Composition and health functional food comprising Lactococcus lactis IDCC 2301 for reducing blood glucose levels

Abstract

The present invention relates to a composition and a health functional food containing Lactococcus lactis IDCC 2301 which is effective in lowering blood sugar, wherein the composition contains Lactococcus lactis IDCC 2301 [Accession No.: ATCC BAA-2834].

Description

Technology Field

[0001] The present invention relates to a composition and a health functional food containing Lactococcus lactis IDCC 2301, which is effective in lowering blood sugar. Background Technology

[0002] Postprandial blood glucose elevation is a physiological phenomenon in which blood glucose concentration temporarily increases after carbohydrate intake.

[0003] To maintain blood glucose homeostasis, glucose uptake and glucose utilization in peripheral tissues must be efficient, and in particular, insulin signaling in adipocytes and muscle cells plays a crucial role in regulating blood glucose metabolism.

[0004] Among the insulin signaling pathways, the IRS-1 / Akt / GLUT4 pathway is closely related to glucose uptake. When IRS-1 and Akt are activated by insulin stimulation, GLUT4 moves to the cell membrane, increasing glucose uptake.

[0005] On the other hand, TNF-α, an inflammatory cytokine, is known as a representative factor that induces insulin resistance and can inhibit glucose uptake-related cellular responses through increased IRS-1 Ser307 phosphorylation, decreased Akt activity, and decreased GLUT4 expression, as shown in Figure 1. Prior art literature

[0006] Korean Patent Registration No. 10-2928714 (February 23, 2026) The problem to be solved

[0007] The objective of the present invention is to provide a composition and a health functional food containing Lactococcus lactis IDCC 2301, which is effective in lowering blood sugar. means of solving the problem

[0008] The objective of the present invention is achieved by including Lactococcus lactis IDCC 2301 [Accession No.: ATCC BAA-2834] in a composition effective for lowering blood sugar.

[0009] The above Lactococcus lactis IDCC 2301 may be in the form of heat-treated dead bacteria.

[0010] The above composition can promote glucose consumption by restoring the expression of GLUT4 (Glucose transporter 4) in fat cells.

[0011] The above composition can inhibit Ser307 phosphorylation of IRS-1 in the insulin signaling pathway and restore Akt phosphorylation.

[0012] The above composition can improve insulin resistance induced by TNF-α (Tumor necrosis factor-alpha).

[0013] The objective of the present invention is achieved by including Lactococcus lactis IDCC 2301 [Accession No.: ATCC BAA-2834] in a health functional food effective for lowering blood sugar.

[0014] The above Lactococcus lactis IDCC 2301 may be in the form of heat-treated dead bacteria.

[0015] The objective of the present invention is achieved by including Lactococcus lactis IDCC 2301 [Accession No.: ATCC BAA-2834] in a pharmaceutical composition for the prevention and treatment of diabetes mellitus.

[0016] The above Lactococcus lactis IDCC 2301 may be in the form of heat-treated dead bacteria. Effects of the invention

[0017] According to the present invention, a composition and a health functional food containing Lactococcus lactis IDCC 2301, which is effective in lowering blood sugar, are provided. Brief explanation of the drawing

[0018] Figure 1 is a schematic diagram of the IRS-1 / Akt / GLUT4 signaling pathway in TNF-α-induced insulin-resistant 3T3-L1 adipocytes, and Figure 2 shows the effect of a heat-treated dead bacterial sample on the cell viability of differentiated 3T3-L1 adipocytes in an experimental example of the present invention, and Figure 3 shows the effect of heat-treated dead bacterial samples on glucose consumption in TNF-α-treated 3T3-L1 adipocytes, and Figures 4a to 4c show the effect of heat-treated dead bacteria samples on the IRS-1 / Akt / GLUT4 signaling pathway of TNF-α-treated 3T3-L1 adipocytes. Specific details for implementing the invention

[0019] The present invention will be described in more detail below with reference to the drawings.

[0020] The attached drawings are merely examples to specifically explain the technical concept of the present invention, and therefore the scope of the present invention is not limited thereto. Additionally, the sizes or spacing, etc., of the drawings may be depicted differently from the actual sizes to clearly explain the relationships between the components.

[0021] The present invention relates to a composition comprising lactic acid bacteria having activity for the effect of lowering blood sugar.

[0022] The composition may be a food composition, a health functional food composition, or a pharmaceutical composition.

