KR20000050331A - A Veterinary Preparation for Preaenting and Treating Diseases of Livestock With Mineral Salt Chitosan Oligosaccharide Derivatives as an Effective Component - Google Patents

Abstract

PURPOSE: A prophylactic and/or a treatment agent for livestock diseases are provided which comprises a therapeutically effective amount of mineral-chitosan oligosaccharides derivatives as an effective ingredient. The livestock diseases caused by 26 strains of E. coli having a different serotype are prevented or treated by improving the absorptivity of mineral and the antibacterial activities. CONSTITUTION: A prophylatic and a treatment agent comprise the chitosan oligosaccharides substituted with a mineral such as Ca, Fe, Zn as an effective ingredient, wherein the mineral-chitosan oligosaccharides derivatives has at least 2000 Da of an average molecular weight, saccharides are distributed in the range of 1-16 and Ca, Fe, Zn are substituted to the chitosan oligosaccharides by 10-30%(w/w), 4-30%(w/w), 1-30%(w/w) or higher, respectively. To treat livestock diseases, the agent is orally administered with being added to foods such as feed-stuff and drinking water at concentration of 0.0001-100% and an effective concentration thereof to be administered is 0.00001-0.5%(w/w) as a prophylatic.

Description

A Veterinary Preparation for Preaenting and Treating Diseases of Livestock With Mineral Salt Chitosan Oligosaccharide Derivatives as an Effective Component}

The present invention is for the treatment of livestock disease treatment and livestock, including mineral salt-chitosan oligosaccharide (hereinafter, calcium salt-chitosan oligosaccharide substituted with Ca, Zn and Fe, zinc salt-chitosan oligosaccharide, iron salt-chitosan oligosaccharide) derivatives. It relates to the prevention and treatment of livestock diseases aimed at eradication of livestock pathogenic microorganisms that potentially cause clinical disease in the environment.

In order to prevent disease outbreaks in the specification of livestock, host livestock should be minimally exposed to disease germs in the breeding environment. However, the most aggressive disease prevention measures include growth inhibition or killing of pathogens that can cause infections in and out of livestock. In addition, the diseased animals are trying to find a substance that can be effective by being absorbed into the body quickly and effectively distributed through the body's biological metabolism and distributed to the target tissue. It is true that known antimicrobial agents have great disadvantages that, despite short-term clear efficacy, cause expression of resistance and safety problems for humans in long-term use. Therefore, the development of a material that can replace the synthetic synthetic antibiotics currently in use is an urgent practical task.

Therefore, it is possible not only to control the physical properties to use the normal metabolism of absorption in the body of the animal and to suppress or kill pathogens that cause disease in the body during the metabolic process, but also to improve disease resistance through the activation of the immune system. As a result, the development of natural antimicrobial materials that can reduce the mortality rate of livestock and improve productivity indicators, such as weight gain rate, has great significance for the development of the livestock industry.

The development of natural antimicrobial substance should basically identify the cause of livestock disease, deterioration factors of breeding environment, defensive factors of defensiveness, and type and occurrence of disease by breeder. The only major pathogens infecting livestock today, including bacteria, fungi, viruses and parasites, are limited to bacterial diseases such as ceft iofur, ciprofloxacin, doxycycline and oploxacin ( ofloxacine, cefazoline, amikacin, amoxaxicilline, streptomycin, streptomycin, norfloxacin, cephalozo1in, norfloxacine, norfloxacine, Various types of synthetic antimicrobials are in use, including neomycin, thiamulin, lincomycin, spectinomycin, and sulfonamide.

These drugs are enteritis that causes diarrhea in major livestock such as pigs, cattle and chickens through oral or intramuscular injection. It causes sepsis, navelitis, peritonitis, dermatitis, and cystitis (algae) and is used as a treatment for "Escherichia coli" infections, including the 0157: H7 isolate. In addition, these antimicrobial agents are used for the prevention and treatment of the following pathogens, which cause respiratory diseases in cattle and pigs, which cause pneumonia, and the pathogen of poultry cholera with high morbidity and mortality. Pasteurella spp "," Salmonella spp "showing various systemic diseases such as enteritis, pneumonia, sepsis and encephalitis as a common pathogen, and" Streptococcus "causing arthritis and encephalitis as well as primary or secondary pathogens. spp) ", livestock skin and mucous membranes, as well as livestock everywhere, depending on the strain, such as" Staphyloccus spp "that causes toxins when infecting the human body.

In addition, in breeding cows, the increase of somatic cells caused by Escherichia coli diarrhea and Staphylococcus aureus bacteria is severe and damage to cattle farms is serious.

In addition, Aspergilus spp, a feed-injectable pathogenic fungus that produces aflatoxin, which causes pneumonia and hemorrhagic causes when invading mucosal tissues of animals, causes massive damage. Difficulty breathing and rarely, diarrhea, loss of appetite, and weakness are observed. When brain lesions occur, neurological symptoms include convulsions, amnesia and paralysis.

In the cattle, pig and poultry industries, which are the major subsidiaries of domestic livestock industry, the bacterial and fungal diseases listed above are prevalent, and the pathogenesis by these pathogens is mainly focused on young livestock. It is estimated that it accounts for most of 600 billion won (96%), the disease damage of livestock production.

