KR100321932B1 - Prevention and treatment medicine of mammitis of a milky cow containing the effective component of chitosan and chitosan oligomar. - Google Patents

Abstract

PURPOSE: Provided is a prophylactic and therapeutic agent containing chitosan and chitosan-oligosaccharide as an active ingredient for mastitis in a milch cow. CONSTITUTION: A prophylactic and therapeutic agent containing chitosan and chitosan-oligosaccharide as an active ingredient is characterized by adding 0.5-0.0001 wt.% of chitosan-oligosaccharide having 1-16 glycones(66.3-97.5%), acetylation rate of 75-100%, and molecular weight of 104-2310 to a livestock feed. It is dissolved in water and orally administered into a milch cow.

Description

{Prevention and treatment medicine of mammitis of a milky cow containing the effective component of chitosan and chitosan oligomar.}

This patent relates to a mastitis treatment of cows using chitosan or chitosan oligosaccharide as an active ingredient.

Chitin, chitosan manufacturing part

Chitin has a chemical formula of those in which the C-2 hydroxyl group of the glucose residue of cellulose is substituted in the acetylamino group, and the deacetylate of chitin is called chitosan, but in reality, glucosamine residues as binding points with biological substances such as proteins And the chemical formula of chitin and chitosan is similar to cellulose. Chitin is widely abundant in nature, especially complexes with proteins in invertebrates and fungi, and similar to the cellulose present in the plant system to form the exoskeleton and the shell of the organism. These chitosans are non-toxic, natural macromolecular cations that are involved in the synthesis and degradation of organisms but do not cause environmental pollution. In recent years, research on them as a potential resource for use has shown that environmental wastewater flocculants, heavy metal adsorbents, and seeds for increased yields. Research is being actively conducted on coatings, protein recovery agents, and wound healing agents. Therefore, the study on the application of chitin, which has limited solubility in most solvents except for special solvents, to prepare chitin as chitosan has been made in various ways to control the degree of deacetylation. As the relationship between the purity and the molecular weight is reversed, there is a limit to the method of supplementing and controlling it freely.

Mastitis Treatment Part

The increase in national income due to the continuous growth of the national economy has brought a big change in the consumption of livestock foods, including the rapid increase in the consumption of milk and dairy products in relation to the quantitative and qualitative improvement of dietary standards. Korea's milk supply and demand shows a huge increase in per capita milk consumption: 0.1kg in 1962, 1.6kg in 1970, 11kg in 1980, and 43kg in 1990. This increase in milk consumption has led to an increase in the proportion and role of milk in food, which has increased the desire of consumers to be hygienic, safe, and higher quality milk. In addition, it is essential to increase price competitiveness by improving productivity of dairy farmers to actively cope with import and opening of dairy products and to maintain continuous growth.

The quality of crude oil is determined not only by nutritional ingredients such as milk fat and protein, but also by factors related to the health of the cow's breast, such as the total bacterial count in milk and somatic cell count. The milk of cows infected with mastitis not only contains bacteria but also dramatically changes in milk components such as an increase in somatic cell counts, thereby significantly reducing the quality of milk. Therefore, it is essential to prevent and treat mastitis, which is one of the most frequently encountered diseases in cows, in order to improve the quality of milk in both nutrition and hygiene.

Mastitis of cows is an inflammation of the mammary gland tissue caused by various microbial infections that are widely distributed in the natural world. It acts as an infectious agent for healthy cows as well as reducing acid flow and lowering oil quality. It causes huge economic loss, including malfunction and death. In addition, economic losses due to mastitis, such as cow culling, treatment costs, reduced sales value, increased labor costs, and replacement cow costs, are 37% of the annual net profit factors for dairy farming, 14% of mastitis, and livelihood and lifespan. Considering -9%, the success of dairy farming is directly related to mastitis control. For example, when 50% of milking cows are infected with mastitis, the economic loss due to reduced annual flow rate is about 36 billion won. If all other expenses are included, the total loss is KRW 67 billion per year. In terms of economic losses per city, it is about 450,000 won. Among them, the loss due to reduced flow accounts for 70% of the damage from mastitis. In the United States, the damage is estimated to be about 250,000 won per cow per year.

