JP5439638B2 - Mastitis treatment - Google Patents

Description

  The present invention relates to a therapeutic agent for bovine mastitis. The therapeutic agent of the present invention contains nisin as an active ingredient.

  Bovine mastitis is a serious disease that affects the profitability of dairy management. The treatment for mastitis is a prophylactic treatment for healthy cows to remove causative bacteria and reduce the risk of infection and epidemic, and treatment for affected cattle. It is roughly divided into treatment, and measures by different means are being studied.

  For treatment, agents containing ingredients effective against causative bacteria are used, and the main route of administration is injection into the breast. For example, Patent Document 1 describes a mastitis therapeutic agent containing chitin as an active ingredient. Patent Document 2 describes a mastitis therapeutic agent for animals characterized by containing a cypress family plant. Patent Document 3 describes a method for treating mastitis, which comprises using a chemotherapeutic agent and chitin as a therapeutic agent for livestock mastitis and administering the chitin into the breast. Patent Document 4 describes an antibacterial agent containing gassericin A produced by a specific method as an active ingredient and applicable to the treatment of mastitis caused by Staphylococcus aureus. Patent Document 5 describes an injectable agent for mastitis with improved elution of the main agent from a base comprising a main agent such as cefazoline, a medium-chain fatty acid monoglyceride, and an oily base. . Patent document 6 describes that mastitis, which is a Staphylococcus aureus infection of the mammary gland, can be treated with cyclic dinucleotides to attenuate the toxicity of microbial pathogens and to inhibit biofilm formation, The administration of effective antibiotics in the treatment is described.

  As an injection for the treatment of mastitis, an antibiotic containing an active ingredient has been put into practical use. However, a period of drug suspension (shipment suspension period) of about one week is required for antibiotic treatment.

  On the other hand, nisin is a bacteriocin produced by lactic acid bacteria, and is an antibacterial peptide consisting of 34 amino acid residues including abnormal amino acids such as unsaturated amino acids and lanthionine. In addition to being incorporated into foods, Nisin has been studied for its effective use in the medical and hygiene fields.

  There have been several reports regarding the use of this nisin for the treatment of mastitis. For example, Patent Document 7 describes a pharmaceutical composition for the treatment or prevention of mastitis in a mammal comprising a lanthionine-containing antimicrobial peptide or a pharmaceutically acceptable acid addition salt thereof. In addition, a report on the antibacterial effect of nisin in vitro against five types of bacteria causing mastitis (Non-Patent Document 1), and nisin Z with a single dose of 2500,000 IU dissolved in physiological saline, There is a report (Non-patent Document 2) that a treatment rate close to that obtained when gentamicin was used was obtained by intramammary injection.

JP-A-6-9409 Japanese Unexamined Patent Publication No. 7-109227 Japanese Patent Laid-Open No. 2001-39877 JP 2005-80636 A WO2006 / 077856 gazette Special Table 2007-500697 JP-T 9-512711

J. Dairy Sci. 72, 3342-3345 (1989) J. Dairy Sci. 90, 3980-3985 (2007)

  Treatment of bovine mastitis with antibiotics includes food safety issues such as antibiotics in milk, environmental contamination due to the emergence of resistant bacteria, and the transition to chronic mastitis due to resistant bacteria. There's a problem. Treatment with antibiotics is said to have a lower actual cure rate for mastitis prevalence.

  In addition, many conventional mastitis injections are generally those in which the main agent is simply turbid in an oily base material, which is considered to be one of the reasons that the main agent has low diffusion / dispersibility and insufficient therapeutic effect. Even in the above-described trial of treatment using nisin as an active ingredient, no examination has been made from the viewpoint of stability as a preparation or drug delivery in the breast.

  The present inventors have a mastitis treatment comprising nisin as an active ingredient as an effective ingredient as a method of treating mastitis that is less harmful to the environment such as the appearance of resistant bacteria, is safer, and has a high therapeutic effect. We have been eagerly investigating agents. In developing a mastitis therapeutic agent, the present inventors have clarified the effective concentration of nisin as a therapeutic agent, formulated an elution promoting component (water-soluble binder component) and formulated an appropriate interface. We also considered improving the diffusion and dispersibility of the main agent from the oily base material by selecting the activator. As a result, the present invention was completed.

