KR20230119870A - Method for preparing a composition for improving brain function and food and pharmaceutical composition for improving brain function - Google Patents

Abstract

The present invention relates to a method for manufacturing a composition for improving a brain function, in which Lactobacillus gallinarum KJM-ALOE2112 (KCTC 14824BP), a new lactic acid bacterium isolated from aloe, produces a large amount of sialic acid, which undergoes fermentation, thereby having a preventive or therapeutic effect on dementia, and to food and a pharmaceutical composition for improving the brain function including the composition for improving the brain function.

Description

Method for preparing a composition for improving brain function and food and pharmaceutical composition for improving brain function

In the present invention, Lactobacillus gallinarum KJM-ALOE2112, KCTC 14824BP, a novel lactic acid bacterium isolated from aloe, produces a large amount of sialic acid, which is fermented to have a preventive or therapeutic effect on dementia. It relates to a method for preparing a composition for improving brain function and a food and pharmaceutical composition for improving brain function including the composition for improving brain function.

BACKGROUND OF THE INVENTION Due to the development of medical technology and the improvement of living standards, the average lifespan of a human being has almost doubled over the past half century, and as a result, the proportion of the elderly population to the total population has rapidly increased. According to the 2005 data released by the National Statistical Office, the current average life expectancy of Koreans is 78.5 years, and the proportion of the population aged 65 or older already reached about 7% in 2000, indicating that Korea is rapidly entering an aging society. In addition, it is predicted that Korea will enter an aged society in 2018 and a super-aged society in which the proportion of the elderly population exceeds 20% in 2026.

Along with the rapid increase in the elderly population, the proportion of patients with cognitive impairment and dementia, including various geriatric chronic diseases, is expected to increase. The proportion of the elderly reached 8.8% of the population aged 65 or older. Dementia, which is the biggest problem among geriatric diseases, is a brain syndrome characterized by cognitive impairment caused by degenerative changes in the brain, cerebrovascular disorders, inflammatory disorders of the brain, and metabolic diseases. Cognitive dysfunction not only impairs the independence and quality of life of patients with dementia, but also increases the nursing burden of caregivers, so the need for prevention or treatment of brain diseases is increasing. Accordingly, studies on health functional foods and medicines with brain function improvement such as cerebral circulation, metabolism improving drugs or dementia treatments are being actively conducted, and functionalities that can prevent and treat cognitive dysfunction including dementia as society is gradually aging. The demand for development of materials and food is increasing.

Alzheimer's disease is a neurodegenerative disease of the central nervous system and is the most common form of dementia. Neurotransmitters such as acetylcholine, dopamine, sialic acid, and γ-aminobutyric acid (GABA) in the process of reducing cognitive function and damaging nerve cells in patients with Alzheimer's disease Abnormalities in the transport, production, secretion, and elimination processes play an important role. These neurotransmitters decrease with aging, and memory impairment, a major symptom of dementia, is known to be caused by severe damage to the cholinergic system, which plays an important role in memory and learning. In particular, the symptoms of cognitive decline in Alzheimer's disease are aggravated by damage to nerve cells that produce acetylcholine and increased activity of acetylcholinesterase (AChE), an enzyme that degrades acetylcholine. Dementia targets various pathways as well as its various causes. Representatively, because memory declines due to a decrease in cholinergic activity in brain tissue, the action of acetylcholine, a neurotransmitter, to activate the cholinergic action of the brain neuronal junction. Many studies have been conducted using cholinergic agonists, acetylcholine precursors, and AChE enzyme inhibitors that inhibit the activity of AChE.

There are drugs that can enhance the function of acetylcholine nerve cells according to various mechanisms of action, such as agonists for muscarine acetylcholine receptors, acetylcholine production promoters, and acetylcholinesterase (AChE) inhibitors. has been developed In fact, most Alzheimer's disease drugs currently used in various countries are acetylcholinesterase inhibitors, including tacrine and donepezil. In addition, rivastigmin and galatamine are also used.

Currently, the development of more effective agents (including health functional food materials) to improve cognitive function or memory decline as a degenerative cranial nerve disease is at a higher level than ever. In the end, research is needed to develop an introduction with a new multi-functional effect targeting the mechanism of brain nerve cells for the onset of dementia.

