Clostridium perfringens spore formation inhibitor

Abstract

According to the present invention, provided is a Clostridium perfringens sporulation inhibitor that contains at least one inorganic compound at such a concentration as to cause the precipitation of C. perfringens in vivo, wherein the inorganic compound is a salt of at least one selected from the group consisting of K, Na, Mg, Ca and Fe and the inorganic compound is coated with a protective layer containing a hardened vegetable oil.

Description

Technical Field

[0001] The present invention relates to an inhibitor of spore formation of Clostridium perfringens.

Background Art

[0002] Necrotic enteritis (NE) in the poultry industry is an important disease that causes significant economic losses ($20 billion US / year) (Non-Patent Document 1). Due to necrotic enteritis, the growth of broilers is greatly reduced, and in the worst case, it leads to death. In NE, the mucosa of the small intestine becomes necrotic, and a yellow-brown or bile-colored pseudomembrane forms. In potential NE, ulcers like depressions form on the mucosal surface (Non-Patent Document 2). Necrotic enteritis is known to be caused by the Gram-positive bacterium Clostridium perfringens. C. perfringens is known to be a commensal bacterium in the intestinal tract of poultry (Non-Patent Document 3). The vegetative cells of C. perfringens are bacilli (1 x 10 μm), but when the environment deteriorates, they form spores (spherical with a diameter of 1 μm). It is known that NetB toxin (such as NetB) is secreted during spore germination, which is a factor causing enteritis (Non-Patent Document 4, Fig. 1). Therefore, to control the toxin production of C. perfringens, it is necessary to understand the life cycle of C. perfringens. C. perfringens is classified into types according to the types of toxins produced (Non-Patent Document 5). In 2008, two new types were defined from type A. C. perfringens that produces the toxin NetB (Necrotic Enteritis Toxin B-like) was defined as type F, and C. perfringens that produces the toxin CPE (Clostridium Perfringens Enterotoxin) was defined as type G. NetB has been reported to be a major cause of NE (Non-Patent Document 6).

[0003] Furthermore, although C. perfringens lacks flagella, it is known to move by extending and retracting its pili (Type-IV-pillar: T4P) and adhere to the intestinal tract of host animals (Non-Patent Literature 7). The structure and mechanism of T4P pili in Gram-positive bacteria, including C. perfringens, have also been reported, and it is known that they utilize ATP energy to extend and retract the tip of the T4P (Non-Patent Literature 8, Fig. 1). There are also reports on the mechanism of action of Clostridium species in the intestinal tract. Clostridium species secrete mucin-degrading enzymes, which break down mucin in the mucus layer, causing thinning, and then adhere to epithelial cells, inducing cell death and inflammation by disrupting tight junctions (Non-patent document 9, Figure 1). One method for controlling necrotizing enterocolitis in poultry involves the use of growth-promoting antibiotics (AGPs: Antibiotic Growth Promoters). Bacitracin methylenedisalicylic acid and aviramycin are used as AGPs in poultry (Non-Patent Literature 10, 11). However, due to environmental impact and the emergence of antibiotic-resistant bacteria, AGP use has been restricted globally. In Europe, they were completely banned in 2006, and in the United States, antibiotics used for both humans and animals, such as tylosin and colistin, have also been banned. Environmentally friendly AGP alternatives that address these challenges are needed. One method that does not utilize antibiotics is to use nano-iron oxide as an inhibitor of Clostridium difficle spore formation (Patent Document 1). The method disclosed therein involves nano-iron oxide directly acting on Clostridium difficle spores to suppress germination, but the document does not describe the effect on C. difficle vegetative cells. The Clostridium difficile described in Patent Document 1 differs from Clostridium perfringens phenotypically, chemically, and phylogenetically, and also possesses flagella; therefore, in 2016, it was named Clostridioides difficile (Non-Patent Document 12). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 6385573 [Non-patent literature]

[0005] [Non-Patent Document 1] Poult. Sci. (2019) vol.98, 128-135 [Non-Patent Document 2] Poult. Sci. (1992) vol.71, pp1145-1153 [Non-Patent Document 3] Avian Diseases (2001) vol.45 pp887-896 [Non-Patent Document 4] Microbiology and Molecular Biology Reviews (2015) vol.79, No.1, pp19-37 [Non-Patent Document 5] Anaerobe (2018) vol.53 pp5-10 [Non-Patent Document 6] PloS Pathogens (2008) vol.4, Issue 2, e26 [Non-Patent Document 7] Trends in Microbiology (2020) vol.28(5) pp340-348 [Non-Patent Document 8] Microbiology and Molecular Biology Reviews (2013) vol.77, No.3, pp323-341 [Non-Patent Document 9] Journal of the Japanese Society of Internal Medicine (2016), Vol. 106, No. 3, pp. 466-471 [Non-Patent Document 10] Poult. Sci. (2003) vol.82, pp360-363 [Non-Patent Document 11] Avian Patho. (2016) vol.45, No.3, pp365-369 [Non-Patent Document 12] Anaerobe (2016) v40 pp95-99 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Therefore, the present invention aims to provide an antibiotic alternative capable of preventing or treating Clostridium perfringens infection, such as necrotizing enterocolitis. The present invention also aims to provide supplements and feeds containing such alternatives. The present invention also aims to provide a method for administering such alternatives. [Means for solving the problem]