[0023] In the present invention, "blood glucose lowering" may also be expressed as anti-hyperglycemic activity, improvement of hyperglycemia, blood glucose regulation, maintenance of blood glucose homeostasis, promotion of glucose metabolism, improvement of symptoms of impaired glucose metabolism, suppression of postprandial blood glucose rise (prevention of postprandial blood glucose spikes, improvement of postprandial blood glucose spikes, alleviation of postprandial blood glucose spikes), reduction of blood glucose rise, prevention of blood glucose rise, suppression of blood glucose rise, or improvement of long-term blood glucose indicators (reduction of glycated hemoglobin (HbA1c) levels).

[0024] If the composition of the present invention is a pharmaceutical composition, it may be effective in preventing, improving, or treating diabetes.

[0025] In the present invention, "diabetes" includes type 1 diabetes, type 2 diabetes, prediabetes, impaired glucose tolerance, and gestational diabetes.

[0026] In addition, the composition of the present invention may be effective in preventing, improving, or treating chronic complications caused by high blood sugar.

[0027] "Chronic complications" in the present invention include metabolic syndrome, diabetic microvascular complications, diabetic macrovascular complications, or diabetic foot ulcers.

[0028] The lactic acid bacteria of the present invention includes Lactococcus lactis IDCC 2301 [Accession No.: ATCC BAA-2834].

[0029] Lacticase Bacillus rhamnosus IDCC 3201 has the effect of lowering blood pressure.

[0030] Lacticase Bacillus rhamnosus IDCC 3201 can promote glucose consumption by restoring the expression of GLUT4 (Glucose transporter 4) in adipocytes, inhibit Ser307 phosphorylation of IRS-1 in the insulin signaling pathway and restore Akt phosphorylation, or improve insulin resistance induced by TNF-α (Tumor necrosis factor-alpha).

[0031] In the composition, Lacticaseibacillus rhamnosus IDCC 3201 may be in the form of live bacteria, dead bacteria, or lysate. In the composition, Lacticaseibacillus rhamnosus IDCC 3201 may be in the form of supernatant. In the case of dead bacteria, it may be dead bacteria, particularly those killed by heat treatment. Heat treatment may be performed at 80 to 120°C or 90 to 110°C for 10 minutes to 2 hours, 10 minutes to 1 hour, or 20 minutes to 1 hour. At the time of heat treatment, the strain may be in the form of a suspension.

[0032] The composition of the present invention may include lactic acid bacteria other than Lacticase Bacillus rhamnosus IDCC 3201.

[0033] In the composition, Lacticase Bacillus rhamnosus IDCC 3201 may be 1% to 100% by weight, 10% to 90% by weight, 1% to 10% by weight, or 0.1% to 50% by weight.

[0034] In a food composition or health functional food, Lacticase Bacillus rhamnosus IDCC 3201 may be 1% to 100% by weight, 10% to 90% by weight, 1% to 10% by weight, or 0.1% to 50% by weight.

[0035] A food composition or a health functional food may contain Lacticase Bacillus rhamnosus IDCC 3201 in an amount of 0.1 billion to 100 billion or 100 million to 10 billion CFU / g.

[0036] The food composition (or health functional food) of the present invention includes compositions for all forms of food, such as functional food, nutritional supplement, health food, and food additives. Food compositions of the above types can be prepared in various forms according to conventional methods known in the art. For example, health foods can be prepared in the form of gum containing lactic acid bacteria, vitamin complexes, tea, juice, and drinks, and food compositions containing Lacticase Bacillus rhamnosus IDCC 3201 as an active ingredient can be consumed by granulating, encapsulating, or powdering.

[0037] The functional food composition of the present invention may include ingredients that are typically added during food manufacturing, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, it may additionally include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, licorice extract, etc.

[0038] The food composition may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, it may contain extracts for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination.

[0039] As another example, the formulation of the food composition can be in the form of powder, granule, pill, tablet, or capsule, as well as any form of general food or beverage.

[0040] There are no specific restrictions on the types of food, and examples of food include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and may include all food in the conventional sense.

[0041] Generally, when manufacturing food, beverages, or health functional foods, the amount of Lacticase Bacillus rhamnosus IDCC 3201 used may be 0.1 to 50% by weight, 0.1 to 20% by weight, or 0.1 to 10% by weight. However, in the case of long-term consumption for the purpose of health and hygiene or health control, the above amount may be less than the above range.

