KR20210001000A - manufacturing method of low molecular weight chitosan using solution phase plasma - Google Patents

Abstract

The present invention relates to a method for manufacturing low molecular weight chitosan using solution plasma generated by using a unipolar power source. More specifically, the present invention relates to a method for rapidly manufacturing low molecular weight chitosan from chitosan without modifying the chemical structure of chitosan through solution plasma.

Description

Manufacturing method of low molecular weight chitosan using solution phase plasma {manufacturing method of low molecular weight chitosan using solution phase plasma}

The present invention relates to a method for producing low-molecular chitosan using a fluid plasma generated by a unipolar power source, and more particularly, to a low-molecular chitosan from chitosan without changing the chemical structure of chitosan through fluid plasma. It relates to a method that can be manufactured.

Chitosan is a major component of the exoskeleton of crustaceans such as crab, squid, and shrimp shells. It is a carbohydrate with more than 5,000 D-glucosamine repeatedly bound and more than 1 million molecular weight. Alternatively, chitin present in the shellfish is deacetylated, and is a linear natural polymer polysaccharide to which glucosamine is bound. Chitosan has received a lot of attention as a material that can be used and applied in various fields such as food and medicine such as drug carriers, wound dressing materials, and antibacterial agents.

Chitosan has properties such as gelation, rapid crosslinking, and water solubility, and has various physiological activities, so it has been considered to have the potential for multipurpose use. However, its molecular weight is so large that it dissolves only in acidic solutions, so its application range is very narrow.

On the other hand, low-molecular chitosan, a decomposition product of chitosan, is well soluble in a wide range of pH solvents and has excellent biomedical properties. For example, low-molecular chitosan has been found to have a wide range of high functionalities, including anti-tumor activity, oral insulin preparations, gastric ulcer treatment, immunity enhancement, antibacterial activity, cholesterol control, and calcium absorption promotion. In spite of these excellent advantages of chitosan, it still requires a lot of time, process steps, and cost to produce low-molecular chitosan, and harmful solvents or materials used during the process remain, and thus have low yield and purity.

In order to solve this problem, methods for manufacturing low-molecular chitosan have been studied in various fields.As a result, chemical methods using strong acids, microbial processes, treatment processes by ultrasonic or microwave, hydrochloric acid pyrolysis process, and methods using plasma have been developed. come.

In the case of a microbial process, an excessive amount of enzymes or microorganisms are required, a long reaction time and reaction conditions (metal catalyst, temperature, pH, etc.) are required. There is a problem that the chitosan product is expensive, and the final produced low molecular weight chitosan product is contaminated by enzymes or microorganisms (Non-Patent Document 1).

For chemical methods. A document in which a large amount of salt and 30% H ₂ O ₂ were reacted for a long time under acidic conditions (360 minutes) to decompose chitosan from 200 kDa to 9 kDa has been known (Non-Patent Document 2). In addition, studies on producing low-molecular chitosan by a physical method using ultrasonic waves, microwaves, and plasma in gas have also been known (Non-Patent Document 3). However, these methods inevitably require expensive devices and equipment, and require a very high frequency to be applied, and operation costs are very high due to frequent replacement of the vibrator. In addition, there are limitations that manufacturing is possible only under limited conditions, there are problems that the recovery rate is not high compared to the difficult conditions, and the operation time is long. On the other hand, the hydrochloric acid pyrolysis process has a relatively short reaction time compared to the microbial treatment process, but the glucosamine production rate is low, and the operating cost increases due to the temperature of 80°C or higher for 8 hours or more.

Therefore, there is a need to develop a method that can easily and efficiently manufacture low-molecular chitosan at a lower cost.

Non-Patent Document 1. Xie, H., Jia, Z., Huang, J & Zhang, C. (2011). Preparation of low molecular weight chitosan by complex enzymes hydrolysis. Inter. J. Chem., 3(2), 180-186. Non-Patent Document 2. Chang, K.L., Tai, M.C & Chang, F.H. (2001). Kinetics and products of the degradation of chitosan by hydrogen peroxide. J.Agri.Food. Chem., 49, 4845-4851. Non-Patent Document 3. Yue, W., Yao, P., Wei, Y., & Mo, H. (2008). Synergetic effect of ozone and ultrasonic radiation on degradation of chitosan. Poly. Degrad. Stabili, 93, 1814-1821.

The present inventors have made diligent efforts to discover a new method for producing low-molecular chitosan. As a result, a strategy using fluid plasma generated by a unipolar power source was devised, and the present invention was completed by confirming that low-molecular chitosan can be produced quickly and efficiently from polymer chitosan using this.

Accordingly, an object of the present invention is to provide a method for producing low molecular weight chitosan quickly and efficiently using fluid plasma.

Another object of the present invention is to provide a low-molecular chitosan having a high swelling ratio, antimicrobial activity and antioxidant activity prepared by the above-described manufacturing method, and an antibacterial composition comprising the same.

According to an exemplary aspect of the present invention, it relates to a method for producing a low molecular weight chitosan comprising the following steps.

1) preparing a chitosan solution by dissolving a polymer chitosan in an acetic acid solution; And

2) a fluid plasma reaction step of generating a plasma in the chitosan solution to generate a low molecular weight chitosan for 15 to 120 minutes; Including, wherein the fluid plasma reaction step includes a voltage of a unipolar power supplied to an electrode exposed to the chitosan solution It may be 700 to 900V, frequency 20 to 40 KHz.

After the step 2), 3) filtering and drying the low-molecular chitosan after irradiation with fluid plasma. It may further include.

The fluid plasma reaction step may be a voltage of 800V and a frequency of 30 KHz of unipolar power supplied to the tungsten electrode exposed to the chitosan solution.

The fluid plasma reaction step may be 15 to 120 minutes.

According to another exemplary aspect of the present invention, it relates to a low molecular weight chitosan prepared according to the above manufacturing method.

The low molecular weight chitosan may have a molecular weight of 4.6 × 10 ³ Da to 7.8 × 10 ³ Da, an antimicrobial activity of 80 to 150 μg/ml, an antioxidant activity of 58 to 75%, and a swelling ratio of 0.2 to 2 mg/mg. .

The low-molecular chitosan may have a solubility of 80 to 99% in an acidic condition (pH <6) is maintained even in a neutral and alkaline range (pH> 7).

Another exemplary aspect of the present invention relates to an antimicrobial composition comprising chitosan prepared according to the above manufacturing method.

