Compositions comprising probiotics, and methods of using the same

Abstract

To provide a method for reinforcing viability of probiotics when being subjected to heat.SOLUTION: A composition includes probiotics in a mixture with an effective amount of an amphipathic material that reinforces viability of probiotics in the composition when the composition is subjected to heat.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The field of the present invention relates to compositions and uses of probiotics containing Pediococcus acidilactici bacteria.

Background Art

[0002] Probiotics are beneficial microorganisms that naturally exist in the digestive (GI) tracts of humans and animals. In 2001, the World Health Organization defined probiotics as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host" (Joint FAO / WHO Expert Consultation Report, 2001). Health effects associated with many probiotics, such as the reduction of symptoms resulting from antibiotic-induced diarrhea, acute diarrhea, traveler's diarrhea, allergies, respiratory and urinary tract infections, inflammatory bowel disease, irritable bowel syndrome, colon and bladder cancer, and rheumatoid arthritis, have been reported. So far, Bifidobacterium and Lactobacillus are commercially available probiotics. However, these bacteria are sensitive to air exposure, elevated temperature, and gastric acid.

[0003] Pediococcus acidilactici is a plant-derived probiotic widely applied as a sausage seasoning for human consumption and as an animal feed additive to improve animal health. Furthermore, P. acidilactici has been reported to be able to stimulate antibody production against parasitic infection of coccidiosis in broiler chickens and ovalbumin antibody production in horses vaccinated with ovalbumin (Furr et al., Journal of Equine Veterinary Science, 34: 1156-1163 (2014)). Proliferation of both T cells and B cells was detected in rats fed a Pediococcus-based probiotic, a mixture of P. acidilactici and Sacchromyces boulardii.

[0004] One of the difficulties in formulating probiotics in foods and pharmaceutical / nutritional supplements for administration to subjects is the incompatibility of the product and the product manufacturing process, particularly with sterilization or treatment under harsh conditions such as heat. These harsh conditions can cause a significant loss of viability of the probiotics. The inability to effectively sterilize the product in the presence of probiotics without significantly losing viability results in additional processing steps in the manufacturing process, which increases complexity and cost.

[0005] Therefore, there is a need for new compositions and methods containing probiotics such as Pediococcus acidilactici, particularly methods for enhancing the viability of probiotics when subjected to heat.

[0006] This preliminary knowledge is provided for informational purposes only. No admission is necessarily intended, nor should it be construed, that any of the foregoing information constitutes prior art against the present invention. SUMMARY OF THE INVENTION

[0007] It is understood that both the above summary of the embodiments and the following detailed description are exemplary and, thus, do not limit the scope of the embodiments.

[0008] According to a non-limiting embodiment, in one aspect, the present invention provides a composition comprising a probiotic in a mixture with an effective amount of an amphiphilic substance that enhances the viability of the probiotic in the composition when the composition is subjected to heat.

[0009] In another aspect, the present invention provides a container suitable for containing a food product that includes a composition comprising a probiotic in a mixture with an effective amount of an amphiphilic substance that enhances the viability of the probiotic in the composition when the composition is subjected to heat.

[0010] In another aspect, the present invention provides a container suitable for containing a food product, wherein a part or surface of the container is coated with an effective amount of Pediococcus acidilactici.

[0011] In another aspect, the present invention provides a method for enhancing the viability of a probiotic in a composition subjected to heat, the method comprising: i) adding an effective amount of an amphiphilic substance to a composition comprising a probiotic; and ii) subjecting the composition to heat, whereby the viability of the probiotic in the composition subjected to heat is enhanced. Thereby, the viability of the probiotic in the composition subjected to heat is enhanced.

[0012] In another aspect, the present invention provides a method for treating a disease or condition characterized by inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of the Pediococcus acidilactici probiotic composition described herein.

[0013] In some embodiments, administration of Pediococcus acidilactici probiotics increases the number of anti-inflammatory M2 macrophage cells in a subject, thereby treating a disease or condition. In some embodiments, administration of Pediococcus acidilactici probiotics increases IL-10 production in a subject, thereby treating a disease or condition. In some embodiments, administration of Pediococcus acidilactici probiotics decreases the levels of IL-6 and / or IL-23 in a subject, thereby treating a disease or condition.

[0014] In some embodiments, the disease or condition characterized by inflammation is selected from the group consisting of malignant tumors (cancer), arthritis, cardiovascular disease, hepatitis, infection, wound healing, pancreatitis, gastroesophageal reflux disease, diabetes, inflammatory bowel disease, peptic ulcer disease, bronchitis, cholecystitis, appendicitis, bursitis, dermatitis, asthma, autoimmune disease, pelvic inflammatory disease, gout, trauma, foreign body infection, burns, dental treatment, tendinitis, rhinitis, mucositis, and exposure to toxins such as chemicals and alcohol.

[0015] In some embodiments, the Pediococcus acidilactici probiotic is the NRRL B-50517 strain. In some embodiments, the subject is human.

[0016] In some embodiments, the subject is administered more than 1.0×10 9 cfu of the probiotic. In some embodiments, the subject is administered more than 4.0×10 9 cfu of the probiotic.

[0017] In some embodiments, the subject is administered one or more additional therapeutic agents. In some embodiments, the subject is administered one or more chemotherapy (anticancer) agents and / or radiation therapy in combination with the Pediococcus acidilactici probiotic.

[0018] In some embodiments, the subject is not administered another therapeutic agent. In some embodiments, the subject is not administered another probiotic.

[0019] In another aspect, the present invention provides a composition comprising a Pediococcus acidilactici probiotic. In some embodiments, the Pediococcus acidilactici is the NRRL B-50517 strain. In some embodiments, the composition is a pharmaceutical composition.

[0020] It is understood that both the foregoing summary and the following detailed description are exemplary and thus do not limit the scope of the invention. Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, while the detailed description and specific examples represent specific embodiments of the invention, various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art from this detailed description, and it should be understood that the detailed description is provided for purposes of illustration only.

Brief Description of the Drawings

[0021] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0022]

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Mode for Carrying Out the Invention

[0023] The present invention is partly based on the surprising discovery that the viability of probiotics such as Pediococcus acidilactici can be enhanced when the composition is combined with an effective amount of an amphiphilic substance when the composition is subjected to heat. The present invention is also partly based on the surprising discovery that an effective amount of Pediococcus acidilactici probiotics can treat diseases or conditions characterized by inflammation.

[0024] Here, embodiments of the present invention are referred to in detail, which serve to explain the principles of the present invention in conjunction with the drawings and the following examples. These embodiments are described in sufficient detail to enable those skilled in the art to practice the present invention, and it is understood that other embodiments may be utilized and that structural, biological, and chemical changes may be made without departing from the spirit and scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0025] For the purpose of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include the plural and vice versa. If any of the definitions set forth below conflict with the use of that term in any other document incorporated herein by reference, the following definitions shall always govern for the purpose of interpreting this specification and its associated claims, unless the contrary meaning is clearly intended (e.g., in the document in which the term was originally used). When used in conjunction with the term "comprising" in the claims and / or this specification, the use of the word "a" or "an" may mean "one", but is also not inconsistent with the meanings of "one or more", "at least one", and "one or more than one". The use of the term "or" in the claims is used to mean "and / or" unless it is clearly indicated to refer only to alternatives or the alternatives are mutually exclusive, but this disclosure supports definitions that refer only to alternatives and "and / or". When used in this specification and the claims (s), the term "comprising" (and any form of comprising such as "comprise" and "comprises") shall be inclusive or open-ended and shall not exclude additional, unrecited elements or method steps. Further, when the term "comprising" is used in the description of one or more embodiments, one of ordinary skill in the art can understand that in some specific cases, the embodiment(s) may alternatively be described using the phrases "consisting essentially of" and / or "consisting of". As used herein, the term "about" means up to ±10% of the numerical value of the number with which it is used.

[0026] Those skilled in the art may refer to general reference documents for a detailed description of known techniques or equivalent techniques discussed herein. These documents include, for example, Current Protocols in Molecular Biology (Ausubel et al., eds., John Wiley & Sons, N.Y. and its appendices), Current Protocols in Immunology (Coligan et al., eds., John Wiley & Sons, N.Y. and its appendices), Current Protocols in Pharmacology (Enna et al., eds., John Wiley & Sons, N.Y. and its appendices), and Remington: The Science and Practice of Pharmacy (Lippincott Williams & Wilkins, 21st edition (2005)).

[0027] Heat-resistant probiotic composition and production method In one embodiment, the present invention provides a composition comprising a probiotic in a mixture with an effective amount of an amphiphilic substance that enhances the viability of the probiotic in the composition when the composition is subjected to heat. The composition comprises an effective amount of a probiotic when suitable for administration to a subject.

[0028] In another embodiment, the present invention includes a method for producing a probiotic composition having improved viability upon reaction with heat, the method comprising adding an effective amount of an amphiphilic substance to a composition comprising a probiotic.

[0029] In another embodiment, the present invention includes a method for enhancing the viability of a probiotic in a composition subjected to heat, the method comprising i) adding an effective amount of an amphiphilic substance to a composition comprising a probiotic; and ii) subjecting the composition to heat. Thereby, the viability of the probiotics in the composition subjected to heat is enhanced.

[0030] The probiotics are not particularly limited. In some embodiments, the probiotics are mixed with an amphiphilic substance and a lyophilized fermentation medium. In some embodiments, the probiotics are Pediococcus acidilactici. The Pediococcus acidilactici probiotics that can be used according to the present invention are not limited. In some embodiments, Pediococcus acidilactici is a strain that can survive above 65°C and can grow under aerobic and anaerobic conditions and at pH 1 to 6.2. In some embodiments, the present Pediococcus acidilactici is a strain deposited in the Agricultural Research Service (ARS) Patent Culture Collection as NRRL B-50517. The NRRL B-50517 strain is described in U.S. Application No. 13 / 676,579, which is incorporated herein by reference.

[0031] In some embodiments, the Pediococcus acidilactici for use in the present invention can be selected for its resistance to elevated temperature, low pH values, and aerobic and anaerobic conditions.

[0032] In some embodiments, the composition contains more than about 1.0×10 9 cfu of probiotics. In some embodiments, the composition contains more than 4.0×10 9 cfu of probiotics.

[0033] Amphiphilic molecules are molecules that have both a polar part and a non-polar part in their structure. Amphiphilic molecules generally have a hydrophobic part of the molecule that faces the hydrophobic phase and a hydrophilic part that faces the aqueous layer.

[0034] Some of the chemical compounds characterized by these molecules are essential for the acceptance of biological and industrial processes. In some embodiments, the amphiphilic substance includes lecithin, peanut butter, almond butter, soy butter, or cookie butter as a carrier for probiotics such as Pediococcus acidilactici NRRL B-50517 fermented culture bacteria. In some embodiments, an excess of amphiphilic substance is used, which can protect the P. acidilactici NRRL B-50517 fermented culture bacteria from severe dry heat or moist heat treatment. Lecithin is phosphatidylcholine and can include a natural mixture of neutral and polar fats derived from plants and / or animals. It is a poor water-soluble but excellent emulsifier. In an aqueous solution, its phospholipids can form either liposomes, bilayer sheets, micelles, or lamellar structures depending on the hydration and temperature. This results in surfactants of the type normally classified as amphiphilic.

[0035] In some embodiments, the amphiphilic substance is a lipid. The hydrophilic characteristic is derived from the presence of polar or charged groups such as carbohydrates, phosphato, carboxylic acids, sulfato, amino, sulfhydryl, nitro, hydroxy, and other similar groups. Hydrophobicity can be imparted by including nonpolar groups, which include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted by one or more aromatic, alicyclic, or heterocyclic group(s). Examples of amphiphilic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids. Representative examples of phospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus, such as sphingolipids, the sphingoglycolipid family, diacylglycerol, and β-acyl-oxy acids, also fall into the group designated as amphiphilic lipids.

[0036] In some embodiments, the amphiphilic substance includes lecithin, peanut butter, almond butter, soy butter, or cookie butter. In some embodiments, the amphiphilic substance is sunflower lecithin.

[0037] The "effective amount" of the amphiphilic substance described in this specification is an amount that can enhance the viability of probiotics in response to heat, as compared to the viability of probiotics in the absence of the amphiphilic substance. In some embodiments, the viability of the probiotics is enhanced by at least about 2-fold as compared to a composition lacking an effective amount of the amphiphilic substance. In some embodiments, the viability is enhanced by at least about 25%, 50%, 75%, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold. In some embodiments, the viability is enhanced by at least about 10-fold as compared to a composition lacking an effective amount of the amphiphilic substance.

