Probiotic composition having effects of improving whey protein-decomposing ability, amino acid-producing ability, and lactose-decomposing ability

Abstract

The present application relates to a mixed strain composition having a whey protein-decomposing ability and a lactose intolerance-improving effect, and more specifically, to a mixed strain composition capable of decomposing whey protein into branched chain amino acids (BCAA), arginine, phenylalanine, beta-glucopyranoside, beta-glucopyranoside, beta-glucopyranoside, beta-glucopyranoside, beta-glucopyranoside, and beta-glucopyranoside. The invention relates to a mixed strain composition of lactobacillus reuteri LM1071 and lactobacillus gasseri LM1065, which can be used for preventing and treating lactose intolerance and can be used for preventing and treating lactose intolerance. The composition comprising a mixed strain of Lactobacillus reuteri LM1071 and Lactobacillus gasseri LM1065 according to the present application has excellent whey protein decomposition ability, and can generate branched chain amino acids (valine, leucine, isoleucine), arginine, and phenylalanine, which can contribute to muscle formation and prevent muscle loss, by decomposing whey protein. The mixed strain composition has excellent beta-galactosidase activity and thus can decompose lactose, and thus the mixed strain composition can be applied to a food for improving dyspepsia, diarrhea, abdominal distension and the like due to the uptake of protein, a health functional food, a modified milk powder and the like, and can be used as a food for improving dyspepsia, diarrhea, abdominal distension and the like due to the uptake of protein, a health functional food, a modified milk powder and the like.

Description

Technical Field

[0001] The present application relates to a mixed strain composition having the ability to decompose whey protein and the effect of improving lactose intolerance. More specifically, the present application relates to a Limosilactobacillus rueteri LM1071 (Accession No.: KCCM12650P) and Lactobacillus gasseri LM1065 (Accession No.: KCCM13018P) mixed strain composition capable of decomposing whey protein into branched-chain amino acids (BCAAs), arginine, and phenylalanine and improving lactose intolerance. Background Art

[0002] The increase in the number of obese people due to sarcopenia caused by aging, Westernized eating habits, and reduced physical activity is of great significance in the field of health science. Protein intake helps maintain muscle while resisting sarcopenia caused by aging, and it is well known that sufficient protein intake at all ages can reduce the disease risks associated with sarcopenia and obesity.

[0003] Representative dietary sources of protein can be divided into animal proteins such as milk, eggs, beef, pork, chicken, and fish, and plant proteins such as soybeans and cereals. Milk protein among protein sources has the advantage of excellent essential amino acid composition, and whey protein concentrate (WPC), which is prepared by concentrating whey, a liquid by-product separated during the production process from milk to cheese or casein, by more than 80%, has the advantages of high bioavailability and fast absorption, and is thus often ingested as a protein supplement.

[0004] It has been reported that whey protein concentrate has various physiological activity effects such as forming muscle in the body, preventing gout, regulating blood cholesterol levels, enhancing immunity, promoting bone growth, and preventing obesity, and is rich in essential amino acids. In particular, amino acids belonging to branched-chain amino acids (BCAAs) such as valine, leucine, and isoleucine are essential amino acids in maintaining normal protein metabolism balance and muscle synthesis, and have the advantages of being able to reduce exercise-induced muscle damage and promote muscle protein synthesis.

[0005] Since dietary proteins such as concentrated whey protein are macromolecules, they need to be broken down into low-molecular substances after ingestion. In this regard, in the body, the peptide chains of proteins are broken down with the participation of various digestive enzymes, such as pepsin in the stomach, trypsin and chymotrypsin in the small intestine, and are ultimately utilized in the form of amino acids by being absorbed by the villi of the small intestine. During this process, the enzymes secreted by intestinal microorganisms present in the digestive organs also affect the breakdown and absorption of proteins. Intestinal microorganisms can secrete enzymes that humans cannot generate or synthesize. Therefore, depending on the composition of intestinal microorganisms and the intake of probiotics, the types and contents of amino acids generated from the breakdown of dietary proteins may change significantly. It has been reported that arginine among the amino acids generated after protein breakdown can increase the value of nitric oxide in the blood, remove lactic acid and ammonia in muscles generated during exercise, and increase blood flow for promoting glucose absorption, and can help restore muscle fibers through the contractility and activation of satellite cells around muscles. In addition, it has been reported that phenylalanine, one of the essential amino acids, can increase muscle strength by stimulating the synthesis of muscle proteins and assist in exercise performance. Therefore, depending on the types of amino acids generated after protein breakdown, it also has an impact on aspects such as exercise performance and post-exercise recovery.

[0006] On the one hand, ingesting protein can produce various physiological effects such as muscle growth and immune enhancement. However, since it is a macromolecule, depending on the health condition of an individual's digestive organs, it may induce indigestion, and phenomena such as flatulence, abdominal distension, increased exhaust, and increased odor of defecation may occur due to the gas generated during the protein breakdown process.

[0007] Concentrated whey protein, which is widely used as a protein supplement, is a high-protein food with a protein content of 70% - 85%. Therefore, ingesting the above-mentioned concentrated whey protein may cause indigestion and abdominal distension. In addition, concentrated whey protein contains approximately 5% lactose. However, in the case of lactose intolerance due to the lack of digestive enzymes for breaking down lactose, that is, the lack of lactase, after ingesting concentrated whey protein, abdominal pain, diarrhea, vomiting and other gastrointestinal dysfunctions may occur because lactose cannot be broken down in the digestive organs.

[0008] Although lactose intolerance may also occur in cases of small intestine diseases, small intestine injuries, or resection of the small intestine, most cases are caused by congenital lactase deficiency. It is reported that the global population with lactose intolerance is about 67%, making it a very common disease. However, due to significant ethnic differences, the prevalence is about 10% in Europe, while in Korea, approximately 75% of the population suffers from lactose intolerance. In addition, the expression rate of lactose intolerance increases with age. It is reported that among Koreans, about 25% of school-age children have lactose intolerance, and about 75% of adults are affected. Moreover, there are gender differences in lactose intolerance. Based on Korean adults, the prevalence is 80% in men and 73% in women, with a higher expression rate in men.