[0007] The inventors have found that when birds are given an inorganic compound that allows C. perfringens to precipitate in a vegetative cell state, spore formation can be suppressed. This method allows C. perfringens to be excreted from the body as feces before it produces toxins, thus suppressing Clostridium perfringens infection. Since C. perfringens is also a commensal bacterium in the human intestinal tract, the inorganic compound can be used not only in animals such as birds, but also as a supplement to prevent or treat Clostridium perfringens infection in humans, or to alleviate the symptoms of the infection. In other words, this application provides the following inventions. 1. A Clostridium perfringens spore formation inhibitor containing at least one inorganic compound at a concentration that causes Clostridium perfringens to precipitate in vivo, wherein the inorganic compound is at least one salt selected from the group consisting of K, Na, Mg, Ca, and Fe, and the inorganic compound is coated with a protective layer containing a plant-hydrogenated oil. 2. The spore formation inhibitor according to claim 1, wherein the inorganic compound is a salt of an anion formed from an acid selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, lignosulfonic acid, silicic acid, lactic acid, citric acid, gluconic acid, succinic acid, fumaric acid, iodine, and iodic acid. 3. The spore formation inhibitor according to claim 1 or 2, wherein the inorganic compound is at least one selected from the group consisting of KCl, NaCl, MgCl2, CaCl2, and FeCl2. 4. The spore formation inhibitor according to any one of 1 to 3 above, wherein the inorganic compound is KCl. 5. A spore formation inhibitor according to any one of items 1 to 4 above, further comprising a polysaccharide. 6. The spore formation inhibitor according to 5, wherein the polysaccharide is at least one selected from the group consisting of pullulan, xanthan gum, guar gum, carrageenan, arabic gum, pectin, carboxymethylcellulose, chondroitin, tara gum, locust bean gum, alginates (sodium salt, potassium salt, calcium salt, or ammonium salt), alginate esters, and mixtures thereof. 7. The spore formation inhibitor according to item 5, wherein the polysaccharide is arabic gum. 8. The spore formation inhibitor according to any one of 1 to 7, wherein the inorganic compound and, if necessary, the polysaccharide are coated with a protective layer containing a hydrogenated plant oil. 9. The spore formation inhibitor according to 8, wherein the hydrogenated vegetable oil is a hydrogenated oil of rapeseed oil, linseed oil, safflower oil, sunflower oil, soybean oil, corn oil, peanut oil, cottonseed oil, sesame oil, rice oil, olive oil, palm oil, palm kernel oil, or coconut oil. 10. The spore formation inhibitor according to any one of 1 to 7 above, wherein the inorganic compound and the polysaccharide optionally contained therein are protected by two layers of rapeseed hardened oil and benzoic acid resin. 11. A supplement for preventing or treating Clostridium perfringens infection, containing the spore formation inhibitor according to any one of 1 to 10 above. 12. The supplement according to 10 or 11 above, containing an inorganic compound at a concentration at which an inorganic compound can be administered at a dose of 1 mg / kg body weight / day or more. 13. The supplement according to 10 above, wherein the Clostridium perfringens infection is necrotizing enteritis. 14. A feed containing the spore formation inhibitor according to any one of 1 to 8 above. 15. The feed according to 14 above, further containing a conventional feed, and the concentration of the inorganic compound is 0.013 mmol / kg or more based on the mass of the conventional feed. 16. A method for preventing or treating Clostridium perfringens infection in non-human animals, comprising administering an inorganic compound of at least one salt selected from the group consisting of K, Na, Mg, Ca, and Fe to the intestinal tract of the non-human animal. 17. A method for suppressing spore formation of Clostridium perfringens in vitro, comprising incubating at an effective concentration of 500 mM or more of an inorganic compound of at least one salt selected from the group consisting of K, Na, Ca, and Fe to suppress spore formation.

Effects of the Invention

[0008] According to the present invention, it is possible to prevent the vegetative cells of C. perfringens from changing into spores. According to the present invention, it is also possible to prevent or treat necrotic inflammation derived from C. perfringens. According to the present invention, it is also possible to exhibit a growth promoting effect on livestock while preventing or treating Clostridium perfringens infection.

Brief Description of the Drawings

[0009] [Figure 1] Figure 1 shows the results of a sedimentation experiment of Clostridium perfringens ATCC10873. [Figure 2] Figure 2 shows the results of observation experiments on agglutination and cell shape of three strains of Clostridium perfringens (ATCC10873, SM101, and CNEOP004). [Figure 3] Figure 3 is a phase-contrast microscope image of a Clostridium perfringens SM101 spore. [Figure 4] Figure 4 shows a phase-contrast microscope image of Clostridium perfringens SM101 cocci after the addition of KCl (500 mM). [Figure 5] Figure 5 shows the results of agglutination experiments when KCl (500 mM) w / 1% AG and NaCl (500 mM) w / 1% AG were added to a mixed bacterial cell of Clostridium perfringens ATCC10873, Salmonella enterica IAM1648, and E. coli MG1655KCl. [Figure 6] Figure 6 shows the enteric coating test results for Coated-Arabic Gum. [Figure 7] Figure 7 shows the results of the elution test for Coated-KCl. [Modes for carrying out the invention]

[0010] [Abbreviation] AG: Arabic Gum AGP: Anti-biotic growth promoter BMD: Bacitracin Methylene Disalicylate BWG: Body Weight Gain Cp: ​​Clostridium perfringens DW: distilled water (pure water) NE: Necrotic enteritis NT:non treatment (no treatment, no additives) OD:optical density