[0038] In some embodiments, the ratio (w / w) of the amphiphilic substance to the probiotics ranges from about 1:10 to about 25:1. In some embodiments, the ratio (w / w) of the amphiphilic substance to the probiotics ranges from about 1:1 to about 10:1. In some embodiments, the ratio (w / w) of the amphiphilic substance to the probiotics is about 5:1. In some embodiments, the ratio (w / w) of the amphiphilic substance to the probiotics is about 10:1. In some embodiments, the ratio of the amphiphilic substance can be higher than 25:1. For example, in some embodiments, peanut butter powder and / or lecithin can be treated as a sugar carrier. In some embodiments, up to 1 part of probiotics can be produced per 25 parts of lecithin or peanut butter, but the probiotics can be blended with the sugar carrier as desired. This means that 1 part of probiotics can be mixed without problem with 100 parts (or even more) of lecithin or peanut butter powder.

[0039] In some embodiments, the amphiphilic substance is mixed with the probiotic as a dry powder. In some embodiments, the amphiphilic substance acts as a carrier and is combined with the probiotic and an oil to form a lipid paste that makes the probiotic more resistant to heat, such as dry heat treatment or liquid heat treatment, such as sterilization for food processing. The oil is not particularly limited and may include edible oil. In some embodiments, the oil may include plant-derived oils such as olive oil, coconut oil, soybean oil, fish oil, sunflower oil, canola oil, corn oil, peanut oil, and other vegetable oils, as well as animal-derived oils such as butter and lard. In some embodiments, the oil is olive oil. In some embodiments, the ratio (w / w) of amphiphilic substance to probiotic to oil ranges from about 1:10:0.1 to about 10:1:25. In some embodiments, the ratio (w / w) of amphiphilic substance to oil ranges from about 1:0.5 to about 0.5:1. In some embodiments, the ratio (w / w) of amphiphilic substance to oil is about 1:1.

[0040] The nature of the heat is not necessarily limited and may include dry heat, steam, and liquid heat treatment. Liquid heat means that the product is immersed in a warm liquid such as water and the heat treatment is carried out with the liquid.

[0041] In some embodiments, the composition is subjected to dry heat. The dry heat treatment can be achieved using an oven or a hot plate without using water and steam. In some embodiments, the heating can start at a first temperature and end at a second desired temperature. In some embodiments, the composition is immediately subjected to dry heat at the desired temperature without a heating step. In some embodiments, the desired temperature of the dry heat is at least 50°C. In some embodiments, the dry heat is at least about 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or 100°C. In some embodiments, the dry heat is in the range of 65 - 100°C. The composition can be exposed to one or more heat treatments at one or more different temperatures for a period of time after being subjected to dry heat at the first temperature for a period of time.

[0042] The exposure period to heat is not particularly limited. In some embodiments, the composition is immediately subjected to heat and then immediately removed from the heat. In some embodiments, the composition is subjected to heat for at least about 1 second. In some embodiments, the composition is subjected to heat for at least about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or at least 2 hours. In some embodiments, the composition is subjected to heat for at least about 10 minutes. In some embodiments, the composition is subjected to dry heat at a first temperature and then the composition is subjected to dry heat at a second temperature. In some embodiments, the first temperature is about 50 - 65°C and the second temperature is about 85°C or higher. In some embodiments, the composition is subjected to heat at the first temperature for at least about 1 second to at least about 2 hours and subjected to heat at the second temperature for at least about 1 second to at least about 2 hours. In some embodiments, the composition is subjected to the first temperature overnight. In some embodiments, the composition is subjected to the second temperature overnight. In some embodiments, the composition is subjected to heat at the first temperature for about 30 minutes and subjected to heat at the second temperature for about 10 minutes. In some embodiments, the composition is subjected to heat at the first temperature for about 30 minutes and subjected to heat at the second temperature for about 30 minutes. In some embodiments, the purpose of multiple heat treatments at different temperatures is to ensure that the sample is at an appropriate temperature for the treatment at the second temperature, which is due to the fact that it may take time for the material to warm up from room temperature to, for example, 85°C. In some embodiments, the composition is pre-treated at 65°C for between 30 minutes and overnight and then transferred to 85°C for the treatment time to achieve the heat treatment at the appropriate temperature.

[0043] In some embodiments, the composition is subjected to liquid heat. In some embodiments, the liquid heat is at least about 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C. In some embodiments, the liquid heat is at least about 50 - 65°C. In some embodiments, the liquid heat is in the range of about 65°C to about 95°C. In some embodiments, the liquid heat is at least about 70°C, 75°C, or 80°C. In some embodiments, the liquid heat is at least about 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, or 95°C.

[0044] In some embodiments, the composition is immediately subjected to liquid heat and then removed from the heat. In some embodiments, the composition is subjected to liquid heat for at least about 1 second. In some embodiments, the composition is subjected to liquid heat for at least about 1 second to about 1 hour. In some embodiments, the composition is subjected to liquid heat for at least about 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds, 6 seconds, 7 seconds, 8 seconds, 9 seconds, 10 seconds, 20 seconds, 30 seconds, 40 seconds, 50 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, or at least 1 hour. In some embodiments, the composition is subjected to heat for at least about 5 minutes. In some embodiments, the composition is subjected to heat for at least about 10 minutes.

[0045] In some embodiments, the composition is contacted with a food, a pharmaceutical, or a nutraceutical and subjected to heat with the product. In some embodiments, the heated food, pharmaceutical, or nutraceutical is dispensed into a container containing the composition or otherwise combined with the composition. In some embodiments, the heated food, pharmaceutical, or nutraceutical is contacted immediately after heat treatment without taking a large cooling step prior to combining the product with the probiotic composition. Thus, in some embodiments, subjecting the composition to the heat step of part ii) involves contacting the composition with a food, a pharmaceutical, or a nutraceutical that has already been subjected to heat prior to contact with the composition. In some embodiments, the composition will be present within a container into which the heated food, pharmaceutical, or nutraceutical is dispensed.

[0046] Container In some embodiments, the invention further provides a container suitable for containing a food, a pharmaceutical, or a nutraceutical comprising the composition described herein. In some embodiments, the container is made of a material including glass, plastic, paper / carton, aluminum, or a dried plant fruit shell. The composition and shape of the container are not limiting, provided that they are suitable for containing a food, a pharmaceutical, or a nutraceutical. In some embodiments, the container is coated with the probiotic composition described herein.

[0047] In some embodiments, the present container is coated with an effective amount of Pediococcus acidilactici. In some embodiments, the Pediococcus acidilactici is the NRRL B-50517 strain. In some embodiments, a composition comprising a probiotic such as Pediococcus acidilactici containing the NRRL B-50517 strain and an amphiphilic substance such as the sunflower lecithin provided herein is coated on the surface or a part of the present container. In some embodiments, the present container further contains food, dietary supplement, or medicine. In some embodiments, the food includes oil, vinegar, yogurt, fruit products, apple sauce, dairy products, beverages, candies, snack foods, juices, or food for desserts, such as cookies, cakes, JELLO, fruit bars, fruit custards, or tiramisus. In some embodiments, after a heated food, dietary supplement, or medicine is dispensed into the coated container, the container is sealed.

[0048] Treatment method According to a non-limiting exemplary embodiment, in one aspect, the present invention provides a method for treating a disease or condition characterized by inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of a Pediococcus acidilactici probiotic. In some embodiments, the subject is administered the composition described herein. In some embodiments, the present composition comprises the probiotic and the amphiphilic substance described herein.

[0049] As used herein, "treating" and all its forms and tenses (including, for example, "treating", "treated", and "treatment") refer to therapeutic measures. In certain embodiments of the present invention, the subject in need of treatment includes a subject already suffering from a pathological disease or condition of the present invention (including, for example, cancer), in which case the treatment refers to administering a therapeutically effective amount of the composition to the subject (including, for example, a human or other mammal in need of treatment), such that the subject has an improvement in the signs or symptoms of the pathological condition of the present invention. This improvement may be any observable or measurable improvement. For this reason, those skilled in the art will understand that the treatment may improve the patient's condition, but may not result in a complete cure of the disease or pathological condition.

[0050] The subjects treated herein are not limited. In some embodiments, the subject is a mammal, bird, reptile, or fish. Mammals that can be treated in accordance with the present invention include, but are not limited to, humans, dogs, cats, horses, mice, rats, guinea pigs, sheep, cows, pigs, monkeys, apes, etc., which are subjects of diseases or other pathological conditions characterized by inflammation. The terms "patient" and "subject" are used interchangeably. In some embodiments, the subject is a human.

[0051] Pediococcus acidilactici probiotics can be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, or twelve times a day. In some embodiments, Pediococcus acidilactici probiotics are administered four times a day, twice a day, or once a day. In some embodiments, Pediococcus acidilactici probiotics are administered every two hours, every four hours, every six hours, every eight hours, every ten hours, every twelve hours, or every twenty-four hours. In some embodiments, Pediococcus acidilactici probiotics are administered once a day.

[0052] The administration period of Pediococcus acidilactici probiotics can vary for each individual being treated, depending on the individual case and the disease or condition being treated. In some embodiments, the Pediococcus acidilactici probiotics can be administered continuously for a treatment period of several days, weeks, months, or years, or can be administered intermittently. In the latter case, the individual receives Pediococcus acidilactici probiotics for a period of time, then does not receive treatment for a period of time, and then resumes treatment as needed to treat the disease or condition. For example, in some embodiments, the individual being treated receives the Pediococcus acidilactici probiotics of the present invention daily, every other day, every three days, every four days, two days per week, three days per week, four days per week, five days per week, or seven days per week. In some embodiments, the individual receives the Pediococcus acidilactici probiotics for one week, two weeks, three weeks, four weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, one year, or longer.

[0053] In some embodiments, administration of the Pediococcus acidilactici probiotics increases the number of anti-inflammatory M2 macrophage cells in the subject, thereby treating the disease or condition. In some embodiments, the anti-inflammatory M2 macrophage cells increase by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 350%, about 400%, or about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% or more compared to the non-treatment level.

[0054] In some embodiments, administration of Pediococcus acidilactici probiotics increases IL-10 production in a subject, thereby treating a disease or condition. In some embodiments, IL-10 production increases by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 125%, about 150%, about 175%, about 200%, about 250%, about 300%, about 350%, about 400%, or about 450%, about 500%, about 600%, about 700%, about 800%, about 900%, or about 1000% or more compared to non-treatment levels.

[0055] In some embodiments, administration of Pediococcus acidilactici probiotics reduces the levels of IL-6 and / or IL-23 in a subject, thereby treating a disease or condition. In some embodiments, the levels of IL-6 and / or IL-23 are reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% compared to non-treatment levels.

[0056] In some embodiments, administration of Pediococcus acidilactici probiotics increases the number of anti-inflammatory M2 macrophage cells in a subject, and concomitantly increases IL-10 production and optionally decreases IL-6 and / or IL-23 in the subject.

[0057] In some embodiments, the disease or condition characterized by inflammation is selected from the group consisting of malignant tumors (cancer), arthritis, cardiovascular disease, hepatitis, infection, wound healing, pancreatitis, gastroesophageal reflux disease, diabetes, inflammatory bowel disease, peptic ulcer disease, bronchitis, cholecystitis, appendicitis, bursitis, dermatitis, asthma, autoimmune disease, pelvic inflammatory disease, gout, trauma, foreign body infection, burns, dental treatment, tendinitis, rhinitis, mucositis, and exposure to toxins such as chemicals and alcohol.

