Description

HIGH-QUALITY WATER-SOLUBLE CHITOSAN OLIGOSACCHARIDE, COMPOSITION COMPRISING THE SAME AND METHOD FOR THE PREPARATION THEREOF

Technical Field

[1] The present invention relates to high-quality water-soluble chitosan oligosaccharide, a composition comprising the same and a method for the preparation thereof.

[2]

Background Art

[3] Chitin and chitosan are polysaccharides found in nature and have been highlighted as novel functional materials.

[4] Chitin is a polysaccharide composed of lots of λf-acetylglucosamine monomers

(β-1,4 linkage) wherein C-2 hydroxyl(-OH) group of glucopyranose in cellulose is substituted by λf-acetylamide(-NHCOCH ) group. Chitosan is prepared by the deacetylation of chitin.

[5] Chitosan is a very useful biomaterial derived from shellfishes such as shrimps and crabs, and its chemical name is (l→4)2-amino-2-deoxy-β-D-glucosamine. Chitosan has various bioactivities such as reduction of blood-cholesterol level, inhibition of fat intake, anticancer, immunity enhancement, antidiabetic, wound-healing and antibacterial activity. Particularly, chitosan is bound to mucous membranes of oral, nasal and intestinal cavities, and thereby accelerates drug absorption, and researches on the application thereof as an absorbefacient for protein drugs or nonabsorbable drugs in oral and various formulations have been extensively carried out. Further, there were records that chitosan had been taken as foods or medicines for the treatment of diseases in Korea, China and Japan from ancient times.

[6] In recent years, applications and effects of chitosan as various biomaterials and pharmaceutical materials have been identified. However, some problems of chitosan, such as high molecular weight and low solubility, should be solved for successful applications thereof. Most of chitosans derived from chitin has several millions of high molecular weight and low solubility in water or organic solvent. For this reason, chitosan faces difficulties in diverse applications. The broad molecular weight distribution of chitosan makes it difficult to obtain reproducibility of effects and efficacies in the bioactivities and the applications thereof.

[7] Additionally, because chitosan has very stable 1— »4 β-D-glucosamine linkages, processes of chemical separation method thereof are complicated and raise envi-

ronmental pollution. Separation of chitosan to low molecular weight or oligosaccharides using instrumental methods such as liquid chromatography or ion- exchange resin column increases processing costs and decreases unit productivity. Therefore, a novel method for the separation and purification of low molecular weight chitosan or chitosan oligosaccharide in a higher yield by simple physical methods is required.

[8] Up to now, many efforts have been devoted in order to solve the problems of chitosan as described above. Firstly, a method for increasing the water solubility of chitosan by producing chitosan salts using acids such as hydrochloric acid or acetic acid is used. Secondly, various methods for decomposing high molecular weight chitosan to chitosan oligosaccharide having low molecular weight are used. Further, methods for increasing the solubility of chitosan by chemical modification are diversely experimented. As a typical modified chitosan, glycol chitosan introduced with ethylene glycol, chitosan introduced with water-soluble polymer and the like are known.

[9] In the case of chitosan in salt form, the water-solubility thereof is increased.

However, it has high-molecular weight and thereby viscosity is high. Further, because of toxic acidic residues, it has difficulty in its application as biomaterials.

[10] In the case of low-molecular weight chitosan oligosaccharide obtained by enzymatic hydrolysis, acid hydrolysis, hydrolysis, oxidizing disintegration, etc., it has improved solubility and low viscosity in aqueous solution due to low molecular weight and thereby may be used for various applications as biomaterials. However, un- uniformity occurred in disintegration process leads to broad molecular weight distribution, and thereby raises several problems of reproducibility in practical applications. Further, in the case of chemically modified chitosan, it has a drawback of deterioration in the characteristics of chitosan itself.

[11] Related patents are surveyed as follows. Japanese Laid-open Patent Publication No. sho 54-148090 discloses a method for preparing low molecular weight chitosan having a predetermined range of molecular weight by treating chitosan obtained through deacetylation of natural chitin in 0.007 to 0.35% hydrogen peroxide solution of pH 6 to 12.

[12] Japanese Laid-open Patent Publication No. hei 01-256395 discloses a method for preparing low molecular weight chitosan by treating chitosan obtained through deacetylation of natural chitin by an enzyme, wherein enzyme reaction is carried out by continuously adding acid to a reaction system while controlling its addition rate and by keeping pH of the system at a substantially constant level in the range of 3 to 9. The method produces D-glucosamine-free low molecular weight chitosan having a molecular weight ranging from 340 to 50,000 Da.

[13] Japanese Laid-open Patent Publication No. hei 02-200196 discloses a method for preparing low molecular weight chitosan by treating chitosan with at least one of neutral protease and lipase having decomposing ability of chitosan.

