KR102678153B1 - Latilactobacillus curvatus WiKim0094 having the effect of improving cognitive ability or memory and uses thereof - Google Patents

Abstract

The Latilactobacillus cultus WiKim0094 strain according to the present invention is a lactic acid bacterium isolated from kimchi and exhibits excellent cognitive ability and memory improvement effects, so it can be used in food compositions, pharmaceutical compositions, bowel preparation compositions and It can be used in a variety of ways, such as as a lactic acid bacteria starter.

Description

Latilactobacillus curvatus WiKim0094 having the effect of improving cognitive ability or memory and uses thereof}

The present invention relates to Latilactobacillus curbetus WiKim0094, which is isolated from kimchi and has cognitive or memory-improving effects, and a composition for improving cognitive ability or memory containing the same.

Among brain and nervous diseases, dementia in the elderly is the most widely known. In Korea, a dementia patient occurs every 12 minutes, and it is estimated that there are currently 700,000 dementia patients in Korea.

Probiotics is derived from the Greek meaning ‘for life’ and has its origins in an ecological term meaning symbiotic relationships between living things. The meaning of this term went through several revisions and was defined by British microbiologist Fuller in 1989 as 'a single substance that has a beneficial effect by improving the host's intestinal flora when fed to humans or animals in the form of dried bacteria or fermented products. Or, it is defined as ‘live bacteria in the form of complex bacteria’. In order to be recognized as a probiotic, it must be a non-pathogenic strain that is non-toxic to the human body, have acid resistance and bile resistance, and must reach the intestines alive, settle and proliferate. Lactic acid bacteria, known as one of the probiotics, are used in various industries such as the food, pharmaceutical, and cosmetic industries, which have been recognized as GRAS (generally recognized safe) by the U.S. FDA, and are used to adjust flavor and acidity, strengthen mineral ingredients, and health functional foods. Lactic acid bacteria are used as a general term for beneficial microorganisms that consume commonly consumed sugars and produce more than 50% of lactic acid as metabolites and do not produce substances harmful to the human body. These lactic acid bacteria are found in a variety of ways in the intestines of humans and animals, fruits and vegetables, and traditional fermented foods such as Korea's kimchi and soybean paste. There are a total of 19 types of probiotic strains recognized by Korea's Ministry of Food and Drug Safety, including strains of the Lactobacillus genus and Bifidobacterium genus. Probiotic lactic acid bacteria are reported to have beneficial effects such as inflammatory diseases, anti-cancer effects, and improvement of vascular health after entering the body. They are known to become dominant bacteria in the intestines and produce antibiotics to suppress the growth of harmful bacteria such as various pathogenic microorganisms.

In particular, it has a healing effect on brain cells or nerve cells damaged by oxidative stress such as dementia, inhibits harmful bacteria that affect brain diseases such as depression, or promotes the production of neurotransmitters such as serotonin, dopamine and GABA. Probiotics are called psychobiotics.

Probiotics are fermented microorganisms, and functional and medical research is being conducted, and many studies have also revealed the medical functions of substances newly created in foods fermented by microorganisms. In addition, many studies have been conducted on the isolation of useful microorganisms from existing traditional fermented foods, but due to the diversity of experimental methods and the focus on simple basic physiological activity research, integrated research is needed for efficient commercialization.

Moreover, the market for health functional foods related to cognitive/memory improvement is increasing due to the entry into an aging society, and there is a need to expand the market by proving the new health functionality of kimchi starter.

Domestic Public Patent No. 10-2019-0094125

Accordingly, the present inventors attempted to find a lactic acid bacteria strain that showed an effect of improving cognitive ability or memory, and as a result isolated and identified a new lactic acid bacteria strain, Latilactobacillus curbetus WiKim0094, and completed the present invention.

Therefore, the purpose of the present invention is to provide Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) with accession number KCCM13400P.

Another object of the present invention is to provide a food composition for improving cognitive ability or memory containing Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) of accession number KCCM13400P, its culture, its lysate, or its extract as an active ingredient. will be.

Another object of the present invention is to provide a pharmaceutical composition for improving cognitive ability or memory containing Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) of accession number KCCM13400P, its culture, its lysate, or its extract as an active ingredient. It is provided.

In addition, another object of the present invention is to provide a bowel preparation composition containing Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) of accession number KCCM13400P, its culture, its lysate, or its extract as an active ingredient, and a lactic acid bacteria starter for fermentation. It is done.

Hereinafter, the present invention will be described in more detail as follows.

The present invention relates to Latilactobacillus curbetus WiKim0094 and uses thereof.

Latilactobacillus cultus WiKim0094 of the present invention is a new strain derived from cabbage kimchi, a Korean fermented food.

The lactic acid bacteria strain isolated through the examples of the present invention was found to have a nucleic acid sequence of SEQ ID NO: 1 as a result of 16S rRNA base sequence analysis for identification and classification of microorganisms.

Therefore, the microorganism of the present invention having a 16S rRNA base sequence of SEQ ID NO: 1 was named Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) with accession number KCCM13400P, and was registered at the Korea Microbiological Conservation Center on October 6, 2023. It was deposited on date (accession number KCCM13400P).

Latilactobacillus curbetus WiKim0094 of the present invention is a Gram-positive bacterium and a facultative anaerobe capable of growing under both aerobic and anaerobic conditions. It does not form spores, is non-motile, and its cells take the form of bacilli. there is.

