Clostridium perfringens sporulation 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

Inhibitor of sporulation of Clostridium perfringens

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

[0002] Necrotic enteritis (NE) is a serious disease in the poultry industry, resulting in significant economic losses (US$2 billion per year) (Non-Patent Document 1). Necrotic enteritis significantly reduces the growth of broilers and, in severe cases, can lead to death. In NE, the small intestinal mucosa becomes necrotic, forming a yellowish-brown or bile-colored pseudomembrane. In latent NE, pit-like ulcers appear 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 normal inhabitant of the intestinal tract of poultry (Non-Patent Document 3). C. perfringens vegetative cells are rod-shaped (1 x 10 μm), but under adverse environmental conditions, they form spores (spherical, 1 μm in diameter). Upon spore germination, they secrete NetB toxins (NetB, etc.), which are known to cause enteritis (Non-Patent Document 4, Fig. 1). Therefore, to control toxin production by C. perfringens, it is necessary to understand its life cycle. C. perfringens is classified into types based on the type of toxin it produces (Non-Patent Document 5). In 2008, two new types were defined from type A: type F, which produces a toxin called NetB (Necrotic Enteritis Toxin B-like), and type G, which produces a toxin called CPE (Clostridium Perfringens Enterotoxin). NetB has been reported to be the main cause of NE (Non-Patent Document 6).

[0003] Although C. perfringens does not have flagella, it is known to move by extending and retracting its Type IV pili (T4P) and adhere to the intestinal tract of host animals (Non-Patent Document 7). The structure and mechanism of T4P pili of Gram-positive bacteria, including C. perfringens, have also been reported, and it is known that the tip of T4P extends and retracts using ATP energy (Non-Patent Document 8, Fig. 1). The mechanism of action of Clostridium in the intestinal tract has also been reported. Clostridium secretes mucin-degrading enzymes, degrading mucin in the mucus layer, and adhering to epithelial cells through thinned mucus, inducing cell death, and inducing inflammation by disrupting tight junctions (Non-Patent Document 9, Fig. 1). One method for controlling necrotic enteritis in poultry is the use of antibiotic growth promoters (AGPs). Bacitracin methylenedisalicylate and avilamycin are used as AGPs in poultry (Non-Patent Documents 10 and 11). However, due to environmental impacts and the emergence of drug-resistant bacteria, restrictions on the use of antimicrobial propionates (AGPs) have been tightened worldwide. They were completely banned in Europe in 2006, and zoonotic antibiotics such as Tylosin and Colistin have also been banned in the United States. Environmentally friendly AGP alternatives that address these issues are needed. One non-antibiotic method is the use of nano-iron oxide as a sporulation inhibitor for Clostridium difficile (Patent Document 1). The method disclosed therein inhibits germination of Clostridium difficile spores by directly acting on them, but the document does not describe its effect on C. difficile vegetative cells. The Clostridium difficile described in Patent Document 1 is phenotypically, chemically, and phylogenetically distinct from Clostridium perfringens, and was named Clostridioides difficile in 2016 due to the presence of flagella (Non-Patent Document 12).

[0004] Patent No. 6385573

[0005] Poult. Sci. (2019) vol.98, 128-135Poult. Sci. (1992) vol.71, pp1145-1153Avian Diseases (2001) vol.45 pp887-896Microbiology and Molecular Biology Reviews (2015) vol.79, No.1, pp19-37Anaerobe (2018) vol.53 pp5-10PloS Pathogens (2008) vol.4, Issue 2, e26Trends in Microbiology (2020) vol.28(5) pp340-348Microbiology and Molecular Biology Reviews (2013) vol.77, No.3, pp323-341Journal of the Japanese Society of Internal Medicine (2016) Vol. 106 No. 3, pp466-471Poult. Sci. (2003) vol.82, pp360-363Avian Patho. (2016) vol.45, No.3, pp365-369Anaerobe (2016) v40 pp95-99

[0006] Therefore, an object of the present invention is to provide an alternative to antibiotics that can prevent or treat Clostridium perfringens infections, such as necrotizing enterocolitis. Another object of the present invention is to provide a supplement and feed containing such an alternative. Another object of the present invention is to provide a method for administering such an alternative.