[0009] Therefore, probiotics that can promote the digestion of concentrated whey protein, which is widely used as a dietary protein in the digestive organs, break down the ingested concentrated whey protein into branched-chain amino acids and arginine, and can also break down lactose are expected to have great industrial value.

[0010] As an example of developing a probiotic composition with proteolytic ability, there are food compositions and functional health foods including Lactobacillus rhamnosus IDCC 3201 with proteolytic ability (Korean Patent No. 10-2423025), etc. However, there is still a need to develop and study compositions with more excellent effects.

[0011] Therefore, the present inventors dedicated themselves to developing a probiotic composition with more excellent whey protein-degrading ability. As a result, a mixed strain composition of Limosilactobacillus reuteri LM1071 (Deposit No.: KCCM12650P) and Lactobacillus gasseri LM1065 (Deposit No.: KCCM13018P) that can break down whey protein and improve lactose intolerance was developed, and the present invention was completed. Summary of the Invention

[0012] Problems to be Solved by the Invention

[0013] The object of the present application is to provide a mixed strain composition with whey protein-degrading ability and the effect of improving lactose intolerance.

[0014] However, the technical problems to be solved by the present application are not limited to the above-mentioned technical problems, and those skilled in the art can clearly understand other technical problems not mentioned through the following description.

[0015] Solutions for Solving the Problems

[0016] The first aspect of the present application provides a mixed strain with whey protein-degrading ability and the effect of improving lactose intolerance.

[0017] The second aspect of the present application provides a food composition having the ability to decompose whey protein and the effect of improving lactose intolerance, wherein the above food composition contains the mixed strain or its disrupted product, culture, and extract of the first aspect as active ingredients.

[0018] The third aspect of the present application provides a health functional food composition having the ability to decompose whey protein and the effect of improving lactose intolerance, wherein the above health functional food composition contains the mixed strain or its disrupted product, culture, and extract of the first aspect as active ingredients.

[0019] The fourth aspect of the present application provides a modified milk powder composition having the ability to decompose protein and the effect of improving lactose intolerance, wherein the above modified milk powder composition contains the mixed strain or its disrupted product, culture, and extract of the first aspect as active ingredients.

[0020] The fifth aspect of the present application provides a pharmaceutical composition for treating lactose intolerance, wherein the above pharmaceutical composition contains the mixed strain or its disrupted product, culture, and extract of the first aspect as active ingredients.

[0021] Advantages of the Invention

[0022] The composition containing the mixed strain of Lactobacillus reuteri LM1071 (deposit number: KCCM12650P) and Lactobacillus gasseri LM1065 (deposit number: KCCM13018P) in the present application has excellent protein decomposition ability, and can generate branched-chain amino acids (valine, leucine, isoleucine), arginine, and phenylalanine that can contribute to muscle growth and prevent muscle loss by decomposing whey protein. Moreover, β-galactosidase has excellent activity, so it can decompose lactose. Therefore, the above mixed strain composition can be applied to foods, health functional foods, modified milk powders, etc. for improving indigestion, diarrhea, abdominal distension, etc. caused by protein intake. Description of the Drawings

[0023] Figure 1 It is a graph showing the results of comparing and measuring the whey protein decomposition ability of the mixed strain according to the mixing ratio of Lactobacillus reuteri LM1071 strain and Lactobacillus gasseri LM1065 strain.

[0024] Figure 2 It is a graph showing the results of comparing and measuring valine, leucine, isoleucine, and their total amounts according to the mixing ratio of the strains in the experiment for understanding the branched-chain amino acid production ability of the mixed strain of Lactobacillus reuteri LM1071 strain and Lactobacillus gasseri LM1065 strain.

[0025] Figure 3 This is a graph showing the results of comparing and measuring arginine according to the mixing ratio of strains in an experiment conducted to understand the arginine production ability of a mixed strain of Lactobacillus reuteri strain LM1071 and Lactobacillus gasseri strain LM1065.

[0026] Figure 4 This is a graph showing the results of comparing and measuring phenylalanine according to the mixing ratio of strains in an experiment conducted to understand the phenylalanine production ability of a mixed strain of Lactobacillus reuteri strain LM1071 and Lactobacillus gasseri strain LM1065. Detailed implementation mode

[0027] The embodiments of the present application will be described in detail below with reference to the accompanying drawings so that those skilled in the art can easily implement them. However, the present application can be implemented in many different forms, and the present application is not limited to the embodiments described herein. Moreover, in order to clearly illustrate the present application, parts irrelevant to the description are omitted in the drawings, and in the entire specification, similar reference numerals are used for similar parts.

[0028] Throughout the specification of the present application, a certain component being "on" another component means not only the case where a certain component is in contact with another component, but also the case where there is another component between the two components.

[0029] Throughout the specification of the present application, a certain part "including" a certain structural element means that, in the absence of a particularly contrary record, other structural elements may also be included, rather than excluding other structural elements. Terms such as "about" and "substantially" indicating degree used throughout the specification of the present application are used to indicate a value or a meaning close to that value when expressing the inherent manufacturing and material tolerance errors in the mentioned meaning, and are used to prevent unscrupulous infringers from misusing the disclosure of the correct or absolute values mentioned to facilitate the understanding of the present application. The terms "(perform) ~ step" or "step of ~" used throughout the specification of the present application do not mean "step for ~".

[0030] Throughout the specification of the present application, the term "these combinations" included in the Markush type expression means a mixture or combination on one selected from the group consisting of the structural elements described in the Markush type expression, and means including one or more selected from the group consisting of the above structural elements.

[0031] Throughout the specification of the present application, the notation "A and / or B" means "A or B, or A and B".

[0032] Hereinafter, embodiments and examples of the present application will be described in detail with reference to the accompanying drawings. However, the present application is not limited to such embodiments, examples, and the drawings.

[0033] The first aspect of the present application provides a mixed strain having proteolytic ability and an effect of improving lactose intolerance.

[0034] In one embodiment of the present application, the mixing ratio of Lactobacillus mucosae LM1071 strain and Lactobacillus gasseri LM1065 strain in the above mixed strain can be 2:8 to 4:6. Preferably, the mixing ratio of Lactobacillus mucosae LM1071 and Lactobacillus gasseri LM1065 can be 3:7, but is not limited thereto. In one embodiment of the present application, it was confirmed that according to the mixing ratio of each strain, the decomposition ability showed significant differences. In particular, it was confirmed that when the mixing ratio of Lactobacillus mucosae LM1071 strain and Lactobacillus gasseri LM1065 strain is 3:7, it has the most excellent milk protein decomposition ability.

