Patent Application
20220135935
The present disclosure was made with the support of the Ministry of Science and ICT, Republic of Korea, under Project No. 2018R1A2B3004143, which was conducted by the Chonnam National R&BD Foundation in the research project named “Study on Nod2-mediated cell/tissue-specific defense system against pathogenic infection” under management of the National Research Foundation of Korea for the project named “Support project for mainstay researchers”, from Mar. 1, 2018 to 28 Feb. 2022.
This application claims priorities to and the benefits of Korean Patent Application Nos. 10-2019-0022670 and 10-2019-0022671 filed on 26 Feb. 2019 and Korean Patent Application No. 10-2020-0012763 filed on 3 Feb. 2020 in the Korean Intellectual Property Office, the contents of which are incorporated herein in their entireties by reference.
The present disclosure relates to a Lactobacillus sakei CVL-001 strain and an isolation method therefor and, more specifically, to a Lactobacillus sakei CVL-001 strain isolated and identified from kimchi.
In addition, the present disclosure relates to a composition comprising a Lactobacillus sakei CVL-001 strain or a culture liquid thereof for alleviating, preventing, or treating bone diseases or metabolic disease and, more specifically, to a composition comprising a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP, or a culture liquid thereof for alleviating, preventing, or treating bone diseases or metabolic diseases.
Lactic acid bacteria are abundantly found in Korean traditional fermented foods such as kimchi. While existing in symbiosis with each other in the human digestive system, lactic acid bacteria are responsible for degrading fibers and composite proteins to afford important nutrients and maintaining the gut environment at acidic pH to inhibit the proliferation of harmful bacteria such as E. coli or Chlostridium sp. and to alleviate diarrhea and constipation, and play a role in vitamin synthesis and lowering blood cholesterol levels. Particularly, strongly binding to the mucosa and epithelial cells of the gut, lactic acid bacteria make great contribution to intestinal regulation.
Also, lactic acid bacteria are known to enhance the ability of macrophages to recognize and kill harmful gut bacteria by promoting the proliferation of macrophages and to exhibit an immunostimulatory effect by promoting the secretion of immune-related materials (Gabriela peridgon et al. J of food Protection 53: 404-411, 1990: Katsumasa sato et al., Microbiol Immunol., 32(7): 689-698, 1998).
Lactobacillus spp. microorganisms, which perform homo- or heterolactic fermentation, are usually involved in fermenting dairy products and vegetables. Active research has recently been conducted into the development of Lactobacillus spp. microorganisms as probiotics, food additives, etc.
In recent years, the importance of the gut microbiota (hereinafter referred to as “GM”) for both health and disease has been intensively studied. The GM includes trillions of bacteria which collectively contain 150-fold more genes than the human genome. GM is acquired at birth and, although being a distinct entity, has clearly coevolved with the human genome and can be considered a multicellular organ that communicates with and affects its host in numerous ways.
The composition of the GM is modulated by a number of environmental factors such as diet and antibiotic treatments. Molecules produced by the gut bacteria can be both beneficial and harmful and are known to affect endocrine cells in the gut, the enteric nervous system, gut permeability, and the immune system. A perturbed microbial composition has been postulated to be involved in a range of inflammatory conditions, within and outside the gut, including Crohn's disease, ulcerative colitis, rheumatoid arthritis, multiple sclerosis, diabetes, food allergies, eczema and asthma, as well as obesity and metabolic syndrome.
Climacteric women experience various menopausal syndromes. Particularly, a drop in estrogen in women at the time of menopause causes calcium resorption from bones to decrease bone mass. The increased bone loss makes the bone porous, leading to a high risk of osteoporosis. In many causes, it is not easy for women in a menopausal period to recognize post-menopausal symptoms because it takes a significant time for the symptoms to appear after onset of menopause. According to the Korean national health statistics of the Ministry of Health and Welfare, osteoporosis prevalence in persons at 30 years or more of age has been reported to be 1% for men and 9% for women, with 9-fold prevalence in women than men.
Bone fracture caused by osteoporosis accounts for a major health concern and results in a huge economic burden on health care systems. The lifetime risk of any osteoporotic fracture is high in the western world (around 50% for women and 20% for men) and fractures are associated with significant mortality and morbidity. Cortical bone accounts for approximately 80% of the bone in the body. Many studies have shown that cortical bone is the major determinant of bone strength and thereby fracture susceptibility. Bone loss after the age of 65 is mainly due to loss in cortical bone, but not trabecular bone (Lancet, 2010, May 15; 375(9727):1729-36).
The skeleton is remodeled by bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCLs). Macrophage colony stimulating factor (MCSF) increases proliferation and survival of OCLs precursor cells as well as up-regulating expression of receptor activator of nuclear factor-κB (RANK) in OCLs. This allows RANK ligand (RANKL) to bind and start the signaling cascade that leads to OCL formation. The effect of RANKL can be inhibited by osteoprotegerin (OPG), which is a decoy receptor for RANKL.
Furthermore, metabolic diseases such as obesity, diabetes mellitus, and so on have tended to increase due to genetic and environmental factors, along with rapid economic growth and westernized dietary life. While studies for materials preventing and treating the diseases are ongoing, development of various functional foods has been required.
