Patent Application
20180355445
The present invention relates to novel lactic acid bacteria and the use thereof, and more particularly to novel lactic acid bacteria, which is isolated from Kimchi, having various types of physiological activities, such as memory improvement activity, tight junction protein expression inducement activity, antioxidant activity, lipopolysaccharide (LPS) production-inhibitory activity or β-glucuronidase-inhibitory activity, and to food and medicinal uses of novel lactic acid bacteria for preventing, alleviating or treating degenerative brain diseases or cognitive function disorders.
With the rise of aging society, the number of patients with degenerative brain diseases, such as Alzheimer's disease, Parkinson's disease and dementia is rapidly increasing. Alzheimer's disease, the most common degenerative brain disease that causes dementia, slowly develops and gradually deteriorates cognitive functions including memory. While the exact cause of Alzheimer's disease has yet to be found, it is closely associated with aging and is growing with the increase of the older population.
Parkinson's disease is a chronic progressive degenerative disease of the nerve system caused by the loss of dopamine neurons, showing symptoms of muscle cramps, bradykinesia (slowness of movement) and postural instability. Patients with Alzheimer's disease show symptoms of dementia, such as speech impairment and executive function inability while patients with Parkinson's disease show symptoms of dementia, such as lapses in concentration and visual/spatial judgment, executive function disorder and slowness in thinking.
Dementia may be defined as a condition where a physically stable person experiences damage to brain function due to various causes and gradual deterioration of the overall cognitive ability, greatly affecting daily life activities. Cognitive ability herein means various intellectual abilities, such as memory, speech, visual-spatial ability, judgment and abstract thinking. Each of the cognitive abilities is deeply associated with a certain part of brain. About 50% of dementia are a Alzheimer's disease-type, 20 to 30% of dementia are a blood vessel-type and there are other types of dementia, such as alcohol-related dementia, dementia with Lewy bodies, frontotemporal dementia, and Parkinson's disease dementia. Recently, there have been studies that various degenerative brain diseases including dementia are associated with Intestinal Permeability Syndrome or intestinal flora disturbance.
Intestinal Permeability Syndrome and Dementia
The gastrointestinal tract of the human body is composed of mucus and villi, which efficiently absorb nutrient components, but prevent the absorption of pathogenic microorganisms having a high molecular weight or toxins produced by these microorganisms. In addition, the human body has an immune system capable of protecting the body from invasion of external antigens having a high molecular weight. However, due to infection with many pathogenic microorganisms or toxins, excessive stress, intake of foods such as high-fat diets capable of proliferating harmful bacterial living in the gastrointestinal tract, excessive alcohol intake, the abuse of drugs (e.g., antibiotics) and the like, intestinal flora is disturbed, abnormalities in the gastrointestinal tract's immune system occur, and expression of tight junction proteins is inhibited. If expression of tight junction proteins is inhibited, tight junction of intestinal mucosa becomes loosened, and the invasion into the body of large molecules due to the loosened gap and abnormalities in the immune system. Intestinal permeability syndrome is also known as leaky gut syndrome, and refers to a condition in which external such as less digested foods, pathogenic microorganisms, toxins or the like are continuously introduced into blood, because the tight junction barrier system of epithelial cells forming the gastrointestinal tract is not smoothly operated. When intestinal permeability syndrome occurs, external antigens that are generally not absorbed into the body enter the body, thus causing ulcerative colitis, Crohn's disease, liver injury, liver dysfunction, allergic diseases (including asthma), atopy, autoimmune diseases, steatorrhea, digestive absorption disorder, acne, accelerated aging, endotoxemia, intestinal infection, eczema, irritable bowel syndrome, chronic fatigue syndrome, psoriasis, rheumatoid arthritis, pancreatic insufficiency, inflammatory joint diseases or the like. Recently, there have been studies that Intestinal Permeability Syndrome is associated with dementia caused by Parkinson's disease or aging.
Intestinal Flora Disturbance and Dementia
There are many bacteria living in the gastrointestinal tract of the human body. The human body has about 10 trillion normal cells, but has about 100 trillion bacteria which are about 10-fold larger than the normal cells. These bacteria may be divided into beneficial bacteria that help human intestinal health and harmful bacteria that are harmful to human health. The health of human body may be maintained when beneficial bacterial such as Lactobacillus, Bifidobacterium, Streptococcus, Leuconostoc, Pediococcus, Sporolactobacillius and the like are more dominant in the gastrointestinal tract than harmful bacteria. Otherwise, diseases may be caused, such as obesity, intestinal permeability syndrome, liver diseases, accelerated aging, enteritis, accelerated aging, dementia and the like. Increase of harmful microorganisms in the intestinal flora (ex. Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, etc.) boosts the activity of NF-kB in intestinal cells, which can dramatically increase the possibility of degenerative brain diseases, such as Alzheimer's disease and Parkinson's disease, and dementia.
Probiotics are collectively referred to as live microorganisms that improve the host's microbial environment in the gastrointestinal tract of animals, including humans, and have beneficial effects on the host's health. In order to be effective as probiotics, it is necessary to have excellent acid resistance, bile resistance and adherence to epithelial cells, because most of these probiotics should reach the small intestine upon oral administration and must be adhered to the intestinal surface. Lactic acid bacteria are used as probiotics because they play a role in decomposing fibrous and complex proteins to make important nutrients while living in the digestive system of the human body. Lactic acid bacteria have been reported to exhibit effects such as maintenance of intestinal normal flora, improvement of intestinal flora, anti-diabetic and anti-hyperlipidemic effects, inhibition of carcinogenesis, inhibition of colitis, and nonspecific activity of the host's immune system. Among these lactic acid bacteria, Lactobacillus sp. strains are major members of normal microbial communities living in the bowel of the human body and have long been known to be important in maintaining a healthy digestive tract and vaginal environment. Currently, according to the U.S. Public Health Service guidelines, all the Lactobacillus strains deposited with the American Type Culture Collection (ATCC) are classified as ‘Bio-Safety Level 1’, which is recognized as having no known potential risk of causing disease in humans or animals. Meanwhile, lactic acid bacteria of kimchi that are involved in kimchi fermentation have been reported to have immune enhancement effects, antimicrobial effects, antioxidant effects, anti-cancer effects, anti-obesity effects, hypertension preventive effects or constipation preventive effects [Hivak P, Odrska J, Ferencik M, Ebringer L, Jahnova E, Mikes Z. One-year application of Probiotic strain Enterococcus facium M-74 decreases Serum cholesterol levels: Bratisl lek Listy 2005; 106(2); 67-72; Agerholm-Larsen L. Bell M L. Grunwald G K. Astrup A.: The effect of a probiotic milk product on plasma cholesterol: a metaanalysis of short-term intervention studies; Eur J Clin Nutr. 2000; 54(11) 856-860; Renato Sousa, Jaroslava Helper, Jian Zhang, Strephen J Lewis and Wani O Li; Effect of Lactobacillus acidophilus supernants on body weight and leptin expression in rats; BMC complementary and alternative medicine. 2008; 8(5)1-8].
