MXPA06006003A - An antibiotic, compositions containing the antibiotic, and methods for administrating the antibiotic and/or said compositions to livestock - Google Patents

Abstract

An antibiotic comprising a protease-resistant bacteriocin derived from a lactic acid bacterium, and compositions thereof, are disclosed. A feed composition for livestock comprising the antibiotic comprising a protease-resistant bacteriocin derived from a lactic acid bacterium is also disclosed. A method for preventing the growth of human food poisoning-causing bacteria in the stomach and/or intestines of livestock comprising administering the feed composition comprising a protease-resistant bacteriocin derived from a lactic acid bacterium to livestock is disclosed.

Description

AN ANTIBIOTIC, COMPOSITIONS THAT CONTAIN THE ANTIBIOTIC AND METHODS FOR ADMINISTERING ANTIBIOTICS AND / OR SAID COMPOSITIONS TO LIVESTOCK BACKGROUND OF THE INVENTION FIELD OF THE INVENTION In recent years, human food poisoning caused by bacteria belonging to the genus Salmonella, Campylobacter, and the like has increased abruptly. Contamination by these bacteria has also spread to the swine and avian breeding industry. As a countermeasure, in Japan, reverse disinfectants and the like have been used to disinfect chicken pens. Overseas vaccines have been used. However, none of these methods has been able to prevent infection of humans by food poisoning by these bacteria, which remain present in the intestines of cattle. Antibiotics against salmonella are commercially available in the form of sugars, organic acids, antibiotics, and compound formulations. The mechanism of Salmonella infection has already been studied. Salmonella has type I fimbria, which are known to bind to the mannose analogue receptors on the cell surface of the epithelium mucosa in the intestines of cattle, resulting in adherence and infection. In particular, since sugars such as sugar are natural substances, they are highly safe and are anticipated to be highly effective as antibiotics by acting directly on Salmonella bacteria. (Characteristics and Usefulness of Mannan Oligosaccharides, "Friend of the Chicken Rancher," June issue, pp. 14-18 (1996), JP10-215790A, WO 99/08544, JP2001-238608A)). However, the mannose is degraded by the enterobacteria present in cattle, and thus has no effect unless it is administered in large quantities. The development of an antibiotic for the mannose degradation bacterium is necessary (in the inhibitory effect in the Salmonella infection of oligosaccharides in chickens, "Poultry Diseases", Vol. 31, p 113-117 (1995)). In addition, nisin, an antibiotic substance produced by lactic acid bacteria, has been investigated for use against Salmonella and Campylobacter bacteria. Nisin has a broad antibiotic spectrum against gram-positive bacteria, but has low antibiotic properties against gram-negative bacteria (Can J. Microbiol., Vol. 47, pp. 322-331 (2001)). Thus, there are examples of nisin which is used as an antibiotic in combination with chelating agents (Journal of Food Protection, Vol 58 (9), pp. 977-93 (1995)). TSP (Journal of Food Protection, Vol. 61 (7), pp. 839-844 (1998)), lysozyme, and organic acids (WO03 / 005963). However, in the intestines of cattle that carry Salmonella, nisin is degraded by digestive enzymes and thus has no lasting antibiotic activity. Therefore, there is a need in the art to develop an antibiotic that does not degrade.

BRIEF DESCRIPTION OF THE INVENTION Also, objectives of the present invention include providing an antibiotic for cattle that is effective to prevent the growth of bacteria responsible for food poisoning in humans in the digestive tract of cattle, and by extension, a method-to prevent the growth of bacteria that they cause poisoning by human food in the stomach and / or intestines of cattle by administering to the cattle a food composition containing said antibiotic. It has been found that the antibiotic as described above is a protease resistant bacteriocin, and can be administered to cattle. This bacteriocin is isolated from the lactic acid bacteria that are typically present in the stomach and intestinal juices of cattle. In this way, it is possible to prevent the growth of bacteria responsible for food poisoning in the digestive tract of cattle. The present invention was planned based on this discovery. An object of the present invention is to provide an antibiotic comprising a protease resistant bacteriocin isolated from a lactic acid bacterium.

It is a further object of the present invention to provide a composition comprising the antibiotic as described above and suitable excipients. It is a further object of the present invention to provide the composition as described above, wherein said composition is formulated for administration to livestock. It is a further object of the present invention to provide the composition as described above, wherein said lactic acid bacterium belongs to a genus selected from the group consisting of Lactobacillus, Weissella, Pediococcus, Leuconostoc, and combinations thereof. It is a further object of the present invention to provide the composition as described above, wherein said lactic acid bacterium is selected from the group consisting of Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus pentosus, Weissella sp. FERM BP-10474, Weissella cibaria, Weissella confusa, Weissella hellenica, Weissella kandleri, Weissella minor, Weissella paramesenteroides, Weissella thailandensis, Pediococcus pentosaceus, Leuconostoc citreum, Leuconostoc pseudomesenteroid? S, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc mesenteroides. It is a further object of the present invention to provide a food composition comprising the antibiotic as described above.