[0058] As used herein, "cancer" refers to a pathophysiological state in which cells are characterized by unregulated and / or proliferative cell growth and the ability to induce such growth, and includes epithelial and non-epithelial malignancies, such as acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphomas, anal cancer, astrocytoma (including, for example, those of the cerebellum and cerebrum), basal cell carcinoma, bile duct carcinoma, bladder cancer, bone cancer, brainstem glioma, brain tumors (including, for example, ependymoblastoma, medulloblastoma, supratentorial primitive neuroectodermal, optic pathway and hypothalamic gliomas), cerebral astrocytoma / malignant glioma, breast cancer, bronchial adenoma / carcinoid, Burkitt lymphoma, carcinoid tumor (including, for example, those of the gastrointestinal tract), cancer of unknown primary site, central nervous system lymphoma, cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorders, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing tumor, extrahepatic bile duct carcinoma, eye cancer (including, for example, intraocular melanoma, retinoblastoma), gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor (including, for example, extracranial, extragonadal, ovarian), gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, head and neck squamous cell carcinoma, hepatocellular carcinoma, Hodgkin lymphoma, hypopharyngeal cancer, islet cell carcinoma (including, for example, those of the endocrine pancreas), Kaposi sarcoma, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer, lung cancer (including, for example, non-small cell cancer), lymphoma, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous neck cancer of unknown primary, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasmacytoma, mycosis fungoides, myelodysplastic syndrome, myelodysplasia / myeloproliferative disease, myeloma, nasal and paranasal cavity cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cancer, oral cavity cancer, osteosarcoma, oropharyngeal cancer, ovarian cancer (including, for example, ovarian, epithelial ovarian cancer, germ cell tumor), ovarian low malignant potential tumor, pancreatic cancer, paranasal and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasmacytoma / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system lymphoma, prostate cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sézary syndrome, skin cancer (including, for example, non-melanoma or melanoma), small intestine cancer,Primitive neuroectodermal tumors on the tent, T-cell lymphoma, testicular cancer, pharyngeal cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor (including, for example, gestational trophoblastic tumor), rare cancers in children and adults, urethral cancer, endometrial carcinoma of the uterus, uterine sarcoma, vaginal cancer, virus-induced cancer (including, for example, HPV-induced cancer), vulvar cancer, Waldenström macroglobulinemia, Wilms tumor, and cancers in women, etc. are included, but not limited thereto.

[0059] The Pediococcus acidilactici probiotics that can be used according to the present invention are not limited. In some embodiments, Pediococcus acidilactici is a strain that can survive above 65°C and can grow under aerobic and anaerobic conditions and at pH 1 to 6.2. In some embodiments, the present Pediococcus acidilactici is a strain deposited in the Agricultural Research Service (ARS) Patent Culture Collection as NRRL B-50517. The NRRL B-50517 strain is described in U.S. Application No. 13 / 676,579, which is incorporated herein by reference.

[0060] In some embodiments, Pediococcus acidilactici for use in the present invention can be selected for resistance to elevated temperature, low pH values, and aerobic and anaerobic conditions.

[0061] According to the treatment method of the present invention, a "therapeutically effective amount" or "effective amount" is administered to a subject. As used herein, a "therapeutically effective amount" or "effective amount" is an amount sufficient to reduce, suppress, or alleviate one or more symptoms associated with a disease or condition. In some embodiments, the subject is administered more than 1.0×10 9 cfu of probiotics. In some embodiments, the subject is administered more than 4.0×10 9 cfu of probiotics.

[0062] In some embodiments, the subject is administered one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents are those commonly used in the treatment of diseases or conditions characterized by inflammation.

[0063] In some embodiments, the subject is co-administered an anti-inflammatory agent. In some embodiments, the administered Pediococcus acidilactici and the anti-inflammatory agent act synergistically. In some embodiments, the anti-inflammatory agent is a non-steroidal anti-inflammatory drug (NSAID). In some embodiments, the anti-inflammatory agent is selected from the group consisting of antazoline, balsalazide, beclomethasone, betamethasone, budesonide, celecoxib, colchicine, deflazacort, dexamethasone, dexibuprofen, diclofenac, etanercept, etodolac, felbinac, fenoprofen, flumethasone, fluocinolone, flurbiprofen, flurbiprofen, fluticasone, gentamicin, hydrocortisone, ibuprofen, indomethacin, ketoprofen, loteprednol, mefenamic acid, meloxicam, mesalazine, methylprednisolone, mometasone, nabumetone, naproxen, nepafenac, olsalazine, prednisone, rimixolone, sulfasalazine, sulindac, tenoxicam, tiaprofenic acid, triamcinolone, and combinations thereof.

[0064] In some embodiments, the subject is administered one or more anti-cancer agents and / or radiation therapy in combination with Pediococcus acidilactici probiotics for treating cancer in the subject.

[0065] In some embodiments, the anti-cancer agent is abiraterone acetate, Abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), Adrucil (fluorouracil), afatinib dimaleate, Afinitor (everolimus), Aldara (imiquimod), Aldesleukin, alemtuzumab, Alimta (pemetrexed disodium), Aloxi (palonosetron hydrochloride), Amboclorin (chlorambucil), Amboclorin (chlorambucil), aminolevulinic acid, anastrozole, aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Aranone (nelarabine), arsenic trioxide, Arzerra (ofatumumab), asparaginase Erwinia chrysanthemi, Avastin (bevacizumab), axitinib, azacitidine, BEACOPP, Becenum (carmustine), Beleodaq (belinostat), belinostat, bendamustine hydrochloride, BEP, bevacizumab, bexarotene, Bexxar (tositumomab iodine 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, busulfan, Busulfex (busulfan), cabazitaxel, cabozantinib-S-malate, CAF, Campath (alemtuzumab), Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, carboplatin, CARBOPLATIN-TAXOL (carboplatin-paclitaxel), carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CeeNU (lomustine) ceritinib, Celbidine (daunorubicin hydrochloride), Cervarix (recombinant HPV bivalent vaccine), cetuximab, chlorambucil,Chlorambucil-Prednisone, CHOP, Cisplatin, Clafen (Cyclophosphamide), Clofarabine, Clolar (Clofarabine), Chloral (Clofarabine), CMF, Cometriq (Cabozantinib-S-Malic Acid Salt), COPP, COPP-ABV, Cosmegen (Dactinomycin), Crizotinib, CVP, Cyclophosphamide, Cyfos (Ifosfamide), Cyramza (Ramucirumab), Cytarabine, Cytarabine, Liposome, Cytosar-U (Cytarabine), Cytoxan (Cyclophosphamide), Dabrafenib, Dacarbazine, Dacogen (Decitabine), Dactinomycin, Dasatinib, Daunorubicin Hydrochloride, Decitabine, Degarelix, Denileukin Diftitox, Denosumab, Deposite (Cytarabine Liposome), Depoform (Cytarabine Liposome), Dexrazoxane Hydrochloride, Docetaxel, Doxil (Doxorubicin Hydrochloride Liposome), Doxorubicin Hydrochloride, Doxorubicin Hydrochloride Liposome, Dox-SL (Doxorubicin Hydrochloride Liposome), DTIC-Dome (Dacarbazine), Efudex (Fluorouracil), Elitek (Rasburicase), Ellence (Epirubicin Hydrochloride), Eloxatin (Oxaliplatin), Eltrombopag Olamine, Emend (Aprepitant), Enzalutamide, Epirubicin Hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin Mesylate, Erbitux (Bismodegib), Erlotinib Hydrochloride, Elspar (Asparaginase Erwinia Chrysanthemi), Etopophos (Etoposide Phosphate), Etoposide, Etoposide Phosphate, Evacet (Doxorubicin Hydrochloride Liposome), Everolimus, Evista (Raloxifene Hydrochloride), Exemestane, Fareston (Toremifene), Faslodex (Fulvestrant), FEC, Femara (Letrozole), Filgrastim, Fludara (Fludarabine Phosphate), Fludarabine Phosphate, Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), FOLFIRI,FOLFIRI-Bevacizumab, FOLFIRI-Cetuximab, FOLFIRINOX, FOLFOX, Folotyn (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant quadrivalent HPV vaccine), Gardasil 9 (recombinant nonavalent HPV vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride, GEMCITABINE-CISPLATIN (gemcitabine-cisplatin), GEMCITABINE-OXALIPLATIN (gemcitabine-oxaliplatin), gemtuzumab ozogamicin, Gemzar (gemcitabine hydrochloride), Tarceva (afatinib dimaleate), Glivec (imatinib mesylate), Gliadel (carmustine implant), Gliadel Wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Herceptin (trastuzumab), recombinant bivalent HPV vaccine, recombinant nonavalent HPV vaccine, recombinant quadrivalent HPV vaccine, Hycamtin (topotecan hydrochloride), Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Ifex (ifosfamide), ifosfamide, Ifosfamidum (ifosfamide), imatinib mesylate, Imbruvica (ibrutinib), imiquimod, Inlyta (axitinib), Intron A (recombinant interferon alpha-2b), iodine 131 tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, Istodax (romidepsin), ixabepilone, Xeloda (capecitabine), Kadcyla (ado-trastuzumab emtansine), tamoxifen (raloxifene hydrochloride), Kepivance (palifermin), Keytruda (pembrolizumab), Kyprolis (carfilzomib), lanreotide acetate, lapatinib ditosylate, lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), Letrozole,Leucovorin Calcium, Leukeran (Chlorambucil), Leuprolide Acetate, Reblan (Aminolevulinic Acid), Linfolizin (Chlorambucil), Lipodox (Doxorubicin Hydrochloride Liposome), Cytarabine Liposome, Lomustine, Lupron (Leuprolide Acetate), Lupron Depot (Leuprolide Acetate), Lupron Depot-Ped (Leuprolide Acetate), Lupron Depot-3 Month (Leuprolide Acetate), Lupron Depot-4 Month (Leuprolide Acetate), Lynparza (Olaparib), Marqibo (Vincristine Sulfate Liposome), Matulane (Procarbazine Hydrochloride), Mechlorethamine Hydrochloride, Megace (Megestrol Acetate), Megestrol Acetate, Mekinist (Trametinib), Mercaptopurine, Mesna, Mesnex (Mesna), Metazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), Mitomycin C, Mitoxantrone Hydrochloride, Mitozitelex (Mitomycin C), MOPP, Mozobil (Plerixafor), Mustargen (Mechlorethamine Hydrochloride), Mutamycin (Mitomycin C), Myleran (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin-Stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Nelarabine, Neosar (Cyclophosphamide), Neupogen (Filgrastim), Nexavar (Sorafenib Tosylate), Nilotinib, Nivolumab, Norvadex (Tamoxifen Citrate), N Plate (Romiplostim), Obinutuzumab, OEPA, Ofatumumab, OFF, Olaparib, Omacetaxine Mepesuccinate, Oncaspar (Pegaspargase), Ontak (Denileukin Diftitox), Opdivo (Nivolumab), OPPA, Oxaliplatin,Paclitaxel, Paclitaxel Albumin-Stabilized Nanoparticle Formulation, PAD, Palbociclib, Parifermin, Paroxetine Hydrochloride, Pamidronate Disodium, Panitumumab, Paraplatin (Carboplatin), Paraplatin (Carboplatin), Pazopanib Hydrochloride, Pegaspargase, Peginterferon Alfa-2b, PEG-Intron (Peginterferon Alfa-2b), Pembrolizumab, Pemetrexed Disodium, Perjeta (Pertuzumab), Pertuzumab, Platinol (Cisplatin), Platinol-AQ (Cisplatin), Prexasertib, Pomalidomide, Pomalis (Pomalidomide), Ponatinib Hydrochloride, Pralatrexate, Prednisone, Procarbazine Hydrochloride, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopag Olamine), Provege (Sipuleucel-T), Purinethol (Mercaptopurine), Purixan (Mercaptopurine), Radium Chloride 223, Raloxifene Hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant Human Papillomavirus (HPV) Bivalent Vaccine, Recombinant Human Papillomavirus (HPV) Nonavalent Vaccine, Recombinant Human Papillomavirus (HPV) Quadrivalent Vaccine, Recombinant Interferon Alfa-2b, Regorafenib, R-EPOCH, Revlimid (Lenalidomide), Rituxan (Rituximab), Rituximab, Romidepsin, Romiplostim, Rubidomycin (Daunorubicin Hydrochloride), LY294002, Sclerosol Intrapleural Aerosol (Talc), Cetuximab, Sipuleucel-T, Somatuline Depot (Lanreotide Acetate), Sorafenib Tosylate, Sprycel (Dasatinib), STANFORD V, Sterile Talc Powder (Talc), Steri-Talc (Talc), Stivarga (Regorafenib), Sunitinib Malate, Sutent (Sunitinib Malate), Sylatron (Peginterferon Alfa-2b), Cyramza (Cetuximab), Synovir (Thalidomide), Synribo (Omacetaxine Mepesuccinate),TAC, Tafinlar (dabrafenib), talc, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride), Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Temodal (temozolomide), temozolomide, Temsi, Selected from the group consisting of lomustine, thalidomide, salomide (thalidomide), thiotepa, topasar (etoposide), topotecan hydrochloride, tamoxifen, tricel (temsirolimus), tositumomab and iodine I 131 iodine tositumomab, tectect (dexrazoxane hydrochloride), TPF, trametinib, trastuzumab, treanda (bendamustine hydrochloride), trisenox (arsenous acid), tykerb (lapatinib ditosylate), vandetanib, VAMP, vectibix (panitumumab), VeIP, velban (vinblastine sulfate), velcade (bortezomib), versasar (vinblastine sulfate), bemrafenib, bepside (etoposide), viadur (leuprolide acetate), vidaza (azacitidine), vinblastine sulfate, vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, besmodigib, voraxaze (glucarpidase), vorinostat, botrient (pazopanib hydrochloride), wellcovorin (leucovorin calcium), xalkori (crizotinib), xeloda (capecitabine), XELIRI, xgeva (denosumab), xofigo (radium 223 dichloride), xtandi (enzalutamide), yervoy (ipilimumab), ziv-aflibercept, zelboraf (bemrafenib), zevalin (ibritumomab tiuxetan), zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, zoladex (goserelin acetate), zoledronic acid, zolinza (vorinostat), zometa (zoledronic acid), zydelig (idelalisib), dicardia (ceritinib), and zytiga (abiraterone acetate). In some embodiments, the drug is selected from the group consisting of paclitaxel, curcumin, docetaxel, ixabepilone, vinblastine, colchicine, Y-27632 fasudil, SU6656 dasatinib, HDAC inhibitor, ROCK inhibitor, parthenolide, costunolide, and ML-7 jazplakinolide.