In addition to reproductive disorders, mastitis in cows is a problem that occurs in almost all farms, and the rate of mastitis in Korea (as of '97) is 24% (specified basis) and the resulting economic losses turn over 60 billion annually. It can be said that. Therefore, in order to develop the dairy industry by improving the quality of milk and improving price competitiveness, which is directly related to public health, efficient treatment and prevention measures for mastitis are urgently needed.

Due to the variety of causes of mastitis, antibiotics have been widely used for the prevention and treatment of mastitis over the past decades. However, due to the use of antibiotics, resistant strains are increased and treatment rates are reduced. When antibiotics remain in the human body, there is a report that it causes adverse effects. I had to throw it away. Therefore, in recent years, attempts have been made to develop immunopotentiators to increase the resistance to disease of the host by physiological and immunological therapies by avoiding antibiotics for the control of diseases such as mastitis.

Chitosan is an N-deacetylide that has been depleted of acetyl by chemical or biological treatment of chitin, which constitutes the skeleton and shell of crustaceans such as crabs and shrimp, as well as the cell walls of mushrooms and filamentous fungi. Since chitin is insoluble in common solvents, its use is limited and most of it is used as a raw material for chitosan. Chitosan has a polycationic nature and has been used for a long time as a flocculant or dehydrating agent for water treatment.However, when the merits and characteristics of chitin chitosan are revealed, iontophores, enzyme fixatives, cosmetics, medical supplies, and volume modifiers Such uses are becoming more widespread. Recently, in Japan, chitosan was implanted into bovine subcutaneous tissues to examine the effect of wound healing. After 7 days of transplantation, the induction of polymorphonuclear cell (PMN) was induced around chitosan and fibroblast activity was observed. The cells disappeared and the connective tissue was regenerated at the same time as the vasculature around the chitosan fibers, and polykaryocytes derived from macrophages adhered to the chitosan fibers and showed excellent effects on wound healing. Forty-three of 48 (89.6%) reported responding to chitosan without treatment with antibiotics. Recently in Korea, there has been a great interest in product development and research results on chitosan and chitosan oligosaccharides.