It is a further object of the present invention to provide a method for preventing the growth of the bacteria responsible for food poisoning in humans in the stomach and / or intestines of livestock which comprises administering to the cattle the food composition as described above. It is a further object of the present invention to provide the method as described above, wherein said bacteria belong to a genus selected from the group consisting of Salmonella, Campylobacter, Listeria, Escherichia coli, Welsh, and combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows the profiles of the bactericidal effects in Salmonella of several antibiotics used in food.

DETAILED DESCRIPTION OF THE INVENTION The present invention is described in detail below. The antibiotic of the present invention contains a protease resistant bacteriocin derived from lactic acid bacteria, and can be formulated as a composition for administration to cattle, or formulated in the feed for said cattle.

The "cattle" of the present invention includes pigs, as well as poultry, such as chickens, quail, guinea fowl, domestic goose, wild duck, turkeys, dark meat chickens, and the like. Generally, "bacteriocin" refers to an antibiotic protein substance (Klaenhammer, TR, Biochemie 60 (3): 337-349 (1988).) However, the "protease-resistant bacteriocin" of the present invention refers to bacteriocins that they are not degraded by protein degradation enzymes (proteases), in contrast to conventional bacteriocins such as nisin The bacteriocins of the present invention are not degraded by proteases such as the digestive enzymes present in the stomach and intestines of cattle. proteases are pepsin (EC3.4.23.1, EC3.23.2, EC34.4.23.3) and trypsin (EC3.421.4) In addition to being resistant to proteases such as digestive enzymes, the protease-resistant bacteriocin that is employed herein invention is also resistant to, and is not degraded by, proteases derived from the genus Aspergillus, which are used in the preparation of beer and fermentation, as well as proteases derived from meat, which They are used in food processing. Examples of these proteases are "Umamizime G" (Amano Enzyme, Inc.), a protease of the genus Aspergillus that is used in the preparation of beer and fermentation, and cathepsin, a protease of meat that is used in food processing.