However, the fundamental weakness of these currently used synthetic antimicrobial agents is that they can lead to the emergence of new pathogenic resistant bacteria if they are misused or long-term doses of these drugs are used without the accurate confirmation of the main pathogen causing the disease. to be. Moreover, the emergence of these resistant bacteria inevitably leads to an increase in the amount of these drugs used, which can lead to an increase in production costs as well as problems in the livestock body, which can cause serious health risks for the people. Most of the livestock use uses the synthetic synthetic antimicrobial agents listed above, and no particularly effective alternatives have been established to date.

Therefore, the use of "chitosan oligosaccharide derivative material", which is a natural substance that can be substituted for the current synthetic antimicrobial agent, is used as an animal medicine, except in 1992 as a preventive agent for mastitis (Japanese Patent Laid-Open No. 4-169529). The present invention is an attempt to apply to the livestock industry a formulation using the antimicrobial properties, which are inherent in chitosan, but with enhanced functionality.

Chitosan, a polymer of monomeric glucosamine (D-glucosamine), is a weakly acidic polyacetylation of chitin, a polymer of acetylglucosamine (N-acetylglucosamine) extracted from shells of shellfish or fungi such as crabs and shrimps as a polycation. It is a product. As such, chitosan, a natural material that can be reproduced, has been shown to exhibit antimicrobial activity against some bacterial and phytopathogenic fungi strains, and there are many differences in antimicrobial activity depending on molecular weight (Hirano et al, 1989, "Effects of chitosan, pectic acid, lysozyme, and chitinase on the growth of several phytopathogens ", Agri. Biol. Chem., Vol. 53 (11), pp 3065-3066). In addition, chitosan is believed to cause defense mechanisms against pathogens by activating cytokines such as interleukin-1 as well as macrophages with non-specific phagocytosis.

On the other hand, in the case of fish, a specific, immune-responsive reaction in which the antibody is the main factor due to the gentle and temperature-sensitive antibody production reaction is not sufficient as a defense means, and nonspecific defense such as natural aggregates, complement, chitinase, and lysozyme present in the fish The importance of the organization is newly recognized. In particular, lysozyme is known to play an important role in the defense mechanism of higher organisms by showing the lysing effect by cell wall decomposition and in addition to the opsonin action, antiviral action and antiparasitic action (Cheng et al, l977 " Lysozyme like activity in the hemolymph of biomphalaria glabrata challenged with bacteria ", J. Invertebra. Path., Vo 1. 29, pp 170-174). Regardless of the phylogenetic stage, various livestock as well as marine fish have been identified in serum, liver tissue, digestive system and mucus (Murray CK et al, 1976, The immunohistochemical localization of lysozyme in plaice (Pleuronectesplatessa L.) tissues). ", J. Fish Biol., Vol. 9, pp 329-334). These lysozymes are combined with mucus materials containing biologically important substances such as water-soluble substances, immune substances, aggregates, glycolytic enzymes, and polar fats, which are produced from macrophages, to be converted into non-aqueous complex barrier material and invading pathogens. (Fletcher, TC, et al, 1973, "Lysozyme activity in the plaice (Pleuronectes platessa L.)", Experimenta, Vol. 29, pp 1283-1285). Another function of chitosan is tissue compatibility, which plays an important role in the rapid regeneration of damaged tissue. In animal monocyte-derived macrophages, gamma interferon- ) Promotes the secretion of N-acetyl-beta-di-glucosaminase to perform depolymerization of chitosan in monomer units. As a result, lysozyme and en-acetyl-beta-di-glucoxaminase promote the metabolic degradation of conventionally modified chitin and earth acid, so that the monomers of the degradation products are hyaluronate, It is believed to bind to keratinsulfate and chondroitinsulfate, and these molecules are known to help tissue regeneration (Riccardo, A. et al, 1993, "Role and fate of exogeneous chitosans"). in human wound tissues ", in Chitin Enzymology, pp 187-196, RAA Muzzarelli, ed., Eur. Chitin. Soc., Ancona).

In addition, the effect of animal disease prevention and / or treatment of chitosan oligosaccharide derivatives as an active ingredient can not be said to be sufficient, the development of more powerful animal disease prevention and / or treatment is desired.

The present invention has been made to solve the above problems is a livestock disease causing damage caused by poultry such as pigs, cows, cows, chickens, ducks, etc. (1) 26 species of E. coli (Escherica coli) with different serotypes, (2) Listeria. Monocytogenes HPB # 410 (serotype 1 / 2a), L. monocytogenes EGD, L. innocua # 11O3, L. ivanovii # 322. (3) Staphylococcus. Aureus ATCC 29213, (4) Streptococcus agalactiae ATCC 13813, (5) Salmonella typhimurium ATCC 14028, Sal. Gallinarium, Sal. Pul1orum ATCC 9120, (6) Klebsiella pneumoniae ATCC 13883 and fungi (7) Aspergillus, parasiticus KCCM 12699, A. parasiticus 24690, A. parasiticus ATCC 36531, A.parasiticus 15517, A. nidurans 26451, A. flavus A. fumigatus) is to provide a disease prevention and / or treatment for the disease that is better than the existing, and to further suppress the infection or to treat and prevent the disease.

1 is a change in the number of Salmonella bacteria in the feces (0.1 g) per day when 5 mg per day per day laying individual mineral salt chitosan oligo derivatives in laying hens

FIG. 2 shows the effect of weight gain of piglets by schedule when the mineral salt chitosan oligo derivatives were administered to piglets for 29 days.