[0042] The beverage in the food composition according to one embodiment may contain various flavoring agents or natural carbohydrates as additional ingredients, as in a conventional beverage. The natural carbohydrates mentioned above may be monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as taumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used.

[0043] In addition to the above, the food composition according to one embodiment may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, and carbonating agents used in carbonated beverages. Furthermore, the food composition of the present invention may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination.

[0044] The present invention comprises a pharmaceutical composition, wherein the pharmaceutical composition may comprise Lacticase Bacillus rhamnosus IDCC 3201. In the pharmaceutical composition, the content of Lacticase Bacillus rhamnosus IDCC 3201 may be 1 to 100 weight%, 1 to 50 weight%, 1 to 30 weight%, or 1 to 10 weight%.

[0045] The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably via an oral administration method. Suitable dosages of the pharmaceutical composition of the present invention may be prescribed differently depending on factors such as the formulation method, administration method, patient's age, body weight, sex, pathological condition, diet, time of administration, route of administration, excretion rate, and response sensitivity. Typical dosages of the pharmaceutical composition of the present invention are within the range of 0.001-100 mg / kg or 0.001-1000 mg / kg for adults.

[0046] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid dosage forms for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As bases for suppositories, Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc. may be used.

[0047] Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" in the present invention refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects. The effective dose level may be determined based on factors including the patient's health status, severity, drug activity, sensitivity to the drug, method of administration, time of administration, route of administration and elimination rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0048] The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. Taking all of the above-mentioned factors into consideration, it is important to administer an amount that obtains maximum effect with a minimum amount without side effects, and this can be easily determined by a person skilled in the art.

[0049] The dosage of the pharmaceutical composition of the present invention may be determined by a person skilled in the art by taking into consideration the purpose of use, the degree of toxicity of the disease, the patient's age, weight, gender, medical history, or the type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention may be administered at a dose of about 0.1 ng to about 1000 mg / kg per adult, specifically 1 ng to about 100 mg / kg or 1 ng to about 10 mg / kg, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but may be administered once a day or administered several times by dividing the dose.

[0050] As another example, the preferred dosage of the pharmaceutical composition according to the present invention may vary depending on the patient's condition, body weight, degree of disease, drug form, route of administration, and duration, but can be appropriately selected by those skilled in the art. However, for a desirable effect, it may be administered at a dose of 0.0001 to 2,000 mg / kg per day, preferably 0.001 to 2,000 mg / kg. The administration may be performed once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0051] However, since the dosage may vary depending on the route of administration, severity of the disease, gender, body weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0052] The present invention will be explained in more detail below through examples.

[0053] In this example, we aimed to evaluate the potential of Lactococcus lactis IDCC 2301, hereinafter LLA, among heat-killed bacterial samples for glucose metabolism. To this end, we examined the effects of LLA on glucose consumption and the IRS-1 / Akt / GLUT4 signaling pathway in a 3T3-L1 adipocyte model in which an insulin resistance-like state was induced by TNF-α.

[0054] 1. Test Materials and Methods

[0055] 1.1. Preparation of Test Samples

[0056] In this example, a heat-killed bacterial sample prepared from a strain owned by Ildong Bioscience was used. The materials subject to evaluation are as follows.

[0057]

[0058] To obtain heat-killed bacteria samples, each culture was centrifuged at 8,000 × g for 5 minutes to remove the supernatant, and the precipitate was resuspended in phosphate-buffered saline (PBS). The bacterial count of the suspension was 1 × 10⁶ 8 After adjusting to CFU / mL, heat-killed bacteria samples were prepared by heating at 100 ℃ for 30 minutes.

[0059] 1.2. 3T3-L1 Cell Culture and Differentiation

[0060] 3T3-L1 preadipocytes were cultured in DMEM supplemented with 10% newborn calf serum (NCS) and 1% penicillin / streptomycin (P / S) at 37°C and 5% CO2. The medium was replaced every 2-3 days, and the cells were subcultured every 4-5 days.

[0061] 2 × 10⁶ 3T3-L1 preadipocytes for differentiation into adipocytes 5 Cells were seeded at a concentration of cells / mL, and differentiation was induced after the cells reached 100% confluence. The differentiation medium consisted of DMEM high glucose supplemented with 10% fetal bovine serum (FBS) and 1% P / S. On Days 0–2, differentiation induction medium containing 0.5 mM IBMX, 1 μM dexamethasone, and 10 μg / mL insulin was administered, and on Days 2–4, the medium was replaced with one containing 10 μg / mL insulin. On Days 4–8, cells were maintained by replacing only the differentiation medium without insulin.