The protease resistant bacteriocin of the present invention is produced by lactic acid bacteria and is highly safe. This protease-resistant bacteriocin has a bacteriostatic and bactericidal action on bacteria that are responsible for human food poisoning, and are typically present in the stomachs and intestines of cattle. Thus, when this protease-resistant bacteriocin, or a culture solution or culture supernatant containing it, is administered as it is or formulated into a food composition, the bacteriocin is not degraded by the protease. Therefore, the bacteriocin is active and suppresses the growth of bacteria that are responsible for human food poisoning, and are present in the stomach or intestines. The infection of these bacteria from the intestines during the handling of cattle in meat processing is therefore avoided. In addition, this bacteriocin is derived from lactic acid bacteria, and is safer than conventional chemically synthesized compounds, even when consumed by cattle in large quantities. It is also desired from the aspect of livestock health. In the present invention, the term "bacteria responsible for human food poisoning" means bacteria that are constantly present in the stomach and intestines of cattle, and which cause food poisoning for humans when the meat and / or eggs are consumed or handled by humans. Specifically, the "bacteria responsible for human food poisoning" includes bacteria of the genera Salmoneila, Campylobacter, Listeria, Escherichia, Welsh, Yersinia (Yersinia enterocolitica), Pseudomonas aeruginosa, Staphylococcus aureus, and Glostridium. In particular, the "bacteria responsible for human food poisoning" are bacteria of the genus Salmonella and Campylobacter. Bacteria of the genus Salmonella are constantly present in the intestines of livestock, such as pigs and domestic poultry such as chickens. In the processing of livestock, bacteria adhere to meat and eggs. When a piece of meat or an egg to which this bacterium adheres is ingested without having been adequately heated, the result is poisoning by food for humans, which results in severe gastroenteritis, nausea, vomiting and the like. Bacteria of the genus Campylobacter are constantly present in the intestines of domestic poultry such as chickens, and can contaminate chicken meat during meat processing, and can cause food poisoning for humans, resulting in diarrhea, abdominal pain, fever, nausea, vomiting and the like. The protease resistant bacteriocin of the present invention can be efficiently manufactured by culturing the lactic acid bacteria according to the example shown below. The lactic acid bacteria that produce the protease resistant bacteriocin of the present invention have been isolated from fermented foods, and the like. Any lactic acid bacteria with antibiotic activity that can be detected by the screening method described below can be employed, even when they are separated from anything other than fermented foods, or the like. The lactic acid bacteria employed in the present invention preferably belong to the genus Lactobacillus, Weissella, Pediococcus, or Leuconostoc. In particular, preferred examples of lactic acid bacteria belonging to the genus Lactobacillus are: Lactobacillus plantarum, Lactobacillus salivarius, and Lactobacillus pentosus. Preferred examples belonging to the Weissella genus are: Weissella sp. FERM BP-10474, Weissella cibaría, Weissella confusa, Weissella hellenica, Weissella kandleri, Weissella minor, Weissella paramesenteroides, and Weissella thailandensis. A preferred example belonging to the genus Pediococcus is Pediococcus pentosaceus. Preferred examples belonging to the genus Leuconostoc are: Leuconostoc citreum, Leuconostoc pseudomesenteroides, Leuconostoc argentinum, Leuconostoc carnosum, and Leuconostoc mesenteroides. Of the above lactic acid bacteria, the following are particularly preferred for use in the present invention: strain Lactobacillus plantarum JCM1149, strain Lactobacillus salivarius JCM1231, strain Lactobacillus pentosus JCM1558, strains Pediococcus pentosaceus JCM5885 and JCM5890, Weissella sp. FERM BP-10474, strain Weissella cibaría JCM12495, strain Weissella confusa JCM 1093, strain Weissella hellenica JCM10103, strain Weissella kandlerí JCM5817, strain Weissella minor JCM1168, strain Weissella paramesenteroides JCM9890, strain Weissella thailandensis JCM10694, strain Leuconostoc citreum JCM9696, strain Leuconostoc pseudomesenteroides JCM11945 , strain Leuconostoc argentinum JCM11052, strain Leuconostoc carnosum JCM9695, and strain Leuconostoc mesenteroides JCM6124. The bacterial strains mentioned by the deposit numbers "JCM" are stored in the "Japan Collection of Microorganisms" of Riken Bioresource Center (an Independent Administrative Institution), 2- Hirozawa, Wako, Saitama Prefecture, Japan. Weissella sp. FERMBP-10474 was deposited as deposit number FERM BP-10474 in "International Deposit of Patent Organization" of the Institute-Advanced National Industrial Science and Technology (an Independent Administrative Institution), Central 6, 1-1-1 Tsukuba East, Ibaraki Prefecture, Japan, October 31, 2003. Whether or not a given lactic acid bacterium produces the protease resistant bacteriocin (sometimes abbreviated as "PRB") of the present invention can be determined by the following method, for example. That is, in the following method, zones of growth inhibition of an indicator strain are formed when PRB is produced in a culture of lactic acid bacteria. (1) A culture solution of lactic acid bacteria is prepared by a typical culture method for lactic acid bacteria (or by a culture method in which the lactic acid bacteria of the present invention is separated). The culture solution of lactic acid bacteria is adjusted from pH 5.5 to 6.0 with NaOH, separated by centrifugation at 12,000 rpm x 10 min, and filtered through 0.45 μm cellulose acetate with a syringe filter unit disposable (ADVANTEC "Dismic-25 is) to obtain a sample When the antibiotic activity is low, the four-fold concentration is carried out under reduced pressure at room temperature When necessary, a concentration of ten times can be carried out. (2) Listeria innocua ATCC33090T, Bacillus circulans JCM2504T, Bacillus coagulans JCM2257, Micrococcus luteus I F012708, Bacillus subtilis JCM465T, Bacillus subtilis IAM1381, Lactococcus lactis sub sp. lactis ATCC19435, Enterococcus faecium JCM5804T, Enterococcus faecium JCM5803T, Pediococcus pentasaceus JCM5855, Lactobacillus plantarum ATCC14917T and Lactobacillus sakei JCM1157T are used as the indicator strain.Antibiotic activity is measured by the button method on grass, described below, or the viable counting method of bacteria, and the indicator strain having the