[14] In these conventional methods, chitosan is decomposed by acid hydrolysis using hydrochloric acid, phosphoric acid, and nitric acid, etc.; hydrogen peroxide treatment; or enzyme treatment under acidic solution. Then, for the separation of oligosaccharide, methanol (CH OH) is repeatedly treated several times to separate into a soluble part and an insoluble part, and each oligosaccharide is separated by changing ethanol concentration, and subsequently treated by chromatography or ion-exchange resin column.

[15] However, low molecular weight chitosan or chitosan oligosaccharide in these methods is substantially obtained by acid hydrolysis, which requires long time, high costs and aftertreatment and raises toxic and environmental problems. In other words, the conventional methods have several drawbacks of long-time aftertreatment, high costs, environmental problems and low yields, resulting in very low productivity. Thus, the methods have some difficulty in industrial production of chitosan.

[16] Therefore, the inventors have conducted numerous studies in order to obtain chitosan oligosaccharide having low toxicity, high purity, and high solubility with a high yield. As a result, the inventors have identified that high-quality water-soluble chitosan oligosaccharide may be prepared by using a multi-step ultrafiltration membrane and have completed the present invention.

[17]

Disclosure of Invention Technical Problem

[18] An object of the present invention is to provide high-quality water-soluble chitosan oligosaccharide having an average molecular weight of 1,000 to 11,000 Da and a molecular weight distribution of 1 to 1.5.

[19] Another object of the present invention is to provide a method for the preparation of high-quality water-soluble chitosan oligosaccharide.

[20] Another object of the present invention is to provide a composition containing high- quality water-soluble chitosan oligosaccharide.

[21]

Technical Solution

[22] The present invention provides high-quality water-soluble chitosan oligosaccharide having an average molecular weight of 1,000 to 11,000 Da and a molecular weight distribution of 1 to 1.5.

[23] Further, the present invention provides a method for the preparation of high-quality water-soluble chitosan oligosaccharide.

[24] Additionally, the present invention provides a composition containing the high- quality water-soluble chitosan oligosaccharide.

[25]

[26] Hereinafter, the present invention will be described in detail.

[27] The present invention provides high-quality water-soluble chitosan oligosaccharide having an average molecular weight of 1,000 to 11,000 Da and a molecular weight distribution of 1 to 1.5, preferably an average molecular weight of 1,000 to 1,700 Da and a molecular weight distribution of 1.3 to 1.5, an average molecular weight of 2,500 to 3,500 Da and a molecular weight distribution of 1.2 to 1.3, an average molecular weight of 5,500 to 6,700 Da and a molecular weight distribution of 1.15 to 1.2, and an average molecular weight of 8,500 to 11,000 Da and a molecular weight distribution of 1.0 to 1.15.

[28] More preferably, high-quality water-soluble chitosan oligosaccharide according to the present invention has an intrinsic viscosity of 0.020 to 0.250 g/D-sec, a degree of deacetylation of 90 to 95%, a moisture content of 1.0 to 3.0%, and a inorganic content of O to l%.

[29]

[30] The molecular weight and its distribution may be determined by gel permeation chromatography coupled with light scattering detector, and the molecular weight of high-quality water-soluble chitosan oligosaccharide according to the present invention is in the range of 1,000 to 11,000 Da. For reference, it is known that the molecular weight to be absorbed into the human body is below 10,000 Da. Further, a molecular weight distribution closer to 1 means that the chitosan oligosaccharide is successfully separated, and has a uniform range of molecular weight. Accordingly, the molecular weight distribution of high-quality water-soluble chitosan oligosaccharide ranging from 1 to 1.5 may be thought to be uniform in molecular weight.

[31] Further, the intrinsic viscosity of high-quality water-soluble chitosan oligosaccharide according to the present invention is in the range of 0.020 to 0.250 g/ D -sec, which may be determined by a rheometer. In consideration of the relationship with molecular weight, viscosity increases as molecular weight increases. Mark- Houwink constant is calculated by Mark-Houwink equation using molecular weight and intrinsic viscosity, and molecular weight may be obtained by intrinsic viscosity.

[32] Further, the degree of deacetylation of high-quality water-soluble chitosan oligosaccharide according to the present invention is high in the range of 90 to 95%, which may be determined by first derivative UV spectroscopic method. The degree of deacetylation is a criterion for the purity of chitosan. Chitin is converted into chitosan by deacetylation, and thus many kinds of chitosans may be obtained according to the conditions of deacetylation. High degree of deacetylation means that the degree of

conversion from chitin into chitosan is high, and that the properties of chitosan are more strongly expressed than those of chitin. Because the expressed bioactivities are almost derived from chitosan, not from chitin, it can be estimated that the higher the conversion rate from chitin into chitosan is, that is, the higher the degree of deacetylation is, the higher the expression of the bioactivities is.

[33] Further, the moisture content and the inorganic content of high-quality water- soluble chitosan oligosaccharide are in the ranges of 1 to 3% and 0 to 1% respectively, which may be determined by a thermogravimetric analyzer. The components of shellfishes such as calcareous compounds, heavy metals, proteins, etc., may be contained as impurities in chitosan oligosaccharide. Accordingly, it may be estimated that the lower the content of the impurities is, the higher the purity of chitosan is. In this respect, high-quality water-soluble chitosan oligosaccharide according to the present invention having low inorganic and moisture contents may be estimated to have high purity.