[0007] The present inventors found that feeding birds an inorganic compound capable of precipitating C. perfringens in the vegetative cell state inhibited spore formation. This method allows C. perfringens to be excreted from the body in the feces before it produces toxins, thereby inhibiting Clostridium perfringens infection. Since C. perfringens is also a normal inhabitant of the human intestinal tract, the inorganic compound can be used not only in animals such as birds, but also as a supplement for preventing or treating Clostridium perfringens infection in humans, or for alleviating the symptoms of infection. That is, the present application provides the following inventions. 1. 1. A sporulation inhibitor for Clostridium perfringens containing at least one inorganic compound at a concentration sufficient to precipitate Clostridium perfringens 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 hydrogenated vegetable oil. 2. The sporulation inhibitor according to 1 above, wherein the inorganic compound is a salt with 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 sporulation inhibitor according to 1 or 2 above, wherein the inorganic compound is at least one selected from the group consisting of KCl, NaCl, MgCl2, CaCl2, and FeCl2. 4. 4. The sporulation inhibitor according to any one of 1 to 3 above, wherein the inorganic compound is KCl. 5. The sporulation inhibitor according to any one of 1 to 4 above, further comprising a polysaccharide. 6. The sporulation inhibitor according to 5 above, 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, alginate (sodium salt, potassium salt, calcium salt, or ammonium salt), alginate ester, and mixtures thereof. 7. The sporulation inhibitor according to 5 above, wherein the polysaccharide is arabic gum.8. The sporulation inhibitor according to any one of 1 to 7 above, wherein the inorganic compound and optionally contained polysaccharide are coated with a protective layer containing hydrogenated vegetable oil. 9. The sporulation inhibitor according to 8 above, wherein the hydrogenated vegetable oil is 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 sporulation inhibitor according to any one of 1 to 7 above, wherein the inorganic compound and optionally contained polysaccharide are protected by two layers of hydrogenated rapeseed oil and benzoic acid resin. 11. A supplement for preventing or treating Clostridium perfringens infection, comprising the sporulation inhibitor according to any one of 1 to 10 above. 12. The supplement according to 10 or 11 above, comprising the inorganic compound at a concentration that allows the inorganic compound to be administered at a dose of 1 mg / kg body weight / day or more. 13. The supplement according to claim 10, wherein the Clostridium perfringens infection is necrotizing enterocolitis. 14. A feed containing the sporulation inhibitor according to any one of claims 1 to 8. 15. The feed according to claim 14, further containing a 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, the method 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 sporulation of Clostridium perfringens in vitro, the method comprising incubating the non-human animal with 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 more.

[0008] According to the present invention, it is possible to inhibit the transformation of vegetative cells of C. perfringens into spores. According to the present invention, it is also possible to prevent or treat necrotizing inflammation caused by C. perfringens. According to the present invention, it is also possible to prevent or treat Clostridium perfringens infection while exerting an effect on weight gain in livestock.

[0009] Figure 1 shows the results of a sedimentation experiment for Clostridium perfringens ATCC10873. Figure 2 shows the results of an experiment to observe the aggregation and cell shape of three Clostridium perfringens strains (ATCC10873, SM101, and CNEOP004). Figure 3 shows a phase-contrast microscopic photograph of Clostridium perfringens SM101 spores. Figure 4 shows a phase-contrast microscopic photograph of Clostridium perfringens SM101 cocci in the presence of 500 mM KCl. Figure 5 shows the results of an aggregation experiment for a mixture of Clostridium perfringens ATCC10873, Salmonella enterica IAM1648, and E. coli MG1655KCl cells in the presence of 500 mM KCl with 1% Ag and 500 mM NaCl with 1% Ag. FIG. 6 shows the results of the enteric test for Coated-Arabic Gum, and FIG. 7 shows the results of the dissolution test for Coated-KCl.

[0010] [Abbreviations] AG: Arabic Gum AGP: Anti-biotic growth promoter BMD: Bacitracin Methylene Disalicylate BWG: Body Weight Gain Cp: Clostridium perfringens DW: Distilled water NE: Necrotic enteritis NT: Non-treatment (no treatment, no additives) OD: Optical density