[0066] In some embodiments, the subject is administered a composition consisting of, or consisting essentially of, Pediococcus acidilactici probiotics optionally mixed with an amphiphilic substance and / or food, without being administered another therapeutic agent.

[0067] In some embodiments, the subject is administered one or more additional probiotics. In some embodiments, the subject is not administered another probiotic.

[0068] Composition In some embodiments, the invention provides a composition comprising Pediococcus acidilactici probiotics. In some embodiments, the composition comprises Pediococcus acidilactici NRRL B-50517. In some embodiments, the composition comprises an effective amount of Pediococcus acidilactici, comprising Pediococcus acidilactici NRRL B-50517. In some embodiments, the invention provides a composition comprising Pediococcus acidilactici probiotics mixed with an effective amount of an amphiphilic substance and optionally an oil described herein. In some embodiments, the composition comprises a food, a pharmaceutical, or a dietary supplement, in combination with Pediococcus acidilactici probiotics mixed with an effective amount of an amphiphilic substance and optionally an oil.

[0069] In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a pharmaceutical composition comprising an effective amount of Pediococcus acidilactici, including Pediococcus acidilactici NRRL B-50517, which can treat one or more diseases or conditions characterized by inflammation. In some embodiments, the composition is a pharmaceutical composition comprising an effective amount of Pediococcus acidilactici, including an effective amount of an amphiphilic substance and optionally Pediococcus acidilactici NRRL B-50517 mixed with oil, which can treat one or more diseases or conditions characterized by inflammation.

[0070] In some embodiments, the pharmaceutical composition comprises one or more pharmaceutically acceptable carriers or additives. Pharmaceutically acceptable carriers and additives are compatible with other components in the formulation and are biologically acceptable. Pediococcus acidilactici can be provided in combination with a pharmaceutically acceptable carrier, additive, or diluent. Suitable carriers, additives, and / or diluents include, but are not limited to, pharmaceutical grade starch, mannitol, lactose, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose (or other sugars), magnesium carbonate, gelatin, oil, alcohol, surfactant, emulsifier, or water (preferably sterile). The composition may be a mixed preparation of the composition or a combined preparation for simultaneous use, separate use, or sequential use (including administration). The Pediococcus acidilactici can also be administered in sachets that need to be drunk after being added to a cup of water.

[0071] In some embodiments, the composition is suitable for oral administration. In some embodiments, the composition is a tablet, capsule, pill, dragee, suspension, troche, emulsion, aqueous solution, liquid, gel, or syrup. In some embodiments, the composition can be delivered in the form of a functional food and / or beverage, as well as in the form of various supplements.

[0072] The formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets, or tablets, each containing a predetermined amount of Pediococcus acidilactici, in some embodiments, as a powder or granule which may be wetted, spray-dried, or freeze-dried; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water emulsion or a water-in-oil liquid emulsion.

[0073] In some embodiments, tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets are in a free-flowing form such as a powder or granule and can be prepared by compressing in a suitable machine an active ingredient that has been mixed, optionally, with a binder (e.g., povidone, gelatin, hydroxypropylmethylcellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethylcellulose), surfactant, or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert diluent. Tablets may optionally be coated or scored and formulated to provide a desired release profile, for example, by using hydroxypropylmethylcellulose in different proportions to provide sustained or controlled release of the active ingredient therein. Tablets may optionally be provided with an enteric coating to effect release in a part of the digestive tract other than the stomach.

[0074] In some embodiments, the composition can comprise one or more of tablets, pills, capsules, suppositories, solutions, or suspensions, which can contain flavoring or coloring agents for immediate release, delayed release, modified release, sustained release, pulsatile release, or controlled release applications.

[0075] For aqueous suspensions and / or elixirs, the compositions of the invention can be combined with various sweetening or flavoring agents, coloring substances or dyes, emulsifying and / or suspending agents, and diluents such as water, propylene glycol, and glycerin, and combinations thereof.

[0076] In some embodiments, the compositions of the invention are formulated in unit dosage forms for ease of administration and uniformity of dosage. The term "unit dosage form" as used herein refers to physically discrete units suitable as a single dosage for the individual to be treated. In some embodiments, the composition is formulated into discrete dosage units each containing a predetermined "unit dosage" or "unit dose" of one or more active compounds calculated to produce the desired effect in association with the required pharmaceutical carrier.

[0077] In some embodiments, the composition comprises gelatin capsules. In some embodiments, the gelatin capsules contain an effective amount of P. acidilactici NRRL B-50517 fermented culture together with peach fruit powder at a dosage of about 1 to 4000 million CFU.

[0078] Although it is possible to administer Pediococcus acidilactici alone according to the present invention, the Pediococcus acidilactici is typically administered on or in a support as part of a product, in particular as a component of a food, a dietary supplement, or a pharmaceutical formulation. These products typically contain additional components well known to those skilled in the art. In one embodiment, the composition comprises an effective amount of an amphiphilic substance and Pediococcus acidilactici probiotics optionally mixed with oil.

[0079] In one embodiment, the present Pediococcus acidilactici is used herein in foods such as dietary supplements, beverages, or milk-based powders. As used herein, the term "food" is used in a broad sense and encompasses human food and animal food (i.e., feed). In one embodiment, the food is for human consumption.

[0080] The food may be in the form of a solution or a solid, depending on the form of use and / or application and / or administration.

[0081] When used as a food such as a functional food or in its preparation, the compositions of the present invention may be used in combination with one or more of a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable additive, a nutritionally acceptable adjuvant, and a nutritionally active ingredient.

[0082] By way of example, the compositions of the present invention can be used as ingredients for soft drinks, fruit juices or beverages containing whey protein, health teas, cocoa beverages, milk beverages, yogurts and drinking yogurts, cheeses, ice creams, water ices and desserts, confectioneries, biscuits, cakes, and cake bases, snack foods, balanced foods and beverages, fruit fillings, care glazes, chocolate fillings for bakery products, cheesecake-flavored fillings, fruit-flavored cake fillings, sugar icings for cakes and doughnuts, instant bakery filling creams, cookie fillings, ready-made bakery fillings, low-calorie fillings, nutritional beverages for adults, acidified soy / juice beverages, aseptic / retort chocolate beverages, snack foods for snacks, beverage powders, calcium-fortified soy / plain and chocolate milk, and calcium-fortified coffee beverages.

[0083] The composition can further be used as a component in foods such as anti-caking agents for American cheese sauce, grated cheese and shredded cheese, chip dips, cream cheese, dry blended whip topping fat free sour cream, frozen / thawed raw whipped cream, frozen / thawed stable whipped topping, low fat and light natural cheddar cheese, low fat Swiss style yogurt, aerated frozen desserts, compacted ice cream, compacted ice cream of low fat ice cream (soft serve) with easily distinguishable and economical ingredients, barbecue sauce, cheese dip sauce, cottage cheese dressing, dry mix Alfredo sauce, mixed cheese sauce, dry mix tomato sauce, and others.

[0084] As used herein, the term "dairy product" means including a medium composed of milk derived from animals and / or plants. Examples of milk derived from animals include cow, sheep, goat, or buffalo milk. Examples of milk derived from plants include any plant-derived fermentable substances that can be used according to the present invention, particularly those derived from soybeans, rice, or grains.

[0085] In some embodiments, the food used according to the present invention is fermented milk or humanized milk.

[0086] In some embodiments, the composition can be used in relation to the production of yogurt such as fermented yogurt beverages, yogurt, drinkable yogurt, cheese, fermented cream, milk-based desserts, and others.

[0087] Preferably, the composition can further be used as a component in one or more of cheese applications, meat applications, or applications containing protective bacteria.

[0088] The present invention provides a method for preparing a food or food ingredient, the method comprising mixing a composition according to the present invention with another food ingredient.

[0089] In some embodiments, the present invention relates to a product contacted with the composition of the present invention (and optionally other components / ingredients), wherein the composition is used in an amount capable of improving the nutrients and / or health effects of the product.

[0090] As used herein, the term "contacted" refers to directly or indirectly applying the composition of the present invention to a product. Examples of application methods that may be used include treating the product in a material containing the composition, directly applying by mixing the composition with the product, spraying the composition onto the product surface, or immersing the product in a preparation of the composition, but are not limited thereto.

[0091] When the product of the present invention is a food product, the composition of the present invention is preferably mixed with the product. Alternatively, the composition may be included in an emulsion or a raw material of a food product. Further alternatively, the composition may be applied as a seasoning, glaze, coloring mixture, etc.

[0092] For some applications, it is important that the composition be available on or to the surface of the product being acted upon / treated. Thereby, the composition can be made capable of conferring one or more of the favorable characteristics of nutrients and / or health effects.

[0093] The composition of the present invention may be applied to the product to disperse, coat with, and / or impregnate it with a controlled amount of live microorganisms.

[0094] In some embodiments, the composition is used in the fermentation of milk, or sucrose-enriched milk, or a milk medium having sucrose and / or maltose, wherein the medium obtained as a result of containing all the components of the composition (i.e., the previously mentioned microorganisms according to the present invention) contains, as one component, at a suitable concentration, for example, 10 6 ~10 10 cfu per daily dose can be added to yogurt at the concentration in the final product that provides the daily dose. The microorganisms according to the present invention may be used before or after the fermentation of yogurt.

[0095] In some aspects, the microorganisms according to the present invention are used as or in the preparation of animal feed such as livestock feed, particularly poultry (such as chickens) feed, pet food, or pet snacks.

[0096] In some embodiments where the product is a food, the present Pediococcus acidilactici should maintain its effect over the normal "sell-by date" or "expiration date", which is the period during which the food is offered for sale by the retailer. Preferably, the shelf life should extend beyond such dates until the end of the normal freshness period when food spoilage becomes apparent. The time and the desired length of the normal shelf life vary for each food product, and those skilled in the art will recognize that the shelf life varies depending on the type of food product, the size of the food product, the storage temperature, the processing conditions, the packaging material, and the packaging equipment.

[0097] In some embodiments, the composition of the present invention may be used as a food ingredient and / or a feed ingredient. As used herein, the terms "food ingredient" or "feed ingredient" include formulations that can be added or can be added as nutritional supplements to functional foods or food products. The food ingredient may be in the form of a solution or solid, depending on the form of use and / or application and / or administration.

[0098] In some embodiments, the composition of the present invention may be a dietary supplement (also referred to herein as a nutritional supplement) or may be added thereto.

[0099] In some embodiments, the compositions of the present invention may be functional foods or added thereto. As used herein, the term "functional food" means a food that not only provides nutritional effects but can also bring additional beneficial effects to consumers. Thus, a functional food is a normal food in which components or ingredients (such as those described herein) that confer specific functional benefits, such as medical or physiological benefits, in addition to mere nutritional effects, are incorporated. There is no legal definition of functional foods, but most people interested in this area agree that functional foods are foods sold as having specific health effects beyond basic nutritional effects. Some functional foods are dietary supplements. As used herein, the term "dietary supplement" means a food that can not only provide nutritional effects and / or taste satisfaction but can also bring therapeutic (or other beneficial) effects to consumers. Dietary supplements cross the traditional boundary line between food and medicine.

