Probiotic composition having effect of improving ability to degrade soybean protein and amino acid production activity

Abstract

The effects of the present invention include the effect of improving the ability to degrade soybean protein, and production activity of branched-chain amino acids such as valine, isoleucine, and leucine, as well as production activity of other amino acids including threonine, glycine, tyrosine, and lysine. Thus, effects of sarcopenia prevention and antioxidant activity can be obtained, and the effects of maintaining protein metabolism balance and ameliorating sarcopenia through muscle synthesis can also be obtained. In addition, compared to when each of the strains used in a strain mixture was used alone, the strain mixture exhibited improved autoaggregation, hydrophobicity, and adhesion to the intestines. Thus, when the strain mixture of the present invention is ingested, the corresponding strains can remain in the intestines for a long period of time.

Description

Probiotic composition with improved soybean protein decomposition and amino acid production ability

[0001] The present invention relates to a mixed strain composition having soybean protein decomposition ability and amino acid production effect, and relates to a mixed strain of Rimosylactobacillus fermentum LM1020 (KCCM12918P) and Lactobacillus acidophilus LM1060 (KCCM12625P) and a composition containing the same as an effective ingredient.

[0002] The loss of muscle mass with age and the rise in obesity due to Westernized eating habits and reduced activity have significant health implications. Protein intake helps maintain muscle mass while resisting age-related muscle loss. Adequate protein intake across all age groups is known to reduce the risk of diseases associated with sarcopenia and obesity.

[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 grains. Among animal protein sources, milk protein has the advantage of having an excellent composition of essential amino acids such as leucine, isoleucine, and methionine. Whey protein, made by concentrating whey, a liquid byproduct separated from milk during the cheese or casein production process, has high bioavailability and is quickly absorbed, making it a popular protein supplement. However, whey protein is low in dietary fiber and high in fat and cholesterol, so caution is advised when consuming it for weight management. Furthermore, because it contains lactose, individuals with lactose intolerance may lack the enzyme lactase, which breaks down lactose. This can lead to gastrointestinal problems such as abdominal pain, diarrhea, and vomiting after consuming whey protein.

[0004] Recently, as eco-friendly and sustainable eating habits have gained global attention, the development of alternative foods to animal-based ingredients is actively underway, and interest in plant-based proteins is also increasing.

[0005] As an example of a probiotic composition having protein decomposition ability that has been developed in the past, there is a food composition and health functional food (Publication No. 10-2023-0040252) containing Lactobacillus casei IDCC 3451 having protein decomposition ability, but development and research on a composition that can complement the shortcomings when consumed together with the decomposition of plant-derived proteins is still necessary.

[0006] The object of the present invention is to provide a mixed strain having an effect of improving soybean protein decomposition ability or amino acid production ability.

[0007] The present invention provides a mixed strain comprising Lactobacillus fermentum LM1020 (accession number KCCM12918P) and Lactobacillus acidophilus LM1060 (accession number KCCM12625P), wherein the mixed strain has an effect of improving soybean protein decomposition ability.

[0008] In addition, the present invention provides a food composition, a health functional food composition, a formulated milk composition, and a pharmaceutical composition for preventing or improving sarcopenia, which contain at least one of the above mixed strains or their cultures, lysates, and extracts as an active ingredient.

[0009] The effects of the present invention include improving soybean protein decomposition ability and preventing or improving sarcopenia.

[0010] Figure 1 shows the results of confirming the valine, isoleucine, and leucine production ability of the mixed strain according to the present invention according to Example 2 at different mixing ratios.

[0011] Figure 2 shows the results of confirming the threonine, glycine, tyrosine, and lysine production ability of the mixed strain according to the present invention according to Example 3.

[0012] 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.

[0013] Throughout this specification, whenever a part is said to 'include' a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0014] In this specification, 'mixing ratio' means 'Rimoshi Lactobacillus fermentum LM1020: Lactobacillus acidophilus LM1060'.

[0015] Generally, strains used as probiotics are expected to have the same or similar effects, whether they are the strain itself, strain culture, strain lysate, or strain extract.

