 1 to 100 mg of the catalase enzyme per 1 kg of the low molecular weight alginate solution.

The reaction time of the hydrogen peroxide by the enzyme is preferably 1 to 10 minutes. This can significantly shorten the treatment time and remove the concentration of hydrogen peroxide to less than 0.02% compared to the method of removing hydrogen peroxide by adding sodium hypochlorite (NaOCl).

When the post-treatment is completed as described above, a low molecular weight alginate solution containing a low molecular weight alginate can be finally obtained. It is needless to say that the low molecular weight alginate solution can be obtained by evaporating and concentrating the low molecular weight alginate solution with a rotary evaporator to remove distilled water as a solvent and drying it to obtain a powdered low molecular weight alginate.

The low molecular weight alginate obtained through the above-mentioned production process has a weight average molecular weight of 4082 to 14108 Da. Such low molecular weight alginates have excellent antibacterial activity against viruses as well as bacteria and fungi. Especially Escherichia coli , Staphylococcus aureus , Pseudomonas aeruginosa , and the like.

Further, the low molecular weight alginate prepared by the present invention can be widely applied to daily necessities (toothbrush, fabric softener, detergent, etc.) and oral-related products (toothpaste, dental floss, oral cleanser, etc.) because of its excellent antimicrobial activity.

Meanwhile, the above-described manufacturing process can be performed through a manufacturing apparatus that is modularized into one system. Fig. 1 schematically shows a manufacturing apparatus. 1, the production apparatus comprises a dissolution part 10 for dissolving a polymer alginate, a decomposition reaction part 20 for adding ultrasonic waves after addition of hydrogen peroxide solution to the polymer alginate solution, an enzyme for the low molecular weight alginate solution And a post-treatment unit 30 for removing hydrogen peroxide.

The dissolution section 10 has a configuration in which a stirrer is installed in a dissolution tank. Polymer alginate and solvent are added to the dissolution tank and dissolved by stirring to obtain a polymer alginate solution.

The decomposition reaction section 20 has a structure in which a stirrer, an ultrasonic vibrator, and a heater are installed in a reaction tank. After injecting the polymer alginate solution discharged from the melting tank into the reaction tank through the connecting tube, an appropriate amount of hydrogen peroxide solution is put into the reaction tank, and the temperature is controlled by heating the polymer alginate solution with a heater. Then, the polymer alginate is decomposed while applying the ultrasonic wave to the polymer alginate solution through the ultrasonic vibrator while operating the stirrer.

The post-processing unit 30 is made up of a post-processing group. The low molecular alginate solution discharged from the reaction tank is injected into the post treatment tank through the connecting tube, and an appropriate amount of catalase enzyme is put into the post treatment tank to remove the remaining hydrogen peroxide.

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

≪ Preparation of low molecular weight alginate &

In all the experiments below, a polymer alginate (China alginate, China chemical) having a weight average molecular weight of 401,271 Da was used.

First, the polymer alginate was dissolved in distilled water to prepare 10 L of a polymer alginate solution having a concentration of 2.0%. The concentration of hydrogen peroxide in the polymer alginate solution was adjusted to 1.0% by adding 34.5% of hydrogen peroxide (Samchun Chemical) to the prepared polymer alginate solution.

The temperature of the polymer alginate solution was maintained at 50 to 80 ° C, and the polymer alginate was decomposed by applying ultrasonic waves at 48 kHz while stirring to obtain a low molecular weight alginate solution.

The hydrogen peroxide concentration, the apparent viscosity (measured at 25 캜) and the weight average molecular weight of the low molecular weight alginate solution were measured at each temperature, and the results are shown in Tables 1 to 4 and Figs. 2 to 3.

Table 1 below shows the results of the decomposition reaction at 50 ° C.

Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 16hr 22hr Residual H ₂ O ₂ concentration (%) 1,000 0.983 0.975 0.967 0.944 0.905 0.890 0.874 Apparent Viscosity (cPs) 400 260 10.5 3.5 1.0 1.0 1.0 1.0 Weight average molecular weight (Da) 401,271 375,363 298,512 195,726 100,052 66,315 51,053 45,487

Table 2 shows the result of decomposition reaction at 60 占 폚.

Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 16hr 22hr Residual H ₂ O ₂ concentration (%) 1,000 0.980 0.972 0.960 0.929 0.882 0.867 0.851 Apparent Viscosity (cPs) 400 200 7.0 2.5 1.0 1.0 1.0 1.0 Weight average molecular weight (Da) 401,271 307,807 124,681 78,286 42,473 27,748 15,218 14,108

Table 3 below shows the results of the decomposition reaction at 70 占 폚.

Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 16hr 22hr Residual H ₂ O ₂ concentration (%) 1,000 0.978 0.970 0.952 0.913 0.867 0.836 0.820 Apparent Viscosity (cPs) 400 80 5.5 1.0 1.00 1.0 1.0 1.0 Weight average molecular weight (Da) 401,271 301,216 120,263 31,370 14,287 11,260 8,271 7,225

Table 4 shows the result of the decomposition reaction at 80 ° C.

Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 16hr 22hr Residual H ₂ O ₂ concentration (%) 1,000 0.977 0.967 0.944 0.898 0.851 0.805 0.789 Apparent Viscosity (cPs) 400 8.5 4.0 1.0 1.0 1.0 1.0 1.0 Weight average molecular weight (Da) 401,271 122,968 29,211 19,009 9,011 7,876 6,248 4,082

Referring to the results of Tables 1 to 4 and FIGS. 2 to 3, as the decomposition reaction temperature increases, the apparent viscosity of the low molecular weight alginate solution becomes 3.5 cPs at 50 ° C. and 2.5 cPs, 1.0 cPs at 70 ° C, and 1.0 cP at 80 ° C.

The weight average molecular weight of the low molecular weight alginate was decreased with the lapse of the decomposition reaction time, and it was confirmed that the decrease of the weight average molecular weight was gentle after 16 hours. For example, when the decomposition reaction was carried out at 50 ° C, it was measured as 401,271 Da at 0 hours, 375,363 Da at 1 hour, 298,512 Da at 2 hours, 195,726 Da at 4 hours, 100,052 Da at 8 hours, 66,315 Da at 10 hours, 51,053 Da and from 22 hours to 45,487 Da.

The weight average molecular weight decreased with increasing temperature at the same time. When the decomposition reaction was conducted for 22 hours, it decreased to 45,487 Da at 50 캜, 14,108 Da at 60 캜, 7,225 Da at 70 캜, and 4,082 Da at 80 캜.

The concentration of residual hydrogen peroxide decreased by 0.874% at 50 ℃, 0.851% at 60 ℃, 0.820% at 70 ℃, and 0.789% at 80 ℃ after 22 hours of decomposition. The concentration of hydrogen peroxide was 22 0.10% at 50 ℃, 0.149% at 60 ℃, 0.180% at 70 ℃, and 0.211% at 80 ℃, respectively.

According to the experimental results, the decomposition reaction time is preferably from 16 hours to 22 hours, and the temperature is from 50 to 80 ° C.

≪ Hydrogen peroxide removal of low molecular weight alginate solution >

In order to remove hydrogen peroxide remaining in the low molecular alginate solution, catalase enzyme (CAT200, catalase enzyme) was added to 10 ml of low molecular alginate solution to adjust the concentration of catalase enzyme to 10 ppm, and the concentration of residual hydrogen peroxide Respectively. For comparison, 2.4 ml of NaOCl solution was added to 10 ml of low molecular weight alginate solution by chemical removal method.

The low molecular weight alginate solution used in the experiment was subjected to degradation reaction of the polymer alginate solution by applying 48 kHz ultrasonic wave at a temperature of 60 ° C for 0 to 22 hours.

Table 5 below shows the results of the addition of NaOCl solution.

Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 12hr 16hr 22hr NaOCl treatment time (hr) 8 7 5 4 3 2 2 2 2 Residual H ₂ O ₂ concentration (%) ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02

Table 6 shows the results of the addition of catalase enzyme.

Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 12hr 16hr 22hr Enzyme treatment time (min) One One One One One One One One One Residual H ₂ O ₂ concentration (%) 0.20 0.18 0.15 0.08 0.05 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 Enzyme treatment time (min) 10 10 10 10 10 10 10 10 10 Residual H ₂ O ₂ concentration (%) ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02 ≪ 0.02

Referring to the experimental results shown in Tables 5 and 6 above, the removal rate of hydrogen peroxide by catalase enzyme proceeded much faster than the chemical treatment.