[0100] In some embodiments, the present invention provides a composition comprising Pediococcus acidilactici, such as Pediococcus acidilactici NRRL B-50517, and an edible oil. In some embodiments, the edible oil is selected from the group consisting of olive oil, corn oil, EVOO, LTOO, peanut oil, and vegetable oil. In some embodiments, the ratio (w / w) of oil to probiotics in the composition ranges from 1:1 to 10:1. In some embodiments, the probiotics exhibit heat resistance in the oil composition.

[0101] In some embodiments, the present invention provides a composition comprising Pediococcus acidilactici, such as Pediococcus acidilactici NRRL B-50517, combined with a sugar such as sucrose or lactose. In some embodiments, the concentration of the sugar ranges from 0.1% to 50% of the solution.

[0102] In some embodiments, the present invention provides a composition comprising a salt solution such as NaCl in combination with Pediococcus acidilactici such as Pediococcus acidilactici NRRL B-50517. In some embodiments, the concentration of the salt ranges from 0.1% to 20% of the solution.

[0103] In some embodiments, the present invention provides a composition comprising peanut butter in combination with Pediococcus acidilactici such as Pediococcus acidilactici NRRL B-50517. In some embodiments, the ratio (w / w) of peanut butter to probiotics ranges from 1:1 to 10:1.

[0104] Sample Embodiment This section describes exemplary compositions and methods of the present invention presented as a series of paragraphs, some or all of which may be designated in alphanumeric order for clarity and efficiency. Each of these paragraphs may be combined in any suitable manner with the disclosure from other parts of this application, including one or more other paragraphs and / or material incorporated by reference. Some of the following paragraphs explicitly reference other paragraphs, further limiting them, and provide non-limiting examples of some of the suitable combinations.

[0105] 1. A composition comprising an effective amount of a probiotic, the composition being in a mixture with an effective amount of an amphiphilic substance that enhances the viability of the probiotic in the composition when the composition is subjected to heat.

[0106] 2. The composition according to paragraph 1, wherein the probiotic is mixed with the amphiphilic substance as a freeze-dried fermentation medium.

[0107] 3. The composition according to any one of paragraphs 1 to 2, wherein the amphiphilic substance is mixed with the probiotic as a dry powder.

[0108] 4. The composition according to any one of paragraphs 1 to 3, further comprising an oil mixed with the amphiphilic substance and the probiotics.

[0109] 5. The composition according to paragraph 4, wherein the oil is olive oil.

[0110] 6. The composition according to any one of paragraphs 4 to 6, wherein the ratio (w / w) of the amphiphilic substance to the probiotics to the oil is in the range of about 1:10:0.1 to about 10:1:25.

[0111] 7. The composition according to any one of paragraphs 1 to 6, wherein the amphiphilic substance forms a lipid paste and acts as a carrier.

[0112] 8. The composition according to any one of paragraphs 1 to 7, wherein the probiotics is Pediococcus acidilactici.

[0113] 9. The composition according to any one of paragraphs 1 to 8, wherein the probiotics is Pediococcus acidilactici NRRL B-50517.

[0114] 10. The composition according to any one of paragraphs 1 to 9, wherein the amphiphilic substance comprises lecithin, peanut butter, almond butter, soy butter, or cookie butter.

[0115] 11. The composition according to any one of paragraphs 1 to 10, wherein the heat is dry heat.

[0116] 12. The composition according to paragraph 11, wherein the dry heat is at least 65°C.

[0117] 13. The composition according to any one of paragraphs 11 to 12, wherein the dry heat is in the range of 65°C to 95°C.

[0118] 14. The composition according to any one of paragraphs 1 to 13, wherein the composition is subjected to heat for at least 1 second.

[0119] 15. The composition according to any one of paragraphs 1 to 14, wherein the composition is subjected to heat for at least about 10 minutes.

[0120] 16. The composition according to any one of paragraphs 1 to 15, wherein after the composition is subjected to dry heat at a first temperature, the composition is subjected to dry heat at a second temperature.

[0121] 17. The composition according to paragraph 16, wherein the first temperature is about 65°C and the second temperature is about 85°C.

[0122] 18. The composition according to any one of paragraphs 16 to 17, wherein the composition is subjected to heat at the first temperature for at least about 1 second to at least about 30 minutes and is subjected to heat at the second temperature for at least about 1 second to at least about 30 minutes.

[0123] 19. The composition according to any one of paragraphs 16 to 17, wherein the composition is subjected to heat at the first temperature for about 30 minutes and is subjected to heat at the second temperature for about 10 minutes.

[0124] 20. The composition according to any one of paragraphs 16 to 17, wherein the composition is subjected to heat at the first temperature for about 30 minutes and is subjected to heat at the second temperature for about 30 minutes.

[0125] 21. The composition according to any one of paragraphs 1 to 10, wherein the heat is liquid heat.

[0126] 22. The composition according to paragraph 21, wherein the liquid heat is at least about 65°C.

[0127] 23. The composition according to paragraph 21, wherein the liquid heat is at least about 80°C.

[0128] 24. The composition according to paragraph 21, wherein the liquid heat is at least about 82°C.

[0129] 25. The composition according to any one of paragraphs 21 to 24, wherein the composition is subjected to the liquid heat for at least about 1 second.

[0130] 26. The composition according to any one of paragraphs 21 to 24, which is subjected to the heat for at least about 5 minutes.

[0131] 27. The composition according to any one of paragraphs 21 to 24, which is subjected to the heat for at least about 10 minutes.

[0132] 28. The composition according to any one of paragraphs 1 to 27, wherein the viability is enhanced by at least about 2-fold compared to a composition lacking the effective amount of the amphiphilic substance.

[0133] 29. The composition according to any one of paragraphs 1 to 27, wherein the viability is enhanced by at least about 10-fold compared to a composition lacking the effective amount of the amphiphilic substance.

[0134] 30. The composition according to any one of paragraphs 1 to 29, wherein the amphiphilic substance is sunflower lecithin.

[0135] 31. The composition according to any one of paragraphs 1 to 30, wherein the ratio (w / w) of the amphiphilic substance to the probiotics is in the range of about 1:10 to about 25:1.

[0136] 32. The composition according to any one of paragraphs 1 to 31, wherein the ratio (w / w) of the amphiphilic substance to the probiotics is in the range of about 1:1 to about 10:1.

[0137] 33. The composition according to any one of paragraphs 1 to 32, wherein the ratio (w / w) of the amphiphilic substance to the probiotics is about 5:1.

[0138] 34. The composition according to any one of paragraphs 1 to 32, wherein the ratio (w / w) of the amphiphilic substance to the probiotics is about 10:1.

[0139] 35. A container suitable for containing a food product comprising the composition according to any one of paragraphs 1 to 34.

[0140] 36. The container according to paragraph 35, made of a substance including glass, plastic, paper / carton, or aluminum.

[0141] 37. The container according to any one of paragraphs 35 to 36, wherein the composition is coated on the surface or part of the container.

[0142] 38. The container according to any one of paragraphs 35 to 37, further comprising food.

[0143] 39. The container according to paragraph 38, wherein the food includes oil, vinegar, yogurt, fruit products, apple sauce, dairy products, beverages, candies, snack foods, and juices.

[0144] 40. A container suitable for containing food, wherein part or the surface of the container is coated with an effective amount of Pediococcus acidilactici.

[0145] 41. The container according to paragraph 40, wherein the Pediococcus acidilactici is strain NRRL B - 50517.

[0146] 42. The container according to any one of paragraphs 40 or 41, made of a substance including glass, plastic, paper / carton, and aluminum.

[0147] 43. The container according to any one of paragraphs 40 to 43, wherein the Pediococcus acidilactici is coated on the inner surface of the container.

[0148] 44. The container according to any one of paragraphs 40 to 43, further comprising food.

[0149] 45. The container according to paragraph 44, wherein the food includes oil, vinegar, yogurt, fruit products, apple sauce, dairy products, beverages, candies, snack foods, and juices.

[0150] 46. A method for enhancing the viability of probiotics in a composition subjected to heat, comprising: iii) adding an effective amount of an amphiphilic substance to a composition containing probiotics; iv) subjecting the composition to heat, whereby the viability of the probiotics in the composition subjected to heat is enhanced.

[0151] 47. The method according to paragraph 46, wherein the amphiphilic substance is mixed with a lyophilized fermentation medium of the probiotics.

[0152] 48. The method according to any one of paragraphs 46 to 47, wherein the amphiphilic substance is in the form of a dry powder.

[0153] 49. The method according to any one of paragraphs 46 to 48, wherein the amphiphilic substance is mixed with the probiotics and an oil.

[0154] 50. The method according to paragraph 49, wherein the oil is olive oil.

[0155] 51. The method according to any one of paragraphs 49 to 50, wherein the ratio (w / w) of the amphiphilic substance to the probiotics to the oil is in the range of about 1:10:0.1 to about 10:1:25.

[0156] 52. The method according to any one of paragraphs 46 to 50, wherein the amphiphilic substance forms a lipid paste and acts as a carrier.

[0157] 53. The method according to any one of paragraphs 46 to 52, wherein the probiotics is Pediococcus acidilactici.

[0158] 54. The method according to paragraph 2, wherein the probiotics is Pediococcus acidilactici NRRL B-50517.

[0159] 55. The method according to any one of paragraphs 46 to 54, wherein the amphiphilic substance comprises lecithin, peanut butter, almond butter, soybean butter, or cookie butter.

[0160] 56. The method according to any one of paragraphs 46 to 55, wherein the heat is dry heat.

[0161] 57. The method according to paragraph 56, wherein the dry heat is at least 50 °C.

[0162] 58. The method according to any one of claims 55 to 56, wherein the dry heat is in the range of 65 to 95 °C.

[0163] 59. The method according to any one of paragraphs 46 to 58, wherein the composition is subjected to heat for at least 1 second.

[0164] 60. The method according to any one of paragraphs 46 to 59, wherein the composition is subjected to heat for at least about 10 minutes.

[0165] 61. The method according to any one of paragraphs 46 to 60, wherein after the composition is subjected to dry heat at a first temperature, the composition is subjected to dry heat at a second temperature.

[0166] 62. The method according to paragraph 61, wherein the first temperature is about 65 °C and the second temperature is about 85 °C.

[0167] 63. The method according to any one of paragraphs 61 to 62, wherein the composition is subjected to heat at the first temperature for at least about 1 to at least about 30 minutes and subjected to heat at the second temperature for at least about 1 to at least about 30 minutes.

[0168] 64. The method according to any one of paragraphs 61 to 63, wherein the composition is subjected to heat at the first temperature for about 30 minutes and subjected to heat at the second temperature for about 10 minutes.

[0169] 65. The method according to any one of paragraphs 61 to 63, wherein the composition is subjected to heat at the first temperature for about 30 minutes and subjected to heat at the second temperature for about 30 minutes.

[0170] 66. The method according to any one of paragraphs 46 to 55, wherein the heat is liquid heat.

[0171] 67. The method according to paragraph 66, wherein the liquid heat is at least about 50 °C.

[0172] 68. The method according to paragraph 66, wherein the liquid heat is at least about 80 °C.

[0173] 69. The method according to paragraph 66, wherein the liquid heat is at least about 82 °C.

[0174] 70. The method according to any one of paragraphs 66 to 69, wherein the composition is subjected to the heat for at least about 1 second.

[0175] 71. The method according to any one of paragraphs 66 to 70, wherein the composition is subjected to the heat for at least about 5 minutes.

[0176] 72. The method according to any one of paragraphs 66 to 70, wherein the composition is subjected to the heat for at least about 10 minutes.

[0177] 73. The method according to any one of paragraphs 46 to 72, wherein the viability is enhanced by at least about 2 times compared to a composition lacking the effective amount of the amphiphilic substance.

[0178] 74. The method according to any one of paragraphs 46 to 72, wherein the viability is enhanced by at least about 10 times compared to a composition lacking the effective amount of the amphiphilic substance.

[0179] 75. The method according to any one of paragraphs 46 to 74, wherein the amphiphilic substance is sunflower lecithin.

[0180] 76. The method according to any one of paragraphs 46 to 75, wherein the ratio (w / w) of the amphiphilic substance to the probiotics is in the range of about 1:10 to about 25:1.

[0181] 77. The method according to any one of paragraphs 46 to 76, wherein the ratio (w / w) of the amphiphilic substance to the probiotics is in the range of about 1:1 to about 10:1.

[0182] 78. The method according to any one of paragraphs 46 to 77, wherein the ratio (w / w) of the amphiphilic substance to the probiotics is about 5:1.