Korean Patent Publication No. 2019-0046770 Japanese Laid-open Patent No. 2018-064494

Lactobacillus gallinarum KJM-ALOE2112, KCTC 14824BP, a new lactic acid bacterium isolated from aloe, produces a large amount of sialic acid, and fermenting it is for improving brain function, which has preventive or therapeutic effects on dementia. It is intended to provide a method for preparing the composition.

In addition, the present invention is a food or pharmaceutical product that can prevent senile dementia diseases such as Alzheimer's by culturing Lactobacillus gallina room, a novel lactic acid bacterium derived from aloe, and further improving brain function including cognitive function and memory from the fermented product. It is intended to provide a composition.

In order to solve the above problems,

In one embodiment of the present invention, (a) preparing a pre-culture solution of lactic acid bacteria by inoculating lactic acid bacteria isolated from fresh aloe leaves into an MRS medium; (b) preparing an aloe polysaccharide extract fermentation medium by adding MRS medium and bird's nest powder to the aloe polysaccharide extract obtained from aloe; (c) preparing an aloe-derived lactic acid bacteria fermentation broth by inoculating the lactic acid bacteria pre-culture solution into the fermentation medium of the aloe polysaccharide extract and fermenting the solution; (d) tyndallizing the aloe-derived lactic acid bacteria fermentation broth; (e) crushing the lactic acid bacteria in the aloe-derived lactic acid bacteria fermentation broth to form a lactic acid bacteria disruption solution; and (f) mixing the tindalized lactic acid bacteria fermented liquid and the lactic acid bacteria disrupted liquid and then drying to prepare a tindalized aloe-derived lactic acid bacteria fermented product.

Separating the lactic acid bacteria from the fresh aloe leaves in step (a) may include separating the lactic acid bacteria from the fresh aloe leaves; Identifying the isolated lactic acid bacteria; And, it may include the step of culturing the isolated and identified lactic acid bacteria in a medium.

Obtaining the aloe polysaccharide extract in step (b) may include harvesting and pre-treating the aloe; and obtaining a mucilaginous aloe polysaccharide extract from the pretreated aloe.

In step (b), the pH of the aloe polysaccharide extract may be corrected to pH6, and the concentration may be adjusted to 10 to 20°Birx.

In step (c), 1 to 5% by weight of the lactic acid bacteria pre-culture solution may be inoculated and cultured with shaking for 60 to 80 hours to ferment.

The step of tindalizing the lactic acid bacteria fermentation broth derived from aloe in step (d) may include heating the lactic acid bacteria fermentation broth at 60 to 150° C. for 10 to 60 minutes; And rapid cooling to 40 ° C. or less may include killing live lactic acid bacteria in the lactic acid bacteria fermentation broth.

The lactic acid bacteria may be Lactobacillus gallinarum.

The aloe may be one or more species selected from the group consisting of aloe vera, aloe arborescens, and aloe saponaria.

In addition, in one embodiment, the present invention provides a food composition for improving brain function comprising, as an active ingredient, a fermented product of aloe-derived lactic acid bacteria using an aloe-derived lactic acid bacterium, Lactobacillus gallina room strain.

The fermented product of lactic acid bacteria derived from aloe may be included in an amount of 5 to 20 parts by weight based on 100 parts by weight of the food composition for improving brain function.

In addition, in one embodiment, the present invention provides a pharmaceutical composition for improving brain function comprising, as an active ingredient, a fermented product of aloe-derived lactic acid bacteria using an aloe-derived lactic acid bacterium, Lactobacillus gallina room strain.

The aloe-derived lactic acid bacteria fermented product may contain 40 to 99 parts by weight based on 100 parts by weight of the pharmaceutical composition for improving brain function.

The present invention relates to a composition for improving brain function (cognitive function or memory ability) containing, as an active ingredient, a novel fermentation product of aloe lactobacillus with increased sialic acid content, and the novel fermentation product with increased sialic acid content of the present invention. silver is not only excellent in inhibiting the activity of acetylcholine degrading enzyme, which inhibits neurotransmission by destroying acetylcholine, but also has excellent ability to restore memory and cognitive ability in animal models with memory impairment induced by scopolamine. It can be usefully used for the prevention or treatment of degenerative cranial nerve diseases such as Alzheimer's dementia, and can also be usefully used as a health functional food composition or pharmaceutical composition for improving or enhancing deteriorated memory, learning ability and cognitive function.