According to various embodiments of the present invention, the method for preparing low molecular weight (LMW) chitosan depolymerized through fluid plasma enables rapid generation, and does not require addition of chemical additives in the manufacturing process. Low-molecular chitosan has achieved excellent swelling properties, antioxidant and antibacterial properties.

In addition, the method for producing a low-molecular chitosan of the present invention has a great advantage of being able to easily, economically and eco-friendlyly produce suitable biomaterials for application in biomedical fields.

FIG. 1 shows the results of the manufacturing process of low-molecular chitosan using fluid plasma, FIG. 1A is a photograph of a color change (0-120 minutes) of chitosan solution according to plasma discharge, and FIG. 1B is a plasma treatment time. It is a graph showing by measuring the viscosity (cps) of the chitosan solution.
FIG. 2 is a graph showing the measurement of water solubility according to pH of low-molecularized chitosan prepared by fluid plasma (SPP) of Example 1. FIG. At this time, according to the fluid plasma treatment time, it was marked as ▲(0 minutes), ■(15 minutes), ●(30 minutes), △(60 minutes), □(90 minutes), and ○(120 minutes).
Figure 3a relates to the yield of the low-molecular chitosan prepared in Example 1, the ratio of the water-soluble (WS) chitosan in the low-molecular chitosan increased as the fluid plasma treatment time increased (0-120 minutes), insoluble (WI) chitosan Decreased.
3B is an FTIR spectrum of the low-molecular chitosan of Example 1 according to the fluid plasma treatment time, and the peaks (3319, 2668, 1478, 1152 cm ^-1 ) according to the fluid plasma treatment time are shown, the effect of the fluid plasma treatment time This was confirmed to be insignificant.
4 is a graph showing the molecular weight of the low-molecular chitosan prepared in Example 1, measured according to fluid plasma treatment time. The low molecular weight chitosan prepared by treating the fluid plasma for 30 minutes was measured to have a molecular weight of 7.8 × 10 ³ Da, the chitosan without the fluid plasma treatment was 3.0 × 10 ⁵ Da, and the low molecular weight prepared by treating the fluid plasma for 60 minutes. It was confirmed that chitosan was 5.8 × 10 ³ Da, and low-molecular chitosan prepared by treating fluid plasma for 90 minutes had a molecular weight of 4.6 × 10 ³ Da.
5 is a graph showing DLS and zeta potential for the low molecular weight chitosan of Example 1 prepared using fluid plasma with various treatment times.
6 is a graph and table showing the antimicrobial activity against the low molecular weight chitosan of Example 1 using fluid plasma.
7A is a graph showing the analysis of the equilibrium swelling ratio (ES ratio) for the low molecular weight chitosan of Example 1 using fluid plasma.
7B is a graph showing DPPH scavenging activity for the low molecular weight chitosan of Example 1 using fluid plasma.

Hereinafter, various aspects and various embodiments of the present invention will be described in more detail.

According to an aspect of the present invention, a method for producing a low molecular weight chitosan comprising the following steps is disclosed.

1) preparing a chitosan solution by dissolving a polymer chitosan in an acetic acid solution; And

2) A fluid plasma reaction step of generating plasma in the chitosan solution to generate low molecular weight chitosan.

Low molecular weight (LMW) chitosan has attracted great attention in the fields of life, chemistry, medicine, and drug delivery, and the present invention aims to solve the problems of the prior art and provide a method for economically and efficiently manufacturing it.

As in the present invention, when high molecular weight (HMW) chitosan is depolymerized through a fluid plasma process (SPP) to obtain low molecular weight (LMW) chitosan, the chemical structure of chitosan is not altered, and low molecular weight chitosan is effectively obtained. I can.

Specifically, 1) polymer chitosan is dissolved in an acetic acid solution to prepare a chitosan solution. Chitosan is easily soluble in acid, and in particular, it is preferable to use a fluid plasma without lowering the degree of polymerization when using an acetic acid solution having a wide concentration.

The concentration of the chitosan solution is not particularly limited thereto, but the concentration in the solution may be preferably 0.1 to 5% by weight, more preferably 0.5 to 2% by weight. The electric conductivity changes according to the concentration of the chitosan solution, which may affect the size of the resulting low molecular weight chitosan. Therefore, if it is less than 0.5% by weight, the effect of the fluid plasma may increase, and the yield of low-molecular chitosan may be inefficient, and if it exceeds 2% by weight, the viscosity of chitosan increases and depolymerization by the release of the fluid plasma may become difficult. It is preferred to use chitosan solutions in one concentration range.

Thereafter, 2) plasma is generated in the chitosan solution to react with a fluid plasma to generate low molecular weight chitosan.

The fluid plasma (SPP) applied to the present invention is a technology that generates high-density, high-energy plasma in a fluid, synthesizes and disperses it in a single process. It is economical, easy to secure productivity, and can produce more efficient low-molecular chitosan. I can.

In fluid plasma, the flow of ions and electrons different from the application of electric energy in the liquid generates plasma in the liquid, and as the amount of applied electric energy increases, the flow of ions and electrons increases, thereby changing the intensity of the plasma. In the present invention, the generation of plasma is related to the flow of electrons, and electrons are provided to the high molecular chitosan present in the liquid to depolymerize the low molecular weight chitosan.

The fluid plasma device used in the present invention is one that is commonly used in the art, and the fluid plasma reaction device used in the experimental examples of the present invention includes a cylindrical Teflon reactor and a cooling system for maintaining the chitosan solution in the reactor at a constant temperature. A system, a circulation pump, a pair of electrodes installed in the reactor, and a unipolar pulse power supply for supplying power to the electrodes were used. The electrode is made of tungsten material, and the distance between the two electrodes is maintained at about 1 mm.

When power is supplied to the chitosan solution in the reactor, plasma is formed in the solution by electric discharge, and depolymerization occurs from high molecular chitosan to low molecular chitosan. At this time, in order to prevent the temperature of the chitosan solution from rising, the temperature was maintained at 20 ~ 30 ℃ through the cooling system. The reactor and the cooling system are connected, and a circulation pump can be circulated.

Specifically, low molecular chitosan was produced by depolymerization by releasing fluid plasma in the chitosan solution under conditions of 700 to 900 V and 20 to 40 kHz for various times (15 to 120 minutes), most preferably 800 V and a frequency of 30 KHz. have. In the fluid plasma reaction step, a voltage of 800V and a frequency of 30 KHz of power supplied to the electrode exposed to the chitosan solution is most preferable.If the voltage is higher than the voltage and the frequency is low, 2 to 10 times the voltage to produce low molecular chitosan Although it takes more time and takes more time, there is a problem in that chitosan having a molecular weight of 10 times or more is obtained.