Fig. 3 shows the results of daily increase rate test after administration of mineral salt-chitosan oligosaccharide derivatives with feed (7.5 mg, w / w)

The present invention is a livestock disease such as pigs, cows, cows, chickens and ducks that cause damage during livestock raising, that is, (1) 26 species of E. coli with different serotypes separated from feces and tissues of naturally infected animals, (2 Listeria. Monocytogenes HPB # 410 (serotype 1 / 2a), L. monocytogenes EGD, L. innocua # 1103, L. ivanovii # 322, (3) Staphylococcus. Aureus ATCC 29213, (4 ) Streptococcus agalactiae ATCC 13813, (5) Salmonella typhimurium ATCC 14028.Sal. Pullorum ATCC 9120), (6) Klebsiella pneumoniae ATCC 13883 and (7) Aspergillus genus Aspergillus parasiticus KCCM 12699, A. parasiticus 24690, A. parasiticus ATTC 36531, A. parasiticus 15517, A. nidurans 2645, A. flavus, A. fumigatus) Mineral salt chitosan oligosaccharide is isolated to achieve livestock growth effect, The neutral salt chitosan oligo has an average molecular weight of 2,000 or less, the sugar distribution ranges from 1-16, and the Ca substituted for the mineral salt chitosan oligosaccharide is 10% to 30% (w / w), and Fe is 4% to 30% ( w / w) and Zn are prepared by enzymatic digestion by substituting 1-30% (w / w) or more, and the concentration of these agents as a disease prevention agent is 0.00001-0.5% (w / v). As a cure. In order to obtain the effect, it is characterized by being orally administered by mixing 0.001 to 100% in food such as drinking water and feed.

For example, it will be described in detail as follows. However, this example does not limit the present invention.

Example 1

(1) Preparation of calcium salt chitosan oligosaccharides

In order to prepare the calcium salt chitosan oligo salt used in the present invention, the calcium chitin prepared so as to retain the calcium contained in the crab shell in the range of 30% to 40% (w / w) is in the range of 40 to 50% which is a general chitosan manufacturing method. Calcium chitosan was prepared by stirring for 10 hours at 95 ° C in NaOH solution.

Calcium salt chitosan oligosaccharide was prepared by sufficiently dispersing 10 g of powdered low molecular weight calcium chitosan having a deacetylation degree of 92% and an average molecular weight of 1.5 ± 0.3 or less in 1 liter of deionized water.

After 0.5N acetic acid was gradually added dropwise to form a solution, the solution was diluted to the point where pH reached 5, and the diluted acetic acid was stopped. The solution was stirred at room temperature for 24 hours, and the temperature was increased to 50 at the final pH of 5.5 to 6.0. The temperature was raised to ℃, the cellulase was administered 40ml per 100g of chitosan and stirred (150rpm), pH was adjusted to 7 ~ 8 with 5% NaOH solution to remove the remaining acetic acid, then autoclaved (121 ℃) , 15 minutes) to inactivate the enzyme, and then filtered to remove impurities, thin layer chromatography (Thin Layer Chromatography) analysis to confirm the appearance of oligosaccharides, and then lyophilized powder calcium salt chitosan oligosaccharides Obtained. Subsequently, the obtained intermediate powder was dissolved in tertiary deionized water and then acetone was added six times to recrystallize several times to remove the unsubstituted residual minerals. The position of calcium substituent of the finally obtained calcium salt-chitosan oligosaccharide was quantified by NMR analysis and the substitution rate was determined by ICP analysis, and the substitution rate was in the range of 5% to 45%.

(2) Preparation of iron salt chitosan oligosaccharides

The iron salt chitosan oligosaccharide used in the present invention was prepared by sufficiently dispersing 70 g of powdered low molecular weight chitosan having a particle size of 200 mesh, a degree of deacetylation, and an average molecular weight of cps of 1.5 ± 0.3 or less in 1 L of deionized water. FeSO4 for feed addition compared to chitosan was added to 1 to 3 equivalents, and then stirred at room temperature for 24 hours to adsorb the iron to chitosan.Then, the filtrate was filtered and washed thoroughly with normal water between filtrations and dried in a general dryer. Phosphorus iron salt-chitosan was prepared.

10 g of dried iron salt-earthic acid and 10 liters of tertiary deionized water were simultaneously administered to a 2-liter three-necked flask, followed by sufficient dispersion for 3 hours. The solution was slowly added dropwise with 0.5N acetic acid, followed by pH 5 The diluted acetic acid administration was stopped at the end point, and the mixture was stirred at room temperature for 24 hours, the temperature was raised to 50 ° C. at the time when the final pH was 5.5 to 6.0, and 40 ml of 100% chitosan was injected into the cellulase, followed by stirring (150 rpm). The pH was adjusted to 7-8 with a 5% Na0H solution to remove residual acetic acid, followed by high pressure sterilization (121 ° C, 15 minutes) to inactivate the enzyme, and then filtered to remove impurities. Standard layer and thin layer chromatography (Thin Layer Chromatography) analysis was performed to confirm the appearance of oligosaccharides and then lyophilized to obtain powdered iron salt-chitosan oligo. The position of the iron substituent of the obtained iron salt-chitosan oligosaccharide was quantified by NMR analysis and lCP analysis, and the substitution rate was in the range of 2% to 28%.

