KR101972236B1 - New Lactobacillus plantarum isolated from KIMCHI and uses thereof - Google Patents

Abstract

The present invention relates to a novel Lactobacillus isolated from kimchi, and more particularly, to a novel Lactobacillus plantarum HY-08 having activity of preventing dementia or improving cognitive function, and thus can be effectively utilized as a pharmaceutical composition for preventing dementia and as health food for improving cognitive function.

Description

Lactobacillus plantarum isolated from Kimchi and its use (KIMCHI and uses thereof)

The present invention relates to a novel Lactobacillus isolated from kimchi, and more particularly to a novel Lactobacillus plantarum HY-08 having activity for preventing dementia or improving cognitive function.

Probiotics generally refers to foods that contain safe microorganisms that are administered to humans and animals and that do not harm the host and exhibit a variety of health-promoting effects. The market for probiotics using them is worldwide It is widely formed and the size of the market is growing every year. Lactobacillus , which is represented by probiotic , is a food industry that includes fermented dairy products such as cheese, fermented milk, and lactic acid bacteria, and research on probiotics that produce bacteriocin is widely used as a natural food preservative and as a substitute for feed antibiotics It is an industrially important microorganism being applied.

However, lactic acid bacteria having such functionalities used in Korea import most fermentation strains from foreign countries, and it is a fact that there is a limit to the use of products using these common lactic acid bacteria. In addition, depending on the type of lactic acid bacteria, not only the intrinsic activity determined by fermentation of the natural product is shown but the activity specific to each lactic acid bacterium has a specific activity, so that it can be used for food or can be used for raw materials of cosmetics. This other is also true.

Therefore, in order to isolate the lactic acid bacteria that are suitable for Korean market, the present inventors have found lactic acid bacteria Lactobacillus plantarum HY-08 And Lactobacillus plantarum HY-08 was found to be effective in preventing dementia and improving cognitive function.

Korean Patent No. KR10-1476236

It is an object of the present invention to provide a lactic acid bacterium having an effect of preventing or treating dementia.

The present invention also provides a composition for preventing or treating dementia, which comprises the lactic acid bacterium.

In order to achieve the above object, the present invention provides Lactobacillus plantarum HY-08 isolated from kimchi.

In addition, the present invention provides a pharmaceutical composition for preventing or treating dementia comprising Lactobacillus plantarum HY-08 as an active ingredient.

In addition, the present invention provides a health functional food for preventing or cognitive improvement of dementia comprising Lactobacillus plantarum HY-08 as an active ingredient.

Since Lactobacillus plantarum HY-08 of the present invention has been found to be effective in preventing dementia and improving cognitive function, it can be usefully used as a pharmaceutical composition for preventing dementia and a health food for improving cognitive function.

FIG. 1 shows the results of 16S rRNA analysis of Lactobacillus plantarum HY-08, which was examined in the Korean Microorganism Preservation Center.
Figure 2 shows the phylogenetic location of Lactobacillus plantarum HY-08, which was tested in the Korean Microorganism Preservation Center.
FIG. 3 is a diagram showing the morphological appearance of lactic acid bacteria by SEM photograph of Lactobacillus plantarum HY-08. FIG.
FIG. 4 shows experimental results showing the cell concentration of Lactobacillus plantarum HY-08 over time using the MRS medium.
Fig. 5 shows the results of an underwater labyrinth of Lactobacillus plantarum HY-08.
FIG. 6 shows the result of manual avoidance experiment of Lactobacillus plantarum HY-08.

Hereinafter, the present invention will be described in detail.

Lactobacillus plantarum HY-08 (KCCM12103P) of the present invention is a novel microorganism isolated from kimchi.

Lactobacillus plantarum HY-08 of the present invention belongs to Lactobacillus in molecular systematic and biochemical characterization based on the 16s rDNA base sequence, and is a strain belonging to Lactobacillus plantarum subsp. plantarum ACGZ01000098 has a low homology of about 64% and has been identified as a novel lactic acid bacterium. It has been deposited with KCCM12103P on September 01, 2017 at the Korean Microorganism Conservation Center, an international depository.

The Lactobacillus plantarum HY-08 of the present invention is a lactic acid bacterium which is effective for fermentation and functional activity of food among lactic acid bacteria derived from kimchi. It can be used as a functional food composition because it can be edible and has excellent antioxidant and memory improving activity Therefore, it can be used as a pharmaceutical composition for preventing or treating phlegm and as a health functional food for improving cognitive function.

The present invention provides a pharmaceutical composition for preventing or treating dementia comprising Lactobacillus plantarum HY-08 (KCCM11291P) as an active ingredient.