[0011] [Inorganic compounds] The inorganic compound used in this invention is at least one salt selected from the group consisting of K, Na, Mg, Ca, and Fe. The anions constituting the salt are not particularly limited, but examples include anions formed from acids selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, lignosulfonic acid, silicic acid, lactic acid, citric acid, gluconic acid, succinic acid, fumaric acid, iodine, iodic acid, etc. From an economic standpoint, Cl - It is preferable. Specifically, it is preferable that the inorganic compound is at least one selected from the group consisting of KCl, NaCl, MgCl2, CaCl2, and FeCl2. It is more preferable that the inorganic compound contains KCl. It is even more preferable that the inorganic compound is KCl. The concentration of the inorganic compound in the spore formation inhibitor of the present invention is not particularly limited, as long as it is sufficient to cause sedimentation of Clostridium perflingens in vivo without causing aggregation. The concentration required for sedimentation varies depending on the type of inorganic compound or the bacterial cell concentration. For example, when the bacterial cell concentration is such that the optical density at a wavelength of 660 nm is about 2.0, the KCl concentration is 100 mM or higher, the NaCl concentration is 500 mM or higher, the MgCl2 concentration is over 500 mM, and the FeCl2 concentration is 10 mM or higher. The concentration of the inorganic compound may be increased to exert the sedimentation effect, but the effect plateaus at a certain concentration, so from an economic standpoint, a concentration of, for example, 1,000 mM or less is desirable.

[0012] [Polysaccharide] Polysaccharides have been reported to exert an agglutination effect on Gram-negative bacteria (International Publication No. 2019 / 177172), but experiments conducted by the present inventors showed that they did not exert either a sedimentation or agglutination effect on the Gram-positive bacterium Clostridium perfringens (see Examples 1 and 5 below). However, it was found that when combined with the inorganic compound, the sedimentation and agglutination effects of the inorganic compound on C. perfringens were enhanced. In particular, it was found that when the spore formation inhibitor of the present invention exerts an agglutination effect on Gram-positive bacteria, it can also improve the growth rate of livestock. Therefore, the spore formation inhibitor of the present invention may further contain polysaccharides. Polysaccharides that can be used in the present invention include at least one selected from the group consisting of pullulan, xanthan gum, guar gum, carrageenan, arabic gum, pectin, carboxymethylcellulose, chondroitin, tara gum, locust bean gum, alginates (sodium salt, potassium salt, calcium salt, or ammonium salt), alginate esters, and mixtures thereof. Of these, arabic gum, carboxymethylcellulose, guar gum, carrageenan, locust bean gum, and pullulan are preferred from the viewpoint of cost-effectiveness and feed registration status, and arabic gum is more preferred.

[0013] Polysaccharides can be applied to the target either together with or separately from the inorganic compound. For example, as will be described later, when forming a spore formation inhibitor by applying a coating agent to an inorganic compound and a polysaccharide, the inorganic compound and the polysaccharide may be applied together with the coating agent and given to the target as an agent in which the inorganic compound and the polysaccharide coexist in a single spore formation inhibitor, or the inorganic compound may be coated with the coating agent to form a spore formation inhibitor, and then applied to the target together with a polysaccharide coated with the coating agent. The concentration of polysaccharide in the spore formation inhibitor of the present invention can be determined as appropriate, but 0.5 to 3% by mass is preferred from an economic standpoint. 1 to 2% by mass is more preferred. Unless otherwise specified, the unit "%" in this specification represents mass%. The concentration of the inorganic compound required to precipitate Clostridium perflingens in vivo can be lower when used in combination with a polysaccharide than when not used in combination with a polysaccharide. For example, when used in combination with Arabic gum, the precipitation effect can be exerted at a KCl concentration of 50 mM or higher in the spore formation inhibitor of the present invention containing KCl. Preferably, it can be 100 mM or higher (e.g., 100 to 1,000 mM), and more preferably 200 mM or higher (e.g., 200 to 700 mM). The concentration of the inorganic compound can be increased to exert the precipitation effect, but the effect plateaus at a certain concentration, so from an economic standpoint, for example, 500 mM or lower is desirable.

[0014] The spore formation inhibitor of the present invention may contain an excipient. The excipient is not particularly limited as long as it is commonly used to improve molding and is pharmacologically acceptable, but examples include calcium carbonate, silicon dioxide, calcium silicate, zeolite, sorbitol, corn starch, talc, yeast bentonite, rice husk, liquid paraffin, polysaccharides other than polysaccharides that have the property of agglutinating Clostridium perfringens, monosaccharides, disaccharides, etc. When the spore formation inhibitor of the present invention contains an excipient, the amount of the excipient is usually preferably 0.1 to 100 parts by mass per 100 parts by mass of the spore formation inhibitor. The spore formation inhibitor of the present invention may also contain any additives that may be included in supplements or feeds. Examples of additives include amino acids, organic acids, vitamins, color enhancers (carotenoids), flavorings, and probiotics. When the spore formation inhibitor contains any additives, the amount of the additive is usually preferably 0.1 to 100 parts by mass per 100 parts by mass of the spore formation inhibitor.