strongest antibiotic activity is selected. (3) Protease derived from Aspergillus ("Umamizime G" or similar manufactured by Amano Enzyme, Inc.) is used as the enzyme. (4) An amount of 10 to 100 unit / mL of the enzyme described in (3) is added to the sample of (1) and the reaction is carried out by keeping the mixture at 30 ° C for one or more hours. (5) The indicator strain that exhibits the highest antibiotic activity in (2) is plated, 0.01 mL of the sample that has been treated with the enzyme from (4) is added dropwise to a medium where the indicator strain will proliferate, such as MRS, and the culture is carried out for 20 to 24 hours at the optimum temperature for growth of the indicator strain (37 ° C for Listeria innocua, Bacillus coagulans, Enterococcus faecium, and Pediococcus pentosaceus, 30 ° C for others) . Subsequently, the areas of inhibition of growth of the indicator strain are confirmed. The composition of the present invention, characterized by containing the protease resistant bacteriocin, can include the lactic acid bacterial culture solution that produces the protease resistant bacteriocin as it is, and / or can include the dried bacterial product of said protease solution. culture, or may include the culture supernatant. The bacteriocin obtained by the separation and purification of any of these may also be included in the composition of the present invention. A suitable excipient or the like, as described below, may also be added to the composition of the present invention. In summary, an agent that exhibits PRB activity derived from the lactic acid bacterium is sufficient as a component of the composition of the present invention. Upon passage, the activity of the protease-resistant bacteriocin produced from the lactic acid bacteria is present intracellularly, and is secreted extracellularly. The protease resistant bacteriocin produced from a culture solution of lactic acid bacteria can be separated and purified as necessary in accordance with methods commonly employed in this field. Specifically, the bacteriocin can be produced by obtaining a fraction having a protease-resistant bacteriocin activity and by performing ammonium sulfate precipitation, column chromatography, ethanol precipitation, or the like. The lactic acid bacterium employed in the present invention can be cultured using medium components adapted to bacterial strains employed and the production of protease resistant bacteriocin. The use of a culture solution that has been adequately concentrated allows for more efficient additional processing. The lactic acid bacteria can be cultured by typical methods, such as those shown below. A carbon source may be present in the medium employed for the present invention, and may include, whey, starch sugar solution or glucose for food use. A source of nitrogen may also be present in the medium employed for the present invention, and may include products of hydrolysis of whey protein concentrates, corn peptides, soy peptides, commercial flavozone solution materials, distilled alcohol sediments from low quality, or enzyme extracts for food use. Additionally, various organic and inorganic products and articles containing said products, which are required for the growth of lactic acid bacteria and enzymatic production, such as phosphates, magnesium salt, calcium salt, manganese salt, other salts, vitamins and Yeast extracts can be added appropriately to the medium. The temperature and culture period can be set according to the common lactic acid bacteria culture methods; for example, in a stationary culture, the temperature and period for culture may be 30 to 37 ° C and 12 to 36 hours, respectively. - In the present invention, the antibiotic effect in the bacteria responsible for human food poisoning in the stomach and intestines of cattle can be confirmed by inhibiting the growth of the indicator strains and bacteria that cause food poisoning in a solution for treatment with artificial stomach juice containing trypsin, pepsin, and the like, or it can be confirmed by oral administration in vivo to real animals to examine whether or not there is a reduction in the bacteria responsible for human food poisoning in the stomach and intestines. The antibiotic and compositions of the present invention can be used in various forms. Examples are powders, granules, and tablets. Excipients, fillers, and the like can be added appropriately as needed. When a culture solution of lactic acid bacteria is used as the composition of the present invention, the proportion of the lactic acid bacteria of the present invention in the composition can be determined in relation to the amount of bacteria responsible for food poisoning for human in the stomach and intestines of cattle, station, and the like. When the protease resistant bacteriocin is of high purity or the specific activity is high, a small amount is administered, and when the medium itself is administered or the specific activity is low, a high ratio is administered. The time of administration of the antibiotic or composition of the present invention is not specifically limited as long as the antibiotic effect of the present invention is present. Administration is possible at any time. However, feeding is desirable before shipment of livestock or domestic poultry for meat processing. The mixing or formulation of the antibiotic in the livestock feed allows particularly effective administration. The dose of the antibiotic or composition of the present invention that is administered is not specifically limited as long as the effect of the antibiotic of the present invention is present. For example, the dose can be adjusted appropriately based on the lactic acid bacteria employed and the animal to which it is administered so that the effect of the present invention is present. The food composition of the present invention contains the aforementioned antibiotic or composition of the present invention. The ratio of the antibiotic or composition in the food composition is usually 0.1 to 10 weight percent, preferably 2 to 10 weight percent. The food composition is not specifically limited; A commercial product can be used as if, or, in addition to corn, wheat, barley, soybean sediment, and other plant materials, animal flours (MBM), chicken flour, fish paste, and other animal materials can be added appropriately to a commercial product as needed. In addition, carbohydrates, fats, proteins, inorganic substances, (such as calcium, magnesium, sodium and phosphorus), vitamins (such as vitamins A, B1, B2 and D), and several different nutrients may be added as necessary. The present invention is specifically described below through the following non-limiting examples.

