Composition comprising, as active ingredient, lactobacillus plantarum hem20701 exhibiting excellent proteolytic ability, or culture, lysate, or extract thereof

Abstract

The purpose of the present invention includes the provision of a composition exhibiting excellent proteolytic ability and, particularly, exhibiting more improved proteolytic ability if combined with an enzyme. The effects of the present invention include the improvement of proteolytic and digestive abilities by providing products containing enzymes and / or probiotic Lactobacillus plantarum HEM20701 to people who have difficulty digesting proteins.

Description

A composition comprising Lactobacillus plantarum HEM20701 having excellent protein decomposition ability or a culture, lysate, or extract thereof as an active ingredient

[0001] The present invention relates to a composition comprising, as an active ingredient, the Lactobacillus plantarum HEM20701 strain, or a culture, lysate, or extract thereof, which can increase the protein decomposition ability of an enzyme. The strain according to the present invention not only exhibits excellent protein decomposition ability on its own, but also further enhances protein decomposition ability when combined with an enzyme, making it useful in the manufacture of general foods or health functional foods.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0120611, filed September 11, 2023, the entire disclosure of which is incorporated herein by reference.

[0003] As we age, the production of enzymes necessary for digestion decreases. In modern society, factors such as irregular eating habits and stress can impair digestive function, leading to an increase in indigestion. Furthermore, changes in eating habits have led to an increase in the number of people consuming excessive amounts of protein. This, in turn, places a strain on the digestive system, leading to a rise in complaints of discomfort such as indigestion and intestinal disorders.

[0004] Meanwhile, probiotics are live microorganisms that, when consumed in a certain amount, have a beneficial effect by maintaining the intestinal microbial balance of the host (Ministry of Food and Drug Safety, Probiotics Safety Evaluation Guide, 2021). Probiotics sold as health functional foods must regulate intestinal flora and be non-toxic and non-pathogenic.

[0005] Research trends in probiotic products are expanding: 1) from single strains to complex strains; 2) from intestinal function to immune-related diseases (enteritis, atopy), metabolic syndromes (obesity, hypertension, diabetes, etc.); 3) from simple diseases to mental disorders (depression, dementia, etc.); and 4) expanding to new strains like Akkermansia sp. through microbiome research (Choi Hak-jong, 2019). Furthermore, research is being conducted on these products, including functional bioactive substances beneficial to human health generated during the fermentation process of probiotics and prebiotics, and postbiotics derived from all killed microorganisms.

[0006] Among them, probiotics involved in protein digestion promote protein breakdown in the intestines, thereby enhancing nutrient absorption, and induce protease and peptase activity or secrete exogenous enzymes that contribute to protein digestion, thereby providing various benefits for protein digestion and helping to improve problems associated with indigestion.

[0007] According to the 2021 World Institute of Kimchi Analysis Report, the lactic acid bacteria with the most approvals for individual raw materials by lactic acid bacteria species in Korea was Lactobacillus, and among them, the plantarum species received the most approvals, with seven. Furthermore, according to the 2022 World Institute of Kimchi Analysis Report, Lactobacillus plantarum accounted for the largest number of patent applications by lactic acid bacteria, with 68 out of a total of 279. Lactobacillus plantarum, which is widely studied in probiotics, is generally known as a beneficial bacterium in the intestines and plays an important role in the digestive system. Some of these bacteria are known to have protein-decomposing abilities.

[0008] The object of the present invention is to provide a composition having excellent protein decomposition ability, and particularly, exhibiting further enhanced protein decomposition ability when combined with an enzyme.

[0009] The above tasks are only examples, and there may be additional tasks that are within the scope of what a person of ordinary skill can understand.

[0010] Our solution to the problem includes the following aspects:

[0011] The first aspect of the present invention provides Lactobacillus plantarum HEM20701 strain (Lactobacillus plantarumHEM20701; Accession No. KCTC 15550BP).

[0012] The second aspect of the present invention provides a composition for improving protein degradation ability, which comprises at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof as an active ingredient.