[0015] [Covering agent] The coating agent forms a protective layer of the inorganic compound. The coating agent can be any substance capable of forming an enteric coating and is safe for ingestion by livestock or humans, without any particular restrictions. The coating agent may be used alone or in combination of two or more. From the viewpoint of ease of handling and economy, the coating agent is preferably a hydrogenated vegetable oil or a substance commonly used as a tablet coating, such as benzoic acid resin, shellac, zeine, hydroxypropyl methylcellulose, and maltitol. Examples of hydrogenated vegetable oils include rapeseed oil, linseed oil, safflower oil, sunflower oil, soybean oil, corn oil, peanut oil, cottonseed oil, sesame oil, rice oil, olive oil, palm oil, palm kernel oil, or coconut oil. The aforementioned hydrogenated vegetable oil is preferably a hydrogenated rapeseed oil, linseed oil, safflower oil, sunflower oil, soybean oil, corn oil, peanut oil, cottonseed oil, sesame oil, rice oil, olive oil, palm oil, palm kernel oil, or coconut oil. Among the coating agents, hydrogenated rapeseed oil and benzoic acid resin are preferred. The layer of hydrogenated rapeseed oil is preferred because it can dissolve the core in a short time. The layer of benzoic acid resin is preferred because it can dissolve the spore formation inhibitor in neutral to alkaline conditions (after passing through the stomach).

[0016] The coating agent is preferably in an amount of 5 to 90% by mass, and more preferably 20 to 30% by mass, based on the total mass of the spore formation inhibitor of the present invention. The coating agent may also contain any additives that can be contained in animal feed or human pharmaceuticals. The coating may be a single layer or multiple layers of two or more. A multi-layer coating is preferable because it allows for easier control of the elution rate in the body. In particular, it is preferable to use a rapeseed hydrogenated oil layer as the outermost layer and a benzoic acid resin layer as the innermost layer in contact with the inorganic compound, as this allows the coating to dissolve in the intestines rather than the stomach. The spore formation inhibitor of the present invention is preferably dissolved in gastric juice at a rate of less than 60%, and preferably dissolved in intestinal juice at a rate of 70% or higher. Such dissolution rates can be adjusted by using a two-layer or multi-layer membrane, or by controlling the type of coating agent and the thickness of the coating in each layer.

[0017] It is particularly preferable that the inorganic compound is a potassium salt (especially KCl), the polysaccharide is Arabic gum, and the coating agent is at least one selected from the group consisting of hydrogenated rapeseed oil and benzoic acid resin. In particular, it is especially preferable that the inorganic compound is a potassium salt (especially KCl), the polysaccharide is Arabic gum, and the coating agent is a two-layer structure of hydrogenated rapeseed oil and benzoic acid resin. In this case, it is even more particularly preferable that the layer formed from the benzoic acid resin is in contact with the inorganic compound, and the layer formed from hydrogenated rapeseed oil is formed on top of it. Most preferably, the KCl concentration in the spore formation inhibitor is 500 mM, the Arabic gum concentration is 1%, the layer formed from the benzoic acid resin is in contact with the inorganic compound, and the layer formed from hydrogenated rapeseed oil is formed on top of it.

[0018] [Covering method] The method for coating the spore formation inhibitor is not particularly limited, but for example, a coated spore formation inhibitor can be obtained by spraying a coating agent, which has been heated to a temperature higher than its melting point and made liquid, while flowing a powder or granular core in a commercially available fluidized bed spray granulator. It is preferable to make the coated spore formation inhibitor to a size of about 0.05 to 5 mm, as this makes it easier to handle. The temperature at which the coating agent is heated is not particularly limited as long as it is above the melting point of the coating agent, but it is preferable that it is about 5°C to 15°C higher than the melting point of the coating agent.

[0019] The spore formation inhibitor of the present invention can be administered orally to humans or non-human animals, or directly to the intestinal tract of non-human animals. When administered directly to the intestinal tract, there is no need to consider inactivation by stomach acid or irritation to the gastric mucosa, and therefore it is not necessary to coat it with a protective layer containing hydrogenated plant oil. In both cases of oral ingestion and intestinal administration, the amount of inorganic compound to be ingested and the number of administrations are the same as those for the supplement described later.

[0020] 〔supplement〕 The spore formation inhibitor of the present invention can also be used as a supplement for humans or non-human animals. The form of the supplement is not particularly limited and may include tablets, granules, powders, drinks, etc. In addition to the spore formation inhibitor of the present invention, the supplement of the present invention may further contain known components for supplements, such as antioxidants and proteins. The supplement of the present invention is suitable for daily intake. The dosage of the supplement of the present invention varies, for example, depending on the body weight of the recipient. For example, the supplement of the present invention can be formulated so that the recipient can be administered an inorganic compound at a dose of 1 mg / kg body weight / day or more, preferably 1 to 100 mg / kg body weight / day. Typically, the supplement of the present invention is administered 1 to 3 times per day. Target animals for administering the supplement of the present invention include humans, ruminants such as cattle, sheep, and goats, and monogastric animals such as horses, pigs, chickens, dogs, and fish.