In the following examples, Latilactobacillus Curvetus WiKim0094 was selected by confirming the effect of alleviating neuroinflammation, and it was confirmed that Latilactobacillus Curvetus WiKim0094 showed excellent cognitive ability or memory improvement effects.

In addition, in the following examples, it was confirmed that the Latilactobacillus curbetus WiKim0094 strain of the present invention exhibited acid resistance, bile resistance, intestinal adhesion ability, and heat resistance, and was not only advantageous for survival in the intestine but also had excellent distribution stability.

Meanwhile, in order to apply probiotic lactic acid bacteria to food, safety must be ensured, and therefore, antibiotic resistance and hemolytic toxicity of Latilactobacillus Curvetus WiKim0094 were confirmed through safety tests. As a result of determining antibiotic resistance, it was judged to be safe as it showed a level of antibiotic resistance that does not pose a risk to the health of the host by showing sensitivity at a concentration lower than or equal to the EFSA (European Food Safety Authority) breakpoint. The Latilactobacillus curbetus WiKim0094 of the invention did not show hemolysis.

Therefore, the Latilactobacillus cultus WiKim0094 strain of the present invention can be used in various fields as a safe probiotic lactic acid bacteria for improving cognitive ability or memory.

Accordingly, the present invention provides a food composition using Latilactobacillus curbetus WiKim0094 of accession number KCCM13400P, its culture, lysate thereof, or extract thereof as an active ingredient. The food composition may include health functional foods, beverages, bars, etc.

Another aspect of the present invention is a health functional food composition for improving cognitive ability or memory comprising Latilactobacillus curbetus WiKim0094 of accession number KCCM13400P, its culture, lysate thereof, or extract thereof as an active ingredient.

In this specification, the term "health functional food" refers to food manufactured and processed using raw materials or ingredients with functionality useful to the human body in accordance with the Act on Health Functional Food (Article 3, Paragraph 1), and "functional" This means adjusting nutrients to the structure and function of the human body or obtaining useful effects for health purposes such as physiological effects (Article No. 2).

In the present invention, the food composition can be used as a functional food or added to various foods. Foods that can be added to the composition of the present invention include, for example, beverages, vitamin complexes, and health supplements.

The food composition of the present invention may include ingredients commonly added during food production, and includes, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. Examples of the above-mentioned carbohydrates include monosaccharides such as glucose, fructose, etc.; Disaccharides such as maltose, sucrose, oligosaccharides, etc.; and polysaccharides, such as common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents (thaumatin, stevia extract (e.g., 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 manufactured as a drink or beverage, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, and various plant extracts may be additionally included.

The above food composition may additionally contain food additives, and its suitability as a “food additive” is determined by the specifications for the item in accordance with the general provisions and general test methods of the Food Additives Code approved by the Ministry of Food and Drug Safety, unless otherwise specified. and standards.

Items listed in the "Food Additive Code" include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid, natural additives such as subchromic pigment, licorice extract, crystalline cellulose, and guar gum, L- These include mixed preparations such as sodium glutamate preparations, noodle additive alkaline preparations, preservative preparations, and tar coloring preparations.

Foods containing the active ingredient of the present invention include confectionery such as bread, rice cake, dried fruit, candy, chocolate, chewing gum, and jam, ice cream products such as ice cream, frozen confectionery, and ice cream powder, milk, low-fat milk, lactose-digested milk, Dairy products such as processed milk, goat milk, fermented milk, butter milk, concentrated milk, milk cream, butter oil, natural cheese, processed cheese, powdered milk, and whey Processed meat products, processed egg products, meat products such as hamburgers Fish cakes, ham, Fish products such as processed fish products such as sausage and bacon, noodles such as ramen, dried noodles, fresh noodles, fried noodles, deluxe dried noodles, modified overnight noodles, frozen noodles, pasta, fruit drinks, vegetable drinks, carbonated drinks, soy milk, Lactobacillus beverages such as yogurt, beverages such as mixed drinks, seasonings such as soy sauce, soybean paste, red pepper paste, chunjang, cheonggukjang, mixed soybean paste, vinegar, sauces, tomato ketchup, curry, and dressing, but limited to margarine, shortening, and pizza. It doesn't work.

In addition to the above, the composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, It may include carbonating agents used in carbonated beverages. Additionally, the composition of the present invention may include pulp for the production of natural fruit juice, fruit juice beverages, and vegetable beverages. These ingredients can be used independently or in combination.

The beverage composition containing the active ingredient of the present invention has no particular limitations on other ingredients and may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional beverage. Examples of the above-mentioned natural carbohydrates include monosaccharides, (e.g., glucose, fructose, etc.); Disaccharides, (e.g. maltose, sucrose, etc.); and polysaccharides, common sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In another aspect, the present invention provides a pharmaceutical composition for improving cognitive ability or memory comprising Latilactobacillus curbetus WiKim0094 of accession number KCCM13400P, its culture, lysate thereof, or extract thereof as an active ingredient.

In the present invention, “cognitive ability or memory improvement” means preventing or treating cognitive dysfunction or dementia, showing effects such as preventing dementia, delaying dementia, or improving dementia.