Since various bioactive activities of lactic acid bacteria were known, There have recently been a growing number of studies designed to develop a safe and highly functional lactic acid bacteria flora for the human body and turn it into an ingredient of medicinal products or functional foods. For example, the Korean Patent Gazette for registration No. 10-1476236 discloses a pharmaceutical composition for prevention or treatment of dementia, comprising lactobacillus pentosus var. plantarum C29 KCCM11291P flora as an active ingredient. The Korean Patent Gazette for registration No. 10-1087972 discloses the preparation method of lactic acid bacteria ferment that is effective in preventing and treating dementia, comprising (a) a stage to inoculate, culture and ferment lactic acid bacteria selected from lactobacillus sp., enterococcus sp. and bifidobacterium sp. into the medium comprising milk; (b) a stage to remove lactic acid bacteria from the above ferment; and (c) a stage to precipitate separate active ingredients adding a solvent selected from a group consisting of acetone and alcohol having 1 to 6 carbon atoms to the ferment, which had lactic acid bacteria removed. In addition, the Korean Patent Gazette for registration No. 10-1424547 discloses a pharmaceutical composition for preventing or treating degenerative brain diseases comprising the lactic acid bacteria ferment of Lactobacillus fermentum KFRI 164 of Sibjeondaebotang, as an active ingredient. Also, the Korean Patent Gazette for publication No. 10-2015-0047687 discloses a composition for preventing or improving forgetfulness or improving memory, comprising the plant extract that was fermented by adding 0.1 to 10 wt % of glucose and 0.1 to 5 wt % of yeast extract to a plant extract, hot-water extracted from Polygala tenuifolia, white Poria cocos Wolf, and Acoris gramineus, and then inoculating the cultured Lactobacillus plantarum.
And yet, lactic acid bacteria disclosed in the prior art or the product fermented by the same is not enough to be applied as a commercial treatment since it is not highly effective in treating degenerative brain diseases including dementia. As such, it is necessary to screening a certain type of lactic acid bacteria that has the equivalent level of treatment effect for brain diseases compared to commercial treatments, and improves Intestinal Flora Disturbance and Intestinal Permeability Syndrome, thereby developing medicinal products or functional foods.
The present invention has been made under the Background Art as described above, and it is an object of the present invention to provide novel lactic acid bacteria having various physiological activities or functionalities required for probiotics.
In addition, an object of the present invention is to provide a composition that comprises novel lactic acid bacteria or ferment thereof and may be used to alleviate, prevent or treat degenerative brain diseases.
To achieve the above objects, an embodiment of the present invention is Lactobacillus johnsonii comprising 16S rDNA nucleotide sequence represented by SEQ ID NO: 2, which provides the lactic acid bacteria having the memory improvement activity, tight junction protein expression inducement activity, antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity or β-glucuronidase inhibitory activity.
To achieve the above objects, an embodiment of the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases or cognitive function disorder comprising a lactic acid bacteria corresponding to Lactobacillus johnsonii comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 2, a culture of the lactic acid bacteria, a lysate of the lactic acid bacteria or an extract of the lactic acid bacteria as an active ingredient. Still another embodiment of the present invention provides a food composition for preventing or alleviating degenerative brain diseases or cognitive function disorder comprising a lactic acid bacteria corresponding to Lactobacillus johnsonii comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 2, a culture of the lactic acid bacteria, a lysate of the lactic acid bacteria or an extract of the lactic acid bacteria as an active ingredient. Still another embodiment of the present invention provides a food composition for improving memory or learning ability comprising a lactic acid bacteria corresponding to Lactobacillus johnsonii comprising a 16S rDNA nucleotide sequence represented by SEQ ID NO: 2, a culture of the lactic acid bacteria, a lysate of the lactic acid bacteria or an extract of the lactic acid bacteria as an active ingredient.
To achieve the above objects, another embodiment of the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases or cognitive disorder, comprising red bean ferment or extract of the red bean ferment as an active ingredient. Also, another embodiment of the present invention provides a food composition for preventing or alleviating degenerative brain diseases or cognitive function disorder, comprising red bean ferment or extract from the red bean ferment. In addition, another embodiment of the present invention provides a food composition for improving memory or learning ability, comprising red bean ferment or extract from the red bean ferment. In the composition according to another embodiment of the present invention, the above red bean ferment is a product that was fermented with lactic acid bacteria that comprises 16S rDNA nucleotide sequence represented by SEQ ID NO: 2 and corresponds to Lactobacillus johnsonii.
A certain Lactobacillus sp. strain according to the present invention is isolated from kimchi, and thus is highly safe, and has various physiological activities such as memory improvement activity, tight junction protein expression inducement activity, antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity or β-glucuronidase inhibitory activity. Accordingly, a certain Lactobacillus sp. strain according to the present invention may be used as an ingredient of medicinal products or functional foods for preventing, alleviating or treating various diseases through combination of the strain's first effect that alleviates intestinal permeability and the secondary effect that improves learning ability or memory.
As used herein, terms used in the present invention will be defined.
As used herein, the term “cognitive function disorder” means a symptom or disease where cognitive functions, such as memory-processing, cognition or problem-solving do not fully function, specifically including declining working memory, attention and vigilance, linguistic learning and memory, visual learning and memory, and reasoning and problem-solving, namely, executive function, processing speed or social cognition.
As used herein, the term “brain disease” means all types of diseases generated in the brain due to destroyed cranial nerve cells.
As used herein, the term “culture” means a product obtained by culturing a microorganism in a known liquid medium or solid medium, and thus is intended to include a microorganism.
As used herein, the term “ferment” means a product yielded by fermenting the raw ingredient, a subject of fermentation, with microorganism, and a concept comprising microorganism.
As used herein, the term “lactic acid bacteria, etc.” means lactic acid bacteria, and culture, lysate or extract of the lactic acid bacteria.
As used herein, the term “red bean ferment, etc.” means the red bean ferment or extract of the ferment.
As used herein, the terms “pharmaceutically acceptable” and “sitologically acceptable” means neither significantly stimulating an organism nor inhibiting the biological activity and characteristics of an active material administered.
As used herein, the term “preventing” refers to all actions that inhibit symptoms or delay the progression of a particular disease by administrating the composition of the present invention.
As used herein, the term “treating” refers to all actions that alleviate or beneficially change the symptoms of a particular disease by administering the composition of the present invention.
As used herein, the term “alleviating” refers to all actions that at least reduce a parameter related to the condition to be treated, for example, the degree of symptom.