(3) Preparation of zinc salt chitosan oligosaccharides

In the preparation of zinc salt-chitosan oligosaccharide used in the present invention, 10 g of powdered low molecular weight chitosan having a deacetylation degree of 92% and an average molecular weight of cps of 1.5 ± 0.3 or less was sufficiently dispersed in 1 L of deionized water.

After the solution was gradually added by dropping O.5N acetic acid into solution, the diluted acetic acid was stopped at the point where the pH became 5, and the solution was stirred at room temperature for 24 hours, and the temperature was increased to 50 when the final pH was 5.5 to 6.0. After raising the temperature and the cellulase 4ml per 10g chitosan was administered, and stirred for 24 hours (150rpm) to prepare a chitosan oligosaccharide mixture. The pH was adjusted to 7-8 using a 5% NaOH solution to remove the remaining acetic acid, and the ZnS04 feed was administered in an amount of 1 to 3 equivalents compared to the chitosan, followed by reaction at room temperature for 24 hours. Substituting the oligosaccharide, followed by high pressure sterilization (121 ℃, 15 minutes) to deactivate the enzyme, and then filtered to remove impurities, and by performing a thin layer chromatography (Thin Layer Chromatography) analysis to confirm the appearance of oligosaccharides It was then freeze-dried to obtain a powdered zinc salt-chitosan oligosaccharide. Subsequently, the obtained intermediate powder was dissolved in tertiary deionized water, and then acetone was added six times, and the method of recrystallization was repeated several times to remove the unsubstituted residual minerals.

The position of the zinc substituent of the zinc salt-chitosan oligosaccharide finally obtained was quantified by NMR analysis and the substitution rate was determined by ICP analysis, and the substitution rate was within 8% to 37%.

(4) Preparation of mineral salt-chitosan oligosaccharide and separation by sugar

In order to obtain a mineral salt-chitosan oligosaccharide having a high molecular weight distribution with antimicrobial properties, the mineral salt-chitosan oligosaccharide mixture was separated using solubility in methanol, ethanol, isopropyl alcohol, acetone, and the like. That is, while stirring the mineral salt-chitosan oligosaccharide mixed solution dissolved in the general water 0.5 to 6 times the solution of methanol, ethanol, isopropyl alcohol and acetone, etc. gradually administered to separate the precipitate formed at this time to separate them. Mineral salts of different molecular weights and chitosan oligosaccharides are analyzed by qualitative physical properties by conducting thin plate chromatography analysis with a standard material, and gel permeatation chromatography (GPC) analysis system (JASCO, Japan) to confirm molecular weight distribution. ) Was used to quantify the distribution, and finally, the obtained mineral salt-chitosan oligosaccharide-containing solution was lyophilized to obtain a mineral salt-chitosan oligosaccharide powder having excellent physical properties.

The molecular weight distribution of each type of mineral salt-chitosan oligosaccharide separated by sugars is shown in <Table 1>. In other words, the average molecular weight of the mineral salt-chitosan oligosaccharide derivative was 2,000 or less, the degree of acetylacetylation and the range of the sugar ranged from 60% to 100% of the 2 to 16 sugars were mineral salt-chitosan oligosaccharide derivatives. General chitosan oligosaccharides were prepared for use in comparison with Ca, Fe, and Zn-chitosan oligosaccharide derivatives, and a total of four kinds were applied as basic materials of the present invention.

Note) *: The composition of mineral salt-chitosan oligosaccharide was set to 2-16 sugar contents.

**: Mineral salt-chitosan oligo derivative

(4) Selection of livestock disease-causing strains for antimicrobial assays

Animal disease pathogen isolates for antimicrobial assays were selected for the most common pathogens and fungi that are most frequently isolated in livestock raising environments. The pathogens 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 materials on nutrient agar media and McConkey agar media, isolate them, and then identify them by biochemical tests. The pathogenic microorganisms applied to the invention are shown in Tables 2, 2-1 and 2-2.

Table 2 shows the pathogenic isolates that can be treated with mineral salt-chitosan or mineral salt-chitosan oligo of the present invention (Table 2-1, Table 2-2).

(5) Confirmation of preparation and antimicrobial effect of medium for antimicrobial assay

Nutritious broth (NB) was used as a basal medium for culturing bacteria and fungi after autoclaving at 121 ° C. for 15 minutes. The medium preparation for measuring the antimicrobial effect of the agent developed in the present invention on the bacteria causing the livestock disease was adjusted to pH 6.5 in double concentration NB medium (pH 6.5), autoclaved at 121 ° C. for 15 minutes. As a control, 1% solution of chitosan oligosaccharide and each prepared mineral salt-chitosan oligosaccharide isolated sugar and sterilized distilled water were added to prepare each derivative formulation at a final concentration of 0.5% (w / v), 0.1% (w / v), and 0.01. % (w / v), 0.001 and 0.0001% (w / v) were used, and the comparative experimental medium was a non-added NB medium. This culture was inoculated with 0.1% (v.v) of the culture medium cultured overnight, and cultured with rotary shaking for 24 hours.

As a result, when the chitosan oligosaccharide derivative (OCHT) was added, it was rapidly killed after 5 minutes compared to the number of bacteria at the time of the first administration. Poetry did not appear to survive and was confirmed to be completely killed.

(6) Determination of antifungal effect on pathogenic fungi

In order to confirm the antifungal properties of the mold to be confirmed herein, the fungal bacteria cultivated under the same conditions as in Example 5 according to the addition concentration of OCHT, Ca, Fe and Zn salt chitosan oligo by fungus type The culture was incubated at 37 ° C.