Reference Example 1 The method for analyzing the lactic acid bacteria producing the protease-resistant bacteriocin will be described based on the example of separation of the fermented food matsoun. To separate the lactic acid bacteria, 0.5 percent samples of fermented milk matsoun (a type of fermented food) were added to a liquid medium that allows the growth of lactic acid bacteria, such as an MRS medium (Table 1 below) ) and half M17 (table 2 below). The samples were cultivated at 30 to 37 ° C (preculture). The culture was carried out for one, five or ten days respectively. After finishing the culture, the bacteria were cultured on the agar medium described above (1.2 percent) containing 0.5 percent calcium carbonate and the colonies of lactic acid bacteria that grew were harvested.

TABLE 1 TABLE 2 The lactic acid bacteria that were harvested were cultured in the same way in the liquid medium and under the culture conditions as stated above (the general culture). Next, the lactic acid bacteria were inoculated onto plates of MRS agar medium to which "Umamizyme G" (Amano Enzyme, Inc.) prefiltered, a protease derived from Aspergillus orizae, was added, and cultured for 24 hours at 30 ° C. These plates were then layered with the Lactobacilli AOAC medium (Table 3 below), mixed with indicator strains, and cultured for 24 hours at 30 ° C, which resulted in areas of growth inhibition of the indicator strains. .

TABLE 3 Composition of Lactobacilli AOAC Medium Other methods can be used to add the protease, such as 1) mixing the protease with the indicator strain, 2) coating the protease on the agar medium, 3) adding the protease in the course of culturing the lactic acid bacteria colonies (in this procedure, the protease can be added at the start of the culture, during the culture, or once the culture is complete), and 4) after culturing the colonies of lactic acid bacteria, acclimating the bacterial mass or eliminating the bacteria in the culture solution, adding to plates containing the indicator strains an adequate amount of a protease containing the sample, and confirming the formation of the zones of inhibition. The above methods 1) to 4) are given by way of example and not by limitations. The protease is not limited to "Umamizyme G". Next, the protease-resistant bacteriocin activity is evaluated by antibiotic spectral analysis. The antibiotic spectrum is examined by the button method on turf, in which the supernatant of culture solution of a lactic acid bacterium exhibiting antibiotic activity is sequentially diluted and stained on a plate of antibiotic activity, described further below. First, samples of antibiotic activity were prepared. The culture solution of a strain having antibiotic activity harvested by the method described above was separated by centrifugation for 10 minutes at 10,000 rpm, yielding a culture supernatant. The culture supernatant was then passed through a filter to obtain a sterile sample. The sample was then diluted in double steps to prepare a diluted solution 211. Subsequently, when the activity was low, as necessary, the reduced pressure concentration was performed in double steps at room temperature to prepare a dilute 2"3 solution. Then, the mixed indicator strain was cultured on a plate of antibiotic activity.The indicator strains in Table 4 below were grown on TSBYE medium (Table 5 below), TSB medium (Table 6 below), or MRS medium. The genera Bacillus and Micrococcus were cultivated with agitation, while other strains were grown in a stationary manner Bacillus coagulans, Listeria, Pediococcus, and Enterococcus were grown at 37 ° C, the other strains at 30 ° C.

TABLE 4 TABLE 5 Composition of the TSBYE medium TABLE 6 Antibiotic activity plates were also prepared. An amount of 10 mL of MRS agar medium (1.2 percent agar) and 5 mL of a Lactobacilli AOAC agar medium (1.2 percent agar) were separately sterilized on heating at 121 ° C for 15 minutes and maintained at room temperature. 55 ° C. The sterilized MRS agar medium was dispersed in a sterile Petri dish and placed on a clean table for one hour. Next, 50 μL of an indicator strain culture solution was mixed with Lactobacilli AOAC agar medium maintained at 55 ° C and stratified on an MRS plate. The plate was placed on a clean table without the plate cover for 15 minutes to dry the surface. The antibiotically active samples prepared above were added dropwise in 10 μL increments, the covers were placed on the plate, and drying was performed for about 1 hour. The plates were cultured for 20 hours at culture temperatures of several indicator strains and the formation of growth inhibition zones was examined. The antibiotic activity (AU / mL) was defined as follows. Antibiotic activity (AU / mL) = (maximum dilution ratio at which the zones of growth inhibition are formed) X 1, 000/10. The antibiotic spectra of the samples were analyzed in this way, and they were found to be resistant to proteases and had broad antibiotic spectra. The bacteriological properties of the lactic acid strain AJ110263 selected by the method described above were examined. This revealed homology of 98.22 percent (table 7) with the Weissella strain confused ATCC 10881 by the base sequence homology analysis 16s ribosomal DNA (rDNA) (Altshul, SF., Madden, T.F.

Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997), Gapped BLAST and PSI-BLAST: a new generation of protein database research programs. Nucleic Acids Res. 25: 3389-3402). The type cultures in deposit in ATCC are used in an evolution of homology.

TABLE 7 The basic characteristics (table 8) of strain AJ110263 coincided with those of a lactic acid bacterium. It was thought that the fermentation of sugar (consumption of sugar, see table 9) is similar to fermentation by Weissella confusa. However, because the fermentation of L-arabinose differed and less than 100% homology was found with 16SrDNA, it was discovered that this new strain is clearly different from the known strains. This bacterium was named Wissella sp. AJ110263. This bacterium was deposited in the International Deposit of Patent Organizations of the National Institute of Advanced Industrial Science and Technology (an Independent Administration Institution). The access number is FERM BP-10474.