[0035] In one embodiment of the present application, the above mixed strain may have branched-chain amino acid production ability, but is not limited thereto.

[0036] In one embodiment of the present application, the mixing ratio of Lactobacillus mucosae LM1071 strain and Lactobacillus gasseri LM1065 strain in the mixed strain having the above branched-chain amino acid production ability can be 9:1 to 6:4. Preferably, the mixing ratio of Lactobacillus mucosae LM1071 and Lactobacillus gasseri LM1065 can be 9:1, but is not limited thereto. In one embodiment of the present application, it was confirmed that according to the mixing ratio of each strain, the ability to decompose concentrated whey protein to form branched-chain amino acids showed significant differences. In particular, it was confirmed that when the mixing ratio of Lactobacillus mucosae and Lactobacillus gasseri is 9:1, it has the most excellent milk protein decomposition ability.

[0037] In one embodiment of the present application, the above mixed strain may have α-galactosidase and β-galactosidase activities, but is not limited thereto.

[0038] The second aspect of the present application provides a food composition having proteolytic ability and an effect of improving lactose intolerance. The above food composition contains the mixed strain or its disrupted product, culture, extract of the first aspect as an active ingredient. The content repeated with the first aspect of the present application also applies to the food composition of the second aspect of the present application.

[0039] In one embodiment of the present application, the above food composition may contain the mixed strain of the present application, or its culture, disrupted product, extract as an active ingredient, but is not limited thereto.

[0040] As used throughout the specification of this application, the term "food" includes: meat, sausage, bread, chocolate, confectionery, snacks, biscuits, pizza, instant noodles, other noodles, chewing gum, dairy products including ice cream, various seasonings, beverages, tea, drinks, alcoholic beverages, multivitamins, health functional foods, health foods, etc., including all foods in the conventional sense.

[0041] The food of this application can be manufactured by methods conventionally used in the art. When manufacturing the above-mentioned food, it can be manufactured by adding raw materials and ingredients conventionally added in the art. In addition, if the dosage form of the above-mentioned food belongs to a dosage form recognized as food, it can also be manufactured without limitation. The food composition of the present invention can be manufactured into various forms of dosage forms. Different from general drugs, since it uses food as a raw material, it has the advantages of not producing side effects that may occur when taking drugs for a long time, etc., and has excellent portability. Therefore, the food of the present invention can be taken as an adjuvant for enhancing proteolytic ability and improving lactose intolerance.

[0042] The food composition of this application can be taken daily. Therefore, excellent effects can be expected in improving lactose intolerance, and it can be used very effectively.

[0043] The above-mentioned food composition may further include a physiologically acceptable carrier, but the type of the carrier is not particularly limited, and any carrier conventionally used in the technical field can be used.

[0044] In addition, the above-mentioned food composition may include additional ingredients conventionally used in food compositions to improve odor, taste, visual appearance, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. In addition, it may include amino acids such as lysine, tryptophan, cysteine, valine, etc.

[0045] In addition, the above food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), fungicides (bleaching powder and high-strength bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar pigments, etc.), color developers (sodium nitrite, sodium nitrite salts, etc.), bleaching agents (sodium sulfite), seasonings (sodium glutamate (MSG), etc.), sweeteners (saccharin, sodium cyclamate, saccharin, sodium, etc.), flavors (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen tartrate, etc.), enhancers, emulsifiers, thickeners (paste), film-forming agents, gum base, defoaming agents, solvents, modifiers, etc. The above additives can be screened according to the type of food and used in appropriate amounts.

[0046] The mixed strains of Lactobacillus reuteri LM1071 and Lactobacillus gasseri LM1065 of the present application can be directly added or used together with other foods or food ingredients, and can be appropriately used according to conventional methods. The mixing amount of the active ingredients can be appropriately determined according to its intended use (prevention, health care or therapeutic treatment). Generally, when manufacturing foods or beverages, the food composition of the present invention can be added to foods or beverages in an amount of 50 parts by weight or less, specifically 20 parts by weight or less. However, for long-term intake for the purpose of health and hygiene, it can contain a content below the above range, and since there are no problems in terms of safety, the active ingredients can also be used in an amount above the above range.

[0047] As an example of the food composition of the present application, it can be used as a health beverage composition. In this case, like conventional beverages, it can contain various flavoring agents or natural carbohydrates, etc. as additional ingredients. The above natural carbohydrates can be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol, erythritol, etc. For sweeteners, natural sweeteners such as thaumatin and stevia extract can be used; synthetic sweeteners such as saccharin and aspartame can be used. In every 100 mL of the health beverage composition of the present invention, the proportion of the above natural carbohydrates can generally be about 0.01 g to 0.04 g, specifically about 0.02 g to 0.03 g.

[0048] In addition to the above, the health beverage composition may include various nutritional agents, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, pectate, alginic acid, alginate, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerol, alcohols or carbonating agents, etc. In addition, it may include the pulp for manufacturing natural fruit juice, fruit juice beverage or vegetable beverage. Such ingredients can be used alone or in combination. Although the proportion of such additives is not very important, generally, relative to every 100 parts by weight of the health beverage composition of the present invention, the selection range of the above additives is 0.01 part by weight to 0.1 part by weight.

[0049] If the food composition of the present application can show the improvement effects on protein decomposition and lactose intolerance, the mixed strains of Lactobacillus reuteri LM1071 and Lactobacillus gasseri LM1065 of the present application can be included in various weight percentages. Specifically, relative to the total weight of the food composition, the content of the mixed strains of Lactobacillus reuteri LM1071 and Lactobacillus gasseri LM1065 of the present application can be 100 to 0.0001% by weight or 80 to 0.01% by weight, but not limited thereto.

[0050] The third aspect of the present application provides a health functional food composition having the ability to decompose proteins and the improvement effect on lactose intolerance. The above health functional food composition contains the mixed strains or their disrupted products, cultures, extracts of the first aspect as active ingredients. The content repeated in the first aspect and the second aspect also applies to the health functional food composition of the third aspect of the present application.