Chitin, chitosan production method

Known methods for producing chitosan from chitin have been described as chitin to chitosan by Hackman (Study on chitin.I. Enzymatic degradation of chitin and chitin esters.Aust.J. Biol.Sci.7.pp168) in 1954. It is known how to prepare, in this method, the shell is washed and dried at 100 ℃, then treated for 5 hours under 2N hydrochloric acid solution at room temperature, washed and dried to pulverized, the powder obtained is again 2N hydrochloric acid After stirring under the condition of 0 ° C to remove calcium, it was repeatedly treated in 1N NaOH aqueous solution several times at 100 ° C to obtain chitinic acid. The chitosan prepared had a degree of deacetylation of 65 to 81.2%, and there was no mention of molecular weight. However, published in 1992 by Alimuniar et al. (An economical technique for producing chitosan.In Advances in chitin and chitosan, CJ Brine, PAStanford, and JP Zikakis (Ed), pp 627. Elsevier Applied Science, Essex, UK.) According to the data, the deacetylation degree of chitosan prepared from shrimp shells was 56-68%, and the molecular weight was 5,074 cps at the time of preparation, and the molecular weight at the starting point of chitin-to-chitosan was reported to be very high. As a method, after degreasing crab, shrimp, and squid, in order to increase the reaction efficiency, a step of grinding to a size of 0.5 to 3 cm, and then removing minerals such as calcium, 1 to 2 N hydrochloric acid and 90% of formic acid are used. Chitin was prepared by the step of stirring and removing the protein by adjusting the KOH or NaOH solution 1 to 5% and heating at room temperature or high temperature for several hours, and the chitin was prepared. It can be said that chitosan is prepared by subjecting the reaction to be stirred for 0.5 hours to 144 hours under a temperature of 30 ° C. to 150 ° C. under 39 to 55% KOH or NaOH solution, and the deacetylation degree of chitosan is 56 to 68. When the percentage is expressed, the molecular weight viscosity ranges from 60 to 5,074 cps, and the deacetylation degree is 68 to 78%, and the viscosity molecular weight is 117 to 5,110 cps, and the range from 80 to 90% is 186 to 780 cps, 99%. In the range of chitosan, brown change occurred. In other words, the relationship between the molecular weight and the degree of deacetylation did not exceed the range of manufacturers not considered. As a general method of extracting chitin and preparing chitosan, the above-mentioned conventional methods show the disadvantage of breaking the molecular chains of chitin and chitosan. In particular, when chitosan is treated in a high temperature and high alkaline solution for a long time, the degree of deacetylation is increased. At the same time, the color of brown color changes, the molecular weight decreases rapidly, and the deacetylation degree of the chitosan of the polymer is extremely low. In addition, according to the domestic patent application for the manufacturing method related to the present invention, according to Publication No. 95-18054 "Manufacturing method of chitin and chitosan for bioclinical medicine from shrimp shell", high purity, high molecular weight, high acetylation degree from shrimp shell And a method for preparing a chitosan for medical use having a high whiteness, and also a method for preparing a bioclinical chitin and chitosan from prairie humpback shrimp shell is disclosed. 1.5 hours of treatment was performed at -40 ° C to + 30 ° C under a 1-6N hydrochloric acid solution, and 0.5 hour and 6 hours at 90-100 ° C with 10% NaOH solution to prepare biomedical chitin. It is known that the deacetylation degree is over 92% and the viscosity molecular weight is 700 ~ 3,270, but it is beyond the range that follows existing one except temperature part. It was not. In addition, due to the cumbersome process such as the composition of low temperature conditions and the small amount of the dose of the first shellfish shell, the increase in the cost of production and the occurrence of additional cost factors due to the use of organic solvents such as alcohol often used during the manufacturing process and It shows the disadvantage that the production cost is increased according to additional facilities such as recovery. Therefore, the present inventors have solved the above-described problems, and examined the method for obtaining chitosan suitable for the production of high purity, white polymer chitosan. As a result, in order to prevent the cleavage of the molecular chain during chitin production, crab and shrimp shell 2mm fine pulverization, nitrogen gas was added to form the temperature at 80 ~ 100 ℃ condition, stirred for 1 to 3 hours using a diluted 0.5% NaOH solution to remove the protein through the first pretreatment process and filtered After the administration of 5% NaOH solution and reacted again for 1 to 3 hours to remove the residual protein and filtered, followed by a neutral adjustment with 1N HCl solution and washing, followed by 0.5N HCl solution at room temperature The reaction was carried out for 0.5 to 1 hour using a pretreatment process to remove primary calcium, and then newly added 1 to 2 NHCl solution for 1 hour to repeat the reaction. The removal of neural and the reduction of the molecular weight is suppressed, and then the pH neutrality is adjusted and washed to dry under the sunlight conditions using solar light to remove astaxanthin pigments that crab shells have by solar-derived cosmic rays. Oxidation yielded high viscosity chitin with excellent whiteness. Subsequently, it was confirmed that the polymer and high purity chitosan can be obtained by repeating the obtained chitin twice at 90-100 ° C. under 40-50% KOH solution while stirring under nitrogen atmosphere. In addition, an inert gas was injected between the alkali reactions to block the inflow of oxygen, which may cause the breakage of the molecular chain. In other words, in order to obtain the pigment-free polymer chitin in the present invention, a pretreatment step of removing primary protein, calcium and minerals using NaOH and HCl at a small concentration of pH control level was introduced, which was extremely short within 1 hour. The use of time ensures the reduction of molecular weight and does not use the oxidizing agent or organic solvent when removing the pigment, and the color and molecular weight of the color of the crab shell by natural ultraviolet rays during the drying process. High polymer chitin could be obtained. In addition, according to the present invention, in order to obtain a polymer and high purity chitosan, 40-50% KOH solution is added to the obtained chitin and an inert gas to block the inflow of oxygen or the like which may cause the cleavage of the chitosan molecular chain. The primary reaction was carried out using phosphorus nitrogen for 3 to 12 hours to suppress the decrease in molecular weight through heating and stirring, and to prepare the filtered intermediate again KOH at the same concentration to carry out the secondary reaction under nitrogen atmosphere. Half conditions were set so that the reduction of the molecular weight could be suppressed to the maximum and the increase in the degree of deacetylation was ensured. Subsequently, the process of filtration and washing with normal water, but washing repeatedly until the pH becomes neutral upon washing, oxidizing and removing the remaining pigments, which may also remain in natural light, by ultraviolet rays, and simultaneously drying the whiteness. Obtained high purity and high viscosity chitosan obtained.