[34]

[35] Additionally, the present invention provides a method for the preparation of high- quality water-soluble chitosan oligosaccharide, comprising the steps of:

[36] (a) separating water-soluble chitosan oligosaccharide according to molecular weight using ultrafiltration membranes;

[37] (b) removing impurities contained each water-soluble chitosan oligosaccharide obtained, and then freeze-drying;

[38] (c) precipitating the freeze-dried water-soluble chitosan oligosaccharide in an organic solvent, to give powdered water-soluble chitosan oligosaccharide; and

[39] (d) vacuum-drying the powdered water-soluble chitosan oligosaccharide.

[40]

[41] Firstly, in the step (a), chitosan oligosaccharide is separated according to the molecular weight thereof, using ultrafiltration membranes. Ultrafiltration is a filtration having filtering effect between microfilration and reverse osmosis, and separates low molecular weight materials from a solution of high molecular weight materials. An ultrafiltration membrane may be prepared from cellulose, polyacrilonitrile copolymer, polyamide, polysulfone, polyimide, and polyvinylidenefluoride, etc., and have a pore size of 5 to 100 D. Ultrafiltration is useful for the separation of solute having a molecular weight of 1,000 to 5,000 Da, and may be applied for the separation of solute and solvent having above 100 times differences in the molecular weights therebetween.

[42] Water-soluble chitosan oligosaccharide having high molecular weight used in the preparation of high-quality water-soluble chitosan oligosaccharide according to the present invention may be obtained by conventional methods such as enzymatic hydrolysis (Korean Patent No. 291308), and commercialized reagents may also be

used. The step (a) may particularly comprise the following sub-steps of:

[43] (1) separating a filtrate containing water-soluble chitosan oligosaccharide having a molecular weight below 30 kDa from high molecular weight water-soluble chitosan oligosaccharide, using a 30 kDa ultrafiltration membrane;

[44] (2) separating the filtrate containing water-soluble chitosan oligosaccharide having a molecular weight below 30 kDa into water-soluble chitosan oligosaccharide having a molecular weight of 10 to 30 kDa and a filtrate containing water-soluble chitosan oligosaccharide having a molecular weight below 10 kDa, using a 10 kDa ultrafiltration membrane;

[45] (3) separating the filtrate containing water-soluble chitosan oligosaccharide having a molecular weight below 10 kDa into water-soluble chitosan oligosaccharide with a molecular weight of 3 to 10 kDa and a filtrate containing water-soluble chitosan oligosaccharide with a molecular weight below 3 kDa, using a 3 kDa ultrafiltration membrane;

[46] 4) separating the filtrate containing water-soluble chitosan oligosaccharide with a molecular weight below 3 kDa into water-soluble chitosan oligosaccharide with a molecular weight of 1 to 3 kDa and a filtrate containing water-soluble chitosan oligosaccharide with a molecular weight below 1 kDa, using 1 kDa ultrafiltration membrane; and

[47] (5) concentrating the filtrates of the water-soluble chitosan oligosaccharides separated according to molecular weight.

[48] Firstly, an aqueous solution of high molecular weight water-soluble chitosan oligosaccharide is prepared and then passed through a 30 kDa ultrafiltration membrane to remove components having a molecular weight above 30 kDa. The filtrate containing water-soluble chitosan oligosaccharide with a molecular weight below 30 kDa is passed through a 10 kDa ultrafiltration membrane to separate into water-soluble chitosan oligosaccharide having a molecular weight of 10 to 30 kDa and a filtrate containing water-soluble chitosan oligosaccharide with a molecular weight below 10 kDa. The 10 to 30 kDa water-soluble chitosan oligosaccharide is recovered and freeze- dried. Further, the filtrate containing water-soluble chitosan oligosaccharide with a molecular weight below 10 kDa is passed through a 3 kDa ultrafiltration membrane to separate into water-soluble chitosan oligosaccharide with a molecular weight of 3 to 10 kDa and a filtrate containing water-soluble chitosan oligosaccharide with a molecular weight below 3 kDa. The 3 to 10 kDa water-soluble chitosan oligosaccharide is recovered and freeze-dried. Finally, the filtrate containing water-soluble chitosan oligosaccharide with a molecular weight below 3 kDa is passed through a 1 kDa ultrafiltration membrane to separate into water-soluble chitosan oligosaccharide having a molecular weight of 1 to 3 kDa and a filtrate containing water-soluble chitosan

oligosaccharide with a molecular weight below 1 kDa. In the present invention, the ultrafiltration may preferably be performed in an ultrafiltration reactor using a cellulose ultrafiltration membrane. Steps of concentration and the ultrafiltration may preferably be repeated 3 times.