Specifically, in the case of emitting fluid plasma at 900 V or higher, that is, at 1600 V and a frequency of 15 kHz, only chitosan having a molecular weight size of 3.2 × 10 ⁴ Da and 1.9 × 10 ⁴ Da can be obtained even if it is treated for 300 minutes or longer. Chitosan of the size has a disadvantage that the physiological activity is remarkably low or hardly exhibited.

The manufacturing method according to the present invention may be performed in 15 to 120 minutes, preferably 70 to 110 minutes, more preferably 80 to 100 minutes, and most preferably 90 minutes in order to obtain a low molecular weight chitosan.

As a result of analyzing the fluid plasma reaction step at various time intervals (15 minutes to 120 minutes), it was found that the low molecular weight chitosan obtained by treatment with 90 minutes improved water solubility (solubility) and antibacterial activity by 1.5 to 2 times or more in the acidity range. Confirmed. If it is carried out below the above range, little chitosan is produced, and if it exceeds the above range, there is no significant difference in the activity of the low molecular chitosan, which is not preferable in view of economics.

The low-molecular chitosan prepared through the above-described process does not require a separate surfactant, but the low-molecular chitosan prepared in the solution has a polycation and exhibits stability to some extent, and can exhibit excellent properties as a biomaterial in the future.

In addition, it may further include the step of filtering and drying the low-molecular chitosan after step 2) and after 3) irradiation with fluid plasma.

Additionally, in order to recover only the water-soluble low-molecular chitosan, the process of obtaining a precipitate formed by centrifuging the low-molecular chitosan, recovering the supernatant, and adding an organic solvent may be further included.

According to another aspect of the present invention, a low molecular weight chitosan prepared through the above-described manufacturing method is disclosed. As a result of analyzing the low-molecular chitosan prepared according to the above-described preparation method by gel permeation chromatography, the molecular weight gradually decreases with the fluid plasma treatment time.After 30 minutes treatment, the molecular weight of chitosan was 3.0 × 10 ⁵ Da. It was confirmed that it decreased to 7.8 × 10 ³ Da, and 4.6 × 10 ³ Da at 90 minutes treatment. As a result of analyzing dynamic light scattering (DLS) and zeta potential, it was confirmed that chitosan nano-aggregates were formed. Furthermore, the low molecular weight chitosan was confirmed that this has an excellent antimicrobial activity even on gram-negative bacteria as well as Gram-positive bacteria specifically MIC (minimal inhibitory concentration) for Escherichia coli, Staphylococcus aureus, Candida albicans prepared as described above is 80-1200 ㎍ / It was confirmed to be ml. Furthermore, it was confirmed that it had a high antioxidant activity (58-75%) and a swelling ratio of 0.2 to 2.0 mg/mg.

In addition, when conventional chitosan is low-differentiated, low-molecular chitosan exhibits solubility only in acidic conditions (pH <6), whereas water solubility is reduced by 2 to 8 times in neutral and alkaline conditions (pH> 7), and the method according to the present invention. It was confirmed that the low-molecular chitosan prepared as has a great advantage in that it has 80 to 90% or more even in acidic conditions (pH <6) as well as neutral and alkaline ranges (pH> 7). In other words, it can be said that 80-99% of solubility in acidic conditions (pH <6) is maintained even in the neutral and alkaline range (pH> 7).

That is, since the low molecular weight chitosan according to the present invention has high solubility, that is, water solubility in the entire pH range, it can be used in a wide range, and it can be seen that it can be used sustainably as a carrier, biomaterial, and biomedical use.

According to another aspect of the present invention, the low-molecular chitosan prepared through the above-described manufacturing method has excellent antibacterial effect, has antioxidant activity, and has no side effects, so it can be usefully used as an antibacterial composition, an external preparation for skin, and a cosmetic.

The low-molecular chitosan of the present invention is used for the purpose of efficiently removing harmful bacteria and fungi, and is not particularly limited in its formulation, and in addition to the low-molecular chitosan, it may contain one or more active ingredients exhibiting the same or similar function. I can.

The antimicrobial composition may be an external preparation for skin, and specifically, may be prepared as a solution, a suspension, a spray, a patch, a pad, a cream, an ointment, or a gel.

In addition to the active ingredient, the solution may contain conventional excipients, such as solvents, solubilizers, and emulsifiers, such as water, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oil, especially cottonseed oil, peanut oil, camellia oil, aloe vera, glycerin, natural corn berry oil, olive oil, castor oil, almond oil and sesame oil, glycerol, glycerol form alcohol, tetrahydrofur Fatty acid esters of furyl alcohol, polyethylene glycol and sorbitan, or a mixture of these substances may be included, and methyl or propyl-p-hydroxybenzoate or sorbic acid may be included as a preservative.

Suspension agents other than the active ingredient, conventional excipients, such as liquid diluents (e.g., water, ethyl alcohol and propylene glycol, polyethylene glycol), and suspending agents (e.g., ethoxylated isostearyl alcohol, polyoxyethylene sorbitol, sorbitan) Esters, cellulose derivatives and hydrogenated edible oils), microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and injectable esters such as tragacanth, ethyloleate, or mixtures of these substances.

Ointments, creams and gels are not only active compounds, but also conventional excipients, such as animal and vegetable fats, wax paraffin, starch, targacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc and zinc oxide, or these substances. It may contain a mixture of.

In addition, the composition may be a cosmetic composition, and the formulation is not particularly limited and may have a conventional formulation. Specifically, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, essence, nutrition essence, pack, liquid or solid soap, shampoo, It includes cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser and bathing agent.

The cosmetic composition includes fatty substances, organic solvents, solubilizers, thickeners and gelling agents, softeners, antioxidants, suspending agents, stabilizers, foaming agents, fragrances, surfactants, water, ionic or nonionic, in addition to low molecular chitosan Contains adjuvants commonly used in the field of dermatology such as type emulsifiers, fillers, sequestering and chelating agents, preservatives, vitamins, blockers, wetting agents, essential oils, dyes, pigments, hydrophilic or lipophilic actives or lipid vesicles can do.

In addition, the ingredients may be introduced in an amount generally used in the field of dermatology. The low-molecular chitosan may be included in the total weight of the antimicrobial composition of the present invention in preferably 0.00001 to 50 parts by weight, more preferably 0.0001 to 10 parts by weight, and most preferably 0.001 to 0.1 parts by weight.