Antimicrobial Activity and Livestock Application of Chitosan Oligomer Derivatives

Antibacterial properties of chitosan are known to be important factors such as deacetylation degree and molecular weight. In other words, the deacetylation degree was 66%, 79%, 90% and 99%, and the minimum growth inhibition concentration was confirmed in Fusarium solini . As a result, 66% of deacetylated chitosan was 0.11% and 79%. 0.09%, 90% and 99% have an antimicrobial effect of 0.07%. The higher the deacetylation degree, that is, the more amino groups, the better the antimicrobial activity. In particular, more than 90% of deacetylated chitosan showed the best antibacterial effect, but there was no significant difference in the degree of deacetylation. In addition, chitosan adjusted to 2, 50, 160 and 200 centipoise showed the best results at 5 cps, and the lower the viscosity, the better the antimicrobial activity (內 田 泰, 1988, chitin, chitosan antimicrobial activity). , Food Chemical, pp 22-29). In addition, the antimicrobial effect of E. coli differs depending on the difference in the sugar composition of chitosan oligosaccharides. In the report, the sugars contained 2 to 8 sugars, of which 80% to 4 to 6 sugars. In the case of the physical properties occupying, the antimicrobial effect appeared only at the concentration of 0.3% or more, and in the case of containing 2 to 7 sugars as another property value, 70% or more showed the content of 3-4 sugars. In some cases, the antimicrobial effect was reported (Uchida, Y., 1995, Antimicrobial activities of chitosan and its applications, 海 の 台 地誌, 1, pp 51-58).

As a result of the published results related to S. aureus in connection with the present invention, the inhibitory effect on bacterial growth of chitosan derivatives was inhibited in the proliferation of Psudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermis and Proteus vulgaris at the concentration of 0.6-2.5%. , Bacillus subtilis, Escherichia coli and Klebsiella pnmonia were not inhibited at 2.5% concentration. However, partial deacetylated chitin, that is , Gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermis, Bacillus subtilis , Psudomonas aeruginosa, Proteus vulgaris, Escherichia coli, and Klebsiella pnmonia at 0.13-0.5% Inhibition of proliferation has been reported to have a higher bacteriostatic effect than low chitosan with high deacetylation. These results indicate that the amino group exhibits a cationic charge in the deacetylation, and thus N-acetylmuramic acid, sialic acid, and neuramic acid on the cell wall surface. It is presumed to inhibit bacterial growth by electrical interaction with anionic components such as.

The production method of chitosan oligosaccharides invented in this study is not particularly different from those published in Japan, but can be classified into acid hydrolysis methods (Japanese Patent No. 61-21103, 1986) and enzyme methods, which are general manufacturing methods. The former may be considered to be damaged by acid groups which may remain due to acid hydrolysis, and according to the present invention, an enzyme-based method was used. This manufacturing method is a method generally used. In the present invention, the sugar range was set so as to excel in the antimicrobial effect, and this was separated, and the deacetylation degree of chitosan oligosaccharides and the portion where the 1-16 sugar portion occupied the whole in GPC analysis of these molecular weight distribution is 96%. That is, based on these results, the present inventors prepared chitosan-olidosaccharide (OCHT) having a deacetylation degree of 96% and a viscosity close to 1 in this experiment.