TABLE 8 Basic characteristics of the bacterial strain of lactic acid AJ110263 TABLE 9 Fermentation properties of sugar of the lactic acid bacterial strain AJ 110263 The present invention is described through the following modalities. However, the present invention is not limited thereto.

EXAMPLE 1 The lactic acid bacterium Weissella sp. AJ110263 (FERM BP-10474), isolated from matsoun fermented milk, and the lactic acid bacteria Pediococcus pentosaceus JCM5885, Pediococcus pentosaceus JCM5890, Lactobacillus plantarum JCM1149, and Lactobacillus salivarius JCM1231, obtained from type cultures, were pre-cultured and cultivated in MRS liquid medium (see table 1 above). The culture temperature was 30 ° C for Weissella sp. and 37 ° C for the other strains. The above lactic acid bacteria were inoculated and cultured for 24 hours on MRS agar medium plates to which was added "Umamizyme G", a protease derived from Aspergillus, in amounts of 0 μ / ml (none added), 200 u / ml, and 400 u / ml. In culture, 100 ml of MRS medium was added to a 500 ml Sakaguchi flask100 ml of the above culture solution was inoculated, and the medium was stirred 100 times / minute. 1 Subsequently, Lactobacillus sakei strain JGM1157 was used, which does not produce bacteriocin, as an indicator strain, and the Lactobacilli AOAC medium was stratified. These plates were cultured for 24 hours at 30 ° C, resulting in the formation of zones of growth inhibition in the indicator strains (see table 10 below). From these results, it is shown that each of the strains produces protease resistant bacteriocin.

TABLE 10 Lactobacillus sakei JCM1157 was used as an indicator strain. The values in the table indicate the diameters of growth inhibition zones.

EXAMPLE 2 Lactococcus lactis NCD0497 (a bacteria that produces nisin A) and Lactococcus lactis NCIMB702054 (a bacteria that produces nisin Z) were cultured separately in liquid medium of MRS at 30 ° C. In the same manner as in Example 1, the antibiotic evaluation was performed using strain JCM1157 of Lactobacillus sakei as an indicator strain. In place of nisin-producing bacterial strains, 10 μl of "nisin solution A1,000 IU / ml" made by ICN Biomedical was stained on MRS agar medium plates and the previous antibiotic evaluation was performed (without using bacterial strains). ). Although zones of inhibition of growth of the indicator strains were formed in the absence of protease, the antibiotic activity caused by nisin decreased as the protease concentration increased (see Table 11).

TABLE 11 Lactobacillus sakei JCM1157 was used as an indicator strain. The values in the table indicate the diameters of proliferation inhibition zones. ND = not detected EXAMPLE 3 Weissella sp. AJ110263 (FERM BP-10474), Pediococcus pentosaceus JCM5885, Lactococcus lactis NCD0497 (a bacteria that produces nisin A) and strain JCM 1157 of Lactobacillus sakei were grown separately and the culture solutions were centrifuged off for 10 minutes at 10,000 rpm to obtain culture supernatants. After adding 200 U / mL of "Umamizyme G" to the culture supernatants and carrying out the protease treatment for 24 hours, the supernatants were filtered through 0.45 μm cellulose acetate ("DISMIC25CS" made by ADVANTEC) to obtain sterile samples. The lawn button method was used to examine the antibiotic spectra. This revealed that Weissella sp. AJ110263 (FERM BP-10474) and Pediococcus pentosauceus JCM5885 presented antibiotic activity even when treated with protease, unlike the culture solution of nisin and Lactobacillus sakei producing bacteria JCM1157, which do not produce bacteriocin (see table 12). Therefore, it was understood that Weissella sp. AJ110263 (FERM BP-10474) and Pediococcus pentosaceus JCM5885 produced protease-resistant bacteriocin.

TABLE 12 After treating the culture solution with protease, the antibiotic activity was measured by the button method on turf using the culture supernatant. The values indicated antibiotic activity. Antibiotic activity (AU / mL) = Maximum dilution ratio in which circles of inhibition were formed x 1,000 / 10.

ND = not detected.

