KR20200135660A - Novel lactobacillus sakei having serotonin secretion promoting effect and composition comprising the same - Google Patents

Abstract

The present invention relates to novel Lactobacillus sakei VL-002 and a composition comprising the same. Lactobacillus sakei VL-002 according to the present invention exhibits an effect of promoting the secretion of serotonin, and thus can be used in various ways for preventing, ameliorating, or treating mental stress or depression.

Description

A novel Lactobacillus Sakeai strain having a serotonin secretion promoting effect and a composition comprising the same {NOVEL LACTOBACILLUS SAKEI HAVING SEROTONIN SECRETION PROMOTING EFFECT AND COMPOSITION COMPRISING THE SAME}

The present invention relates to a novel Lactobacillus Sakeai VL-002 strain and a composition comprising the same.

Serotonin (5-HT), known as a neurotransmitter and hormone, is distributed in more than 80 to 90% of the human brain as well as the gastrointestinal tract, is produced by enterochromaffin cells, and then secreted into the blood, and then the whole body. It works by circulating. The known strains for promoting serotonin production and secretion are Clostridium ramosum [Scientific Reports 2019, 9, 1177], Spore-forming bacteria [Cell, 2015, 161(2):264- 276], Escherichia coli Nissle 1917 [Scientific Reports 2015, 5, 17324], Lactobacillus plantarum PS128 [Brain research 1631 (2016) 1-12], Lactobacillus brevis SBC8803 [Food and Nutrition Sciences, 2013, 4, 767-771] has been reported.

Depression is known to occur when the amount of serotonin in the human body is insufficient. Drugs that act on serotonin are being developed for the purpose of treating various physical diseases. Currently, domestic antidepressant drugs include a selective serotonin reuptake inhibitor (SSRI) series and a selective norepinephrine reuptake inhibitor (SNRI) series, and SSRI acts to increase serotonin concentration in synapses by preventing the nerve endings from reabsorbing serotonin. In addition to serotonin, selective SNRI enhances two neurotransmitters, norepinephrine.

However, antidepressants have a wide range of effects not only on the central nervous system but also systemically. Pfizer's first SNRI drug, Pfizer's Ifaxa (venlafaxine hydrochloride), has side effects such as nausea and vomiting, and side effects such as high blood pressure risk when taken at high doses.

Meanwhile, according to recent research results, excessive mental stress has been raised as a major cause of depression. The causes of mental stress include social factors related to study, work, marriage, and child-rearing in a complex modern society, and environmental factors such as weather and traffic. Regardless, the incidence of depression in modern people is rapidly increasing (Patent Document 1).

Therefore, it is a very important task to prevent, ameliorate or treat mental stress or depression-related diseases, and among them, the need for research and development of therapeutic agents without side effects is gradually increasing.

KR Registered Patent No. 10-1401612 (2014.05.23.)

Accordingly, the present inventors have tried to find a strain that has an effect of improving, preventing or treating mental stress or depression, and as a result, a novel Lactobacillus sakei having a serotonin secretion promoting effect has been isolated and identified to complete the present invention. .

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of mental stress or depression, comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving mental stress or depression, comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

In addition, another object of the present invention is to provide a composition for enhancing immunity comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

Another object of the present invention is to provide an intestinal medicament composition comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

In addition, another object of the present invention is to provide a fermentation starter comprising Lactobacillus Sakeai VL-002, its culture, its lysate or its extract as an active ingredient.

Another object of the present invention is to provide a feed additive or feed composition comprising Lactobacillus Sakeai VL-002, its culture, its lysate or its extract as an active ingredient.

In addition, another object of the present invention is to provide a probiotic composition comprising Lactobacillus Sakeai VL-002, its culture, its lysate or its extract as an active ingredient.

In order to achieve the object of the present invention as described above, the present invention provides a Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002).

Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002) according to the present invention is a new strain of Lactobacillus sakei derived from a mixed fermentation solution of parsley and mugwort. Although the Lactobacillus Sakeai VL-002 in the present invention was separated and identified from the mixed fermentation solution of parsley and mugwort, the route of obtaining the same is not limited thereto.

Through the examples of the present invention, it was confirmed that the novel microorganisms isolated from the mixed fermentation solution are excellent as probiotics, and show the effect of preventing, improving or treating mental stress or depression. In addition, 16S rRNA sequencing analysis for the identification and classification of microorganisms revealed a strain belonging to Lactobacillus Sakeai having the nucleic acid sequence of SEQ ID NO: 1.