In order to confirm the optimum concentration of enzyme to completely remove hydrogen peroxide, the concentration of hydrogen peroxide was determined by varying the concentration of the enzyme. Catalase enzyme was treated with enzyme at a concentration of 1 ppm (× 100 dilution), 10 ppm (× 1000 dilution), 100 ppm (× 10000 dilution), and the removal rate of hydrogen peroxide was confirmed.

Referring to FIG. 4, it was confirmed that even when the enzyme was treated at a concentration of 100 ppm in a hydrogen peroxide solution having a concentration of 1.0%, it was completely removed to 0.02% or less in 10 minutes.

≪ Analysis of yield and productivity of low molecular weight alginate &

Polymer alginate solution was decomposed by 48kHz ultrasonication at 60 ℃ for 0 ~ 22hours, and catalase enzyme was added at 1% concentration and hydrogen peroxide was removed for 1 min.

100 ml of low molecular weight alginate solution from which hydrogen peroxide had been removed was concentrated by evaporation using a rotary evaporator at 50 ° C to remove distilled water as a solvent and then water was removed at 50 ° C for 48 hours to obtain a powdered low molecular weight alginate.

The yields of the low molecular weight alginate in the powder state according to the decomposition reaction temperature and time of the polymer alginate solution were analyzed and shown in Tables 7 to 10 below.

Table 7 shows the results of the analysis of the yield of the low molecular weight alginate according to the decomposition reaction time at the decomposition reaction temperature of 50 ° C.

 Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 16hr 22hr Sampling Amount (ml) 100 100 100 100 100 100 100 300 Low molecular weight alginate recovery (g) 1.84 1.75 1.73 1.70 1.66 1.64 1.62 20.8 yield(%) 92.0 87.5 86.5 85.0 83.0 82.0 81.0 80.0

Table 8 shows the results of the analysis of the yield of the low molecular weight alginate according to the decomposition reaction time at the decomposition reaction temperature of 60 占 폚.

   Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 16hr 22hr Sampling Amount (ml) 100 100 100 100 100 100 100 300 Low molecular weight alginate recovery (g) 1.84 1.74 1.72 1.68 1.66 1.62 1.60 4.6 yield(%) 92.0 87.0 86.0 84.0 83.0 81.0 80.0 76.7

Table 9 shows the results of the analysis of the yield of the low molecular weight alginate according to the decomposition reaction time at the decomposition reaction temperature of 70 ° C.

   Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 16hr 22hr Sampling Amount (ml) 100 100 100 100 100 100 100 300 Low molecular weight alginate recovery (g) 1.84 1.72 1.70 1.65 1.61 1.58 1.55 4.4 yield(%) 92.0 86.0 85.0 82.5 80.5 79.0 77.5 73.3

Table 10 shows the results of the analysis of the yield of the low molecular weight alginate according to the decomposition reaction time at the decomposition reaction temperature of 80 ° C.

    Decomposition reaction time 0hr 1hr 2 hr 4hr 8hr 10hr 16hr 22hr Sampling Amount (ml) 100 100 100 100 100 100 100 300 Low molecular weight alginate recovery (g) 1.84 1.71 1.69 1.64 1.60 1.57 1.54 4.3 yield(%) 92.0 85.5 84.5 82.0 80.0 78.5 77.0 71.7

Referring to Tables 7 to 10, the yield tended to decrease with increasing decomposition reaction temperature. When the reaction was carried out at 50 ° C for 22 hours, the reaction rate was 80.0%, when the reaction was conducted at 60 ° C for 22 hours, 73.3% for 22 hours reaction and 71.7% for 22 hours reaction at 80 ℃. This shows that mass production is possible because the production efficiency is greatly improved as compared with the existing commercialized process.

The conventional methods of producing low molecular alginates, such as the hydrolysis method, the chemical decomposition method, the enzymatic decomposition method, and the gamma-ray method, are somewhat complicated in the recovery process after the decomposition reaction, resulting in inefficiency in terms of mass production, There is a drawback that the efficiency is low.

On the other hand, the present invention can easily recover the low molecular weight alginate by a simple process of completely removing the remaining amount of hydrogen peroxide in the water by using the catalase enzyme.

