Composition comprising lactobacillus plantarum hem20865 having excellent proteolytic activity or culture, lysate or extract thereof as active ingredient

Abstract

The present invention relates to a composition comprising, as an active ingredient, a Lactobacillus plantarum HEM20865 strain capable of improving the proteolytic activity of an enzyme, or a culture, lysate or extract thereof. The present invention provides Lactobacillus plantarum HEM20865, which is a probiotic strain, or a food composition or a pharmaceutical composition which comprise the strain, to people who have difficulty in protein digestion, and thus the strain or the compositions can be effectively used to improve protein degradation and digestion ability.

Description

A composition comprising Lactobacillus plantarum HEM20865, which has 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 HEM20865 strain, or a culture, lysate, or extract thereof, which can enhance the proteolytic capacity of an enzyme. The strain according to the present invention not only exhibits excellent proteolytic capacity alone, but also further enhances proteolytic capacity when used in combination with an enzyme, enabling it to be effectively utilized in the manufacture of general foods or health functional foods.

[0002] This application claims priority to Korean Patent Application No. 10-2023-0175841, filed December 6, 2023, and Korean Patent Application No. 10-2024-0146706, filed October 24, 2024, the entire contents of which are incorporated herein by reference.

[0003] As we age, the secretion of enzymes necessary for digestion declines, and factors such as irregular eating habits and stress often impair digestive function. Consequently, the number of people experiencing indigestion is increasing. In particular, changes in eating habits, such as excessive protein intake, can put a strain on the digestive system, leading to a rise in complaints of indigestion and intestinal disorders. Probiotics are gaining attention as one way to address these issues.

[0004] Probiotics are live, beneficial microorganisms that inhabit the human intestines or other organs and have a positive impact on health. They primarily include Lactobacillus and Bifidobacterium, and are known to have various health-promoting effects, including improving the intestinal environment and boosting immunity (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 actively underway, including 1) expansion of complex strain-based research, 2) development of personalized probiotics, 3) research related to immune enhancement and metabolic diseases, 4) development of probiotic products for the elderly and infants, 5) probiotics as functional foods and beverages, 6) research on new strains and microbiomes, and 7) research on postbiotics and prebiotics.

[0006] 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, 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. This indicates that Lactobacillus plantarum is becoming an important topic of probiotics research for health promotion.

[0007] The present invention provides a food composition and health functional food comprising the Lactobacillus plantarum HEM20865 strain, which exhibits excellent protein-decomposing ability, particularly when combined with enzymes, and exhibits even greater protein-decomposing ability. The above-described object is merely one example of the present invention, and additional objects may exist that can be achieved by the technical concepts of the present invention.

[0008] The solution to the problem of the present invention includes the following aspects.

[0009] A first aspect of the present invention provides a Lactobacillus plantarum HEM20865 strain (Lactobacillus plantarumHEM20865; Accession No. KCTC 15551BP).

[0010] A second aspect of the present invention provides a composition for improving protein degradation ability, comprising at least one of Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof as an active ingredient.

[0011] A third aspect of the present invention provides a composition comprising at least one of Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof and a protein-decomposing enzyme as active ingredients.

[0012] The fourth aspect of the present invention provides a food composition for improving protein decomposition ability, comprising at least one of Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof as an active ingredient.

[0013] The fifth aspect of the present invention provides a pharmaceutical composition for improving protein degradation, comprising at least one of Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof as an active ingredient.

[0014] The sixth 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 HEM20865 strain or a culture, a lysate, or an extract thereof.

[0015] The seventh aspect of the present invention provides a use for improving protein degradation ability of the Lactobacillus plantarum HEM20865 strain or at least one of a culture, a lysate, and an extract thereof.

[0016] The eighth aspect of the present invention provides the use of at least one of the Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof for producing a protein degradation enhancing agent.

[0017] Such means are merely examples of the present invention, and additional means may exist within the scope understandable to those skilled in the art.

[0018] The present invention can be usefully utilized to improve protein decomposition and digestion ability by providing the probiotic Lactobacillus plantarum HEM20865, or a food composition or pharmaceutical composition containing the strain, to people who have difficulty in digesting proteins.

[0019] The above effects are merely examples, and additional effects may exist within the scope understandable to a person skilled in the art.

[0020] Figure 1 is a diagram showing the amount of protein degradation measured through treatment with four strains of Lactobacillus plantarum (HEM20865, HEM21080, HEM21089, HEM21113) and enzyme alone.

[0021] Figure 2 is a diagram showing the amount of protein degradation measured through four strains of Lactobacillus plantarum (HEM20865, HEM21080, HEM21089, HEM21113) and enzyme combination treatment.

[0022] Figure 3 is a diagram comparing the amount of protein decomposition according to treatment with four strains of Lactobacillus plantarum (HEM20865, HEM21080, HEM21089, HEM21113) and enzymes alone or in combination.