In addition, the composition contains such a low-molecular chitosan as an active ingredient because it is possible to prevent or treat diseases such as food poisoning, candism, typhoid fever, and cholera, which are diseases typically caused by Gram-positive or Gram-negative bacteria showing antibacterial activity. It can be used as a pharmaceutical composition for the prevention or treatment of the above diseases. In addition, the present invention has excellent antibacterial activity against Gram-negative bacteria and Gram-positive bacteria, and by confirming that the antioxidant effect is also excellent, it was found that it can be used as an antioxidant as well as antibacterial use.

The low-molecular chitosan may have antimicrobial activity against any one or more selected from the group consisting of E. coli , S. aureus, and C. albicans .

On the other hand, the method of administration of the composition is not particularly limited, but preferably, it may be injected into an artery or vein, or subcutaneously, rectal, nasal, or any other parenteral administration, and more preferably arterial Alternatively, it is preferable to be injected intravenously, orally or directly into muscle cells.

In addition, the dosage level selected from the composition will depend on the activity of the compound, the route of administration, the severity of the condition being treated and the condition and previous medical history of the patient being treated. However, it is within the knowledge of the art to start with a dose of the compound at a lower level than required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved, and the preferred dosage is age, sex. , It can be determined according to body type and weight. The composition may be further processed prior to being formulated into a pharmaceutically acceptable pharmaceutical formulation, preferably pulverized or ground into smaller particles. Also, the composition will depend on the condition and the patient being treated, but this can be determined non-ingeniously.

Hereinafter, the present invention will be described in more detail through examples, etc., but the scope and contents of the present invention are reduced or limited by the examples below and cannot be interpreted. In addition, if based on the disclosure content of the present invention including the following examples, it is clear that the present invention for which no specific experimental results are presented can be easily carried out by a person skilled in the art, and such modifications and modifications are attached to the patent. It is natural to fall within the scope of the claims.

In addition, the experimental results presented below are only representative of the experimental results of the above examples and comparative examples, and the effects of each of the various embodiments of the present invention that are not explicitly presented below will be described in detail in the corresponding section.

Experimental material

Polymer chitosan powder (Mw: 100-300 kDa, Acros organic, USA) and glacial acetic acid (Sigma Aldrich Co., USA) were used to prepare low molecular weight chitosan. Luria-Bertani medium (LB, yeast extract 0.5%, NaCl 0.5%, tryptone 1%), potato dextrose (PD; potato dectrose 2.4%), yeast peptone medium (YPD, yeast extract 1%, peptone 2%) used for microbial culture , Dextrose 2%) was purchased from Difco (USA). M㎻ller-Hinton agar plates (beef extract 0.2%, acid digest of casein 1.75%, starch 0.15%, agar 1.7%) of the antimicrobial activity test were purchased from Difco (USA). For the analysis of antioxidant activity, 1,1-Diphenyl-2-picryl-hydrazyl (DPPH; Santa Cruz Biotech, Inc., USA) and absolute ethanol (99%, absolute ethanol) were purchased from Sigma Aldrich. All chemical substances used in the present invention were purchased with analytical grade, and ionized water was used.

<Test method 1. Measurement of color and viscosity>

In the case of color, the apparent color change for each manufacturing step was observed and recorded with the naked eye. The viscosity of the solution was measured at room temperature using a viscometer (Vibro SV-100, A & D Co., Japan).

<Test method 2. Water solubility analysis>

The chitosan solution treated with or without fluid plasma was adjusted to pH 3-12 using 5M NaOH solution (Lee and Song, 2014). At this time, the turbidity of the solution was measured at 600 nm in the transmittance mode by spectroscopy (UV-S370, Molecular Device, Germany).

<Experimental Method 3. Yield Measurement>

The yield of low molecular chitosan was measured through fractionation analysis. Specifically, fluid plasma-treated or untreated chitosan (10 ml) was put into a clean centrifuge tube, and the pH was adjusted to pH 7.5 using 5 M NaOH. The formed precipitate was expressed as precipitate I (water-insoluble chitosan), and collected by centrifugation (6,000 rpm) at 4° C. for 20 minutes. Then, the supernatant was transferred to another tube, and the same amount of acetone was added and mixed. The formed precipitate was designated as precipitate II (water-soluble chitosan). The precipitates I and II were dried overnight at 60°C in a convention oven. The yield (%) of precipitates I and II was calculated according to Equation 1 below.

[Equation 1]

Yield of precipitate Ⅰ or Ⅱ (%) = (A / B) × 100

In Equation 1 above,

A is the representative weight of the dried chitosan precipitate I or II,

B is the weight of untreated chitosan.

<Experimental Method 4. FTIR Analysis>

The purity and functional groups of the fluid plasma-treated or untreated chitosan solution were analyzed in the range of 400 cm ^-1 to 4,000 cm ^-1 by FTIR (FTIR-Vertex 8V, Bruker, Germany).

<Experimental method 5. Gel permeation chromatography (GPC)>

The molecular weight (Mw) of the low molecular weight chitosan was determined after plasma treatment by gel permeation chromatography (GPC, Tosch Corp. Japan). The chitosan solution was filtered using a 0.45 μm Millipore membrane, and injected into a 2X TSK gel GMPWx1 column (Waters, USA) equipped with an RI detector (Waters, USA) (3 mg/ml). The sample was separated by using a mixture of 0.5M acetic acid and sodium acetate (1:1) as an eluent at a flow rate of 1.0 ml/min at 40°C. For the molecular weight (Mw) analysis of fluid plasma-treated chitosan, a polysaccharide mixture (Mw: 180-708,000Da) was used as a standard, and a calibration curve was analyzed using EMPOWER software (Waters, USA), and the molecular weight of the sample was determined. Confirmed.

<Experimental Method 6. DLS and Zeta Potential Analysis>

In 2 ml of chitosan solution, it was analyzed with a particle size analyzer to confirm the size of the fine particles in the solution, and the average value of the fine particles was measured from multiple readings in DLS (DLS-Photal, Otsuka Elec., Japan). The zeta potential of 2 ml of chitosan solution is in the range of -200 mA to +400 mA (Zeta Photal, Otsuka Elec., Japan) with constant viscosity (0.86 cps) and electric field (16.22 V / cm) for stability of chitosan solution. Analyzed.