As used herein, the term “administering” means providing the composition of the present invention to a subject by any suitable method. As used herein, the term “subject” means all animals, including humans, monkeys, dogs, goats, pigs or rats, which have a particular disease whose symptoms may be alleviated by administering the composition of the present invention.
As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to treat diseases, at a reasonable benefit/risk ratio applicable to any medical treatment. The pharmaceutically effective amount may be determined depending on factors including the kind of subject's disease, the severity of the disease, the activity of the drug, sensitivity to the drug, the time of administration, the route of administration, excretion rate, the duration of treatment and drugs used in combination with the composition, and other factors known in the medical field.
Hereinafter, the present invention will be described in detail.
One aspect of the present invention is related to novel lactic acid bacteria having various physiological activities.
A lactic acid bacteria according to one embodiment of the present invention is Lactobacillus johnsonii comprising 16S rDNA nucleotide sequence represented by SEQ ID NO: 2, having memory improvement activity, tight junction protein expression inducement activity, antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity or β-glucuronidase inhibitory activity. The Lactobacillus johnsonii is an anaerobic bacillus isolated from kimchi, is positive to gram staining, can survive in a wide temperature range and low pHs, and produces glucosidase. Furthermore, the Lactobacillus johnsonii utilizes D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, maltose, lactose, sucrose, gentibiose and the like as carbon sources. In addition, the Lactobacillus johnsonii is preferably Lactobacillus johnsonii CH32 (accession number: KCCM 11763P).
One aspect of the present invention relates to a composition comprising a particular lactic acid bacteria, etc., as an active ingredient. The composition according to one embodiment of the present invention comprises 16S rDNA nucleotide sequence represented by SEQ ID NO: 2, and also comprises lactic acid bacteria corresponding to Lactobacillus johnsonii, culture, lysate or extract thereof as an active ingredient. The Lactobacillus johnsonii, is an anaerobic bacillus isolated from kimchi and positive to gram staining, and survives in a wide range of temperatures with low PH environment and produces glucosidase. Also, the Lactobacillus johnsonii uses D-glucose, D-fructose, D-mannose, N-acetyl-glucosamine, Maltose, Lactose, Sucrose, Gentiobiose and the like as a carbon source. In addition, the Lactobacillus johnsonii is preferably Lactobacillus johnsonii CH32 (accession number: KCCM 11763P).
In the present invention, a culture of the lactic acid bacteria is a produced by culturing a certain strain or a mixture of strains in a medium. The medium may be selected from known liquid media or solid media, and may be, for example, MRS liquid medium, MRS agar medium or BL agar medium.
The composition according to one embodiment of the present invention may be used to prevent, alleviate or treat degenerative brain diseases or cognitive function disorder, since lactic acid bacteria, etc., the active ingredient, has various types of physiological activities, such as memory improvement activity, tight junction protein expression inducement activity, antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity or β-glucuronidase inhibitory activity. The degenerative brain disease may be Alzheimer's disease, Parkinson's disease, Huntington's disease, or dementia, specifically. Also, the dementia may be selected from a group consists of senile dementia, vascular dementia, Lewy body dementia, frontotemporal dementia, Alzheimer's disease-type dementia, Parkinson's disease-type dementia, Huntington's disease-type dementia, Creutzfeldt-Jacob disease-type dementia, Pick's disease-type dementia, normal pressure hydrocephalus-causing dementia and head injury-causing dementia. Also, the composition according to one embodiment of the present invention may be used to improve memory or learning ability.
Another aspect of the present invention relates to a composition comprising a product fermented by lactic acid bacteria, etc., as an active ingredient. The composition according to another embodiment of the present invention comprises red bean ferment or extract of the same. In the composition according to another embodiment of the present invention, the red bean ferment is a product that the red bean was fermented with lactic acid bacteria corresponding to Lactobacillus johnsonii and comprising 16S rDNA nucleotide sequence represented by SEQ ID NO: 2. In the composition according to another embodiment of the present invention, the technical characteristics of the Lactobacillus johnsonii are as described above, and thus the description thereof is omitted.
Red bean ferment, an active ingredient of the composition according to another embodiment of the present invention, has higher physiological activity than lactic acid bacteria and is more effective in the aspect of functional food and medicinal ingredient, since the ferment comprises both the secondary metabolite produced by the red bean fermentation and lactic acid bacteria used in the red bean fermentation. The composition according to another embodiment of the present invention may be used to prevent, alleviate or treat degenerative brain diseases or cognitive function disorder. The degenerative brain diseases may be Alzheimer's disease, Parkinson's disease, Huntington's disease, or dementia, specifically. Also, the dementias may be selected from a group consists of senile dementia, vascular dementia, Lewy body dementia, frontotemporal dementia, Alzheimer's disease-type dementia, Parkinson's disease-type dementia, Huntington's disease-type dementia, Creutzfeldt-Jacob disease-type dementia, Pick's disease-type dementia, normal pressure hydrocephalus-causing dementia and head injury-causing dementia. Also, the composition according to another embodiment of the present invention may be used to improve memory or learning ability.
In the present invention, the composition may be embodied as a pharmaceutical composition, a food additive, a food composition (particularly, a functional food composition), a feed additive or the like depending on the intended use or aspect. In addition, the content of the lactic acid bacteria, etc. or red bean ferment, etc., as an active ingredient may also be adjusted within a wide range depending on the specific type, intended use or aspect of the composition.
The content of the lactic acid bacteria, etc. or red bean ferment, etc., as an active ingredient in the pharmaceutical composition according to the present invention is not particularly limited. For example, the content may be 0.01 to 99 wt %, preferably 0.5 to 50 wt %, more preferably 1 to 30 wt %, based on the total weight of the composition. In addition, the pharmaceutical composition according to the present invention may further contain, in addition to the active ingredient, additives such as pharmaceutically acceptable carriers, excipients or diluents. Carriers, excipients and diluents, which may be contained in the pharmaceutical composition according to the present invention, include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxylbenzoate, talc, magnesium stearate and mineral oil. In addition, the pharmaceutical composition according to the present invention may further contain, in addition to the lactic acid bacteria, etc. or red bean ferment, etc., one or more active ingredients having the effect of preventing or treating degenerative brain diseases or cognitive function disorder. The pharmaceutical composition according to the present invention may be prepared as formulations for oral administration or formulations for parenteral administration, and the formulations may be prepared using diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like, which are commonly used. Solid formulations for oral administration include tablets, pellets, powders, granules, capsules and the like, and such solid formulations may be prepared by mixing the active ingredient with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose or gelatin. In addition to simple excipients, lubricants such as magnesium stearate or talc may also be used. Liquid formulations for oral administration include suspensions, solutions, emulsions and syrup, and may contain various excipients, for example, wetting agents, flavoring agents, aromatics, preservatives and the like, in addition to water and liquid paraffin which are frequently used simple diluents. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations and suppositories. Propylene glycol, polyethylene glycol, plant oils such as olive oil, injectable esters such as ethyl oleate and the like may be used as non-aqueous solvents or suspending agents. As the base of the suppositories, witepsol, Macrogol, Tween 61, cacao butter, laurin fat, glycerogelatin and the like may be used. Furthermore, the composition may preferably be formulated depending on each disease or component by a suitable method known in the art or the method disclosed in Remington's Pharmaceutical Science (the latest edition), Mack Publishing Company, Easton Pa. The pharmaceutical composition of the present invention may be administered orally or parenterally to mammals, including humans, according to a desired method. Routes for parenteral administration include skin external application, intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, intrathoracic injection or the like. The dose of the pharmaceutical composition of the present invention is not particularly limited as long as it is a pharmaceutically effective amount. The dose may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration mode, excretion rate and the severity of the disease. The daily dose of the pharmaceutical composition of the present invention is not particularly limited, but is preferably 0.1 to 3000 mg/kg based on an active ingredient, more preferably 1 to 2000 mg/kg based on an active ingredient and may be administered once or several times a day.
