HUMAN-DERIVED LACTOBACILLUS FREMENTUM MG4231 OR LACTOBACILLUS FREMENTUM MG4244 STRAIN HAVING ANTI-OBESITY ACTIVITY AND COMPOSITION COMPRISING SAME
The present invention relates to human-derived Lactobacillus fermentum MG4231 or Lactobacillus fermentum MG4244 and to a pharmaceutical composition for the prevention or treatment of obesity-related diseases, containing the same as an active ingredient, and a food composition, quasi-drug composition and functional health food. The Lactobacillus fermentum MG4231 strain or MG4244 strain of the present invention exhibits anti-obesity activity that inhibits lipase enzyme activity, preadipocyte differentiation and neutral lipid accumulation, has high autoaggregation ability to thus show excellent colony formation ability on the epithelial cells of the digestive tract, and is resistant to acid and bile. Thereby, the Lactobacillus fermentum MG4231 strain or the MG4244 strain can be variously utilized in a pharmaceutical composition for the prevention and treatment of obesity-related diseases, and a food composition, quasi-drug composition and functional health food.
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The present invention relates to human-derived Lactobacillus fermentum MG4231 or Lactobacillus fermentum MG4244 and a pharmaceutical composition for the prevention or treatment of obesity-related diseases, containing the same as an active ingredient, and a food composition, quasi-drug composition and functional health food.
BACKGROUNDLactic-acid bacteria have special physiological activity, and are generally regarded as safe bacteria (GRAS). Lactic-acid bacteria are not only used in the production of various fermented foods, but are also widely used in fermented fruit and vegetable products and dairy products having functional and probiotic characteristics. Recently, as consumer demand for natural supplements to replace chemical supplements increases, lactic-acid bacteria are emerging as an alternative thereto. Probiotics are living microorganisms that have a very beneficial effect on the host animal's health and function to improve the balance of intestinal microflora and to enhance the absorption of nutrients. Furthermore, probiotics exhibit antimicrobial activity on pathogenic microorganisms in the intestinal environment. Probiotics include a variety of microorganisms, but the genera Lactobacillus and Bifidobacterium are the most common. In particular, the genus Lactobacillus is typically used in the process of fermentation of dairy products, meat, fruit, vegetables and cereal products.
Obesity is known as a major cause of heart disease, cancer, arthritis and diabetes, and despite the increasing public awareness of obesity, the number of obese patients is constantly increasing. Obesity occurs with an increase in the number of adipocytes and in the lipid content of adipocytes as a result of adipogenesis. Adipocytes play a major role in the synthesis and storage of excess calories into neutral lipids (triglycerides), and adipogenesis results in increased size and number of adipocytes and accelerated intracellular lipid accumulation.
In the past, adipose tissue was thought only to be an energy storage organ that stores excess energy in the form of triacylglycerol and releases it when needed, but in recent years, adipose tissue has come to be accepted as an important endocrine organ that regulates homeostasis of energy by secreting various adipokines such as adiponectin, leptin and resistin (Trends Endocrinol. Metab. 13:18, 2002). Therefore, understanding of the proliferation of adipocytes and the substances secreted from adipocytes and investigation of the in-vivo regulation mechanisms thereof are expected to be the foundation for understanding obesity and a variety of obesity-related diseases and developing effective therapeutic agents. Accordingly, thorough research into the regulation of adipogenesis is ongoing, and the differentiation of preadipocytes in the body is considered to be the main mechanism for increased adipocyte derivation in obese patients. The process of differentiation of preadipocytes into adipocytes has been studied using cells such as 3T3-L1, and various kinds of transcription factors, especially transcription factors known to be involved in adipogenesis, C/EBPs (CAAT enhancer binding proteins), PPARs (peroxisome proliferator activated receptors) and ADD/SREBPs (adipocyte determination and differentiation dependent factor 1/sterol response element binding proteins) are known to be expressed over time and regulate the process thereof (Bart A Jessen et al., Gene, 299, pp 95-100, 2002; Darlington et al., J. Biol. Chem., 273, pp 30057-30060, 1998; Brun R. P. et al., Curr. Opin. Cell. Biol., 8, pp 826-832, 1996). Given the stimulation of hormones such as MDI (isobutylmethylxanthine, dexamethasone and insulin), C/EBP β and δ are the first, transiently expressed, to initiate differentiation into adipocytes (Reusch J. E. et al., Mol. Cell. Biol., 20, pp 1008-1020, 2000). Only the differentiated adipocytes synthesize fatty acids and store neutral lipids (triglycerides). Therefore, the current research trend is focused on the discovery of substances that are capable of suppressing metabolic processes related to adipogenesis as a method for the prevention or treatment of obesity-related metabolic diseases. In other words, attempts have been made to treat obesity through the regulation of adipocytes based on the onset of obesity. These attempts aim at reducing lipid levels by suppressing lipid synthesis or promoting lipolysis and lipid oxidation, and at reducing the number of adipocytes by inhibiting adipogenesis, and transcription factors, proteins and adipokines known to mediate or regulate these processes are emerging as targets for the development of new obesity therapeutic agents. Known obesity therapeutic agents to date include Xenical (Roche Pharmaceuticals, Switzerland), Reductil (Eboth, USA), Exolise (Arkopharma, France), etc., and are largely classified into appetite suppressants, energy consumption accelerators, and lipid absorption inhibitors. Most obesity therapeutic agents are appetite suppressants that suppress the appetite by regulating neurotransmitters associated with the hypothalamus.