[0051] In one embodiment of the present application, the above health functional food composition may contain the mixed strains or their cultures, disrupted products, extracts of the present application as active ingredients, but not limited thereto.

[0052] The term "health functional food" used throughout the specification of the present application refers to a food manufactured and processed using raw materials or ingredients with useful functions for the human body in accordance with the Health Functional Food Act No. 6727. "Functionality" refers to the regulation of nutrients for the structure and function of the human body or the achievement of health benefits such as physiological effects.

[0053] The above-mentioned health functional food refers to a food that has a positive effect of maintaining or enhancing health compared to general food, and a health supplement food refers to a food for the purpose of health assistance. Depending on the situation, terms such as health functional food, health food, and health supplement food can be used interchangeably. Specifically, the above-mentioned health functional food is a food produced by adding the mixed strains of Limosilactobacillus reuteri LM1071 and Lactobacillus gasseri LM1065 of the present application to food materials such as beverages, teas, spices, chewing gums, and biscuits, or by means of encapsulation, powderization, suspension, etc. It means that ingesting the above food can bring specific health effects. However, different from general drugs, since it uses food as the raw material, it has the advantage of not having side effects that may occur when taking drugs for a long time.

[0054] The fourth aspect of the present application provides a modified milk powder composition having proteolytic ability and an effect of improving lactose intolerance, wherein the above-mentioned modified milk powder composition contains the mixed strains or their disruptions, cultures, and extracts of the first aspect as active ingredients. The content repeated from the first aspect to the third aspect also applies to the modified milk powder composition of the fourth aspect of the present application.

[0055] In an exemplary embodiment of the present application, the above-mentioned modified milk powder composition may contain the mixed strains or their disruptions, cultures, and extracts of the present application as active ingredients, but is not limited thereto.

[0056] The fifth aspect of the present application provides a pharmaceutical composition for treating lactose intolerance, and the above-mentioned pharmaceutical composition contains the mixed strains or their disruptions, cultures, and extracts of the first aspect as active ingredients. The content repeated from the first aspect to the fourth aspect also applies to the pharmaceutical composition of the fifth aspect of the present application.

[0057] In an exemplary embodiment of the present application, the above-mentioned pharmaceutical composition may contain the mixed strains or their disruptions, cultures, and extracts of the present application as active ingredients, but is not limited thereto.

[0058] In an exemplary embodiment of the present application, the above-mentioned pharmaceutical composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc.; external medications; adjuvants or sterilized injection solutions according to conventional methods, but is not limited thereto.

[0059] In an exemplary embodiment of the present application, when formulating the above-mentioned pharmaceutical composition, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, or surfactants can be used for formulation, but is not limited thereto.

[0060] In one exemplary embodiment of the present application, the solid dosage forms for oral administration may include tablets, pills, powders, granules, capsules, etc. Such solid dosage forms can be formulated by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin, etc., in the above-mentioned strain-derived components. In addition, for example, in addition to simple excipients, lubricants such as magnesium stearate and talc can also be used, but not limited thereto.

[0061] In one exemplary embodiment of the present application, the liquid dosage forms for oral administration include suspensions, oral liquids, emulsions, syrups, etc. And in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, flavoring agents, preservatives, etc. can be included, but not limited thereto.

[0062] In one exemplary embodiment of the present application, the preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and adjuvants, but not limited thereto. For example, as the above-mentioned non-aqueous solvents or suspensions, vegetable oils such as propylene glycol, polyethylene glycol, and olive oil can be used; injectable esters such as ethyl oleate, etc. can be used, but not limited thereto. For example, as the above-mentioned adjuvants, semi-synthetic fatty acid esters (witepsol), polyethylene glycol, tween 61, cocoa butter, glycerol laurate, glycerogelatin, etc. can be used, but not limited thereto.

[0063] The pharmaceutical composition according to one exemplary embodiment of the present application can be a pharmaceutical composition or a quasi-pharmaceutical composition.

[0064] The term "quasi-pharmaceutical" used throughout the specification of the present application refers to an article that has a milder effect among the articles used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases in humans or animals. For example, according to the provisions of the Pharmacist Law, a quasi-pharmaceutical refers to an article other than those used for the purpose of a drug, including products for treating or preventing diseases in humans and animals, products that have a mild effect on the human body or do not directly act on the human body, etc.

[0065] The above-mentioned quasi-pharmaceutical composition of the present application can be manufactured into dosage forms selected from the group consisting of body cleansers, disinfectant cleansers, cleaning agents, kitchen cleansers, cleaning agents for cleaning, toothpaste, mouthwashes, wet wipes, detergents, soaps, hand sanitizers, hair cleansers, hair conditioners, humidifier fillers, masks, ointments, and filter fillers, but not limited thereto.

[0066] In one exemplary embodiment of the present application, the above-mentioned pharmaceutical composition can be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" in the present application refers to a reasonable benefit / risk ratio applicable to medical treatment or prevention, an amount sufficient to treat or prevent a disease. The effective dose level can be determined based on the severity of the disease, the activity of the drug, the age, weight, health, gender of the patient, the sensitivity of the patient to the drug, the administration time of the composition of the present invention used, the administration route, the excretion rate, the treatment cycle, including factors of drugs used in combination with or together with the composition of the present invention, and factors well-known in other medical fields. The pharmaceutical composition of the present application can be administered alone or in combination with known components that show therapeutic effects on intestinal diseases. Importantly, by considering all the above factors, the amount that can achieve the maximum effect with the least amount without side effects can be administered.

[0067] In one exemplary embodiment of the present application, for the dosage of the above-mentioned pharmaceutical composition, those skilled in the art can determine it by considering the purpose of administration, the severity of the disease, the age, weight, gender, medical history of the patient, or the type of the substance used as the active ingredient, etc. For example, the pharmaceutical composition of the present invention can be administered in an amount of about 0.1 ng to 1000 mg / kg, preferably about 1 ng to 100 mg / kg per adult, and the dosing frequency of the composition of the present application is not limited to this, but can be administered once a day or in multiple divided doses. The above dosage or dosing frequency does not limit the scope of the present application at any level.