Furthermore, the content of the lactic acid bacteria, etc. or red bean ferment, etc., as an active ingredient in the food composition according to the present invention is 0.01 to 99 wt o, preferably 0.1 to 50 wt o, more preferably 0.5 to 25 wt o, based on the total weight of the composition, but is not limited thereto. The food composition of the present invention may be in the form of pellets, powders, granules, infusions, tablets, capsules, liquid or the like, and specific examples of the food may include meats, sausages, breads, chocolates, candies, snacks, confectionaries, pizzas, ramens, other noodles, gums, dairy products including ice creams, various kinds of soups, beverages, teas, functional water, drinks, alcoholic beverages, vitamin complexes and the like, and may include all health foods in a general sense. The food composition of the present invention may further contain sitologically acceptable carriers, various flavoring agents or natural carbohydrates as additional ingredients, in addition to the active ingredient. Additionally, the food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salt, alginic acid and its salt, an organic acid, a protective colloidal thickener, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonating agent used for carbonated drinks and the like. Additionally, the food composition of the present invention may contain fruit flesh for preparing natural fruit juices, fruit juice drinks and vegetable drinks. These ingredients may be used independently or as a mixture. The above-described natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin and sugar alcohols such as xylitol, sorbitol, and erythritol. As a flavoring agent, a natural flavoring agent such as thaumatin or a stevia extract, or a synthetic flavoring agent such as saccharin or aspartame may be used.
Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are merely intended to clearly illustrate the technical characteristics of the present invention and do not limit the scope of the present invention.
1. Isolation and Identification of Lactic Acid Bacteria
(1) Isolation of Lactic Acid Bacteria from Kimchi
Each of Chinese cabbage kimchi, radish kimchi and green onion kimchi was crushed, and the crushed liquid was suspended in MRS liquid medium (MRS Broth; Difco, USA). Next, the supernatant was collected, transferred to MRS agar medium (Difco, USA) and cultured anaerobically at 37° C. for about 48 hours, and then strains that formed colonies were isolated.
(2) Isolation of Lactic Acid Bacteria from Human Feces
Human feces were suspended in GAM liquid medium (GAM broth; Nissui Pharmaceutical, Japan). Next, the supernatant was collected, transferred to BL agar medium (Nissui Pharmaceutical, Japan) and cultured anaerobically at 37° C. for about 48 hours, and then Bifidobacterium sp. strains that formed colonies were isolated.
(3) Identification of Screened Lactic Acid Bacteria
The physiological characteristics and 16S rDNA sequences of the strains isolated from kimchi or human feces were analyzed to identify the species of the strains, and names were given to the strains. Table 1 below the control numbers and strain names of the lactic acid bacteria isolated from Chinese cabbage kimchi, radish kimchi, green onion kimchi and human feces.
Lactobacillus acidophilus
Lactobacillus acidophilus
Lactobacillus acidophilus
Lactobacillus brevis CH4
Lactobacillus curvatus CH5
Lactobacillus brevis CH6
Lactobacillus casei CH7
Lactobacillus planantrum
Lactobacillus sakei CH9
Lactobacillus curvatus CH10
Lactobacillus sakei CH11
Lactobacillus curvatus CH12
Lactobacillus plantarum
Lactobacillus fermentum
Lactobacillus fermentum
Lactobacillus gasseri CH16
Lactobacillus paracasei
Lactobacillus helveticus
Lactobacillus helveticus
Lactobacillus johnsonii
Lactobacillus johnsonii
Lactobacillus johnsonii
Lactobacillus brevis CH23
Lactobacillus paracasei
Lactobacillus kimchi CH25
Lactobacillus gasseri CH26
Lactobacillus paracasei
Lactobacillus pentosus CH28
Lactobacillus pentosus CH29
Lactobacillus reuteri CH30
Lactobacillus sakei CH31
Lactobacillus johnsonii
Lactobacillus sakei CH33
Lactobacillus sakei CH34
Lactobacillus plantarum
Lactobacillus
sanfranciscensis CH36
Bifidobacterium
pseudocatenulatum CH37
Bifidobacterium
pseudocatenulatum CH38
Bifidobacterium
adolescentis CH39
Bifidobacterium
adolescentis CH40
Bifidobacterium
adolescentis CH41
Bifidobacterium animalis
Bifidobacterium animalis
Bifidobacterium bifidum
Bifidobacterium bifidum
Bifidobacterium breve CH46
Bifidobacterium breve CH47
Bifidobacterium breve CH48
Bifidobacterium catenulatum
Bifidobacterium catenulatum
Bifidobacterium dentium
Bifidobacterium infantis
Bifidobacterium infantis
Bifidobacterium infantis
Bifidobacterium longum CH55
Bifidobacterium longum CH56
Bifidobacterium longum CH57
Bifidobacterium longum CH58
Bifidobacterium longum CH59
Bifidobacterium longum CH60
Among the strains shown in Table 1 above, Lactobacillus brevis CH23 was a gram-positive anaerobic bacillus, did not form spores, and could survive even under aerobic conditions. Furthermore, Lactobacillus brevis CH23 survived at 10 to 42° C. and was an acid-resistant strain stable at pH 2 for 2 hours. Furthermore, Lactobacillus brevis CH23 survived even in 2% sodium chloride solution and actively produced glucosidase. In addition, to chemically classify Lactobacillus brevis CH23, the 16S rDNA thereof was analyzed, and as a result, it was shown that Lactobacillus brevis CH23 had a nucleotide sequence of SEQ ID NO: 1. The 16S rDNA nucleotide sequence of Lactobacillus brevis CH23 was identified by BLAST in the Genebank (http://www.ncbi.nlm.nih.gov/), and as a result, a Lactobacillus brevis strain having the same 16S rDNA nucleotide sequence as that of Lactobacillus brevis CH23 was not found, and Lactobacillus brevis CH23 showed a homology of 99% with the 16S rDNA sequence of Lactobacillus brevis strain FJ004.