Therefore, the microorganism of the present invention having the 16S rRNA nucleotide sequence of SEQ ID NO: 1 was named Lactobacillus sakei VL-002, and deposited with the Korea Microbiological Conservation Center on March 28, 2019. (Accession number KCCM12470P).

Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002; accession number KCCM12470P) strain of the present invention is a Gram-positive bacterium, is a facultative anaerobe capable of growing under anaerobic and aerobic conditions, and takes the form of a bacillus.

Lactobacillus Sakeai VL-002 of the present invention has a general intestinal effect of lactic acid bacteria and an immune enhancing effect. It is a well-known fact that Lactobacillus Sakeai has an intestinal effect and immune enhancing effect.

In the present invention, "probiotics" are understood to mean "live microorganisms that have a beneficial effect on the health of the host by improving the intestinal microbial environment of the host in the gastrointestinal tract of animals including humans". Probiotics are living microorganisms with probiotic activity, and when fed to humans or animals in the form of single or multiple strains in the form of dried cells or fermented products, they can have a beneficial effect on the intestinal flora of the host.

In the examples of the present invention, it was confirmed that the Lactobacillus Sakeai VL-002 of the present invention exhibits an effect of promoting serotonin secretion of intestinal chromophilic cells. Lactobacillus Sakeai VL-002 according to the present invention is a probiotic and can be used in various ways for preventing, improving, or treating mental stress or depression, as well as for use in human or animal suits and immunity enhancement.

Accordingly, one embodiment of the present invention provides a composition comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

The Lactobacillus Sakeai VL-002 contained in the composition according to the present invention may exist as dead or live cells, and may also exist in a dried or lyophilized form. Forms of lactic acid bacteria suitable for inclusion in various compositions and methods of formulation are well known to those skilled in the art.

In one embodiment, when the composition of the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention may be prepared using a pharmaceutically suitable and physiologically acceptable adjuvant in addition to the active ingredient, and the adjuvant As the furnace, excipients, disintegrants, sweeteners, binders, coating agents, expanding agents, lubricants, lubricants or flavoring agents may be used.

The pharmaceutical composition may be preferably formulated as a pharmaceutical composition, including at least one pharmaceutically acceptable carrier in addition to the active ingredients described above for administration.

For example, for formulation in the form of tablets or capsules, the active ingredient may be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, and the like. In addition, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders are, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, lacquercanth or sodium oleate, sodium stearate, magnesium stearate, sodium benzo Eate, sodium acetate, sodium chloride, and the like. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, and the like. As acceptable pharmaceutical carriers for compositions formulated as liquid solutions, sterilization and biocompatible ones, saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added if necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

Further, it may be preferably formulated according to each disease or ingredient using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA) as an appropriate method in the field.

In another embodiment, the present invention may be a food composition for preventing or improving mental stress or depression comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

The food composition according to the present invention may be formulated in the same manner as the pharmaceutical composition and used as a functional food or added to various foods. Foods containing the composition of the present invention include, for example, health functional foods, beverages, vitamin complexes, and health supplement foods.

The food composition of the present invention may include ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents and flavoring agents. Examples of the aforementioned carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)] and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is made of drinks and beverages, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included.

The present invention provides a composition for preventing or improving mental stress or depression comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. The mental stress or depression may be due to insufficient amount of serotonin, but is not limited thereto. Here, the insufficient amount of serotonin refers to a case where the amount of serotonin secreted in the body is lower or almost no longer than that of a normal person, or when the amount of serotonin is lowered in the same individual.

The present invention provides a composition for enhancing immunity comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. It is a well-known fact that the lactic acid bacteria of Lactobacillus Sakeai have immune enhancing effects.

In one embodiment, the composition may be a composition for oral administration comprising the Lactobacillus Sakeai VL-002 strain present as live bacteria.

In addition, one embodiment of the present invention provides an intestinal composition comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient. The intestinal composition according to the present invention can be used for the prevention, treatment, and improvement of gastrointestinal diseases in animals including humans, and preferably, the animals include livestock such as cattle, horses and pigs. The'gastrointestinal disease' includes both bacterial infection and inflammatory bowel disease for the stomach, for example, infectious diarrhea caused by pathogenic microorganisms (E. coli, Salmonella, Clostridium, etc.), gastrointestinal inflammation, inflammatory bowel disease, neurogenic enteritis syndrome. , Small intestine microbial hyperplasia, intestinal acute diarrhea, and the like, but are not limited thereto.