The dementia may include Alzheimer type dementia, cerebrovascular dementia, Pick's disease, Kruszfeld-Jacob disease, dementia caused by head injury, and Parkinson's disease.

The composition of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the preparation of pharmaceutical compositions.

The composition of the present invention can be administered orally or parenterally, and it is preferable to select an external or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection, But is not limited thereto.

The composition of the present invention can be formulated into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions according to conventional methods have. Examples of carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose ), Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . The non-aqueous solutions include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, etc. Examples of non-aqueous solutions and suspensions include vegetable oils such as propylene glycol, polyethylene glycol, , Injectable esters such as ethyl oleate, and the like can be used. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the composition is preferably administered at a dose of 0.0001 to 1 g / kg per day, preferably 0.001 to 200 mg / kg per day, but is not limited thereto. The above administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

Further, the present invention provides a health functional food for preventing or cognitive improvement of dementia comprising Lactobacillus plantarum HY-08 (KCCM11291P) as an active ingredient.

Lactobacillus plantarum HY-08 of the present invention is excellent in activity for protection of brain cells and exhibits an improving effect in experiments of underwater maze and passive avoidance, and thus is usefully used as an effective ingredient of a food composition for preventing dementia or improving cognitive function .

There is no particular limitation on the kind of the food. Examples of the food include dairy products including drinks, meat, sausage, bread, biscuits, rice cakes, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice cream, various soups, And a combination thereof, all of which include health foods in a conventional sense.

The Lactobacillus plantarum HY-08 of the present invention can be added directly to the food or can be used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). Generally, the amount of the extract in the health food may be 0.01 to 50% by weight of the total food, and the health beverage composition may be added in a proportion of 0.02 to 5 g, preferably 0.3 to 1 g, based on 100 ml have. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The health functional beverage composition of the present invention has no particular limitation on other components other than those containing Lactobacillus plantarum HY-08 as an essential ingredient in the indicated ratios, and various flavors or natural carbohydrates As a component. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above .

In addition to the above-mentioned foods, the food of the present invention may contain flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and heavies (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the extract of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of such additives is not so critical, but is generally selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the extract of the present invention.

Reference will now be made to the embodiments described hereinafter with respect to characteristic parts of the present invention and manufacturing method thereof. Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited to these examples.

< Example  1> in kimchi Lactobacillus Flora Room HY How to separate -08

For the isolation of novel lactic acid bacteria, sampling was carried out using a cotton swab in commercially available kimchi. Then, a pre-sampled swab was taken on a pre-fabricated MRS Agar medium (Difco, USA) to inoculate the initial microorganism. The inoculated initial samples were cultured at 30 ° C. for 24 hours, and the obtained microorganism colonies were inoculated again onto the MRS Agar medium to obtain strains.

The microorganisms obtained by the above method were submitted to the Korean Microorganism Preservation Center and amplified by polymerase chain reaction (PCR) using a primer of 16 rRNA to identify lactic acid bacteria. The results are shown in FIG. 1 and in SEQ ID NO: 1.

As a result of the Korean Microorganism Preservation Center, the strain obtained in the present invention was confirmed to be a strain in Lactobacillus , and was newly named Lactobacillus plantarum HY-08 (Deposit No. KCCM12103P).

In addition, Lactobacillus plantarum HY-08 isolated from Kimchi according to the results of Fig. 2 showing the phylogenetic location is a well-known Lactobacillus plantarum subsp. plantarum ACGZ01000098 was found to be a novel lactic acid bacterium having a low homology of about 64%.

In addition, SEM analysis was performed for morphological analysis in FIG. 3, and it was confirmed that Lactobacillus plantarum HY-08 was a rod type.

< Example  2> In kimchi Lactobacillus Flora Room HY -08 shredding method

In order to carry out the activity test of the novel lactobacillus Lactobacillus plantarum HY-08, an experimental sample was obtained in the following manner.

Specifically, a culture of Lactobacillus Planta room HY-08 of the novel lactic acid bacteria and then, 10 ⁹ CFU / ml 10 bun 8,000 rpm, as, after the resultant was centrifuged under the conditions of 4 ℃ 1mm diameter ball mill (HK- BA01, SAMHAW CERAMIC COMPANY, KOREA) to obtain a Lactobacillus planta HY-08 disruption product, followed by co-drying to prepare a sample.