Four comparative groups of this culture medium adjusted to 1 mg / ml (dry weight basis) and then added at different concentrations (0.5%, 0.1%, 0.01%, 0.001%) according to Ca, Fe and Zn salt chitosan oligosaccharides. Was set, and as a comparative example, a control without adding each agent was placed. After adding Ca, Fe, and Zn salt-chitosan oligo by concentration, 10 μl of each treatment group was collected for 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, and 120, and the paper was added to the nutrient broth agar medium. After mounting the disc, the fungus was dropped onto the paper and cultured in a 37 ° C. incubator, and the growth rings of the fungus growing after 24 hours of cultivation were measured, and the comparative examples without Ca, Fe and Zn salt chitosan oligo were treated. Growth inhibition was compared. As a result, the addition of Ca, Fe and Zn salt-chitosan oligo showed the killing effect of the fungus within 2 hours, and the results of the killing process are shown in Table 3.

ㆍ-: No measurement.

ㆍ Significance Review: (P <0.01)

ㆍ Average error range: ± 1 ～ ± 5

ㆍ OCHT: General Chitosan Oligo Ca-OCHT: Calcium Salt-Chitosan Oligo Derivative

Fe-OCHT: iron salt-chitosan oligo derivatives

Zn-OCHT: Zinc salt-chitosan oligo derivative

(7) Evaluation of killing effect by time after addition of mineral salt-chitosan oligosaccharide of multiple mixed Escherichia coli induced livestock diarrhea

In the present experiment, the antibacterial effect of the mineral salt-chitosan oligosaccharide derivatives was determined by E. coli present in the environment causing diarrhea of livestock by a variety of Escherichia coli, and in this experiment, the culture medium cultured in NA medium by whole strains (Table 2). The cell precipitate obtained by centrifugation at 15,000 rpm for 30 seconds with a microcentrifuge was washed twice with sterile phosphate buffer solution (pH 6.0) and finally suspended in 1 ml phosphate buffer solution. The E. coli culture suspension was evenly mixed with 1 ml, and then diluted with 2 × 10 ⁷ CFU / ml to assay the antimicrobial effect of mineral salts-chitosan oligosaccharides. Comparative Examples and Examples for the verification thereof were measured in the same manner as in Example 6. The number of viable cell counts is shown in <Table 4> as a result. In other words, the killing rate of bacteria was different depending on the strain and species, but the same rapidly decreased within 1 minute after contact with the microorganism and the mineral salt-chitosan oligosaccharide, and after 46 hours colonies did not appear at all and completely died. It was confirmed that. The difference in the antimicrobial activity of the mineral salt-chitosan oligosaccharide formulations was OCHT <calcium-chitosan oligo derivative <Fe-chitosan oligo derivative <Zn-chitosan oligo derivative.

The results of the comparison of antimicrobial activity were based on OCHT (1), 1.5 times for the calcium-chitosan oligo derivatives, 2.2 times for the iron salt-chitosan oligo derivatives, and 2.6 times for the zinc-chitosan oligo derivatives. Indicated.

ㆍ-: No measurement.

ㆍ Significance Review: (P <0.01), ㆍ Error Range: ± 3 ～ ± 12

ㆍ OCHT: general chitosan oligo, Ca-OCHT: calcium salt-chitosan oligo derivative

Fe-OCHT: iron salt-chitosan oligo derivatives

Zn-OCHT: Zinc salt-chitosan oligo derivative

Effect of Disease Treatment on Pigs with Natural Diseases

Comparative Example 1

In the comparative example of the present application, only the piglets showing diarrhea symptoms among the same piglets showing diarrhea at 1 day of age and 7 to 14 days of birth in the same sow were measured and compared with each other by measuring the survival rate and treatment efficiency according to a conventional method. As an example, it was compared with the result of an Example. The administration conditions of the general antibiotics of the comparative example of the control group was carried out as follows. Sows were injected with cefazoline antibiotics 3 days before and 2 days before delivery, and at the time of delivery, a single antipan was injected. After birth, the piglets were orally administered with Gentamicin (Taesung Microorganisms) once a day. The results of treatment of piglets' diarrhea symptoms were first confirmed as clinical symptoms, and the number of bacteria present in the feces of piglets was confirmed as follows. After confirming the clinical symptoms of the piglets showing the first watery diarrhea symptoms, the anus of the piglets showing diarrhea symptoms was disinfected with alcohol so as to minimize contamination with external bacteria, and feces in the rectum were forcibly collected. In addition, 0.1 g of the collected fecal fraction was accurately aliquoted and dispersed in 1,000 µl of PBS buffer solution, which was diluted 500-fold, 5,000-fold, and 500,000-fold in McConkey or mENDO (Difco) solid medium. Bacterial colonies appearing by counting. In addition, the process of confirming the therapeutic effect by the mineral salt-chitosan oligo derivative was also performed in the same process.

Example 2

According to the administration of the mineral salt-chitosan oligosaccharide, the administration conditions of the preparations related to the present invention were set as follows. In other words, after giving birth to the mineral salt-chitosan oligosaccharide respectively, 0.1 mg / kg body weight was forced orally once a day for 1 day-2 days of age. It carried out similarly to the example.

Example 3

Example 3 was carried out under the same conditions as in Example 2, except that the administration conditions were 2 mg.