The intestinal composition according to the present invention is preferably administered orally. The dosage may vary depending on the type of gastrointestinal disease, the degree of the disease, age, sex, race, treatment or prevention purpose, etc., but generally, 10 million to 100 billion animals can be administered per day based on adults.

Lactobacillus Sakeai VL-002 according to the present invention may be included in medicine, health functional food, food, feed, feed additive or fermentation starter due to this advantageous action.

In the present invention, the food composition containing the strain as an active ingredient may include beverages such as fermented milk. Accordingly, the present invention provides a fermentation starter comprising Lactobacillus Sakeai VL-002, a culture thereof, a lysate thereof, or an extract thereof as an active ingredient.

The fermentation starter may further include lactic acid bacteria in addition to the Lactobacillus Sakeai VL-002 strain. The lactic acid bacteria that are additionally included may be lactic acid bacteria known in the art.

The composition according to the present invention may be used as a feed additive or feed.

When used as a feed additive, the composition may be a high concentration of 20 to 90% or may be prepared in the form of powder or granules. The feed additives are organic acids such as citric acid, humic acid, adipic acid, lactic acid, malic acid, or phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate), polyphenol, catechin, alpha-tocopherol, rosemary Any one or more of natural antioxidants such as extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid may be further included. When used as feed, the composition may be formulated in a conventional feed form, and may include a common feed component.

The feed additives and feed include grains, such as crushed or crushed wheat, oats, barley, corn and rice; Vegetable protein feeds such as feeds based on rape, soybeans, and sunflowers; Animal protein feeds such as blood meal, meat meal, bone meal and fish meal; It may further include a dry component composed of sugar and dairy products, for example, various milk powders and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth accelerators, and the like.

The feed additive may be administered to the animal alone or may be administered in combination with other feed additives in an edible carrier. In addition, the feed additives may be easily administered to animals as top dressings, directly mixed with animal feeds, or in an oral formulation separate from feed. When the feed additive is administered separately from animal feed, it can be prepared in an immediate release or sustained release formulation by combining it with a pharmaceutically acceptable edible carrier as well known in the art. Such edible carriers may be solid or liquid, such as corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the feed additive may be a tablet, a capsule, a powder, a troche or a sugar-containing tablet, or a top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be a gelatin soft capsule, or a formulation of a syrup, suspension, emulsion, or solution.

In addition, the feed additive and feed may contain adjuvants, such as preservatives, stabilizers, wetting or emulsifying agents, solution accelerators, and the like. The feed additive may be used by dipping, spraying, or mixing to add to animal feed.

The feed or feed additive of the present invention can be applied to a number of animal diets, including mammals, poultry and fish.

As the mammal, pigs, cows, sheep, goats, experimental rodents, and experimental rodents, as well as pets (eg, dogs, cats), etc. can be used, and as the poultry, chickens, turkeys, ducks, geese, pheasants, And it may be used for quail and the like, and may be used for trout as the fish, but is not limited thereto.

The amount of the Lactobacillus Sakeai VL-002 strain included in the composition according to the present invention may be about 10 ⁶ to 10 ¹² cfu/g, for example, 10 ⁷ to 10 ¹¹ cfu/g, 10 ⁸ to 10 May be ¹⁰ cfu/g. In the case of administering the strain, it is preferable to administer it in a viable state, and it may be killed before ingestion or administered in an attenuation state. In addition, in the case of manufacturing using a culture supernatant or the like, a sterilization process through a heat treatment process may be additionally performed. The amount of strain and the degree of daily intake required to have the minimum efficacy may vary depending on the body or health condition of the ingestor, but may generally be about 10 ⁶ to 10 ¹² cfu/g, such as 10 ⁷ to 10 ¹¹ cfu/g , 10 ⁸ to 10 ¹⁰ cfu/g.

Advantages and features of the present invention, and a method of achieving them will become apparent with reference to embodiments described below in detail. However, the present invention is not limited to the embodiments disclosed below, but will be implemented in a variety of different forms, and only the present embodiments are intended to complete the disclosure of the present invention, and the general knowledge in the technical field to which the present invention belongs It is provided to completely inform the scope of the invention to those who have it, and the invention is only defined by the scope of the claims.

Lactobacillus Sakeai VL-002 according to the present invention is a lactic acid bacterium isolated from a mixed fermentation solution of parsley and mugwort, and exhibits an effect of promoting the secretion of serotonin from intestinal chromophilic cells. Can be utilized. Furthermore, it can be usefully used as a fermentation starter.