Among the strains shown in Table 1 above, Lactobacillus johnsonii CH32 was a gram-positive anaerobic bacillus, did not form spores, and could survive under aerobic conditions. Furthermore, Lactobacillus johnsonii CH32 survived stably at a temperature of 10 to 45° C., and was an acid-resistant strain stable in pH 2 for 2 hours. Moreover, Lactobacillus johnsonii CH32 actively produced glucosidase, but did not produce β-glucuronidase. In addition, to chemically classify Lactobacillus johnsonii CH32, the 16S rDNA thereof was analyzed, and as a result, it was shown that Lactobacillus johnsonii CH32 had a nucleotide sequence of SEQ ID NO: 2. The 16S rDNA nucleotide sequence of Lactobacillus johnsonii CH32 was identified by BLAST in Genebank (http://www.ncbi.nlm.nih.gov/), and as a result, a Lactobacillus johnsonii strain having the same 16S rDNA nucleotide sequence as that of Lactobacillus johnsonii CH32 was not found, and Lactobacillus johnsonii CH32 showed a homology of 99% with the 16S rDNA sequence of Lactobacillus johnsonii strain JCM 2012.
Among the strains shown in Table 1 above, Bifidobacterium longum CH57 was a gram-positive anaerobic bacillus, did not form spores, and showed very low viability under aerobic conditions. Furthermore, Bifidobacterium longum CH57 was thermally unstable. Furthermore, Bifidobacterium longum CH57 actively produced glucosidase, but did not produce β-glucuronidase. In addition, to chemically classify Bifidobacterium longum CH57, the 16S rDNA thereof was analyzed, and as a result, it was shown that Bifidobacterium longum CH57 had a nucleotide sequence of SEQ ID NO: 3. The 16S rDNA nucleotide sequence of Bifidobacterium longum CH57 was identified by BLAST in the Genebank (http://www.ncbi.nlm.nih.gov/), and as a result, a Bifidobacterium longum strain having the same 16S rDNA nucleotide sequence as that of Bifidobacterium longum CH57 was not found, and Bifidobacterium longum CH57 showed a homology of 99% with the 16S rDNA sequence of Bifidobacterium longum strain CBT-6.
In addition, among the physiological characteristics of Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longum CH57, the carbon source utilization was analyzed using a sugar fermentation by an API kit (model: API 50 CHL; manufactured by BioMerieux's, USA). Table 2 below shows the results of analyzing the carbon source utilization of Lactobacillus brevis CH23; Table 3 below shows the results of analyzing the carbon source utilization of Lactobacillus johnsonii CH32; and Table 4 below shows the results of analyzing the carbon source utilization of Bifidobacterium longum CH57. In Tables 2, 3 and 4, “+” indicates the case in which carbon source utilization is positive; “−” indicates the case in which carbon source utilization is negative; and “±” indicates the case in which carbon source utilization is ambiguous. As shown in Tables 2, 3 and 4 below, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 and Bifidobacterium longum CH57 showed carbon source utilization different from that of other strains of the same species with respect to some carbon sources.
L. brevis
L. brevis
L. brevis
1)
L. brevis
1)
1)Suriasih K., Aryanta WR, MahardikaG, Astawa NM. Microbiological and Chemical Properties of Kefir Made of Bali Cattle Milk. Food Science and Quality Management 2012; 6: 112-22.
L. johnsonii
L. johnsonii
L. johnsonii
2)
L. johnsonii
2)
2)Pridmore RD, Berger B, Desiere F, Vilanova D, Barretto C, Pittet AC, Zwahlen MC, Rouvet M, Altermann E, Barrangou R, Mallet B, Mercenier A, Klaenhammer T, Arigoni F, Schell MA. The genome sequence of the probiotic intestinal bacterium Lactobacillus johnsonii NCC 533. Proc Natl Acad Sci USA. 2004 Feb. 24; 101(8): 2512-7.
B. longum
B. longum
B. longum
3)
B. longum
3)
3)Lukacova D, Karovucova J, Greifova M, Greif G, Sovcikova A, Kohhajdova Z. In vitro testing of selected probiotic characteristics of Lactobacillus plantarum and Bifidobacterium longum. Journal of Food and Nutrition Research 2006; 45: 77-83.
(4) Information on Deposition of Lactic Acid Bacteria
The present inventors deposited Lactobacillus brevis CH23 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depositary authority, on Sep. 1, 2015 under accession number KCCM 11762P. Furthermore, the present inventors deposited Lactobacillus johnsonii CH32 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea), an international depositary authority, on Sep. 1, 2015, under accession number KCCM 11763P. Furthermore, the present inventors deposited Bifidobacterium longum CH57 with the Korean Culture Center of Microorganisms (address: Yurim Building, 45, Hongjenae 2ga-gil, Seodaemun-gu, Seoul, Korea)), an international depositary authority, on Sep. 1, 2015 under accession number KCCM 11764P.
2. Evaluation of the Effect of Lactic Acid Bacteria on Alleviation of Intestinal Damage or Intestinal Permeability
In order to evaluate the effect of the lactic acid bacteria isolated from kimchi or human feces, on the alleviation of intestinal damage or internal permeability, the antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity, β-glucuronidase (harmful intestinal enzyme) inhibitory activity, tight junction protein expression inducement activity and memory improvement activity of the lactic acid bacteria were measured.
Antioxidant Activity
DPPH (2,2-diphenyl-1-picrylhydrazyl) was dissolved in ethanol to a concentration of 0.2 mM to prepare a DPPH solution. A lactic acid bacteria suspension (1×108 CFU/ml) or a vitamin C solution (1 g/ml) was added to 0.1 ml of the DPPH solution and cultured at 37° C. for 20 minutes. The culture was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. Next the absorbance of the supernatant at 517 nm was measured, and the antioxidant activity of the lactic acid bacteria was calculated.
Lipopolysaccharide (LPS) Production Inhibitory Activity
Pathogenic bacteria, such as Escherichia coli, Klebsiella pneumonia and Proteus mirabilis were separated from the elderly subject and cultured. Next, the pathogenic bacteria (1×105 CFU respectively) and lactic acid bacteria (1×105 CFU) were transplanted into 10 of sterile general anaerobic medium (GAM broth; Nissui Pharmaceutical, Japan) and anaerobically cultured for 24 hours.
Next, the culture was sonicated for about 1 hour to disrupt the outer cell membrane of the bacteria, and centrifuged at 5000×g, and the supernatant was collected. Next, the content of LPS (lipopolysaccharide) (which is a typical endotoxin) in the supernatant was measured by a LAL (Limulus Amoebocyte Lysate) assay kit (manufactured by Cape Cod Inc., USA). In addition, in order to evaluate the E. coli proliferation inhibitory activity of the lactic acid bacteria, the culture obtained through the same experiment as described above was diluted 1000-fold and 100000-fold and cultured in DHL medium, and then the number of E. coli, Klebsiella pneumonia and Proteus mirabilis was counted.