1 shows a manufacturing process of tindalized aloe lactic acid bacteria fermented powder according to an embodiment of the present invention.
2 is a photograph of lactic acid bacteria colonies formed on Aloe vera BCP-MRS (a) and BS medium (b) according to an embodiment of the present invention.
3 is a photograph taken by FE-SEM of morphological characterization of aloe-derived lactic acid bacteria according to an embodiment of the present invention.
4 shows the nucleotide sequence (forward blast) of 16S rRNA of aloe-derived lactic acid bacteria according to an embodiment of the present invention.
5 shows the nucleotide sequence (reverse blast) of 16S rRNA of aloe-derived lactic acid bacteria according to an embodiment of the present invention.
6 shows a phylogenetic tree (Lactobacillus gallinarum) of aloe-derived lactic acid bacteria according to an embodiment of the present invention.
7 is a microorganism safety deposit certificate of aloe-derived lactic acid bacteria (Lactobacillus gallinarum KJM-ALOE2112) according to an embodiment of the present invention.
8 is a microorganism patent deposit of aloe-derived lactic acid bacteria (Lactobacillus gallinarum KJM-ALOE2112, KCTC 14824BP) according to an embodiment of the present invention.
9a and 9b are analysis of sialic acid components before (FIG. 9a) and after (FIG. 9b) fermentation of aloe-derived lactic acid bacteria according to an embodiment of the present invention (KOTITI Testing & Research Institute official test report, fluorescent label kit and HPLC-FLD) It is an official test report showing the results.
10 is a graph showing the results of in vitro brain nerve cell proliferation evaluation using the MTT assay according to an embodiment of the present invention.
11 is a graph and chart showing the results of in vitro antioxidant activity evaluation using the DPPH assay according to an embodiment of the present invention.
12 is a result of HPLC evaluation of oxidized glutathione (GSSG) (a) and reduced glutathione (GSH) (b) according to an embodiment of the present invention.
13 is a graph showing the change in the slope of the absorbance with respect to a certain amount of GSH according to an embodiment of the present invention.
14 is a result of HPLC evaluation of total sialic acid in vitro cell influx according to an embodiment of the present invention.
15 is a result of evaluating the in vivo efficacy for AChE activity according to an embodiment of the present invention.
16 is a graph showing the evaluation of spatial memory recovery through the Morris water maze test according to an embodiment of the present invention.
17 is a photograph of histologically analyzing the in vivo efficacy evaluation for amyloid-β (Aβ) activity according to an embodiment of the present invention.

Since the present invention can make various changes and have various embodiments, specific embodiments will be illustrated in the drawings and described in detail in the detailed description.

However, this is not intended to limit the present invention to specific embodiments, and should be understood to include all modifications, equivalents, and substitutes included in the spirit and scope of the present invention.

In the present invention, the term "comprises" or "has" is intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or more other features It should be understood that the presence or addition of numbers, steps, operations, components, parts, or combinations thereof is not precluded.

In the present invention, “sialic acid” is a general term for acyl derivatives of neuraminic acid having a nine-carbon skeleton, and it is said that more than 20 kinds of derivatives are currently known in the animal world and more than 30 kinds of derivatives exist in the natural world. It is known. Among them, sialic acid, also known as N-acetylneuraminic acid, is widely present as a component of oligosaccharides, polysaccharides, glycoproteins, and glycolipids in animal brains, organs, tissues, blood, mucus, milk, etc. It is absent in lactic acid bacteria, but is relatively widely distributed in microorganisms such as lactic acid bacteria. In addition, sialic acid is a component of glycoproteins or glycolipids on the cell surface of cell membranes and is located at the end to exert various biological functions, and various physiological functions have been elucidated due to recent active research. For example, sialic acid is synthesized from fructose-6-phosphate via N-acetylamannoseamine in mammals. Sialic acid is a component of ganglioside or glycoprotein in the brain, and is particularly abundant in the brain or central nervous system, and its amount increases rapidly during infancy. It is known. However, in the case of neonates and the elderly, it was found through animal experiments that the activity of UDP-N-acetylglucosamine-2'-epimerase, which synthesizes N-acetylmannoseamine, is weak. And it has been found to be helpful in brain development of the elderly. In particular, sialic acid has been found to be effective in the treatment of Alzheimer's disease by acting as an inhibitor of butyrylcholinesterase (BuChE). In general, sialic acid forms N-acetylgalactosamine (GalNAc) through a glycosidic bond, and this compound plays an important role in the proper functioning of synapses and is known to help improve memory.

