Probiotic composition having effect of improving soybean protein decomposition ability and amino acid production ability

Abstract

The effects of the present invention include the ability to decompose soybean protein, the ability to produce valine, isoleucine, and leucine, which are branched chain amino acids, and the ability to produce threonine, glycine, tyrosine, and lysine, which are other amino acids, are improved. As a result, it is possible to achieve the effects of preventing sarcopenia and antioxidant activity, and also achieve the effect of improving sarcopenia by maintaining a protein metabolic balance and muscle synthesis. Furthermore, the self-aggregation property, hydrophobicity, and intestinal adhesion of the mixed strain are improved compared to the case in which each strain used in the mixed strain is used alone, and therefore, when ingestion of the mixed strain of the present application, it is possible to obtain the effect of long-term retention of the corresponding strain in the intestinal tract.

Description

Technical Field

[0001] The present application relates to a mixed strain composition having the ability to decompose soybean protein and produce amino acids, and relates to a mixed strain of Lactobacillus fermentum LM1020 (KCCM12918P) and Lactobacillus acidophilus LM1060 (KCCM12625P), and a composition containing them as active ingredients. Background Art

[0002] The increasing prevalence of obesity due to aging-related muscle loss, Westernized eating habits, and decreased physical activity is of great significance in health science. It is well known that protein intake helps maintain muscle mass while counteracting aging-related muscle loss. Adequate protein intake can reduce the risk of sarcopenia and obesity-related diseases in all age groups.

[0003] Representative dietary sources of protein can be divided into animal proteins such as milk, eggs, beef, pork, chicken and fish, and plant proteins such as soybeans and cereals. Among animal protein sources, milk protein has the advantages of excellent essential amino acid components such as leucine, isoleucine and methionine, and whey protein has the advantages of high bioavailability and rapid absorption. Therefore, it is widely consumed as a protein supplement. Among them, whey protein is made by concentrating the liquid by-product (whey) separated from milk during the cheese or casein production stage. However, whey protein lacks dietary fiber and is high in fat and cholesterol, so you may need to pay attention to its intake when managing your weight. Moreover, because it contains lactose, if you lack the enzyme (lactase) that breaks down lactose and suffer from lactose intolerance, after taking whey protein, lactose cannot be broken down in the digestive organs, which may cause gastrointestinal disorders such as abdominal pain, diarrhea and vomiting.

[0004] In recent years, as environmentally friendly and sustainable eating habits have attracted worldwide attention, the development of alternative foods to animal-based ingredients is being actively carried out, and attention to plant proteins is increasing.

[0005] As examples of existing probiotic compositions with protein-degrading capabilities, there are food compositions including Lactobacillus casei IDCC 3451 with protein-degrading capabilities and health functional foods (Publication No. 10-2023-0040252), etc. However, there is still a need to develop and research compositions that can make up for the shortcomings of decomposing plant-derived proteins when taken together. Summary of the Invention

[0006] Problems to be solved by the invention

[0007] The object of the present invention is to provide a mixed bacterial strain having an effect of improving soybean protein decomposition ability or amino acid production ability.

[0008] Solutions for solving problems

[0009] The present invention provides a mixed strain, which is a mixed strain comprising Lactobacillus fermentum LM1020 (preservation number KCCM12918P) and Lactobacillus acidophilus LM1060 (preservation number KCCM12625P), wherein the mixed strain has an effect of improving the ability to decompose soybean protein.

[0010] In addition, the present invention provides a food composition, a health functional food composition, a formula milk powder composition, and a pharmaceutical composition for preventing or improving sarcopenia, which contain any one or more of the above-mentioned mixed strains or their cultures, lysates, and extracts as active ingredients.

[0011] Effects of the Invention

[0012] The effects of the present invention include an effect of improving the ability to decompose soybean protein and an effect of preventing or improving sarcopenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The results of confirming the valine, isoleucine, and leucine production capabilities of the mixed strains according to the present application at different mixing ratios according to Example 2 are shown.