EXAMPLE 4 Culture solutions of the various lactic acid bacteria shown in table 13 - Weissella sp. AJ110263 (FERM BP-10474), Pediococcus pentosaceus JCM5885, Lactobacillus plantarum JCM1149, Lactobacillus salivarius JCM1231, Leuconostoc citreum JCM9698, Leuconostoc pseudomesenteroides JCM9696 and JCM11045, Lactococcus lactis NCIMB702054 (a bacterium that produces nisin Z) - were treated enzymatically in the same manner as in Example 3, and antibiotic activity was measured through the button method on turf using Bacillus subtilis as the indicator strain. In the same way as in Example 3, "Umamizyme G" was used. In addition, 100 units / mL of a-amylase derived from Bacillus subtilis (Wako Junyaku) were added to the culture solutions of the lactic acid bacteria, and reacted for more than one hour at 30 ° C. Likewise, using Bacillus subtilis IAM1381 as the indicator strain, antibiotic activity was measured through the button method on turf to verify the effect of a-amylase on antibiotic activity. As shown in Table 13, the culture solutions of Weissella sp. AJ110263 (FERM BP-10474), Weissella cibaria JCM12495, Weissella confusa JCM1093, Weissella hellenica JCM10103, Weissella kandleri JCM5817, Weissella minor JCM1168, Weissella paramesenteroides JCM9890, We / '// sse to thatlandensis JCM 10694, Pediococcus pentosaceus JCM5885, Lactobacillus plantarum JCM 1149 Lactobacillus salvarius JCM1213, Lactobacillus pentosus JCM1558, Leuconostoc citreum JCM9698, Leuconostoc pseudomesenteroides JCM9696 and JCM11045 , Leuconostoc argentinum JCM11052, Leuconostoc carnosum JCM9695, and Leuconostoc mesenteroides JCM6124 showed antibiotic activity even when treated with protease. In this way, it was determined that each of these strains produces protease resistant bacteriocin. This confirmed that the antibiotic activity of these culture solutions decreased when treated with α-amylase. The residual activity of Table 13 was calculated as follows: antibiotic activity (AU / mL) = maximum dilution ratio in which zones of inhibition were formed x1, 000/10 x (diameter of the inhibition circle of treatment sample with enzyme / diameter of the control inhibition circle).

TABLE 13 EXAMPLE 5 Evaluation of antibiotic activity after treatment with artificial gastric and intestinal juices (a) Culture of lactic acid bacteria Lactococcus lactis NCIMB8780 (a strain that produces nisin A), Lactococcus lactis NCIMB702054 (a strain that produces nisin Z), Weissella sp. AJ110263 (a strain that produces PRB), Lactobacillus salivaríus JCM1231 (a strain that produces PRB), and Lactobacillus plantarum ATCC14917 (indicator strain) were cultured at 30 ° C and Lactobacillus gasser JCM1131 (a strain that does not produce bacteriocin) and Pediococcus pentosaceus JCM5885 (a strain that produces PRB) were cultured statically at 37 ° C in MRS medium for 24 hours. (b) Treatment with artificial gastric and intestinal juices After adding 0.2 percent NaCl and 0.2 percent pepsin (1: 5,000) to the culture solution of the lactic acid bacteria - to adjust the pH to 2, the solution culture was treated with protease for 2 hours at 41 ° C (treatment with artificial gastric juice). After adding 0.2 percent trypsin (1: 5,000) to adjust the pH to 6, the culture solution was treated with protease for 2 hours at 41 ° C (treatment with artificial intestinal juice). During the course of these treatments, lactic acid and caustic soda were used as pH adjusters. The previous digestive enzyme treatment was performed to prepare the following samples: a culture solution (broth A) containing the bacterial mass of the lactic acid bacteria cultured in MRS, a sterile supernatant (sob.A) obtained by centrifugally separating the broth A for 10 minutes at 10,000 rpm and pass it through 0.45 μm of a cellulose acetate filter ("DISMIC25CS" made by ADVANTEC), a digestive enzyme treatment solution (broth B), and a sterile supernatant of the B broth (sob B). (c) Lactobacillus plantarum antibiotic activity test ATCC14917, unaffected by lactic acid, was used as an indicator strain. An amount of -50 μl of this bacterium was plated on a "GAM" agar plate (Nissui Pharmaceutical Co., Ltd.). The surface of this plate was well dried, after which it was stained with 10 μL of the samples prepared in paragraph (b) above and cultured for 24 hours at 30 ° C. The formation of zones of growth inhibition was confirmed. The results are shown below in table 14. The numbers in the table indicate the diameter (mm) of the zones of growth inhibition. When subjected to treatment with artificial gastric and intestinal juices, nisin lost its antibiotic activity while the protease-resistant bacteriocin retained its antibiotic activity.

TABLE 14 Nd = not detected EXAMPLE 6 Salmonella Proliferation Inhibition Test (Live Bacteria Counting Evaluation) (a) Sample preparation The methods set forth in (a) and (b) of Example 5 were employed to culture lactic acid bacteria, perform treatments with artificial gastric and intestinal juices, and prepare samples of sterile supernatants. (b) Salmonella growth inhibition test (live bacteria count evaluation) Salmonella enteritidis NBRC3313 precultured with trypticase soy casein (made by BBL) was suspended at 105 bacteria / mL in TSBYE medium. An amount of 10 percent culture supernatant of lactic acid bacteria was added and the inhibitory effect on the proliferation of Salmonella was evaluated. Compared with the non-addition system and culture supernatant of bacteria that do not produce bacteriocin, the supernatant of bacteria that produce protease-resistant bacteriocin significantly inhibited the proliferation of Salmonella. See table 15.