The present invention provides the following.
1) An infusion for mastitis treatment comprising nisin, an oily base, and a food acceptable surfactant.
2) The agent according to 1), in which powdered nisin is dispersed in an oily base.
3) The agent according to 2), wherein the surfactant has an HLB of 10 to 17 (preferably 12 to 16, more preferably 14 to 16).
4) Nisin is powdered using a water-soluble binder (preferably dextrin);
The water-soluble binder / nisin weight ratio is 0.10 / 100 to 50/100 (preferably 0.50 / 100 to 25/100, more preferably 1.0 / 100 to 6.0 / 100);
The surfactant content is 0.10 to 10.0% (preferably 0.25 to 4.0%, more preferably 0.50 to 2.0%);
The agent according to 3), wherein the content of nisin powder is 1.0 to 40% (preferably 2.5 to 30%, more preferably 5 to 25%).
5) As described in any one of 1) to 4) for administering nisin as a daily dose of 500,000 IU or more (preferably 1 million to 20 million IU, more preferably 2 million to 10 million kIU) Agent.
6) A method for treating mastitis, comprising a step of administering the agent according to any one of 1) to 5) to a subject (excluding a human).
7) The treatment method according to 6), wherein the subject is a mastitis cow, and the administration is repeated 1 to several times a day for 2 to 7 days continuously or discontinuously.

FIG. 1-1 is a graph and a photograph showing the particle size distribution of a nisin granulated product and a control (non-granulated). Fig. 1-2 is a graph and a photograph showing the hygroscopicity of Nisin granulated product. Fig. 1-3 is a photograph showing the dispersibility of Nisin granulated product in oil base. Fig. 1-4 is a graph showing the storage test results of Nisin granulated product. FIG. 2 is a photograph showing the formulation characteristics (milk affinity test results) of the product of the present invention. A: KP Rack-5G (manufactured by Fort Dodge Co., Ltd.), B: S.P. Milk Soft Syringe L “Mitaka” (manufactured by Mitaka Pharmaceutical Co., Ltd.), Prototype: Powder product prepared in the examples (freeze-dried product) FIG. 3-1 is a graph showing the therapeutic effect of the product of the present invention (latent mastitis 250 IU injection) in terms of the number of bacteria in milk. FIG. 3-2 is a graph showing the therapeutic effect (potential / 2.5 million IU injection) of the product of the present invention in terms of the number of somatic cells in milk. FIG. 3-3 is a graph showing the remaining antibacterial activity after intramammary injection of the product of the present invention (latency / 2.5 million IU injection). FIG. 3-4 is a graph showing the therapeutic effect (potential / 7.5 million IU injection) of the present invention in terms of the number of bacteria. Fig. 3-5 is a graph showing the therapeutic effect (potential: 7.5 million IU injection) of the product of the present invention in terms of the number of somatic cells Fig. 3-3 is a graph showing residual antibacterial activity after intramammary injection of the product of the present invention (potential: 7.5 million IU injection). FIG. 3-7 is a graph showing the therapeutic effect (clinical type, 7.5 million IU injection) of the present invention in terms of the number of bacteria. Fig. 3-8 is a graph showing the therapeutic effect (clinical type, 7.5 million U injection) of the product of the present invention in terms of the number of somatic cells.

  The injection for mastitis treatment of the present invention contains nisin.

  In the present invention, “nisin” includes nisin A, nisin Z, and nisin Q unless otherwise specified. In the composition of the present invention, preferably nisin A or Z, more preferably nisin A is used.

Nisin A is produced by the lactic acid bacterium Lactococcus lactis and has already been used as a food additive in more than 50 countries including Europe and America. Nisin Z is a bacteriocin similar to nisin A. Of the 34 amino acid residues constituting nisin A, the 27th from the N-terminus is nisin A, whereas nisin Z is an asparagine residue. It differs only in that it is a residue.

  In the present specification, among nisins, nisin A is particularly described as an example, but the description also applies to other nisins unless otherwise indicated.

  In the present specification, regarding the various additives to be added to the therapeutic agent, when it is referred to as “food-acceptable”, unless otherwise indicated, the safety when the additive is taken orally has been clarified. This refers to cases where it is considered that there is no problem in taking a certain amount orally. Additives acceptable as food include ingredients that are acceptable in oral medicine, ingredients that are approved for use in the lips or oral cavity, and additives listed in Appendix 1 of the Food Sanitation Law Enforcement Regulations (designated additives) ), Additives listed in the existing additive list (existing additives), and generally used for food and drink as food and used as additives (general food additives).