However, the conventional therapeutic agents cause side effects such as heart disease, respiratory disease, nervous system disease and the like, and are low in sustainability of the efficacy thereof, and it is thus necessary to develop more improved obesity therapeutic agents. Moreover, there are few therapeutic agents that have satisfactory therapeutic effects without side effects among the currently developed products, and the development of new obesity therapeutic agents is required.
Meanwhile, a great deal of effort has been made to reduce blood cholesterol levels using lactic-acid bacteria, which are considered safe microorganisms. Lactic-acid bacteria have been reported to have effects such as maintenance of normal intestinal flora, improvement of intestinal flora, inhibition of carcinogenesis, inhibition of colitis, and nonspecific activity of the host's immune system. In particular, strains of the genus Lactobacillus are major members of the normal microflora in the intestines of the human body, and have long been known to be important for maintaining a healthy digestive system and vaginal environment. In accordance with the U.S. Public Health Service guidelines, all Lactobacillus strains currently deposited at the American Type Culture Collection (ATCC) are classified as ‘Bo-safety Level 1’, meaning that the potential risk of causing disease in humans or animals is unknown. Although lactic-acid bacteria are known to have superior immune response regulation effects and superior anticancer and antioxidant effects through previous studies, research into the effects of Lactobacillus strains on inhibiting adipogenesis and lipid accumulation is insufficient.
SUMMARY Technical ProblemTherefore, the present inventors have conducted studies for the effects of Lactobacillus strains on inhibiting adipogenesis and lipid accumulation and have ascertained that human-derived Lactobacillus fermentum MG4231 and MG4244 have anti-obesity activity, thus culminating in the present invention.
Accordingly, an objective of the present invention is to provide a human-derived Lactobacillus fermentum MG4231 or Lactobacillus fermentum MG4244 and a pharmaceutical composition for the prevention or treatment of obesity-related diseases, containing the same as an active ingredient, and a food composition, quasi-drug composition and functional health food.
Technical SolutionIn order to accomplish the above objective, the present invention provides a human-derived Lactobacillus fermentum MG4231 strain (Accession number: KCTC 13593BP) or Lactobacillus fermentum MG4244 strain (Accession number: KCTC 13594BP).
In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of obesity-related diseases containing the above strain.
In addition, the present invention provides a food composition for the prevention or amelioration of obesity-related diseases containing the above strain.
In addition, the present invention provides a functional health food for the prevention or amelioration of obesity-related diseases containing the above strain.
In addition, the present invention provides a quasi-drug composition for the prevention or amelioration of obesity-related diseases containing the above strain.
Advantageous EffectsAccording to the present invention, a Lactobacillus fermentum MG4231 strain or MG4244 strain exhibits anti-obesity activity that inhibits lipase enzyme activity, preadipocyte differentiation and triglyceride accumulation, has high autoaggregation ability to thus show excellent colony formation ability on the epithelial cells of the digestive tract, and is resistant to acid and bile. Thereby, the Lactobacillus fermentum MG4231 strain or the MG4244 strain can be variously utilized in a pharmaceutical composition for the prevention and treatment of obesity-related diseases, and a food composition, quasi-drug composition and functional health food.