β-Glucuronidase Inhibitory Activity
0.1 ml of 0.1 mM p-nitrophenyl-β-D-glucuronide solution, 0.2 ml of 50 mM phosphate buffered saline and 0.1 ml of a lactic acid bacteria suspension (prepared by suspending of a lactic acid bacteria culture in 5 ml of physiological saline) were placed in a reactor and subjected to an β-glucuronidase enzymatic reaction, and 0.5 ml of 0.1 mM NaOH solution was added to stop the reaction. Next, the reaction solution was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected. Then, the absorbance of the supernatant at 405 nm was measured.
Tight Junction Protein Expression Inducement Activity
Caco2 cells obtained from the Korean Cell Line Bank were cultured in RPMI 1640 medium for 48 hours, and then the cultured Caco2 cells were dispensed to each well of a 12-well plate at a density of 2×106 cells/well. Next, each well was treated with 1 μg of LPS (lipopolysaccharide) or a combination of 1 μg of LPS (lipopolysaccharide) and 1×104 CFU of lactic acid bacteria and incubated for 24 hours. Next, the cultured cells were collected from each well, and the expression level of tight junction protein ZO-1 in the cells was measured by an immunoblotting method.
Memory Improvement Activity
SH-SY5Y cells obtained from the Korean Cell Line Bank were cultured in DMEM medium, in which 10% of FBS and 1% of antibiotics were added, and dispensed to each well of a 12-well plate at a density of 2×106 cells/well. Next, along with lactic acid bacteria (1×104 CFU/), LPS (lipopolysaccharide), separated from Proteus mirabilis, was added to each well at the concentration of 0.2 mg/
and cultured, and then the level of inhibition on NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activity and expression level of α-synuclein were measured by the immunoblotting method. NF-κB is known as a substance that causes tissue damage by inflammation reaction and aging-related diseases like Alzheimer's disease, while α-synuclein is known as a substance that causes Parkinson's disease.
The antioxidant activity, lipopolysaccharide (LPS) production inhibitory activity, β-glucuronidase inhibitory activity and tight junction protein expression inducement activity of the lactic acid bacteria isolated from kimchi or human feces were measured, and the results of the measurement are shown in Tables 5, 6 and 7 below. As shown in Tables 5, 6 and 7 below, Lactobacillus curvatus CH5, Lactobacillus sakei CH11, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32, Bifidobacterium pseudocatenulatum CH38 and Bifidobacterium longum CH57 had excellent antioxidant activity, strongly inhibited lipopolysaccharide (LPS) production and β-glucuronidase activity, strongly induced the expression of tight junction protein, strongly inhibited the NF-κB activity, and strongly inhibited the expression of α-synuclein. These lactic acid bacteria have an excellent antioxidant effect, have an excellent effect of inhibiting the enzymatic activity of intestinal flora's harmful bacteria associated with inflammation and carcinogenesis, inhibit the production of endotoxin LPS (lipopolysaccharide) produced by intestinal flora's harmful bacteria, and induce the expression of tight junction protein. Thus, these lactic acid bacteria can improve intestinal permeability syndrome. Also, the lactic acid bacteria can alleviate Alzheimer's disease and Parkinson's disease, as the lactic acid bacteria inhibit not only the production of LPS (lipopolysaccharide), an endotoxin of intestinal microorganisms, such as K. pneumoniae, E. coli and P. mirabilis, associated with Alzheimer's disease or Parkinson's disease, but also the production or activity of substances inducing neurodegeneration. In particular, the lactic acid bacteria are expected to bring a synergistic effect through various functions, such as alleviation of Intestinal Permeability Syndrome and inhibition on the production of endotoxin and the activity or production of substances inducing neurodegenerative.
K. pneupomiae
E. coli
P. mirabilis
L. acidophilus
L. acidophilus
L. acidophilus
L. brevis
L. curvatus
L. brevis
L. casei
L. planantrum
L. sakei
L. curvatus
L. sakei
L. curvatus
L. plantarum
L. fermentum
L. fermentum
L. gasseri
L. paracasei
L. helveticus
L. helveticus
L. johnsonii
K. pneupomiae
E. coli
P. mirabilis
L. johnsonii
L. johnsonii
L. brevis
L. paracasei
L. kimchi
L. gasseri
L. paracasei
L. pentosus
L. pentosus
L. reuteri
L. sakei
L. johnsonii
L. sakei
L. sakei
L. plantarum
L. sanfranciscensis
B. pseudocatenulatum
B. pseudocatenulatum
B. adolescentis
B. adolescentis
K. pneupomiae
E. coli
P. mirabilis
B. adolescentis
B. animalis
B. animalis
B. bifidum
B. bifidum
B. breve
B. breve
B. breve
B. catenulatum
B. catenulatum
B. dentium
B. infantis
B. infantis
B. infantis
B. longum
B. longum
B. longum
B. longum
B. longum
B. longum
(1) Selection of Lactic Acid Bacteria for Experiment to Measure Improvement Effect for Cognitive Ability
The following 13 types of lactic acid bacteria were selected as lactic acid bacteria for the experiment to measure improvement effect for cognitive ability among a total of 60 types of lactic acid bacteria isolated from Kimchi or human feces.
Lactobacillus acidophilus CH3, Lactobacillus curvatus CH5, Lactobacillus sakei CH11, Lactobacillus fermentum CH15, Lactobacillus johnsonii CH21, Lactobacillus brevis CH23, Lactobacillus johnsonii CH32, Lactobacillus plantarum CH35, Bifidobacterium pseudocatenulatum CH38, Bifidobacterium adolescentis CH41, Bifidobacterium longum CH56, Bifidobacterium longum CH57, Bifidobacterium longum CH59
(2) Passive Avoidance Test and the Result
In the Passive Avoidance test apparatus, which is divided into the first and second space, there is a guillotine-like door that connects the two spaces. By using lightings, the first space was kept bright while the second space was kept dark. On the floor of the second space that was kept dark was placed an electric grid that flowed 0.5 mA of electric shock for three seconds in the case of the animal moving to the dark space.
The selected 13 types of lactic acid bacteria were each suspended in physiological saline at the concentration of 1×1010 CFU/, and then the suspension was administered to mice of the test animal (5-week-old ICR male mice from Raonbio), at the dosage of 0.1
(equivalent to 1×109 CFU of lactic acid bacteria) on a daily basis for three days. Also, 0.1
of physiological saline were administered to the mice corresponding to the normal group and memory impairment group on a daily basis for three days. The mice corresponding to the positive control group were administered with 5 mg/kg (body weight) of the positive control drug, donepezil (treatment for Alzheimer's disease and Alzheimer's disease-type dementia), on a daily basis for three days. The number of mice per the experimental group was 9.