TABLE 15 EXAMPLE 7 The bactericidal effect of Salmonella in food (a) Culture of lactic acid bacteria Weissella sp. AJ110263 (a bacterium that produces PRB) was cultured for 24 hours at 30 ° C in MRS medium to which 0.1 percent L-Cys and 0.1 percent L-Met were added. The culture solution was used in the following tests as a solution containing protease-resistant bacteriocin. (b) Preparation of food artificially contaminated with Salmonella The strain of Salmonella enteritidis (SE) KTE-61 (resistant to rifampicin) was cultured for 24 hours at 37 ° C in medium. infusion of brain and heart (Difco). An amount of 300 mL of this culture solution (living bacteria count of 109 cfu / mL) was added to 6 kg of commercial feed combination (non-chemical commercial feed for broilers "BroAce F2") and mixed in a mixer to prepare food contaminated with SE. (c) Addition of antibiotic Separately, a total of four segments were prepared in the form of a segment obtained by adding 0.2 percent commercial antibiotic "Bio-Add" (0.2 percent formic acid + propionic acid) to the artificially contaminated food with Salmonella in paragraph (b), a segment obtained by adding 2 percent of a solution containing protease-resistant bacteriocin to it, and a segment without added antibiotic (control). (d) Antibiotic evaluation Over time, each food segment was sampled and. Illuyó with phosphate buffer to measure the live bacteria count. For counting live bacteria, the diluted solution was rubbed on MLCB agar medium (Nissui Pharmaceutical Co., Ltd.) to which 0.1 mg / ml rifampicin was added and cultured for 24 hours at 37 ° C. As indicated in Figure 1 below, the solution containing PRB of lactic acid bacteria showed immediate, stable, and good bactericidal results in Salmonella in food. After 30 hours, an antibiotic effect was maintained that exceeds that of the commercial antibiotic Bio-Add.

EXAMPLE 8 Campylobacter Proliferation Inhibition Test (Live Bacteria Counting Evaluation) (a) Sample preparation Bacteria of lactic acid were cultured and filtered through the method set forth in (a) of Example 5 to prepare a sterile supernatant. (b) Campylobacter proliferation inhibition test (live bacteria count evaluation) The pre-cultured Campylobacter jejuni strain 702 was suspended in 10 6 cells / ml in Brucella medium and 1 percent of the culture supernatant of the bacteria was added. lactic acid. The live bacteria count of Campylobacter was made using CCDA medium.

Compared with the non-addition (control) system, the PRB-producing bacterial supernatant markedly inhibited the proliferation of Campylobacter, as shown in Table 16. TABLE 16 n.d. = not detected

Claims (10)

NOVELTY OF THE INVENTION CLAIMS

1. - An antibiotic comprising a protease resistant bacteriocin isolated from a lactic acid bacterium.

2. A composition comprising the antibiotic of claim 1 and one or more suitable excipients.

3. The composition according to claim 2, further characterized in that said composition is formulated for administration to livestock.

4. A composition comprising a lactic acid bacterium belonging to a genus selected from the group consisting of Lactobacillus, Weissella, Pediococcus, Leuconostoc, and combinations thereof.

5. The composition according to claim 4, further characterized in that said lactic acid bacterium is selected from the group consisting of Lactobacillus plantarum, Lactobacillus salivarius, Lactobacillus pentosus, Weissella sp. FERM BP-10474, W? Issella cibaria, Weissella confusa, Weissella hellenica, Weissella kandleri, Weissella minor, Weissella paramesenteroides, Weissella thailandensis, Pediococcus pentosaceus, Leuconostoc citreum, Leuconostoc pseudomesenteroides, Leuconostoc argentinum, Leuconostoc carnosum, Leuconostoc mesenteroides, and combinations of same.

6. A food composition comprising the antibiotic of claim 1.

7. A food composition comprising the composition of claim 4.

8. The use of the composition of claim 2 to prepare a medicine to prevent growth of bacteria responsible for poisoning by food for human in the stomach and / or cattle intestines.

9. The use of the composition of claim 4 for preparing a medicament for preventing the growth of bacteria responsible for human food poisoning in the stomach and / or intestines of cattle.

10. The use as claimed in claims 8 or 9, wherein said bacteria are of the sort selected from the group consisting of Salmonella, Campylobacter, Listeria, Escherichia coli, Welsh, and combinations thereof.