The present invention pertains to a human-derived Lactobacillus fermentum MG4231 strain (Accession number: KCTC 13593BP) or Lactobacillus fermentum MG4244 strain (Accession number: KCTC 13594BP).
Hereinafter, a detailed description will be given of the present invention.
The Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain is a novel strain having anti-obesity activity. As used herein, the term “Lactobacillus” refers to a bacterium that ferments sugars widely distributed in nature and thus acquires energy to generate a large amount of lactic acid and morphologically shows polymorphism as gram-positive non-spore bacillus. Microorganisms belonging to the genus Lactobacillus include Lactobacillus fermentum (L. fermentum), Lactobacillus plantarum (L. plantarum), Lactobacillus brevis (L. brevis), etc. The present inventors have identified the Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain having anti-lipase activity, activity of inhibiting preadipocyte differentiation or activity of inhibiting triglyceride accumulation as follows.
In order to isolate Lactobacillus strains according to the present invention, strains were isolated from the vaginas of healthy Korean women. There are numerous Lactobacillus strains in healthy vaginas and the distribution thereof is known to vary depending on the race, age and environment. Among the isolated strains, two strains having the highest anti-obesity activity were selected and identified to be Lactobacillus fermentum.
The above strain was named Lactobacillus fermentum MG4231 and was deposited with the accession number KCTC 13593BP at the Korea Culture Center of Microorganisms, and another strain was named Lactobacillus fermentum MG4244 and was deposited with the accession number KCTC 13594BP.
In an embodiment of the present invention, it was confirmed that the newly isolated Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain has an excellent anti-obesity effect.
The Lactobacillus fermentum MG4231 strain or the Lactobacillus fermentum MG4244 strain is characterized by having at least one activity selected from the group consisting of anti-lipase activity, activity of inhibiting preadipocyte differentiation and activity of inhibiting triglyceride accumulation.
Also, the strain of the present invention is characterized in that it has acid resistance and bile resistance. In an embodiment of the present invention, it was confirmed that the Lactobacillus fermentum MG4231 strain or the Lactobacillus fermentum MG4244 strain maintains high viability at a pH of 2 to 4, corresponding to strong acid conditions containing pepsin. Also, in an embodiment of the present invention, it was confirmed that the Lactobacillus fermentum MG4231 strain or the Lactobacillus fermentum MG4244 strain maintains high viability even upon treatment with bile salts containing pancreatin at a pH of 7 to 8.
The Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain according to the present invention exhibits superior autoaggregation ability, making it possible to prevent the removal of probiotics due to intestinal spasms and to effectively form colonies on the epithelial cells of the digestive tract in the intestines.
In addition, the present invention pertains to a pharmaceutical composition for the prevention or treatment of obesity-related diseases containing the above strain.
The pharmaceutical composition contains, as an active ingredient, the Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain of the present invention. The above strain is preferably contained in an amount of 0.01 to 95 parts by weight, and more preferably 1 to 80 parts by weight, based on 100 parts by weight of the pharmaceutical composition. If the amount of the strain is less than 0.01 parts by weight, dosing efficiency may decrease. On the other hand, if the amount thereof exceeds 95 parts by weight, there may be difficulties in formulation thereof.
According to the present invention, the pharmaceutical composition containing the Lactobacillus fermentum MG4231 strain or the Lactobacillus fermentum MG4244 strain as the active ingredient may be directly used as a composition that has the effect of preventing, ameliorating or treating obesity-related diseases due to inhibition of lipase enzyme activity, inhibition of preadipocyte differentiation or inhibition of triglyceride accumulation, and may also be used as an adjuvant.
The pharmaceutical composition according to the present invention may include a pharmaceutically effective amount of a Lactobacillus fermentum MG4231 strain or a Lactobacillus fermentum MG4244 strain alone, or may further include one or more pharmaceutically acceptable carriers, excipients or diluents. As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to exhibit the effect of preventing, ameliorating or treating obesity-related diseases due to, for example, inhibition of lipase enzyme activity, inhibition of preadipocyte differentiation or inhibition of triglyceride accumulation. The term “pharmaceutically acceptable” refers to a composition which is physiologically acceptable and does not cause allergic reactions such as gastrointestinal disorders, dizziness, etc., or reactions similar thereto when administered to humans.