[0011] [Inorganic Compound] The inorganic compound used in the present invention is at least one salt selected from the group consisting of K, Na, Mg, Ca, and Fe. The anion constituting the salt is not particularly limited, but examples thereof 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 economical viewpoint, Cl -is preferred. Specifically, the inorganic compound is preferably at least one selected from the group consisting of KCl, NaCl, MgCl2, CaCl2, and FeCl2. It is more preferred that the inorganic compound includes KCl. It is even more preferred that the inorganic compound is KCl. The concentration of the inorganic compound in the sporulation inhibitor of the present invention is not particularly limited as long as it is a concentration sufficient to precipitate Clostridium perfringens in vivo without causing agglutination. The concentration required for precipitation varies depending on the type of inorganic compound or the bacterial cell concentration. For example, when the bacterial cell concentration has an optical density of about 2.0 at a wavelength of 660 nm, the KCl concentration is 100 mM or more, the NaCl concentration is 500 mM or more, the MgCl2 concentration is more than 500 mM, and the FeCl2 concentration is 10 mM or more. Although the inorganic compound concentration may be increased to exert a precipitation effect, the effect plateaus at a certain concentration, and therefore, from an economical viewpoint, it is desirable to use an inorganic compound concentration of, for example, 1,000 mM or less.

[0012] [Polysaccharides] Polysaccharides have been reported to have a flocculating effect on Gram-negative bacteria (WO 2019 / 177172). However, experiments conducted by the present inventors showed that they did not have a sedimentation or flocculation effect on the Gram-positive bacterium Clostridium perfringens (see Examples 1 and 5 described below). However, when combined with an inorganic compound, it was found that the sedimentation and flocculation effects of the inorganic compound on C. perfringens were enhanced. In particular, it was found that when the sporulation inhibitor of the present invention has a flocculation effect on Gram-positive bacteria, it can also improve the weight gain effect of livestock. Therefore, the sporulation inhibitor of the present invention may further contain a polysaccharide. Examples of 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. Among these, from the viewpoints of cost-effectiveness and feed registration status, arabic gum, carboxymethyl cellulose, guar gum, carrageenan, locust bean gum, and pullulan are preferred, with arabic gum being more preferred.

[0013] The polysaccharide can be administered to a subject together with the inorganic compound or separately. For example, as described below, when a sporulation inhibitor is formed by applying a coating agent to an inorganic compound and a polysaccharide, the inorganic compound and the polysaccharide may be combined and applied with the coating agent, resulting in a single sporulation inhibitor in which the inorganic compound and the polysaccharide coexist, and then administered to the subject. Alternatively, a sporulation inhibitor may be prepared by applying a coating agent to the inorganic compound, and then the resulting sporulation inhibitor and the polysaccharide coated with the coating agent may be administered to the subject. The concentration of the polysaccharide in the sporulation inhibitor of the present invention can be determined as appropriate, but from an economical standpoint, a concentration of 0.5 to 3% by mass is preferred. 1 to 2% by mass is more preferred. Unless otherwise specified, the unit "%" in this specification refers to % by mass. The concentration of the inorganic compound required to precipitate Clostridium perfringens in vivo can be lowered 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 achieved when the KCl concentration in the sporulation inhibitor of the present invention containing KCl is 50 mM or higher. The concentration is preferably 100 mM or more (e.g., 100 to 1,000 mM), and more preferably 200 mM or more (e.g., 200 to 700 mM). The inorganic compound concentration may be increased to exert a sedimentation effect, but the effect reaches a plateau at a certain concentration, so from an economical point of view, the concentration is preferably 500 mM or less.

[0014] The sporulation inhibitor of the present invention may contain an excipient. The excipient is not particularly limited as long as it is pharmacologically acceptable and is commonly used to improve moldability. 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 aggregating Clostridium perfringens, monosaccharides, disaccharides, etc. When the sporulation inhibitor of the present invention contains an excipient, the amount of excipient is typically preferably 0.1 to 100 parts by mass per 100 parts by mass of the sporulation inhibitor. The sporulation inhibitor of the present invention may also contain any additive that can be contained in a supplement or feed. Examples of additives include amino acids, organic acids, vitamins, color enhancers (carotenoids), flavorings, probiotics, etc. When the sporulation inhibitor contains an optional additive, the amount of the optional additive is usually preferably 0.1 to 100 parts by mass per 100 parts by mass of the sporulation inhibitor.