  The agent of the present invention is preferably in the form of an ointment or suspension (suspension) in which a fine solid active ingredient is dispersed in a liquid to semi-solid base. In order to form such a dosage form, nisin needs to be pulverized (solidified, dried). The powder nisin that can be used in the agent of the present invention may be in the form of powder or granulated. Granulated nisin is preferable in that it is excellent in workability during preparation of the preparation and is considered to have excellent dispersibility in the base.

  In order to granulate nisin or granulate, various existing means in the pharmaceutical or food field used for the same purpose can be used. For example, there are a spray drying (spray drying) method and a freeze drying (freeze drying) method. The process for pulverization is not particularly limited, but according to the spray drying method, a granulated product having good dispersibility in an oily base can be obtained. Granulation can be performed by repeating the spray drying process, but repeated heat treatment may reduce the antimicrobial activity of nisin. In order to make the antibacterial activity of nisin above a certain level, it is effective to concentrate the purified nisin solution for spray drying in advance. According to the freeze-drying method, the particle size may become non-uniform and may become lumps (thickened by moisture absorption) and may be difficult to disperse uniformly in the base, but aseptic There is an advantage that it is easy to perform. In order to uniformly disperse, homogenization such as homogenization may be effective.

  As for the size of nisin powder, it is considered that the smaller the particle size when dispersed in the base, the better the dispersion stability in the oil base, so that it separates during storage and forms a precipitate. There are few and it is thought that it is favorable. It is also speculated that the immediate release of the active ingredient in the subject at the time of administration will be improved.

  The size of the powdered nisin before being dispersed in the base may be preferably large to some extent mainly from the viewpoint of workability. The particle size of the powdered nisin is not particularly limited as long as the dispersion stability in an oily base and clinical release are good, but for example, 70% by number, preferably 80%, More preferably, if 90% or more of the powder is distributed in a particle size range of 20 to 100 μm, there is no particular problem in using it for the agent of the present invention.

  In the case of nisin powdering, it is preferable to use a binder (binder). Nisin can be pulverized without using a binder, but it has high hygroscopicity and is difficult to handle because it becomes cocoon-like due to moisture in the air. In addition, the component specification of the commercial product (food additive) of Nisin is a mixture of an antibacterial polypeptide and sodium chloride containing a component derived from a non-fat milk medium or a sugar medium. Contains 50% or more sodium chloride. This manufacturing method is based on concentrating a fermentation liquid, adding sodium chloride, and spray-drying the obtained salting-out precipitation.

  From the viewpoint of facilitating release of nisin from the agent, it is preferable to perform granulation using a water-soluble binder.

  Examples of binders include food-acceptable dextrin, cyclodextrin, starch, skim milk powder, whey powder, gum arabic, gelatin, hydroxypropylcellulose, methylcellulose, carboxymethylcellulose, ethylcellulose, and polyvinylpyrrolidone. It is preferable to use a water-soluble but low hygroscopic material. From such a viewpoint, an example of a particularly preferable binder is dextrin.

  The amount of the binder added can be appropriately determined by those skilled in the art, but is 0.1 to 10% (w / w), preferably 0.5 to 7% (w / w), more preferably, with respect to the nisin solution. It can be 1 to 5% (weight / weight).

  The amount of binder added can also be 0.10 / 100 to 50/100, preferably 0.50 / 100 to 25/100, more preferably 1.0 / 100 to 6.0 / 100, as a weight ratio of water-soluble binder / nisin. . In any case, from the viewpoint of ensuring workability and yield during production, the amount of binder used is preferably 3.0 / 100 or more, and it is intended to design a high nisin activity value in the preparation. In some cases, it can be reduced to less than 3.0 / 100, for example 1.6 / 100.

  The content of nisin powder with respect to the total weight of the agent of the present invention can be 1.0 to 40%, preferably 2.5 to 30%, more preferably 5 to 25%. In addition, regarding the component contained in an agent by this invention, when referring to a content and ratio, it is a value based on a weight except the case where it showed especially.

  The agent of the present invention can be used for daily administration of nisin of 500,000 IU or more, preferably 1 million to 20 million IU, more preferably 2 million to 10 million IU. Below this, the clinical effect of one to several doses per day is difficult to be observed. According to the study by the present inventors, the treatment effect tended to be higher when the nisin content was increased. In the present invention, the term “for one day” refers to the amount for one day to be administered to one chamber unless otherwise specified. The necessary daily dose may be given to each quarter of a cow.