[0014] Figure 2 The results of Example 3, which confirm the production capabilities of threonine, glycine, tyrosine, and lysine of the mixed strains according to the present application at different mixing ratios, are shown. DETAILED DESCRIPTION

[0015] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily implement them. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. In addition, to clearly illustrate the present application in the drawings, portions not relevant to the description are omitted, and similar reference numerals are used throughout this specification for similar portions.

[0016] Throughout the specification of the present application, when a part is said to “comprise” an element, unless otherwise stated, it means that other elements may also be included, rather than excluding other elements.

[0017] In this specification, the "mixing ratio" refers to "Lactobacillus fermentum LM1020:Lactobacillus acidophilus LM1060".

[0018] Generally speaking, for bacterial strains used as probiotics, it is expected that the above-mentioned strains themselves, strain cultures, strain lysates, and strain extracts all have the same or similar effects.

[0019] The soy protein used in this specification is described. Among plant proteins, soy protein contains a variety of physiologically active ingredients, has the advantage of preventing muscle loss, and is rich in dietary fiber. Compared with animal protein, the content of saturated fat and carbohydrates is lower, so it is effective for weight management and is widely used as a supplement in plant protein sources. In addition, it is well known that compared with whey protein, soy protein has a higher content of some amino acids such as arginine, phenylalanine and tryptophan, which is very necessary for the formation of muscle tissue. When continuously ingested, it reduces the low-density lipoprotein (LDL) value in the blood and increases the high-density lipoprotein (HDL) value in the blood, thereby helping to improve the cholesterol value. In addition, it is well known that compared with other plant proteins such as pea protein, it is cheaper and does not have the peculiar smell of plant-derived protein.

[0020] Dietary proteins, including soy protein, are high-molecular substances and therefore need to be broken down into lower-molecular substances after ingestion. Various digestive enzymes, such as pepsin in the stomach and trypsin and chymotrypsin in the small intestine, participate in this process, breaking down the peptide chains of the protein, which are ultimately absorbed as amino acids by the villi of the small intestine. During this process, enzymes secreted by intestinal microorganisms present in the digestive organs also affect the breakdown and absorption of protein. However, since intestinal microorganisms can secrete enzymes that humans cannot biosynthesize, the types and amounts of amino acids produced from the breakdown of dietary protein can vary greatly depending on the composition of the intestinal microbiome and the intake of probiotics.

[0021] Among the amino acids produced after protein breakdown, valine, isoleucine, and leucine, which belong to the branched-chain amino acids (BCAAs), are essential amino acids for maintaining normal protein metabolic balance and muscle synthesis. They have the advantages of reducing muscle damage caused by exercise and promoting muscle synthesis. In particular, leucine is an amino acid that plays a signaling role in the muscle biosynthesis pathway and helps to improve sarcopenia in the elderly.

[0022] In addition to branched-chain amino acids, specific amino acids such as glycine, a component of creatine that helps with athletic performance, threonine that contributes to skeletal muscle protein synthesis, tyrosine that helps improve athletic performance, and lysine that helps skeletal muscle growth and inhibits protein breakdown can improve muscle formation and athletic performance.

[0023] On the other hand, soy protein generally has a lower amino acid content, including branched-chain amino acids, than whey protein, and is absorbed more slowly. Therefore, even though it is widely used as a plant-based dietary protein, soy protein currently lacks advantages over whey protein in terms of immediate muscle regeneration and the prevention or improvement of sarcopenia.

[0024] Therefore, if the digestion and decomposition of soy protein can be promoted and the ability to generate amino acids (branched-chain amino acids, etc.) from the ingested soy protein can be improved, muscle regeneration and other effects can be achieved by ingesting soy protein, which is not inferior to whey protein.