Obesity prevalence is increasing around the world while an interest in treating obesity has been rising due to metabolic complications caused by obesity. Anti-obesity drugs currently used are generally designed to suppress appetite by inducing satiety or to induce weight loss by lowering fat absorption. Satiety is induced by increasing a concentration of norepinephrine or serotonin at the synapses of the neurotransmitters or by stimulating serotonergic receptors or adrenergic receptors.
However, medicines developed for treatment of obesity exhibit serious adverse effects, compared to their pharmacological effects. There is therefore an urgent need for development of a substance that has a novel mechanism of action leading to an excellent anti-obesity effect with little side effects.
Through intensive and thorough research, the present inventors succeeded in isolating and identifying a Lactobacillus sakei strain from kimchi samples collected from all parts of Korea and found that the Lactobacillus sakei strain isolated from kimchi and a culture liquid thereof have the effects of inhibiting osteoclast differentiation, alleviating osteoporosis diseases, inhibiting adipocyte differentiation, and suppressing weight gain.
A purpose of the present disclosure is therefore to provide a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP.
Another purpose of the present disclosure is to provide a culture liquid of a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP.
A further purpose of the present disclosure is to provide a method for isolation of a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP, the method comprising a step of culturing a kimchi extract.
A still further purpose of the present disclosure is to provide a Lactobacillus sakei CVL-001 strain, deposited under accession number KCTC13816BP, which has the activity of alleviating, preventing, or treating a bone disease or a metabolic disease.
A still another purpose of the present disclosure is to provide a culture liquid of a Lactobacillus sakei CVL-001 strain, deposited under accession number KCTC13816BP, which has the activity of alleviating, preventing, or treating a bone disease or a metabolic disease.
A yet another purpose of the present disclosure is to provide pharmaceutical composition for prevention or treatment of a bone disease or a metabolic disease, the composition comprising a culture liquid of a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP.
A yet further purpose of the present disclosure is to provide a food composition for alleviation of a bone disease or a metabolic disease, the composition comprising a culture liquid of a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP.
An additional purpose of the present disclosure is to provide a use of a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP in alleviating, preventing, and treating a bone disease or a metabolic disease.
The present disclosure relates to a Lactobacillus sakei CVL-001 strain, an isolation method therefor, a composition comprising a Lactobacillus sakei CVL-001 strain or a culture liquid thereof for alleviating, preventing, or treating a bone disease or a metabolic disease.
The present inventors isolated strains from kimchi extracts, made a selection of Gram-positive, catalase-negative strains therefrom, and identified the selected strain as Lactobacillus sakei CVL-001. In addition, the strain or a culture liquid thereof was identified to inhibit osteoclast differentiation and have a therapeutic effect on a bone disease, with a suppression of weight gain and a decrease in blood glucose level observed in mice of high fat diet-induced obesity.
Below, a detailed description will be given of the present disclosure.
An aspect of the present disclosure pertains to a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP.
The strain may include the 16S rRNA sequence represented by SEQ ID NO: 1.
Another aspect of the present disclosure pertains to a culture liquid of a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP. The strain may include the 16S rRNA sequence represented by SEQ ID NO: 1.
Another aspect of the present disclosure pertains to a method for isolating a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP, the method comprising a step of culturing a kimchi extract.
The kimchi extract may be a kimchi-ground juice obtained by grinding kimchi and filtering the ground kimchi. For example, the kimchi extraction may be a diluted kimchi juice prepared by adding water to the kimchi-ground juice, but is not limited thereto.
The culturing step may include a first culturing step for incubating the kimchi extract; and a second culturing step for culturing a strain isolated in the first culturing step.
The first culturing step may be conducted in a medium containing 0.5 to 2% of calcium carbonate or 0.004 to 0.006% of bromocresol purple to discriminate lactic acid bacteria, for example, in a medium containing calcium carbonate, but with no limitations thereto.
The first culturing step may be conducted at 20 to 32° C. for 36 to 60 hours, for example, at 30° C. for 48 hours, but with no limitations thereto. Subsequently, colonies with a halo were picked and subjected to the second step.
The second culturing step may be a step in which lactic acid bacteria are selected from among the picked colonies and cultured. In detail, it may be a step in which selection is made of a Gram-positive, catalase-negative lactic acid bacterium from the isolated strains.
The second culturing step may be conducted at 20 to 32° C. for 12 to 36 hours, for example, at for 30° C. for 24 hours, but with no limitations thereto.
Another aspect of the present disclosure pertains to a Lactobacillus sakei CVL-001 strain, deposited under accession number KCTC13816BP, which has the activity of alleviating, preventing, or treating a bone disease. The strain may be isolated from kimchi, but with no limitations thereto.
A further aspect of the present disclosure pertains to a culture liquid of a Lactobacillus sakei CVL-001 strain, deposited under accession number KCTC13816BP, which has the activity of alleviating, preventing, or treating a bone disease.
A still further aspect of the present disclosure pertains to a pharmaceutical composition comprising a Lactobacillus sakei CVL-001 strain, deposited under accession number KCTC13816BP, or a culture liquid thereof for preventing or treating a bone disease.