  The daily dose can be administered once to divided into several times (for example, 3 times). One dose can be one agent (for example, one syringe) or a plurality of agents. It is desirable to use 1 agent at a time.

  The amount or activity of nisin can be expressed in terms of activity value (IU) or weight by HPLC area ratio based on a standard product. For example, as described in Food Additives Analysis in Foods (Ministry of Health, Labor and Welfare), using Nisin standard products distributed by the Japan Standards Association, comparison of HPLC peak areas or minimum inhibitory circle concentration test method Can be measured. One unit of nisin titer corresponds to 0.025 μg of the antimicrobial polypeptide containing nacin A. In the examples of the present specification, Nisin A manufactured by Ohm Dairy Co., Ltd. is used, and the activity of this purified Nisin product (aqueous solution) is 50 to 100 kIU / mL.

  The agent of the present invention is based on an oily base. The oily base that can be used in the present invention may be oil that is liquid at normal temperature or fat that is solid at normal temperature, and is preferably acceptable as a food. Examples of oily bases that can be used in the agent of the present invention include vegetable oils and fats such as olive oil, soybean oil, rapeseed oil, sunflower oil, palm oil, palm oil, corn oil, peanut oil, sesame oil, Castor oil, bran oil; animal fat foods such as lard, beef tallow, lanolin; processed vegetable oil; edible hardened oil; beeswax; petrolatum, liquid paraffin; and mixtures thereof. Particularly preferred examples include mixtures of petrolatum or liquid paraffin, beeswax, and vegetable oils (eg olive oil, peanut oil, coconut oil).

  In the agent of the present invention, the amount of the oily base as a base can be determined by those skilled in the art in consideration of the concentration of all other components to be included in the agent. 99 to 50%, preferably 97 to 60%, more preferably 95 to 75%.

  The agent of the present invention contains a food-acceptable surfactant. The surfactant has an effect of stabilizing the dispersibility of nisin in the base, and can improve the elution of nisin from the oily base. Surfactants are sometimes referred to as dispersants, emulsifiers, and suspending agents because of their action.

  The surfactant that can be used in the present invention is preferably a surfactant that does not reduce the bactericidal efficacy of nisin. In addition, those acceptable for medical or food hygiene are preferable, and examples thereof include surfactants that are permitted as food additives. Examples of surfactants acceptable as food additives include glycerin fatty acid esters, polyglycerin esters (diglycerin monocaprate, diglycerin monolaurate, diglycerin monostearate, diglycerin monooleate, triglycerin monolaurate, Decaglycerin monolaurate, decaglycerin monostearate, hexaglycerin condensed ricinoleate), organic acid monoglyceride (acetic acid monoglyceride, citric acid monoglyceride, citric acid monoglyceride, diacetyltartaric acid monoglyceride, succinic acid monoglyceride), sorbitan fatty acid ester, propylene glycol fatty acid ester , Lecithin (lecithin, enzymatically decomposed lecithin), sucrose stearate, sucrose palmitate, sucrose myristi Acid ester, sucrose oleic acid ester, sucrose lauric acid ester, sucrose behenic acid ester, sucrose erucic acid ester sucrose mixed fatty acids (oleic acid, palmitic acid, stearic acid) esters.

  Examples of the agent of the present invention include glycerin fatty acid ester, sucrose fatty acid ester, stearoyl calcium lactate, sorbitan fatty acid ester (for example, sorbitan trioleic acid ester (span), etc.), propylene glycol fatty acid ester, polysorbates (for sorbitan fatty acid ester). Those obtained by condensation of about 20 molecules of ethylene oxide, polysorbate 20, polysorbate 60, polysorbate 65, polysorbate 80) can be used particularly preferably. From the viewpoint of not reducing the activity and stability of nisin and being stable in the acidic region where nisin is stable, examples of particularly preferred surfactants are polysorbates.