Also, the pharmaceutical composition according to the present invention may be formulated into oral dosage forms, such as powder, granule, tablet, capsule, suspension, emulsion, syrup, and aerosol formulations, as well as formulations for external use, suppositories, and sterile injectable solutions, in accordance with typical methods. Suitable formulations known in the art are preferably those disclosed in the literature (Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton Pa.). The carriers, excipients or diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like. When the pharmaceutical composition is formulated, typical excipients or diluents such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, and the like may be used. A solid formulation for oral administration may include tablets, pills, powders, granules, capsules, and the like, and such a solid formulation may be prepared by mixing the above composition with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. An oral liquid formulation may include suspensions, solutions, emulsions, or syrups, and may include not only simple diluents, such as water or liquid paraffin, but also various excipients, for example, wetting agents, sweeteners, fragrances, preservatives and the like. A parenteral formulation may include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc. As non-aqueous solvents or suspensions, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable esters such as ethyl oleate, and the like may be used. As the base of the suppositories, Witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin and the like may be used.
The pharmaceutical composition of the present invention may be administered in a therapeutically effective amount, corresponding to the amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as determined by researchers, veterinarians, doctors or other clinicians, i.e. the amount that induces relief of the symptoms of the disease or disorder being treated. It will be apparent to those skilled in the art that the therapeutically effective amount and frequency of administration for the pharmaceutical composition of the present invention vary depending on the desired effect. Therefore, the optimal dose to be administered may be easily determined by those skilled in the art, and may be adjusted depending on various factors, such as the type of disease, the severity of disease, the amounts of the active ingredient and other ingredients in the composition, the type of formulation, and the patient's age, weight, general state of health, gender and diet, time of administration, route of administration, rate of secretion of the composition, duration of treatment and drugs used simultaneously therewith. For the desired effect, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, preferably 1 to 200 mg/kg/day, and may be administered once or several times a day.
The pharmaceutical composition of the present invention may be administered to a subject through various routes. All modes of administration may be considered, for example, oral, rectal, intravenous, intramuscular, subcutaneous, intrauterine epidural or intracerebroventricular injection.
The amount of the Lactobacillus fermentum MG4231 strain or the Lactobacillus fermentum MG4244 strain in the pharmaceutical composition according to the present invention may be appropriately selected depending on the absorbability of the active ingredient in the body, the excretion rate, and the subject's age, gender, and condition.
The pharmaceutical composition according to the present invention may be used alone, or may be used in combination with other appropriate therapeutic methods (e.g. surgery, radiation therapy, hormonal therapy, chemotherapy and the use of biological response modifiers, etc.), which are usually effective in enhancing the treatment of obesity-related diseases, depending on the choice of those skilled in the art.
The obesity-related disease of the present invention may be at least one selected from the group consisting of, for example, fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, arteriosclerosis and lipid-related metabolic syndrome, but the present invention is not limited thereto.
As used herein, the term “preventing” or “prevention” may refer to any action that inhibits or delays the onset of obesity-related diseases by administering to the subject a composition for preventing or treating obesity-related diseases according to the present invention.
As used herein, the term “treating” or “treatment” may refer to any action that alleviates or eliminates the symptoms of obesity-related diseases by administering the composition according to the present invention to a subject suspected of developing an obesity-related disease.
As used herein, the term “ameliorating” or “amelioration” may refer to any action that at least reduces the parameters associated with the condition being treated, e.g. the extent of symptoms.
As used herein, the term “subject” may refer to any animal, including a human, who has or is likely to develop an obesity-related disease.
In addition, the present invention pertains to a food composition for the prevention or amelioration of obesity-related diseases containing the above strain.
In the food composition according to the present invention, the kind of food is not particularly limited, and may include all foods in the typical sense. Unlimited examples of foods to which the above substance may be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes. When the composition is used as a food additive, the composition may be added alone or may be used in combination with other foods or food ingredients, and may be appropriately used through a typical method.
As used herein, the term “food” refers to a natural product or processed product containing one or more nutrients, and preferably indicates, as a usual meaning, the state in which it may be directly eaten through predetermined processing, and the food composition is intended to include all of foods, food additives, functional health foods and beverages.