<Analysis of molecular structure of alginate>

FT-IR (Shidmazu 8601PC) and H ¹ -NMR were measured to analyze the molecular structure characteristics when the polymer alginate was made low-molecular-weight, and are shown in FIGS. 5 and 6, respectively. Deuterium Oxide (Aldrich) was used for the NMR analysis.

Polymer alginate solution was decomposed by 48kHz ultrasonic wave at 50 ~ 80 ℃ for 22 hours. Then catalase enzyme was added at 1% concentration and hydrogen peroxide was removed for 1 minute. The low molecular alginate solution was evaporated at 50 ℃ by rotary evaporator The concentrated solution was filtered to remove distilled water, and the water was removed at 50 ° C for 48 hours. The low molecular weight alginate powder was used as a sample.

Referring to FIG. 5, it was observed that a peak generated at 1650 cm ^-1 (C = O) was split into two peaks, although no peaks were generated or disappearing due to the decomposition reaction temperature. It was found that a new carbonyl group was formed as it was cut off. In FIG. 5, 'native sodium alginate' is a polymer alginate having a weight average molecular weight of 401,281 Da.

The results of H ¹ -NMR in FIG. 6 confirm that the intensity of the peak increases with increasing temperature at 8.5 ppm. This means that the hydroxycarboxylic acid produced by the hydrogen peroxide breaks 1,4-glycosidic bonds of the alginate salt, thereby increasing the formation of HCOO- (formate). 6, 'raw-sodium alginate' is a polymer alginate having a weight average molecular weight of 401,281 Da.

<Analysis of antibacterial properties of low molecular weight alginate>

In order to analyze the antimicrobial properties of polymer alginate in low molecular weight, the antimicrobial activity of E. coli, Staphylococcus aureus and P. aeruginosa was checked by microbial activity and then the minimum inhibitory concentration was measured by Korea Institute of Construction and Environment.

The antimicrobial activity of alginate is determined by the method of KS J 4206: 2008 by inoculating and culturing the test specimens and control specimens with the isolates (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa), shaking them in a certain amount of liquid to extract the cultured bacteria, The number of bacteria present is measured and the rate of decrease in the antimicrobial test piece is calculated and measured.

Bacterial reduction rate [%] = [(B-A) / B] 100

(A: the number of bacteria recovered from a test piece cultured through a certain contact time after inoculation, and B: the number of bacteria recovered from a control piece cultured through a certain contact time after inoculation)

A more specific method of evaluating the antimicrobial activity of alginate is as follows: 1.00 g to 0.05 g of the alginate prepared in the sterilized 100 ml tube is mixed with 100 ml of the medium, and then the optimal culture (1.3 × 10 ⁶ to 1.8 × 10 ⁶ CFU / ml) were inoculated and cultured at 37 ° C for 24 hours. Then, 20 μl of 10 ² to 10 ³ diluted samples were plated on a solid medium, and cultured at 37 ° C for 24 hours. And the antimicrobial activity was measured.

A low molecular weight alginate having a weight average molecular weight of 14,108 Da and a low molecular weight alginate having a weight average molecular weight of 4,082 Da were used as test specimens used in the experiment, and a polymer alginate having a weight average molecular weight of 401,271 Da was used as a control sample.

Table 11 below shows the results of antibacterial test of alginate according to each molecular weight when the concentration of alginate is 1.0%.

       Weight average molecular weight (Da) 401,271 14,108 4,082 Microbial reduction rate (%) for E. coli 10.1 99.9 99.9 Bacterial Reduction Rate for Staphylococci (%) 9.3 99.9 99.9 Bacterial reduction rate against Pseudomonas aeruginosa (%) 4.4 99.9 99.9

Referring to the results shown in Table 11, the microbial reduction rate at the 1.00% concentration of polymer alginate having a weight average molecular weight of 401,271 Da was 10.1% for E. coli, 9.3% for Staphylococci and 4.4% for P. aeruginosa , And low molecular weight alginates having a weight average molecular weight of 14,108 Da and 4,082 Da were significantly improved in antibacterial activity by 99.9%. For reference, FIG. 7 shows the result of the antibacterial activity of the polymer alginate having a weight average molecular weight of 401,271 Da.