Saito et al. (1994) reported that there were many differences in the effective antimicrobial concentrations of chitosan by strains in the antimicrobial test of Streptococci using chitosan extracted from squid. Although a minority, the difference in antimicrobial activity due to chitosan oligosaccharide was sometimes recognized. That is, it was determined that there is a difference in the antibacterial activity according to the species. Taken together, the antimicrobial mechanism of chitosan against E. coli is due to the fact that the amino group of chitosan specifically interferes with the growth of fungi by binding to the cell wall of pathogens. Presumably, the effect on the structure of the cell surface and the inhibitory effect on DNA formation during the metabolism of bacteria is emerging, but it is not yet clear. Therefore, the identification of the mechanism in the future is considered to be a very important task to expand the application range of chitosan. Chitin and chitosan are almost nontoxic when administered orally and are digested by chitinase and chitosanase secreted by enterobacteriaceae, and are absorbed as oligosaccharides in the small intestine and are transferred to blood and tissues. Chitin oligosaccharides and chitosan oligosaccharides act on macrophages in the blood to induce lysozyme to decompose the cell wall of pathogens and play an important role in the prevention of bacterial infections. Recently, in Japan, chitosan has been used in veterinary clinic for the purpose of anti-inflammatory action and wound healing against various purulent diseases occurring in cows, cats, dogs and the like.

It is an object of the present invention to provide a prophylactic agent for inhibiting the production of mastitis in cows and a mastitis treatment in livestock farms.

1 is an outline of a method for producing chitosan oligosaccharides.

2 a to d is a data table analyzing the molecular weight distribution by time GPC after enzyme treatment of chitosan oligosaccharides prepared for the prevention and treatment of cow mastitis.

3 is an enlarged photograph showing the antimicrobial effect of sugar composition and added concentration of chitosan or chitosan oligosaccharide by paper disc method (sensitivity) for pathogenic bacteria.

The chitosan oligosaccharide has a sugar content of 1 to 16 sugars of 66.3% to 97.5%, a deacetylation degree of 75% to 100%, and a molecular weight of 104 to 2310 as an active ingredient, and then mixed with 0.5 to 0.0001wt% of feed or drinking water. It is a mastitis treatment of dairy cow, characterized in that it is dissolved and orally administered to the cow.

Preparation of Chitosan Oligosaccharides Using Chitosanase

The preparation of chitosan oligosaccharides used in the present invention was prepared in four kinds of deacetylation degrees of 75%, 85%, 96% and 100%, and four kinds of total low molecular weight chitosan of 10 g or less with a mean molecular weight of cps1.5 ± 0.3. Sufficiently dispersed in. The different weak acids, 0.5N acetic acid, or hydrochloric acid or lactic acid, were slowly added to each other in a reactor to be liquefied to prepare a chitosan-acid salt into which a hydrochloric acid group was introduced. Dilute acetic acid to the end point was pH 4-5. Or stop the administration of hydrochloric acid or lactic acid, and stirred at room temperature for 24 hours to raise the temperature to 50 ℃ when the final pH is 5.5 to 6.0, chitosanase ( Bacillus pumilus BN-262, 曉 津 水産 化學 工業 ( Note) and Japan) were used.

The purity of chitosanase used in real time was 3,000-20,000U / g, and chitosan degrading coenzyme produced by Bacillus pumilus BN-262. In addition, an enzyme having a physical property of about 31,000 (SDS-PAGE method) was selected and used. That is, after adding 0.011 g of enzyme to 1 g of chitosan and stirring at 150 rpm for 24 hours at the same temperature, the temperature was stirred for 30 minutes at 80 ° C. for 1 hour to remove the activity of the enzyme, followed by adjustment for 12 hours. The precipitated enzyme was centrifuged (15,000 rpm, 15 minutes) to completely remove the precipitate. It was concentrated and lyophilized to prepare several chitosan oligosaccharides.