[0013] The third aspect of the present invention provides a composition comprising at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof and a protein-decomposing enzyme as active ingredients.

[0014] The fourth aspect of the present invention provides a general food composition comprising at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof as an active ingredient.

[0015] The fifth aspect of the present invention provides a health functional food composition for improving protein decomposition ability, comprising at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof as an active ingredient.

[0016] The sixth aspect of the present invention provides a pharmaceutical composition for improving protein degradation, comprising at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof as an active ingredient.

[0017] The seventh aspect of the present invention provides a method for improving protein degradation, comprising administering to a subject in need thereof at least one of the Lactobacillus plantarum HEM20701 strain or a culture, a lysate, or an extract thereof.

[0018] The eighth aspect of the present invention provides a use for improving protein degradation ability of at least one of the Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof.

[0019] The ninth aspect of the present invention provides the use of any one or more of the Lactobacillus plantarum HEM20701 strain or a culture, lysate or extract thereof for producing a protein degradation enhancing agent.

[0020] The above means are only examples, and additional means may exist within the scope understandable to a person skilled in the art.

[0021] The effect of the present invention includes providing a product containing the probiotic Lactobacillus plantarum HEM20701 to people who have difficulty digesting proteins, thereby improving their ability to break down and digest proteins. The product may be provided together with a proteolytic enzyme.

[0022] The above effects are only examples, and additional effects may exist within the scope understandable to a person skilled in the art.

[0023] Figure 1 is a diagram showing the protein degradation amount of three strains of Lactobacillus plantarum (HEM20701, HEM21089, HEM21113) and the enzyme-only treatment group.

[0024] Figure 2 is a diagram showing the protein degradation amount of three strains of Lactobacillus plantarum (HEM20701, HEM21089, HEM21113) and the enzyme combination treatment group.

[0025] Figure 3 is a diagram showing the amount of protein decomposition in groups treated with three strains of Lactobacillus plantarum (HEM20701, HEM21089, HEM21113) alone or in combination with enzymes.

[0026] Figure 4 is a diagram showing the multiple of protein and total protein degradation in each of the enzyme-only treatment group, the Lactobacillus plantarum HEM20701-only treatment group, and the Lactobacillus plantarum HEM20107 and enzyme combination treatment group compared to the control group.

[0027] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0028] Throughout this specification, when it is said that an element is "on" another element, this includes not only cases where the element is in contact with the other element, but also cases where another element exists between the two elements.

[0029] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless otherwise stated.

[0030] The terms "about," "substantially," and the like used throughout this specification are used to mean at or near the numerical values ​​when manufacturing and material tolerances inherent to the meanings stated are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that state precise or absolute values ​​to aid understanding of this specification. The terms "step of doing" or "step of" used throughout this specification do not mean "step for doing."

[0031] Throughout this specification, the term "combination(s) thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0032] Throughout this specification, references to “A and / or B” mean “A or B, or A and B.”

[0033] The term "culture solution" used throughout the present specification includes a substance obtained by culturing the strain of the present invention in a known liquid medium or solid medium, and may be used interchangeably with "culture."

[0034] The term "food" as used throughout this specification includes meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods, and includes all foods in the conventional sense.

[0035] The term "health functional food" used throughout this specification refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body as defined in Act No. 6727 on Health Functional Foods, and "functionality" means obtaining a beneficial effect for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body.

[0036] The food of the present invention can be manufactured using methods commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. Furthermore, the food formulation of the present invention can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various forms, and unlike general drugs, it has the advantage of not having side effects that can occur with long-term use of drugs because it uses food as a raw material, and it is highly portable.

[0037] The term "pharmaceutical composition" used throughout the present specification may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, or sterile injectable solutions, according to conventional methods, but may not be limited thereto.

[0038] The first aspect of the present invention provides Lactobacillus plantarum HEM20701 strain. Lactobacillus plantarum HEM20701 strain is a strain derived from adult feces. The strain was deposited with the Korea Center for Biological Resources (KCTC) on August 14, 2023, under the accession number KCTC 15550BP. The strain according to the present invention may promote protein degradation.