[0021] 〔feed〕 The spore formation inhibitor of the present invention can be given directly to non-human animals such as livestock, or it can be mixed with excipients or diluents such as corn, soybean flour, rice bran, fish meal, or brewer's yeast to form animal feed. The feed of the present invention may also contain any additives that may be included in conventional feeds. The feed of the present invention is suitable for daily intake. The amount of feed intake will vary depending on the size of the livestock, but for example, in the case of chickens, it is desirable to provide the inorganic compound at a rate of approximately 0.013 to 2.7 mmol / kg (1 to 340 ppm), preferably 0.14 to 1.4 mmol / kg (10 to 170 ppm), compared to conventional feed other than the spore formation inhibitor. Furthermore, if a spore formation inhibitor containing polysaccharides is included in conventional feed, the intake amount will increase accordingly. For example, when the feed contains 133 ppm of Arabic gum as a polysaccharide, it is desirable to provide inorganic compounds in an amount of approximately 0.013 to 1.4 mmol / kg (1 to 170 ppm), preferably 0.14 to 0.7 mmol / kg (10 to 85 ppm), and more preferably 0.54 to 0.8 mmol / kg (40 to 60 ppm) relative to the conventional feed. In this specification, "ppm" means "ppm by mass". In this specification, "livestock" means animals kept by humans. Specifically, this includes ruminants such as cattle, sheep, and goats, and monogastric animals such as horses, pigs, chickens, dogs, and fish. It is particularly preferable to feed the feed of the present invention to monogastric animals. The method of feeding the spore formation inhibitor of the present invention is not particularly limited. [Examples]

[0022] Example 1. Bacterial cell sedimentation experiment Clostridium perfringens ATCC10873 was cultured for 24 hours under anaerobic conditions at 37°C on a GAM plate (Nissui Pharmaceutical Co., Ltd., modified GAM broth "Nissui"). Anaerobic culture was performed using the anaerobic culture kit "Anelopack" manufactured by Mitsubishi Gas Chemical Company. The obtained culture was suspended in 4 mL of pure water produced using a Merck Millipore pure water production system, and the optical density (OD) of the suspension was adjusted to approximately 2.0 at a wavelength of 660 nm. To a vial containing 0.5 mL of this suspension, the precipitates listed in Table 1 were added at the concentrations listed in Table 1, and Clostridium perfringens was brought into contact with each substance. Arabic Gum ("AG") was used as a positive control. The vials were left standing at 37°C for 8 hours. During this time, the optical density of the samples was continuously measured automatically.

[0023] <Rating> A precipitating agent was deemed to have a precipitating effect if the decrease in optical density of the sample 8 hours after the addition of the precipitating agent was more than twice the decrease in optical density of the sample without the precipitating agent after 8 hours (i.e., the decrease in optical density due to natural sedimentation of bacterial cells).

[0024] <Results and Discussion> The results are shown in Table 1 and Figure 1. While AG exhibited a sedimentation effect against Gram-negative bacteria, it did not have a sedimentation effect against the Gram-positive bacterium C. perfringens. In the absence of AG, sedimentation was observed with KC1 at concentrations of 100 mM or higher, NaCl at 500 mM, or FeCl2 at 10 mM or higher. This suggests that the cations reduced the repulsive force by altering the surface potential of the Clostridium perfringens cells. MgCl2 (500 mM), which did not exhibit a sedimentation effect in the absence of AG, did exhibit a sedimentation effect when used in combination with AG.

[0025] [Table 1]

[0026] Example 2. Experiment to observe agglutination and bacterial cell shape. The same experiment as in Example 1 was performed using three strains of Clostridium perfringens [ATCC10873 (toxin-producing Type A), SM101 (toxin-producing Type F: CPE toxin producer), and CNEOP004 (toxin-producing Type G: netB toxin producer), all of which are rod-shaped bacteria]. However, the optical density of the samples was not measured. Samples were collected 8 hours after the addition of the precipitating substance, and the presence or absence of aggregation and the cell shape of each bacterial strain were observed using an upright microscope (OLYMPUS, model: BX50). <Result> The results are shown in Table 2 and Figure 2. Aggregate (AG), which exhibited an agglutination effect against Gram-negative bacteria, did not exhibit an agglutination effect against Clostridium perfringens. When KC1 or NaCl was added to the sample, in the absence of AG, the shape of the bacterial cells changed from rods to cocci, but the cells themselves did not agglutinate. In the presence of KC1 or NaCl and AG, the bacterial cells agglutinated in a cocci state. When MgC12 was added to the sample, in the absence of AG, the shape remained rod-shaped and no agglutination was observed. However, in the presence of AG, agglutination of Clostridium perfringens was observed. When CaC12 and FeC12 were added to the samples, Clostridium perfringens agglutinated in rod-shaped form in the absence of AG.

[0027] [Table 2]

[0028] Example 3. Measurement of the surface potential of Clostridium perfringens. The same experiment as in Example 1 was conducted using three strains of Clostridium perfringens (ATCC10873, SM101, and CNEOP004). However, the optical density of the samples was not measured. Eight hours after adding the precipitating agent, the sample was collected, diluted 100-fold, and the surface potential of Clostridium perfringens cells was measured using a Malvern Zetasizer Nano (model: Nano-ZS). The surface potential of the precipitating agent before and after its addition was also measured (the concentration used for measurement was matched to the concentration used for the surface potential measurement of the sample). <Results and Discussion> The results are shown in Tables 3 and A. Clostridium perfringens did not agglutinate when Arabic Gum or KCl were added individually, but agglutination occurred when both were present. This is thought to be because the bacterial cell surface is negatively charged in water, and the addition of KCl increased the negative charge of the bacterial cell surface potential. Furthermore, the presence of Arabic Gum brought the surface potential to neutral, leading to mutual interference between the bacterial cell, KCl, and Arabic Gum, resulting in agglutination (Table 3). In the case of strain ATCC10873, when MgC12 was added, the bacterial cell surface potential initially became neutral, and then, upon addition of Arabic Gum, a change (negative charge) was observed again in the bacterial cell surface potential (Table 3-1). This suggests that the bacterial cell, MgC12, and Arabic Gum mutually interfered with each other, leading to agglutination. On the other hand, in SM101 and CNEOP004, when MgC12 was added, the cell surface potential shifted significantly towards neutral, suggesting aggregation (Tables 3-2 and 3-3). When CaC12 (20 mM or higher) and FeC12 (10 mM or higher) were added, respectively, in the absence of AG, the cell surface potential shifted significantly towards neutral, and the cells and CaC1 2、 It is thought that the bacterial cells and FeC12 interacted with each other and aggregated (Table 3).