Preparation of Chitosan Oligosaccharides Using Cellulase

It was prepared herein using Cellulase (ZEANZYME, Jeans care LTD., England). That is, 4 g of deacetylation degree of 75%, 85%, 96% and 100% of the chitosan oligosaccharide used in the present invention, and 4 g of the low molecular weight chitosan with the average molecular weight of cps1.5 ± 0.3 or less of 4 kinds in total, 1 L of deionized water Sufficiently dispersed in. To each solution, 0.5N acetic acid or hydrochloric acid or lactic acid, each of which is slightly different, was gradually added dropwise in different reactors to prepare a chitosan-acid salt to which hydrochloric acid groups were introduced. The pH was 4 to 5 diluted to the end point. Stop the administration of acetic acid or hydrochloric acid or lactic acid, stir at room temperature for 24 hours, raise the temperature to 50 ° C when the final pH reaches 5.5 to 6.0, administer 40 ml of cellulase per 100 g of chitosan, and then 150 rpm for 24 hours. The reaction was carried out, and the stirring was stopped for 24 hours, the pH was adjusted to 7-8 with 5% NaOH solution to remove residual acetic acid or lactic acid or hydrochloric acid, and the resultant was filtered to remove impurities, followed by standard material and thin plate glow. After confirming the appearance of oligosaccharides by thin layer chromatography (Thin Layer Chromatography) analysis, and then lyophilized to obtain a powdery chitosan oligosaccharides.

Chitosan Oligosaccharides

Chitosan oligosaccharide mixtures were separated using methanol or ethanol or isopropyl alcohol or acetone to obtain chitosan oligo derivatives having a high molecular weight distribution. That is, 0.5 to 6 times methanol or ethanol or isopropyl alcohol or acetone organic solvent was gradually administered in different beakers while stirring the chitosan oligosaccharide solution dissolved in ordinary water, and the precipitates formed at this time were separated and separated. . Chitosan oligosaccharides with different molecular weights were analyzed for qualitative physical properties by standard chromatography and thin plate chromatography. In order to confirm the molecular weight distribution, GPC (gel permeatation chromatography) analysis system (JASCO, Japan) was used to determine the distribution. The remaining amount of the remaining hydrochloric acid group was titrated using AgNO ₃ titration method. The finally obtained chitosan oligosaccharide-containing solution was concentrated and lyophilized to obtain chitosan oligosaccharide powder. The molecular weight distribution of each type of chitosan oligosaccharide separated by sugar is shown in <Table 1>. That is, the average molecular weight of the total chitosan oligosaccharide was 2,000 or less, the deacetylation degree was set at 96%, and the chitosan oligosaccharide was composed of three kinds of chitosan oligosaccharides having physical properties of 66.3% to 97.5% of 1-16 sugars. Separated.

Table 1. Chitosan oligosaccharide (hereinafter referred to as COS) used in the present invention

Composition

Note) *: The composition of chitosan oligosaccharide was determined to be 1-16 sugars.

Selection of mastitis-inducing strains for antimicrobial assay

Livestock disease pathogen isolates for the antimicrobial assay were selected as the most common pathogens isolated from cattle breeding conditions when cow mastitis occurs. Mastitis-causing strains were isolated from small intestine contents, feces, and lesion tissues of animals that were referred to an autopsy at the College of Veterinary Medicine, Chonnam National University. The basic method of securing strains was to isolate the test material on nutrient agar plates, isolate them, and then identify them by biochemical tests. As a pathogenic isolate name that can be treated with chitosan and chitosan oligosaccharides of the present invention, a total of 10 species were used as a material of the present invention, including 9 isolates isolated from standard strains and livestock feces (Table 2).

Table 2. Livestock mastitis-induced pathogenicity that can be treated with chitosan or chitosan oligosaccharides of the invention

List of germs

Preparation and Culture of Mastitis Medium for Antimicrobial Assay

Nutritious broth (NB) was autoclaved at 121 ° C. for 15 minutes and then used as a basal medium for bacterial culture. The medium preparation for this culture was adjusted to pH6.5 in 2 times the concentration of autoclaved NB medium (pH6.5) at 121 ° C. for 15 minutes, and then a 1% solution containing chitosan oligosaccharide isolated sugar and sterilized distilled water were added. The final concentrations of chitosan oligosaccharides were 0.5% (w / v), 0.1% (w / v), 0.01% (w / v), 0.001% and 0.0001% (w / v). Medium was used. This culture was inoculated with 0.1% (v / v) of the culture medium incubated overnight, followed by rotation shaking culture for 24 hours.