[0039] The second aspect of the present invention provides a composition for improving protein degradation ability, which comprises at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof as an active ingredient.

[0040] The third aspect of the present invention provides a composition for improving protein degradation, comprising at least one of the Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof and a protein-degrading enzyme as active ingredients. When the composition is treated in combination with an enzyme, a higher protein degradation improvement effect can be observed compared to when the composition or strain is treated alone.

[0041] The fourth aspect of the present invention provides a general food composition comprising at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof as an active ingredient.

[0042] The fifth aspect of the present invention provides a health functional food composition for improving protein decomposition ability, comprising at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof as an active ingredient.

[0043] The sixth aspect of the present invention provides a pharmaceutical composition for improving protein degradation, comprising at least one of Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof as an active ingredient.

[0044] The compositions according to aspects 2 through 6 can be manufactured into a dosage form that can be ingested by an individual. Furthermore, each composition can be used in conjunction with a proteolytic enzyme. The term "individual" herein should be interpreted to include all animals capable of exhibiting increased protein decomposition activity, such as humans, monkeys, pigs, cows, and chickens. Furthermore, the term "formulation" herein includes any product manufactured using methods commonly used in the art, and may be in the form of, for example, powders, tablets, liquid capsules, etc.

[0045] The seventh aspect of the present invention provides a method for improving protein degradation, comprising administering to a subject in need thereof at least one of the Lactobacillus plantarum HEM20701 strain or a culture, a lysate, or an extract thereof.

[0046] The eighth aspect of the present invention provides a use for improving protein degradation ability of at least one of the Lactobacillus plantarum HEM20701 strain or a culture, lysate, or extract thereof.

[0047] The ninth aspect of the present invention provides the use of any one or more of the Lactobacillus plantarum HEM20701 strain or a culture, lysate or extract thereof for producing a protein degradation enhancing agent.

[0048] The characteristics of each aspect can be applied to other aspects within the same or similar range.

[0049] Hereinafter, implementation examples and embodiments of the present invention will be described in detail with reference to the attached drawings. However, the present invention may not be limited to these implementation examples and embodiments and drawings.

[0050] Example 1. Comparison of protein degradation capacity when treated with three Lactobacillus plantarum species and enzyme alone.

[0051] The three strains used in this example were all Lactobacillus plantarum, HEM20701 isolated from adult feces, HEM21089 isolated from lemon, and HEM21113 isolated from tomato. All strains were cultured in MRS medium containing 10% skim milk as a cryoprotectant, and strains with a bacterial count of 1.0E+11 CFU / g or more were used in the experiment.

[0052] The enzyme used in this experiment was fermented enzyme powder. The fermented enzyme powder is a mixture of fruit and vegetable mixture (resistant maltodextrin, carrot concentrate, tart cherry concentrate, Bacillus amyloliquefaciens, chicory extract powder, apple concentrate, fructooligosaccharide, 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, frost seaweed, Job's tears, glutinous rice, sorghum, Lactobacillus plantarum, Lactobacillus casei, Pediococcus acidilactici, Bacillus), and BioCore Optimum K.

[0053] To obtain samples, the PMAS experiment was conducted as follows. In this specification, PMAS (Personalized Pharmaceutical Meta-Analysis Screening) refers to a technology useful for screening personalized intestinal environment improvement substances as a method for forming an identical / similar intestinal environment in vitro. In this experiment, by utilizing the characteristic of the PMAS experiment of forming an identical / similar intestinal environment in vitro, four types of proteins were added to the medium, and when the strain and enzyme were treated, the protein content in the medium was confirmed to decrease, thereby screening for a strain and enzyme combination showing a high protein degradation amount. Detailed experimental methods are in accordance with registered patents Nos. 10-2227382 and 10-2124474.