[0062] 1.3. Cytotoxicity Assessment

[0063] An MTT assay was performed to determine cytotoxicity based on the test material and treatment conditions.

[0064] Differentiated 3T3-L1 adipocytes were treated with 10 ng / mL of TNF-α and either 2 mM of metformin or each heat-killed bacterial sample. After 24 hours of treatment, MTT reagent was added, and the resulting formazan crystals were dissolved in DMSO, after which the absorbance was measured at 570 nm. Cell viability was calculated as a percentage relative to the untreated control.

[0065] 1.4. Evaluation of Glucose Consumption

[0066] A glucose assay was performed to evaluate the effects of LLA and candidate materials on glucose consumption. Insulin resistance-like states were induced in differentiated 3T3-L1 adipocytes by treating them with TNF-α 10 ng / mL. Subsequently, metformin or heat-killed bacterial samples were added, and the cells were cultured for 24 hours. After culture, the residual glucose concentration in the medium was measured using a glucose assay kit. A lower residual glucose concentration in the medium was interpreted as an increase in apparent glucose consumption by the cells.

[0067] 1.5. Western Blot Analysis

[0068] Western blot analysis was performed to investigate the effects of LLA on the IRS-1 / Akt / GLUT4 signaling pathway. Differentiated 3T3-L1 adipocytes were lysed with RIPA buffer, and proteins were extracted. Equal amounts of protein were separated by SDS-PAGE and transferred to a PVDF membrane. Subsequently, protein expression and phosphorylation levels were analyzed using antibodies against IRS-1, p-IRS-1 Ser307, Akt, p-Akt, GLUT4, and GAPDH. Protein band intensity was quantified using ImageJ software.

[0069] 1.6. Statistical Analysis

[0070] All results were expressed as the mean ± standard deviation of three independent replicates. Statistical significance was analyzed using Dunnett's multiple-comparison test following one-way ANOVA. Cytotoxicity was evaluated against the untreated control, while glucose consumption and Western blot analysis were compared against the TNF-α treated group. Statistical significance was determined based on a p < 0.05 threshold.

[0071] 2. Test Results

[0072] 2.1. Results of Cytotoxicity Evaluation

[0073] As a result of evaluating cytotoxicity following treatment with heat-killed bacterial samples and metformin, no distinct cytotoxicity was observed in any of the treatment groups used in the test (Fig. 2, Effect of heat-killed bacterial samples on cell viability of differentiated 3T3-L1 adipocytes. Cell viability was evaluated via the MTT assay after 24 hours of treatment. Data are presented as mean ± SD. Statistical significance was analyzed using Dunnett's test compared to the untreated control group). Cell viability was maintained at over 80% in all TNF-α, metformin, TH, LLA, and RH treatment groups. Therefore, the treatment conditions used in this study are considered to be within a range that does not induce clear cytotoxicity in 3T3-L1 adipocytes. Based on these results, subsequent evaluations of glucose consumption and mechanism analysis were performed within the concentration range where no cytotoxicity was observed.

[0074] 2.2. Glucose Consumption Evaluation Results

[0075] In the TNF-α treated group, the residual glucose concentration in the medium increased compared to the untreated control. This is interpreted as a decrease in glucose consumption by 3T3-L1 adipocytes due to TNF-α treatment. On the other hand, in the group treated with heat-killed bacterial samples, a decreasing trend in the residual glucose concentration in the medium was observed compared to the TNF-α alone treatment group. In particular, 5 × 10 6 Under the cells / mL treatment conditions, TH, LLA, and RH all showed an effect in reducing the residual glucose concentration increased by TNF-α treatment. The apparent glucose consumption increase rates for TH, LLA, and RH were 22.8%, 23.7%, and 19.0%, respectively (Fig. 3, Effect of heat-treated inactivated bacterial samples on glucose consumption of TNF-α-treated 3T3-L1 adipocytes. Residual glucose concentration in the culture medium was measured after 24 hours of treatment with TNF-α and heat-treated inactivated bacterial samples. Data are presented as mean ± SD. Statistical significance was analyzed using Dunnett's test in comparison to the TNF-α treatment group. *p < 0.05, **p < 0.01, ***p < 0.001).

[0076] Among the three candidate materials, LLA showed the highest apparent trend of increased glucose consumption. Accordingly, LLA was selected as the subject for the subsequent analysis of the IRS-1 / Akt / GLUT4 signaling pathway.