[0015] [Coating Agent] The coating agent forms a protective layer for the inorganic compound. Any substance capable of forming an enteric coating and safe for consumption by livestock or humans can be used without particular limitations. The coating agent may be used alone or in combination of two or more. From the viewpoints of ease of handling and economy, preferred coating agents are hydrogenated vegetable oils or substances commonly used as tablet coating agents, such as benzoic acid resin, shellac, zein, 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, and coconut oil. The hydrogenated vegetable oil is preferably 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. Hydrogenated rapeseed oil and benzoic acid resin are particularly preferred as coating agents. A layer of hydrogenated rapeseed oil is preferred because it allows the core to elute in a short period of time. A layer of benzoic acid resin is preferred because it allows the sporulation inhibitor to elute in neutral or alkaline conditions (after passing through the stomach).

[0016] The coating agent is preferably present in an amount of 5 to 90% by mass, more preferably 20 to 30% by mass, based on the total mass of the sporulation inhibitor of the present invention. The coating agent may also contain any additives that can be contained in feed or human medicines. The coating may be a single layer or two or more layers. A multi-layer coating is preferred because it is easier to control the dissolution rate in the body. In particular, a coating in which the outermost layer is a layer of hardened rapeseed oil and the innermost layer in contact with the inorganic compound is a layer of benzoic acid resin is preferred because the coating agent dissolves in the intestine but not in the stomach. The dissolution rate of the sporulation inhibitor of the present invention in gastric juice is preferably less than 60%, and the dissolution rate in intestinal juice is preferably 70% or more. Such dissolution rates can be achieved by using a two-layer or multi-layer film, or by controlling the type and thickness of the coating agent in each layer.

[0017] It is particularly preferred that the inorganic compound is a potassium salt (particularly 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. Among these, it is particularly preferred that the inorganic compound is a potassium salt (particularly 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 preferred that a layer formed from benzoic acid resin is in contact with the inorganic compound, and a layer formed from hydrogenated rapeseed oil is formed thereon. It is particularly preferred that the sporulation inhibitor has a KCl concentration of 500 mM, an arabic gum concentration of 1%, a layer formed from benzoic acid resin is in contact with the inorganic compound, and a layer formed from hydrogenated rapeseed oil is formed thereon.

[0018] [Coating Method] The method for coating the sporulation inhibitor is not particularly limited, but for example, a coated sporulation inhibitor can be obtained by spraying a liquid coating agent heated to a temperature higher than the melting point while fluidizing powdered or granular cores in a commercially available fluidized bed spray granulator. It is preferable to make the coated sporulation inhibitor approximately 0.05 to 5 mm in size, 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 equal to or higher than the melting point of the coating agent, but is preferably approximately 5 to 15°C higher than the melting point of the coating agent.

[0019] The sporulation inhibitor of the present invention can be orally ingested by humans or non-human animals, or directly administered to the intestinal tract of non-human animals. When administered directly to the intestinal tract, there is no need to consider inactivation by gastric acid or irritation to the gastric mucosa, so there is no need to coat the compound with a protective layer containing hydrogenated vegetable oil. In both oral and intestinal administration, the amount and frequency of administration of the inorganic compound to be ingested by the subject are the same as those of the supplements described below.

[0020] [Supplements] The sporulation inhibitor of the present invention can also be formulated as a supplement for humans or non-human animals. The form of the supplement is not particularly limited, and examples include tablets, granules, powders, and drinks. In addition to the sporulation inhibitor of the present invention, the supplement of the present invention may further contain known supplement ingredients, such as antioxidants and proteins. The supplement of the present invention is suitable for continuous daily administration. The intake amount of the supplement of the present invention varies depending on, for example, the body weight of the subject to which it is administered. For example, the supplement of the present invention can be formulated so that a subject receiving the supplement can receive a dose of 1 mg / kg body weight / day or more, preferably 1 to 100 mg / kg body weight / day of the inorganic compound. Typically, the supplement of the present invention is administered one to three times per day. Subjects to which the supplement of the present invention is administered include humans, ruminants such as cows, sheep, and goats, and monogastric animals such as horses, pigs, chickens, dogs, and fish.