In the present invention, “cognitive dysfunction” refers to decreased attention, decreased learning ability, decreased memory, and impaired perception. Memory impairment is a common symptom. In most cases, memory for past events is intact, but the ability to remember and learn new information is impaired, resulting in learning difficulties.

The cognitive dysfunction or dementia may include Alzheimer's disease, vascular dementia, Parkinson's disease, mild cognitive impairment, senile dementia, frontotemporal dementia, etc.

When the composition according to the present invention is used as a pharmaceutical composition, the pharmaceutical composition of the present invention is prepared using pharmaceutically suitable and physiologically acceptable adjuvants in addition to Latilactobacillus Curvetus WiKim0094 of the accession number KCCM13400P. The auxiliary agent may be an excipient, disintegrant, sweetener, binder, coating agent, swelling agent, lubricant, lubricant, or flavoring agent.

For administration, the pharmaceutical composition may be preferably formulated as a pharmaceutical composition containing one or more pharmaceutically acceptable carriers in addition to the active ingredients described above.

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, etc. Additionally, if desired or necessary, suitable binders, lubricants, disintegrants and coloring agents may also be included in the mixture. Suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tracacance or sodium oleate, sodium stearate, magnesium stearate, sodium benzoate. Includes sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methylcellulose, agar, bentonite, xanthan gum, etc. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include those that are sterile and biocompatible, such as saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and One or more of these ingredients can be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets.

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 lactic acid bacteria starter for fermentation consisting of Latilactobacillus cultus WiKim0094 or a culture thereof with accession number KCCM13400P.

As used herein, the term "lactic acid bacteria starter" refers to the first start of fermentation in which lactic acid is produced through the decomposition of lactose in milk components by lactic acid bacteria. Normally, this means using it after cultivating and proliferating lactic acid bacteria stored as starter to produce an inducing effect product. In the present invention, it means using it after cultivating and proliferating Latilactobacillus Curvetus WiKim0094.

In addition, Latilactobacillus Curvetus WiKim0094, accession number KCCM13400P of the present invention, has the general intestinal regulation effect and immune-boosting effect of lactic acid bacteria.

Therefore, the present invention provides a bowel preparation composition containing Latilactobacillus curbetus WiKim0094 of accession number KCCM13400P, its culture, its lysate, or its extract as an active ingredient.

The bowel preparation composition according to the present invention can be manufactured and administered in various formulations and methods, and can be prepared by using only Ratylactobacillus curbetus WiKim0094 with accession number KCCM13400P; its culture; Or Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus delbruecki ssp. Bulgaricus (Lactobacillus delbrueckii ssp. bulgaricus), Lactobacillus helveticus, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus Lactobacillus reuteri, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactococcus lactis, Enterococcus faecium, Enterococcus faecalis , Streptococcus thermophilus, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum and Bifidobacterium animalis ssp. . A mixture containing one or more probiotics selected from Bifidobacterium animalis ssp. lactis is mixed with a carrier and flavoring agent commonly used in the pharmaceutical field to form tablets, troches, capsules, It can be manufactured and administered in the form of elixir, syrup, powder, suspension, or granule. The carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, and suspending agents. The administration method may be oral, parenteral, or application, but oral administration is preferred. Additionally, the administered dose can be appropriately selected depending on the absorption rate, inactivation rate, and excretion rate of the active ingredient in the body, and the age, gender, and condition of the recipient.

The amount of Latilactobacillus curbetus WiKim0094 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 based on one time. It may be ¹⁰ cfu/g. When administering a strain, it is preferable to administer it in a live state, and it can be killed before ingestion or administered in an attenuated state. In addition, when manufacturing using culture supernatant, etc., it may additionally undergo a sterilization process through a heat treatment process. The amount of strain and daily intake required to have minimal efficacy may vary depending on the body or health status of the ingestor, but may generally be about 10 ⁶ to 10 ¹² cfu/g, for example, 10 ⁷ to 10 ¹¹ cfu/g. , it may be 10 ⁸ to 10 ¹⁰ cfu/g.

The advantages and features of the present invention and methods for achieving them will become clear with reference to the embodiments described in detail below. However, the present invention is not limited to the embodiments disclosed below and will be implemented in various different forms. The present embodiments are merely provided to ensure that the disclosure of the present invention is complete and to provide common knowledge in the technical field to which the present invention pertains. It is provided to fully inform those who have the scope of the invention, and the present invention is only defined by the scope of the claims.

The present invention relates to Latilactobacillus curbetus WiKim0094, a strain isolated from kimchi that has the effect of improving memory/cognitive ability, and can be used to formulate compositions for preventing and treating cognitive dysfunction containing the strain as an active ingredient. It can be used in functional foods such as starters for fermented foods.