[0068] The pharmaceutical composition of the present application is not particularly limited, but can be administered through routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, pulmonary administration, rectal administration, etc. However, when administered orally, it can also be administered in an unformulated form. Since the above-mentioned mixed strains of Lactobacillus reuteri LM1071 and Lactobacillus gasseri LM1065 may be deformed or decomposed by gastric acid, the oral composition can be administered in the oral cavity in a form coated with an active agent or in a formulated form that protects it from decomposition in the stomach or in the form of an oral patch. In addition, the above composition can be administered through any device that can move the active substance to the target cells.

[0069] Hereinafter, the present invention will be described in more detail through the examples of the present application. However, the following examples are only shown to help understand the present application, and the content of the present application is not limited to the following examples.

[0070]

Examples

[0071] Example 1 Screening of Strains with Excellent Caseinolytic Ability

[0072] To screen strains with excellent milk protein decomposition ability, skim milk powder and agar were used to prepare a plate medium, and the milk protein decomposition ability of each strain was confirmed. 25 g of skim milk powder and 15 g of agar were separately dissolved in 250 mL and 500 mL of distilled water, sterilized, and then the two solutions were mixed and dispensed into petri dishes in 20 mL aliquots. After cooling at room temperature and confirming the solidification of the agar, it was used. As a positive control group, pancreatin was used, and it was prepared by dissolving pancreatin powder at a concentration of 0.01% (w / v) (100 μg / mL) in a simulated intestinal buffer solution (0.5% NaCl, 0.03% CaCl 2 , 0.06% KCl, 0.06% NaHCO 3 ) and then used.

[0073] The strains used in strain screening are shown in Table 1. They were cultured 3 times at 12-hour intervals and then used to evaluate the decomposition ability. The inoculation concentration was 0.1% v / v each time of culturing, and the culture temperature of the strains was adjusted to 37 ± 3 °C. The culture solution of each strain was treated in the agar medium made of skim milk powder in 4 μL aliquots and left at room temperature for 30 minutes to allow the culture solution to fully penetrate into the medium. Then, it was cultured in an incubator at 37 °C for 24 hours. Subsequently, the formation of a clear zone, the area of the clear zone, and the transparency were measured using the Image J program. The area of the clear zone was obtained by measuring the area of the protein-decomposed and transparent part and converted into mm 2 . For transparency, the gray value of the clear zone and the gray value of the medium were measured, and the protein decomposition index was calculated using the following formula. All experiments were repeated 3 times, and comparisons were made by calculating the average value of each result.

[0074] Protein decomposition index = (A × B) / 100

[0075] A: Area of the clear zone (mm 2 )

[0076] B: Transparency of the clear zone (%) = 100 - (gray value of the clear zone / gray value of the medium) × 100

[0077] The results confirmed that not all probiotics can decompose milk proteins, but only specific probiotics have excellent milk protein decomposition ability. By considering the protein decomposition index, strains L5 (Lactobacillus gasseri LM1065 strain (KCCM13018P)) and L7 (Limosilactobacillus reuteri LM1071 strain (KCCM12650P)) were screened out from probiotics of various sources as strains with excellent milk protein decomposition ability (refer to [Table 1] and [Table 2]).

[0078] [Table 1]

[0079] Strains for comparing milk protein decomposition ability

[0080] Abbreviation Strain name Source B1 Bifidobacterium animalis lactis Infant feces B2 Bifidobacterium bifidum Human milk B3 Bifidobacterium longum Infant feces L1 Lactobacillus rhamnosus Cheese L2 Lactobacillus rhamnosus Infant feces L3 Lactiplantibacillus plantarum Pickled vegetables L4 Lactobacillus acidophilus Adult feces L5 Lactobacillus gasseri Human milk L6 Limosilactobacillus fermentum Fermented batter L7 Limosilactobacillus reuteri Human milk S1 Streptococcus thermophilus Dairy products

[0081] [Table 2]

[0082] Comparison of protein decomposition index for each probiotic

[0083] Distinguish Area of the clear zone Transparency of the clear zone Protein decomposition index Agar medium - - - <![CDATA[Positive control group 1) > 44.2 21.6 9.6 B1 - - - B2 - - - B3 25.1 6.2 1.6 L1 - - - L2 34.2 11.6 4.0 L3 34.1 10.7 3.6 L4 73.6 7.7 3.5 L5 44.2 9.8 4.3 L6 47.9 7.8 3.8 L7 38.5 21.1 8.1 S1 - - -

[0084] 1) 0.01% Trypsin

[0085] Example 2 Change in protein decomposition ability according to the mixing ratio of Limosilactobacillus reuteri and Lactobacillus gasseri

[0086] In order to evaluate the protein decomposition ability based on the mixing ratio of Limosilactobacillus reuteri and Lactobacillus gasseri, which were screened as probiotics with excellent protein decomposition ability in Example 1 above, a medium was prepared using skim milk powder and agar by the same method as in Example 1, and the milk protein decomposition ability was confirmed using the plate medium method. As a positive control group, a bromelain solution dissolved in acetate buffer at a concentration of 5% w / v at pH 4.5 was used.

[0087] For the two strains screened in Example 1 as having excellent proteolytic ability, after culturing three times at 12-hour intervals by the same method as in Example 1, they were used to evaluate the proteolytic ability. As shown in Table 3, the mixing ratios of the two strains were altogether made into 9 combinations, and the proteolytic abilities were compared. After mixing each strain according to the mixing ratio, 10 μL was inoculated into the skim milk powder medium and left at room temperature for 30 minutes to allow the culture solution to fully penetrate into the medium. Then, after culturing in a 37 °C incubator for 48 hours, the area and transparency of the clear zone were determined by the same method as in Example 1, and the protein decomposition index was calculated. After calculating the predicted value of the synergistic effect using the Colby formula, the actual measured value and the predicted value of the synergistic effect were compared.