<Test method 7. Antimicrobial activity of low molecular chitosan (MIC)>

E. coli, S. aureus ( bacteria), C. albicans (yeast) The antimicrobial activity of low-molecular chitosan (0-2400 μg/ml) was analyzed using three microbial pathogens. MIC for pathogens was measured in the same manner as commonly used in the industry.

<Test Method 8. Swelling ratio analysis>

After treatment with fluid plasma, a certain amount of dried chitosan was thoroughly mixed with 2 ml of dH ₂ O, and immersed at room temperature for 2 hours. It was held inclined for 30 minutes to remove excess water from the soaked pieces. Equilibrium swelling (ES, equilibrium swelling) ratio (ratio) was calculated according to Equation 2 below.

[Equation 2]

In Equation 2,

Swollen gell _weight is the mass of expanded chitosan,

Dried gel _weight is the mass of dried chitosan.

<Experimental Method 9. DPPH Analysis>

Chitosan treated with fluid plasma and chitosan not treated with plasma were prepared, respectively, and their antioxidant activity was confirmed through DPPH free radical scavenging analysis. The percentage of free radical scavenging activity was calculated through Equation 3 below.

[Equation 3]

DPPH inhibition (%)=[(Abs _control -Abs _sample )/(Abs _control )] x 100

In equation 3,

Abs _control is the absorbance of chitosan without fluid plasma treatment,

Abs _sample is the absorbance of low-molecular chitosan prepared by fluid plasma treatment.

Example

<Example 1. Preparation of low molecular weight chitosan using fluid plasma>

A chitosan solution was prepared by dissolving polymeric chitosan (0.5% w/v) in 1 N acetic acid (acetic acid, glacial). 300 ml of the chitosan solution was placed in a Teflon reactor. The Teflon reactor was used in which two tungsten electrodes were provided in a juxta-position with an interval of 1 mm. The reactor was connected to a unipolar power supply (EnerPulse 10HV, EN Technology, Korea), and the polymer chitosan solution was discharged with constant stirring for 0-120 minutes at a frequency of 800 voltage and 30 kHz. A cooling system (Water chhiller, TRC 2000, Korea) was connected to the reactor. This is to minimize evaporation of the solution during the process. Finally, a chitosan solution depolymerized through the above process was prepared by filtering through a membrane having 0.45 μm pores (Millipore, USA).

<Experimental Example 1. Depolymerization of chitosan using fluid plasma>

The polymer chitosan is not easily soluble in water, and the fluid plasma conditions (800V and 30kHz) were selected by confirming that it is the most optimal condition for the low molecular weight of the polymer chitosan.

FIG. 1 shows the results of the manufacturing process of low-molecular chitosan using fluid plasma, FIG. 1A is a photograph of a color change (0-120 minutes) of chitosan solution according to plasma discharge, and FIG. 1B is a plasma treatment time. It is a graph showing by measuring the viscosity (cps) of the chitosan solution.

As shown in FIG. 1A, it was confirmed that the color of the solution was changed according to the manufacturing process of the low molecular weight chitosan of Example 1. In particular, it was confirmed that a color change was evidently caused as the fluid plasma process proceeded. Specifically, it was confirmed that the chitosan solution was pale yellow before plasma treatment (0 minutes), and gradually became yellow (15 minutes), golden brown (60 minutes), and dark brown in the last 120 minutes.

When the gas plasma of O ₂ and N ₂ , not fluid plasma, was treated with the chitosan solution at high voltage (300 and 400 W), the solution phase changed from brown to brown, and finally, a brown colored solution was obtained.

As a result of examining the viscosity of the chitosan solution that caused the fluid plasma discharge as shown in FIG. 1B, it was confirmed that the chitosan solution (control group) not treated with plasma had a viscosity higher (4.80 cps) than the low-molecular chitosan obtained by treatment with plasma. The viscosity of the chitosan solution subjected to plasma discharge for 15 minutes decreased rapidly to 2.1 cps. Thereafter, it was confirmed that the chitosan solution rapidly decreased to 1.8 cps at 30 minutes, 1.5 cps at 60 minutes, 1.2 cps at 90 minutes, and 0.9 cps at 120 minutes according to the plasma treatment time.

It was confirmed that the viscosity decreased by 2.7 times (62.5%) and 5.3 times (81.2%), respectively, compared to the control (untreated chitosan), when the molecular weight was reduced for 30 minutes and 120 minutes through a fluid plasma using unipolar power. .

<Experimental Example 2. Analysis of water solubility of low-molecular chitosan using fluid plasma>

To investigate the water solubility of the low-molecular chitosan prepared from Example 1. In addition, the overall analysis of the change in water solubility of chitosan during the entire process of fluid plasma treatment was attempted.

FIG. 2 is a graph showing the measurement of water solubility according to pH of low-molecularized chitosan prepared by fluid plasma (SPP) of Example 1. FIG. At this time, according to the fluid plasma treatment time, it was marked as ▲(0 minutes), ■(15 minutes), ●(30 minutes), △(60 minutes), □(90 minutes), and ○(120 minutes).

As shown in FIG. 2, the aqueous solubility of the polymeric chitosan was soluble only at an acidic pH (<6), but it was confirmed that the low molecular weight chitosan of Example 1 exhibited excellent solubility even at an alkaline pH (>7).

Specifically, the low-molecular chitosan prepared by fluid plasma treatment for 15 or 30 minutes was dissolved at pH 3.0 at pH 7.5. It was confirmed that the solubility of the low-molecular chitosan prepared by fluid plasma treatment for 60 minutes was maintained up to the alkali pH range (7-9). On the other hand, it was found that the low-molecular chitosan prepared by fluid plasma treatment for 90 and 120 minutes showed solubility of 70% and 82% or more, respectively, in a wide pH range. Therefore, as the treatment time of the fluid plasma increases, the solubility of the low-molecular chitosan increases, which is associated with a decrease in the chain length and intermolecular interactions of the low-molecular chitosan.

<Experimental Example 3. Yield Analysis of Low Molecular Chitosan Using Fluid Plasma>

Through fractionation analysis, the yield of water-soluble (WS) chitosan and insoluble (WI) chitosan in the low-molecular chitosan of Example 1 was to be quantitatively analyzed.

Figure 3a relates to the yield of the low-molecular chitosan prepared in Example 1, the ratio of the water-soluble (WS) chitosan in the low-molecular chitosan increased as the fluid plasma treatment time increased (0-120 minutes), insoluble (WI) chitosan Decreased.

