A composition containing Lactobacillus plantarum HEM20701 or its culture, lysate, or extract as an active ingredient, which has excellent protein degradation properties.

Abstract

The object of the present invention is to provide a composition that exhibits excellent protein degradation capabilities, and in particular, even better protein degradation capabilities when combined with enzymes. The effect of the present invention is to improve protein breakdown and digestion capacity by providing a product containing the enzyme and / or probiotic Lactobacillus plantarum HEM20701 to people who experience difficulty digesting proteins.

Description

[Technical Field]

[0001] The present invention relates to a composition comprising, as an active ingredient, Lactobacillus plantarum HEM20701 strain capable of increasing the proteolytic activity of an enzyme, or a culture, disrupted product or extract thereof. The strain according to the present application not only has excellent proteolytic activity when used alone, but also further enhances proteolytic activity when treated in combination with an enzyme, and thus can be advantageously used in the production of general foods or health functional foods.

[0002] The present application claims priority based on Korean Patent Application No. 10-2023-0120611 filed on September 11, 2023, and all contents disclosed in the specification and drawings of said application are incorporated herein by reference. [Background Art]

[0003] As people age, the secretion of enzymes required for digestion decreases. Particularly in modern society, factors such as irregular eating habits and stress reduce digestive function, leading to an increase in the number of patients with indigestion. In addition to this, the number of people who consume excessive protein due to changes in eating habits is increasing, which imposes a burden on digestive function, and tends to increase the number of people complaining of discomfort such as indigestion and intestinal diseases.

[0004] Meanwhile, probiotics refer to live microorganisms that exert beneficial effects by maintaining the balance of host intestinal microorganisms when ingested in a certain amount (Ministry of Food and Drug Safety, Probiotics Safety Evaluation Guide, 2021). Probiotics marketed as health functional foods must regulate intestinal flora, be non-toxic and non-pathogenic.

[0005] Research trends in probiotic products include: 1) expansion from existing single strains to complex strains; 2) expansion from intestinal regulation to immune-related diseases (colic, atopic dermatitis) and metabolic syndromes (obesity, hypertension, diabetes, etc.); 3) expansion from simple diseases to mental illnesses (depression, dementia, etc.); and 4) expansion to new strains such as Akkermansia sp. through microbiome research (Choi Hak-jeong, 2019). Furthermore, research is progressing on postbiotics, which are functional bioactive substances beneficial to human health produced during the fermentation process from probiotics to prebiotics, as well as all dead microorganisms.

[0006] Among these, probiotics, which are involved in protein digestion, promote the breakdown of proteins in the intestines, enhance nutrient absorption, induce protease and peptase activity, or secrete exenzymes that contribute to protein digestion. In this way, they offer various benefits to protein digestion and help alleviate problems associated with indigestion.

[0007] According to the 2021 World Kimchi Institute analysis report, the genus Lactobacillus received the most individual certifications for raw materials among domestic lactic acid bacteria species, with the plantarum species receiving the most certifications (7). Furthermore, according to the 2022 World Kimchi Institute analysis report, Lactobacillus plantarum accounted for the largest number of patent applications, with 68 out of a total of 279 patent applications. Thus, Lactobacillus plantarum, which is extensively studied in probiotics, is generally known as a beneficial bacterium in the intestines and plays an important role in the digestive system. It is also known that some of these bacteria possess the ability to break down proteins. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The object of the present invention is to provide a composition that exhibits excellent protein degradation capabilities, and in particular, shows even better protein degradation capabilities when combined with enzymes.

[0009] The issues mentioned above are merely examples, and there may be other issues that are within the realm of understanding for the average engineer. [Means for solving the problem]

[0010] The means for solving the problems of this invention include the following aspects.

[0011] The first aspect of this application provides the Lactobacillus plantarum HEM20701 strain (deposit number KCTC15550BP).

[0012] A second aspect of the present application provides a composition for improving protein degradation, comprising one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract as an active ingredient.

[0013] A third aspect of the present application provides a composition comprising one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract, and a proteolytic enzyme as an active ingredient.

[0014] The fourth aspect of this application provides a general food composition containing one or more of the Lactobacillus plantarum HEM20701 strain or its culture, crushed product, or extract as an active ingredient.