[0016] Here, we explain the soy protein used in this specification. Among plant-based proteins, soy protein has various physiologically active components, which are beneficial in preventing muscle loss. It is also rich in dietary fiber and has lower saturated fat and carbohydrate content than animal-based proteins, making it effective for weight management. Therefore, it is widely used as a supplement among plant-based protein sources. Furthermore, soy protein has a higher content of certain amino acids, such as arginine, phenylalanine, and tryptophan, compared to whey protein, making it essential for muscle tissue production. Regular intake is known to help improve cholesterol levels by reducing low-density lipoprotein (LDL) levels and increasing high-density lipoprotein (HDL) levels. Furthermore, it is known to be less expensive than other plant-based proteins, such as pea protein, and to be free of the off-flavor characteristic of plant-based proteins.

[0017] Dietary proteins, including soy protein, are high-molecular-weight substances and must be broken down into low-molecular-weight substances after ingestion. Within the body, various digestive enzymes, such as pepsin in the stomach and trypsin and chymotrypsin in the small intestine, are involved in breaking down the peptide chains of proteins, ultimately converting them to amino acids that are absorbed from the villi of the small intestine. During this process, enzymes secreted by intestinal microorganisms present in the digestive tract also affect the breakdown and absorption of proteins. Since intestinal microorganisms can secrete enzymes that humans cannot synthesize, the types and content of amino acids produced through the breakdown of dietary proteins can vary significantly depending on the composition of the intestinal microflora and the intake of probiotics.

[0018] Among the amino acids produced after protein breakdown, valine, isoleucine, and leucine, which belong to branched-chain amino acids (BCAA), are essential amino acids for maintaining normal protein metabolism balance and muscle synthesis. They have the advantage of reducing muscle damage caused by exercise and promoting muscle synthesis. In particular, leucine is an amino acid that can help improve sarcopenia in the elderly population by playing a signaling role in the muscle biosynthesis pathway.

[0019] In addition to branched-chain amino acids, certain amino acids can increase muscle building and exercise performance, such as glycine, a component of creatine that helps with exercise performance; threonine, which contributes to skeletal muscle protein synthesis; tyrosine, which can help improve exercise performance; and lysine, which can help with skeletal muscle growth and protein breakdown inhibition.

[0020] Meanwhile, soy protein generally has a lower amino acid content, including branched-chain amino acids, than whey protein, and its absorption rate is also slower. Despite its widespread use as a plant-based dietary protein, soy protein is currently at a disadvantage compared to whey protein in promoting immediate muscle regeneration and preventing or improving sarcopenia.

[0021] Therefore, if we can promote the digestion and breakdown of soy protein and improve the ability to produce amino acids (branched-chain amino acids, etc.) from ingested soy protein, it is expected that we will be able to obtain effects such as muscle regeneration from ingesting soy protein just as much as from whey protein.

[0022] Accordingly, the present inventors developed a mixed strain invention including Lactobacillus fermentum LM1020 (Accession No. KCCM12918P) and Lactobacillus acidophilus LM1060 (Accession No. KCCM12625P) that can improve the disadvantages of consuming soy protein, which has a low digestion and absorption rate compared to whey protein and a relatively low content of branched-chain amino acids.

[0023] Hereinafter, 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 drawings and embodiments.

[0024] The present invention provides a mixed strain comprising Lactobacillus fermentum LM1020 (accession number KCCM12918P) and Lactobacillus acidophilus LM1060 (accession number KCCM12625P), wherein the mixed strain has an effect of improving soybean protein decomposition ability.

[0025] In addition, the present invention provides a composition comprising various forms of the above mixed strains as an active ingredient, wherein the various forms include at least one of the mixed strains or a culture, a lysate, and an extract thereof, and the composition includes a food composition, a health functional food composition, a formulated milk composition, and a pharmaceutical composition for preventing or improving sarcopenia.

[0026] The term 'food' 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 conventional foods.

[0027] 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. In addition, the food formulation 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. In addition, it is highly portable, so it can be consumed as a supplement.

[0028] The above food composition may additionally include a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the technical field of the present invention may be used.

[0029] Additionally, the food composition may include additional ingredients commonly used in food compositions to improve odor, taste, appearance, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), etc.

[0030] As an example, the food composition of the present invention can be used as a health beverage composition, and in this case, various natural carbohydrates or sweeteners, etc., can be contained as additional ingredients, like regular beverages. The above natural carbohydrates can be monosaccharides, disaccharides, polysaccharides, sugar alcohols, etc. The sweetener can be a natural sweetener such as thaumatin or stevia extract, or a synthetic sweetener such as saccharin or aspartame.

[0031] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. Furthermore, the composition may contain fruit pulp for the production of natural fruit juice, fruit juice beverages, or vegetable beverages. These ingredients may be used independently or in combination.