[0021] [Feed] The sporulation inhibitor of the present invention can be fed directly to non-human animals such as livestock, or can be combined with an excipient or diluent such as corn, soybean flour, rice bran, fish meal, or brewer's yeast to form feed. The feed of the present invention may also contain any additives that can be contained in conventional feed. The feed of the present invention is suitably ingested daily. The amount of feed intake varies depending on the size of the livestock. For example, in the case of chickens, the daily intake of the sporulation inhibitor is preferably about 0.013 to 2.7 mmol / kg (1 to 340 ppm), preferably 0.14 to 1.4 mmol / kg (10 to 170 ppm), of inorganic compounds relative to the conventional feed excluding the sporulation inhibitor. Furthermore, when a sporulation inhibitor containing a polysaccharide is added to conventional feed, the intake amount is correspondingly higher. For example, when the polysaccharide content is 133 ppm of gum arabic, the inorganic compound is preferably added to a conventional feed at a concentration 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). As used herein, "ppm" means "ppm by mass." As used herein, "livestock" refers to living creatures kept by humans. Specific examples include ruminants such as cows, sheep, and goats, as well as 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. There are no particular limitations on the method of feeding the sporulation inhibitor of the present invention.

[0022] Example 1. Bacterial Cell Sedimentation Experiment. Clostridium perfringens ATCC10873 was cultured on GAM plates (Nissui Modified GAM Broth, manufactured by Nissui Pharmaceutical Co., Ltd.) under anaerobic conditions at 37°C for 24 hours. Anaerobic culture was performed using the Mitsubishi Gas Chemical Company's Anaeropack anaerobic culture kit. The resulting culture was suspended in 4 mL of purified water prepared using a Merck Millipore water purification system, and the optical density (OD) of the suspension was adjusted to approximately 2.0 at 660 nm. To a vial containing 0.5 mL of this suspension, the precipitating substances listed in Table 1 were added at the concentrations listed in Table 1, allowing Clostridium perfringens to come into contact with each substance. Arabic gum (AG) was used as a positive control. The vial was then incubated at 37°C for 8 hours. During this time, the optical density of the sample was continuously and automatically measured.

[0023] <Evaluation> A precipitating substance was judged to have a sedimentation effect if the decrease in optical density of the sample 8 hours after addition of the precipitating substance was at least twice the decrease in optical density of the sample without addition of the precipitating substance (i.e., the decrease in optical density due to natural sedimentation of the bacterial cells).

[0024] <Results and Discussion> The results are shown in Table 1 and Figure 1. AG, which had a sedimentation effect on Gram-negative bacteria, did not have a sedimentation effect on the Gram-positive bacterium C. perfringens. In the absence of AG, sedimentation was confirmed with KCl 1 100 mM or more, NaCl 500 mM, or FeCl2 10 mM or more. This is thought to be because the cations changed the surface potential of Clostridium perfringens cells, thereby reducing the repulsive force. MgCl2 (500 mM), which did not have a sedimentation effect in the absence of AG, exhibited a sedimentation effect when used in combination with AG.

[0025]

[0026] Example 2: Aggregation and Cell Shape Observation Experiments Using three Clostridium perfringens strains [ATCC10873 (toxigenic Type A), SM101 (toxigenic Type F: CPE toxin producer), and CNEOP004 (toxigenic Type G: netB toxin producer), all bacilli], a similar experiment to Example 1 was conducted. However, the optical density of the samples was not measured. Eight hours after the addition of the precipitating substance, samples were collected, and the presence or absence of aggregation and cell shape of each strain were observed using an upright microscope (OLYMPUS, model: BX50). <Results> The results are shown in Table 2 and Figure 2. AG, which exerted an agglutinating effect on Gram-negative bacteria, did not exert an agglutinating effect on Clostridium perfringens. When KC1 or NaCl was added to the sample in the absence of AG, the cell shape changed from bacilli to cocci, but the cells themselves did not aggregate. In the presence of KC1 or NaCl and AG, the bacterial cells aggregated into cocci. When MgC12 was added to the sample, the shape remained rod-shaped and no aggregation was observed in the absence of AG, but aggregation of Clostridium perfringens was observed in the presence of AG. When CaC12 and FeC12 were added to the sample, Clostridium perfringens aggregated into rod-shaped bacteria in the absence of AG.