1 is a graph confirming the acid resistance of the Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002) strain.
Figure 2 is a graph confirming the bile resistance of the strain Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002).
Figure 3 shows the effect of promoting the secretion of serotonin Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002) strain.

Hereinafter, the present invention will be described in detail through examples. The following examples are merely illustrative of the present invention, and the scope of the present invention is not limited to the following examples.

[Example]

Example 1: Identification of Lactobacillus Sakeai strain and measurement of enzymatic activity

The Lactobacillus Sakeai strain was isolated from a mixed fermentation solution of parsley and mugwort, and a single colony obtained by spreading on MRS medium was collected with loops and cultured in MRS broth. DNA extraction and 16S rRNA sequencing were identified by requesting Macrogen. Bacterial identification was performed through the Basic Local Alignment Search Tool (BLAST) search engine of the National Center for Biotechnology Information (NCBI, www.ncbi.nlm.nih.gov) for 16S rRNA sequences. As a result of 16S rRNA sequencing analysis for the identification and classification of microorganisms, the strain isolated through the examples of the present invention, the strain having the nucleic acid sequence of SEQ ID NO: 1 was used as Lactobacillus sakei VL-002 ( Lactobacillus sakei VL- 002), and deposited with the Korea Microorganism Conservation Center on March 28, 2019 (accession number: KCCM12470P).

<SEQ ID NO: 1> 16S rRNA of Lactobacillus sakei VL-002

ACGACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGCACTCTCGTTTAGATTGAAGGAGCTTGCTCCTGATTGATAAACATTTGAGTGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTAAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAAAACCTAACACCGCATGGTGTAGGGTTGAAAGATGGTTTCGGCTATCACTTTAGGATGGACCCGCGGTGCATTAGTTAGTTGGTGAGGTAAAGGCTCACCAAGACCGTGATGCATAGCCGACCTGAGAGGGTAATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAACTCTGTTGTTGGAGAAGAATGTATCTGATAGTAACTGATCAGGTAGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATC CTTTGACCACTCTAGAGATAGAGCTTTCCCTTCGGGGACAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGCCGGTGAGGTAACCCTTCGGGGAGCCAGCCGTCTAAGGTGGGACAGATGATTAGGGTGAA

Lactobacillus Sakeai using API ZYM kit The enzymatic activity of VL-002 was measured, and it was confirmed that it has enzymatic activities of alpha-glucosidase and beta-glucosidase (Table 1). In Table 1, when each enzyme is used, it is indicated as +, when not used, it is indicated as -, and if it shows a weak reaction, it is indicated as W.

Enzyme L. sakei VL-002 Alkaline phosphatase (alkaline phosphatase) W Esterase (C4) + Esterase lipase (C8-lipases) + Lipase (C14) W Leucine arylamidase + Valine arylamidase + Cystine arylamidase + Trypsin - Alpha-chymotrypsin - Acid phosphatase + Naph-AS-BI-phosphohydrolase
(naphthol-AS-BI-phosphohydrolase) - Alpha-galactosidase - Beta-galactosidase (β-galactosidase) + Beta-glucuronidase - Alpha-glucosidase + Beta-glucosidase + N-acetyl-beta-glucosaminedase
(N-acetyl-β-glucosaminidase) + Alpha-mannosidase - Alpha-fucosidase -

Symbols: +, positive; -, negative; w, weak reaction

Example 2: Measurement of acid resistance and bile resistance of Lactobacillus Sakeai VL-002

In order to check the resistance of lactic acid bacteria isolated from natural vegetable fermentation broth to stomach acid, the initial number of bacteria was 2×10 ⁹ cfu/ml (OD 2.0 ≒ 1×10 ⁹ cfu/ml) in MRS broth adjusted to pH 2.5 to 1%. After inoculation and incubation at 37° C. for 3 hours, the absorbance was measured at 600 nm, and a strain having 100% or more was selected as a strain having excellent acid tolerance. Bile tolerance is the same method as acid resistance by inoculating 1% of lactic acid bacteria in a bile salt medium prepared by adding Oxgall (Difco, Sparks, MD, USA) 0.6 (w/v)% and measuring the absorbance at 600 nm. The strain of 100% or more was selected as a strain having excellent bile resistance.

Acid resistance (%) = [O.D. Experimental group (3h) / O.D. Control (0h)] × 100

Bile resistance (%) = [O.D. Experimental group (3h) / O.D. Control (0h)] × 100

As a result of measuring the acid resistance and bile resistance of the L. sakei VL-002 strain, it was confirmed that both the acid resistance and bile resistance were excellent (FIGS. 1 and 2).