The bone disease may be selected from osteoporosis, bone metastatic cancer, osteomalacia, rickets, fibrous osteitis, adynamic bone disease, metabolic disease, and periodontal disease, for example, osteoporosis, but with no limitations thereto.
The culture liquid may be a supernatant obtained by culturing the Lactobacillus sakei CVL-001 strain and removing the same, a concentrate thereof, a fraction thereof, or a lyophilisate thereof.
The culture liquid may be obtained as a result of culturing the Lactobacillus sakei CVL-001 strain for 12 to 30 hours, 12 to 24 hours, 18 to 30 hours, or 18 to 24 hours, for example, 22 to 26 hours, but with no limitations thereto. With a threshold given of 24 hours, the inhibitory activity against osteoclast differentiation is not increased in a culturing time-dependent manner when the culturing time exceeds the threshold.
For culturing the Lactobacillus sakei CVL-001 strain, Alpha-MEM medium may be utilized.
In detail, BME (Basal Media Eagle) medium, which is obtained as a result of a study on essential nutrients, inorganic salts, and vitamins necessary for cell growth, is a fundamental composition for MEM and DMEM, which are both widely used. MEM (Minimum Essential Media Eagle) medium is higher in amino acid concentration than BME and contains Earle's salts for buffering actions of CO2/NaHCO3. According to types of cells, a medium further supplemented with non-essential amino acids or a medium containing Hank's salts instead of Earle's salts may be used. Being supplemented with amino acids, vitamins, etc., Alpha MEM (MEM, Alpha modification) is useful for DNA transformation in animal cells.
The composition may contain the culture liquid at a concentration of 50 to 400, 50 to 350, 50 to 300, 50 to 250, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 150 to 400, 150 to 350, 150 to 300, 150 to 250, 180 to 400, 180 to 350, or 180 to 300 μl/ml, for example, 180 to 250 μl/ml, but without limitations thereto.
The culture liquid may have a pH of 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5, or 7.0 to 8.0, for example, a pH of 7.0 to 7.5, but is not limited thereto.
As the lactic acid bacteria grow, metabolites such as lactic acid, etc. are produced to make the culture liquid acidic, which falls within a pH range unsuitable for cell culturing. On the basis of this situation, a pH of 7.4 is the most suitable for cell culturing.
The prevention and treatment of a bone disease could be achieved by inhibiting osteoclast differentiation, but with no limitations thereto.
A still another aspect of the present disclosure pertains to a pharmaceutical composition comprising a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP or a culture liquid thereof for prevention or treatment of a metabolic disease.
The metabolic disease may be at least one selected from the group consisting of obesity, diabetes mellitus, hypertension, hyperlipidemia, cardiovascular disease, and hyperinsulinemia.
The pharmaceutical composition may comprise the Lactobacillus sakei CVL-001 strain at a concentration of 5×105 to 5×1012 CFU/ml, 5×106 to 5×1011 CFU/ml, or 5×107 to 5×1010 CFU/ml, for example, 5×108 to 5×109 CFU/ml, but with no limitations thereto.
The culture liquid may be obtained by culturing the Lactobacillus sakei CVL-001 strain for 12 to 30 hours, 12 to 24 hours, 18 to 30 hours, or 18 to 24 hours, for example, 22 to 26 hours, but with no limitations thereto. With a threshold given of 24 hours, the effect of suppressing weight gain or reducing blood glucose levels is not significantly increased in a culturing time-dependent manner when the culturing time exceeds the threshold.
The culture liquid may be a culture supernatant obtained by culturing the Lactobacillus sakei CVL-001 strain and removing the same, a concentrate thereof, a fraction thereof, or a lyophilisate thereof.
The culture liquid may have a pH of 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5, or 7.0 to 8.0, for example, a pH of 7.0 to 7.5, but is not limited thereto.
As the lactic acid bacteria grow, metabolites such as lactic acid, etc. are produced to make the culture liquid acidic, which falls within a pH range unsuitable for cell culturing. On the basis of this situation, a pH of 7.4 is the most suitable for cell culturing.
The pharmaceutical composition of the present disclosure may be used as a pharmaceutical composition comprising a pharmaceutically effective amount of the Lactobacillus sakei CVL-001 strain culture liquid and/or a pharmaceutically acceptable carrier.
As used herein, the term “pharmaceutically effective amount” refers to an amount that allows the efficacy or activity of the Lactobacillus sakei CVL-001 strain culture liquid to be sufficiently achieved.
The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present disclosure may be any one that is usually used in formulations. Examples of the carrier include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. In addition to the carriers, the pharmaceutical composition of the present disclosure may further contain a lubricant, a humectant, a sweetener, a flavorant, an emulsifier, a suspending agent, a preservative, and so on.
The pharmaceutical composition according to the present disclosure may be administered to mammals including humans via various routes. All possible routes of administration may be contemplated, including, for example, oral, dermal, intravenous, intramuscular, subcutaneous, and other routes, with preference for an oral route.
A suitable dose of the pharmaceutical composition of the present disclosure may vary depending on pharmaceutical formulation methods, administration methods, the patient's age, body weight, sex, severity of diseases, diet, administration time, administration route, an excretion rate and sensitivity. Physicians with average skill may easily determine and diagnose dosage level of medicine effective for desirably treating or preventing target disorders or diseases.