[0027]

[0028] Example 3 Measurement of Clostridium perfringens Cell Surface Potential An experiment was conducted under the same conditions as in Example 1 using three Clostridium perfringens strains (ATCC10873, SM101, and CNEOP004). However, the optical density of the samples was not measured. Eight hours after the addition of the precipitating substance, samples were collected and diluted 100-fold. The surface potential of Clostridium perfringens cells was then measured using a Malvern Zetasizer Nano (model: Nano-ZS). The surface potential of the precipitating substance before and after its addition was also measured (note that the concentration during measurement was adjusted to the concentration during surface potential measurement of the sample). <Results and Discussion> The results are shown in Tables 3 and A. Clostridium perfringens did not aggregate when either Arabic Gum or KCl was added alone, but did aggregate when both were present. This is because the bacterial cell surface is negatively charged in water. Addition of KCl increased the negative charge on the bacterial cell surface potential, and the addition of Arabic gum neutralized the surface potential. This suggests that the bacterial cells, KCl, and Arabic gum interacted with each other, resulting in aggregation (Table 3). In the case of the ATCC10873 strain, the bacterial cell surface potential temporarily became neutral when MgC12 was added, but the addition of Arabic gum caused the bacterial cell surface potential to change again (to negative charge) (Table 3-1). This suggests that the bacterial cells, MgC12, and Arabic gum interacted with each other, resulting in aggregation. In contrast, the addition of MgC12 significantly shifted the bacterial cell surface potential toward neutral, resulting in aggregation (Tables 3-2 and 3-3) in SM101 and CNEOP004 strains. When CaCl2 (20 mM or more) and FeCl2 (10 mM or more) were added, the cell surface potential significantly shifted to the neutral side in the absence of AG. 2、 It is believed that the bacteria and FeC12 interfered with each other, causing aggregation (Table 3).

[0029]

[0030]

[0031] Example 4: Phase-contrast microscopic observation of Clostridium perfringens cocci upon addition of KCl. Generally, under stressful conditions, there are numerous reports that E. coli, Campylobacter, and Helicobacter pylori change their shape to a cocci form known as a "coccoid form," and it has also been reported that Clostridium also becomes spherical (International Journal of Current Microbiology and Applied Sciences (2016), 5(7), 210-223). The spore formation process requires cell division, which requires the necessary nutrients. It has been reported that Clostridium perfringens spores, like Clostridium difficile, transition from a phase-bright spore to a phase-dark spore before becoming vegetative cells (J. Am. Chem. Soc. (2014) v136 pp14498-14504). Because pure water containing no nutrients was used in Example 2, the cocci observed in Example 2 were expected to be "coccoid forms" rather than spores. Therefore, we investigated whether the cocci (1 μm in diameter) formed when KCl (0.5 M) was added to Clostridium perfringens were vegetative cells or spores. Spores glow white under a phase-contrast microscope (phase-bright spores) because their water content is reduced and DNA and other components are concentrated. Phase-dark spores are observed before germination into vegetative cells, but are dark under a phase-contrast microscope. Vegetative cells are pale in color and show black spots within the cells under a phase-contrast microscope. For reference, spores of Clostridium perfringens SM101 strain were observed under a phase-contrast microscope (Figure 3). In Figure 3, the oval, shining white spores are phase-bright spores, and the dark, similarly shaped spores are phase-dark spores.Fragments of vegetative bacteria were also observed.On the other hand, in Example 2, when KCl (500 mM) was added to Clostridium perfringens SM101 strain, the coccoid bacteria that formed were observed under a phase-contrast microscope (Fig. 4). Similar to the vegetative bacillus, they were pale in color and had black spots inside the cells, suggesting that they were not spores. Therefore, it is believed that the change in 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. Aggregation Observation Experiment of Enterobacteria Aggregation of enterobacteria other than Clostridium perfringens was observed in the same manner as in Example 2. The selected enterobacteria were Gram-positive bacteria Lactobacillus casei ATCC393 and Bifidobacterium animalis JCM1190, and Gram-negative bacteria Salmonella enterica IAM1648 and E. coli MG1655. Lactobacillus casei ATCC393 and Bifidobacterium animalis JCM1190 were cultured on MRS plates (manufactured by Difco), E. coli MG1655 on LB plates (manufactured by BD), and Salmonella enterica IAM1648 on NB plates (manufactured by Difco), all at 37°C. Bifidobacterium animalis JCM1190 was cultured under anaerobic conditions, similar to Clostridium perfringens, using the Mitsubishi Gas Chemical Company's Anaeropack anaerobic culture kit. Other bacteria were cultured under aerobic conditions. The results are shown in Table 4. For reference, the results for Clostridium perfringens ATCC10873 in Example 2 are also shown. The agglutination effect of KCl in the presence of Arabic Gum was specific to Clostridium perfringens among Gram-positive bacteria, and did not affect Lactobacillus casei or Bifidobacterium animalis. The Gram-negative bacteria Salmonella enterica and E. coli were agglutinated by Arabic Gum. These results are similar to those reported in WO2019 / 177172. None of the Gram-negative bacteria aggregated with KCl alone, but did aggregate when combined with Arabic Gum. Therefore, it was suggested that the combined use of KC1 and Arabic Gum specifically agglutinated the harmful bacteria Clostridium perfringens, Salmonella enterica, and E. coli.FeCl2 also exhibited aggregating effects on the Gram-positive bacteria Clostridium perfringens, Lactobacillus casei, and Bifidobacterium animals.