Example 3: Evaluation of intestinal adhesion ability of Lactobacillus Sakeai VL-002

In order to confirm the intestinal adhesion ability to Lactobacillus sakei VL-002, the method of Haghshenas et al. (2015) was modified and carried out using Caco-2 cells. Caco-2 cells were cultured in a DMEM medium (10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL streptomycin) in a 37°C, 5% CO ₂ incubator for 3 days. Lactobacillus sakei VL-002 strain was incubated at 37°C for 18 hours, then the cells (1×10 ⁸ cfu/ml (OD 1.0) were recovered, washed twice with PBS (phosphate buffer solution, Gibco)) and diluted in RPMI 1640 medium. Then, 1 mL of lactic acid bacteria was inoculated into Caco-2 cells cultured at 3×10 ⁵ cells/well in a 12-well plate, followed by incubation for 3 hours in a 5% CO ₂ incubator at 37°C. , Cells in the wells are washed three times with PBS (phosphate buffer solution, Gibco) to remove non-adherent lactic acid bacteria and then microscopic observation and Trypsin-EDTA (Hyclone, Logan, UT, USA) 200 After dispensing μL, the desorbed Caco-2 cells were appropriately diluted, inoculated on an MRS agar plate, and cultured at 37°C for 18 hours, and then the number of viable cells was confirmed. Attachment ability was compared using L. rhamnosus GG as a positive control.

Intestinal adhesion rate (%)= {viable cell count/initial cell count} × 100

Compared to the positive control, L. rhamnosus GG, it was confirmed that the intestinal adhesion ability was higher in the L. sakei VL-002 strain (Table 2).

Strain name Confirmed species
(identified species) Inoculation (cfu/mL) Adhesion (cfu/mL) VL-002 L. sakei 38.7×10 ⁶ 12.6×10 ³ LGG L. rhamnosus GG 106.3×10 ⁶ 1.2×10 ³

Example 4: Measurement of promoting serotonin secretion of Lactobacillus Sakeai VL-002

1) Preparation of freeze-dried sample of culture supernatant of Lactobacillus sakei VL-002 strain

Of Lactobacillus sakei VL-002 strain Plant medium replacing MRS and MRS at an initial concentration of 1×10 ⁸ cfu/ml to 1×10 ¹⁰ cfu/ml (food grade): hydrolysed soy protein 15 g, yeast extract (yeast extract) 10g, D-glucose monohydrate 20g, tween 80 1g, sodium acetate 5g, magnesium sulfate 0.1g, manganese sulfate 0.05g, dipotassium phosphate 2g] incubated at 30°C for 24 hours at 72 hours and centrifuged for 30 minutes at 4,000 rpm. After separating the culture supernatant, -80°C using a freeze dryer Freeze-dried at for 72 hours. The freeze-dried samples were stored at -80°C and used for experiments.

2) Measurement of promotion of serotonin secretion in sample

Changes in the promotion of serotonin secretion of RIN-14B cells, an enterochrome affinity-like cell line, were performed using a serotonin ELISA kit, (abcam). That is, after dispensing RIN-14B cells into 24-well plates at 3×10 ⁵ cells/ml, incubating for 72 hours, removing the medium, 0.1% BSA and 2 μM fluoxetine were included. Washed with KRPH buffer. 10mg/ml, 20mg/ml, or 50mg/ml of the lyophilized sample prepared in 1) above and 100μM or 300μM of cinnamaldehyde (CA) were dissolved in KRPH buffer containing 0.1% BSA and 2 μM fluoxetine, respectively, and then cells 0.25 ml each was treated at 37° C. for 30 minutes. Thereafter, centrifugation was performed at 4000 rpm for 5 minutes, and the supernatant was taken, and 100 μl was added to each well of the serotonin ELISA kit, followed by the assay procedure, and the amount of serotonin was measured at 405 nm. Cinnamic aldehyde, which promotes the secretion of serotonin in RIN-14B cells, was used as a positive control.

As a result of measuring the amount of serotonin in the culture supernatant of Lactobacillus sakei VL-002 strain, when the L. sakei VL-002 culture supernatant was treated, the amount of serotonin increased and the effect of promoting serotonin secretion was confirmed (FIG. 3 ).