[0025] In response to this, the inventors have developed a mixed strain comprising Lactobacillus fermentum LM1020 (deposit number KCCM12918P) and Lactobacillus acidophilus LM1060 (deposit number KCCM12625P). The mixed strain can improve the shortcomings of soy protein, which has a lower digestion and absorption rate than whey protein and a relatively low content of branched-chain amino acids, when ingesting soy protein.

[0026] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings. However, the present application is not limited to these drawings and embodiments.

[0027] The present application provides a mixed strain, which is a mixed strain comprising Lactobacillus fermentum LM1020 (deposit number KCCM12918P) and Lactobacillus acidophilus LM1060 (deposit number KCCM12625P), wherein the mixed strain has an effect of improving the ability to decompose soy protein.

[0028] In addition, the present application provides a composition comprising various forms of the above-mentioned mixed strains as active ingredients, wherein the various forms include any one or more of the mixed strains or their cultures, lysates, and extracts, and the composition includes a food composition, a health functional food composition, a formula milk powder composition, and a pharmaceutical composition for preventing or improving sarcopenia.

[0029] The term "food" as used throughout this application specification refers to meat, sausage, bread, chocolate, candy, fast food, biscuits, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, oral medications, alcoholic beverages, vitamin complexes, health functional foods and health foods, etc., including all conventional foods.

[0030] The food of the present application can be prepared using conventional methods in this area. When prepared as described above, the raw materials and ingredients commonly added in this area can be added to prepare. In addition, the dosage form of the above-mentioned food can be prepared without restriction as long as it is identified as the dosage form of food. The food composition of the present invention can be prepared into various forms of dosage forms, and is different from general medicines, using food as raw material, therefore having the advantages of not producing the side effects that may occur when taking medicines for a long time, and excellent portability, therefore can be used as an adjuvant for intake.

[0031] The food composition may further contain a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any conventional carrier in the art may be used.

[0032] In addition, the above-mentioned food composition may include additional ingredients commonly used in food compositions to improve smell, taste, vision, etc. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folic acid, panthotenic acid, etc. may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu) may be included.

[0033] As an example of the food composition of the present application, it can be used as a health drink composition. In this case, similar to conventional beverages, it can contain various natural carbohydrates or sweeteners as additional ingredients. The above-mentioned natural carbohydrates can be monosaccharides, disaccharides, polysaccharides, sugar alcohols, etc. Sweeteners can use natural sweeteners such as thaumatin and stevia extract; synthetic sweeteners such as saccharin and aspartame, etc.

[0034] In addition to the above, the health drink composition may also contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectin acid, pectinates, alginic acid, alginates, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. Furthermore, it may contain fruit pulp used to prepare natural fruit juices, fruit drinks, or vegetable drinks. These ingredients may be used alone or in combination.

[0035] The term "health functional food" as used throughout this specification refers to foods prepared and processed using raw materials or ingredients that have functional properties beneficial to the human body in accordance with the Health Functional Foods Act No. 6727 of Korea. "Functionality" means that the food has a beneficial effect on the structure and function of the human body, such as regulating nutrients or exerting physiological effects.

[0036] The above-mentioned health functional food refers to a food that has a positive health maintenance or enhancement effect compared to general food, and a health supplement food refers to a food for the purpose of health supplementation. Depending on the circumstances, the terms health functional food, health food and health supplement food can be used interchangeably. Specifically, the above-mentioned health functional food refers to a food material added to beverages, tea, spices, chewing gum, biscuits, etc., or prepared in the form of encapsulation, powder, suspension, etc. When ingested, although it means a certain effect on health, unlike general medicines, food is used as a raw material, so it has the advantage of not producing side effects that may occur when taking medicines for a long time.

[0037] The term "pharmaceutical composition" as used throughout the present application can be used in the form of oral medication such as powder, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, ointments, suppositories or sterile injections according to conventional methods, but is not limited thereto.