[0054] In the PMAS experiment, the incubation time was set to 0 and 24 hours to compare before and after incubation. The treatment groups consisted of enzyme alone, strain alone, and strain and enzyme combination treatment groups. To ensure equal viable cell counts, the strains were treated to 9.0E+09 CFU / g in each well. The control group was not treated with strain or enzyme. Four types of proteins were also dissolved in the medium and treated so that 2.5 mg was added to each well.

[0055] PMAS experiments were repeated twice to account for non-homogenization of proteins or enzymes, and cultures were performed in an anaerobic chamber. After culturing for 0 and 24 hours, samples were removed from the anaerobic chamber and centrifuged at room temperature (20°C) for 10 minutes to separate the resulting supernatant. The supernatant sample was diluted 1 / 10 in distilled water (Fisher) and used for protein measurement. Protein measurement was performed using Quick Start. TM The Bradford Protein Assay (Bio-Rad) kit was used according to the protocol. After subtracting the background and average values ​​from the measured values, the protein concentration value was determined based on the standard curve concentration. The protein concentration value was multiplied by a dilution factor of 10 since a 1 / 10 diluted sample was used. After that, all protein concentration values ​​were normalized by the adjustment factor so that the background value was calculated to be the amount of protein added. At this time, the protein measurement value was used as the average value of two replicates, and to calculate the amount of protein degradation, the value after 24 hours of incubation was subtracted from the value after 0 hours of incubation and the value was displayed in a graph.

[0056] When three Lactobacillus plantarum strains, HEM20701, HEM21089, and HEM21113, were treated alone with enzymes, the degree of protein degradation in four proteins (skim milk, soybean protein, pea protein, and gluten) was compared with the control group. As a result, the Lactobacillus plantarum HEM20701 strain showed a higher protein degradation amount than the other Lactobacillus plantarum strains (Fig. 1, Table 1).

[0057] In skim milk, the protein decomposition amount was high in the following order: HEM20701, HEM21113, HEM21089 strains; in soybean protein, HEM20701, HEM21089, HEM21113 strains; in pea protein, HEM20701, HEM21089, HEM21113 strains; and in gluten, HEM20701, HEM21089, HEM21113 strains. That is, in all cases, it was confirmed that the HEM20701 strain according to the present invention had the highest degree of protein decomposition.

[0058]

[0059] Comparison of protein degradation when treated with strains alone

[0060] - If resolution does not exist, the resolution is indicated as -

[0061] - Protein degradation amount (㎍ / mL): Calculate the protein degradation amount by subtracting the values ​​measured after 0 hours of culture and 24 hours of culture.

[0062] - Standard deviation: Standard deviation of two replicate values

[0063] - p-value: t-test of each enzyme and strain compared to the control protein degradation amount (p-value < 0.05 is indicated with *)

[0064] Example 2. Comparison of protein degradation capacity when treated with three Lactobacillus plantarum strains and enzyme combination.

[0065] The detailed experimental process is the same as Example 1.

[0066] The degree of protein degradation in four proteins [skim milk / soybean protein / pea protein / gluten] was compared with the control group by combining enzymes with three Lactobacillus plantarum strains, HEM20701, HEM21089, and HEM21113. As a result, when Lactobacillus plantarum HEM20701 strain was mixed with enzymes, a higher amount of protein degradation was observed than when other Lactobacillus plantarum strains and enzymes were mixed (Fig. 2, Table 2).

[0067] In skim milk, the protein degradation amount was highest in the following order: HEM21113, HEM20701, HEM21089 strains; in soybean protein, HEM20701, HEM21089, HEM21113 strains; in pea protein, HEM20701, HEM21089, HEM21113 strains; and in gluten, HEM20701, HEM21113, HEM21089 strains. In most cases, the HEM20701 strain claimed herein was confirmed to have the highest degree of protein degradation.

[0068] These results suggest that protein degradation is enhanced not only when treated with the strain alone but also when combined with enzymes, due to the interaction between the strain and the enzyme (Fig. 3, Table 2). Furthermore, we confirmed that protein degradation varies across strains, suggesting that this could serve as an important indicator for the development of probiotics with enhanced degradation of target proteins.