As shown in FIG. 3A, as the fluid plasma treatment time increased, the yield of insoluble (WI) chitosan decreased, and the yield of water-soluble (WS) chitosan increased.

Polymeric chitosan before fluid plasma treatment contained 98% of insoluble chitosan and 2% of water-soluble chitosan, but as a result of low-differentiation according to Example 1, the yield of water-soluble chitosan increased to 87.5% (60-120 min. ). From this result, it was found that fluid plasma effectively induces the low molecular weight of chitosan and significantly increases the water-soluble chitosan content. Moreover, it was confirmed that this change in yield was maintained repetitively and reproducibly. Therefore, the low molecular weight manufacturing method of the present invention has an advantage in that the yield for obtaining water-soluble chitosan is constant, and thus process design is easy using this. However, in the case of conventional techniques for low molecular weight chitosan, the yield of water-soluble chitosan was not constant due to the insoluble chitosan remaining in the solution.

<Experimental Example 4. Analysis of Chemical Changes of Low Molecular Chitosan Using Fluid Plasma>

In order to confirm the chemical change of the low-molecular chitosan of Example 1 prepared by fluid plasma treatment with various treatment times (0-120 minutes), the IR spectrum was measured.

3B is an FTIR spectrum of the low-molecular chitosan of Example 1 according to the fluid plasma treatment time, and the peaks (3319, 2668, 1478, 1152 cm ^-1 ) according to the fluid plasma treatment time are shown, the effect of the fluid plasma treatment time This was confirmed to be insignificant.

As shown in FIG. 3B, 2668, 1478 and 1152 cm ^-1 peaks represent NH ₂ bending and -COC-glycosidic bonds between glucosamine and N-acetyl glucosamine of chitosan, indicating the purity and chemical properties of chitosan. The low-molecular chitosan of Example 1 prepared with fluid plasma did not differ significantly from each other according to treatment time. In other words, it means that the molecular structure of chitosan is continuously decreased by the fluid plasma treatment, whereas the chemical structure is not changed at all.

In addition, the 3319 cm ^-1 peak corresponds to a peak in which the NH ₂ and OH group stretching vibrations in chitosan and the intensity of the -NH ₂ band in 1578 cm ^-1 are combined, and these clearly indicate the purity of chitosan. have. As a result, a sharper peak was observed at 1578 cm ^-1 bending vibration, indicating high deacetylated chitosan. The broad peaks at 1030 cm ^-1 and 1070 cm ^-1 indicate CO expansion and contraction vibrations in chitosan. As in the previous description, the chemical structure of chitosan did not change at all during the low-molecularization through fluid plasma.

<Experimental Example 5. Molecular Weight of Low Molecular Chitosan Using Fluid Plasma>

The molecular weight (Mw) of the low-molecular chitosan prepared from Example 1 was confirmed through GPC analysis.

4 is a graph showing the molecular weight of the low-molecular chitosan prepared in Example 1, measured according to fluid plasma treatment time. The low molecular weight chitosan prepared by treating the fluid plasma for 30 minutes was measured to have a molecular weight of 7.8 × 10 ³ Da, the chitosan without the fluid plasma treatment was 3.0 × 10 ⁵ Da, and the low molecular weight prepared by treating the fluid plasma for 60 minutes. It was confirmed that chitosan was 5.8 × 10 ³ Da and low molecular chitosan prepared by treating fluid plasma for 90 minutes had a molecular weight of 4.6 × 10 ³ Da.

As shown in FIG. 4, a high molecular weight chitosan having a molecular weight of 3.0 × 10 ⁵ Da was prepared as low molecular chitosan by treating a fluid plasma for 30, 60, and 90 minutes, and the low molecular chitosan was of 7,852 Da, 5,865 Da, and 4,681 Da, respectively. It was found to have a molecular weight. By using the above-described method, it was possible to obtain a low molecular weight chitosan of 7,852 Da from 30 minutes, which can be said to be the shortest period compared to conventional methods of producing low molecular chitosan. In addition, it was confirmed that the low-molecular chitosan prepared through the above-described method exhibits remarkably superior physiological activity compared to the low-molecular chitosan prepared by the conventional method.

As described above, decreasing the molecular weight of chitosan leads to an increase in the degree of deacetylation (DD; %). For example, a chitosan having a molecular weight of 7.4 × 10 ⁴ Da has a degree of deacetylation (DD) of 95.4%, and a chitosan having a molecular weight of 3.6 × 10 ⁴ Da has a degree of deacetylation (DD) of 98%. The high degree of deacetylation (DD) is considered an important characteristic of chitosan-based biomaterials.

<Experimental Example 6. Analysis of DLS and Zeta Potential of Low Molecular Chitosan Using Fluid Plasma>

5 is a graph showing DLS and zeta potential for the low molecular weight chitosan of Example 1 prepared using fluid plasma with various treatment times.

As shown in FIG. 5, the low molecular weight chitosan prepared through fluid plasma formed nano-aggregate, and the size of chitosan nanomaterial gradually decreased as the fluid plasma treatment time (0-120 min) increased. Was confirmed.

Chitosan nanoparticles can be synthesized using a polyanionic reducing agent (tripolyphosphate) through an ionic gelation method, but when preparing low-molecular chitosan according to the manufacturing method of the present invention as shown in FIG. 5, any reducing agent in chitosan (polycationic) It can be seen that nano-sized low-molecular chitosan can be produced without also using (polyanionic).

The average size of the formed low-molecular chitosan particles was found to be from 273.8 ± 42.9 ㎚ (30 minutes) to 142.3 ± 25.8 ㎚ (60 minutes), 127.6 ± 26.2 ㎚ (90 minutes), and 53.8 ± 4.3 ㎚ (120 minutes). The nano-sized low-molecular chitosan of Example 1 has a low zeta potential (-35.16 to -7.49 mV), thereby forming an electrostatic repulsive force between the particles, thereby ensuring the stability of the particles in the solution.

In order to further secure the stability of the low-molecular chitosan of Example 1, a chemical stabilizer may be used, but it is more preferable to mix it with anionic biopolymers such as proteoheparan sulfate and extracellular polymers.

From the above results, it can be seen that the manufacturing method (fluid plasma) of the present invention has a great advantage in being able to directly generate nano-sized low molecular chitosan or low molecular chitosan nanoparticles. The nano-sized low-molecular chitosan prepared in this way is a biocompatible material and can be used for various purposes because it exhibits various physiological activities.