[0015] The fifth aspect of this application provides a health functional food composition for improving protein degradation, comprising one or more of the Lactobacillus plantarum HEM20701 strain or its culture, crushed product, or extract as an active ingredient.

[0016] The sixth aspect of this application provides a pharmaceutical composition for improving protein degradation, comprising one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract as an active ingredient.

[0017] A seventh aspect of the present application provides a method for improving protein degradation, comprising the step of administering one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract to an individual in need.

[0018] The eighth aspect of this application provides applications for improving the protein degradation ability of one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract.

[0019] The ninth aspect of this application provides one or more uses of Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract for producing a protein degradation improving agent.

[0020] The methods described above are merely examples, and there may be other methods that are within the realm of understanding for the average engineer. [Effects of the Invention]

[0021] The effect of the present invention is to improve protein breakdown and digestion capacity by providing a product containing the probiotic Lactobacillus plantarum HEM20701 to people who have difficulty digesting proteins. The product may also be provided together with a proteolytic enzyme.

[0022] The effects described above are merely examples, and there may be other effects that are within the realm of understanding for the average engineer. [Brief explanation of the drawing]

[0023] [Figure 1]It is a diagram showing the amount of proteolysis in three strains of *Lactobacillus plantarum* (HEM20701, HEM21089, HEM21113) and an enzyme-only treatment group. [Figure 2] It is a diagram showing the amount of proteolysis in the combined treatment group of three strains of *Lactobacillus plantarum* (HEM20701, HEM21089, HEM21113) and an enzyme. [Figure 3] It is a diagram showing the amount of proteolysis in treatment groups of three strains of *Lactobacillus plantarum* (HEM20701, HEM21089, HEM21113) alone or in combined treatment with an enzyme. [Figure 4] It is a diagram showing the fold changes of protein and total proteolysis amounts respectively for the enzyme-only treatment group, the *Lactobacillus plantarum* HEM20701-only treatment group, and the combined treatment group of *Lactobacillus plantarum* HEM20107 and the enzyme relative to the control group. MODES FOR CARRYING OUT THE INVENTION

[0024] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings so that a person having ordinary knowledge in the technical field to which the present application pertains can easily implement the embodiments. However, the present application can be embodied in various different forms, and is not limited to the embodiments described herein. In the drawings, portions irrelevant to the description are omitted to clearly explain the present application, and similar reference numerals are assigned to similar parts throughout the entire specification.

[0025] Throughout the specification of the present application, when it is stated that one member is positioned "on" another member, this includes not only the case where one member is in contact with another member, but also the case where another member exists between the two members.

[0026] In the entirety of the specification of this application, when a part "includes" a certain component, this means that, unless otherwise stated, it may include other components rather than excluding them.

[0027] Throughout this specification, terms of degree such as “about” and “substantially” are used in reference to the manufacturing and material tolerances inherent to the meaning referred to, either numerically or in a sense close to such numerical values, to prevent unscrupulous infringers from unfairly exploiting disclosures that refer to precise or absolute numerical values ​​to aid in understanding this application. Throughout this specification, terms of degree such as “~(to) step” or “~ step” are not meant to mean “~ step for.”

[0028] Throughout the specification of this application, the term “these combinations” as used in the Markush expression means one or more mixtures or combinations selected from the group of components described in the Markush expression, and means including one or more selected from the group of components.

[0029] Throughout the specification of this application, the phrase "A and / or B" means "A or B, or A and B."

[0030] The term "culture medium" as used throughout the specification of this application includes the substance obtained by culturing the bacterial strain of this application in a publicly known liquid medium or solid medium, and may be used interchangeably with "culture product."

[0031] As used throughout the specification of this application, the term "food" includes all foods in the ordinary sense, such as meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen and other noodle products, gums, dairy products including ice cream, various soups, beverages, teas, energy drinks, alcoholic beverages, vitamin complexes, functional foods, and health foods.

[0032] The term "health functional food" as used throughout the specification of this application means a food manufactured and processed using raw materials or components that have functional properties useful to the human body, as defined in Act No. 6727 on Health Functional Foods. "Functionality" means obtaining effects useful for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body.

[0033] The food product of this invention can be manufactured by methods commonly used in the industry, and during the manufacturing process, raw materials and components commonly added in the industry may be added. Furthermore, the dosage form of the food product may be manufactured without restriction as long as it is a dosage form recognized as a food product. The food composition of the present invention may be manufactured in various dosage forms, and unlike general pharmaceuticals, it is made from food as a raw material, thus having the advantage of not having side effects that may occur with long-term use of pharmaceuticals, and is highly portable.