On the second day after administering the drug, the above mice were placed in the first space which was bright, observed for 20 seconds, and allowed to move into the second space that was kept dark by opening the guillotine-like door. The mice, which did not move to the second space for 60 seconds after the door was opened, were excluded from the experiment. Once the mice move to the second space, the guillotine-like door is closed, and the mice receive the electric shock at 0.5 mA through the grid on the floor for three seconds. The mice are forced to remember the shock.
On the third day after administering the drug, an hour after administering lactic acid bacteria or positive control drug, 1 mg/kg (body weight) of scopolamine (cholinergic blocking drug inducing cognitive ability disorder or memory damage), dissolved in distilled water, was administered in the abdomen while the normal group was administered with physiological saline in the abdomen. The experiment was conducted by using Passive Avoidance experiment apparatus after 30 minutes of administration of scopolamine. With 10 seconds of observation, the guillotine-like door is opened, the time that the mice in each experimental group took to have all of their four feet inside the second space was measured up to 300 seconds, whose result is shown in Table 8. The result in Table 8 shows that longer latency time means better performance in learning passive avoidance and recovering short-term memory.
Lactobacillus acidophilus CH3
Lactobacillus curvatus CH5
Lactobacillus sakei CH11
Lactobacillus fermentum CH15
Lactobacillus johnsonii CH21
Lactobacillus brevis CH23
Lactobacillus johnsonii CH32
Lactobacillus plantarum CH35
Bifidobacterium pseudocatenulatum CH38
Bifidobacterium adolescentis CH41
Bifidobacterium longum CH56
Bifidobacterium longum CH57
Bifidobacterium longum CH59
As shown in Table 8, when model animals, induced to have cognitive ability disorder or memory impairment by scopolamine, were administered with Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 or Bifidobacterium longum CH57, latency time significantly increased compared to the memory impairment group administered with only scopolamine, and especially, administration of Lactobacillus johnsonii CH32 showed better effect than the administration of donepezil, a commercial treatment.
(3) Y-Maze Test and the Result
Apparatus used in the Y-maze test consists of three arms with each being 42 cm in length, 3 cm in width and 12 cm in height. The three arms, made from black polyvinyl resin, were placed at an angle of 120 degrees.
The selected 13 types of lactic acid bacteria were each suspended in physiological saline at the concentration of 1×1010 CFU/, and then the suspension was administered to mice of the test animal (5-week-old ICR male mice from Raonbio), at the dosage of 0.1
(equivalent to 1×109 CFU of lactic acid bacteria) on a daily basis for three days. Also, 0.1
of physiological saline were administered to the mice corresponding to the normal group and memory impairment group on a daily basis for three days. The mice corresponding to the positive control group were administered with 5 mg/kg (body weight) of the positive control drug, donepezil (treatment for Alzheimer's disease and Alzheimer's disease-type dementia), on a daily basis for three days. The number of mice per experimental group was 9.
An hour after the final administration of lactic acid bacteria or positive control drug, 1 mg/kg (body weight) of scopolamine (cholinergic blocking drug inducing cognitive ability disorder or memory impairment), dissolved in distilled water, was administered in the abdomen while the normal group was administered with physiological saline in the abdomen. half an hour after the administration of scopolamine, the mouse was carefully placed in one of the three arms, A, B, and C, in the Y-maze and allowed to freely move for eight minutes, and then the arm in which the mouse moved was recorded. In the experiment, the arm was recorded as a case in which the test animal moved only when the entire body including the tail was inside the arm, or when the mouse moved back in. In the case of the mouse moving into each arm sequentially, one point (actual alteration) was allocated. Alteration behavior is defined as a case where the mouse moved in all of the three arms sequentially. The experimental result was calculated by the formula below (Sarter, M. et al., Psychopharmacology., 94, pp491-495, 1998), as shown in Table 9 below.
Spontaneous alteration (%)=actual alternation/maximum alternation×100 (maximum alteration: the total number of entries−2)
Table 9 below shows that greater alternation behavior (unit: %) means better recovery of learning and spatial memory.
Lactobacillus acidophilus CH3
Lactobacillus curvatus CH5
Lactobacillus sakei CH11
Lactobacillus fermentum CH15
Lactobacillus johnsonii CH21
Lactobacillus brevis CH23
Lactobacillus johnsonii CH32
Lactobacillus plantarum CH35
Bifidobacterium pseudocatenulatum CH38
Bifidobacterium adolescentis CH41
Bifidobacterium longum CH56
Bifidobacterium longum CH57
Bifidobacterium longum CH59
As shown in Table 9, when model animals, induced to have cognitive ability disorder or memory impairment by scopolamine, were administered with Lactobacillus brevis CH23, Lactobacillus johnsonii CH32 or Bifidobacterium longum CH57, alternation behavior significantly increased compared to the memory impairment group administered with only scopolamine, and especially, administration of Lactobacillus johnsonii CH32 showed excellent effect equivalent to the administration of donepezil, a commercial treatment.
4. Preparation of Red Bean Ferment by Using Lactic Acid Bacteria and Measurement of Improvement Effect of the Red Bean Ferment for Cognitive Ability
(1) Preparation of Red Bean Ferment
Lactobacillus johnsonii CH32 was cultured in the edible TS medium and centrifuged at 10,000 gf for 20 minutes to yield Lactobacillus johnsonii CH32 biomass. The yielded Lactobacillus johnsonii CH32 biomass was washed with physiological saline twice and suspended in 100 of physiological saline to prepare the suspension of Lactobacillus johnsonii CH32 biomass. Next, 10 g of finely crushed red bean was added to 90
of suspension of Lactobacillus johnsonii CH32 biomass, which was then cultured for 24 hours to ferment red bean. Next, the red bean ferment liquid was freeze-dried to yield red bean ferment.
(2) Passive Avoidance Test and the Result
Except the type and amount of administered drug, Passive Avoidance test was conducted in the same way as stated earlier herein, and the result is shown in Table 10 below. As shown in Table 10, administration of the red bean suspension or Lactobacillus johnsonii CH32 (daily dosage: 2×108 CFU/mouse) failed to significantly improve the damaged memory while the administration of the red bean ferment significantly improved the damaged memory.
Lactobacillus johnsonii CH32
(3) Y-Maze Test and the Result
Except the type and amount of administered drug, Y-maze test was conducted in the same way as stated earlier herein, and the result is shown in Table 11 below. As shown in Table 11, administration of the red bean suspension or Lactobacillus johnsonii CH32 (daily dosage: 2×108 CFU/mouse) to the mouse failed to significantly improve the damaged memory while the administration of the red bean ferment significantly improved the damaged memory.