Determination of Antimicrobial Effects by the Paper Disk Method of Chitosan Oligosaccharides Prepared by Sugars

In order to determine the antimicrobial activity of COS according to the type of livestock disease-inducing bacteria to be identified in the present invention, nutritional broth was prepared in a 250 ml flask, inoculated with bacteria (E. coli), and incubated at 37 ° C. These bacteria culture medium was adjusted to 2 x 10 ⁷ CFU / ㎖, and then 0.5 ml of the aliquot was plated on nutrient agar agar medium, chitosan oligosaccharides prepared according to the paper disk method by sugar concentration (0%: no addition group, 0.5%, 0.1%, 0.05%, 0.01%, 0.001%, 0.00004%), the growth inhibitory ring formed by incubating at 37 ℃, 24 hours in an incubator by measuring the antimicrobial effect of each concentration of sugar It is shown in <Figure 3>. That is, the chitosan oligosaccharide mixture of 1 to 6 sugars was confirmed to have no antimicrobial effect regardless of the concentration, and the chitosan oligosaccharide mixture of 7 to 14 sugars and 1 to 16 sugars was confirmed to have an antimicrobial effect when added at 0.0004% or more.

Antimicrobial Effects of Mastitis-inducing Bacteria on Chitosan Oligo Derivatives

In order to determine the antimicrobial activity of each concentration of COS according to the mastitis-inducing bacteria to be identified in the present invention, the cultured bacteria were cultured in nutrient broth medium at 37 ° C. These bacterial culture solutions were adjusted to 2 × 10 ⁷ CFU / mL, and then, as an example, COS-1 prepared in Example 20-3 was used for standard strains and field isolate strains (Tables 3-1 to 3-4). -2 shows the antibacterial effect against the outdoor isolate strains (Table 3-5 to Table 3-8) and COS-3 against the field isolate strains (Tables 3-9 to 3-11) under laboratory conditions. That is, five treatment groups in which COS was added by concentration (0.5%, 0.1%, 0.01%, 0.001%, 0.0001%) were set, and as a comparative example, a control group without COS was provided. Cap test tube containing 990 μl of phosphate buffered solution after adding 10 μl of each treatment group at 5 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 18 hours and 24 hours after adding COS. After mixing well, 50 μl of this was plated on nutrient agar medium and cultured for one day to count colonies. At the same time, the antimicrobial effects of COS added at the same time when the bacterial counts were added were compared with each other (Table 3, 3-1 to 3-11). As a result, when chitosan oligosaccharide (COS) was added, the rapid death occurred after 5 minutes compared to the number of bacteria at the first administration, and there was a slight difference due to the type of bacteria, and more than 12 hours had passed. Surviving colonies did not appear and were confirmed to have been completely killed.

Table 3. After adding chitosan oligosaccharides Staphylococcus. aureus Results of measuring the killing effect of bacteria by time and concentration (first dose: 2x10 ⁷ CFU / ml)

Table 3-1. Standard strain Staphylococcus aureus Antimicrobial Effects on ATCC 29213

Table 3-2. Outdoor Separation (Cow Small Intestine Separation) Staphylococcus. aureus Antimicrobial effect against

Table 3-3. Outdoor Separation (Cow Colon Separation) Staphylococcus. aureus Antimicrobial effect against

Table 3-4. Field separation (from cow feces) Staphylococcus. aureus Antimicrobial effect against

Table 3-5. Outdoor detachment (from cow feces) Staphylococcus. aureus Antimicrobial effect against

Table 3-6. Field separation (from chicken subcutaneous tissue) Staphylococcus. aureus Antimicrobial effect against

Table 3-7. Outdoor Separation (Separation from Chicken Intestine) Staphylococcus. aureus Antimicrobial effect