Figure 1 shows the results of 16S rRNA identification of Latilactobacillus curbetus WiKim0094.
Figure 2 shows the measurement results of acid resistance (A), bile resistance (B), intestinal adhesion ability (C), and heat resistance (D) of Latilactobacillus cultus WiKim0094.
Figure 3 shows the results of antibiotic resistance investigation of Latilactobacillus curbetus WiKim0094.
Figure 4 shows the hemolytic measurement results of Latilactobacillus curbetus WiKim0094.
Figure 5 shows the results of treating glial cells inflamed by LPS with culture medium of Latilactobacillus curbetus WiKim0094 strain, confirmed through immunofluorescence staining.
Figure 6 shows the results of treating glial cells inflamed by LPS with the culture medium of the Latilactobacillus curbetus WiKim0094 strain of the present invention and counting the number of glial cells through immunofluorescence staining.
Figure 7 shows the results of measuring Nitrite secreted from glial cells in which inflammation was induced through LPS to confirm the effect of Latilactobacillus cuvetus WiKim0094 on controlling neuroinflammation.
Figure 8 shows the results of confirming the spatial learning and short-term memory improvement effects of kimchi lactic acid bacteria using a Y-maze after orally administering Latilactobacillus curbetus WiKim0094 strain to memory loss mice administered scopolamine [PBS: Normal group not administered scopolamine, SCO: group administered scopolamine alone, DNPZ: group administered donepezil, WiKim0094: group treated with Latilactobacillus curbetus WiKim0094 strain].
Figure 9 shows the effect of improving learning ability and long-term memory for fear stimuli in a passive avoidance test after orally administering Latilactobacillus curbetus WiKim0094 strain to memory loss mice administered scopolamine [PBS: Scopola Min-untreated normal group, SCO: scopolamine-only administered group, DNPZ: donepezil-administered group, WiKim0094: Latilactobacillus curbetus WiKim0094 strain treated group].
Figure 10 shows the effect of Latilactobacillus Curvetus WiKim0094 strain on scopolamine-induced hyperactivity through open field analysis after oral administration of Latilactobacillus Curvetus WiKim0094 strain to mice with memory loss administered with scopolamine. The inhibitory effect was confirmed [PBS: normal group not administered scopolamine, SCO: group administered scopolamine alone, DNPZ: group administered donepezil, WiKim0094: group treated with Latilactobacillus curbetus WiKim0094 strain].
Figure 11 shows the results of pH and acidity analysis of Latilactobacillus cultus WiKim0094 starter kimchi.

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. Isolation and identification of Latilactobacillus curvatus WiKim0094

The present inventor isolated kimchi lactic acid bacteria from kimchi. The kimchi sample was crushed, and the crude solution of the kimchi extract was spread on MRS agar medium (Difco) and cultured at 30°C for more than 24 hours. Single colonies formed after culture were subcultured to select single colonies, and the selected colonies were finally identified through 16S RNA base sequence analysis.

As a result of analyzing the phylogenetic tree microorganism by analyzing the base sequence of the 16S rRNA of the selected strain, it was identified as a microorganism of the present invention with a 16S rDNA base sequence of SEQ ID No: 1, and was identified as Ratylactobacillus curbetus WiKim0094. It was named and deposited at the Korea Microbial Conservation Center on October 6, 2023 (accession number KCCM13400P) [Figure 1].

SEQ ID No: 1

ctcaggacgaacgctggcggcgtgcctaatacatgcaagtcgaacgcactctcgttagattgaagaagcttgcttctgattgataacatttgagtgagtggcggacgggtgagtaacacgtgggtaacctgccctaaagtgggggataacatttggaaacagatgctaataccgcataaaacctagcaccgcatggtgcaaggttgaaagatggtttcggctatcactttaggatggacccgcggtgcattagttagttggtgaggtaaaggctcaccaagaccgtgatgcatagccgacctgagagggtaatcggccacactgggactgagacacggcccagactcctacgggaggcagcagtagggaatcttccacaatggacgaaagtctgatggagcaacgccgcgtgagtgaagaaggttttcggatcgtaaaactctgttgttggagaagaacgtatttgatagtaactgatcaggtagtgacggtatccaaccagaaagccacggctaactacgtgccagcagccgcggtaatacgtaggtggcaagcgttgtccggatttattgggcgtaaagcgagcgcaggcggtttcttaagtctgatgtgaaagccttcggctcaaccgaagaagtgcatcggaaactgggaaacttgagtgcagaagaggacagtggaactccatgtgtagcggtgaaatgcgtagatatatggaagaacaccagtggcgaaggcggctgtctggtctgtaactgacgctgaggctcgaaagcatgggtagcaaacaggattagataccctggtagtccatgccgtaaacgatgagtgctaggtgttggagggtttccgcccttcagtgccgcagctaacgcattaagcactccgcctggggagtacgaccgcaaggttgaaactcaaaggaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgaagcaacgcgaagaaccttaccaggtcttgacatcctttgaccactctagagatagagctttcccttcggggacaaagtgacaggtggtgcatggttgtcgtcagctcgtgtcgtgagatgttgggttaagtcccgcaacgagcgcaacccttattactagttgccagcatttagttgggcactctagtgagactgccggtgacaaaccggaggaaggtggggacgacgtcaaatcatcatgccccttatgacctgggctacacacgtgctacaatggatggtacaacgagtcgcgagaccgcgaggtttagctaatctcttaaaaccattctcagttcggattgtaggctgcaactcgcctacatgaagccggaatcgctagtaatcgcggatcagcatgccgcggtgaatacgttcccgggccttgtacacaccgcccgtcacaccatgagagtttgtaacacccaaagccggtgaggtaaccttcgggagccagccgtctaaggtgggacagatgattagggtgaagtcta

The general mycological characteristics of Latilactobacillus curbetus WiKim0094 are that it is a Gram-positive bacterium, a facultative anaerobic that can grow under both aerobic and anaerobic conditions, does not form spores, is non-motile, and its cells are not similar to bacilli. It is taking shape. Additional characteristics were observed through experiments using the API 50 CH kit for identification of lactic acid bacteria from Biomerieux, and the results are shown in Table 1 below.