  According to the study by the present inventors, HLB (Hydrophlic Lipophilic Balance) is used as a surfactant from the viewpoint of stabilizing the emulsion when an agent for treating mastitis containing nisin is constituted as a W / O type emulsion. The value should be relatively low, preferably in the range of 3-6, and an example of a particularly preferred surfactant was sorbitan monooleate. However, it was found that the dispersibility of nisin powder decreases when a surfactant having a low HLB is used when the powder is dispersed. Therefore, the HLB of the surfactant particularly suitable for use in the agent of the present invention in the form of dispersed powder is 10 to 17, preferably 12 to 16, and more preferably 14 to 16. Examples of highly safe surfactants having a suitable HLB value and designated as food additives include polysorbates having strong hydrophilicity and O / W type emulsifiers. The HLB value of polysorbate 20 is 16.7, the HLB value of polysorbate 60 is 14.9, the HLB value of polysorbate 65 is 10.5, and the HLB value of polysorbate 80 is 15.0.

  The content of the surfactant is 0.10 to 10.0%, preferably 0.25 to 4.0%, more preferably 0.50 to 2.0%, based on the total weight of the agent.

  The agent of the present invention is a dosage form suitable for injection into the breast from the nipple mouth. The injection syringe can be filled so that the injection is easy. Moreover, it can also be made into the form of the kit for preparing such an injection. The amount suitable for one administration can be appropriately designed by those skilled in the art, but in the case of cattle, it can be 0.10 to 500 g, preferably 1.0 g to 50 g, more preferably 2.0 g to 8.0 g. .

  The agent of the present invention is not particularly limited with respect to the number of administrations, and can be administered for only one day, or may be repeatedly administered for 2 to 7 days that are continuous or discontinuous.

  The agent of the present invention may contain other ingredients that are acceptable as food or acceptable as pharmaceuticals or veterinary drugs. For example, antioxidants, colorants, preservatives and the like. In order to stabilize nisin, it is preferable to contain methionine and / or thioctic acid.

  The agent of the present invention can be used for the treatment of mastitis. In the present invention, “mastitis” refers to inflammation of mammary tissue caused by bacterial infection in mammals (particularly cattle), unless otherwise indicated. Mastitis is broadly divided into subclinical mastitis, in which there is no abnormality in the appearance of the breast or milk, but inflammation in the mammary gland, and clinical mastitis, in which the appearance abnormality can be visually confirmed. The Clinical mastitis accounts for more than 20% of all dairy cow diseases, and latent mastitis is said to affect more dairy cows.

Examples of bacteria that cause mastitis include the following:
(1) Staphylococcus genus bacteria (staphylococci): Staphylococcus aureus ( Staphylococcus aureus ), CNS (Coagulase Negative Staphylococus, coagulase negative staphylococci);
(2) Streptococcus spp. (Streptococcus): Streptococcus agalactiae ( Lactococcus lactis ), Streptococcus dysgalactie ( Lactococcus lactis ), Streptococcus uberis , Streptococcus bovis ;
(3) Escherichia coli ;
(4) Klebsiella bacteria;
(5) Pseudomonas bacteria: Pseudomonas aeruginosa (Pseudomonas aeruginosa);
(6) Micrococcus bacteria;
(7) Corynebacterium genus: Corynebacterium bovis , Corynebacterium pyogenes
Note that staphylococci, Streptococcus bacteria (Streptococci), Micrococcus bacteria, Corynebacterium bacteria are Gram-positive, Escherichia coli (E. coli), Klebsiella (Klebsiella) bacteria, Pseudomonas bacteria are gram-negative.

Staphylococcus aureus is one of the causative bacteria that are most prone to cause refractory mastitis, among the causative bacteria of mastitis. CNS is not as strong infectious as Staphylococcus aureus, but because it is a resident bacteria such as the bovine body surface, it often causes mastitis due to opportunistic infections, etc., and should be emphasized. CNS includes Staphylococcus intermedius , Staphylococcus hyicus , Staphylococcus xylosus , Staphylococcus epidermidis ( Staphylococcus epidermidis ) and the like.

  When inflammation occurs in the mammary gland, the hydrogen ion concentration (pH) and the amount of chlorine in the milk change, and the lysosomal enzyme N-acetyl β-D-glucosaminidase (NAGase) in milk rises. Cell number increases. Therefore, the NAGase concentration or the number of somatic cells in milk may be used as a criterion for determining whether or not mastitis. Normally, a healthy cow has a somatic cell count of 100,000 / ml or less, but if it exceeds 200,000 / ml, it is considered that there is some kind of inflammation.