Table 12 below shows the results of antibacterial tests according to the concentrations of low molecular weight alginates having a weight average molecular weight of 14,108 Da.

                   Sample concentration (%) 1.00 0.50 0.10 0.05 Microbial reduction rate (%) for E. coli 99.9 99.9 3.3 3.1 Bacterial Reduction Rate for Staphylococci (%) 99.9 99.9 5.7 1.5 Bacterial reduction rate against Pseudomonas aeruginosa (%) 99.9 99.9 6.5 2.0

Referring to the results shown in Table 12, the microbial reduction rate was 99.9% for E. coli, 99.9% for Staphylococci, and 99.9% for P. aeruginosa at a concentration of 0.50% of low molecular weight alginate having a weight average molecular weight of 14,108 Da, At the concentration of 0.10%, the microbial reduction rate was found to be 3.3% for E. coli, 5.7% for Staphylococci, and 6.5% for P. aeruginosa and the minimum inhibitory concentration was 0.50%.

8A to 8C are photographs showing results of antibacterial experiments at low molecular weight alginate salt concentrations of 1.00%, 0.50% and 0.10% having a weight average molecular weight of 14,108 Da.

Table 13 below shows the results of antibacterial tests according to the concentration of low molecular weight alginate having a weight average molecular weight of 4,082 Da.

                   Sample concentration (%) 1.00 0.50 0.10 0.05 Microbial reduction rate (%) for E. coli 99.9 99.9 99.9 1.6 Bacterial Reduction Rate for Staphylococci (%) 99.9 99.9 99.9 4.5 Bacterial reduction rate against Pseudomonas aeruginosa (%) 99.9 99.9 99.9 4.1

Referring to the results shown in Table 13, the microbial reduction rate was 99.9% for Escherichia coli, 99.9% for Staphylococci, and 99.9% for Pseudomonas aeruginosa at a concentration of 0.10% of low molecular weight alginate having a weight average molecular weight of 4,082 Da, At the concentration of 0.05%, the microbial reduction rate was found to be 0.10% for the microorganism, 1.6% for the Escherichia coli, 4.5% for the Staphylococcus and 4.1% for the Pseudomonas aeruginosa.

9A to 9C are photographs showing results of antibacterial experiments at low molecular weight alginate salt concentrations of 1.00%, 0.50% and 0.10% having a weight average molecular weight of 4,082 Da.

As a result of the above-mentioned antibacterial test results, the low molecular weight alginate having a weight average molecular weight of 4,082 Da prepared according to the present invention has no conventional antimicrobial activity at a concentration of 1.0% of a conventional molecular weight aliquot of 401,271 Da, And 99.9%, respectively. Therefore, it was found that 0.10% should be included in order to give antimicrobial properties to daily necessities (toothbrush, fabric softener, detergent, etc.) and oral related products (toothpaste, dental floss, oral cleanser, etc.).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation. Accordingly, the true scope of protection of the present invention should be determined only by the appended claims.

The low molecular weight alginate prepared according to the present invention is excellent in antibacterial activity against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa, Can be widely applied.

10: dissolution part 20: decomposition part
30: Post-

Claims (4)

Dissolving the polymer alginate in distilled water to obtain a polymer alginate solution having a concentration of 2.0% (w / w);
A decomposition reaction step of adding hydrogen peroxide solution to the polymer alginate solution and then decomposing the polymer alginate into a low molecular weight alginate by irradiation with 48 kHz ultrasonic waves;
And a post-treatment step of adding a catalase enzyme to the low molecular weight alginate solution obtained in the decomposition reaction step to remove the hydrogen peroxide contained in the low molecular weight alginate solution for 1 to 10 minutes,
In the decomposition reaction step, the polymer alginate solution is irradiated with the ultrasonic wave for 16 to 22 hours while the temperature of the polymer alginate solution is maintained at 50 to 80 DEG C,
Wherein the post-treatment step is performed at a concentration of 1 to 100 ppm (mg / kg) of the catalase enzyme in the low molecular weight alginate solution. delete delete The method according to claim 1, wherein the low molecular weight alginate has a weight average molecular weight of from 4082 to 14108 Da and is characterized by having antimicrobial activity against Escherichia coli , Staphylococcus aureus and Pseudomonas aeruginosa , (Method for preparing low molecular weight alginate).

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