Lactobacillus johnsonii CH32
5. Measurement of Improvement Effect of Lactic Acid Bacteria for Cognitive Ability by Using Model Animals Induced to have Memory Impairment by Escherichia coli
(1) Inducement of Memory Impairment and Administration of Drug
Escherichia coli K20 separated from the animal with impaired memory was administered to the mouse to induce memory impairment, and the improvement effect of lactic acid bacteria for cognitive ability was measured by conducting Y-maze test and Passive Avoidance test.
In more detail, Escherichia coli K20 was administered to a mouse (5-week-old ICR male mice from Raonbio) that was fed for a week in the animal laboratory at the dosage of 1×109 CFU on a daily basis for five days to induce memory impairment. In the meantime, the normal group was administered with physiological saline instead of Escherichia coli K20. From the next day of the final administration of Escherichia coli K20, lactic acid bacteria suspension (lactic acid bacteria suspended in physiological saline at the concentration of 1×1010 CFU/) was administered to the mouse induced to have memory impairment at the dosage of 0.1
(equivalent to 1×109 CFU of lactic acid bacteria) on a daily basis for five days. Also, the normal group and memory impairment-induced group were administered with 0.1
of physiological saline on a daily basis for five days. In addition, lactic acid bacteria suspension (lactic acid bacteria suspended in physiological saline at the concentration of 1×1010 CFU/
) was administered to the normal group at the dosage of 0.1
(equivalent to 1×109 CFU of lactic acid bacteria) on a daily basis for five days, which was used as a positive control group. The number of mice per experimental group was 9.
(2) Y-Maze Test
An hour after the final administration of drug, the mouse was carefully placed in one of the three arms, A, B, and C, in the Y-maze and allowed to freely move for eight minutes, and then the arm in which the mouse moved was recorded. In the experiment, the arm was recorded as a case in which the test animal moved only when the entire body including the tail was inside the arm, or when the mouse moved back in. In the case of the mouse moving into each arm sequentially, one point (actual alteration) was allocated. Alteration behavior is defined as a case where the mouse moved in all of the three arms sequentially. The experimental result was calculated by the formula below (Sarter, M. et al., Psychopharmacology., 94, pp491-495, 1998).
Spontaneous alteration (%)=actual alternation/maximum alternation×100 (maximum alteration: the total number of entries−2)
(3) Passive Avoidance Test
On the second day after administering the drug, the above mice were placed in the first space which was bright, observed for 20 seconds, and allowed to move into the second space that was kept dark by opening the guillotine-like door. The mice, which did not move to the second space for 60 seconds after the door was opened, were excluded from the experiment. Once the mice move to the second space, the guillotine-like door is closed, and the mice receive the electric shock at 0.5 mA through the grid on the floor for three seconds. The mice are forced to remember the shock.
An hour after the final administration of drug, the experiment was conducted by using the Passive Avoidance test apparatus. With 10 seconds of observation, the guillotine-like door is opened, the time that the mice in each experimental group took to have all of their four feet inside the second space was measured up to 300 seconds.
(3) Measurement of the Expression Level of Nerve Growth-Promoting Factor
Two hours after the final administration of drug, hippocampus was separated from the mice of each experimental group, and the expression level of BDNF (Brain-Derived Neurotrophic Factor), known as a nerve growth-promoting factor, and the activity level of CREB (Cydic AMP Response Element-Binding), known as a memory-improving transcription factor, were measured.
(4) Experimental Result
From
6. Measurement of Improvement Effect of Lactic Acid Bacteria for Cognitive Ability by Using Model Animals with Induced Memory Impairment by Proteusmirabilis
Proteus mirabilis K21 separated from the animal with damaged memory was administered to the mice to induce memory impairment (similar symptoms to Parkinson's disease), and Y-maze test was conducted to measure the improvement effect of lactic acid bacteria for cognitive ability.
In more detail, Proteus mirabilis K21 was administered to a mouse (5-week-old ICR male mice from Raonbio) that was fed for a week in the animal laboratory at the dosage of 1×109 CFU on a daily basis for five days to induce memory impairment. In the meantime, the normal group was administered with physiological saline instead of Proteus mirabilis K21. From the next day of the final administration of Proteus mirabilis K21, lactic acid bacteria suspension (lactic acid bacteria suspended in physiological saline at the concentration of 1×1010 CFU/) was administered to the mouse induced to have memory impairment at the dosage of 0.1
(equivalent to 1×109 CFU of lactic acid bacteria) on a daily basis for five days. Also, the normal group and memory impairment-induced group were administered with 0.1
of physiological saline on a daily basis for five days. In addition, donepezil (treatment for Alzheimer's disease and Alzheimer's disease-type dementia), a positive control drug, was administered to the positive control group at the dosage of 5 mg/kg (body weight) on a daily basis for five days. The number of mice per experimental group was 9.
An hour after the final administration of drug, the mouse was carefully placed in one of the three arms, A, B, and C, in the Y-maze and allowed to freely move for eight minutes, and then the arm in which the mouse moved was recorded. In the experiment, the arm was recorded as an arm in which the test animal moved only when the entire body including the tail was inside the arm, or when the mouse moved back in. In the case of the mouse moving into each arm sequentially, one point (actual alteration) was allocated. Alteration behavior is defined as a case where the mouse moved in all of the three arms sequentially. The experimental result was calculated by the formula below (Sarter, M. et al., Psychopharmacology., 94, pp491-495, 1998), as shown in Table 12 below.
Spontaneous alteration (%)=actual alternation/maximum alternation×100 (maximum alteration: the total number of entries−2)
Table 12 below shows that greater alternation behavior (unit: %) means better recovery of learning and spatial memory.
Proteus mirabilis K21
mirabilis K21 and donepezil
Lactobacillus acidophilus CH3
Lactobacillus curvatus CH5
Lactobacillus sakei CH11
Lactobacillus fermentum CH15
Lactobacillus johnsonii CH21
Lactobacillus brevis CH23
Lactobacillus johnsonii CH32
Lactobacillus plantarum CH35
Bifidobacterium pseudocatenulatum CH38
Bifidobacterium adolescentis CH41
Bifidobacterium longum CH56
Bifidobacterium longum CH57
Bifidobacterium longum CH59
As shown in Table 12, administration of Lactobacillus johnsonii CH32 to model animals induced to have memory impairment by Proteus mirabilis K21 flora showed better effect than the administration of donepezil, a commercial treatment.
Although the present invention has been described above with reference to the examples, the scope of the present invention is not limited to these examples, and various modifications are possible without departing from the scope and idea of the present invention. Therefore, the scope of protection of the present invention should be interpreted to include all embodiments falling within the appended claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2015-0130124 | Sep 2015 | KR | national |
| 10-2016-0001312 | Jan 2016 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2016/009961 | 9/6/2016 | WO | 00 |