[0029] [Table 3]

[0030] [Table A]

[0031] Example 4. Phase-contrast microscopy observation of Clostridium perfringens cocci upon KCl addition. In general, under stressful environments, there are numerous reports that E. coli, Campylobacter, and Helicobacter pylori change shape into a coccoid form called "coccoid-form," and spheroidization has also been reported in Clostridium (International Journal of Current Microbiology and Applied Sciences (2016), 5(7), 210-223). The spore formation process requires cell division, which in turn requires the necessary nutrients. Clostridium perfringens spores, like those of Clostridium difficile, have been reported to transition from phase-bright spores to phase-dark spores before becoming vegetative cells (J. Am. Chem. Soc. (2014) v136 pp14498-14504). In Example 2, since pure water without nutrients was used, it was expected that the cocci observed in Example 2 were in the "coccoid form" rather than spores. Therefore, we investigated whether the cocci (1 μm in diameter) that formed when KCl (0.5 M) was added to Clostridium perfringens were vegetative cells or spores. Spores have a reduced water content and concentrated DNA, so they glow white under a phase-contrast microscope (Phase-bright spores). Phase-dark spores are observed before germination into vegetative cells, and appear darker under a phase-contrast microscope. Vegetative cells appear lighter in color under a phase-contrast microscope, and black spots can be seen inside the cell. For reference, spores of Clostridium perfringens strain SM101 were observed under a phase-contrast microscope (Figure 3). In Figure 3, the bright white oval shapes are phase-bright spores, while the darker, similarly shaped spores are phase-dark spores. In addition, fragments of vegetative fungi were observed. On the other hand, in Example 2, when the spherical bacteria that formed when KCl (500 mM) was added to the Clostridium perfringens SM101 strain were observed using a phase-contrast microscope (Figure 4), it was inferred that they were not spores, as they were pale in color and had black spots inside, similar to the rod-shaped cells which are vegetative cells. Therefore, it is thought that the change in the shape of Clostridium perfringens cells due to the addition of KCl, etc., occurred in the vegetative cell state without the formation of spores.

[0032] Example 5. Experiment to observe the aggregation of intestinal bacteria For intestinal bacteria other than Clostridium perfringens, agglutination of each strain was observed in the same manner as in Example 2. The selected intestinal bacteria were the Gram-positive bacteria Lactobacillus casei ATCC393 and Bifidobacterium animalis JCM1190, and the Gram-negative bacteria Salmonella enterica IAM1648 and E. coli MG1655. Lactobacillus casei ATCC393 and Bifidobacterium animalis JCM1190 were cultured at 37°C on MRS plates (Difco), E. coli MG1655 on LB plates (BD), and Salmonella enterica IAM1648 on NB plates (Difco). Bifidobacterium animalis JCM1190 was cultured under anaerobic conditions, similar to Clostridium perfringens, using the "Anelopack" anaerobic culture kit from Mitsubishi Gas Chemical Company. The other bacteria were cultured under aerobic conditions. The results are shown in Table 4. For reference, the results for Clostridium perfringens ATCC10873, as performed in Example 2, are also included. The agglutination effect of KCl in the presence of Arabic Gum was specific to Clostridium perfringens among Gram-positive bacteria, but did not show agglutination activity for Lactobacillus casei or Bifidobacterium animalis. Gram-negative bacteria Salmonella enterica and E. coli agglutinated with Arabic Gum. This result is similar to that reported in WO2019 / 177172. None of the Gram-negative bacteria agglutinated with KCl alone, but they agglutinated when used in combination with Arabic Gum. Therefore, it is suggested that the combined use of KCl and Arabic Gum specifically agglutinates Clostridium perfringens, Salmonella enterica, and E. coli, which are considered harmful bacteria. Furthermore, FeCl2 showed agglutination activity against Gram-positive bacteria such as Clostridium perfringens, Lactobacillus casei, and Bifidobacterium animals.

[0033] [Table 4]

[0034] Example 6. Experiment to observe the aggregation of three types of harmful intestinal bacteria. Similar to Example 5, three strains of Clostridium perfringens ATCC10873, Salmonella enterica IAM1648, and E. coli MG1655 were cultured. Suspensions of each strain were prepared using the same method as in Example 1. After adjusting the optical density (OD) of each suspension to approximately 0.6-0.7 at a wavelength of 660 nm, the suspensions of the three bacterial strains were mixed, the mixed cells were collected by centrifugation, and the mixture was adjusted in vials to a final optical density of approximately 2.0 (at a wavelength of 660 nm). A mixed solution of 500 mM KCl and 1% Arabic Gum, and a mixed solution of 500 mM NaCl and 1% Arabic Gum were added to the vials and brought into contact with the mixed cells. Each vial was left to stand at 37°C, and the optical density was automatically and continuously measured. The results are shown in Figure 5. It was confirmed that the combination of 500 mM KCl and 1% Arabic Gum, and the combination of 500 mM NaCl and 1% Arabic Gum, can rapidly agglutinate three types of harmful intestinal bacteria.