Examples of the food to which the composition of the present invention may be added include various foods, beverages, gum, candy, tea, vitamin complexes, functional foods, and the like. Additionally, the food in the present invention may include special nutritional products (e.g., prepared dry milk, young and infant foods, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed pastes, etc.), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kinds of kimchi, sliced vegetables preserved in soy sauce or soybean paste, etc.), beverages (e.g. fruits, vegetable drinks, soymilk, fermented beverages, ice creams, etc.), natural seasonings (e.g. ramen soups, etc.), vitamin complexes, alcoholic beverages, liquors and other dietary supplements, but the present invention is not limited thereto. The foods, beverages or food additives may be prepared through typical manufacturing methods.
In particular, the present inventors have ascertained that the Lactobacillus fermentum MG4231 strain or the Lactobacillus fermentum MG4244 strain is effective at preventing, ameliorating or treating obesity-related diseases due to the inhibition of lipase enzyme activity, the inhibition of preadipocyte differentiation or the inhibition of triglyceride accumulation. Thus, the food composition may be used directly in all foods, including fermented foods.
In addition, the present invention pertains to a functional health food for the prevention or amelioration of obesity-related diseases containing the above strain.
As used herein, the term “functional health food” may refer to any food manufactured and processed by extracting, concentrating, refining, and mixing specific ingredients that are used as raw materials or are contained in food materials to support health, and also to a food that is designed and processed so that bioregulatory functions such as biological defenses, regulation of biorhythms, prevention of and recovery from diseases, etc. may be sufficiently exerted on the living body by the above ingredients, and is responsible for performing a function related to the prevention of disease or the restoration of health.
When the Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain according to the present invention is used in a functional health food, it may be added alone or may be used in combination with other foods or food ingredients, and it may be appropriately selected and used as needed. Also, the amount of the Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain that is added may be appropriately determined depending on the end use. The Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain according to the present invention is biosafe and exhibits the effect of increasing the activity in proportion to the concentration thereof, and thus it is obvious that it may be used in an appropriate amount without limitation to a specific range.
Moreover, there is no particular limitation on the types of functional health food in which the Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain according to the present invention may be used. Examples thereof may include ramen, other noodles, beverages, tea, drinks, alcoholic beverages, soups, meat, sausages, breads, chocolates, candy, confectionary, pizza, gum, dairy products including ice cream, vitamin complexes, etc. In particular, Lactobacillus fermentum MG901 or Lactobacillus plantarum MG989 according to the present invention is very effective at preventing, ameliorating or treating obesity-related diseases due to ability to resist gastric acid or bile acid in the digestive tract, intracellular adhesion and inhibition of lipase enzyme activity, inhibition of preadipocyte differentiation or inhibition of triglyceride accumulation, and is especially suitable for producing a variety of lactic-acid-bacteria fermented milk or fermented products. Examples of the fermented milk functional health food include yogurt, Calpis, cheese, butter, and the like, and examples of the fermented product may include tofu, soybean paste, fermented soybean paste, jelly, kimchi, and the like. The fermented milk or fermented product may be easily manufactured by replacing only the strain in the typical manufacturing method with the Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain according to the present invention.
Furthermore, the Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain according to the present invention may be mixed with known additives and other suitable auxiliary ingredients that may be typically contained in a functional health food, depending on the choice of those skilled in the art. The known additives also include other microorganisms that may be used in combination with the strain according to the present invention.
In addition, the present invention pertains to a quasi-drug composition for the prevention or amelioration of obesity-related diseases containing the above strain.
The strain of the present invention may be added to the quasi-drug composition for the prevention or amelioration of obesity-related diseases. When the Lactobacillus fermentum MG4231 strain or Lactobacillus fermentum MG4244 strain of the present invention is used as the quasi-drug additive, the above strain may be added alone or may be used in combination with other quasi-drug ingredients, and may be appropriately used through typical methods. The amount of the active ingredient that is mixed may be appropriately determined depending on the end use (prevention, health or therapy).
A better understanding of the present invention will be given through the following examples and preparation examples. However, these examples and preparation examples are merely set forth to illustrate the present invention but are not to be construed as limiting the present invention.
Example 1. Isolation of Strains Used and Preparation of SamplesSamples were taken using swabs from the vaginal linings of healthy women in Ewha Womans University Mokdong Hospital, refrigerated and transported to the laboratory. Kits with samples taken using swabs were immediately spread on a Rogosa SL (Difco, USA) medium and cultured at 37° C. for 48 hr under anaerobic conditions. When colonies were formed in the medium, each colony was spread on the Rogosa SL medium and the purely isolated colonies were finally selected through gram staining (positive), morphological (bacillus) observation, and biochemical characteristics (catalase negative). Final strains frozen at −70° C. with 10% (v/v) glycerol were used in the examples below.