Example 2. Evaluation of acid resistance, bile resistance, heat resistance and intestinal adhesion ability

1) Evaluation of acid resistance and bile resistance of Latilactobacillus curbetus WiKim0094 strain

Resistance to acids and bile salts, such as those found in the intestinal environment, of the Latilactobacillus curbetus WiKim0094 strain was confirmed.

After culturing the Latilactobacillus cultus WiKim0094 strain for 16 hours, it was washed twice in phosphate buffered saline (PBS), and the initial bacterial count was adjusted to 1 to ⁵ . To confirm resistance to acidity, Latilactobacillus curbetus WiKim0094 was inoculated into MRS liquid medium adjusted to pH 2.5 and pH 3.0 using 1N HCl, and cultured for 1 hour at 30°C. The culture was serially diluted in PBS and spread on an MRS agar plate to measure the number of viable cells. The number of viable bacteria before adjusting pH was used as a control.

The strain survival rate to confirm acid resistance was measured using Equation 1 below.

[Equation 1]

Strain survival rate (%) = [(Log index value of experimental group/Log index value of control group) × 100]

The results are shown in Figure 2(A). As a control for lactic acid bacteria, Lactobacillus rhamnosus GG (ATCC53103)[LGG], which is generally used as a commercial strain, was used, and the acid resistance stability of Lactobacillus curbetus WiKim0094 strain was confirmed in MRS liquid medium at pH 2.5 and pH 3.

To analyze resistance to bile acids, oxgall (Difco, USA) was inoculated into MRS liquid medium with adjusted concentrations of 0.1%, 0.3%, and 0.5%, and the number of viable cells was measured after culturing at 30°C for 2 hours. The number of viable bacteria before adding Oxgall was used as a control, and to evaluate resistance to bile acids, the strain survival rate was measured using Equation 1.

The results are shown in Figure 2(B). Lactobacillus rhamnosus GG (ATCC53103), which is commonly used as a commercial strain, was used as a lactic acid bacteria control. Latilactobacillus curbetus WiKim0094 strain showed a similar level of bile resistance as LGG, the lactic acid bacteria control, at all tested oxgall concentrations.

2) Evaluation of attachment ability of Latilactobacillus curbetus WiKim0094 strain

Intestinal attachment ability is one of the important elements that probiotics must have in order to pass through gastric acid and bile and attach to the final destination, the intestines, to exert efficacy. To analyze the intestinal adhesion ability of the Latilactobacillus cultus WiKim0094 strain, the HT-29 cell line, a colonic epithelial cell line purchased from the Korea Cell Line Bank, was used. After washing the HT-29 cells that formed a monolayer 5 times with PBS, lactic acid bacteria were suspended in RPMI-1640 medium without antibiotics, inoculated into a 6-well plate where HT-29 cells were growing, and incubated at 37°C. It was incubated for 2 hours. After the culture was completed, it was washed three times using PBS to remove non-adhered lactic acid bacteria and check the adhesion ability following washing. After washing with water, 0.25% Trypsin-EDTA was dispensed to remove the attached cells, serially diluted in PBS, and then smeared on an MRS agar plate to confirm the number of viable cells. The intestinal attachment rate to confirm the intestinal attachment ability was measured using Equation 2 below.

[Equation 2]

Intestinal attachment rate (%) = [(Log index value of number of attached viable bacteria/Log index value of number of inoculated viable cells) × 100]

The results are shown in Figure 2(C). LGG, which is generally used as a commercial strain, was used as a lactic acid bacteria control.

3) Heat resistance evaluation of Latilactobacillus curbetus WiKim0094 strain

The resistance to heat of the Latilactobacillus cultus WiKim0094 strain was confirmed by inoculating WiKim0094 in MRS liquid medium maintained at 40°C and 50°C, incubating for 10 minutes, and measuring the number of viable bacteria.

The strain survival rate to confirm thermal stability was measured using Equation 3 below.

[Equation 3]

Survival rate (%) = [(Log index value of viable cell count after heat treatment / Log index value of viable cell count inoculated) x 100]

The results are shown in Figure 2(D).

Example 3. Antibiotic resistance testing

Strains with antibiotic resistance may carry an antibiotic resistance gene (horizontal transfer) on a plasmid, and in this case, horizontal transfer of the antibiotic resistance gene is possible. In other words, when a strain containing an antibiotic resistance gene is used as a starter for food fermentation, a probiotic activator, or a probiotic, the antibiotic resistance gene may be transferred to humans, or the resistance gene may be transferred to pathogenic bacteria within the host, resulting in a detrimental effect on the host's health. According to reports that antibiotic resistance characteristics of newly developed strains, even if they are GRAS grade lactic acid bacteria, are being required to be investigated.

Accordingly, antibiotic resistance of Latilactobacillus curbetus WiKim0094 was investigated by measuring the minimum inhibitory concentration (MIC) for each type of antibiotic. The antibiotic resistance standard determined the susceptibility or resistance of Latilactobacillus curbetus WiKim0094 to several antibiotics based on the breakpoint of the European Food Safety Authority (EFSA). The results are shown in Figure 3.