[0034] The term "pharmaceutical composition" as used throughout this specification may be, but is not limited to, formulations prepared by conventional methods into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, topical preparations, suppositories, or sterile injection solutions.

[0035] The first aspect of this application provides the Lactobacillus plantarum HEM20701 strain. The Lactobacillus plantarum HEM20701 strain is derived from the feces of an adult. The strain was deposited with the Japan Biomedical Resource Center (KCTC) on August 14, 2023, under deposit number KCTC15550BP. The strain relating to this application may also promote protein degradation.

[0036] A second aspect of the present application provides a composition for improving protein degradation, comprising one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract as an active ingredient.

[0037] A third aspect of the present application provides a composition for improving protein degradation, comprising one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract, and a proteolytic enzyme as active ingredients. When the composition is processed in parallel with the enzyme, a higher protein degradation improvement effect is obtained compared to when the composition or strain is processed alone.

[0038] The fourth aspect of this application provides a general food composition containing one or more of the Lactobacillus plantarum HEM20701 strain or its culture, crushed product, or extract as an active ingredient.

[0039] The fifth aspect of this application provides a health functional food composition for improving protein degradation, comprising one or more of the Lactobacillus plantarum HEM20701 strain or its culture, crushed product, or extract as an active ingredient.

[0040] The sixth aspect of this application provides a pharmaceutical composition for improving protein degradation, comprising one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract as an active ingredient.

[0041] The compositions relating to the second through sixth aspects may be prepared as formulations that can be ingested by individuals. Each composition may also be used in conjunction with proteolytic enzymes. Here, "individual" should be interpreted to include all animals from which a protein-degrading effect can be obtained, such as humans, monkeys, pigs, cattle, and chickens. Here, "formulation" includes all those manufactured by methods commonly used in this industry, and may be in the form of powders, tablets, liquid capsules, etc.

[0042] A seventh aspect of the present application provides a method for improving protein degradation, comprising the step of administering one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract to an individual in need.

[0043] The eighth aspect of this application provides applications for improving the protein degradation ability of one or more of the Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract.

[0044] The ninth aspect of this application provides one or more uses of Lactobacillus plantarum HEM20701 strain or its culture, lysate, or extract for producing a protein degradation improving agent.

[0045] The characteristics of each aspect may also apply to other aspects within the same or similar scope.

[0046] The following describes in detail the embodiment and examples of the present application with reference to the attached drawings. However, the present application is not limited to these embodiment and examples and drawings.

[0047] Example 1. Comparison of protein degradation rates for three Lactobacillus plantarum species and enzyme-only treatments. The three bacterial strains used in this example were all Lactobacillus plantarum: HEM20701 isolated from adult feces, HEM21089 isolated from lemons, and HEM21113 isolated from tomatoes. All strains were cultured in MRS medium containing 10% skim milk as a cryoprotectant, and only strains with a bacterial count of 1.0E+11 CFU / g or higher were used in the experiment.

[0048] The enzyme used in this experiment was a fermented enzyme powder. This fermented enzyme powder is a mixture of fruits and vegetables (indigestible maltodextrin, carrot concentrate, tart cherry concentrate, Bacillus amyloliquefaciens, chicory extract powder, apple concentrate, fructooligosaccharides, red beet concentrate, white grape concentrate, mango concentrate, pomegranate concentrate, cabbage concentrate, broccoli concentrate, Jerusalem artichoke extract powder, grapefruit extract powder), organic skim milk powder, purified water, grain yogurt (brown rice, corn, domestic wheat, black rice, barley, black soybeans, Job's tears, glutinous rice, sorghum, Lactobacillus plantarum, Lactobacillus casei, Pediococcus acidilactici, Bacillus), and Biocore Optimum K.

[0049] To obtain samples, the PMAS experiment was conducted in the following manner. In this specification, PMAS (Personalized Pharmaceutical Meta-Analysis Screening) refers to a method for creating identical / similar intestinal environments outside the body, and is a technique useful for screening personalized intestinal environment improving substances. In this experiment, utilizing the PMAS experiment's characteristic of creating identical / similar intestinal environments outside the body, we screened for combinations of bacterial strains and enzymes that exhibited high protein degradation by adding four types of proteins to the culture medium and treating the bacterial strains and enzymes, and confirming that the protein content in the culture medium decreased. Detailed experimental methods are in accordance with Korean Registered Patent Nos. 10-2227382 and 10-2124474.