Thereafter, the culture broth resulting from culturing the strains at 37° C. for 18 hr was centrifuged at 1,100×g at 4° C. for 3 min and then washed two times with phosphate-buffer saline (PBS, pH 7). The washed Lactobacillus strains were lyophilized, re-suspended to a concentration of 10 mg/ml with PBS, and homogenized using a sonicator (VCX 400, Sonics & Materials Inc., CT, USA). The homogenized Lactobacillus strains were centrifuged at 1,100×g at 4° C. for 15 min and were used as samples in the following examples.
Example 2. Confirmation of Strains Having Obesity Prevention and Treatment Effects 2.1 Confirmation of Strains Having Anti-Lipase ActivityIn order to select strains having superior obesity prevention and treatment effects among 221 strains obtained in Example 1, anti-lipase activity was measured. In particular, excellent anti-lipase activity is capable of inhibiting the lipid absorption in the intestines, which is effective at preventing obesity. Specifically, 14 strains having high anti-lipase activity were first selected. In order to select strains having the greatest obesity prevention and treatment effects among the 14 strains, experiments were performed to measure anti-lipase activity with porcine pancreatic lipase (Sigma, USA). The sample was diluted to a concentration of 0.1 mg/ml, placed in a plate together with a 0.167 mM p-nitrophenyl palmitate (PNP; Sigma, USA) solution, a 0.061 M Tris-HCl buffer (pH 8.5) and 0.3 mg/ml lipase, and allowed to react at 25° C. for 10 min. After the reaction, absorbance was measured at 405 nm. A control experiment was performed by replacing the sample with a solvent. Thereafter, anti-lipase activity was calculated using the following equation. The results are shown in Table 1 below.
Anti-lipase activity (%)={1−(absorbance of sample/absorbance of control)}×100
As is apparent from Table 1, the strains MG4227, MG4231 and MG4244 exhibited the greatest anti-lipase activity in that order. Thus, the anti-lipase activity of the above 4 strains was found to be 10% or more, indicating that the lipid absorption in the intestines could be inhibited, resulting in excellent prevention and treatment of obesity.
2.2 Confirmation of Strains Having Activity of Inhibiting Preadipocyte DifferentiationIn order to select strains having superior obesity prevention and treatment effects among the strains obtained in Example 1, experiments were performed to evaluate the ability to inhibit differentiation of preadipocytes into adipocytes. In order to induce differentiation of 3T3-L1 preadipocytes, the preadipocytes were seeded at 1.25×105 cells in each well of a 96-well plate. After 2 days, the medium was exchanged so that the cells were completely fused on the 4th day. A 10% FBS DMEM was treated with MDI (0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone and 1 μg/ml insulin solution), after which, in the experimental group, the medium was added with 1,000 μg/ml of the culture supernatant of the strains obtained in Example 1. As a positive control, differentiation was induced by the addition of 100 μM baicalin, which is known to be excellent in inhibiting adipogenesis. Adipogenesis was completed after 6 days, and the accumulation of lipids produced in the cells was measured using Oil Red O (Sigma, USA), which specifically reacted with lipid drops produced in the cells. After the completion of adipogenesis, the medium was removed, and the cells were washed two times with PBS and fixed with 10% formalin at 4° C. for 1 hr. The fixed cells were washed two times with 60% isopropanol (in PBS) and stained with a 0.5% Oil Red O solution for 30 min at room temperature. After the staining process, removal of the dye solution and washing two times with distilled water were performed. For quantitative determination, isopropyl alcohol was added to the well, which had been completely dried by removing distilled water therefrom, and then absorbance was measured at 520 nm. Based on the results of measured absorbance, the amount of produced lipid drops was compared with MDI-treated group, whereby the extent of inhibition of adipogenesis was analyzed. The analysis results are shown in Table 2 below.