As a result of determining susceptibility or resistance to antibiotics based on the break point for Lactobacillus presented by EFSA, the concentrations showing susceptibility to 9 types of antibiotics were different, but susceptibility was shown at concentrations lower than or equal to the EFSA breakpoint. As a result, it was judged to be safe because it showed antibiotic resistance at a level that did not pose a risk to the host's health, and there was no antibiotic resistance in the host.

Example 4. Confirmation of hemolytic activity of WiKim0094 strain

In order for the Latilactobacillus cuvetus WiKim0094 to be used as a raw material or additive in food, safety without hemolytic toxicity to the human body is required. Therefore, the hemolytic property of Latilactobacillus curbetus WiKim0094 was tested in relation to whether it can induce destruction or decomposition of red blood cells.

The hemolysis was confirmed by smearing Latylactobacillus cuvetus WiKim0094 on blood solid medium supplemented with 7% horse blood (Oxoid, England), culturing it at 30°C for 48 hours, and determining hemolysis by whether or not a transparent ring was formed around the bacterial cells. and the results are shown in Figure 4.

As shown in FIG. 4, it was confirmed that Latilactobacillus curbetus WiKim0094 did not induce a hemolytic reaction because it did not produce any clear rings, which are caused by destruction of red blood cells around the bacterial cells, in the hemolytic test.

Therefore, the Latilactobacillus cuvetus WiKim0094 was confirmed to have no harmful effects on the human body, such as hemolysis, and was therefore confirmed to be safe when applied to food.

Example 5. Confirmation of neuroinflammation inhibitory effect of Latilactobacillus curbetus WiKim0094 strain

1) Preparation of Latilactobacillus Curvetus WiKim0094 strain culture medium

The Latilactobacillus cultus WiKim0094 strain was cultured in MRS medium at 30°C for 24 hours, and then the cells were centrifuged at 8,000 rpm for 5 minutes and rinsed with PBS to remove remaining medium components. This ^was again inoculated into DMEM (Dulbecco's Modified Eagle's Medium, Hyclone, USA) at 1 The pH of the supernatant was corrected to 7.2 and then filtered using a syringe filter (0.22 μm pore size) before use.

2) Primary culture of glial cells

The scalp of a 2-day-old Sprague-Dawley rat was disinfected with alcohol, and the cerebrum was quickly removed without damage. The extracted cerebrum was immersed in cold HBSS (Hank's Balanced Salt Solution) solution, the cerebral cortex was separated under a dissecting microscope, and a single cell suspension was made using a Pasteur pipette, which was then cultured for 10 days. For glial cell culture, 10% fetal bovine serum (Hyclone, USA), 10% horse serum (Hyclone, USA), and 1% penicillin/streptomycin (Gibco, USA) were used. DMEM medium containing was used, and cultured at 37°C in an incubator containing 5% CO ₂

3) Confirmation of the neuroinflammation control effect of Latilactobacillus curbetus WiKim0094 strain

In order to confirm the effect of suppressing neuroinflammation of the Latilactobacillus Curvetus WiKim0094 strain culture medium prepared through the above example, glial cell inflammation was induced with LPS (lipopolysaccharide) and then the Latilactobacillus Curvetus WiKim0094 strain culture medium was treated. did. Glial cells were distributed in a ²⁴ -well plate at a concentration of 1.0 2 hours after LPS treatment, the culture medium of Latilactobacillus curbetus WiKim0094 was treated to 1/5 the volume of glial cell medium.

It was confirmed through immunostaining chemistry that the activity of glial cells (microglia and astrocytes) was significantly reduced when the culture medium of the Latilactobacillus cultus WiKim0094 strain was treated, even though neuroinflammation was induced with LPS (Figure 5). , the number of cells per unit area was counted and shown in Figure 6. At this time, the selective markers were Iba-1 (ionized calcium-binding adapter molecule 1) and GFAP (Glial fibrillary acidic protein) for microglia and astrocytes, respectively.

Additionally, the amount of nitrate in the cell culture medium of glial cells was analyzed using a griess assay (Promega, USA) and is shown in Figure 7. As a result of treatment with the culture medium of the Latilactobacillus cultus WiKim0094 strain, it was confirmed that the increase in nitric oxide (NO) production caused by LPS was suppressed.

In other words, the above results confirmed that neuroinflammation was alleviated from the WiKim0094 strain culture medium.

Example 6. Cognitive and memory improvement effect of Latilactobacillus curbetus WiKim0094 strain in animal model

1) Creation of cognitive decline animal model

7-week-old male mice (C57BL/6) were allowed to acclimate in the breeding room for 1 week and divided into 4 groups of 8 mice each. From 30 days before scopolamine injection until the end of the behavioral analysis, 200 ul of PBS was orally administered to the negative and positive control groups 6 times a week, and the Latilactobacillus Curvetus treated group was administered the Latilactobacillus Curvetus WiKim0094 strain. After culturing in MRS medium at 30°C for 24 hours, the cells were centrifuged at 8,000 rpm for 5 minutes, rinsed with physiological saline to remove remaining medium components, and incubated at 1×10 ⁹ CFU/200 ul using physiological saline. The number of bacteria was quantified and administered to experimental animals six times a week until the behavioral analysis was completed.