[0088] Predicted value of synergistic effect = (A + B) - (A × B / 100)

[0089] A: Measured value of proteolytic ability of Lactobacillus mucosae

[0090] B: Measured value of proteolytic ability of Lactobacillus gasseri

[0091] The protein decomposition index was measured for the two screened strains at a single or a total of 9 mixing ratios. The results showed that, compared with bromelain as the positive control group, the mixed strains, regardless of the mixing ratio, had more excellent proteolytic ability. In addition, under all mixing ratio conditions, the measured protein decomposition index was higher than the predicted value of the synergistic effect, thus proving the synergistic effect brought by the mixture of Lactobacillus mucosae and Lactobacillus gasseri. However, according to the mixing ratio of each strain, the decomposition ability showed significant differences. Especially when the mixing ratio of Lactobacillus mucosae and Lactobacillus gasseri was 3:7, the milk protein decomposition ability was optimal (refer to

Figure 1

Table 3

[0092]

Table 3

[0093] Change in proteolytic ability according to the mixing ratio of Lactobacillus mucosae and Lactobacillus gasseri

[0094]

[0095] 1) Lactobacillus mucosae

[0096] 2) Lactobacillus gasseri

[0097] Example 3 Change in the ability to produce branched-chain amino acids after the decomposition of concentrated whey protein according to the mixing ratio of Lactobacillus mucosae and Lactobacillus gasseri

[0098] After culturing a mixed composition of Lactobacillus reuteri and Lactobacillus gasseri together with concentrated whey protein, the content change of branched-chain amino acids (valine, isoleucine, leucine) generated by decomposing concentrated whey protein was analyzed using a high-performance liquid chromatography / photodiode array detector system.

[0099] The culture medium containing concentrated whey protein was prepared by dissolving 12.5 g of concentrated whey protein powder in 500 mL of sterilized water, injecting it into 50 mL conical tubes in portions of 30 mL each, and then sterilizing it at 65 °C for 30 minutes to produce a 2.5% (w / v) concentrated whey protein culture medium. After culturing the strains 3 times according to the method of Example 1 above, the cells were recovered and diluted to 8 log CFU / ml in phosphate buffered saline, and then a single strain or a mixed strain was inoculated into the concentrated whey protein culture medium at a rate of 0.1% v / v per portion. Subsequently, it was cultured in an incubator at 37 ± 3 °C for 72 hours. After 72 hours, the culture solution was centrifuged (4000 rpm, 15 minutes), and the supernatant was used as an analysis sample.

[0100] 5 mL of the above analysis sample was placed in a test tube, concentrated at a temperature of 110 °C under a nitrogen atmosphere, uniformly pulverized, dissolved in 1 ml of 0.1 N hydrochloric acid aqueous solution, and then homogenized by vortex mixing. After that, free amino acids in the concentrated sample were extracted in an ultrasonic water bath for 15 minutes. The free amino acid extract was centrifuged and the supernatant was filtered through a filter, and then the free amino acids were analyzed using a high-performance liquid chromatography / photodiode array detector system. For the analysis method, 0.1% formic acid aqueous solution and 0.1% formic acid acetonitrile solution were used as the mobile phase, and an appropriate gradient elution was provided. For the chromatographic column, analysis was carried out by using Zorbax Eclips AAA (4.6 mm ID * 150 mm, 5 μm) from Agilent.

[0101] As a result, extremely trace amounts of branched-chain amino acids below the calibration limit were detected in the concentrated whey protein, while a large amount of branched-chain amino acids were detected in the culture solution obtained by culturing concentrated whey protein and probiotics together. It was confirmed that both Lactobacillus reuteri and Lactobacillus gasseri can decompose concentrated whey protein to generate branched-chain amino acids, and when the two strains were mixed, the branched-chain amino acids further increased, and the content of branched-chain amino acids changed greatly according to the mixing ratio of the strains. The proteolytic ability in skim milk powder confirmed in Example 2 was the most excellent when the mixing ratio of Lactobacillus reuteri and Lactobacillus gasseri was 3:7, while the ability to decompose concentrated whey protein to form branched-chain amino acids was the most excellent at a ratio of 9:1 (seeFigure 2 ), and [Table 4].

[0102] [Table 4]

[0103] Difference in branched-chain amino acid production capacity according to the mixing ratio of Lactobacillus reuteri and Lactobacillus gasseri

[0104]

[0105]

[0106] 1) Lactobacillus reuteri

[0107] 2) Lactobacillus gasseri

[0108] Example 4 Changes in the production capacity of arginine and phenylalanine after the decomposition of concentrated whey protein according to the mixing ratio of Lactobacillus reuteri and Lactobacillus gasseri

[0109] After culturing the mixed composition of Lactobacillus reuteri and Lactobacillus gasseri together with concentrated whey protein, the content changes of arginine and phenylalanine produced by decomposing concentrated whey protein were analyzed by a high-performance liquid chromatography / photodiode array detector system in the same manner as in Example 3.

[0110] As a result, in the culture solution prepared from concentrated whey protein, a trace amount of arginine below the calibration limit was detected, and for phenylalanine, although it was above the calibration limit, a small amount was also detected. In the culture solution in which concentrated whey protein and probiotics were cultured together, a large amount of arginine and phenylalanine were detected. It was confirmed that both Lactobacillus reuteri and Lactobacillus gasseri can decompose concentrated whey protein to produce arginine and phenylalanine, and the amount further increases when the two strains are mixed, and the content of arginine changes greatly according to the mixing ratio of the strains. The branched-chain amino acid production capacity in concentrated whey protein confirmed in Example 3 was the most excellent when the mixing ratio of Lactobacillus reuteri and Lactobacillus gasseri was 9:19, and the production capacities of arginine and phenylalanine were also the most excellent at a ratio of 9:1 (see [, [, [Table 5] and [Table 6]). Figure 3 [, Figure 4 [, [Table 5] and [Table 6]).