Example 4

Example 4 was carried out under the same conditions as in Example 2, except that the administration conditions were 4 mg.

Example 5

Example 5 was carried out under the same conditions as in Example 2, except that the administration conditions were 7.5 mg.

Example 6

Example 6 was carried out under the same conditions as in Example 2, except for the precocious diarrhea symptoms appearing around 7 to 14 days of the birth age of the piglets.

Example 7

In Example 7, each mineral salt-chitosan oligosaccharide was administered orally once daily with 0.1 mg / kg body weight for 7 to 14 days old piglets.

Example 8

Example 8 was treated similarly to Example 7 except administering 2 mg.

Example 9

Example 8 was treated in the same manner as in Example 7 except that 4 mg was administered.

Example 10

Example 10 was treated in the same manner as in Example 7, except that 7.5 mg was administered.

Test of effectiveness as a treatment for cow mastitis

Determination of Laboratory Antimicrobial Effects of Mastitis-inducing Bacteria on Mineral Salts-Chitosan Oligo Derivatives

Comparative Example 2

Comparative Example 2 was carried out as follows to confirm the therapeutic effect on cow mastitis.

First, Staphylococcus aureus, a pathogen that represents cow mastitis, was excreted and isolated according to Hagan and Bruner's Microbiology and Infectious Disease of Domestic Animals (8th edition, pp171-196, 1982). Laboratory antimicrobial effect was assayed.

That is, the cultured bacteria were cultured in a nutrient broth medium at 37 ° C. to examine the antimicrobial activity of the mineral salt-chitosan oligo derivatives by the concentration of the mastitis-inducing bacteria to be identified in the present invention. These bacterial culture solutions were adjusted to 2 x 10 ⁷ CFU / mL (Table 5), and then the antimicrobial effect was performed under laboratory conditions against standard strains (Table 5-1) and field isolate strains (Table 5-2). . That is, the mineral salt-chitosan oligo derivatives were set to five treatment groups in which concentrations (0.5%, 0.1%, 0.01%, 0.001%, 0.0001%) were added, and as a comparative example, the control group was not added. After adding the mineral salt-chitosan oligo derivative, 10 μl of each treatment group was collected at 5 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 18 hours, and 24 hours. After mixing well in a cap test tube containing phosphate buffer solution, 50 μl of this was plated on nutrient agar medium and cultured for one day to count colonies.

As a result, in the case of addition of the mineral salt-chitosan oligo derivative, the rapid killing occurred after 5 minutes compared to the number of bacteria at the time of the first administration. Poetry did not appear to survive and was confirmed to be completely killed.

Results of Somatic Cell Reduction in Field Cows

Therapeutic Effect on Dairy Cow Diarrhea

Example 11

In the present invention, in order to test the treatment effect in naturally infected livestock, the calf causing the diarrhea by selecting only the iron salt or zinc salt chitosan oligo derivative (Table 1) excellent in the antimicrobial effect of the experimental results antibacterial effect test (weight: 60 kg) The treatment effect was tested on cows with a body weight of 650 kg.

For this purpose, the experimental group for testing the therapeutic effect was set as follows.

That is, the comparative example was set to three groups as a comparison experiment group 6 to 8 individuals of the adult cow, Example is selected from the calf diarrhea group and adult cow experimental group showing the symptoms of diarrhea minerals-3 to adult cow 5, 3, 2 mg per horse for 5 days, 3.2 mg for 5 days for calf, and 7.5 mg for 5 days for adult cows were compared. . As a comparative example in connection with the present invention, only the general animal antibiotics causing diarrhea were administered. In other words, in the comparative example, cefazoline antibiotics were injected for 5 days showing diarrhea, and gentamicin [Sungsung microorganism] was orally administered once a day once a day, but diarrhea symptoms did not improve. In the case of treatment with only mineral salt-3, diarrhea was treated as a clinical symptom (Table 6).

a: diarrhea induced, b: adult cows 3.2 mg / head / day for 5 days

c: 3.2 mg / head / day administration for 5 days in calves

d: 7.5 mg / head / day for 5 days in adult cows

Test of effectiveness as a treatment for cow mastitis

Antimicrobial Effect in Laboratory

Comparative example

Comparative Example was carried out as follows to confirm the therapeutic effect on cow mastitis.

First, Staphylococcus aureus, a pathogen showing cow mastitis, was isolated from feces in accordance with Hagan and Bruner's Microbiology and Infectious Disease of Domestic Animals (8th edition, pp171-196, 1982). Was assayed.

In other words, the bacteria cultured in order to determine the antimicrobial activity according to the chitosan preparations and the concentration of the additive according to the mastitis-inducing bacteria to be identified in the present invention was cultured in nutrient broth medium 37 ℃. These bacterial culture solutions were adjusted to 2 × 10 ⁷ CFU / mL), and the standard strain and the isolated strain were subjected to antimicrobial effects under laboratory conditions (Table 7). That is, five treatment groups in which chitosan-oligoderivatives were added at different concentrations (0.5%, 0.1%, 0.01%, 0.001%, 0.0001%) were set. After adding chitosan oligoderivatives, take 10µl of each treatment group for 1 hour, 4 hours, 8 hours and 24 hours, mix well in a cap test tube containing 990µl phosphate buffer, and then smear 50µl onto nutrient agar medium. The colonies were counted by culturing for one day. As a result, in the case where the chitosan oligo derivative was added, the rapid killing occurred after 1 hour compared to the number of bacteria at the time of initial administration, and the difference was somewhat different due to the type of bacteria. In the case of mineral salt-1,2,3, the colonies that survived after 18 hours after treatment at concentrations of 0.001% or more were confirmed to be completely killed. However, in the case of treatment with mineral salt-0 in the standard strain and the outdoor isolate strain, antimicrobial effect was not recognized as compared to the control group. In addition, in the case of mineral salt-1, the antimicrobial efficacy was sharply decreased when added at a concentration of 0.1% or more, but the antimicrobial effect was rapidly increased when the concentration was less than 0.1%. In the case of chitosan oligosaccharides, the molecular weight of the chitosan oligosaccharide was first considered to inhibit the antimicrobial mechanism of the oligosaccharides related to antimicrobial activity by coating on the bacterial membrane or wall. However, when the molecular weight is small, the opposite phenomenon is shown.