After 30 days of oral administration of physiological saline and lactic acid bacteria, scopolamine (1mg/kg) was injected intraperitoneally every day for 10 days to create a mouse model of cognitive decline, and the cognitive ability and memory of Latilactobacillus curbetus WiKim0094 strain was created. The improvement effect was evaluated.

Hereinafter, the first group (PBS) in the experiment is a control group to confirm the degree of memory decline induced by scopolamine, and is a normal group that was orally administered saline solution for 30 days and then intraperitoneally injected with PBS for 10 days. ,

Group 2 is a negative control group in which memory impairment was induced by orally administering physiological saline for 30 days and then intraperitoneally injecting scopolamine at 1 mg/kg for 10 days.

Group 3 is a positive control group that was administered oral saline solution for 30 days and then intraperitoneally injected with scopolamine (1mg/kg) and Donepezil (3mg/kg), a drug known to improve memory, for 10 days. . At this time, donepezil was injected 30 minutes before scopolamine was injected.

Group 4 was orally administered Latilactobacillus curbetus WiKim0094 strain for 30 days, and then intraperitoneally injected scopolamine at 1mg/kg for 10 days to recognize Latilactobacillus curbetus WiKim0094 strain. This is an experimental group that confirmed the effect of improving ability and memory.

2) Confirmation of spatial learning and short-term memory improvement effects

The Y-maze was conducted on the 6th day of scopolamine injection to confirm the effect of improving spatial learning and short-term memory. The Y-maze device consists of three passages (8 cm wide, 30 cm long, and 14 cm high). Each is arranged at a constant angle of 120° to each other.

The mouse was placed at the end of one passage and then allowed to freely roam the Y maze for 8 minutes. Each passage is marked as passage 1, 2, and 3, and the number of times the mouse explores each passage and the order and number of times it enters each passage are recorded to determine alteration (example: 123, 321, 231) and new region (example: 123, 113). ) The frequency of visiting the previous region (example: 121) and the same region (example: 122,111) was calculated.

The spontaneous alteration (%) was calculated and expressed using the following equation 4 by counting the cases in which all three passages were entered without overlapping.

[Equation 4]

Spontaneous alteration (%) = total number of alterations/(total number of aisle entries - 2) × 100

As a result, the scopolamine control group showed an average of 44.28% of voluntary change behavior, and in the case of the Ratilactobacillus Curvetus WiKim0094-administered group, short-term memory and spatial memory abilities were confirmed to be improved to 57.94% (FIG. 8). In addition, it was confirmed that the group showed similar effects to the group administered donepezil, a known dementia treatment.

3) Confirmation of improvement in learning ability and long-term memory for fearful stimuli through passive avoidance experiment

The passive avoidance experiment was conducted on the 7th and 8th days of scopolamine injection to confirm the effect of improving learning ability and long-term memory for fearful stimuli. Pre-training was performed using equipment consisting of two boxes separated by a sliding door. , training, and testing were carried out in three stages. Pre-training allowed all animals to recognize light and dark boxes without electric shock. Training was conducted 30 minutes after pre-training, and when the animal entered the dark box, the sliding door was closed and an electric shock (0.5 mA) was given to learn about fear stimulation. In the test stage, 24 hours after training, the time at which the animal first entered the dark box was analyzed, and it was confirmed that there was no significant difference between groups in all test groups during learning.

As a result of a memory test conducted 24 hours after learning, the time taken to first enter the dark box was significantly reduced in the scopolamine-treated group (control group, group 2) compared to the first group (normal group). This means that memory impairment was effectively induced by scopolamine. While the negative control group (SCO) first entered the dark box at an average of 138.6 seconds, the WiKim0094 administration group was observed to first enter the dark box at an average of 291.4 seconds (FIG. 9). In addition, it was confirmed that the group showed similar effects to the group administered donepezil, a known dementia treatment.

4) Confirmation of the effect of suppressing hyperactivity induced by scopolamine

It has been known that scopolamine not only causes memory impairment but also anxiety disorders such as hyperactivity. Therefore, an open field analysis was performed to evaluate the improvement effect of Ratylactobacillus curbetus WiKim0094 on hyperactivity induced by scopolamine. At this time, the animal's behavior was monitored using the TSE Multi Conditioning system (TSE systems, USA) Actimot video tracking system. The video camera was installed in the center of the ceiling, 100 cm high from the floor, and was able to take pictures of each plastic box for open field testing (width 45 cm, length 45 cm, height 40 cm). Mice were acclimatized in the plastic box for 1 minute, and their behavior was recorded for the next 10 minutes. The center zone of the plastic box was designated as 15 cm wide and 15 cm tall.

As a result of the open field analysis (FIG. 10), compared to the negative control group (SCO), the average distance explored on the open field in the group ingesting Latilactobacillus curbetus WiKim0094 was 11,856 cm, compared to the average distance moved by the negative control group, 14,824 cm. As a result, hyperactivity was reduced, and the time the animal rested without showing any movement during the entire analysis time was 30.81% in the negative control group, whereas in the group that consumed Latilactobacillus Curvetus WiKim0094, it was 47.83%. It was confirmed that hyperactivity symptoms caused by scopolamine drug were significantly improved in the WiKim0094 intake group.