[0023] Figure 4 is a diagram comparing the multiples of protein decomposition amounts of skim milk, soy protein, pea protein, gluten, and total protein for the enzyme-only treatment group, the Lactobacillus plantarum HEM20865-only treatment group, and the Lactobacillus plantarum HEM20865 and enzyme combination treatment group, respectively, compared to the control group.

[0024] Below, with reference to the attached drawings, embodiments of the present invention are described in detail so that those skilled in the art can easily implement them. However, the present invention may 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 of description, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0025] 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.

[0026] 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.

[0027] The terms "about," "substantially," and the like used throughout this specification are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings mentioned are presented, and are used to prevent unscrupulous infringers from unfairly exploiting the disclosure, which contains precise or absolute numerical values ​​to aid understanding of the present invention. The terms "step of doing" or "step of" used throughout this specification do not mean "step for doing."

[0028] 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.

[0029] Throughout this specification, references to “A and / or B” mean “A or B, or A and B.”

[0030] The term "culture solution" used throughout this 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."

[0031] 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.

[0032] 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" refers to obtaining a useful effect for health purposes, such as regulating nutrients or physiological effects for the structure and function of the human body.

[0033] 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, any formulation of the food recognized as a food can be manufactured without limitation. The food composition of the present invention can be manufactured in various forms, and unlike general drugs, it has the advantage of being free from side effects that can occur with long-term use of drugs since it uses food as a raw material, and is highly portable.

[0034] A first aspect of the present invention provides a Lactobacillus plantarum HEM20865 strain. The Lactobacillus plantarum HEM20865 strain is derived from sikhye (Korean rice drink). The strain was deposited with the Korea Center for Biological Resources (KCTC) on August 14, 2023, under the deposit number KCTC 15551BP. The strain according to the present invention may promote protein degradation.

[0035] A second aspect of the present invention provides a composition for improving protein degradation ability, comprising at least one of Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof as an active ingredient.

[0036] A third aspect of the present invention provides a composition for improving protein degradation, comprising at least one of Lactobacillus plantarum HEM20865 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.

[0037] In the present invention, the enzyme is a biocatalyst that increases the speed of metabolism by lowering the activation energy of a chemical reaction by forming an enzyme-substrate complex by binding to a substrate, and specifically, may be a protein-decomposing enzyme, and in one embodiment of the present invention, a fermentation enzyme was used, but is not limited thereto.

[0038] The fourth aspect of the present invention provides, in one embodiment, a food composition for improving protein degradation ability, comprising at least one of Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof as an active ingredient.

[0039] The fifth aspect of the present invention provides, in one embodiment, a pharmaceutical composition for improving protein degradation ability, comprising at least one of Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof as an active ingredient.

[0040] The composition according to the second to fifth aspects of the present invention may be manufactured into a dosage form that can be ingested by an individual. The term "individual" herein should be interpreted to include all animals capable of exhibiting increased protein degradation, such as humans, monkeys, pigs, cows, and chickens. Furthermore, the term "formulation" herein includes any preparation manufactured by a method commonly used in the art, and may be in the form of, for example, powder, tablets, or liquid capsules.

[0041] The sixth 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 HEM20865 strain or a culture, a lysate, or an extract thereof.

[0042] The seventh aspect of the present invention provides a use for improving protein degradation ability of the Lactobacillus plantarum HEM20865 strain or at least one of a culture, a lysate, and an extract thereof.

[0043] The eighth aspect of the present invention provides the use of at least one of the Lactobacillus plantarum HEM20865 strain or a culture, lysate, or extract thereof for producing a protein degradation enhancing agent.

[0044] The characteristics of each aspect can be applied to other aspects within the same or similar range.

[0045] Hereinafter, embodiments and examples 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 embodiments and examples and drawings.

[0046] Example 1. Comparison of protein degradation capacity between four Lactobacillus plantarum strains and enzyme treatment alone.

[0047] The four strains used in this example were all Lactobacillus plantarum, including HEM20865 isolated from Sikhye, HEM21080 isolated from Chestnut, 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.

[0048] The enzyme used in this experiment was a mixture of commercially available fermented enzyme powder [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), Biocore Optimum K].

[0049] The PMAS experiment for sample acquisition 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 by creating an identical / similar intestinal environment in vitro. In this experiment, utilizing the core of PMAS technology, which is creating an identical / similar intestinal environment in vitro, four types of proteins were added to the medium, and then treated with specific strains and enzymes. Through this, a decrease in the protein content in the medium was confirmed, and a strain and enzyme combination showing a high protein degradation rate was selected. For details on the experimental method, refer to Korean Patent Nos. 10-2227382 and 10-2124474.

[0050] 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.