  Regarding “mastitis” in the present specification, “treatment” refers to a treatment or growth that suppresses the degree of growth of at least one causative bacterium in a subject that has mastitis, unless otherwise indicated. Treatment to prevent the disease from being caused (bacteriostatic), treatment to reduce the number of bacteria to a problem-free level, preferably treatment to reduce the causative bacteria to a problem-free level, more preferably treatment to reduce to a detection limit or less (bactericidal) Point to. If the state below the detection limit is maintained even after one week from the last administration date, it can be determined that mastitis has been completely cured.

  A person skilled in the art can confirm the degree of detection or presence of causative bacteria by a conventional method. For example, the milk collected from the mammary gland is diluted stepwise with sterilized physiological saline, applied to the agar medium by plate smearing, cultured at 37 ° C for 48 hours, and colonies formed on the agar medium Count the number and calculate the number of bacteria (cfu / ml) in the collected milk.

  If the number of somatic cells in the milk is used as a reference instead of or in combination with the number of bacteria, the level at which a therapeutic effect can be obtained is 500,000 / ml or less, preferably 400,000 / m1 or less More preferably, it is 30 / 10,000 ml or less, more preferably 200,000 / ml or less, still more preferably 180,000 / ml or less, and most preferably 100,000 / ml or less.

  The number of somatic cells can be measured by a person skilled in the art by a conventional method. For example, milk collected from the mammary gland can be diluted as necessary and / or stained with propidium iodide or the like and measured with a dedicated cell number measuring device such as a flow cytometer.

  NAGase can also be used as a reference instead of or in combination with the number of bacteria or somatic cells.

  The agent of the present invention is an injection and is administered by injection from the mouth of the nipple. The time of administration can be appropriately set by those skilled in the art. Since safe ingredients are used as food, it can be administered during lactation. In addition to administration during lactation, it is possible to administer high concentrations of drugs in the breast for a long time during dry periods without milking.

  Preparation of the agent of the present invention can be appropriately carried out by those skilled in the art. For example, thioctic acid, a nisin stabilizer, is added to an oily base, dissolved by heating, cooled, and then added with a surfactant. Depending on the condition, it can be prepared by adding sterile powdered nisin sterilized and stirring with a homogenizer. Oily base materials and surfactants are sterilized by dry heat at 120 ° C for 2 hours as necessary.

  The agents of the present invention containing methionine and thioctic acid have been confirmed to be stable for at least 4 months at 25 ° C. Moreover, when it disperse | distributes to milk and an affinity was evaluated, since it disperse | distributes favorably compared with the existing product, it is thought that it is excellent also for delivery in a breast.

  The agent of the present invention can exert an effect on any of the above-mentioned mastitis-causing bacteria, but typical breasts such as staphylococci (Staphylococcus aureus, coagulase-negative staphylococci) streptococci and Escherichia coli. It is particularly effective against flame-causing bacteria. The agent of the present invention can completely cure latent mastitis caused by these bacteria and relatively mild clinical mastitis.

  The agent of the present invention is nisin whose active ingredient is clear as food safety, so even if nisin is contained in milk obtained from the administered subject (eg, milk), If it is below an appropriate level, it can be said that there are fewer problems than in the case of injections containing other antibiotics as active ingredients. Nisin residue in milk can be measured by a person skilled in the art according to a standard method.For example, the solution is diluted stepwise, and a prescribed inhibition circle prepared by the agarwell method, the disk method, or the cup method is used. The formed dilution solution with the minimum concentration can be calculated as 1 unit, and the reciprocal of the dilution rate can be calculated as antibacterial activity (AU; Arbitrary Unit).

  According to the dosage form of the present invention, when Nisin A 2.5 million IU was administered 3 times a day for 2 consecutive days to cattle with latent mastitis, it could be completely cured, and from the last administration date (day 0), 3 to On the fourth day, the residual activity in the milk obtained can be below the limit of detection. In addition, with the dosage form of the present invention, when Nisin A 2.5 million IU was administered 3 times a day for 2 days to cattle with mild clinical mastitis, it can be completely cured and from the last administration date (Day 0) On the 4th to 5th days, the residual activity in the milk obtained can be below the detection limit.

[Production of Nisin powder]
1． Binder screening Using a spray dryer (yamato pulvis GB22), using the following binder as the binder under conditions of inlet temperature 150-160 ° C, outlet temperature 80-90 ° C, air volume 0.3-0.5m ³ / min ( 3% against nisin solution), workability, adhesion to the chamber (affects the chamber through which the sample passes, affects yield), and the hygroscopicity of the powdered product.