Example 7: Fermentation characteristics of kimchi using Latilactobacillus cultus WiKim0094 as a starter

1) Strain preparation and starter kimchi production

The strain Latilactobacillus cultus WiKim0094 was applied to kimchi, a fermented lactic acid bacteria food, to confirm its potential as a probiotic food.

The strain Latilactobacillus cultus WiKim0094 was cultured in MRS liquid medium for 24 hours and then centrifuged (10,000 × g, 10 minutes). The resulting cells were washed twice with sterilized water and used as starter for kimchi. The cabbage was divided into four pieces, pickled in 16.7% (w/v) sea salt (Shinan, Korea) brine at room temperature for 5 to 7 hours, washed three times in running water, and dehydrated at 4°C for 12 hours. The prepared pickled cabbage and auxiliary ingredients were mixed well in the mixing ratio shown in Table 2 below to prepare kimchi. That is, Latilactobacillus Curvetus WiKim0094 prepared by the above method was added to the kimchi seasoning prepared at the mixing ratio in Table 2 to obtain 10 ⁷ CFU per 1 g of kimchi, and mixed with pickled cabbage to prepare starter kimchi. As a control, kimchi prepared under all the same conditions was used, except for the addition of Latilactobacillus cultus WiKim0094. The prepared kimchi was stored at 6 ± 0.5°C for 28 days in 500g pouch packs (polyethylene resin), obtained at intervals of 7 days, ground with a hand blender for 2 minutes, and then filtered through sterilized gauze to prepare kimchi liquid. , was used to analyze fermentation characteristics.

Table 2 above shows the mixing ratio for producing kimchi.

2) pH and acidity analysis of kimchi

The pH of kimchi was measured with a pH meter (Orion 3-star, Thermo Scientific, Waltham, MA, USA), and the acidity was measured using the AOAC method by mixing 10 mL of kimchi liquid with 0.1N NaOH solution until pH reached 8.3. It was defined as the consumption amount, which was converted to lactic acid content and expressed as total acid content.

[Equation 5]

Total acid content (%) = consumption of 0.1 N NaOH solution (mL) × factor of 0.1 N NaOH solution × factor of organic acids equivalent to 1 mL of 0.1 N NaOH solution (0.009 for lactic acid)

As shown in Figure 11, the pH and acidity at 6 ± 0.5°C in the control group (kimchi without added starter) showed pH 4.02 and acidity 1.55% on the 28th day of fermentation, while the kimchi with Latilactobacillus curvatus WiKim0094 added as a starter The pH and acidity were pH 4.08 and acidity 1.30%, which showed that it had a fermentation delay effect compared to kimchi without starter.

3) Analysis of number of lactic acid bacteria and seed dominance rate in kimchi

To confirm the dominance of the lactic acid bacteria Latilactobacillus cuvetus WiKim0094 added to kimchi in the microbial environment in kimchi, the shape of lactic acid bacteria colonies formed on 2% CaCO ₃ -MRS solid medium were observed, gram stained, and selected using an optical microscope. Then, the occupancy rate of the added Latilactobacillus curbetus WiKim0094 among the lactic acid bacteria was measured by comparing the 16S rRNA sequence (Table 3).

[Table 3]

Table 3 above shows the results of measuring the occupancy rates of lactic acid bacteria and spawn in kimchi prepared using Latilactobacillus cultus WiKim0094 as a starter and in the control group (kimchi without added starter) without using the starter. In the microbial environment in kimchi manufactured using Latilactobacillus cultus WiKim0094 as a starter, the total number of bacteria and the number of lactic acid bacteria were ^2.0x107 CFU/mL and 2.0x107CFU/mL on day 0 of storage, and ^2.0x107 CFU/mL on day 28 of fermentation. It showed 4.1x10 ⁷ CFU/mL and 4.6x10 ⁷ CFU/mL. Among the lactic acid bacteria in kimchi, the dominance rate of Latilactobacillus curbetus WiKim0094 used as a starter was found to be maintained at 87.4% from 91.07% on day 0 of fermentation to 87.4% until day 28 of fermentation.

Korea Center for Microbial Conservation (KCCM) KCCM13400P 20231006

Sequence list electronic file attached

Claims (10)

Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) strain with accession number KCCM13400P, which has the effect of improving cognitive ability or memory.
delete According to claim 1,
Strain with 16S rRNA indicated as SEQ ID NO: 1.
A food composition for improving cognitive ability or memory comprising Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) of accession number KCCM13400P according to claim 1, its culture, its lysate, or its extract as an active ingredient.
According to claim 4,
The food is a composition that is a health functional food.
According to claim 4,
A composition wherein the food is a beverage, bar, or fermented milk.
A pharmaceutical composition for improving cognitive ability or memory comprising Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) of accession number KCCM13400P according to claim 1, its culture, its lysate, or its extract as an active ingredient.
According to claim 7,
A pharmaceutical composition wherein the cognitive ability or memory improvement includes one or more selected from the group consisting of dementia prevention, dementia delay, and dementia improvement.
A bowel preparation composition comprising Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) of accession number KCCM13400P according to claim 1, its culture, its lysate, or its extract as an active ingredient.
A lactic acid bacteria starter for fermentation containing Latilactobacillus curvatus WiKim0094 ( Latilactobacillus curvatus WiKim0094) of accession number KCCM13400P according to claim 1 or a culture thereof as an active ingredient.