[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 in accordance with the Health Functional Food Act ('HEALTH FUNCTIONAL FOODS ACT' of Korea No. 6727), and 'functionality' means 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 above health functional food refers to a food that has a more active health maintenance or promotion effect than general food, and health supplement food refers to a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and health supplement may be used interchangeably. Specifically, the above health functional food refers to a food that is prepared by adding the present invention to food materials such as beverages, tea, spices, gum, and confectionery, or by manufacturing it in the form of capsules, powder, suspension, etc., and means that when consumed, it brings about a specific health effect. However, unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs because it uses food as a raw material.

[0034] The term 'pharmaceutical composition' used throughout the present specification may be used in the form of oral medications such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, ointment, suppository, or sterile injection according to conventional methods, but is not limited thereto.

[0035] A pharmaceutical composition according to one embodiment of the present invention may be a pharmaceutical composition or a quasi-drug composition.

[0036] The term 'quasi-drug' used throughout this specification refers to products that have a milder effect than pharmaceutical products among products used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases in humans or animals. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products other than those used for pharmaceutical purposes, and include products used for treating or preventing diseases in humans or animals, and products that have a mild effect on the human body or do not act directly.

[0037] In one embodiment of the present invention, the pharmaceutical composition may be administered in a pharmaceutically effective amount, wherein the 'pharmaceutically effective amount' means an amount sufficient to treat or prevent a disease at a reasonable benefit-to-risk ratio applicable to medical treatment or prevention. The level of the effective amount may be determined according to factors including 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 time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, drugs used in combination 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 invention may be administered alone or in combination with a component known to exhibit a therapeutic effect on a known intestinal disease. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without side effects.

[0038] The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered via routes such as intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, transdermal patch administration, oral administration, intranasal administration, intrapulmonary administration, and rectal administration, depending on the intended purpose. However, when administered orally, it may be administered in an unformulated form, and since the present invention may be denatured or decomposed by gastric acid, the oral composition may be administered orally in a form that coats the active agent or is formulated to protect it from decomposition in the stomach, or in the form of an oral patch. In addition, the composition may be administered by any device that allows the active agent to move to the target cell.

[0039] Example 1. Selection of strains with excellent soybean protein decomposition ability

[0040] To select strains with excellent soy protein degradation ability, a culture medium was prepared using isolated soy protein, and the soy protein degradation ability of each strain was confirmed. The soy protein culture medium was prepared by dissolving 10 g of isolated soy protein powder and 1 g of glucose in 1 L of distilled water, dispensing 10 mL each into 15 mL conical tubes, sterilizing, and cooling to room temperature before use. A 5% w / v bromelain solution in acetate buffer (pH 4.5) was used as a positive control.

[0041] The strains used for strain selection are as shown in Table 1, and each strain was cultured three times at 12-hour intervals and then used for the degradation evaluation. The inoculum concentration for each culture was 0.1% v / v, and the strain culture temperature was adjusted to 37±3℃. After culture, the culture solution of each strain was centrifuged (10,000 rpm, 10 min) to recover the cells, and after washing twice with phosphate-buffered saline solution, the cells were resuspended in phosphate-buffered saline solution to 8 log CFU / mL to prepare samples and use.

[0042] After inoculating 100 μL of the above bacterial cell sample into 10 mL of soybean protein culture medium, the protein was quantified and cultured in an incubator at 37±3℃ for 48 hours. The protein was quantified and the protein amount before and after culture was compared to evaluate the soybean protein decomposition ability of each strain.

[0043] Protein quantification was performed using a BCA assay kit. 2 mg of bovine serum albumin was dissolved in 1 mL of distilled water to prepare a standard, which was then diluted with distilled water to 1 mg / mL, 0.5 mg / mL, 0.25 mg / mL, and 0.125 mg / mL. These were used as standard materials at various concentrations, along with distilled water (0 mg / mL). 100 μL of culture samples were collected and diluted (10-fold) in 900 μL of distilled water. 20 μL of the standard samples and diluted culture samples at concentrations of 0 to 2 mg / mL were dispensed into 96-well plates, and 160 μL of the BCA reagent in the kit were added to each well. After incubating in the dark for 30 minutes in an incubator at 37±3°C, the absorbance was measured at 560 mm. A standard curve was created using the absorbance results for each concentration of the standard sample, and the absorbance values ​​of each culture were entered into the standard curve to measure the amount of protein. Protein degradation capacity was expressed as the value obtained by subtracting the protein quantification value after 48 hours of incubation from the protein quantification value before incubation and multiplying the result by 100. Hereinafter, in the tables of this specification, - means 0.