[0111] [Table 5]

[0112] Difference in arginine production capacity according to the mixing ratio of Lactobacillus reuteri and Lactobacillus gasseri

[0113]

[0114] 1)Lactobacillus mucosae Roy

[0115] 2) Lactobacillus gasseri

[0116] [Table 6]

[0117] Difference in phenylalanine production capacity according to the mixing ratio of Lactobacillus mucosae Roy and Lactobacillus gasseri

[0118]

[0119] 1) Lactobacillus mucosae Roy

[0120] 2) Lactobacillus gasseri

[0121] Example 5 Changes in enzyme activity according to the mixing ratio of Lactobacillus mucosae Roy and Lactobacillus gasseri

[0122] To evaluate the enzyme activities of Lactobacillus mucosae Roy and Lactobacillus gasseri and the changes in enzyme activity according to their mixing ratio, an API ZYM kit was used. The cells of Lactobacillus mucosae Roy or Lactobacillus gasseri cultured 3 times by the method of Example 1 above were recovered, and according to the usage method of the API ZM kit, they were prepared using a phosphate buffer saline solution so that the turbidity of MacFarland reached 5 (9.18 log CFU / Ml). Then, the samples were processed at 65 μl per portion on the test strip in the kit and cultured in an incubator at 37 °C for 2 hours, and then treated with ZYM reagent and the activity of each enzyme was confirmed by a color reaction. The degree of change in the color reaction was expressed as a value of 0 to 5, a negative reaction was expressed as "-", a reaction with the maximum intensity (40 nanomoles) was expressed as "+++++", and in addition, the intermediate values of 30, 20, 10, and 5 nanomoles were expressed as ++++, +++, ++, and +.

[0123] The evaluation of enzyme activity using the API ZYM kit was carried out on single strains of Lactobacillus mucosae Roy and Lactobacillus gasseri, a 3:7 mixed composition with the most excellent caseinolytic ability, and a 9:1 mixed composition with the most excellent ability to generate branched-chain amino acids and arginine from concentrated whey protein. As a result, it was confirmed that the enzyme activities of the two individual strains were different in the activities of some lipid metabolism enzymes, protein metabolism enzymes, and sugar metabolism enzymes, and the enzyme activity also changed partially due to the mixing of the strains (refer to [Table 7]).

[0124] The results of enzyme activity evaluation confirmed that both strains belong to safe strains that do not express the activity of β-glucuronidase, which is an enzyme that generates amines and toxic substances during metabolism and is involved in colorectal cancer caused by intestinal mucosal damage and the induction of various diseases.

[0125] The activity of β-galactosidase, which is used to decompose lactose, was only confirmed in the strain of Lactobacillus mucosae, while Lactobacillus gasseri did not show the activity against β-galactosidase. However, when the two strains were mixed, regardless of the mixing ratio, the enzyme activity of β-galactosidase was confirmed to be the maximum value. Thus, it was confirmed that when the two strains were mixed, the protein decomposition ability could be improved, the production of branched-chain proteins could be promoted by decomposing concentrated whey proteins, and the disadvantages caused by the ingestion of high protein could be improved by digesting lactose.

[0126] In addition, it is well known that when α-galactosidase is lacking in the intestine, non-digestible oligosaccharides such as raffinose and stachyose reach the large intestine without being digested and act as fermentation substrates, and anaerobic bacteria in the large intestine generate excessive methane, carbon dioxide, and hydrogen, causing a feeling of bloating, and increasing the nitrogen emission in feces, causing bad odors. It was confirmed that although Lactobacillus gasseri does not have the activity of α-galactosidase, when mixed with Lactobacillus mucosae, it can maintain the activity of α-galactosidase, thus solving the problems such as bloating and stinky feces.

[0127] On the one hand, it is well known that mannosidase and flucosidase inhibit the formation of carbohydrates and have a negative impact on energy intake, but neither of the two strains showed the activity of flucosidase. However, it was confirmed that Lactobacillus gasseri has the activity of α-mannosidase, which has a negative impact on energy metabolism, but did not show the activity of α-mannosidase in the mixed composition with Lactobacillus mucosae. In addition, the activities of leucine arylamidase and valine arylamidase, which affect protein synthesis, did not change significantly in the mixed composition. Therefore, it was confirmed that the mixed composition of the present invention does not have a negative impact on energy metabolism and can have a positive impact on protein synthesis in the body.

[0128]

Table 7

[0129] Evaluation of the combined enzyme activity of Lactobacillus reuteri and Lactobacillus gasseri

[0130]

[0131]

[0132] Example 6 Acid tolerance, bile acid tolerance, auto - aggregation ability, and intestine adhesive property of the mixed strain

[0133] To predict the survival rate of Lactobacillus reuteri and Lactobacillus gasseri in the digestive organs, the acid tolerance, bile acid tolerance, auto - aggregation ability, and intestine adhesive property were evaluated, and the changes in acid tolerance, bile acid tolerance, auto - aggregation ability, and intestine adhesive property according to the mixing ratio of Lactobacillus reuteri and Lactobacillus gasseri were evaluated. When evaluating, the Lacticaseibacillus rhamnosus GG strain (ATCC 53103) was treated as a comparison strain, and the results were compared.

[0134] To evaluate acid tolerance, pepsin was dissolved in MRS broth medium at pH 2.5 with 0.1N hydrochloric acid to reach 0.3% (w / v), and it was injected into 15 - mL conical tubes at 9 mL per portion to prepare the medium for acid tolerance evaluation and use. The Lactobacillus reuteri strain and Lactobacillus gasseri strain cultured three times according to the method of Example 1 or mixtures with different ratios were injected into the acid - tolerance evaluation medium at 1 mL per portion, then cultured in a 37 °C incubator for 2 hours, and the viable cell counts before and after culture were measured by the plate medium method. Then, the acid tolerance was evaluated by the following formula.

[0135] Acid tolerance (%)=(A / B)×100%

[0136] A: Initial bacterial count (log CFU / mL)

[0137] B: Bacterial count after 2 hours (log CFU / mL)

[0138] To evaluate bile acid resistance, bile powder was dissolved in MRS broth medium to reach 0.3% (w / v), and 9 mL of each portion was separately injected into 15 mL conical tubes to prepare the medium for evaluating bile acid resistance. The Limosilactobacillus reuteri strain and Lactobacillus gasseri strain cultured three times according to the method of Example 1 above or a mixture of different ratios were separately injected into the medium for evaluating bile acid resistance at 1 mL per portion, then cultured in a 37 °C incubator for 24 hours, the viable cell count before and after culture was measured, and then the bile acid resistance was evaluated by the following formula.