[49] In the step (b), impurities in each water-soluble chitosan oligosaccharide separated in the step (a) are removed, and then freeze-drying is performed. In detail, the solution of water-soluble chitosan oligosaccharide is passed through a filter, preferably a 0.8 D filter to remove impurities and then freeze-dried. Freeze-drying is preferably performed at -40°C for 24 hrs.

[50] Subsequently, in the step (c), the water-soluble chitosan oligosaccharide separated according to molecular weight and freeze-dried in the step (b) is precipitated in an organic solvent, to give powdered water-soluble chitosan oligosaccharide. The organic solvent used in this step is a mixed solvent of acetone and ether, preferably in the ratio of 50:50 by volume.

[51] In the step (d), the powdered water-soluble chitosan oligosaccharide obtained in the step (c) is vacuum-dried. Vacuum-drying is preferably performed at 10 to 13 torr for 48 hrs.

[52] The above-prepared high-quality water-soluble chitosan oligosaccharide has an average molecular weight of 1,000 to 11,000 Da, a molecular weight distribution of 1 to 1.5, an intrinsic viscosity of 0.020 to 0.250 g/D-sec, a degree of deacetylation of 90 to 95%, a moisture content of 1.0 to 3.0%, and a inorganic content of 0 to 1%. These values indicate that water-soluble chitosan oligosaccharide is successfully separated according to molecular weight from high molecular weight water-soluble chitosan oligosaccharide, and the purity thereof is high.

[53] Therefore, the high-quality water-soluble chitosan oligosaccharide according to the present invention may be usefully applied in various fields, for example pharmaceutical applications or functional health foods.

[54]

[55] The present invention further provides a composition including high-quality water- soluble chitosan oligosaccharide.

[56] A composition according to the present invention may further comprise one or more active substances having the same or similar function to that of high-quality water- soluble chitosan oligosaccharide.

[57] A composition according to the present invention may be prepared by additionally comprising one or more pharmaceutically acceptable carriers suitable for administration. A pharmaceutically acceptable carrier may be saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and their mixture. Other conventional additives such as antioxidant, buffer

solution, bacteriostat, etc. may be added, if necessary.

[58] Further, a composition may be formulated into pills, capsules, granules, tablets or injection formulations such as aqueous solution, suspension, emulsion, etc. by additionally comprising diluents, dispersing agents, surfactants, binders and lubricants. Furthermore, a composition may preferably be formulated according to diseases or components by appropriate methods known in the art or methods disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18 , 1990).

[59] A composition according to the present invention may be parenterally administrated

(for example, in the form of intravenous, subcutaneous, intraperitoneal or topical application) or orally administrated. A dosage may be controlled according to the patient's condition such as weight, age, sex, health condition, diet, administration time, administration route, excretion ratio, and disease severity, etc. Preferably, a daily dosage may be in the range from 0.01 to 100 D/D, which may be administrated at one time or divided into several doses.

[60] A composition according to the present invention may be a functional health food.

[61] In the case that high-quality water-soluble chitosan oligosaccharide according to the present invention is used as food additives, it may be added alone or in an admixture with other food or food ingredients by conventional methods. The amount of effective ingredients to be mixed may be properly controlled according to its purpose (prevention, health or curative treatment). In the case of formulations such as foods or beverages, high-quality water-soluble chitosan oligosaccharide according to the present invention may be added in an amount below 100 parts by weight, preferably below 50 parts by weight based the total raw material. However, in the case of long- term intake for the improvement of health and sanitation or for the regulation related to health, the added amount may be below this range. Because of no problem in safety, the effective ingredient may be used in the amount above this range.

[62] Foods are not limited to special kinds. Foods, to which chitosan oligosaccharide may be added, are meat, sausage, bread, chocolate, candies, snacks, cookies, pizza, instant noodles, other noodles, chewing gums, dairy products such as ice creams, various kinds of soup, drinking water, tea, drink products, alcoholic beverage and vitamin complex, etc. including all kinds of health foods in conventional means.

[63] Health-improving beverage composition according to the present invention may additionally comprise various flavorings or natural carbohydrates. Natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; and polysaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol and erythritol, etc. As a flavoring, natural flavorings such as thaumatin and stevia extracts, or synthetic flavorings such as saccharin and aspartame

may be used.

[64] In addition, a composition according to the present invention may further comprise various kinds of nutritional supplements, vitamins, electrolytes, flavorings, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective- colloid thickners, pH regulators, stabilizers, preservatives, glycerin, alcohols and carbonating agents used in carbonated beverages, etc. Further, a composition according to the present invention may comprise fruit flesh used for natural fruit juices, fruit juice drinks and vegetable drinks. These ingredients may be used alone or in combination thereof. The ratio of these additives may not be important. However it may generally be selected to be in the range of 0.01 to 0.1 parts by weight to 100 parts by weight of the composition.

Advantageous Effects

[65] The high-quality water-soluble chitosan oligosaccharide according to the present invention has an average molecular weight of 1,000 to 11,000 Da, a molecular weight distribution of 1 to 1.5, an intrinsic viscosity of 0.020 to 0.250 g/D-sec, a degree of deacetylation of 90 to 95%, a moisture content of 1.0 to 3.0%, and a inorganic content of O to l%.