< Experimental Example  1> Lactobacillus Flora Room HY Optimal culture method of -08

Lactobacillus plantarum HY-08 isolated in Example 1 was cultured in MRS medium to determine the optimal incubation time to match the characteristics of the strain. The culture medium was prepared by adding 55% MRS medium and 0.5% L-cysteine hydrochloride to the culture medium. The incubation temperature was incubated at 36 ° C. and the culture was carried out under anaerobic conditions in the sealed form. Therefore, the culture was carried out at a minimum stirring speed (50 to 60 rpm), and the volume of the flask was 400 ml in a 1 L flask for 72 hours.

Lactobacillus plantarum HY-08 The cell concentration of the strain was checked with time.

As a result, it was confirmed that when the cells were cultured up to 100 hours, the cell concentration gradually decreased after the amount of about 2.11 × 10 ⁸ was present, and it was confirmed that the fermentation should be continued for a maximum of 100 hours (FIG.

< Experimental Example  2> Lactobacillus Flora Room HY -08 Antioxidative Activity Analysis

Antioxidant activity of Lactobacillus plantarum HY-08 was analyzed. Antioxidant activities were measured by DPPH scavenging activity and reducing power. Add 0.5 mL of 0.5 mM DPPH solution (Abs. EtOH soln.) To 1 mL of ethanol, 10 μL of sample (Lactobacillus plantarum HY-08) and 990 μL of 100 mM sodium acetate buffer (pH 5.5) After 5 min of reaction in the dark room, the concentration of residual radical was measured at 517 nm using UV spectrophotometer. As and Ac were calculated by substituting the absorbance values of the experimental group and the control group, respectively.

As a result, as shown in the following Table 1, it was confirmed that Lactobacillus plantarum HY-08 had high antioxidative activity.

Lactic acid bacteria name DPPH free radical scavenging ratio (%) Reducing power (O.D.) Lactobacillus Flora Room HY-08 45.13 0.512

< Experimental Example  3> Lactobacillus Flora Room HY -08 Cerebral Nerve Cell Protection Activity Analysis

HT22 cells were cultured in DMEM medium supplemented with 10% FBS and incubated in a 5% CO ₂ and 37 ° C incubator at 37 ° C. for 30 minutes in order to analyze the neuronal cell protection activity of Lactobacillus plantarum HY-08. Lt; / RTI &gt; HT22 cells were cultured in 48-well plates at a density of 6.7 × 10 ⁴ cells / 300 μl to measure neuronal cell protection activity. After 24 hours and 24 hours, the Lactobacillus plantarum HY-08 fragment prepared in Experimental Example 2, 50 μM tetramethylchromancarboxylic acid (Trolox, positive control) and 2 mM glutamate were added, and 5 And incubated for 24 hours in an incubator with% CO ₂ and 37 ° C. After incubation, the medium was removed and treated with 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT) and absorbance was measured at 570 nm. Brain cell protection activity was expressed as relative protection (%).

As a result, as shown in Table 2, it was confirmed that Lactobacillus plantarum HY-08 strain had a brain-protective activity in HT22 cell line derived from mouse hippocampus.

Groups Relative protection (%) Control (control) 100 Tetramethylchromanecarboxylic acid (Trolox) 86.15 Lactobacillus Flora Room HY-08
500 ug / mL 30.22 1000 ug / mL 39.87

< Experimental Example  4> Lactobacillus Flora Room HY -08 underwater labyrinth experiment analysis

The rats used in this experiment were 4 weeks old SPF male ICR mice. After the acquisition, the adaptation period in the breeding environment was completed for one week. The animals were weighed during the adaptation period and the selected animals were uniformly distributed to the average weight of each group. The animals were given a temperature of 22 ± 2 ℃, a relative humidity of 50 ± 5%, a ventilation frequency of 10 to 15 times / hr, an illumination time of 12 hours (lighting at 7.5 o'clock in the morning from 7.5 o'clock in the afternoon), and an illumination condition of 150 to 300 Lux.

The underwater maze test of Lactobacillus plantarum HY-08 was performed using an EthoVision Maze test system (Noldus Information Technology, Wageningen, Netherlands). When the animal remembers the markers around the water tank and visits the esophagus located in a certain place in the water tank for more than 10 seconds, the escape latency is set to escape latency twice a day Mean escape latency was defined as the mean value.

Experiments were carried out twice a day for 4 consecutive days. If the animal was not found within 120 seconds, the animal was placed on the escape pod for 10 seconds. After the experiment, the incubation period was set to 120 seconds. Samples were administered daily for 4 days starting on the second day, and scopolamine (1 mg / kg) was dissolved in physiological saline and subcutaneously injected 30 minutes before the training to induce dementia. One hour before the administration of scopolamine, Oral doses of Flora Room HY-08 samples (200, 400 mg / kg) and the positive control donepezil (1 mg / kg) were administered to determine the effect of scopolamine on dementia induced by scopolamine.