The pharmaceutical composition of the present disclosure may be formulated into a unit dose form and a multi-dose form according to methods that can be implemented by a person skilled in the art, using a pharmaceutically acceptable carrier and/or vehicle. In this regard, the formulation may be in the form of a solution in an oil or aqueous medium, a suspension or an emulsion, an extract, a pulvis, a granule, a tablet, a capsule, or a gel (e.g., hydrogel) and may further comprise a dispersant or a stabilizer.
A yet further aspect of the present disclosure pertains to a food composition comprising a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP or a culture liquid thereof for alleviation of a bone disease.
The bone disease may be selected from osteoporosis, bone metastatic cancer, osteomalacia, rickets, fibrous osteitis, adynamic bone disease, metabolic disease, and periodontal disease, for example, osteoporosis, but with no limitations thereto.
The culture liquid may be a supernatant obtained by culturing the Lactobacillus sakei CVL-001 strain and removing the same, a concentrate thereof, a fraction thereof, or a lyophilisate thereof.
The culture liquid may be obtained as a result of culturing the Lactobacillus sakei CVL-001 strain for 12 to 36 hours, 12 to 30 hours, 12 to 24 hours, 18 to 36 hours, 18 to 30 hours, or 18 to 24 hours, for example, 22 to 26 hours, but with no limitations thereto.
The composition may contain the culture liquid at a concentration of 20 to 400, 20 to 350, 20 to 300, 20 to 250, 50 to 400, 50 to 350, 50 to 300, 50 to 250, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 150 to 400, 150 to 350, 150 to 300, 150 to 250, 180 to 400, 180 to 350, or 180 to 300 μl/ml, for example, 180 to 250 μl/ml, but without limitations thereto.
The culture liquid may have a pH of 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5, or 7.0 to 8.0, for example, a pH of 7.0 to 7.5, but is not limited thereto.
The alleviation of a bone disease could be achieved by inhibiting osteoclast differentiation, but with no limitations thereto.
A yet another aspect of the present disclosure pertains to a health functional food composition comprising a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP or a culture liquid thereof for alleviation of a metabolic disease.
The metabolic disease may be at least one selected from the group consisting of obesity, diabetes mellitus, hypertension, hyperlipidemia, cardiovascular disease, and hyperinsulinemia.
The health functional food composition may comprise the Lactobacillus sakei CVL-001 strain at a concentration of 5×105 to 5×1012 CFU/ml, 5×106 to 5×1011 CFU/ml, or 5×107 to 5×1010 CFU/ml, for example, 5×108 to 5×109 CFU/ml, but with no limitations thereto.
The culture liquid may be obtained by culturing the Lactobacillus sakei CVL-001 strain for 12 to 36 hours, 12 to 30 hours, 12 to 24 hours, 18 to 36 hours, 18 to 30 hours, or 18 to 24 hours, for example, 22 to 26 hours, but with no limitations thereto.
The culture liquid may be a culture supernatant obtained by culturing the Lactobacillus sakei CVL-001 strain and removing the same, a concentrate thereof, a fraction thereof, or a lyophilisate thereof.
The culture liquid may have a pH of 6.5 to 8.5, 6.5 to 8.0, 6.5 to 7.5, 7.0 to 8.5, or 7.0 to 8.0, for example, a pH of 7.0 to 7.5, but is not limited thereto.
When used as a food additive, the food composition of the present disclosure invention may be added as it is or may be used in combination with another food or food ingredient, and can be suitably applied in conventional manners. In preparing foods or beverages, the food composition may be generally added in an amount of 15% by weight or less and preferably in an amount of 10% by weight, based on the weight of the raw materials.
No particular limitations are imparted to types of foods to which the food composition can be applied. Examples of the foods added include meats, sausages, breads, chocolate, candies, snacks, confectionery, pizza, ramen, other types of noodles, gums, dairy products including ice creams, various types of soups, beverages, teas, drinks, alcohol drinks and vitamin complexes, and in a common sense, all types of foods.
The beverages may contain various flavorants or natural carbohydrates as additional ingredients. Examples of the natural carbohydrates include monosaccharides, such as glucose and fructose, disaccharides, such as maltose and sucrose, natural sweeteners, such as dextrin and cyclodextrin, and synthetic sweeteners, such as saccharin and aspartame. The amount of the natural carbohydrates can be appropriately determined by a person skilled in the art.
In addition, the composition of the present disclosure may contain various nutrients, vitamins, electrolytes, flavorants, colorants, pectic acid or salts thereof, alginic acid or salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated drinks, etc. Moreover, the food composition of the present disclosure may contain fruit flesh for the preparation of natural fruit juices, fruit juice beverages and vegetable juices. These components may be used alone or in combination. The contents of these additives in the composition can be appropriately selected by a person skilled in the art.
The present disclosure relates to a Lactobacillus sakei CVL-001 strain and an isolation method therefor. The strain can be isolated from kimchi and, as such, can be advantageously used in a probiotic agent or a food additive.
In addition, the present disclosure relates to a composition comprising a Lactobacillus sakei CVL-001 strain or a culture liquid thereof for alleviating, preventing, or treating a bone disease. Exhibiting the effect of inhibiting osteoclast differentiation, alleviating osteoporotic diseases, and suppressing adipogenic differentiation and weight gain, the Lactobacillus sakei strain isolated kimchi and a culture liquid thereof can find advantageous applications in alleviating, preventing, or treating osteoporosis or obesity disease.