[66] High-quality water-soluble chitosan oligosaccharide according to the present invention separated according to the molecular weight thereof using ultrafiltration membranes has very narrow molecular weight distribution and high purity, and exhibits little cytotoxicity, thereby being usefully applied in various fields, for example pharmaceutical applications or functional health foods.

[67]

Brief Description of the Drawings

[68] FIG. 1 is a NMR spectrum for fraction 4 passed through an 1 kDa ultrafiltration membrane.

[69] FIG. 2 is a NMR spectrum for fraction 3 passed through a 3 kDa ultrafiltration membrane and not passed through an 1 kDa ultrafiltration membrane.

[70] FIG. 3 is a NMR spectrum for fraction 2 passed through a 10 kDa ultrafiltration membrane and not passed through a 3 kDa ultrafiltration membrane.

[71] FIG. 4 is a NMR spectrum for fraction 1 passed through a 30 kDa ultrafiltration membrane and not passed through a 10 kDa ultrafiltration membrane.

[72] FIG. 5 is a GPC chromatogram for high-quality water-soluble chitosan oligosaccharide according to the present invention.

[73] FIG. 6 shows the intrinsic viscosity of fraction 4 of high-quality water-soluble chitosan oligosaccharide according to the present invention.

[74] FIG. 7 shows the intrinsic viscosity of fraction 3 of high-quality water-soluble

chitosan oligosaccharide according to the present invention. [75] FlG. 8 shows the intrinsic viscosity of fraction 2 of high-quality water-soluble chitosan oligosaccharide according to the present invention. [76] FlG. 9 shows the intrinsic viscosity of fraction 1 of high-quality water-soluble chitosan oligosaccharide according to the present invention. [77] FlG. 10 shows the relationship between molecular weight and intrinsic viscosity of high-quality water-soluble chitosan oligosaccharide according to the present invention. [78] FlG. 11 is a TGA diagram for fraction 4 of high-quality water-soluble chitosan oligosaccharide according to the present invention. [79] FlG. 12 is a TGA diagram for fraction 3 of high-quality water-soluble chitosan oligosaccharide according to the present invention. [80] FlG. 13 is a TGA diagram for fraction 2 of high-quality water-soluble chitosan oligosaccharide according to the present invention. [81] FlG. 14 is a TGA diagram for fraction 1 of high-quality water-soluble chitosan oligosaccharide according to the present invention. [82] FlG. 15 shows the result of cytotoxicity assay of high-quality water-soluble chitosan oligosaccharide according to the present invention. [83]

Best Mode for Carrying Out the Invention [84] The present invention will be described in more detail with reference to the following exemplary embodiments. However, these examples are provided for the purpose of illustration only and should not be construed as limiting the scope of the invention, which will be apparent to one skilled in the art. [85] [86] <Example 1> Preparation of high-quality water-soluble chitosan oligosaccharide according to the present invention [87] 1-1. Preparation of chitosan oligosaccharide

[88] For the preparation of high-quality water-soluble chitosan oligosaccharide according to the present invention, high molecular weight water-soluble chitosan oligosaccharide obtained from high molecular weight chitosan (Korean Patent No.

291308) was used. [89] Firstly, 2,800 £ of distilled water and 150 D of high molecular weight chitosan were poured into 3 ton-enzyme reactor equipped with an agitator. The mixture was agitated at 40°C, 150 rpm for 2 hrs to be swollen and then was adjusted to pH 5.0 with lactic acid (Aldrich, USA). [90] To this solution, a solution of 300,000 units of chitosanase (endo-type chitosanase derived from Bacillus sp. strain, Mw 45,000 Da, Amicogen, Inc., Korea) in 500 D of

distilled water was added and then stood for 30 min. Then, hydrolysis was performed for 36 hrs under agitation at 120 rpm.

[91] Subsequently, the solution was passed through a 1 D prefilter and 0.7 D zetafilter to remove unreacted materials, and then concentrated.

[92] Next, the concentrate was sterilized by high temperature short time method(HTST) and spray-dried to give high molecular weight water-soluble chitosan oligosaccharide.

[93] The product was dissolved in distilled water to 75 g/£, and stored at below 4 °C before being used in the following example 1-2.

[94]

[95] 1-2. Preparation of high-quality water-soluble chitosan oligosaccharide according to the present invention

[96] For the preparation of high-quality water-soluble chitosan oligosaccharide having various molecular weights and narrow molecular weight distribution, the high molecular weight water-soluble chitosan oligosaccharide obtained in Example 1-1 was used.

[97] Firstly, high molecular weight chitosan oligosaccharide and chitosanase (Mw

45,000 Da) were removed using a 30 kDa ultrafiltration membrane.