[0050] In the PMAS experiment, the culture time was set to 0 hours and 24 hours to compare the pre- and post-cultivation states. The treatment groups consisted of groups treated with the enzyme alone, the bacterial strain alone, and groups treated with a combination of the bacterial strain and the enzyme. To ensure the same viable cell count, each well was treated with a bacterial strain at a concentration of 9.0E+09 CFU / g. The control group was not treated with any bacterial strain or enzyme. Four types of proteins were also dissolved in the culture medium at a concentration of 2.5 mg per well and treated.

[0051] The PMAS experiment was repeated twice to account for the heterogeneity of proteins or enzymes, and cultured in an anaerobic chamber. After culturing for 0 hours and 24 hours, the samples were removed from the anaerobic chamber and centrifuged at room temperature (20°C) for 10 minutes to separate the supernatant. The supernatant sample was diluted 1 / 10 with distilled water (Fisher) and used for protein measurement. Protein measurement was performed using Quick Start. TM The procedure was performed using the Bradford Protein Assay (Bio-Rad) kit according to the protocol. After subtracting the blank value and mean value from all measured values, the protein concentration value was determined according to the standard curve concentration. Since a 1 / 10 diluted sample was used, the protein concentration value was multiplied by 10, which is the dilution factor. Then, all protein concentration values ​​were normalized by an adjustment factor so that the blank value was calculated to be the amount of added protein. At this time, the protein measurement value was repeated twice and the average value was used. In order to calculate the amount of protein degradation, the value from 0 hours of culture to 24 hours of culture was subtracted and the values ​​were represented as a graph.

[0052] When three Lactobacillus plantarum strains (HEM20701, HEM21089, and HEM21113) were treated with enzymes individually, the degree of protein degradation in four proteins [skim milk, soybean protein, pea protein, and gluten] was compared with a control group. As a result, the Lactobacillus plantarum HEM20701 strain showed a higher degree of protein degradation compared to the other Lactobacillus plantarum strains (Figure 1, Table 1).

[0053] In skim milk, the HEM20701, HEM21113, and HEM21089 strains showed the highest levels of protein degradation. In soybean protein, the HEM20701, HEM21089, and HEM21113 strains showed the highest levels of protein degradation. In pea protein, the HEM20701, HEM21089, and HEM21113 strains showed the highest levels of protein degradation. In other words, in all cases, the HEM20701 strain related to this application showed the highest degree of protein degradation.

[0054] [Table 1]

[0055] Comparison of protein degradation rates when treated with individual bacterial strains. -If no resolution exists, the resolution amount is indicated by -. -Protein degradation amount (μg / mL): Calculate the protein degradation amount by subtracting the measured values ​​from the 0-hour to 24-hour incubation period. - Standard deviation: Standard deviation of the two repeated values -p-value: t-test results comparing each enzyme and bacterial strain to the protein degradation amount in the control group (p-value < 0.05 is indicated with *).

[0056] Example 2. Comparison of protein degradation rates for three Lactobacillus plantarum species and enzyme combined treatment. The detailed experimental procedure is the same as in Example 1.

[0057] Three Lactobacillus plantarum strains, HEM20701, HEM21089, and HEM21113, were combined with enzymes, and the degree of protein degradation in four proteins [skim milk, soybean protein, pea protein, and gluten] was compared with a control group. As a result, when Lactobacillus plantarum strain HEM20701 was mixed with the enzyme, a higher degree of protein degradation was observed compared to when the other Lactobacillus plantarum strains were mixed with the enzyme (Figure 2, Table 2).

[0058] In skim milk, the HEM21113, HEM20701, and HEM21089 strains showed the highest levels of protein degradation. In soybean protein, the HEM20701, HEM21089, and HEM21113 strains showed the highest levels of protein degradation. In pea protein, the HEM20701, HEM21089, and HEM21113 strains showed the highest levels of protein degradation. In gluten, the HEM20701, HEM21113, and HEM21089 strains showed the highest levels of protein degradation. In most cases, the HEM20701 strain claimed in this application was found to have the highest degree of protein degradation.