[0033]

[0034] Example 6: Observation of Aggregation of Three Intestinal Malicious Bacteria Similar to Example 5, three strains, Clostridium perfringens ATCC10873, Salmonella enterica IAM1648, and E. coli MG1655, were cultured. A suspension of each strain was 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 660 nm, the three bacterial suspensions were mixed, the mixed bacterial cells were collected using a centrifuge, and the final optical density (OD) was adjusted to approximately 2.0 (at 660 nm) in a vial. 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 vial and allowed to come into contact with the mixed bacterial cells. Each vial was placed 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, were able to rapidly aggregate three types of bad intestinal bacteria.

[0035] Example 7. Preparation of Coated Sporulation Inhibitor for Feed Arabic gum (Wako Pure Chemical Industries, Ltd.) and potassium chloride (Wako Pure Chemical Industries, Ltd.) were used as core materials, and hydrogenated rapeseed oil (melting point 67°C) and Benzoin Resin (Chuo Koryo Co., Ltd.) were used as coating agents. A predetermined amount of coating agent, which had been liquefied by heating above the melting point, was sprayed onto powdered or granular cores to obtain a coated sporulation inhibitor for feed. Arabic gum was used as a single-layer coating, with 25 parts by weight of hydrogenated rapeseed oil coated on 75 parts by weight of core material. Hereinafter, this additive will be referred to as "Coated-Arabic Gum." Meanwhile, KCl was used as a two-layer coating, with 2.23 parts by weight of Benzoin Resin as the first layer (inner layer) and 13 parts by weight of hydrogenated rapeseed oil as the second layer (outer layer) on 84.77 parts by weight of core material. Hereinafter, this additive will be referred to as "Coated-KCl."

[0036] Example 8. Enteric Coating Test of Coated Arabic Gum (Artificial Gastric Fluid Treatment) 0.2% NaCl and 0.2% pepsin (from Porcine Stomach Mucosa, 1:5,000, 2,500 units / mg) were added to purified water prepared using a Merck Millipore water purification system, and the pH was adjusted to 2. The Coated Arabic Gum prepared in Example 7 was then added and subjected to enzyme treatment at 37°C for 2 hours. The enteric coating was evaluated by automatically and continuously measuring the optical density during this period. Note that the "2 hours" represents the time from when the feed reaches the chicken's stomach until it passes through. (Artificial Gastric Fluid Treatment) After the artificial gastric fluid treatment, 0.2% trypsin (from Porcine Pancreas, 1:5,000, 4,500 units / mg) was added, the pH was adjusted to 6, and the enzyme treatment was performed at 37°C for 2 hours. The enteric coating was evaluated by automatically and continuously measuring the optical density during this period. The "two hours" refers to the time it takes for the feed to pass through the chicken's intestines after reaching them. Optical density (OD) for both treatments was measured using a Shimadzu UVmini-1240 spectrophotometer (at a wavelength of 190 nm). Hydrochloric acid and sodium hydroxide were used as pH adjusters in both treatments. The results are shown in Figure 6. The dissolution rate was suppressed to approximately 60% or less two hours after the start of gastric fluid treatment, while it exceeded 90% in the intestinal fluid treatment. These results demonstrate that good release control can be achieved with the sample coated with hydrogenated rapeseed oil.

[0037] Example 9. Dissolution Test of Coated-KCl The coated-KCl prepared in Example 7 was added to purified water produced using a Merck Millipore water purification system, and a dissolution test was performed at 37°C. The dissolution rate was determined by continuous, automated measurement of optical density during the test. 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. While the dissolution rate was suppressed to approximately 50% or less within 2 hours after the start of treatment, it reached 80% in the intestinal fluid treatment. These results demonstrate that good release control can be achieved with samples coated with a two-layer coating of Benzoin Resin and hydrogenated rapeseed oil.