[98] Subsequently, the chitosan oligosaccharide solution passed through a 30 kDa ultrafiltration membrane was separated according to molecular size using a 10 kDa ultrafiltration membrane. The fraction 1 of chitosan oligosaccharide having an average molecular weight of 8,500 to 11,000 Da was obtained by separating the filtrate passed through a 30 kDa membrane and not passed through a 10 kDa membrane.

[99] Subsequently, the fraction 2 of water-soluble chitosan oligosaccharide having an average molecular weight of 5,500 to 6,700 Da, the fraction 3 having an average molecular weight of 2,500 to 3,500 Da and the fraction 4 having an average molecular weight of 1,000 to 1,700 Da were obtained by using 3 kDa and 1 kDa ultrafiltration membranes.

[100] In the above steps, the ultrafiltration was performed in an ultrafiltration reactor

(Amicon 8400, USA) equipped with a cellulose ultrafiltration membrane (Millipore, USA). 350 D of the chitosan oligosaccharide solution was added to the ultrafiltration reactor, and nitrogen was supplied(supply pressure : 3.5 Df/D) to concentrate to 1/7 of the initial volume. The ultrafiltration membranes used in these steps were those having MWCO (molecular weight cut-off) of 30 kDa (YM30), 10 kDa (YMlO), 3 kDa (YM3) and 1 kDa (YMl) respectively.

[101] Each concentrated high-molecular- weight chitosan oligosaccharide was dissolved again in distilled water to be 350 D, and then concentrated in the same procedure as described above. These procedures were repeated 3 times.

[102] Final high-quality chitosan oligosaccharide solution was obtained from the ultra-

filtration reactor. The solution was passed through a 0.8 D filter to remove impurities formed in the separation process and then freeze-dried at -40°C, for 24 hrs.

[103] The freeze-dried chitosan oligosaccharide was dissolved again in distilled water and then precipitated into a mixed solvent of acetone/ether (50:50 in volume ratio). The powdered chitosan oligosaccharide obtained was vacuum-dried (pressure: 10 - 13 torr) for 48 hrs, to give final high-quality water-soluble chitosan oligosaccharide powder.

[104]

[105] 1-3. Analysis of high-quality water-soluble chitosan oligosaccharide according to the present invention

[106] Structural analysis of high-quality water-soluble chitosan oligosaccharide separated according to molecular weight in the above Example 1-2 was performed by a nuclear magnetic resonance equipment (400 mHz H-NMR,Bruker, Germany).

[107] Absolute molecular weight and molecular- weight distribution thereof were determined by a GPC-MALS (gel permeation chromatography-multi angle light scatteringXDAWN DSP and OPTILAB DSP, Wyatt, USA).

[108] Solution viscosity and intrinsic viscosity of high-quality water-soluble chitosan oligosaccharide dissolved in a mixed solvent (0.1M acetic acid and 0.2M NaCl) were determined by a rheometer (Bohlin Instruments, UK), and the relationship between viscosity and molecular weight was calculated. Mark-Houwink constants K and a were calculated by the following Math Figure 1, Mark-Houwink equation, using absolute molecular weight and intrinsic viscosity.

[109]

[110] MathFigure l

Mark-Houwinkequation: [x\]=KMa

[111] (Wherein, [η] is intrinsic viscosity, M is molecular weight, K and a are constants for polymer solution.)

[112]

[113] Degree of deacetylation was determined by UV first order differential calculus using a UV spectrophotometer (UV 1601, Shimadzu, Japan).

[114] The moisture content and the inorganic content of the high-quality water-soluble chitosan oligosaccharide were determined by a TGA (thermogravimetric analyzer)(TGA951, TA Instruments, USA). The determination was performed while heating up to 800 °C, at a heating rate of 10 °C/min, in air flow of 50 D/min.

[115] The results were shown in Tables 1 and 2, and Figs. 1 to 14.

[116] As shown in Figs. 1 to 4, the results of ¹ H-NMR were analyzed and all the peak characteristics of chitosan were checked. It was identified that high-purity chitosan was obtained without the decomposition thereof in enzymatic hydrolysis, ultrafiltration and

purification procedures.

[117] [118] Table 1 Characteristics of high-quality water-soluble chitosan oligosaccharide

[119] [120] As shown in Table 1 and Fig. 5,it is identified by the average molecular weight that water-soluble chitosan oligosaccharide was separated according to molecular weight using ultrafilration membranes. Additionally, it was identified by the low average molecular weight distribution that the separation was successfully achieved.

[121] As shown in Table 1 and Figs. 6 to 9, the intrinsic viscosity of the high-quality water-soluble chitosan oligosaccharide separated increases as the molecular weight thereof increased. This result accorded with the theory that intrinsic viscosity increased as molecular weight increased.

[122] Additionally, as shown in Table 1, the degree of deacetylation was very high in the range of 90 to 95%, which meant that high-quality water-soluble chitosan oligosaccharide was obtained.

[123] [124] Table 2

Viscosity of high-quality water-soluble chitosan oligosaccharide solution according to the concentrations thereof

[125] [126] As shown in Table 2, the viscosity of high-quality water-soluble chitosan oligosaccharide solution increased as the concentration thereof increased.