As is apparent from Table 2, among 14 Lactobacillus strains, MG4244 exhibited the highest ability to inhibit adipogenesis of 71.90%, and MG4270, MG4261, MG5055, MG5033, MG5087 and MG4231 showed a relative lipid content of 81.40% or less. Accordingly, it was confirmed that the use of the above 7 lactic-acid bacteria was effective at inhibiting the differentiation of preadipocytes into adipocytes. In particular, considering that the baicalin-treated group as the positive control shows a relative lipid content of 81.40%, it was confirmed that the 7 strains described above showed a lower content, thereby exhibiting a high ability to inhibit adipogenesis.
2.3 Confirmation of Strains Having No CytotoxicityIn order to select strains not only having superior obesity prevention and treatment effects but also having no cytotoxicity among the strains obtained in Example 1, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay for evaluating whether 14 strains having superior obesity prevention and treatment effects in Examples 2.1 and 2.2 showed cytotoxicity on 3T3-L1 cells was performed. Here, 3T3-L1 cells used for experiments were purchased from the American Type Culture Collection (ATCC, MD, USA). The cells were cultured under conditions of supply of 5% CO2/95% air at 37° C. using a Dulbecco's modified Eagle's medium (DMEM, Invitrogen, NY, USA) containing 10% fetal bovine serum (FBS, Invitrogen, NY, USA) and 1% penicillin-streptomycin (Gibco, NY, USA). The 3T3-L1 cells cultured under the above conditions were seeded at a concentration of 16×104 cells in each well of a 96-well plate and cultured for 24 hr, after which the medium was removed. Thereafter, 100 μl of a new DMEM was added with the sample diluted at 1,000 μg/ml, cultured for 24 hr, added with 20 μl of a 5 mg/ml MTT (Sigma, USA) solution, and cultured at 37° C. for 4 hr. Thereafter, the culture supernatant was removed, 200 μl of dimethyl sulfoxide (DMSO) was added, and then absorbance was measured at 546 nm using a microplate reader (Bio-Rad Model 550; Hercules, Calif., USA) to analyze whether 14 strains were cytotoxic. The analysis results are shown in Table 3 below.
As is apparent from Table 3, all of the 14 Lactobacillus strains exhibited MTT assay results of 80 or more, indicating that treatment with a concentration of 1,000 μg/ml did not affect the viability of the cells and thus 14 Lactobacillus strains were not cytotoxic. Therefore, it was concluded that the Lactobacillus strains having superior anti-obesity activity as above are not cytotoxic, and thus highly useful in many applications.
Example 3. Identification of Strains Having Obesity Prevention and Treatment EffectsMG4231 and MG4244 strains, which are high not only in anti-lipase activity but also in activity of inhibiting differentiation of preadipocytes among the strains having superior obesity prevention and treatment effects as confirmed in Example 2, were finally selected as strains having obesity prevention and treatment effects, and experiments for identification thereof were performed. The selected strains were cultured in an MRS liquid medium at 37° C. for 24 hr and subjected to gram staining to observe morphological characteristics with a phase-contrast microscope. In order to identify the selected Lactobacillus strains, 16S rRNA gene sequencing was carried out, and gene amplification was conducted using universal rRNA gene primers (27F and 1492R), and each procedure was performed through Sol-Gent Co. (Daejeon, Korea). Thereafter, the analyzed 16S rRNA sequencing results were compared with a GenBank database using Basic Local Alignment Search Tool (BLAST) analysis (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of National Center for Biotechnology Institute (NCBI), and identified. Sequence alignment was carried out through PHYDIT 3.1, after which a phylogenetic tree was created through a neighbor-joining method using MEGA 5.1 software. The results of identification of the two strains are summarized in Table 4 below.
As is apparent from Table 4, MG4231 and MG4244 were identified to be Lactobacillus fermentum.