[0183] 79. The method according to any one of paragraphs 46 to 77, wherein the ratio (w / w) of the amphiphilic substance to the probiotics is about 10:1.

[0184] 80. The method according to any one of paragraphs 46 to 79, wherein the composition is contacted with food and subjected to heat together with the food.

[0185] 81. The method according to paragraphs 46 to 79, wherein subjecting the composition to the heat step in part ii) includes contacting the composition with food, and the food has already been subjected to heat before the contact with the composition.

[0186] 82. A method for treating a disease or condition characterized by inflammation in a subject in need thereof, the method comprising administering to the subject an effective amount of Pediococcus acidilactici probiotics.

[0187] 83. The method according to paragraph 82, wherein the method includes administering to the subject the composition according to any one of claims 1 to 34.

[0188] 84. The method according to paragraph 83, wherein the composition further comprises food, a dietary supplement, or a pharmaceutical.

[0189] 85. The method according to any one of paragraphs 82 to 84, wherein the disease or condition is selected from the group consisting of malignant tumor (cancer), arthritis, cardiovascular disease, hepatitis, infection, wound healing, pancreatitis, gastroesophageal reflux disease, diabetes, inflammatory bowel disease, peptic ulcer disease, bronchitis, cholecystitis, appendicitis, bursitis, dermatitis, asthma, autoimmune disease, pelvic inflammatory disease, gout, trauma, foreign body infection, burns, dental treatment, tendinitis, rhinitis, mucositis, and exposure to toxins such as chemicals and alcohol.

[0190] 86. The method according to any one of paragraphs 82 to 85, wherein the Pediococcus acidilactici probiotic is the NRRL B-50517 strain.

[0191] 87. The method according to any one of paragraphs 82 to 86, wherein the subject is human.

[0192] 88. The method according to any one of paragraphs 82 to 87, wherein the subject is administered more than 1.0×10 9 cfu of the probiotic.

[0193] 89. The method according to any one of paragraphs 82 to 88, wherein the subject is administered more than 4.0×10 9 cfu of the probiotic.

[0194] 90. The method according to any one of paragraphs 82 to 89, wherein the subject is administered one or more additional therapeutic agents.

[0195] 91. The method according to any one of paragraphs 82 to 89, wherein the subject is not administered another therapeutic agent.

[0196] 92. The method according to any one of paragraphs 82 to 91, wherein the Pediococcus acidilactici probiotic increases the number of anti-inflammatory M2 macrophage cells in the subject.

[0197] 93. The method according to any one of paragraphs 82 to 92, wherein the subject exhibits increased IL-10 production.

[0198] 94. The method according to any one of paragraphs 82 to 93, wherein the subject exhibits a reduced level of IL-6 and / or IL-23.

[0199] 95. The method according to any one of paragraphs 82 to 94, wherein the disease is cancer.

[0200] 96. The method according to paragraph 95, wherein the subject is administered one or more chemotherapeutic agents and / or radiation therapy in combination with Pediococcus acidilactici probiotics.

[0201] 97. The method according to any one of paragraphs 82 to 94, wherein the disease is pancreatitis.

[0202] 98. A composition comprising an effective amount of Pediococcus acidilactici probiotics for use in the method according to any one of paragraphs 82 to 97.

[0203] 99. The composition according to paragraph 98, wherein the Pediococcus acidilactici probiotics are formulated as tablets.

[0204] 100. The composition according to paragraph 98, wherein the Pediococcus acidilactici probiotics are formulated as capsules.

[0205] 101. The composition according to any one of paragraphs 98 to 100, comprising peach fruit powder as a flavoring agent.

[0206] 102. The composition according to any one of paragraphs 98 to 101, wherein the Pediococcus acidilactici probiotics are the NRRL B-50517 strain.

[0207] The present invention is further illustrated by the following examples. These examples are provided to assist in the understanding of the present invention and should not be construed as limitations thereof.

Examples

[0208] Example 1 - Administration of P. acidilactici stimulates the innate immune response in animals This example explains the effect of P. acidilactici administration on the innate immune response in rats. [Table 1] *: Sprague Dawley rats (9 weeks old) were fed Harlan #7012 rat chow ad libitum. The probiotic was a Pediococcus-based probiotic (Imagilin, Frederick, MD). The groups were a control group (without probiotic), a low dose (1×10 9 cfu), a medium dose (2×10 9 ), and a high dose (10×10 9 ) (n = 10). The animals were given 2 grams of food mixed with the probiotic at 11:00 am. Thereafter, the chow was fed ad libitum from 8:00 pm to 11:00 am. Water was provided ad libitum for 24 hours. Tail blood samples were analyzed for total blood cell counts.

[0209] When rats were administered 2.0×10 9 ~10×10 9 cfu of Pediococcus-based probiotic for 15 days, the amount of macrophages increased by 150% - 180% compared to that in rats not administered the probiotic. The increase in macrophages indicates that Pediococcus-based probiotics can stimulate the innate immune response in rats. Interestingly, when rats were fed a small amount (1.0×10 9 ) of Pediococcus-based probiotic, the number of macrophages was similar to the amount of macrophages from the control. This shows that a sufficient amount of Pediococcus-based probiotic is necessary to stimulate the innate immune response, such as an increase in the amount of macrophage cells.

[0210] Example 2 - Cytokine production in human subjects is stimulated by administration of P. acidilactici. This example illustrates the effect of administration of P. acidilactici on cytokine production in human subjects.

[0211] Regarding the innate immune response, macrophages can be broadly divided into two groups: pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. M2 macrophages also refer to macrophages that function in constructive processes such as wound healing and tissue repair, as well as macrophages that neutralize harmful immune system activation through the production of anti-inflammatory cytokines such as interleukin-10 (IL-10). [Table 2] *: Serum samples were collected from five volunteers before administration of Pediococcus acidilactici NRRL B-50517 probiotics and after administration of 4 billion cfu of Pediococcus probiotics per day for 45 days. The serum samples were analyzed using a Luminex-based multiplex assay (EMD Millipore; Milliplex) designed to measure biomarkers associated with the pro-inflammatory IL-6 and the anti-inflammatory IL-10.

[0212] After all 5 volunteers were administered Pediococcus probiotics for 45 days, they exhibited a significantly increased anti-inflammatory IL-10 activity (an increase of 158% - 422%). In contrast, the effects on pro-inflammatory IL-6 showed inconsistent results, with decreased activity in 3 volunteers and increased activity in 2 volunteers. These results indicate that the administration of Pediococcus probiotics in human subjects can more than double the anti-inflammatory IL-10 activity. Together with the results showing an increase in macrophages in rats treated with Pediococcus-based probiotics, these results indicate that Pediococcus-based probiotics can enhance the innate immunity of humans and animals. The innate immune responses of humans and animals treated with Pediococcus exhibit an increase in M2 macrophages and anti-inflammatory IL-10.

[0213] Example 3 - Effect of P. acidilactici NRRL B - 50517 supplementation for weight management: Subject randomization double-blind This weight management study was a 12-week supplementation of the probiotic strain Pediococcus acidilactici NRRL B-50517 in 30 adult participants in a controlled randomized double-blind trial. Body fat percentage was measured at the start and end of the trial by bioelectrical impedance analysis (BIA). Levels of the pro-inflammatory biomarkers interleukin-6 (IL-6) and interleukin-23 (IL-23) were determined using blood samples collected before the start and after the end of the trial. Appetite, energy levels, bowel movements, stool quality, bloating, and gas were monitored throughout the trial period using weekly questionnaires. Specific weight loss and anti-inflammatory effects of P. acidilactici are described here for the first time. Daily supplementation with 4 billion CFU of P. acidilactici resulted in an average decrease in body fat percentage of 0.86 ± 0.42% in the probiotic group and an increase of 0.28% ± 0.19 in the control group (p = 0.0264). The ratio of pro-inflammatory IL-6 differed by 0.61 ± 0.22 and 3.06 ± 0.87 in the probiotic and control groups, respectively (p = 0.0295), and the pro-inflammatory IL-23 ratio was 0.65 ± 0.14 in the probiotic group and 1.71 ± 0.38 in the control group (p = 0.0068).

[0214] Methods and Materials Participants in this trial were selected on a volunteer basis, and age, gender, and BMI were equal across treatment groups. Volunteers were instructed not to change their normal eating patterns or exercise habits during the trial. The probiotics were tested in subject groups divided as follows: 20% normal weight status (18.5 - 24.99), 47% overweight (25 - 29.99), and 33% obese (over 30).

[0215] Before the start of the supplementation period, participants underwent a comprehensive physical examination, including bioelectrical impedance analysis to determine body fat percentage and blood tests to quantify IL-6 and IL-23 levels. The same test procedures were repeated at the end of the trial. Over 12 weeks, 30 participants were administered either two gelatin capsules once daily containing a compound of fermented P. acidilactici NRRL B-50517 combined with peach fruit powder, reaching a dose of 4 billion CFU of Pediococcus probiotics per day, or a placebo treatment of two capsules containing only peach powder. The safety of the probiotics was analyzed in terms of their effects on appetite, energy levels, bowel movements, stool quality, bloating, and gas. As part of a weekly questionnaire, participants were asked to rate their experience of these symptoms on an arbitrary scale of 1 to 5, with 1 being the mildest and 5 being the most severe.

[0216] Results These results are the first to show that supplementing the diet of overweight or obese individuals with the probiotic Pediococcus acidilactici NRRL B-50517 modifies body fat storage and affects the concentration of inflammatory biomarkers associated with the pathology of obesity. The clear differences in body fat percentage and between IL-6 and IL-23 levels observed between the placebo group and the 5051 treatment group at the end of the trial indicate the effect of the probiotic in assisting weight loss without traditional dietary changes or exercise. The results are shown in Figures 1 - 3. Error bars for all three graphs indicate that the respective values of body fat percentage, IL-6, and IL-23 for the placebo group and the probiotic treatment group are not within one standard deviation of each other, thus suggesting a significant difference between the two.

[0217] The effect of P. acidilactici 5051 probiotics on body fat percentage is shown in Figure 1. Participants were administered either two gelatin capsules once a day containing a compound of P. acidilactici NRRL B - 50517 fermented culture bacteria combined with peach fruit powder, which reached a dose of 4 billion CFU of Pediococcus probiotics per day, or two capsules containing only peach powder. Body fat percentage was determined by bioelectrical impedance analysis (BIA) test. The values are based on a double - blind field evaluation of Pediococcus probiotics for a total of 30 volunteers for 3 months of treatment. P = 0.0264, t = 2.4073, placebo group median = 0.280 ± 0.190 (SEM); NutriLots™ median = - 0.864 ± 0.418 (SEM). The significantly greater difference in body fat percentage observed in the probiotic treatment group (NutriLots) compared to the placebo group after 3 months of treatment may indicate that P. acidilactici NRRL B - 50517 supplementation can accelerate weight loss without changing diet or exercise patterns.

[0218] The effect of Pediococcus probiotic supplementation on the inflammatory biomarkers IL - 6 and IL - 23. The effect of P. acidilactici 5051 probiotics on IL - 23 is shown in Figure 2. Blood samples were collected from each participant before the start of the test and at the end of the treatment period, either with placebo or probiotics, and changes in the presence of IL - 6 and IL - 23 were determined. A significant decrease in both IL - 6 and IL - 23 was observed in the Pediococcus probiotic treatment group. The values are based on a double - blind field evaluation of Pediococcus probiotics for a total of 30 volunteers for 3 months of treatment. P = 0.0295, t = 2.4239, placebo median = 3.058 ± 0.867 (SEM); NutriLots™ median = - 0.612 ± 0.221 (SEM). The lower ratio of IL - 23 in the probiotic treatment group (NutriLots) suggests that 5051 has the ability to reduce obesity - related inflammation.

[0219] The effect of P. acidilactici 5051 probiotics on IL-6 is shown in Figure 3. The values are based on a double-blind field evaluation of Pediococcus probiotics for a total of 30 volunteers over 3 months of treatment. P = 0.0068, t = 3.0194, placebo median = 1.714 ± 0.377 (SEM); NutriLots (trademark) median = -0.648 ± 0.137 (SEM). The decreased ratio of IL-6 in the probiotic treatment group (NutriLots) indicates that 5051 has the ability to reduce obesity-related inflammation.

[0220] Discussion In this study, 12 weeks of P. acidilactici NRRL B-50517 probiotic treatment resulted in a significant decrease in body fat percentage, interleukin 6 and 23 when administered to participants with various BMIs (Figures 1, 2, and 3). Consistent results across the board indicate that the power of the P. acidilactici effect is not limited to individuals in the obese body weight state, but is the same for individuals with lower BMIs. Most previous studies have shown the efficacy of LAB probiotic treatment only for obese subjects. When other probiotic strains were not effective in reducing the presence of obesity-related inflammation, 5051 decreased the levels of both interleukin 6 and 23 compared to the placebo group.