  A dextrin having less adhesion to the chamber and having a good weight yield, and having an optimal hygroscopic property and water solubility of the powder was selected as a binder to be used below. As negative data, skim milk powder was not dissolved in the purified nisin A solution and precipitated, resulting in poor workability. The whey powder was less adhered to the chamber, but the powder product was highly hygroscopic. Cyclodextrins were scattered during the powdering process, and the weight yield was very poor.

2． Manufacture of spray-dried products (granulated products) Using a spray dryer (yamato pulvis GB22), dextrin as binder (150-160 ° C, outlet temperature 80-90 ° C, air flow 0.3-0.5m ³ / min) A nisin A spray-dried product (non-granulated product and granulated product) was produced using a 3% nisin solution and a weight ratio of 3/100 as dextrin / nisin. Non-granulated products were produced by spray drying only once. The granulated product was produced by repeating the spray-drying process repeatedly until a certain size was achieved, that is, by repeating the process of spraying the liquid again on the once dried powder and drying. Note that the amount of binder used was determined to be 3% against the liquid, focusing on two points: adhesion to the chamber and yield. At 3% or less, there was a lot of adhesion to the chamber and the weight yield decreased.

  The particle size distribution and appearance are shown in FIG.

  The spray-dried product (granulated product) obtained was tested for hygroscopicity, dispersibility in an oily base, and stability of antibacterial activity by a conventional method. The results are shown in Figs. 1-2-4. By using a granulated product, the hygroscopicity was lowered, and the workability and storage stability were improved. Moreover, the granulated product had a high specific gravity, the amount was reduced, and scattering was reduced. In addition, there is an advantage that the particles are large and are less likely to become lumpy when added to an oily base. Further, the granulated product has improved antibacterial activity stability compared to the untreated product.

3． Production of freeze-dried product (powder product) Pre-freeze dextrin (1.6% purified solution, 1.6 / 100 weight ratio as dextrin / nisin) as a binder in nisin A purified solution, and then freeze-dried (Kyowa Vacuum) Using a dryer RLE-103, Yamato freeze dryer DC41A), freeze-dried products (powder products) were produced under the conditions of a drying time of 5 to 8 days and a final temperature of 20 ° C. (treatment amount 2 L). Here, the amount of binder used was determined to be 1.6% with emphasis on the nisin activity value. Although the adhesion to the container cannot be ignored with this amount, the maximum amount of activity was secured by setting the amount of the binder within a range where the necessary amount could be secured.

  3 million IU / g of powdered sterile nisin A could be obtained.

[Manufacture of Nisin Combination Mastitis Treatment Agent]
1． Emulsion type <Formulation>
An emulsion-type nisin compound was produced at the compounding ratio shown in the table below.

<Manufacturing procedure>
(1) A surfactant and thioctic acid are mixed in an oil base and heated.
(2) Mix Blue No. 1 and methionine into nisin solution and warm.
(3) The nisin solution is gradually added to the oily base with stirring.
(4) After emulsification, cool rapidly with stirring, and finish stirring after 5 minutes.

2． Ointment type (granulated product used)
<Combination>
An ointment-type nisin compound was produced at the compounding ratio shown in the table below.

<Manufacturing procedure>
(1) Mix thioctic acid with oil base and warm.
(2) Blue No. 1, methionine is uniformly dispersed, and then an emulsifier is added and mixed.
(3) Add powdered nisin A and mix to prevent lumps.
(4) Stir with a homogenizer. (10,000rpm, 1min)
3． Ointment type (powder product)
<Combination>
An ointment-type nisin compound was produced at the compounding ratio shown in the table below.

<Process>
(1) Add thioctic acid to the oil base and sterilize by dry heat at 120 ° C for 2 hours.
(2) Sterilize each of blue No. 1 and emulsifier with dry heat at 120 ℃ for 2 hours.
(3) Cool while mixing the oily base, add Blue No. 1 and disperse uniformly.
(4) Furthermore, an emulsifier is mixed.
(5) Add powdered sterile nisin A and mix.
(6) Stir with a homogenizer. (3,500rpm, 2min)
Four. Evaluation of activity and stability The nisin activity per dose of each agent produced above was summarized.

  The reason why the activity of the ointment type with a blending amount of 20% was increased was that a high concentration of purified nisin was used.