[0059] These results suggest that protein degradation is improved not only when the bacterial strain is treated alone, but also when it is treated in combination with an enzyme, due to the interaction between the bacterial strain and the enzyme (Figure 3, Table 2). Furthermore, it was confirmed that different bacterial strains have different protein degradation capabilities, which suggests that this can be used as an important indicator in the development of probiotics that improve the degradation of target proteins.

[0060] [Table 2]

[0061] Comparison of protein degradation rates during combined enzyme treatment of bacterial strains. -If no resolution exists, the resolution amount is indicated by -. -Protein degradation amount (μg / mL): Calculate the protein degradation amount by subtracting the measured values ​​from the 0-hour to 24-hour incubation period. - Standard deviation: Standard deviation of the two repeated values -p-value: t-test results comparing each enzyme and bacterial strain to the protein degradation amount in the control group (p-value < 0.05 is indicated with *).

[0062] Example 3. Lactobacillus plantarum HEM20701 strain that enhances the protein-degrading ability of enzymes. As confirmed in Examples 1 and 2, the Lactobacillus plantarum HEM20701 strain exhibited considerably high protein degradation capabilities even when alone, and showed even higher protein degradation capabilities when interacting with enzymes. When the degradation amount was expressed as a percentage based on the amount of protein detected at 0 hours of culture, the Lactobacillus plantarum HEM20701 strain showed a high degradation capability of approximately 15%, although there were differences depending on the type of protein compared to other identical strains. Furthermore, when the strain and enzyme were combined, the protein degradation capability increased by 5-17% compared to treatment alone (Table 3).

[0063] Figure 4 shows the protein degradation levels of skim milk, soybean protein, pea protein, and gluten, as well as the total protein degradation levels of the four proteins, as multiples compared to the control group's protein degradation levels, with the enzyme, HEM20701, and the HEM20701 and enzyme mixture being the baseline. In the case of the Lactobacillus plantarum HEM20701 strain and enzyme mixture, the degradation levels of the four proteins and the total protein were 3.8 to 4.5 times higher than the control group, indicating the best protein degradation capability. In the case of Lactobacillus plantarum HEM20701 strain alone, the degradation levels were 2.6 to 3.6 times higher than the control group, showing higher protein degradation capability than the enzyme group for all proteins.

[0064] Therefore, it has been confirmed that the bacterial strain relating to this application can promote protein degradation on its own, and can further promote protein degradation when used in combination with an enzyme.

[0065] [Table 3]

[0066] Protein degradation analysis results (%) -If no resolution exists, the resolution amount is indicated by -. -Based on the protein amount detected at 0 hours of culture, the percentages of degradation by the strain alone and the percentages of degradation by the strain plus enzymes are displayed. -The HEM20701 strain exhibits approximately 15% higher protein resolution compared to other identical strains, depending on the type of protein. When treated with enzymes, protein resolution increases by 5-17% compared to treatment alone.

[0067] JPEG2026532619000005.jpg10972

Claims

1. Lactobacillus plantarum HEM20701 strain (deposit number KCTC15550BP).

2. The strain is the strain according to claim 1, which promotes protein degradation.

3. The strain is the strain according to claim 1, which improves the degrading ability of proteolytic enzymes.

4. A composition for improving protein degradation, comprising as an active ingredient one or more of the bacterial strains, cultures, crushed products, or extracts thereof described in any one of claims 1 to 3.

5. A composition for improving protein degradation, comprising one or more of the bacterial strains, cultures, crushed products, or extracts thereof described in any one of claims 1 to 3, and a proteolytic enzyme as an active ingredient.

6. A general food for improving protein decomposition, comprising one or more of the bacterial strains, cultures, crushed products, or extracts thereof described in any one of claims 1 to 3 as an active ingredient.

7. A health functional food for improving protein decomposition, comprising one or more of the bacterial strains, cultures, crushed products, or extracts thereof described in any one of claims 1 to 3 as an active ingredient.

8. A pharmaceutical composition for improving protein degradation, comprising one or more of the bacterial strains, cultures, crushed products, or extracts thereof described in any one of claims 1 to 3 as an active ingredient.

9. A method for improving protein degradation, comprising the step of administering one or more of the strains, cultures, crushed products, or extracts thereof described in any one of claims 1 to 3 to an individual in need of such strains.

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