[0139] Bile acid resistance (%) = (A / B) × 100%

[0140] A: Initial bacterial count (log CFU / mL)

[0141] B: Bacterial count after 24 hours (log CFU / mL)

[0142] To evaluate self - cohesion, the Limosilactobacillus reuteri strain or Lactobacillus gasseri strain cultured three times according to the method of Example 1 above was centrifuged (10000 rpm, 10 minutes), and the precipitated cells were recovered, then washed three times with phosphate - buffered saline. After that, each cell sample was diluted in phosphate - buffered saline to adjust the absorbance at 600 nm to reach 0.5 ± 0.05 and prepared. For the mixed compositions with different mixing ratios, they were prepared as follows: after mixing the cell samples of individual strains according to each ratio, the mixed cells were diluted in phosphate - buffered saline to adjust the absorbance at 600 nm to reach 0.5 ± 0.05 and prepared. The prepared samples were separately injected into 15 mL conical tubes at 5 mL per portion, then cultured in a 37 °C water bath for 24 hours, the absorbance before and after culture was measured, and the self - cohesion was evaluated by the following formula.

[0143] Self - cohesion (%) = (1 - B / A) × 100%

[0144] A: Initial absorbance

[0145] B: Absorbance after 24 hours

[0146] To evaluate intestinal adhesion ability, human - derived intestinal epithelial cells (HT - 29 cells) were used. The cells were cultured in a 75 mL T - flask until the concentration of intestinal epithelial cells reached 1.5×10 6 cells / 12 mL. After confirming that more than 50% of the cells in the flask aggregated, they were adjusted to 1.0×10 5They were respectively injected into a 24-well plate at a rate of cells / well / mL. Then, the culture medium was replaced with RPMI medium containing 10% fetal bovine serum at intervals of 2 days, and the formation of a monolayer was confirmed or not.

[0147] To treat the strains in the HT-29 cell monolayer, after culturing Lactobacillus mucosae and Lactobacillus gasseri 3 times according to the method of Example 1, centrifugation was performed at 10,000 rpm for 10 minutes to recover the bacteria precipitated at the bottom. The recovered bacteria were washed 3 times with phosphate-buffered saline and prepared, and were adjusted with RPMI medium without fetal bovine serum to make the final bacterial count reach 8 log CFU / mL.

[0148] The culture solution of the intestinal epithelial cells with a monolayer formed was removed. After washing the intestinal epithelial cells 3 times with phosphate-buffered saline (DPBS) used as a cell culture buffer solution, the prepared strain or strain mixed composition was treated at a rate of 1 mL per portion, and it was cultured in an incubator at 37 °C with the atmospheric composition adjusted by 5% carbon dioxide for 2 hours. After 2 hours, the culture solution was removed, washed 2 times with phosphate-buffered saline (DPBS), and then treated with a solution in which Triton-X was dissolved at 0.1% (v / v) in purified water at a rate of 1 mL per portion, and the cells and bacteria were recovered. For the evaluation of the intestinal adhesion ability activity, after determining the viable cell counts before and after culture by the plate culture method, the calculation was performed using the following formula.

[0149] Intestinal adhesion ability = (A / B) × 100%

[0150] A: Initial bacterial count (log CFU / mL)

[0151] B: Bacterial count after 2 hours (log CFU / mL)

[0152] As a result of the study, compared with the comparative strains, Lactobacillus mucosae and Lactobacillus gasseri were more excellent in acid resistance, self-cohesion, and intestinal adhesion ability. When the strains were mixed at different ratios, it showed a tendency that the acid resistance, self-cohesion, and intestinal adhesion ability changed according to the mixing ratio. However, compared with the comparative strains, more excellent activities were shown at all mixing ratios (refer to [Table 8]).

[0153]

Table 8

[0154] Differences in acid resistance, bile acid resistance, self-cohesion, and intestinal adhesion ability according to the mixing ratio of Lactobacillus mucosae and Lactobacillus gasseri

[0155]

[0156]

[0157] 1) Lactobacillus reuteri

[0158] 2) Lactobacillus gasseri

[0159] Generally speaking, it can be confirmed that the mixed strains of the present application or the composition of the culture containing the above-mentioned mixed strains have excellent probiotic properties, such as intestinal survival rate and adhesion ability. Since they can decompose milk proteins, they have the ability to generate valine, leucine, and isoleucine, which are branched-chain amino acids, and generate arginine and phenylalanine from concentrated whey protein. Moreover, because they have the activity of the enzyme related to preventing gas formation in the intestine, α-galactosidase activity, and the activity of the enzyme for decomposing lactose, β-galactosidase activity, when taking protein supplements, they have an effect on alleviating side effects related to protein digestion and muscle growth.

[0160] The above description of the present invention is exemplary. Those skilled in the art to which the present application pertains can understand that it can be easily deformed into other specific forms without changing the technical idea or essential technical features of the present application. Therefore, it should be understood that the above-described embodiments are exemplary in all aspects and not restrictive. For example, each structural element described as a single type can be implemented in a dispersed manner, and similarly, the structural elements described in a dispersed form can be implemented in a combined manner.

[0161]

[0162]

[0163]

[0164]

Claims

1. A mixed strain of Limosilactobacillus reuteri LM1071 (deposit number: KCCM12650P) and Limosilactobacillus gasseri LM1065 (deposit number: KCCM13018P) with the ability to decompose whey protein and the effect of improving lactose intolerance.

2. The mixed strain according to claim 1, wherein, the mixing ratio of Limosilactobacillus reuteri LM1071 and Limosilactobacillus gasseri LM1065 in the mixed strain is 2:8 to 4:

6.

3. The mixed strain according to claim 1, wherein, the mixed strain has the ability to produce branched-chain amino acids, arginine or phenylalanine.

4. The mixed strain according to claim 3, wherein, the mixing ratio of Limosilactobacillus reuteri LM1071 and Limosilactobacillus gasseri LM1065 in the mixed strain is 9:1 to 6:

4.

5. The mixed strain according to claim 1, wherein, the mixed strain has α-galactosidase and β-galactosidase activities.

6. A food composition comprising any one or more of the mixed strain according to claim 1 or its culture, disrupted product, extract as an active ingredient.

7. A health functional food composition comprising any one or more of the mixed strain according to claim 1 or its culture, disrupted product, extract as an active ingredient.

8. A modified milk powder composition comprising any one or more of the mixed strain according to claim 1 or its culture, disrupted product, extract as an active ingredient.

9. A pharmaceutical composition for treating lactose intolerance or sarcopenia comprising any one or more of the mixed strain according to claim 1 or its culture, disrupted product, extract as an active ingredient.

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