[0069]

[0070] Comparison of protein degradation capacity when combined with strain enzymes

[0071] - If resolution does not exist, the resolution is indicated as -

[0072] - Protein degradation amount (㎍ / mL): Calculate the protein degradation amount by subtracting the values ​​measured after 0 hours of culture and 24 hours of culture.

[0073] - Standard deviation: Standard deviation of two replicate values

[0074] - p-value: t-test of each enzyme and strain compared to the control protein degradation amount (p-value < 0.05 is marked with *)

[0075] Example 3. Lactobacillus plantarum HEM20701 strain that increases the protein decomposition ability of enzymes.

[0076] As confirmed in Examples 1 and 2, the Lactobacillus plantarum HEM20701 strain showed a significantly high protein decomposition ability even on its own, and showed an even higher protein decomposition ability when interacting with enzymes. When the decomposition amount was expressed as a % based on the amount of protein detected at 0 hours of culture, the Lactobacillus plantarum HEM20701 strain showed a high decomposition ability of about 15% compared to other strains of the same type, although there were differences depending on the type of protein. In addition, when the strain and enzyme were combined, the protein decomposition ability increased by 5 to 17% compared to the single treatment (Table 3).

[0077] The total protein amount, which is the sum of the decomposition amounts of each protein of skim milk, soybean protein, pea protein, and gluten and the four types of proteins, was expressed as a multiple of the protein decomposition amounts of enzyme, HEM20701, and HEM20701 and enzyme mixture based on the protein amount of the control group (Fig. 4). In the case of the Lactobacillus plantarum HEM20701 strain and the enzyme mixture, the decomposition amount of the four types of proteins and the total protein amount was 3.8 to 4.5 times higher than the control group, showing the best decomposition ability. In the case of Lactobacillus plantarum HEM20701 strain alone, the decomposition amount was 2.6 to 3.6 times higher than the control group, showing a higher protein decomposition ability than the enzyme group for all proteins.

[0078] Therefore, it was confirmed that the strain according to the present invention can promote protein degradation alone, and can further promote protein degradation when used together with an enzyme.

[0079]

[0080] Protein degradation analysis results (%)

[0081] - If resolution does not exist, the resolution is indicated as -

[0082] - The amount of protein detected at 0 hours of culture is expressed as a % of the amount of decomposition by strain alone and the amount of decomposition by strain + enzyme.

[0083] - The HEM20701 strain showed a higher protein degradation ability of about 15% compared to other strains of the same type, and when treated with enzymes, the protein degradation ability increased by 5-17% compared to treatment alone.

[0084]

Claims

1. Lactobacillus plantarum HEM20701 strain (Lactobacillus plantarumHEM20701; Accession No. KCTC 15550BP).

2. In paragraph 1, The above strain is a strain that promotes protein degradation.

3. In paragraph 1, The above strain is a strain that improves the decomposition ability of a protein-decomposing enzyme.

4. A composition for improving protein decomposition ability, comprising as an effective ingredient at least one of the strains of any one of claims 1 to 3 or their cultures, fragments, or extracts.

5. A composition for improving protein decomposition ability, comprising at least one of the strains of any one of claims 1 to 3 or their cultures, fragments, and extracts, and a protein decomposition enzyme as active ingredients.

6. A general food for improving protein decomposition ability, comprising as an active ingredient at least one of the strains of any one of clauses 1 to 3 or their cultures, fragments, or extracts.

7. A health functional food for improving protein decomposition ability, comprising as an active ingredient at least one of the strains of any one of clauses 1 to 3 or their cultures, fragments, or extracts.

8. A pharmaceutical composition for improving protein degradation, comprising as an active ingredient at least one of the strains of any one of claims 1 to 3 or their cultures, fragments, or extracts.

9. A method for improving protein degradation, comprising a step of administering to an individual in need thereof at least one of the strains of any one of claims 1 to 3 or their cultures, fragments, or extracts.

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