  When the stability of the active ingredient was evaluated by the storage stability test (25 ° C.) for the ointment type with a blending amount of 10%, it was confirmed that it was stable for at least 4 months.

  Also, when dispersed in milk, prepared in the present product A (KP-rack-5G, Fort Dodge Co., Ltd.), existing product B (SP milk soft syringe L "Mitaka", Mitaka Pharmaceutical Co., Ltd.) and this example. The emulsion type produced oil droplets and did not disperse, while the ointment type both dispersed well (Figure 2).

[Clinical effect of Nisin combination mastitis treatment agent]
1． Effect on latent mastitis (Nisin A 2.5 million IU / day)
Latent mastitis 11 cases cow, in particular S. Suffering intermedius were cases 4 cases, S. Hyicus 1 case, S. Epidermidis 1 case, S. Xylosus 1 case, other CNS 1 case, Micrococcus spp 1 In one case, one corynebacterium bacterium and one streptococci were treated with the ointment type (spray-dried) therapeutic agent of Example 2 once a day (Nisin A 2.5 million IU) for 2 consecutive days.

As a result, in all cases, reduction and body cell number reduction of bacterial numbers is observed, 5 cases (S. Intermedius 3 cases, S. Hyicus one case, Micrococcus spp 1 case) has been completely cured (below, Fig. 3-1, Fig. 3-2).

  The remaining period of antibacterial activity in milk was 3 to 4 days (table below, Fig. 3-3).

2． Effect on latent mastitis (Nisin A 7.5 million IU / day)
Latent mastitis 7 cases of cows, in particular S. Suffering intermedius were cases 2 cases, S. Epidermidis 1 case, S. Hyicus 1 case, S. Simulans 1 case, S. Xylosus 1 case, corynebacterium genus bacteria 1 Cases were injected with the same ointment-type injections as described above three times a day (Nisin A 7.5 million U) for 2 consecutive days.

As a result, in all cases, reduction marked decrease and the number of somatic cells of bacteria count of up to about 100,000 / ml are observed, S. Intermedius 2 cases, S. Epidermidis 1 case, S. Hyicus 1 cases cure (Table below, Fig. 3-4, Fig. 3-5).

  Moreover, the remaining period of the antibacterial activity at this time was 4 to 5 days (the following table, FIG. 3-6).

3． Effect on clinical mastitis (Nisin A 7.5 million U / day)
6 cases of relatively mild clinical mastitis affected by CNS, streptococci, E. coli etc. in dairy cows, in particular 1 case affected by S. intermedius , 1 case of Stp . Bovis, 1 case of unknown, E. coli Two cases, Klebsiella genus bacteria, were injected intramammary with 3 ointments of the same type as above (Nisin A 7.5 million U) for 2 consecutive days.

As a result, in all cases, there was a marked decrease in the number of bacteria and a decrease in the number of somatic cells, and 5 cases except for Klebsiella bacteria infection were completely cured (table below, Fig. 3-7, Fig. 3-8). .

Four. Summary of clinical trials The agent of the present invention is effective in latent mastitis caused by coagulase-negative staphylococci (CNS), the main causative agent of latent mastitis in dairy cows, and treatment with higher nisin A content is effective The effect was high. The product of the present invention was effective for relatively mild clinical mastitis caused by CNS, streptococci and E. coli. It has been found that the agent of the present invention containing nisin as an active ingredient can be effectively used for the treatment of mastitis, particularly for the treatment of latent mastitis and mild clinical mastitis.

Claims (6)

An infusion for mastitis comprising nisin, an oily base, and a food acceptable surfactant ,
The above-mentioned agent in which nisin powder is dispersed . HLB of the surfactant is 10 to 17, agent according to claim 1. Nisin is powdered with a water-soluble binder;
The water-soluble binder / nisin weight ratio is from 0.10 / 100 to 50/100;
The content of the surfactant is 0.10 to 10.0%;
The content of nisin powder is from 1.0 to 40%, agent according to claim 2. The agent according to any one of claims 1 to 3 , for administering nisin of 500,000 IU or more as one day. A method for treating mastitis, comprising a step of administering the agent according to any one of claims 1 to 4 to a subject (excluding a human). 6. The method of treatment according to claim 5 , wherein the subject is a cow with mastitis, and the administration is repeated 1 to several times a day for 2 to 7 days continuously or discontinuously.

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