[0221] The safety of this probiotic was confirmed in a separately published trial conducted in parallel with this study. No significant differences in appetite, bowel movements, bloating, stool quality, energy levels, or gas were observed between the placebo group and the probiotic treatment group during the start and end of the study period as scored by the participants.

[0222] These findings have a great impact on the future treatment and prevention of metabolic diseases. Since the majority of cases of chronic conditions such as cardiovascular disease (CVD) and type 2 diabetes occur in parallel with obesity, improving the management of this disease in particular has the potential to greatly reduce the incidence of several other significant threats to public health.

[0223] Adipocytes have hitherto been thought to function only as a container for storing excess calories in the form of triglycerides, but have been found to play complex roles in metabolism, immunity, and cancer (Calabro P, Yeh ETH. 2007. Obesity, Inflammation, and Vascular Disease: the role of the adipose tissue as an endocrine organ. Subcellular Biochemistry. 42:63-91). White adipocytes secrete proteins including cytokines and hormone-like factors such as adiponectin, leptin, and resistin. This phenomenon has been of particular interest because these molecules are involved in vascular and metabolic complications (Calabro P, Yeh ETH. 2007. Obesity, Inflammation, and Vascular Disease: the role of the adipose tissue as an endocrine organ. Subcellular Biochemistry. 42:63-91). In most obese patients, low-grade inflammation of white adipose tissue (WAT) resulting from chronic activation of innate immunity leads to an increased likelihood of the ultimate development of insulin resistance, glucose intolerance, and diabetes predisposition (Bastard JP, Maachi M, Lagathu C, Kim MJ, Caron M, Vidal H, Capeau J, Feve B. 2006. Recent advances in the relationship between obesity, inflammation, and insulin resistance. Eur. Cyt. Net. 17(1):4-12). Macrophage infiltration of obese WAT acts as a source of pro-inflammatory cytokines and further contributes to the cause of insulin resistance. On the other hand, the circulating levels of adiponectin, an insulin-sensing effector highly expressed in WAT, are lower in obese subjects than in normal-weight subjects. WAT in these individuals overproduces and secretes increased levels of numerous inflammatory molecules, including IL-6, another regulator of insulin sensitivity.Therefore, the inflammatory-promoting causes of obesity and the systemic manifestation of insulin resistance are closely related and linked by the regulation of WAT.

[0224] The regulation of calorie extraction from food substances can be considered as a potential mechanism of the probiotic effect on the results shown in this specification. The composition of the human gut microbiota has been consistently related as a determinant of body weight as a result of its important role in nutrient acquisition and its role in heat collection and regulation (Tennyson CA, Friedman G. 2008. Microecology, obesity, and probiotics. Curr. Opin. Endocr. Diab. Obes. 15(5):422-7, DiBaise JK, Zhang H, Crowell MD, Krajmalnik-Brown R, Decker GA, Rittmann BE. 2008. Gut microbiota and its possible relationship with obesity. Mayo Clinic Proceedings. 83(4):460-69.). There is a reason to believe that the optimization of the targeted microbiota by the introduction of probiotics has the potential as a new therapeutic agent in the treatment of metabolic diseases. The conscious editing of the microbiota can be the key to modulating the imbalance between calorie intake and consumption resulting from the obese state.

[0225] The lack of change in the reported scores for appetite in both the placebo group and the probiotic treatment group indicates no effect on possible dietary pattern changes or the satiety hormone leptin.

[0226] In reducing serum IL-6 concentration and at the same time decreasing body fat percentage, P. acidilactici 5051 can increase insulin sensitivity and reduce overall systemic inflammation, so it can be presumed to contribute to reducing the risk of type 2 diabetes. Since IL-6 is also related to vascular damage in obese individuals, the reduced serum levels of cytokines are likely to reduce the risk of CVD (Calabro P, Yeh ETH. 2007. Obesity, Inflammation, and Vascular Disease: the role of the adipose tissue as an endocrine organ. Subcellular Biochemistry. 42:63 - 91). The inflammatory activity in obese individuals that increases according to WAT macrophage infiltration can be presumed to decrease when body fat disappears.

[0227] The results shown here reflect what was observed in the analysis of postoperative outcomes in morbidly obese individuals. After bariatric surgery, patients showed clinically significant decreases in IL-6, triglycerides, cholesterol, LDL, glucose, and insulin that correlated with BMI, thereby demonstrating the existence of a relationship between body weight and the inflammatory profile and further elucidating the relationship between BMI and biochemical parameters of chronic metabolic and vascular pathologies (Illan-Gomez F, Gonzalvez-Ortega M, Orea-Soler I, Alcaraz-Tafalla MS, Aragon-Alonso A, Pascual-Diaz M, Perez-Paredes M, Lozano-Almela ML. 2012. Obesity and inflammation: change in C-reactive protein, tumor necrosis factor-alpha and interleukin-6 after bariatric surgery. Obes. Surg. 22:950 - 55).

[0228] The significant decrease in IL-23 concentration observed in the probiotics treatment group is also a strong indicator of the decrease in disease risk. IL-23 / IL-17 is strongly associated with the activation of signaling pathways leading to tumor formation and carcinogenesis pathways. Since stimulation of the IL-23 / IL-17 axis was observed in obese women independent of increases in abdominal fat, insulin resistance, leptin, or MIF levels, it is reasonable to assume that it is due to diet and behavioral patterns associated with the manifestation of obesity rather than obesity itself (Sumarc-Dumanovic M, Stevanovic D, Ljubic A, Jorga J, Simic M, Stamenkovic-Pejkovic D, Starcevic V, Trajkovic V, Micic D. 2009. Increased activity of interleukin-23 / interleukin-17 proinflammatory axis in obese women. Int. J. Obes. 33:151-56).

[0229] Conclusion In summary, the probiotic P. acidilactici NRRL B-50517 showed a decreasing effect on body fat percentage, IL-6, and IL-23, suggesting its beneficial effects on weight management and metabolic diseases. Based on the evidence presented in this study, Pediococcus acidilactici NRRL B-50517 was found to be effective in reducing body fat and inflammation in individuals desiring weight loss.

[0230] Example 4 - Effects of Pediococcus-based probiotics on dogs and cats suffering from pancreatitis A 14-year-old spayed female Toy Poodle with significant abdominal pain, vomiting, and anorexia was diagnosed as potentially having pancreatitis. Ultrasonography showed high-fat echoes from the stomach to the duodenum, and serum analysis showed a spec cPL (canine pancreatic-specific lipase) of 432 ug / L. The dog was immediately switched to a low-fat diet and treated with 100 mg of KAMOSTAAL100 twice a day, but the spec cPL remained high, above 400, and approximately 2.5 months later reached 610 ug / L, accompanied by diarrhea and vomiting. At that point, the dog was treated with 200 mg of a Pediococcus-based probiotic twice a day in addition to the current treatment of a low-fat diet and 100 mg of KAMOSTAAL100 twice a day. Interestingly, not only did the diarrhea and vomiting stop, but the spec cPL returned to normal at 163 ug / L within approximately 6 weeks. For this reason, treatment with both the Pediococcus-based probiotic and KAMOSTAAL100 was stopped. However, approximately 3 months later, the dog relapsed and the spec cPL increased to 276 ug / L. At this point, the dog was treated with 200 mg of the Pediococcus-based probiotic only twice a day. Treatment was continued for approximately 7 months while well controlling diarrhea, vomiting, and anorexia, and the spec cPL was shown to be normal at 108 ug / L (Figure 4).

[0231] Spec cPL (Canine Pancreatic Lipase Specific) and spec fPL (Feline Pancreatic Lipase Specific) are the normal spec cPL in dogs and are well-established assays for pancreatitis in dogs and cats. In healthy dogs and cats, spec cPL is less than 200 ug / L and spec fPL is 0.7 - 3.5 ug / L. Dogs and cats are considered to have pancreatitis when spec cPL exceeds 400 ug / L and feline spec fPL exceeds 5.4 ug / L. Based on this criterion, Pediococcus probiotics were applied to 2 dogs and 1 cat, of which both dogs had spec cPL exceeding 600 ug / L and the cat had spec fPL of 50 ug / L and suffered from pancreatitis. All of these dogs and cats not only had vomiting and diarrhea stopped, but also had spec cPL and spec fPL controlled and returned to normal. Furthermore, 2 dogs that potentially had pancreatitis due to elevated spec cPL (303 ug / L and 205 ug / L) were also treated.

[0232] Example 5 - Influence of Pediococcus probiotics given to dogs suffering from cancer undergoing chemotherapy Four dogs with various cancers undergoing chemotherapy were treated by dosing with Pediococcus probiotics. After a short-term treatment with Pediococcus probiotics, these dogs experienced improvement in symptoms. *: Chemotherapy and Pediococcus probiotic administration treatment were carried out at Daktari Animal Hospital Central, Torri and Yaizu.

Table 3

[0233] Example 6 - Production of Pediococcus acidilactici NRRL B - 50517 First, the strain required for the fermentation process was selected from bacteria frozen in a -70°C freezer. The cultured bacteria were grown in a sterilized medium bottle. Once grown, samples were taken and the cleanliness and basic phenotypical purity were confirmed. If it was clean and the cells matched the first Gram stain smear, permission was given to prepare the medium for the tank (fermentation broth). Before inoculation, the inoculation material tank was CIP (Clean In Place) with caustic and acidic solutions. After discharging, mixing, and before filling with the broth fermentation components that are sterilized, the tank was sterilized. The tank medium was sterilized at 220°F - 250°F for 30 minutes to one and a half hours depending on the volume of the tank. Next, the temperature was lowered to 85 - 95°F and the inoculation material was seeded into the tank. When the inoculated tank had grown, the tank was cooled to 55 - 65°F. After cooling, samples were taken for repeated purity checks. If release was approved, the steps for tank preparation and inoculation were repeated. The product would inoculate the tank with the grown and approved inoculation material bottle. After growing all the desired tanks, the purity check was repeated, and then a centrifuge was prepared to concentrate the cells into a concentrated liquid. The concentrated liquid culture was put into a sterilized storage tank. A sterilized liquid cryoprotectant solution was added to the centrifuged culture.

[0234] This was homogenized in the storage tank by a stirrer. Once homogenized, the culture was pumped into a sterilized kettle (functional aliquot for freezing or pelletizing of the product) and pelletized in a liquid nitrogen bath. Once completed, the frozen pellets were lyophilized or freeze dried. After drying, the freeze-dried pellets were milled into fine powder. After taking the ground culture, homogenizing it to ensure uniformity, the culture was sampled for quality assurance by morphological, physiological, 16S rRNA DNA sequence, and high-temperature stress assays. The product was taken out of the blender used to homogenize the material, then put into bags and stored in a cool place at room temperature.

[0235] Example 7 - Formulation and Testing of Pediococcus acidilactici NRRL B - 50517 in Food As knowledge of the health benefits of probiotics spreads and the demand for probiotic - infused foods continues to grow, food companies face a series of new difficulties when beginning collaborations with biotechnology companies. First, one or more probiotic strains need to be selected from a large number of available options. Ideally, the bacteria selected should: 1. be able to survive any manufacturing stress such as high - heat treatment, 2. possess compatibility with the chemical and physical properties of the desired food matrix, 3. maintain viability in the food during the product's shelf - life after incorporation, and 4. be resistant to destruction by digestive mechanisms in order to confer its health benefits on the host. Many probiotic strains (such as Lactobacillus and Bifidobacterium), which are common in commercially available supplements, do not effectively meet these requirements and are thus not suitable for industrial food production. Their use is very limited in this context due to a lack of the critical high - heat resistance necessary for survival in recently sterilized foods. The instability of these strains at room temperature can present further problems in transportation and storage for grocery stores and potential consumers. As facultative anaerobes - obligate anaerobes, Lactobacillus and Bifidobacterium are particularly vulnerable to loss of viability upon any exposure to oxygen, further reducing their potential for incorporation into foods. More versatile and reliable strains are sought for the formulation of effective probiotic - infused foods.

[0236] Pediococcus acidilactici NRRL B - 50517 is a uniquely formulated powder composed of bacterial strains that can withstand a wide range of temperatures, osmotic pressures, and oxygen exposures. Probiotics, which are originally durable microorganisms isolated from plant materials, have been proven to be capable of surviving in a wide variety of foods under various environmental conditions and heat - treatment procedures.