The present disclosure is concerned with a Lactobacillus sakei CVL-001, deposited under accession number KCTC13816BP, having activity of alleviating, preventing, or treating a bone disease.
A better understanding of the present disclosure may be obtained via the following examples which are set forth to illustrate, but are not to be construed as limiting the present disclosure.
Unless stated otherwise, “%”, used to indicate concentrations of particular substances, stands for (wt./wt.) % for solid/solid, (wt./vol.) % for solid/liquid, and (vol./vol.) % for liquid/liquid throughout the specification.
Napa cabbage kimchi just after being prepared was collected from all parts of Korea. While being stored at 6±0.5° C. from week 0 to week 5, the collected kimchi was used as kimchi samples for isolating lactic acid bacteria. A hand blender (Hanil Co, Korea) was used to grind 500 g of the kimchi, followed by filtration through sterile gauze. The filtrate was diluted with sterile water in a serial manner from 101 to 107 fold. Finally, the 107-fold diluted kimchi juice was spread over an agar.
The diluted kimchi juice was spread over an MRS agar (Difco Co., France) containing 2% of calcium carbonate (CaCO3) and incubated at 30° C. for 48 hours before picking up colonies with halo. The picked colonies were subjected to Gram staining (Gram stain kit, BD Co., USA) and catalase testing (Biomerieux Co., France) to tentatively select Gram-positive, catalase-negative colony as lactic acid bacteria.
The lactic acid bacterium strains thus isolated were inoculated into an MRS broth, incubated at 30° C. for 24 hours, and added with 25% (v/v) of glycerol to prepare glycerol stocks which were stored at −70° C. until use.
The finally selected lactic acid bacterium Lactobacillus sakei CVL-001 was identified by 16S rRNA sequencing. After purely isolated, the strains spread on MRS plates were delivered via refrigerated parcel service to SolGent (Daejeon, Korea) in which the strains were analyzed by 16S rRNA sequencing with a universal primer set of 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′).
The isolated strain Lactobacillus sakei CVL-001 was determined to have the 16S rRNA coding nucleotide sequence consisting of a total of 1,439 bp shown in Table 1, below.
Lactobacillus
sakei CVL-001
With respect to the nucleotide sequence obtained above, sequence similarity search was run with the Blast program (www.ncbi.nlm.nhi.gov) for the registered database (GenBank database). As a result, the strain was finally identified as Lactobacillus sakei LZ217 because of a similarity of 99% therewith.
In light of the results obtained above, that is, morphological traits, sugar metabolism ability, and 16S rRNA sequencing results, the Lactobacillus sakei CVL-001 strain with excellent mannitol productivity and viability (predominancy) in kimchi juice was identified as Lactobacillus sakei LZ217.
The isolated strain was termed Lactobacillus sakei CVL-001 and deposited with the Korean Collection for Type Culture located at 823, Shinjung-dong, Jeongeup City, Jeollabuk-do, Korea.
A culture for use in application to cells was obtained by culturing Lactobacillus sakei CVL-001 for 24 hours in alpha-MEM medium, centrifuging the cell culture, and adjusting the separated supernatant into a pH of 7.4.
Mouse macrophages were seeded at a density of 2×105 cells/well into 12-well plates and incubated for 24 hours. After the medium was changed with a medium supplemented with fetal bovine serum (hereinafter referred to as “FBS”), 1% penicillin-streptomycin (hereinafter referred to as “PS”), and macrophage colony stimulating factor (hereinafter referred to as “M-CSF”), the cells were incubated with the culture liquid (50, 100, and 200 μl/ml) for 2 hours. Then, the cells were treated with 100 ng/ml RANKL (receptor activator of nuclear factor kappa-B ligand) for 24 hours and allowed to differentiate for 6 days in the manner described above.
Subsequently, a chromogenic substrate for the cytochemical enzyme marker tartrate-resistant acid phosphatase (hereinafter referred to as “TRAP”) was added to stain the nuclei. Multinucleated cells with three or more nuclei were observed and imaged.
When applied to macrophages, RANKL binds to RANK to cause differentiation into TRAP positive cells. Stimulation of TRAP-positive cells with an inflammatory factor such as RANKL, TNF-α, etc. renders cell fusion into multinuclear TRAP positive cells.
As shown in
Examination was made of the MAPK (mitogen-activated protein kinase) and NF-κB signaling mechanism when mouse macrophages were treated with RANKL and the culture liquid. To this end, Mouse macrophages were seeded at a density of 4×105 cells/well into 12-well plates and incubated for 24 hours. After the medium was changed with a medium supplemented with FBS, 1% PS, and M-CSF, the Lactobacillus sakei CVL-001 strain culture liquid (100 μl/ml) was incubated for 2 hours. Then, the cells were treated with 100 ng/ml RANKL for 0, 5, 15, and 30 min.