[0035] Example 7. Preparation of a coated spore formation inhibitor for animal feed. Arabic Gum (manufactured by Wako Pure Chemical Industries, Ltd.) and KCl (manufactured by Wako Pure Chemical Industries, Ltd.) were used as core materials, and hydrogenated rapeseed oil (melting point 67°C) and benzoin resin (manufactured by Chuo Koryo Co., Ltd.) were used as coating agents. A coated spore formation inhibitor for feed was obtained by spraying a predetermined amount of the coating agent, which had been liquefied by heating to a temperature higher than its melting point, onto the powdered or granular core. Arabic Gum was used as a single-layer coating, with 25 parts by mass of hydrogenated rapeseed oil covering 75 parts by mass of core material. Hereafter, this additive will be referred to as "Coated-Arabic Gum". On the other hand, KCl was used as a two-layer coating, with 2.23 parts by mass of benzoin resin as the first layer (inner layer) and 13 parts by mass of hydrogenated rapeseed oil as the second layer (outer layer) covering 84.77 parts by mass of core material. Hereafter, this additive will be referred to as "Coated-KCl".

[0036] Example 8. Enteric coating test of Coated Arabic Gum (Artificial gastric juice treatment) Pure water produced using a Merck Millipore pure water production system was mixed with 0.2% NaCl and 0.2% pepsin (from Porcine stomach Mucosa, 1:5,000, 2,500 units / mg), adjusted to pH 2, and then Coated-Arabic Gum prepared in Example 7 was added. Enzyme treatment was carried out at 37°C for 2 hours. Enteric coating was evaluated by automatically and continuously measuring the optical density during this time. Note that "2 hours" refers to the time it takes for the feed to reach and pass through the chicken's stomach. (Artificial Intestinal Fluid Treatment) After treatment with artificial gastric fluid, 0.2% trypsin (from Porcine Pancreas, 1:5,000; 4,500 units / mg) was added, the pH was adjusted to 6, and enzyme treatment was carried out at 37°C for 2 hours. Enteric coating was evaluated by automatically and continuously measuring the optical density during this period. Note that "2 hours" is the time it takes for the feed to reach the chicken's intestines and pass through them. The optical density in both treatments was measured as turbidity (Optical density [OD], wavelength 190 nm) using a Shimadzu UVmini-1240 spectrophotometer. Hydrochloric acid and sodium hydroxide were used as pH adjusters in both treatments. The results are shown in Figure 6. The elution rate was suppressed to approximately 60% or less two hours after the start of gastric juice treatment, while it exceeded 90% in intestinal juice treatment. From these results, it was found that good release control was possible with samples coated with hydrogenated rapeseed oil as a coating agent.

[0037] Example 9. Dissolution test of Coated KCl Coated-KCl prepared in Example 7 was added to pure water produced using a Merck Millipore pure water production system, and an elution test was conducted at 37°C. The elution rate was measured by automatically and continuously measuring the optical density during this time. The optical density in both treatments was measured as turbidity (Optical density [OD], wavelength 190 nm) using a Shimadzu UVmini-1240 spectrophotometer. The results are shown in Figure 7. The elution rate was suppressed to approximately 50% or less after 2 hours from the start, while it reached 80% in the intestinal fluid treatment. From these results, it was found that good release control could be achieved with samples coated with a two-layer coating of benzoin resin and hydrogenated rapeseed oil as coating agents.

[0038] Example 10. Clostridium perfringens infection test The Coated-KCl prepared in Example 7 was added to the feed described in Hofacre, CL, et al., Avian Dis. 1998;42(3):579-84 so that the amount of core material was 40 ppm, thereby obtaining a feed composition. The experiment was commissioned to SPRG and conducted in the United States. In a free-range chicken house, 25 day-old broiler chicks were placed in one section and fed with the specified feed composition. A total of 500 chicks were reared for 6 weeks from the time of feeding. The test conditions were as follows: On Day 0, a commercially available coccidia attenuated vaccine (0.007 mL vaccine / bird) was administered. Subsequently, on Days 14, 15, and 16, after 4 hours of fasting and 2-3 hours of water deprivation, Clostridium perfringens was administered at a dose of 1 x 10⁶. 8 The contents were added to a drinker to achieve a concentration of cfu / mL, and then water was added. We evaluated the mortality rate and intestinal damage score from necrotic enteritis (NE) during the rearing period. The intestinal damage score from NE was assigned as follows: 0 = none, 1 = mild, 2 = moderate, 3 = severe, and the mean score was calculated. The control group (without additives) was used as the control (labeled "Challenge Control" in Table 5). Bacitracin methylenedisalicylate (BMD) was used as the positive control. BMD is known to be effective against necrotizing enterocolitis caused by Clostridium perfringens and is widely used as an antimicrobial growth promoter (AGP), being one of the most widely used AGPs in the United States. BMD was added to the feed described in the Hofacre literature above to a concentration of 55 ppm by volume to create the positive control feed composition. Commercially available BMD (Zoetis' BMD®, uncoated) was used as is. The results are shown in Table 5. BMD reduced mortality by 13.2% compared to the Challenge Control. Coated-KCl reduced mortality by 3.8% compared to the Challenge Control. The NE lesion score was significantly improved compared to the Challenge Control (Control: 0.75, BMD: 0.30, Coated-KCl: 0.52).