[127] As shown in Fig. 10, Mark-Houwink constants K and a, corresponding to the inclination and intersection point of the axis respectively, were able to obtain by log-log fitting molecular weight values and intrinsic viscosity values. K and a values, for the high-quality water-soluble chitosan oligosaccharide in a mixed solvent of 0.1M acetic acid and 0.2M NaCl, were 4.2446x10 and 1.1754 respectively. The molecular weights of chitosan, which were neither known nor measured, were able to calculate using these constant values and the intrinsic viscosity thereof.

[128] Additionally, as shown in Table 1 and Figs. 11 to 14, the high-quality water-soluble chitosan oligosaccharide contained 1 to 3% moisture and trace amount of ash, indicating that the amount of impurities was very low.

[129] Consequently, it was identified from the above results that the high-quality water- soluble chitosan oligosaccharide prepared in the example embodiment was successfully separated according to molecular weight and the purity thereof was very high.

[130] [131] Experimental Example 1> Cytotoxicity and cell proliferation assays for high- quality water-soluble chitosan oligosaccharide according to the present invention

[132] For the application of high-quality water-soluble chitosan oligosaccharide according to the present invention to living matters and pharmaceutical materials, the effects on cytotoxicity and cell proliferation were investigated.

[133] The cytotoxicity of the high-quality water-soluble chitosan oligosaccharide according to its molecular weight was investigated by MTT assay. [134] Caco-2 (human colon cancer cell lines) cells were plated in 96- well microplates at density of about 1x10 cells per well, and incubated in DMEM supplemented with 10% FBS, at 37 °C, for 24 hrs to be firmly attached onto the plates.

[135] Meanwhile, each fraction of high-quality water-soluble chitosan oligosaccharide obtained in the above example embodiment was dissolved in PBS 7.4 solution containing 10% FBS to prepare diverse concentrations (6.25, 12.5, 25, 50, 100 and 200 D/D).

[136] After 24 hr incubation, cell culture medium in the plates was removed. Then, the high-quality water-soluble chitosan oligosaccharide solution was added into the plates

and cultures were maintained in an incubator for 2 hrs.

[137] After 2 hr incubation, the chitosan solution was removed. The cells were washed with physiological saline solution and further stabilized for 4 hrs in a fresh culture medium. Subsequently the cells were incubated for 4 hrs in the incubator with cell culture medium containing 0.5 D/D MTT.

[138] After 4 hr incubation, the cell culture medium was removed. The formazan crystals formed by the activity of mitochondria in a cell were dissolved with 100 D DMSO (dimethylsulfoxide).

[139] Relative cell viability was calculated by the following Math Figure 2 using the optical density at 570 D(OD ) of the solution in DMSO. A group containing only cell- free medium was used as a negative control group, and another group without treatment of high-quality water-soluble chitosan oligosaccharide was used as a positive control group.

[140] [141] MathFϊgure 2

Relative cell viability = [ (OD ₅₇₀ of the test group - OD ₅₇₀ of the negative control group) / (OD ₅₇₀ of the positive control group - OD ₅₇₀ of the negative control group) ]

[142] [143] The results were shown in Table 3 and Fig. 15. [144] [145] Table 3

Cell viability affected by treatment of high-quality water-soluble chitosan oligosaccharide according to the concentrations thereof

[146] [147] As shown in Table 3 and Fig. 15, when the initial cell count was 1, it was identified that the fractions 4 and 3 had no cytotoxicity regardless the concentration thereof.

Further, the fractions 2 and 1 exhibited little cytotoxicity in low concentrations, but the cell viability decreased as the concentration thereof increased. The fraction 2 showed cell viability of 0.75 at high concentration, and the fraction 1 showed cell viability of 0.54 at considerably high concentration of 100 D/D.

[148] Consequently, it was identified from the above results that chitosan oligosaccharide exhibited high cytotoxicity as the molecular weight thereof increased. Nevertheless, considering that toxicity is negligible when cell viabilityis above 0.5, the high-quality water-soluble chitosan oligosaccharide prepared may be estimated to have no cytotoxicity. Therefore, it was identified that high-quality water-soluble chitosan oligosaccharide according to the present invention might be usefully applied in various fields, for example pharmaceutical applications or functional health foods.

[149]

[150] Hereinafter, formulation examples of the composition according to the present invention are described.

[151]

[152] <Formulation Example 1> Pharmaceutical formulations

[153] 1-1. Preparation of powder

[154] High quality water-soluble chitosan oligosaccharide according to the present invention 2 g

[155] Lactose 1 g

[156]

[157] Powder product was prepared by mixing the above ingredients and filling an airtight package therewith.

[158]

[159] 1-2. Preparation of tablet

[160] High quality water-soluble chitosan oligosaccharide according to the present invention 100 D

[161] Corn starch 100 D

[162] Lactose 100 D

[163] Magnesium stearate 2 D

[164]

[165] Tablets were prepared by mixing the above ingredients and tabletting by a conventional method.