<Experimental Example 7. Antimicrobial Activity of Low Molecular Chitosan Using Fluid Plasma>

In order to measure the antimicrobial activity of the low-molecular chitosan of Example 1 using fluid plasma, the MIC was analyzed for two types of bacteria and pathogenic yeast, such as representative Gram-negative bacteria and Gram-positive bacteria.

6 is a graph and table showing the antimicrobial activity against the low molecular weight chitosan of Example 1 using fluid plasma.

As shown in FIG. 6, the MIC of the low-molecular chitosan (Example 1) prepared by treatment with fluid plasma for 15-120 minutes was measured, and it was found to be 80 μg/ml in E. coli , a representative gram-negative bacterium.

For S. aureus , the MIC of low-molecular chitosan prepared by fluid plasma treatment for 15 to 60 minutes was 160 µg/ml, and the MIC of low-molecular chitosan prepared by fluid plasma treatment for 90 to 120 minutes was 80 µg/ml. Confirmed.

Gram-positive bacteria are more resistant to antimicrobial agents than Gram-negative bacteria due to their thick cell walls. Nevertheless, it can be seen that the low-molecular chitosan prepared through the production method of the present invention has remarkably excellent antibacterial activity against not only Gram-negative bacteria but also Gram-positive bacteria.

The low-molecular chitosan prepared by the method of the present invention forms nanoaggregates, which have polycation characteristics. Therefore, the low-molecular chitosan prepared by the production method of the present invention interacts with the negatively charged surface of the bacterial cell in a short time (30 minutes) and eventually destroys the cell membrane and kills it. In addition, the amino groups of the low-molecular chitosan are stacked on the cell surface and bound to DNA, thereby inhibiting mRNA synthesis and interfering with bacterial metabolism, thereby exerting antibacterial activity.

It was confirmed that the antimicrobial activity of the low-molecular chitosan prepared by conventional microbial processes or chemical methods, etc., varies variably according to the synthesis method. In particular, in most cases, no antimicrobial activity was observed, and even if the antimicrobial activity was present, the level was remarkably low. Chitooligosaccharides are known to exhibit much weaker antibacterial activity than low molecular weight chitosan.

For C. albicans , the MIC of chitosan prepared by fluid plasma treatment for 15 to 30 minutes was measured to be 1,200 μg/ml. The MIC of the low-molecular chitosan prepared by fluid plasma treatment for 60 to 120 minutes was measured as 300 µg/ml and 150 µg/ml.

This is higher than the antifungal activity of high molecular weight chitosan, and it can be seen that it is 2 to 4 times higher than the previously reported antifungal activity of low molecular weight chitosan (MIC: 4.8 mg/ml). That is, the low molecular weight chitosan prepared according to the present invention can be said to be the most effective antibacterial agent.

Meanwhile, the low-molecular chitosan prepared through fluid plasma as in the present invention has a harmful effect on bacteria and fungi, and it can be seen that it can be used very effectively to kill bacterial pathogens and inhibit growth at a low concentration.

<Experimental Example 8. Swelling ratio of low-molecular chitosan using fluid plasma>

It was intended to compare the equilibrium swelling (ES) ratio of the low-molecular chitosan (Example 1) prepared according to the treatment time of the fluid plasma. ES ratio was calculated based on the water-soluble (WS) chitosan of Example 1.

7A is a graph showing an analysis of the equilibrium swelling ratio (ES ratio) for the low molecular weight chitosan of Example 1 using fluid plasma.

As shown in Fig. 7a, the equilibrium swelling ratio is dependent on the concentration and time of chitosan. It was confirmed that the low-molecular chitosan prepared through fluid plasma for 15 minutes can hold 2 mg/mg of water, which is the highest ES. The beam was confirmed. The low molecular weight chitosan prepared by treatment with fluid plasma for 30 minutes and 60 minutes was confirmed to have 0.9 mg/mg and 0.1 mg/mg, respectively. The low molecular weight chitosan prepared by treating fluid plasma for 90 minutes and 120 minutes was completely dissolved in water due to its high water solubility. The above-described equilibrium swelling ratio is an index indicating that it can be used as a biomaterial for sustainable treatment. Therefore, it can be seen that the low-molecular chitosan produced through the fluid plasma of the present invention also has excellent persistence, and thus can be used as a biomaterial for various treatments.

<Experimental Example 9. Antioxidant Activity of Low Molecular Chitosan Using Fluid Plasma>

In order to analyze the antioxidant activity of the low-molecular chitosan of Example 1 using fluid plasma, it was confirmed through DPPH analysis.

7B is a graph showing DPPH scavenging activity for the low-molecular chitosan of Example 1 using fluid plasma.

As shown in FIG. 7B, the polymer chitosan not treated with fluid plasma was only 51%, but the low molecular chitosan prepared by fluid plasma treatment showed a DPPH free radical scavenging activity of 58 to 75%. It was confirmed that the DPPH scavenging activity of the low-molecular chitosan prepared with fluid plasma (Example 1) was relatively faster than that of the conventional pharmaceutical compound (takes 30 to 300 minutes) (5 minutes). It is believed that this is because when the low molecular weight chitosan of the present invention is produced, fluid plasma is processed, thereby releasing sugar.

Ascorbic acid was used as a positive control of the present invention, and it was confirmed that the low-molecular chitosan of the present invention was significantly faster and exhibited higher antioxidant activity.

<Experimental Example 10. Comparison of the characteristics of known techniques and the present invention>

In order to compare the conventionally known low-molecular chitosan production technology and the low-molecular chitosan production technology of the present invention, each characteristic is summarized and shown in Table 1 below.