*: Number of bacteria in each experimental group identified before administration of initial mineral salts, x 10 ⁷ CFU / mL

-Standard deviation range between experiments: ± 4∼18%

** mineral salt-0: 16 sugar branch content of sugar composition is less than 10%,

** mineral salt-1: 16 sugar branch content of sugar composition is less than 30%,

** mineral salt-2: 16 sugar branch content of sugar composition is less than 50%,

** mineral salt-3: 16 sugar branch content of the sugar composition is less than 100%.

Effective treatment of cow mastitis

Example 11

Example 11 is a result of testing the somatic cell reduction effect by the treatment of cow mastitis caused by Staphylococcus aureus bacteria, milk cows (mean body weight: 650 ± 30 kg) Using the Fosomatic somatic cell tester, the chitosan oligos of Ca, Fe, and Zn salts were orally forced to orally administered 7.5 mg of mineral salt chitosan oligos per kg body weight once a day for 7 days. Existing somatic cells were examined. Analytical milk was collected only from cows that were determined to have somatic cell counts in grades 2 and 3, and in order to reduce the test error between samples, five or more milks were removed from the same nipple, and 50 ml were collected in a sterile tube. Immediately transported in refrigerated state and measured. The results are shown in Table 8 in terms of percentage reduction after treatment to the number of somatic cells before administration of each mineral salt chitosan oligo.

Example 12

Example 12 was carried out in the same manner as in Example 11 except that the dosage was changed to 5 mg.

Example 13

Example 13 was carried out in the same manner as in Example 11, except that the dosage was changed to 3.5 mg.

Poultry disease treatment effect

Example 14

Example 14 as a result of assaying the therapeutic effect on Salmonella-infected laying hens, the use of the mineral salt chitosan oligosaccharide according to the present invention as an anti-Smonellase agent for laying hens showing an average weight of 2.5kg as follows Determined.

First, the broiler farms of the examples were carried out on the same farm, and clinically, the disease symptoms were randomly selected from three chickens estimated to be Gamboro, infectious bronchitis, infectious vaginal bronchitis, combined respiratory syndrome, and E. coli diarrhea, Hagan and Bruner's According to the Microbiology and Infectious Disease of Domestic Animals (8th edition, pp74-87, 1982), the clinical symptoms were pathologically confirmed, indicating that the disease was caused by typical Salmonella infection. In comparison with the administration of the mineral salt chitosan oligosaccharide, the 10 individuals and the treatment group were separated and segregated 20 individuals separately, and 7.5 mg of the mineral salt chitosan preparation per kg of chicken was mixed in the drinking water once a day. Treatment was for days. The results showed that clinical symptoms improved, and at the same time it was determined that the feces showed normal findings and were treated. After the administration of the mineral salt-chitosan preparation, the number of bacteria present in the laying hens was determined as follows. After confirming the clinical symptoms of the first laying hen, the feces in the rectum of the laying hen were forcibly collected. Then, 0.1 g of the collected fecal fraction was accurately collected and dispersed in 1,000 μl of PBS buffer solution, and the bacterial colonies which appeared by diluting them 500 times, 5,000 times, and 500,000 times in SS solid medium were counted.

Example 15

Example 15 was carried out in the same manner as in Example 14 except that the dosage was changed to 5 mg.

Example 16

Example 16 was carried out in the same manner as in Example 13, except that the dose was changed to 3.5 mg.

-: Severe disease, ++: Vitality recovery, +++: Vitality

Figure 1. Changes of Salmonella bacteria in feces (0.1 g) in certain feces (SS solid media, 37 ° C for 24 hours incubation) when 5 mg / day of mineral crab chitosan oligo derivatives were given to laying hens.

Increased Livestock Growth Rate

Example 16

Herein, piglets administered with mineral salt-chitosan oligo were treated with oral administration of 7.5 mg once a day from day 1 to day 4 to day 5, and from day 5 to 5% mineral salt chitosan oligo derivative in 50: After diluting to 1 (w / v) and mixing it with the pig feed indefinitely, the pigs were not fed chitosan oligosaccharide derivatives after weighing continuously for 7 days after checking the weight of the pigs before the first administration. Compared with the increase rate was confirmed for a total of 29 days.

Herein, the sow birth experience, which was set as an experimental group for measuring the rate of increase of livestock, was selected as a pig having a relatively low number of births between 1 and 4 mountains, and used as a material for the piglets given in the example. -A group of 9-12 piglets were selected at the time of average birth from the same farm and the same sows of the comparison group not administered chitosan derivatives, respectively. The results are shown in <Fig. 2>.