[166]

[ 167] 1-3. Preparation of capsule

[168] High quality water-soluble chitosan oligosaccharide according to the present invention 100 D

[169] Corn starch 100 D

[170] Lactose 100 D

[171] Magnesium stearate 2 D

[172]

[173] Capsules were prepared by mixing the above ingredients and filling a gelatin capsule by a conventional method.

[174]

[ 175] <Formulation Example 2> Foods

[176] Foods including the high-quality water-soluble chitosan oligosaccharide according to the present invention were prepared as follows.

[177] 2-1. Preparation of cooking spices

[178] Health-improving cooking spices were prepared by adding high-quality water- soluble chitosan oligosaccharide according to the present invention in an amount of 0.2 to 10 parts by weight.

[179]

[ 180] 2-2. Preparation of tomato ketchup or sauce

[181] Health-improving tomato ketchup or sauce was prepared by adding high-quality water-soluble chitosan oligosaccharide according to the present invention in an amount of 0.2 to 1.0 parts by weight to tomato ketchup or sauce.

[182]

[183] 2-3. Preparation of flour foods

[184] Health-improving flour foods were prepared by adding high-quality water-soluble chitosan oligosaccharide according to the present invention in an amount of 0.1 to 5.0 parts by weight to flour and then making breads, cakes, cookies, crackers or noodles with the flour.

[185]

[ 186] 2-4. Preparation of soup and gravies

[187] Health-improving meat products, noodle soup or gravies was prepared by adding high-quality water-soluble chitosan oligosaccharide according to the present invention in an amount of 0.1 to 1.0 parts by weight to meat products, soup or gravies.

[188]

[ 189] 2-5. Preparation of ground beef

[190] Health-improving ground beef was prepared by adding high-quality water-soluble chitosan oligosaccharide according to the present invention in an amount of 10 parts by weight of to ground beef.

[191]

[192] 2-6. Preparation of dairy products

[193] Health-improving dairy products were prepared by adding high-quality water- soluble chitosan oligosaccharide according to the present invention in an amount of 0.1

to 1.0 parts by weight to milk and then making butter or ice creams from the milk.

[194]

[195] 2-7. Preparation of mixed cereal food [196] Brown rice, barley, glutinous rice and jobs tears were alpha-transformed by a conventional method and dried. Then, the mixture was parched and pulverized to 60 mesh powder.

[197] Black soybean, black sesame and wild sesame were steamed by a conventional method and dried. Then, the mixture was parched and pulverized to 60 mesh powder. [198] The high-quality water-soluble chitosan oligosaccharide according to the present invention was condensed under reduced pressure in a vacuum concentrator and then dried with a spray hot air dryer. The dried product was pulverized to 60 mesh powder.

[199] Grain powders, seed powders and high-quality water-soluble chitosan oligosaccharide powder according to the present invention were mixed in the following Table 4.

[200] [201] Table 4

[202] [203] <Formulation Example 3> Beverages [204] 3-1. Preparation of carbonated beverages [205] 5 to 10% of sucrose, 0.05 to 0.3% of citric acid, 0.005 to 0.02% of caramel and 0.1 to 1% of vitamin C were mixed, and 79 to 94% of distilled water was added thereto to give syrup. The syrup was sterilized at 85 to 98°C, for 20 to 180 sec. and then mixed with cooled water in the ratio of 1 :4. 0.5 to 0.82% of carbonated gas was injected thereto, to give a carbonated beverage including high-quality water-soluble chitosan oligosaccharide according to the present invention.

[206]

[207] 3-2. Preparation of health-improving beverages

[208] High-quality water-soluble chitosan oligosaccharide according to the present invention and subsidiary ingredients such as liquid fructose (0.5%), oligosaccharide (2%), sucrose (2%), salt (0.5%) and water (75%) were uniformly mixed, and then pasteurized. The pasteurized product was packed in small containers such as glass bottles, pet bottles, etc., to give a health-improving beverage.

[209]

[210] 3-3. Preparation of vegetable juice

[211] Health-improving vegetable juice was prepared by adding 0.5 g of high-quality water-soluble chitosan oligosaccharide according to the present invention to 1,000 D tomato or carrot juice.

[212]

[213] 3-4. Preparation of fruit juice

[214] Health-improving fruit juice was prepared by adding 0.1 g of high-quality water- soluble chitosan oligosaccharide according to the present invention to 1,000 D apple or grape juice.

[215]

[216] Although the invention has been described in detail herein, it should be understood that the invention is not limited to the embodiments herein disclosed. Various changes, substitutions and modifications may be made thereto by those skilled in the art without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

[217]

Industrial Applicability

[218] High-quality water-soluble chitosan oligosaccharide according to the present invention separated according to the molecular weight thereof using ultrafiltration membranes has very narrow molecular weight distribution and high purity, and exhibits little cytotoxicity, thereby being usefully applied in various fields, for example pharmaceutical applications or functional health foods.