[0077] 2.3. Analysis Results of IRS-1 / Akt / GLUT4 Signaling Pathway

[0078] In the TNF-α treated group, GLUT4 expression decreased compared to the untreated control. This suggests that glucose uptake-related signals were inhibited by TNF-α. Conversely, in the LLA treated group, a recovery of GLUT4 expression, which had decreased due to TNF-α, was observed. According to the report, GLUT4 expression levels decreased to 49.7% compared to the untreated control in the TNF-α alone treated group, while recovering to 125.2% in the LLA treated group (Fig. 4a; GLUT4 expression was analyzed after treatment with TNF-α and heat-treated dead bacterial samples).

[0079] In addition, IRS-1 Ser307 phosphorylation increased in the TNF-α treated group. The increase in IRS-1 Ser307 phosphorylation is interpreted as a change associated with the inhibition of insulin signaling. In the LLA treated group, the p-IRS-1 Ser307 / IRS-1 ratio decreased compared to the TNF-α alone treated group (Fig. 4b, LLA reduced the p-IRS-1 Ser307 / IRS-1 ratio increased by TNF-α). The p-IRS-1 Ser307 / IRS-1 ratio increased to 165.9% in the TNF-α treated group compared to the untreated control, and decreased to 105.3% in the LLA treated group.

[0080] In the case of Akt phosphorylation, the p-Akt / Akt ratio in the TNF-α treated group decreased to 64.5% compared to the untreated control. On the other hand, in the LLA treated group, the p-Akt / Akt ratio recovered to 93.6% (Fig. 4c, LLA restored the p-Akt / Akt ratio inhibited by TNF-α). These results suggest that LLA may act to lower IRS-1 Ser307 phosphorylation and restore Akt activity and GLUT4 expression in an insulin resistance-like state induced by TNF-α.

[0081] Data in Figures 4a to 4c are presented as mean ± SD. Statistical significance was analyzed using Dunnett's test in comparison to the TNF-α treatment group. *p < 0.05, **p < 0.01, ***p < 0.001.

[0082] As described above, in the examples, the potential of heat-killed Lactococcus lactis IDCC 2301, LLA, for blood glucose metabolism was evaluated in a cell-based model.

[0083] TNF-α is a representative inflammatory cytokine that induces insulin resistance and can cause reduced glucose consumption, inhibition of Akt signaling, and reduced GLUT4 expression in adipocytes. In this example, it was confirmed that the residual glucose concentration in the medium increased and the levels of GLUT4 and p-Akt decreased in the TNF-α treated group. This indicates that a TNF-α-induced insulin resistance-like model was appropriately formed.

[0084] In the LLA-treated group, the residual glucose concentration in the medium, which had increased due to TNF-α treatment, decreased. This suggests the possibility that LLA acted to restore glucose consumption reduced by TNF-α. Furthermore, LLA induced the restoration of GLUT4 expression, a decrease in p-IRS-1 Ser307, and a restoration of the p-Akt / Akt ratio. These changes support the possibility of improved glucose metabolism associated with the IRS-1 / Akt / GLUT4 signaling pathway.

[0085] As shown in the results of the examples, heat-killed Lactococcus lactis IDCC 2301, LLA, showed a tendency to restore glucose consumption in a TNF-α-induced insulin resistance-like 3T3-L1 adipocyte model. In addition, LLA demonstrated a tendency to improve the IRS-1 / Akt / GLUT4 signaling pathway inhibited by TNF-α. Specifically, upon LLA treatment, GLUT4 expression was restored, the p-IRS-1 Ser307 / IRS-1 ratio decreased, and the p-Akt / Akt ratio was restored. Therefore, LLA can be considered as a candidate for a functional ingredient, excipient, or auxiliary ingredient as a heat-killed postbiotic material with potential related to blood glucose metabolism.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 delete Claim 6 A health functional food effective for lowering blood sugar, comprising Lactococcus lactis IDCC 2301 [Accession No.: ATCC BAA-2834]. Claim 7 In claim 6, the above Lactococcus lactis IDCC 2301 is a health functional food in the form of heat-treated dead bacteria. Claim 8 A pharmaceutical composition for the prevention and treatment of diabetes mellitus, comprising Lactococcus lactis IDCC 2301 [Accession No.: ATCC BAA-2834]. Claim 9 In claim 8, the above Lactococcus lactis IDCC 2301 is a pharmaceutical composition in the form of a heat-treated dead bacterium.

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