And, the daily increase rate was measured every day compared to the administration group for the comparative example that did not administer the mineral salt-chitosan oligo derivatives, and as a result, the daily increase rate was 21.51% (FIG. 3).

Figure 2. Effect of weight gain of certain piglets after 29 days administration of various mineral salt chitosan oligo derivatives to pigs (dose, 2mg / dose per day)

Control: Mineral salt chitosan oligosaccharide administration group

OCHT: General Chitosan Oligo Derivative

Ca-OCHT: calcium salt chitosan oligo derivative

Fe-OCHT: Iron salt chitosan oligo derivatives

Zn-OCHT: Zinc Salt Chitosan Oligo Derivative

Figure 3. After increasing the mineral salt-chitosan oligosaccharide derivatives with feed (7.5 mg, w / w)

Control: No administration of mineral salt-chitosan oligo derivatives

G-D: mineral salt-chitosan oligo derivative administration group

Comparison of antimicrobial effects with general antibiotics

Example 17

Example 17 was purchased from the common antibiotic Oxytettacycline-HCl (OTC, Korea, Samyang Pharmacy, Lot No. 9503-1) commercialized using E. coli 0157: K88ac species and compared with the mineral salt chitosan oligo derivatives Antimicrobial activity was compared. In other words, only the iron salt chitosan oligo derivatives were selected from the OTC and mineral salt chitosan oligo derivatives, and the comparative experiments were performed. w), 0.1%, 0.01%, 0.001%, 0.0001% of the medium was administered, 2x10 ¹¹ CFU / ㎖ E. coli cultures were mixed 1 hour after, 13 hours, 16 hours after the iron salt chitosan oligo derivatives and OTC In addition to comparing the antimicrobial effect with the iron salt chitosan oligo derivatives pH 4,8 and pH 6.75 were also tested for the difference in the antimicrobial effect on the pH effect of the iron salt chitosan oligo derivative formulations. The results are shown in <Table 10>.

**: OTC (Oxytetracycline HCl, OTC 200g / Total 1kg)

2,000 ↑: Displayed when the bacteria count is excessive

Coliforms: 0157: K88ac

Natural oral chitosan, oral administration of derivatives substituted with oligosaccharides and mineral salts to provide excellent antimicrobial effects to livestock, thereby enhancing the absorption efficiency of minerals essential for the living body and functional agents that have enhanced antibacterial properties. As a conventional antimicrobial agent, it does not continue to be resistant to bacteria and remains very effective in preventing livestock diseases from infecting diseases, increasing the growth rate, and treating them without harming the health of consumers.

Claims (17)

Animal disease infection prevention agent that contains mineral salt-chitosan oligosaccharide derivative Animal disease treatment agent with active ingredient of mineral salt-chitosan oligosaccharide derivative Livestock growth rate enhancer containing mineral salt-chitosan oligosaccharide derivatives as an active ingredient [Claim 2] The livestock disease infection prevention agent according to claim 1, wherein the average molecular weight of the mineral salt-chitosan oligosaccharide derivative formulation has a distribution of 2,000 or less. The agent for treating animal diseases according to claim 2, wherein the average molecular weight of the mineral salt-chitosan oligosaccharide derivative formulation has a distribution of 2,000 or less. 4. The animal livestock growth rate enhancer according to claim 3, wherein the average molecular weight of the mineral salt-chitosan oligosaccharide derivative formulation has a distribution of 2,000 or less. The method of claim 1, wherein the mineral salt-chitosan oligosaccharide derivatives range from 0.1 mg to 10 mg in a feed or drinking water mixed with oral administration for livestock disease infection prevention agent The method of claim 2, wherein the mineral salt-chitosan oligosaccharide derivatives range from 1 mg to 10 mg in a feed or drinking water to treat livestock diseases, characterized in that oral administration According to claim 3, Mineral salt-chitosan oligosaccharide derivatives range from 1 mg to 10 mg livestock growth rate enhancer, characterized in that oral administration by mixing with feed and drinking water The livestock disease of Claim 1 or 4 which targets the livestock disease-causing strains of bacteria, such as Escherichia coli, Listeria genus, Staphylococcus aureus, Streptococcus, Salmonella, Krebgiella, and mold such as Aspergillus. Infection Prevention The livestock disease of Claim 2 or 5 which targets the livestock disease-causing strains of bacteria, such as Escherichia coli, Listeria genus, Staphylococcus aureus, Streptococcus, Salmonella, Creepziella, and fungi, such as Aspergillus. Infection Prevention The agent for increasing the livestock growth rate of fungi such as bacteria such as Escherichia coli, Listeria spp., Staphylococcus aureus, Streptococcus, Salmonella spp. And Creepziella spp. 5. The livestock disease prevention agent according to claim 1 or 4, wherein the livestock is pig, cow, cow, chicken, or duck. The agent for treating a livestock disease infection according to claim 2 or 5, wherein the livestock is pig, cow, cow, chicken, or duck. 8. A livestock growth rate enhancer according to claim 3 or 7, wherein the livestock is pig, cow, cow, chicken, duck. 13. The animal treatment according to claim 2, wherein the livestock is cow. The method of claim 1, 2, or 3, wherein the mineral salt chitosan oligosaccharide derivative is substituted for iron salt, calcium salt or zinc salt in the treatment of livestock diseases, characterized in that the substitution rate of 2% to 45%