After the medium was removed, proteins were extracted with a protein lysis buffer and quantitated and 30 μg of the proteins was separated in SDS-PAGE gel. The proteins were transferred onto a membrane, incubated with primary and secondary antibodies (p-jnk (Cell Signaling Technology, #9251S), p-p38 (Cell Signaling Technology, 9211S), p-ERK (Santa Cruz, sc-7383), Ikbalpha (Cell Signaling Technology, 9242S), p-p65 (Cell Signaling Technology, 3031 S)), sequentially. Color development was made using a kit (BIO-RAD, ECL solution detection kit) and measured by a device (Chemidoc).
RANKL binds to RANK to upregulate the expression of MAPK and NF-κB via TRAF6 and induce the activation of various transcription factors, leading to the promotion of osteoclast differentiation. Examination was thus made to see whether the Lactobacillus sakei culture liquid intervenes with the mechanisms to inhibit osteoclast differentiation. In the regard, phosphorylation of the MAPK proteins ERK, JNK, and p38 was analyzed by western blotting.
As a result, increased phosphorylation levels of JNK, P38, and ERK were observed 5 and 15 min after incubation with RANKL whereas the MAPK proteins were dephosphorylated by treatment with the culture liquid. In addition, inhibition was observed on both the degradation of IkB alpha and the phosphorylation of p65.
As is understood from the data of
After being treated with the Lactobacillus sakei culture liquid, the mouse macrophages were assayed for gene expression of TRAP, DC-STAMP, Cathepsin K, and NFATc1, which are genes involved in osteoclastogenesis, through real-time PCR. Bone marrow-derived macrophages (hereinafter referred to as “BMDMs”) were seeded at a density of 1×105 cells/well into 12-well plates and incubated for 24 hours. Next, the cells were treated with a FBS-free medium supplemented with 1% PS only for 1 hour and then with 100 μl/ml Lactobacillus sakei culture liquid for 2 hours, followed by incubation with RANKL for 24 hours. In this manner, the cells were subjected to differentiation for 3 days before RNA isolation from the cells with the aid of TRIzol solution. On the basis of RNA quantitation, RT premix was used to synthesize cDNA which was then amplified by real-time PCR using primers.
The primers used were designed for Mouse TRAP, DC-STAMP, Cathepsin K, and NFATc1, which were assayed in relative amounts to the control gene GAPDH.
Macrophages undergo differentiation into TRAP-positive, multinucleated osteoclasts when treated When treated with RANKL, which binds to RANK. In addition, treatment with RANKL activates NFATc1, which is a transcription factor playing an important role in differentiation into osteoclasts, and upregulates the expression of TRAP, Cathepsine K, and DC-STAMP, which are involved in osteoclastogenesis.
As can be seen in
C57BL/6 female mice (7 weeks old) were housed in plastic cages maintained at a temperature of 22±2° C. and a relative humidity of 50±10% with a photoperiod of 12-h light and 12-h dark. The mice were acclimated to the same environment for about one week before ovariectomy.
The mice were anesthetized using an intramuscular injection of Zoletil and Lumpun, after which the ovariectomy region was shaved and sterilized. A 1-cm incision was made on the skin. Then, the ovary was detected along the uterus with care to exert no injuries on other organs and ligated with a suture so as to resect the opposite ovaries. After ovariectomy, the organs were each repositioned within the abdomen and the incision was sutured with a thread. From day 10, a therapeutic substance was administered for 8 weeks.
Administration of lactic acid bacteria: After pre-culture and main culture, Lactobacillus sakei was divided into aliquots of 1×107, 1×108, and 1×109 cells/ml and orally administered.
Administration of culture liquid: After pre-culture and main culture, Lactobacillus sakei was divided into aliquots of 1×107, 1×108, and 1×109 cells/ml. The cell aliquots were 10-fold diluted and cultured in an MRS medium at 30° C. for 24 hours. The cells were settled by centrifugation, and the culture liquid supernatant was collected and adjusted into a pH of 7.4 before oral administration at a dose of 200 μl.
Test groups were divided as follows:
C57BL/6 mice; 80 heads
(1) G1: non-ovariectomized sham, 10 heads, MRS medium (control) administered
(2) G2: non-ovariectomized sham, 10 heads, culture liquid administered
(3) G3: non-ovariectomized sham, 10 heads, Lactobacillus sakei administered at a dose of 1×109 cells/ml
(4) G4: ovariectomized test group (OVX), 10 heads, MRS medium (control) administered
(5) G5: ovariectomized test group (OVX), 10 heads, culture liquid (1/4 diluted) administered
(6) G6: ovariectomized test group (OVX), 10 heads, culture liquid (1/2 diluted) administered
(7) G7: ovariectomized test group (OVX), 10 heads, culture liquid (stock) administered
(8) G8: ovariectomized test group (OVX), 10 heads, Lactobacillus sakei administered at a dose of 1×107 cells/ml
(9) G9: ovariectomized test group (OVX), 10 heads, Lactobacillus sakei administered at a dose of 1×108 cells/ml
(10) G10: ovariectomized test group (OVX), 10 heads, Lactobacillus sakei administered at a dose of 1×109 cells/ml
Bone mineral densities were measured using microcomputed tomography (micro-CT), which can provide high-definition images than simple radiography or computed tomography.