[0044] Protein decomposition (%) = (AB)x100

[0045] A: Protein quantification before culture

[0046] B: Protein quantification after 48 hours of culture

[0047] When comparing the soy protein decomposition ability of various strains under the same conditions, not all strains could decompose soy protein, and the decomposition ability varied by strain. Among the strains used in the evaluation, the soy protein decomposition ability of the LP2 Lactobacillus acidophilus LM1060 strain and the LP7 Rimosylactobacillus fermentum LM1020 strain was confirmed to be higher than that of strains belonging to other genera, and in particular, the Rimosylactobacillus fermentum LM1020 strain was confirmed to have a superior decomposition ability than that of another Rimosylactobacillus fermentum strain in the same genus, LP6 (Table 2).

[0048] Strains used for comparison of soy protein decomposition ability Abbreviation Strain Origin ATCC 53103 Lacticazei Bacillus rhamnosus Human LP1 Lactobacillus gasseri Breast milk LP2 Lactobacillus acidophilus LM1060 Adult feces LP3 Lactibacillus plantarum Kimchi LP4 Limosi Lactobacillus reuteri Breast milk LP5 Limosi Lactobacillus reuteri Adult feces LP6 Limosi Lactobacillus fermentum Cheese LP7 Limosi Lactobacillus fermentum LM1020 Fermented dough

[0049] Soybean protein decomposition rate (%) Positive control - 5% Bromelain 20.42±0.50 ATCC 5310 314.40±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

[0050] Comparison of soy protein decomposition rates of each probiotic

[0051] Example 2. Confirmation of valine, isoleucine, and leucine production ability

[0052] A mixed composition of Limosylactobacillus fermentum LM1020 and Lactobacillus acidophilus LM1060 was cultured with soy protein, and the changes in the content of valine, isoleucine, and leucine, which are branched-chain amino acids produced by decomposing soy protein, were analyzed using a high-performance liquid chromatograph / photodiode array detector system.

[0053] The strains were cultured three times in the same manner as in Example 1, and then the cells were harvested and inoculated into soy protein medium as single or mixed strains. The cells were then cultured in an incubator at 37±3°C for 72 hours, and after 72 hours, the culture solution was centrifuged (4,000 rpm, 15 minutes) and the supernatant was used as the analysis sample.

[0054] 5 mL of the above analysis sample was placed in a test tube, concentrated at 110℃ under a nitrogen atmosphere, finely ground, dissolved in 1 mL of 0.1 N hydrochloric acid aqueous solution, and homogenized by vortex stirring. After that, free amino acids in the concentrated sample were extracted for 15 minutes in an ultrasonic bath. The free amino acid extract was centrifuged, and the supernatant was filtered. The free amino acids were analyzed using a high-performance liquid chromatograph / photodiode array detector system. The analysis method was performed using a 0.1% formic acid aqueous solution and a 0.1% formic acid acetonitrile solution as the mobile phase and an appropriate gradient elution was performed, and the column used was Agilent's Zorbax Exlips AAA (4.6 mm ID * 150 mm, 5 μm). After analysis, the synergistic effect prediction value was calculated using the Colby formula, and the actual measured value was compared with the predicted synergistic effect value.

[0055] As a result, when the two strains were mixed, an improvement in productivity was confirmed compared to when a single strain was used in all combination ratios (CFU ratios), except for some combination ratios regarding valine (Fig. 1, Table 3).

[0056] Comparison of branched-chain amino acid production capacity according to the mixing ratio of Limosylactobacillus fermentum LM1020 and Lactobacillus acidophilus LM1060A 1) B 2)Measured value (μg / mL) Predicted value (μg / mL) ValineIsoleucineLeucineTotalValineIsoleucineLeucineTotal100%0%24.922.417.765.0----90%10%72.6188.3127.2388.024.322.517.264.080%20%91.0302.9183.1577.023.822.716.863.370%30%49.0231.0134.4414.323.423.016.562.960%40%63.5262.0167.4492.823.223.416.262.950%50% 0.0210.0115.9325.923.024.016.063.140%60%7.9345.8165.1590.223.024.715.963.630%70%60.5278.4131.4470.223.125.515.864.420%80%51.8260.5129.2441.523.426.515.765.610%90%56.5243.2131.4431.023.727.615.767.00%100%24.228.815.868.8----