division Way fair
time Molecular weight of chitosan before and after (Da) Physiological activity Announcement Technology 1 Chemical: H ₂ O ₂ (30% H ₂ O ₂ , pH 3.9-4.0) 360 minutes 2.0 × 10 ⁵ to
9.0 × 10 ³ ND ^a Announcement Technology 2 Chemical:HCl (0.05 M, 65 ℃, pH 3.5) 1800 minutes 2.0 × 10 ⁵ to
7.3 × 10 ³ ND ^a Announcement Technology 3 Biological; pectinase(A. niger, 100 ℃, 10 min, pH 12) 360 minutes 9.0 × 10 ⁴ to
2.0 × 10 ⁴ ND ^a Announcement Technology 4 Biological;cellulase(1%, 50 ℃) 1440 minutes 5.1 × 10 ⁵ to
1.1 × 10 ³ ND ^a Announcement Technology 5 Physical;sonication(550 W, 40 kHz; 100% duty cycle) 30 minutes 6.6 × 10 ⁵ to
6.5 × 10 ⁴ ND ^a Announcement Technology 6 Physical;Microwave(600W, 70 ℃) 40 minutes 6.5 × 10 ⁵ to
4.1 × 10 ⁴ Antifungal activity
MIC:100-400 μg.mL ^-1 Announcement Technology 7 Physical;Plasma(Ozonation: 400W, N _2, O ₂ gases) 400 minutes 5.9 × 10 ⁴ to
3.3 × 10 ⁴ ND ^a The present invention Physical;Solution plasma process(800 V, 30 kHz under ambient conditions) 30 minutes 3.0 x 10 ⁵ to
7.8 x 10 ³ Antibacterial activity (MIC): 80-150㎍.㎖ ^-1
Antioxidant activity (DPPH): 58-75%
Swelling ratio: 0.2-2mg.mg ^-1 60 minutes 3.0 x 10 ⁵ to
5.8 x 10 ³ 90 minutes 3.0 x 10 ⁵ to
4.6 x 10 ³

a: ND, not determined

Known Technology 1: Chang, K.L., Tai, M.C & Chang, F.H. (2001). Kinetics and products of the degradation of chitosan by hydrogen peroxide. J.Agri.Food. Chem., 49, 4845-4851.

Known Technology 2: Kasaai, MR., Arul, J & Charlet G. (2013), Fragmentation of Chitosan by Acids. The Sci. World J., Article ID 508540, 11 pages. http://dx.doi.org/10.1155/2013/508540.

Announced Technology 3: Kittur, F.S., Kumar, A.B.V., & Tharanathan, R.N. (2003). Low molecular weight chitosan-preparation by depolymerization with Aspergillus niger pectinase, and charaterization. Carbohydr. Polym., 338: 1283-1290.

Known Technology 4: Xie, H., Jia, Z., Huang, J & Zhang, C. (2011). Preparation of low molecular weight chitosan by complex enzymes hydrolysis. Inter. J. Chem., 3(2), 180-186.

Known Technology 5: Yue, W., Yao, P., Wei, Y., & Mo, H. (2008). Synergetic effect of ozone and ultrasonic radiation on degradation of chitosan. Poly. Degrad. Stabili, 93, 1814-1821.

Known Technology 6: Li, K., Xinga, R., Liua, S., Qina, Y., Menga, Z. & Li, P. (2012). Microwave-assisted degradation of chitosan for a possible use in inhibiting crop pathogenic fungi. Inter. J. Biol. Macromol., 51, 767-773.

Known Technology 7: Lee, K & Song, K. (2014). Effect of plasma power on degradation of chitosan. Korean J. Chem. Eng., 31(1), 162-165.

It can be seen that the method for producing low-molecular chitosan of the present invention was performed in the shortest time among the prior art. Among the conventional techniques, chitosan (5.5 × 10 ⁵ Da) was reduced to 1.9 × 10 ⁴ Da when the bipolar power supply was treated for 300 minutes, and chitosan (7.4 × 10 ⁴ Da) was reduced to N ₂ and O ₂ Gas plasma was treated at a maximum of 400 W for 5 minutes to prepare a low molecular chitosan of 3.6 × 10 ⁴ Da.

When the above-described experimental results are summarized, it can be seen that the method for producing low-molecular chitosan of the present invention is faster, safer, and more effective than the conventional method for producing low-molecular chitosan. It was confirmed that the low-molecular chitosan production method of the present invention produced low-molecular chitosan (7.8 × 10 ³ Da) by depolymerizing high molecular chitosan (3.0 × 10 ⁵ Da) in a short time. The depolymerization rate was confirmed through the solubility and yield of water-soluble chitosan, and the size of the low-molecular chitosan prepared according to the present invention was about 44 ± 10.6 to 127 ± 26.2 nm, and it was confirmed that it has adequate stability.

Therefore, it can be seen that fluid plasma is an effective manufacturing method for preparing high molecular weight chitosan into low molecular weight chitosan, and it was confirmed that the low molecular weight chitosan prepared by the present invention has significantly improved therapeutic effect than that of high molecular weight chitosan. Furthermore, it can be seen that it has significantly better swelling ratio, water solubility in a wide acidity range, antibacterial activity, and antioxidant effects than those of the low-molecular chitosans prepared by the conventional manufacturing method. Specifically, the low-molecular chitosan prepared by the production method of the present invention has 0.2-2 mg/mg equilibrium swelling ratio (ES ratio), 58-75% radical scavenging activity. Therefore, the low molecular weight chitosan produced through the present invention can be used as a variety of nano-biomaterials having biomedical properties through the above-described characteristics because the size of the particles is predictable.

Claims (8)

1) preparing a chitosan solution by dissolving a polymer chitosan in an acetic acid solution; And
2) a fluid plasma reaction step of generating a plasma in the chitosan solution to generate low-molecular chitosan for 15 to 120 minutes; including,
The fluid plasma reaction step is a method of producing a low-molecular chitosan, characterized in that the voltage of a unipolar power source supplied to the electrode exposed to the chitosan solution is 700 to 900V and a frequency of 20 to 40 KHz. The method of claim 1,
After step 2) 3) filtering and drying the low-molecular chitosan after irradiation with fluid plasma. A method for producing low-molecular chitosan, further comprising: The method of claim 1,
The fluid plasma reaction step is a method for producing low molecular chitosan using a liquid plasma reaction, characterized in that the voltage of a unipolar power supply supplied to the tungsten electrode exposed to the chitosan solution is 800V and a frequency of 30 KHz. The method of claim 1,
The method for producing a low molecular weight chitosan, characterized in that the fluid plasma reaction step is 90 minutes. Low molecular weight chitosan prepared according to claim 1. The method of claim 5,
The low molecular weight chitosan has a molecular weight of 4.6 × 10 ³ Da to 7.8 × 10 ³ Da, an antimicrobial activity of 80 to 150 μg/ml, an antioxidant activity of 58 to 75%, and a swelling ratio of 0.2 to 2 mg/mg. Low molecular weight chitosan. The method of claim 5,
The low-molecular chitosan is a low-molecular chitosan, characterized in that 80 to 99% solubility in acidic conditions (pH <6) is maintained even in the neutral and alkaline range (pH> 7). An antimicrobial composition comprising a low-molecular chitosan prepared according to claim 1.

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