[0038] The pharmaceutical composition according to one embodiment of the present application may be a pharmaceutical composition or a quasi-drug composition.

[0039] The term "quasi-drug" as used throughout this application is intended to refer to an item that is used for the purpose of diagnosing, treating, ameliorating, alleviating, treating, or preventing human or animal diseases and has an effect weaker than that of a pharmaceutical. For example, under the Pharmaceutical Affairs Act, a quasi-drug is an item other than a pharmaceutical, including products used to treat or prevent human or animal diseases and products that have little or no direct effect on the human body.

[0040] According to an embodiment of the present application, the above-mentioned pharmaceutical composition can be administered in a pharmaceutically effective amount, wherein "pharmaceutically effective amount" refers to an amount sufficient to treat or prevent a disease with a reasonable benefit or risk ratio applicable to medical treatment or prevention. The effective dosage standard can be determined based on the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the administration time of the composition of the present invention used, the route of administration and the excretion rate, the treatment time, the factors including the drugs used in conjunction with or simultaneously with the composition of the present invention used, and other factors well-known in the medical field. The pharmaceutical composition of the present application can be administered alone or in combination with well-known ingredients that show therapeutic effects on known intestinal diseases. Taking all the above factors into account, it is important to administer the amount that obtains the maximum effect in the minimum amount without side effects.

[0041] The pharmaceutical composition of the present application is not particularly limited thereto, but according to purpose, can be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration and other approaches. However, when administered orally, it can be administered in a non-preparation form, and because the application may be denatured or degraded because of gastric acid, therefore, for oral compositions, active agents can be coated or administered in the form of a preparation to prevent degradation in the stomach or in the form of an oral patch. In addition, the above composition can be administered by any device that can move to target cells according to the active substance.

[0042] Example 1. Screening of strains with excellent soybean protein decomposition ability

[0043] In order to select the bacterial strain that soy protein decomposition ability improves the effect excellence, prepare culture fluid with isolated soy protein, and confirm the soy protein decomposition ability of each bacterial strain.For soy protein culture fluid, 10g isolated soy protein powder and 1g glucose are dissolved in 1L distilled water, and are injected in 15mL conical tube (conical tube) with 10mL, use after sterilization and room temperature cooling.As positive control group, used the bromelain solution in the acetate buffer solution that is dissolved in pH 4.5 with 5%w / v.

[0044] The strains used for strain selection are shown in Table 1. Each strain was cultured three times at 12-hour intervals and then used for evaluation of decomposition capacity. The inoculum concentration for each culture was 0.1% v / v, and the culture temperature was adjusted to 37±3°C. After incubation, the culture broth of each strain was centrifuged (10,000 rpm, 10 minutes) to recover the cells. After washing twice with phosphate-buffered saline, the cells were dissolved in phosphate-buffered saline to a concentration of 8 log CFU / mL to prepare samples for use.

[0045] 100 μL of the bacterial sample was inoculated into 10 mL of soy protein culture medium, and the protein was quantified. After 48 hours of incubation in a 37±3°C incubator, the protein was quantified and the protein amounts before and after incubation were compared to evaluate the soy protein decomposition ability of each strain.

[0046] Protein quantification was performed using a BCA assay kit. A standard substance was prepared by dissolving 2 mg of bovine serum albumin in 1 mL of distilled water. The standard substance was then diluted with distilled water to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL, and used with distilled water (0 mg / mL) to produce different concentrations. 100 μL of each culture medium sample was diluted (10-fold) in 900 μL of distilled water and used. 20 μL of the standard substance at a concentration of 0 to 2 mg / mL and the diluted culture medium sample were dispensed into a 96-well plate. After treating each well with 160 μL of the BCA reagent provided in the kit, the plate was incubated in the dark at 37 ± 3°C for 30 minutes, and the absorbance was measured at 560 nm. A standard curve was created using the absorbance results of the different concentrations of the standard substance. The absorbance values of each culture medium were substituted into the standard curve to determine the protein amount. The protein degradation ability was expressed by subtracting the protein quantitative value after 48 hours of culture from the protein quantitative value before culture and multiplying the result by 100. In the tables of this specification, - represents 0.