[0237] The survival of P. acidilactici NRRL B - 50517 in sucrose solutions in the concentration range of 10 - 50% indicates the resistance of the probiotic to osmotic pressure (Table 3). Weaker bacteria are likely to lose their viability in solutions with relatively high osmotic pressure, but P. acidilactici NRRL B - 50517 retains a significantly stable cell count even at the highest concentration tested. Similar results were obtained in lactose solutions within the same concentration range over 9 days (Table 4). In solutions of sterilized water, 0.1 - 20% NaCl, and solutions combining NaCl and sucrose, P. acidilactici NRRL B - 50517 maintained significant cell viability for up to 1 week in all the samples assayed, demonstrating the ability of the probiotic to adapt to numerous chemical environments (Table 5).

Table 4

[0238] Conclusion: P. acidilactici NRRL B - 50517 maintains its viability in sucrose solutions in the concentration range of 10 - 50%, indicating its resistance to high - osmotic - pressure environments.

Table 5

[0239] Conclusion: Over 9 days, P. acidilactici NRRL B-50517 maintained a very stable viable count in 10% - 50% lactose solutions.

Table 6

[0240] When assayed on a large scale for viability, 5051 (registered trademark) maintained the viable count (CFU / g) for up to 113 days after incorporation into peanut butter when stored at room temperature, showing high storage stability (Table 6). This probiotic also showed similar good results when heated to 85 °C in peanut butter and then stored at room temperature. The stability was also constant when the probiotic was incorporated into purée heated to 85 °C, and the viable count remained within 1 logarithm over 29 days of storage in the refrigerator (Table 7).

Table 7

[0241] Conclusion: P. acidilactici NRRL B-50517 showed a high bacterial count (CFU / g) in peanut butter at room temperature over 113 days, indicating that products containing both components can maintain high storage stability. Even when stored at 37 °C, the mixture of peanut butter and P. acidilactici NRRL B-50517 showed similarly high viability over 22 days and decreased in the viability test from day 22 to day 113.

Table 8

[0242] Conclusion: Over a two-week period after high heat treatment, P. acidilactici NRRL B-50517 maintained high viability in peanut butter, supporting its compatibility with commercially produced nut products containing P. acidilactici NRRL B-50517. PB1 ingredients: Roasted peanuts, sugar, hydrogenated vegetable oil for separation prevention (cottonseed oil, soybean oil, and rapeseed oil), salt.

[0243] Hazelnut spread ingredients: Sugar, vegetable oil (palm oil and rapeseed oil), hazelnuts, cocoa powder, skim milk, whey, lactose, sunflower lecithin (emulsifier), natural vanilla flavor.

[0244] Testing P. acidilactici NRRL B - 50517 in five types of heat - treated oils (corn oil, EVOO, LTOO, peanut oil, and vegetable oil) yielded results similar to those observed in peanut butter. Two of the oils, EVOO and corn oil (Table 8), showed excellent survival rates even after continuous exposure to 85 °C for 30 minutes (Tables 9 and 10). The apparent durability of probiotics in oil particularly promotes their use in traditional food preparation techniques involving heat.

Table 9

[0245] Conclusion: P. acidilactici NRRL B - 50517 maintains a high viable cell count (CFU / g) in various commercially available oils at room temperature after heat treatment.

Table 10

[0246] Conclusion: P. acidilactici NRRL B-50517 can survive high heat treatment in EVOO and produce a high viable cell count even after 30 minutes at 85°C. This probiotic is likely to be compatible with various food preparation techniques, including those involving heating.

Table 11

[0247] Conclusion: P. acidilactici NRRL B-50517 can survive high heat treatment in corn oil and produce a high viable cell count even after 30 minutes at 85°C. This probiotic is likely to be compatible with various food preparation techniques involving heating.

[0248] P. acidilactici NRRL B-50517 can survive after being subjected to a heat treatment of 85°C in different foods mimicking the sterilization procedures used in the food industry, dispensed into different containers together with P. acidilactici NRRL B-50517 (Tables 11 and 12), and retain its viability in products with diverse physicochemical properties over weeks or months. This provides a new approach for introducing live probiotics into foods.

Table 12

[0249] Conclusion: After incorporation into either vanilla pudding or chocolate pudding heated at 85 °C under conditions similar to sterilization, P. acidilactici NRRL B-50517 maintains a very stable cell count for approximately one month when stored at refrigerator temperature.

Table 13

[0250] **All viability tests were performed by serially diluting the mixture of P. acidilactici NRRL B-50517 + heat-treated food in 0.1% saline, plating on MRS, incubating overnight, and then counting the plates. The survival rate was calculated as the ratio to the saline + P. acidilactici NRRL B-50517 control (0.2 g of 1 B / g of P. acidilactici NRRL B-50517 added to 20 mL of 0.1% saline at room temperature).

[0251] Conclusion: P. acidilactici NRRL B-50517 maintains viability in various liquid and solid matrices after high heat treatment and shows high compatibility for incorporation into many different foods after sterilization or other similar high heat sterilization procedures.

[0252] Example 8 - Increase in probiotic viability under heat treatment by applying an amphiphilic product as a carrier Amphiphilic molecules are molecules that have both a polar and a nonpolar part in their structure. Chemical compounds characterized by these molecules are essential for the acceptance of biological and industrial processes.

[0253] In this test, amphiphilic products such as lecithin, peanut butter, almond butter, soy butter, and cookie butter are applied as alternative carriers for the fermented culture of Pediococcus acidilactici NRRL B-50517. Excessive amphiphilic products can protect the fermented culture of P. acidilactici NRRL B-50517 from harsh dry heat or moist heat treatment. After mixing the dry powder of the amphiphilic product and the freeze-dried fermented culture of the bacteria with oil, the inherent properties of the amphiphilic product form a lipid paste, which functions as a carrier for the fermented culture of P. acidilactici and can withstand dry heat treatment or hot water treatment similar to sterilization for food processing.

[0254] There are several approaches for including probiotics in sterilized food / sample products. The desired approach is to include the probiotics together in the food / sample components and then perform a sterilization process that may include hot water treatment. Alternatively, the food / sample components can be dispensed into the probiotic composition. This can be practiced by taking out the hot water-treated juice / liquid after it has reached the specified sterilization temperature and immediately combining it with a mixture of the probiotics and the amphiphilic product. For this reason, the following experiments applied hot water heat treatment to the mixture of the probiotics and the amphiphilic product for 5 minutes and 10 minutes immediately after the hot water / liquid reached the desired temperature, and then assays were performed to determine the number of live probiotics after these heat treatments. In these efforts, it can be shown that the amphiphilic product combined with the probiotics can withstand heat treatment such as the sterilization process.

[0255] Lecithin is defined as phosphatidylcholine and represents a natural mixture of neutral and polar fats derived from plants and / or animals. It is a poor water-soluble but excellent emulsifier. In aqueous solution, its phospholipids can form either liposomes, bilayer sheets, micelles, or lamellar structures depending on the hydration and temperature. This results in a type of surfactant that is usually classified as amphiphilic.

[0256] In this test, amphiphilic products such as lecithin, peanut butter, almond butter, soy butter, and cookie butter are applied as alternative carriers for the fermented culture of Pediococcus acidilactici NRRL B-50517. An excessive amount of amphiphilic products can protect the fermented culture of P. acidilactici NRRL B-50517 from severe dry heat or moist heat treatment. After mixing the dry powder of the amphiphilic product and the freeze-dried fermented culture of the bacteria with oil, the inherent properties of the amphiphilic product can form a lipid paste, carrying the fermented culture of P. acidilactici and making it resistant to heat such as hot water treatment.

Table 14

[0257] 1.75 g of sunflower lecithin and 0.2 g (500 million cfu / g) of 1.75 g of P. acidilactici NRRL B-50517 (1 billion cfu / g) powder were transferred onto weighing paper. The samples were heated at 65 °C for 30 minutes and then cooled for 10 minutes. Other samples treated at 65 °C were transferred to 85 °C for 10 minutes and 30 minutes and cooled for 10 minutes. The temperature was confirmed with a digital probe. After dissolving in 5 ml of physiological saline, appropriate dilution was performed, 100 μl was plated on MRS agar plates, and incubated overnight at 37 °C for viability calculation.

[0258] Results: By applying lecithin as a carrier for freeze-dried fermented P. acidilactici bacteria, the survival of bacteria under different heat treatments was improved 5 to 10 times compared to that of only heat-treated freeze-dried fermented P. acidilactici bacteria.

Table 15

[0259] Results: The mixture of P. acidilactici NRRL B-50517 and sunflower lecithin was resuspended as a paste with olive oil and immersed in water for heat treatment at 80 °C or 82 °C. When the heated bacterial paste was removed from the hot water bath when it reached 80 °C, more than 37% viable bacteria were detected when the treated bacterial paste product was transferred to room temperature for the cooling process. With a similar heat treatment up to 82 °C, about 8.0% viable bacteria were detected. These heat treatment processes involve applying probiotics after the product has undergone a sterilization process and directly dispensing these hot products into the P. acidilactici fermented bacteria combined with an amphiphilic product in a container.

Table 16

[0260] Results: The mixture of lecithin and freeze-dried fermented bacteria of P. acidilactici was stable when stored for more than 90 days at either room temperature or 37 °C. The viability of P. acidilactici remained at a similar viable count throughout storage at both room temperature and 37 °C.

Table 17

Table 18

Table 19

[0261] Example 9 - Influence of Different Amphiphilic Products as Carriers for Probiotics on Heat Treatment In this example, the effects of different amphiphilic products were tested for their ability to enhance the heat resistance of Pediococcus acidilactici.

Table 20

[0262] A 0.5 g (1 billion cfu / g) mixture of peanut butter and P. acidilactici NRRL B-50517 was placed in an Eppendorf-type test tube and placed at 95°C for 1 minute, 2.5 minutes, 5 minutes, and 10 minutes, or placed at 85°C for 1 minute, 2.5 minutes, 5 minutes, 10 minutes, and 20 minutes. The temperature was confirmed with a digital probe. 500 μl of physiological saline was added, vortexed, diluted, and 100 μl of the desired dilution was plated on an MRS plate at 37°C overnight for viability calculation. The survival rate was obtained by dividing the number of viable bacteria during heat treatment by the number of viable bacteria at room temperature (without heat treatment).

Table 21

[0263] A 0.5 g (1 billion cfu / g) mixture of P. acidilactici NRRL B-50517 fermented culture and peanut butter was placed in a 1.5 ml test tube containing 1 ml of distilled water. The 1.5 ml test tube was placed on a hot plate. After taking 5 minutes or until the temperature reached 80°C, 1 minute, 2.5 minutes, and 5 minutes were taken. The temperature was confirmed with a digital probe. After heat treatment, the sample was vortexed, diluted, and 100 μl of the desired dilution was plated on an MRS plate at 37°C overnight for viability calculation. The survival rate was obtained by dividing the number of viable bacteria during heat treatment by the number of viable bacteria at room temperature (without heat treatment).

Table 22

Table 23

[0264] Although what is currently considered to be the preferred embodiments of the present invention has been shown and described, those skilled in the art will understand that other and further embodiments can be made without departing from the spirit and scope of the invention described in this application, and that all such modifications within the intended scope of the claims shown herein are included in this application. All patents and publications mentioned and / or cited herein are incorporated by reference to the same extent as if each individual publication was specifically and individually indicated as being incorporated by reference in its entirety.

Claims

**Claim 1** A container suitable for containing food, wherein a part or the surface of the container is coated with an effective amount of *Pediococcus acidilactici*, and the *Pediococcus acidilactici* is in a state of being mixed with an effective amount of an amphiphilic substance to form a composition that enhances the viability of probiotics. **Claim 2** The container according to claim 1, wherein the *Pediococcus acidilactici* is strain NRRL B-50517. **Claim 3** The container according to any one of claims 1 to 2, wherein the container is made of a substance including glass, plastic, paper / carton, aluminum, or a dried plant fruit shell. **Claim 4** The container according to any one of claims 1 to 3, wherein the composition is coated on the surface or a part of the container. **Claim 5** The container according to any one of claims 1 to 4, further including food. **Claim 6** The container according to claim 5, wherein the food includes oil, vinegar, yogurt, fruit products, applesauce, dairy products, beverages, candies, snack foods, juices, dessert foods, cookies, cakes, JELLO, fruit bars, fruit custards, or tiramisus.

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