After completion of the experiment, the mouse legs were fixed with formalin, and analyzed for bone mineral density in the Laboratory Animal Center of Daegu-Gyeongbuk Medical Innovation Foundation.
Ovariectomized mice are known to decrease in bone mineral density due to estrogen inefficiency and as such, are widely used for studying osteoporosis diseases.
As can be seen in
9-1. Preparation of Lactobacillus sakei CVL-001 Culture Liquid
Lactobacillus sakei CVL-001 was cultured at a density of 1×108 CFU/ml in DMEM medium for 24 hours, followed by centrifugation. The supernatant thus obtained was adjusted into a pH of 7.4 to afford a Lactobacillus sakei CVL-001 culture liquid.
9-2. Adipose Differentiation Using Undifferentiated Preadipocyte Strain 3T3-L1
Preadipocyte 3T3-L1 purchased from ATCC (American type culture collection, USA) was maintained by passage in DMEM supplemented with 10% bovine calf serum (BCS) and 1% PS. The day at which the cells reached 100% confluence after being seeded into plates was designated day −2. While the cells were cultured for an additional two days, the cell cycle was arrested at G1 phase in all the cells cultured in the plates.
For day 0, the cells were allowed to start differentiation in the presence of IBMX (3-isobutyl-1-methylxanthine), dexamethasone, and insulin (MDI). On day 2, the medium was changed with a medium containing insulin only. From day 4, the medium was changed with 10% fetal bovine serum (FBS)-supplemented medium every two days until differentiation.
9-3. Assay for Inhibitory Activity of Lactobacillus sakei CVL-001 Culture Liquid Against Adipocyte Differentiation (Oil Red O Staining)
Adipocyte differentiation was induced when bovine calf serum (BCS) in the medium composition was substituted with FBS. 3T3-L1 cells were seeded at a density of 5×105 cells/well into 12-well plates containing a medium supplemented with 10% FBS and 1% PS. On two days after the cells reached 100% confluence, that is, on day 0, the cells were treated with the culture liquid (12.5, 25.0, and 50.0%) for 2 hours and then with the adipogenesis inducer MDI.
The culture liquid was prepared as a result of culturing L. sakei at a concentration of 1×108 cfu/ml in DMEM (High glucose)+FBS (10%)+PS (1%) medium for 24 hours, and dilution of the culture liquid was conducted with DMEM medium.
On day 2, the cells were incubated with the culture liquid (12.5, 25.0, and 50.0%) for 2 hours while the medium was changed, followed by treatment with insulin. On day 4, the cells were incubated with the culture liquid (12.5, 25.0, and 50.0%) while the medium was changed. The same procedure was repeated every two days until differentiation.
When differentiated adipocytes were observed, the medium was changed with 4% formalin to fix the cells for 10 min. For adipocyte staining, the cells were washed twice with D.W. and incubated with a mixture of 6:4 Oil red O staining: D.W. for 30 min. Washing with D.W. was followed by microscopic observation. Then, Oil red O was extracted with 100% isopropanol and quantitative analysis was performed by reading absorbance at 510 nm.
As is understood from the data of
Taken together, the data imply that the Lactobacillus sakei CVL-001 culture liquid can inhibit adipogenic differentiation.
10-1. Preparation of Live Lactobacillus sakei CVL-001
Live bacteria to be applied to mice were prepared. In brief, the bacteria were cultured for 24 hours at 30° C. in MRS agar plates, and a single colony thus formed was picked and pre-cultured in MRS broth. The pre-culturing was conducted in 5 ml of the medium at 30° C. for 24 hours while shaking at 150 rpm. Subsequently, a 10-fold dilution of the culture was amplified for 3 hours in the same conditions.
In order to keep a balance between cell counts, the cultures finally obtained were diluted in PBS until the optical density at 600 nm (OD600) reached 0.6 as measured by a spectrophotometer. The O.D. value accounts for the existence of the lactic acid bacteria at a concentration of 0.89×109 CFU/ml. With the administration of 200 μl to each mouse (1×108 CFU/mouse, 1×109 CFU/mouse) in mind, live bacterial media were prepared by centrifugation at 3,000 RPM for 15 min.
10-2. Preparation of Lactobacillus sakei CVL-001 Culture Liquid
An MRS medium in which the amplification of Lactobacillus sakei had been completed was 10-fold diluted was incubated at 30° C. for 24 hours while shaking at 150 rpm. After centrifugation at 3,000 RPM for 15 min, the supernatant was separated and adjusted into a pH of 7.4. Filtration through 0.45 μm membrane filter paper was conducted before storage at 4° C.
10-3. Assay for Anti-Obesity Activity of Lactobacillus sakei CVL-001 Strain and Culture Liquid (In Vivo)
To C57BL/6 male mice at 7 weeks of age were administered the live bacteria (single daily dose 200 μl/subject), the culture liquid (single daily dose 200 μl/subject), and a high fat diet. Subsequently, the mice were weighed every two weeks. At week 11, the mice were measured for blood sugar levels. At week 14, an autopsy was performed on the mice, after which blood was sampled and the organs were weighed.