[0047] Protein degradation capacity (%) = (AB) × 100

[0048] A: Protein quantitative value before culture

[0049] B: Protein quantification after 48 hours of culture

[0050] Comparison of the soy protein-degrading abilities of various strains under the same conditions revealed that not all strains could degrade soy protein, with different strains possessing varying degrading abilities. Among the strains used for evaluation, Lactobacillus acidophilus LM1060 (LP2) and Lactobacillus fermentum LM1020 (LP7) were confirmed to have higher soy protein-degrading abilities than strains corresponding to other genera. Furthermore, Lactobacillus fermentum LM1020 was found to have superior degrading abilities compared to LP6, another Lactobacillus fermentum strain from the same genus (Table 2).

[0051]

Table 1

[0052] Strains used to compare soy protein degradation capabilities

[0053] abbreviation strains source ATCC 53103 Lactobacillus rhamnosus human body LP1 Lactobacillus gasseri breast milk LP2 Lactobacillus acidophilus LM1060 Adult feces LP3 Lactobacillus plantarum Xin Qi LP4 Lactobacillus reuteri breast milk LP5 Lactobacillus reuteri Adult feces LP6 Lactobacillus fermentum cheese LP7 Lactobacillus fermentum LM1020 yeast dough

[0054]

Table 2

[0055] the difference Soy protein decomposition rate (%) Positive control group - 5% bromelain 20.42±0.50 ATCC 53103 14.40±3.27 LP1 13.96±3.62 LP2 14.41±2.08 LP3 14.20±1.96 LP4 8.86±0.63 LP5 - LP6 - LP7 17.40±0.50

[0056] Comparison of soy protein decomposition rates of various probiotics

[0057] Example 2. Confirmation of Valine, Isoleucine, and Leucine Production Capacity

[0058] A mixed composition of Lactobacillus fermentum LM1020 and Lactobacillus acidophilus LM1060 was incubated with soy protein, and the levels of valine, isoleucine, and leucine, branched-chain amino acids produced by decomposing the soy protein, were analyzed using a high-performance liquid chromatography / photodiode array detector system.

[0059] After culturing the strains three times using the same method as in Example 1, the cells were recovered and inoculated into soy protein medium as either a single strain or a mixed strain. The cultures were then incubated in an incubator at 37±3°C for 72 hours. After 72 hours, the culture broth was centrifuged (4000 rpm for 15 minutes), and the supernatant was used as an analytical sample.

[0060] 5 mL of the above analysis sample was placed in a test tube, concentrated at 110°C under a nitrogen atmosphere, and then finely ground. The sample was dissolved in 1 mL of 0.1N hydrochloric acid solution and vortexed for homogenization. The free amino acids in the concentrated sample were then extracted in an ultrasonic bath for 15 minutes. The free amino acid extract was centrifuged, and the supernatant was filtered through a filter. The free amino acids were then analyzed using a high-performance liquid chromatography / photodiode array detector system. The analysis method was as follows: 0.1% formic acid aqueous solution and 0.1% acetonitrile formic acid solution were used as the mobile phase, providing an appropriate gradient elution, and analysis was performed using an Agilent Zorbax Exlips AAA (4.6 mm ID*150 mm, 5 μm) column. After analysis, the predicted value of the synergistic effect was calculated using the Colby formula, and the actual measured value was compared with the predicted value of the synergistic effect.

[0061] As a result, when the two strains were mixed, the productivity improvement effect was confirmed in all combination ratios (CFU ratios) except for some combination ratios related to valine, compared with the case of using a single strain ( Figure 1 , Table 3).