[0051] PMAS experiments were performed twice to account for non-homogenization of proteins or enzymes, and cultures were performed in an anaerobic chamber. Samples were incubated for 0 and 24 hours, then 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 in distilled water (Fisher) and used for protein measurement. Protein measurement was performed according to the protocol using the Quick Start™ Bradford Protein Assay (Bio-Rad) kit. After subtracting the background and average values ​​from the measured values, the protein concentration values ​​were determined based on the standard curve concentrations. The protein concentration values ​​were multiplied by a factor of 10 to account for the 1 / 10 diluted sample used. All protein concentration values ​​were normalized by the adjustment factor, calculating the background value to be equal to the added protein amount. At this time, the protein measurement value was the average of two repetitions, and to calculate the amount of protein decomposition, 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.

[0052] When four Lactobacillus plantarum strains, HEM20865, HEM21080, HEM21089, and HEM21113, were treated alone with enzymes, the degree of protein degradation in four proteins (skim milk / soybean protein / pea protein / gluten) was compared with the control group. As a result, the Lactobacillus plantarum HEM20865 strain showed a higher protein degradation amount than the other Lactobacillus plantarum strains (Fig. 1, Table 1).

[0053] In skim milk, the protein decomposition amount was high in the following order: HEM21113, HEM20865, HEM21089, HEM21080 strains; in soybean protein, HEM20865, HEM21089, HEM21080, HEM21113 strains; in pea protein, HEM20865, HEM21089, HEM21080, HEM21113 strains; and in gluten, HEM20865, HEM21089, HEM21080, HEM21113 strains. That is, in most cases, it was confirmed that the HEM20865 strain according to the present invention had the highest degree of protein decomposition.

[0054]

[0055] - If resolution does not exist, the resolution is indicated as -

[0056] - Normalize to baseline

[0057] - 0 hour of culture - Subtract the 24 hour culture measurement value to display the protein degradation amount in a graph.

[0058] - Protein measurements use the average of two replicates.

[0059] - Standard deviation is the standard deviation of two replicate values

[0060] *: t-test of strain or enzyme compared to control group, p-value < 0.05

[0061] Example 2. Comparison of protein degradation capacity when four Lactobacillus plantarum species and enzyme combination treatment were used.

[0062] The detailed experimental process is the same as Example 1.

[0063] 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 four Lactobacillus plantarum strains HEM20865, HEM201080, HEM21089, and HEM21113. As a result, when Lactobacillus plantarum HEM20865 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).

[0064] In skim milk, the protein degradation amount was high in the following order: HEM21113, HEM20865, HEM21080, HEM21089 strains; in soybean protein, HEM20865, HEM21089, HEM21080, HEM21113 strains; in pea protein, HEM20865, HEM21089, HEM21080, HEM21113 strains; and in gluten, HEM20865, HEM21113, HEM21080 HEM21089 strains. In most cases, the HEM20865 strain claimed in the present invention was confirmed to have the highest degree of protein degradation.

[0065] 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 (Figure 3, Table 3). 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.

[0066]

[0067] - If resolution does not exist, the resolution is indicated as -

[0068] - Normalize to baseline

[0069] - 0 hour of culture - Subtract the 24 hour culture measurement value to display the protein degradation amount in a graph.

[0070] - Protein measurements use the average of two replicates.

[0071] - Standard deviation is the standard deviation of two replicate values

[0072] *: t-test of strain or enzyme compared to control group, p-value < 0.05

[0073] Example 3. Lactobacillus plantarum HEM20865 strain that increases the protein decomposition ability of enzymes.

[0074] As confirmed in Examples 1 and 2, the Lactobacillus plantarum HEM20865 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 HEM20865 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 1 to 11% compared to the single treatment (Table 3).

[0075] 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, HEM20865, and HEM20865 and enzyme mixture based on the protein amount of the control group (Fig. 4). In the case of the Lactobacillus plantarum HEM20865 strain and enzyme mixture, the decomposition amount of the four types of proteins and the total protein amount was 3.0 to 3.9 times higher than the control group in the decomposition amount, showing the best decomposition ability. In the case of Lactobacillus plantarum HEM20865 strain alone, the decomposition amount was 2.4 to 3.1 times higher than the control group, showing a higher protein decomposition ability than the enzyme group in all proteins.

[0076] 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.

[0077]

[0078] - 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.

[0079] - If resolution does not exist, the resolution is indicated as -

[0080] - The HEM20865 strain showed a higher protein degradation ability of about 15% compared to other strains of the same type, depending on the protein type, and when treated with enzymes, the protein degradation ability increased by 1-11% compared to treatment alone.

[0081]

Claims

1. Lactobacillus plantarum HEM20865 strain (Lactobacillus plantarumHEM20865; Accession No. KCTC 15551BP).

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

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

4. 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, or extracts as an effective ingredient.

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 food composition for improving protein decomposition ability, comprising as an effective ingredient any one of claims 1 to 3 or at least one of a culture, a fragment, or an extract thereof.

7. A pharmaceutical 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, or extracts as an effective ingredient.

8. A method for improving protein decomposition ability, comprising a step of administering to a subject in need thereof at least one of the strains of any one of claims 1 to 3 or a culture, fragment or extract thereof.

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