Table 3-8. Outdoor separation (chicken colon separation) Staphylococcus. aureus Antimicrobial effect against

Table 3-9. Outdoor Separation (Separation from Chicken Intestine) Staphylococcus. aureus Antimicrobial effect against

Table 3-10. Outdoor separation (from chicken feces) Staphylococcus. aureus Antimicrobial effect against

Table 3-11. Outdoor separation (from chicken feces) Staphylococcus aureus Antimicrobial effect against

Chitosan oligosaccharides for the treatment of mastitis In vivo )>

After intraperitoneal administration of chitosan oligosaccharides S. aureus Investigation of defense against

Looking at the existing announcements using chitosan and chitosan oligosaccharides against S. aureus , a pathogen associated with the present invention, it was previously reported to confirm the laboratory antimicrobial effect by Uchida et al., When using general chitosan S. aureus is When the concentration of 0.05% or more was added, the antimicrobial effect appeared. Also, similarly to the present invention, the chitosan oligosaccharide prepared by decomposing the enzyme was distributed in the laboratory conditions with the formulation having the physical properties of 3-8 sugars. The antimicrobial effect was verified that the concentration of antimicrobial effect on S. aureus was more than 0.6%. However, these experimental results were limited only in laboratory conditions, and the effect in animal experiments was not verified. However, the chitosan oligosaccharide formulations used in the present invention compared the laboratory antimicrobial effect using COS-3 prepared in Example 20-3, and applied to animal experiments to confirm the survival water to assay the therapeutic effect. There is no published example. Therefore, in the present invention, to confirm the therapeutic effect against S. aureus , which is the most incidence, infectious, and low treatment rate among the mastitis causative organisms, which causes the largest economic loss to dairy farmers, 0.5 to 2 mg per head intraperitoneally in mice 7 On the last 8 days after daily injection, 5 × 10 ³ CFU strains per ml were challenged intraperitoneally. As a result, in the control group not administered chitosan, 9 out of 10 eyes died, but the chitosan oligosaccharide administration group showed 70-100% survival rate according to the concentration difference, and the protective effect against S. aureus was recognized (Table 4).

Table 4. Chitosan oligosaccharide was intraperitoneally administered to the mouse for 7 days S. aureus Investigation of defense against

* 5 × 10 ⁸ CFU / ml

Therapeutic Effect of Chitosan Oligosaccharides on Mastitis Cows in vivo ) black

In the present invention, COS-3 prepared in Example 20-3 in consideration of high digestibility and economic efficiency of chitin chitosan in the case of ruminants and the report of the above-mentioned in order to confirm the efficacy of chitosan as a mastitis treatment agent based on the above-mentioned facts Sanctions were administered at a rate of 15g per head. In other words, milk cows infected with mastitis were mixed orally with feed once a day for 7 days, and then milk was collected on the 7th and 14th days after the initial administration to examine the effect of mastitis. It was. As a result, as shown in <Table 5>, the somatic cell count was 278 million before chitosan oligosaccharide administration, but showed a decrease effect of 29.0% to 1.97 million on the 7th day after the first administration, and a high somatic cell number reduction effect of 43.6% on the 14th day after administration. Indicated.

Table 5. Investigation of Somatic Cell Reduction Effect by Chitosan Sanction Supplementation

* Somatic cell count after administration-Somatic cell count before administration / Somatic cell count before administration × 100

The therapeutic effect of chitosan oligosaccharides for mastitis has the antimicrobial effect of directly acting on and killing the bacteria causing mastitis, and chitosan oligosaccharides indirectly enhance PMN cell migration and macrophage activity in blood and tissues to remove bacteria. It has a healing effect on damaged and damaged breast tissue.

Claims (1)

The chitosan oligosaccharide has a sugar content of 1 to 16 sugars of 66.3% to 87.5%, a deacetylation degree of 75% to 100%, and a molecular weight range of 104 to 2310 as an active ingredient, which is mixed with 0.5 to 0.0001wt% of feed or in drinking water. The agent for the treatment of mastitis of cows, characterized by dissolution and oral administration to cows.