[0062]

Table 3

[0063] Comparison of branched-chain amino acid production ability according to the mixing ratio of Lactobacillus fermentum LM1020 and Lactobacillus acidophilus LM1060

[0064]

[0065] 1) Lactobacillus fermentum LM1020 2) Lactobacillus acidophilus LM1060

[0066] Example 3. Confirmation of Threonine, Glycine, Tyrosine, and Lysine Production Capacity

[0067] After incubating a mixed composition of Lactobacillus fermentum LM1020 and Lactobacillus acidophilus LM1060 with soy protein, the free amino acid content generated by soy protein degradation was analyzed using a high-performance liquid chromatography / photodiode array detector system in the same manner as in Example 2. Based on the analysis results, the predicted synergistic effect value was calculated using the Colby equation, and the actual measured value was compared with the predicted synergistic effect value.

[0068] As a result, except for some combination ratios related to tyrosine, in all combination ratios (CFU ratios), the improvement effect of the production capacity of threonine, glycine, tyrosine, and lysine was confirmed compared with the case of using a single strain ( Figure 2 , Table 4).

[0069]

Table 4

[0070] Comparison of the production capacity of threonine, glycine, tyrosine, and alanine according to the mixing ratio of Lactobacillus fermentum LM1020 and Lactobacillus acidophilus LM1060

[0071]

[0072] 1) Lactobacillus fermentum LM1020 2) Lactobacillus acidophilus LM1060

[0073] In summary, it was confirmed that the mixed strain of the present application, or a composition containing a culture of the mixed strain, can improve protein decomposition ability and can improve the ability to produce valine, leucine, isoleucine or threonine, glycine, tyrosine and lysine as branched-chain amino acids from soybean protein.

[0074]

[0075]

Claims

1. A mixed strain, wherein: The mixed strain is a mixed strain comprising Lactobacillus fermentum LM1020 (preservation number KCCM12918P) and Lactobacillus acidophilus LM1060 (preservation number KCCM12625P), The mixed strain has an effect of improving the ability to decompose soybean protein.

2. The mixed strain according to claim 1, wherein The mixed strain has an effect of improving amino acid production ability.

3. The mixed strain according to claim 2, wherein The amino acid is selected from any one or more of the group consisting of isoleucine, leucine, threonine, glycine and lysine, The mixing ratio of Lactobacillus fermentum LM1020 to Lactobacillus acidophilus LM1060 in the mixed strain is 9:1 to 1:

9.

4. The mixed strain according to claim 3, wherein The amino acids also include valine, The mixing ratio of Lactobacillus fermentum LM1020 to Lactobacillus acidophilus LM1060 in the mixed strain is 9:1 to 6:4 or 3:7 to 1:

9.

5. The mixed strain according to claim 3, wherein The amino acids also include tyrosine, The mixing ratio of Lactobacillus fermentum LM1020 to Lactobacillus acidophilus LM1060 in the mixed strain is 9:1 to 2:

8.

6. The mixed strain according to claim 3, wherein The amino acids also include valine and tyrosine, The mixing ratio of Lactobacillus fermentum LM1020 to Lactobacillus acidophilus LM1060 in the mixed strain is 9:1 to 6:4 or 3:7 to 2:

8.

7. A food composition, wherein The food composition comprises any one or more of the mixed strain or the culture, lysate and extract thereof according to claim 1 as an effective ingredient.

8. A health functional food composition, wherein: The health functional food composition comprises any one or more of the mixed strain or the culture, lysate and extract thereof according to claim 1 as an effective ingredient.

9. A formula milk powder composition, wherein: The formula milk powder composition comprises any one or more of the mixed strain or the culture, lysate and extract thereof according to claim 1 as an active ingredient.

10. A pharmaceutical composition for preventing or improving sarcopenia, wherein: The pharmaceutical composition for preventing or improving sarcopenia comprises any one or more of the mixed strain or its culture, lysate and extract according to claim 1 as an active ingredient.

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