Information on the groups used for the strain test was as follows:
G1: Control group (normal diet fed), PBS (phosphate buffered saline) orally administered (n=6)
G2: Control group, 109 L. sakei orally administered (n=6)
G3: High fat group (high fat diet fed), PBS orally administered (n=8)
G4: High fat group, 108 L. sakei orally administered (n=8)
G5: High fat group, 109 L. sakei orally administered (n=8)
Information on groups used for culture liquid testing was as follows:
G1: Control group (normal diet fed), MRS orally administered (n=6)
G2: Control group, culture liquid (stock) orally administered (n=6)
G3: High fat group (high fat diet fed), MRS orally administered (n=8)
G4: High fat group, culture liquid (1/2 diluted) orally administered (n=8)
G5: High fat group, culture liquid (stock) orally administered (n=8)
11-1. Change in Body Weight by Lactobacillus sakei CVL-001 Strain or Culture Liquid Thereof (In Vivo)
Oral administration of a high fat diet including 60% of fats is known to further increase the weight of mice than that of a normal diet. Thus, high fat diet-induced obesity animal models are widely used for studies into obesity diseases.
As can be seen in
L. sakei 109
L. sakei 108
L. sakei 109
As shown in
As is understood from data of
11-2. Fasting Blood Sugar Test in Obesity-Induced Mice after Administration of Lactobacillus sakei CVL-001 Strain and Culture Liquid
After being fasted for 12 hours from p.m. 9 before the day of experiment to a.m. 9 on the day of experiment, mice were subjected to a blood sugar test. The high fat-fed mouse animal models were measured to maintain high fasting blood sugar levels. Tests for experimental groups were conducted in the same conditions as in Example 10-3, and the groups to which the strain and the culture liquid were administered are listed in Tables 8 and 9, respectively.
As can be seen in Table 8, the high fat-fed group G3 maintained a significantly high fasting blood glucose level, compared to the normal diet-fed group G1 in this experiment. Particularly, blood glucose levels in G4 and G5 to which the Lactobacillus sakei CVL-001 strain had been administered at different doses, together with the high fat diet, were lower than that of G3 and similar to that of the normal diet-fed group G1.
As can be seen in Table 9, a significantly high fasting blood glucose level was maintained in the high fat diet-fed group G3, compared to the normal diet-fed group G1 in this experiment. Particularly, the blood glucose level in G5 to which the Lactobacillus sakei CVL-001 culture liquid stock had been fed, together with the high fat diet was observed to be low, compared to that of the culture liquid stock-administered group G3, and similar to that of the normal diet-fed group G1.
11-3. Glucose Tolerance Test in Obesity-Induced Mice after Administration of Lactobacillus sakei CVL-001 Strain and Culture Liquid
Tests for experimental groups were conducted in the same conditions as in Example 10-3, and mice were subjected to a glucose tolerance test after being fasted for 12 hours from p.m. 9 before the day of experiment to a.m. 9 on the day of experiment. A PBS solution containing 10% glucose, sterilized by 0.2 μm filtration) was intraperitoneally administered (27-gauge sterile needle) at a dose of 2 mg (glucose/g. volume (μl)=body weight (g)×20) to each mouse. The mice were housed again in cages and measured for blood glucose level at time intervals of 0, 30, 60, 90, and 120 min. For the glucose test, blood was taken from tails. Measurements are given in Tables 10 and 11 and
As shown in Table 10 and
L. sakei 10{circumflex over ( )}9
L.sakei 10{circumflex over ( )}8
L. sakei 10{circumflex over ( )}9
As can be seen in Table 11 and
11-4. Insulin Tolerance Test in Obesity-Induced Mice after Administration of Lactobacillus sakei CVL-001 Strain and Culture Liquid
Tests for experimental groups were conducted in the same conditions as in Example 10-3, and mice were subjected to an insulin tolerance test after being fasted for 4 hours from a.m. 9 to p.m. 1 on the day of experiment. To each mouse, 2 mg/ml insulin (in D.W. adjusted into a pH of 3 by diluted HCl) was intraperitoneally administered (27-gauge sterile needle) at a dose of 1 U (0.03846 mg/kg. Volume (ul)=Body weight (g)×20). The mice were housed again in cages and measured for blood glucose level at time intervals of 0, 30, 60, 90, and 120 min. For the glucose test, blood was taken from tails.
L. sakei sup
L. sakei sup(1/2)
L. sakei sup
As shown in Table 12 and
sakei 10{circumflex over ( )}9
L. sakei 10{circumflex over ( )}8
sakei 10{circumflex over ( )}9
As can be seen in Table 13 and
The present disclosure relates to a Lactobacillus sakei(Lactobacillus sakei) CVL-001 strain and an isolation method therefor and, more specifically, to a Lactobacillus sakei CVL-001 strain isolated and identified from kimchi.
In addition, the present disclosure relates to a composition comprising the Lactobacillus sakei CVL-001 strain or a culture liquid thereof for alleviation, prevention, or treatment of a bone disease and, more specifically, to a composition comprising a Lactobacillus sakei CVL-001 strain deposited under accession number KCTC13816BP or a culture liquid thereof for alleviation, prevention, or treatment of a bone disease.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2019-0022670 | Feb 2019 | KR | national |
| 10-2019-0022671 | Feb 2019 | KR | national |
| 10-2020-0012763 | Feb 2020 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2020/002612 | 2/24/2020 | WO | 00 |