Example 4. Evaluation of Acid Resistance and Bile Resistance of Selected Lactobacillus StrainsSince resistance to acid and bile is regarded as important in order to serve as a probiotic, experiments were performed to evaluate the acid resistance and bile resistance of Lactobacillus fermentum MG4231 and MG4244 at a low pH (a pH of 2.0 to 4.0) and in bile. In order to evaluate the resistance of a Lactobacillus strain to artificial gastric acid and artificial bile simulating the intestinal environment, experiments were carried out through a Maragkoudakis method. The selected Lactobacillus strains were cultured for 18 hr, centrifuged at 2,000×g at 4° C. for 5 min, washed two times with PBS, suspended at a concentration of 108 CFU/ml, and used for resistance experiments. In order to evaluate the resistance to artificial gastric acid, PBS (pH 2, 3, 4) was added with pepsin (Sigma-Aldrich, USA) at a final concentration of 3 g/L, further added with the Lactobacillus suspension, and cultured at 37° C. for 3 hr, after which the number of viable cells was counted. For measurement of artificial bile salt resistance, PBS (pH 7, 8) was added with pancreatin (Sigma-Aldrich, USA) at a final concentration of 1 g/L, further added with the Lactobacillus suspension, and cultured at 37° C. for 4 hr, after which the number of viable cells was counted. The results of evaluation of acid resistance of the selected strains are shown in
As shown in
As shown in
Therefore, it was concluded that Lactobacillus fermentum MG4231 and MG4244 have high resistance to low pH and bile salt, which is the most important requirement of probiotics.
Example 5. Evaluation of Autoaggregation Ability of Selected Lactobacillus StrainWhen the strain has good autoaggregation ability, it is useful as a probiotic because it prevents the removal of probiotics due to intestinal spasms and effectively forms colonies on the epithelial cells of the digestive tract in the intestines. Experiments were performed to evaluate the autoaggregation ability of selected Lactobacillus fermentum MG4231 and MG4244. The selected Lactobacillus strains were cultured at 37° C. for 18 hr, centrifuged at 4,000×g at 4° C. for 5 min, washed two times with PBS, and re-suspended to a final concentration OD600 of 1.0, after which 5 ml of the suspension was shaken for 10 sec and allowed to stand for 5 hr, and the autoaggregation ability was evaluated. Immediately after the start of the experiment (A0) and after 5 hr (A), respectively, 0.1 ml of the supernatant was taken and mixed with 0.9 ml of PBS, after which absorbance was measured at 600 nm (A0, A), and the autoaggregation ability was calculated using the following equation. The results are shown in
Autoaggregation ability (%)=(A0−A)/A0×100
As shown in
Experiments were performed to evaluate whether the selected strains Lactobacillus fermentum MG4231 and MG4244 are able to inhibit the accumulation of neutral lipids (triglycerides). The Lactobacillus fermentum MG4231 and MG4244 strains were cultured, centrifuged and lyophilized, after which 10 mg of the lyophilized strain powder (1×108 CFU) was quantified, dissolved in 1 ml of distilled water and then filtered. The untreated group was a control, and the group treated with only MDI (0.5 mM 3-isobutyl-1-methylxanthine), which may activate adipogenesis, and the group in which differentiated 3T3-L1 adipocytes were treated with MDI and the filtered strain (LFS) in amounts of 50 and 100 μl were subjected to TG assay for measuring the amount of triglyceride produced. The results measured above were compared with those of the MDI-treated group to analyze the extent of inhibition of triglycerides. The analysis results are shown in
As shown in
[Accession number]
Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology
Accession number: KCTC13593BP
Date of deposit: 20180720
Name of Depositary Institution: Korea Research Institute of Bioscience and Biotechnology
Accession number: KCTC13594BP
Date of deposit: 20180720
Claims
1-6. (canceled)
7. A method for treating an obesity related disease, comprising:
- administering to a subject in need thereof a composition comprising an effect amount of a vaginal-lining-derived Lactobacillus fermentum MG4231 strain (Accession number: KCTC 13593BP) and at least one excipient
8. The method of claim 7, wherein the obesity-related disease is at least one selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, a cardiovascular disease, arteriosclerosis and a lipid-related metabolic syndrome.
9. The method of claim 7, wherein the composition is a pharmaceutical composition.
10. The method of claim 7, wherein the composition is a food composition.
11. The method of claim 7, wherein the composition is a functional health food composition.
12. The method of claim 7, wherein the composition is a quasi-drug composition.
13. The method of claim 7, wherein the vaginal-lining-derived Lactobacillus fermentum MG4231 strain inhibits neutral lipid accumulation, acid resistance, bile resistance or autoaggregation ability.
14. A composition comprising a vaginal-lining-derived Lactobacillus fermentum MG4231 strain (Accession number: KCTC 13593BP) and at least one excipient.
Type: Application
Filed: Sep 30, 2019
Publication Date: Mar 5, 2020
Applicant:
Inventors: Nam Soo PAEK (Seoul), Chang Ho KANG (Chungcheongbuk-do)
Application Number: 16/587,311