Korea Microorganism Conservation Center (overseas) KCCM12470P 20190328

<110> Vegut LAB Inc. <120> NOVEL LACTOBACILLUS SAKEI HAVING SEROTONIN SECRETION PROMOTING EFFECT AND COMPOSITION COMPRISING THE SAME <130> P19E10C0384 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1496 <212> RNA <213> Unknown <220> <223> Lactobacillus sakei VL-002 <220> <221> gene <222> (1)..(1496) <400> 1 acgacgctgg cggcgtgcct aatacatgca agtcgaacgc actctcgttt agattgaagg 60 agcttgctcc tgattgataa acatttgagt gagtggcgga cgggtgagta acacgtgggt 120 aacctgccct aaagtggggg ataacatttg gaaacagatg ctaataccgc ataaaaccta 180 acaccgcatg gtgtagggtt gaaagatggt ttcggctatc actttaggat ggacccgcgg 240 tgcattagtt agttggtgag gtaaaggctc accaagaccg tgatgcatag ccgacctgag 300 agggtaatcg gccacactgg gactgagaca cggcccagac tcctacggga ggcagcagta 360 gggaatcttc cacaatggac gaaagtctga tggagcaacg ccgcgtgagt gaagaaggtt 420 ttcggatcgt aaaactctgt tgttggagaa gaatgtatct gatagtaact gatcaggtag 480 tgacggtatc caaccagaaa gccacggcta actacgtgcc agcagccgcg gtaatacgta 540 ggtggcaagc gttgtccgga tttattgggc gtaaagcgag cgcaggcggt ttcttaagtc 600 tgatgtgaaa gccttcggct caaccgaaga agtgcatcgg aaactgggaa acttgagtgc 660 agaagaggac agtggaactc catgtgtagc ggtgaaatgc gtagatatat ggaagaacac 720 cagtggcgaa ggcggctgtc tggtctgtaa ctgacgctga ggctcgaaag catgggtagc 780 aaacaggatt agataccctg gtagtccatg ccgtaaacga tgagtgctag gtgttggagg 840 gtttccgccc ttcagtgccg cagctaacgc attaagcact ccgcctgggg agtacgaccg 900 caaggttgaa actcaaagga attgacgggg gcccgcacaa gcggtggagc atgtggttta 960 attcgaagca acgcgaagaa ccttaccagg tcttgacatc ctttgaccac tctagagata 1020 gagctttccc ttcggggaca aagtgacagg tggtgcatgg ttgtcgtcag ctcgtgtcgt 1080 gagatgttgg gttaagtccc gcaacgagcg caacccttat tactagttgc cagcatttag 1140 ttgggcactc tagtgagact gccggtgaca aaccggagga aggtggggac gacgtcaaat 1200 catcatgccc cttatgacct gggctacaca cgtgctacaa tggatggtac aacgagttgc 1260 gagaccgcga ggtttagcta atctcttaaa accattctca gttcggattg taggctgcaa 1320 ctcgcctaca tgaagccgga atcgctagta atcgcggatc agcatgccgc ggtgaatacg 1380 ttcccgggcc ttgtacacac cgcccgtcac accatgagag tttgtaacac ccaaagccgg 1440 tgaggtaacc cttcggggag ccagccgtct aaggtgggac agatgattag ggtgaa 1496

Claims (12)

Lactobacillus sakei VL-002 of accession number KCCM12470P { Lactobacillus sakei VL-002}. Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002; accession number KCCM12470P), a culture thereof, a lysate thereof or a pharmaceutical composition for the prevention or treatment of mental stress or depression, comprising as an active ingredient. The method of claim 2,
The mental stress or depression is a pharmaceutical composition that appears due to insufficient amount of serotonin. The method of claim 2,
The composition is a pharmaceutical composition for oral administration. Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002; accession number KCCM12470P), a culture thereof, a lysate thereof, or a food composition for preventing or improving mental stress or depression, comprising as an active ingredient. The method of claim 5,
The mental stress or depression is a food composition that is due to insufficient amount of serotonin. The method of claim 5,
The food is a health functional food, food composition. Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002; accession number KCCM12470P), a culture thereof, a lysate thereof or a composition for enhancing immunity comprising an extract thereof as an active ingredient. Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002; accession number KCCM12470P), its culture, its lysate, or an intestinal composition comprising an extract thereof as an active ingredient. Fermentation starter comprising Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002; accession number KCCM12470P), its culture, its lysate or its extract as an active ingredient. A feed additive or feed composition comprising Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002; accession number KCCM12470P), its culture, its lysate or its extract as an active ingredient. A probiotic composition comprising Lactobacillus sakei VL-002 ( Lactobacillus sakei VL-002; accession number KCCM12470P), its culture, its lysate or its extract as an active ingredient.

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