[0039] [Table 5]

[0040] Example 11. Growth Expansion Test The Coated-KCl and Coated-Arabic Gum prepared in Example 7 were added to the basal feed without feed additives shown in Table 6, so that the core material amounts were 40 ppm by volume and 100 ppm by volume, respectively, to obtain feed compositions.

[0041] [Table 6]

[0042] The test chicks were day-old male broiler chicks (UK Chunky) of a breed specifically for broilers, selected for their weight of 38-46g. All test groups were fed the basic feed without feed additives shown in Table 6 in a free-range chicken house. Three replicates of 50 chicks each were raised for three weeks from the time of weaning, ensuring a nearly even weight distribution. To introduce environmental stress, approximately 1 liter of water per group was sprayed onto the entire floor once daily during the rearing period, resulting in a wet floor environment. The test groups were: 1) Control (Non-treatment), 2) Positive control (BMD 55 ppm), 3) Coated-Arabic Gum (133 ppm), and 4) Coated-Arabic Gum (133 ppm) + Coated-KCl (47 ppm). Body Weight Gain (BWG) was evaluated during the rearing period. The results are shown in Table 7. The results are shown with the negative control set to 100. Compared to the Control (Non-treatment) group, BMD showed improved weight gain across all timeframes (0-21 days old). On the other hand, while the combined Coated-Arabic Gum and Coated-KCl group did not show a significant difference compared to the Control group, the weight gain effect became apparent with growth, and was observed in the 15-21 day age range. Since the fish are typically shipped at 6-7 weeks of age, a sufficient weight gain effect was expected.

[0043] [Table 7]

Claims

1. A Clostridium perfringens spore formation inhibitor comprising at least one inorganic compound at a concentration that causes Clostridium perfringens to precipitate in vivo, wherein the inorganic compound is at least one salt selected from the group consisting of K, Na, Mg, Ca, and Fe, and the inorganic compound is coated with a protective layer containing a plant-hydrogenated oil.

2. The spore formation inhibitor according to claim 1, wherein the inorganic compound is a salt of an anion formed from an acid selected from the group consisting of hydrochloric acid, sulfuric acid, phosphoric acid, carbonic acid, lignosulfonic acid, silicic acid, lactic acid, citric acid, gluconic acid, succinic acid, fumaric acid, iodine, and iodic acid.

3. The inorganic compound is KCl, NaCl, MgCl 2 CaCl 2 , and FeCl 2 The spore formation inhibitor according to claim 1 or 2, which is at least one selected from the group consisting of the following.

4. The spore formation inhibitor according to claim 1, wherein the inorganic compound is KCl.

5. The spore formation inhibitor according to claim 1, further comprising a polysaccharide.

6. The spore formation inhibitor according to claim 5, wherein the polysaccharide is at least one selected from the group consisting of pullulan, xanthan gum, guar gum, carrageenan, arabic gum, pectin, carboxymethylcellulose, chondroitin, tara gum, locust bean gum, alginates (sodium salt, potassium salt, calcium salt, or ammonium salt), alginate esters, and mixtures thereof.

7. The spore formation inhibitor according to claim 5, wherein the polysaccharide is Arabic gum.

8. The spore formation inhibitor according to claim 1, wherein the inorganic compound and optionally included polysaccharides are coated with a protective layer containing a hydrogenated plant oil.

9. The spore formation inhibitor according to claim 8, wherein the hydrogenated vegetable oil is a hydrogenated rapeseed oil, linseed oil, safflower oil, sunflower oil, soybean oil, corn oil, peanut oil, cottonseed oil, sesame oil, rice oil, olive oil, palm oil, palm kernel oil, or coconut oil.

10. The spore formation inhibitor according to claim 1, wherein the inorganic compound and optionally included polysaccharides are protected by two layers of rapeseed hydrogenated oil and benzoic acid resin.

11. A supplement for preventing or treating Clostridium perfringens infection, comprising the spore formation inhibitor described in claim 1.

12. The supplement according to claim 10 or 11, comprising an inorganic compound in a concentration that allows for the administration of an inorganic compound in a dose of 1 mg / kg body weight / day or more.

13. The supplement according to claim 10, wherein Clostridium perfringens infection is necrotizing enterocolitis.

14. A feed containing the spore formation inhibitor described in claim 1.

15. The feed according to claim 14, further containing conventional feed, wherein the concentration of the inorganic compound is 0.013 mmol / kg or more, based on the mass of the conventional feed.

16. A method for preventing or treating Clostridium perfringens infection in a non-human animal, comprising administering an inorganic compound of at least one salt selected from the group consisting of K, Na, Mg, Ca, and Fe to the intestinal tract of the non-human animal.

17. A method for inhibiting spore formation of Clostridium perfringens in vitro, comprising incubating an inorganic compound of at least one salt selected from the group consisting of K, Na, Ca, and Fe at an effective concentration of 500 mM or higher.

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