[0057] 1) Rimosylactobacillus fermentum LM1020 2) Lactobacillus acidophilus LM1060

[0058] Example 3. Confirmation of threonine, glycine, tyrosine, and lysine production ability

[0059] After culturing a mixed composition of Limosylactobacillus fermentum LM1020 and Lactobacillus acidophilus LM1060 with soy protein, the change in the content of free amino acids produced by decomposing soy protein was analyzed using a high-performance liquid chromatograph / photodiode array detector system in the same manner as in Example 2. The analysis results were used to calculate a predicted synergy effect using the Colby formula, and then the actual measured value was compared with the predicted synergy effect.

[0060] As a result, we confirmed the improvement in threonine, glycine, tyrosine, and lysine production compared to when a single strain was used in all combination ratios (CFU ratios), except for some combination ratios for tyrosine (Fig. 2, Table 4).

[0061] Comparison of threonine, glycine, tyrosine, and alanine production according to the mixing ratio of Limosylactobacillus fermentum LM1020 and Lactobacillus acidophilus LM1060 A 1) B 2) Measured value (μg / mL)Predicted value (μg / mL)ThreonineGlycineTyrosineLysineThreonineGlycineTyrosineLysine100%0%12.25.27.217.7----90%10%66.0172.226.9203.411.95.17.217.280%20%65.5151.632.1232.411.65.07.316.870%30%52.7134.822.2182.511.45.07.316.560%40%60.6149.224.4211.111.24.97.316.25 0%50%47.1127.611.9175.811.04.87.416.040%60%72.1162.534.3254.010.84.87.515.930%70%52.5128.324.2207.910.74.77.515.820%80%46.8122.816.8209.010.64.77.615.710%90%51.1132.70.0196.510.54.67.715.70%100%10.54.67.815.8----

[0062] 1) Rimosylactobacillus fermentum LM1020 2) Lactobacillus acidophilus LM1060

[0063] In summary, it was confirmed that the mixed strain of the present invention or a composition including a culture of the mixed strain can improve protein decomposition ability and improve the ability to produce branched-chain amino acids such as valine, leucine, isoleucine, or threonine, glycine, tyrosine, and lysine from soybean protein.

[0064]

[0065]

Claims

1. In a mixed strain containing Limosylactobacillus fermentum LM1020 (accession number KCCM12918P) and Lactobacillus acidophilus LM1060 (accession number KCCM12625P), The above mixed strain is a mixed strain having an effect of improving soybean protein decomposition ability.

2. In paragraph 1, The above mixed strain is a mixed strain having an effect of improving amino acid production ability.

3. In paragraph 2, The above amino acid is at least one selected from the group consisting of isoleucine, leucine, threonine, glycine, and lysine, A mixed strain, wherein the mixing ratio of the above mixed strains, Rimosylactobacillus fermentum LM1020: Lactobacillus acidophilus LM1060, is 9:1 to 1:

9.

4. In paragraph 3, The above amino acids additionally include valine, A mixed strain, wherein the mixing ratio of the above mixed strains, Rimosylactobacillus fermentum LM1020: Lactobacillus acidophilus LM1060, is 9:1 to 6:4 or 3:7 to 1:

9.

5. In paragraph 3, The above amino acids additionally include tyrosine, A mixed strain, wherein the mixing ratio of the above mixed strains, Rimosylactobacillus fermentum LM1020: Lactobacillus acidophilus LM1060, is 9:1 to 2:

8.

6. In paragraph 3, The above amino acids additionally include valine and tyrosine. A mixed strain, wherein the mixing ratio of the above mixed strains, Rimosylactobacillus fermentum LM1020: Lactobacillus acidophilus LM1060, is 9:1 to 6:4 or 3:7 to 2:

8.

7. A food composition containing at least one of the mixed strains of paragraph 1 or their cultures, fragments, or extracts as an effective ingredient.

8. A health functional food composition containing at least one of the mixed strains of paragraph 1 or their cultures, fragments, or extracts as an effective ingredient.

9. A formulated milk composition containing at least one of the mixed strains of paragraph 1 or their cultures, fragments, or extracts as an effective ingredient.

10. A pharmaceutical composition for preventing or improving muscle atrophy, comprising at least one of the mixed strains of paragraph 1 or their cultures, fragments, or extracts as an effective ingredient.

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