As a result, the improvement activity of the scopolamine - induced memory impairment was confirmed in the underwater maze experiment in all experimental groups. In particular, it was confirmed that escape latency (time to visit target in water) was gradually reduced during 4 days of experiment period (FIG. 5).

< Experimental Example  5> Lactobacillus Flora Room HY -08 passive avoidance experiment analysis

To evaluate the effect of Lactobacillus plantarum HY-08 on learning and memory, a manual avoidance test was performed using a GEMINITM Avoidance System (San Diego Instrument, San Diego, Calif., USA). If you put an animal in a bright room of a manual evasive box divided into a dark room and a bright room, you will go to a dark room and then an electric shock for 2 seconds. After 24 hours of electric shock, when the animal is placed back in a bright room, the electric shock of the dark room is memorized, suppressing the nature of going to the dark room. At this time, the step through latency in the bright room is measured Respectively.

Samples were administered orally and scopolamine was subcutaneously administered. Lactobacillus plantarum HY-08 samples (200, 400 mg / kg) and the positive control donepezil (1 mg / kg) were orally administered 120 minutes before the experiment. After 90 minutes, the control group was subcutaneously administered with physiological saline Was administered scopolamine (1 mg / kg) subcutaneously. After 30 minutes, administer the scopolamine. After 24 hours, conduct the same experiment and measure the time that the mouse moves to the dodge box by escape latency time. If there was no movement for 180 seconds, the experiment was stopped.

As a result, the activity of improving the memory damage induced by scopolamine of Lactobacillus plantarum HY-08 was confirmed by passive avoidance experiment (Fig. 6).

Korea Microorganism Conservation Center (overseas) KCCM12103P 20170901

<110> SEOWON UNIVERSITY INSTITUTE OF INDUSTRY-ACADEMY COLLABORATION <120> New Lactobacillus plantarum isolated from KIMCHI and uses thereof <130> PN1708-286 <160> 1 <170> KoPatentin 3.0 <210> 1 <211> 1420 <212> DNA <213> Unknown <220> <223> Lactobacillus plantarum HY-08 <400> 1 tgattggtgc ttgcatcatg atttacattt gagtgagtgg cgaactggtg agtaacacgt 60 gggaaacctg cccagaagcg ggggataaca cctggaaaca gatgctaata ccgcataaca 120 acttggaccg catggtccga gcttgaaaga tggcttcggc tatcactttt ggatggtccc 180 gcggcgtatt agctagatgg tggggtaacg gctcaccatg gcaatgatac gtagccgacc 240 tgagagggta atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc 300 agtagggaat cttccacaat ggacgaaagt ctgatggagc aacgccgcgt gagtgaagaa 360 gggtttcggc tcgtaaaact ctgttgttaa agaagaacat atctgagagt aactgttcag 420 gtattgacgg tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata 480 cgtaggtggc aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggtttttta 540 agtctgatgt gaaagccttc ggctcaaccg aagaagtgca tcggaaactg ggaaacttga 600 gtgcagaaga ggacagtgga actccatgtg tagcggtgaa atgcgtagat atatggaaga 660 acaccagtgg cgaaggcggc tgtctggtct gtaactgacg ctgaggctcg aaagtatggg 720 tagcaaacag gattagatac cctggtagtc cataccgtaa acgatgaatg ctaagtgttg 780 gagggtttcc gcccttcagt gctgcagcta acgcattaag cattccgcct ggggagtacg 840 gccgcaaggc tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg 900 tttaattcga agctacgcga agaaccttac caggtcttga catactatgc aaatctaaga 960 gattagacgt tcccttcggg gacatggata caggtggtgc atggttgtcg tcagctcgtg 1020 tcgtgagatg ttgggttaag tcccgcaacg agcgcaaccc ttattatcag ttgccagcat 1080 taagttgggc actctggtga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc 1140 aaatcatcat gccccttatg acctgggcta cacacgtgct acaatggatg gtacaacgag 1200 ttgcgaactc gcgagagtaa gctaatctct taaagccatt ctcagttcgg attgtaggct 1260 gcaactcgcc tacatgaagt cggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa 1320 tacgttcccg ggccttgtac acaccgcccg tcacaccatg agagtttgta acacccaaag 1380 tcggtggggt aaccttttag gaaccagccg cctaagtgac 1420

Claims (5)

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