[0038] Example 10. Clostridium perfringens infection test. The Coated-KCl prepared in Example 7 was added to the diet described in Hofacre, CL, et al., Avian Dis. 1998;42(3):579-84 at a concentration of 40 ppm to obtain a feed composition. The experiment was outsourced to SPRG, Inc. and conducted in the United States. In a free-range chicken coop, 25 day-old broiler chicks were fed the feed composition in one section. A total of 500 birds were fed in 20 replicates for 6 weeks from the time of feeding. The test conditions were as follows: on Day 0, a commercially available attenuated coccidiosis vaccine (0.007 mL vaccine / bird) was administered. After 4 hours of fasting and 2-3 hours of water deprivation on Days 14, 15, and 16, Clostridium perfringens was administered at a concentration of 1 x 10 8The animals were placed in drinkers at a concentration of 0.05 cfu / mL and provided with water. Necrotic enteritis (NE)-related mortality and NE-related intestinal damage scores were assessed during the rearing period. NE-related intestinal damage scores were assigned a scale of 0 = none, 1 = mild, 2 = moderate, and 3 = severe, and the mean scores were calculated. The untreated group served as the control ("Challenge Control" in Table 5). Bacitracin methylene disalicylate (BMD) was used as a positive control. BMD is known to be effective against necrotic enteritis caused by Clostridium perfringens. It is also widely used as an antibacterial growth promoter (AGP), and is one of the most widely used AGPs in the United States. BMD was added to the feed described in the Hofacre publication at 55 ppm by volume to create a positive control feed composition. A commercially available BMD (Zoetis BMD®, uncoated) was used as is. The results are shown in Table 5. BMD reduced mortality by 13.2% compared with challenge controls. Coated-KCl reduced mortality by 3.8% compared with challenge controls. NE lesion scores were significantly improved compared with challenge controls (control: 0.75, BMD: 0.30, coated-KCl: 0.52).

[0039]

[0040] Example 11. Test of weight gain effect The Coated-KCl and Coated-Arabic Gum prepared in Example 7 were added to the basal feed containing no feed additives shown in Table 6 so that the amounts of the core material were 40 ppm by volume and 100 ppm by volume, respectively, to obtain feed compositions.

[0041]

[0042] The test chicks were day-old male broiler chicks (UK Chunky) weighing 38–46 g. All test groups were fed the additive-free basal diet listed in Table 6 in a free-range chicken coop. Each group consisted of 50 chicks, and each group was divided into three groups, with three replicates of 50 chicks each, for three weeks from the time of feeding. To create environmental stress, the chicks were reared on wet floors, with approximately 1 L of water sprayed on the floor once daily throughout the rearing period. 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 at 100. Compared to the control (non-treatment), BMD showed improved weight gain across all intervals (0-21 days of age). On the other hand, while no significant difference was observed in the combined Coated-Arabic Gum and Coated-KCl group compared to the control, the weight gain effect became apparent as the animals grew, and a weight gain effect was observed in the interval (15-21 days of age). Since pigs are usually shipped at 6-7 weeks of age, a sufficient weight gain effect was expected.

[0043]

Claims

1. A Clostridium perfringens spore formation inhibitor containing at least one inorganic compound at a concentration capable of precipitating Clostridium perfringens 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 vegetable hardened oil, said spore formation inhibitor.

2. The spore formation inhibitor according to claim 1, wherein the inorganic compound is a salt with 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 at least one selected from the group consisting of 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.

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, carboxymethyl cellulose, 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, if included, the polysaccharide are coated with a protective layer containing vegetable hardened oil.

9. The spore formation inhibitor according to claim 8, wherein the vegetable hardened oil is a hardened oil of rapeseed oil, linseed oil, safflower oil, sunflower oil, soybean oil, corn oil, peanut oil, cottonseed oil, sesame oil, rice bran 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, if included, the polysaccharide are protected by two layers of rapeseed hardened oil and benzoic resin.

11. A supplement for preventing or treating Clostridium perfringens infections, containing the spore formation inhibitor according to claim 1.

12. The supplement according to claim 10 or 11, 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 claim 10, wherein the Clostridium perfringens infection is necrotizing enteritis.

14. A feed containing the spore formation inhibitor according to claim 1.

15. The feed according to claim 14, further containing a 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 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 inhibiting 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.