In the present invention, the "edible bird's nest" is a nest made of saliva and feathers secreted by a small sea petrel called 'Aerodramus funiphagus', which mainly lives in Southeast Asia, and has been since the Ming Dynasty of China. It is a traditional Chinese food consumed. Bird's nest is not only rich in vitamins, hormones and fatty acids, but also contains various nutritional components such as proteins, carbohydrates, mineral salts, amino acids, and sialic acid. In particular, the mechanism of cognitive function improvement in bird's nest has been found to be covalently bound to form N-acetyl galactosamine (GalNAc), a compound important for the proper functioning of cranial nerve synapses, in sialic acid in bird's nest extract, and GalNAc also has brain function ( It has been shown to improve memory and cognitive function. In addition, sWGA and GSLII were negative in the hippocampal subfields of all brains studied, while GalNAc and GluNAc, which are sialic acid substances, were positive. It has been shown to be effective in dementia and Alzheimer's, etc.). It is widely known as a suitable material for extracting sialic acid, as there is 2,000 times the amount of sialic acid in the bird's nest and 200 times that of royal jelly. The carbohydrate component of bird's nest has various pharmacological effects on the human body. Among them, sialic acid, which is contained at a concentration of about 60 to 160 mg/kg, is neuraminic acid with a 9-carbon skeleton. It is a generic term for acyl derivatives of , and more than 20 species are currently known in the animal kingdom.

In the present invention, "lactic acid bacteria" have been used for a long time, and beneficial effects such as intestinal function, anticancer effect, immune enhancing effect, and neurotransmitter production of lactic acid bacteria have been reported. In addition, during the fermentation process, various metabolites such as lactic acid and a number of organic acids are produced (antibacterial substances such as bacteriocin having antibacterial effects, antioxidant substances, immunoactive substances, neurotransmitters such as GABA and sialic acid, etc.) It is known to play an important role in affecting flavor and product life or regulating human physiological activity and health functions. In addition, as probiotics, it has been reported to play various roles such as improving intestinal flora, immunomodulating action, lowering cholesterol, inhibiting the growth of pathogenic bacteria, and improving lactose intolerance while living in the human intestine. Research on various physiologically active substances produced by lactic acid bacteria is being actively carried out, and it is being used as a variety of materials in the fields of health functional food, cosmetics, and pharmaceuticals.

Aloe has been widely used in the treatment of diseases as a folk remedy for thousands of years, both in the East and the West. In the Orient, aloe is called Nohoe, and it was first listed as a chastity item in 'Gaebo Herb' in the Song Dynasty of China, and the medicinal effects of aloe are described as 'clearing the eyes, treating convulsions, and controlling the five senses'. In the meantime, many studies on aloe have been conducted, and in studies using aloe extract, the treatment effect on hypotension, the accumulation of calcium / phosphorus in bones, the treatment effect of arteriosclerosis, the treatment effect of leg ulcers and skin diseases, the treatment effect of focal pulmonary tuberculosis, Composition of anti-inflammatory ingredients, therapeutic effect on eye disease, role as a moisturizer in cosmetics, immune action, weight control action, brain nerve cell protection and learning ability improvement action, antioxidant action, prostatitis and psoriasis treatment effect, etc. The research was reported and a new lactic acid bacterium, Lactobacillus gallina room, which lives in aloe was discovered, and excellent pharmacological efficacy could be expected.

Therefore, in the present invention, the fermented product of Lactobacillus gallina room, a novel aloe lactic acid bacterium with increased sialic acid content, was tested in a passive avoidance test (SAMP8), an animal model for memory and learning impairment known as an animal model for dementia. test) and grading score of senescence, neurotransmitters and their metabolites, the ratio of metabolites to neurotransmitters, and the activity of acetylcholinesterase were evaluated to obtain significant results. It provides a method for preparing a composition for improving brain function (cognitive function or memory ability) and a food and pharmaceutical composition.

Specifically, the present invention includes the steps of (a) preparing a pre-culture solution of lactic acid bacteria by inoculating lactic acid bacteria isolated from fresh aloe leaves into an MRS medium; (b) preparing an aloe polysaccharide extract fermentation medium by adding MRS medium and bird's nest powder to the aloe polysaccharide extract obtained from aloe; (c) preparing an aloe-derived lactic acid bacteria fermentation broth by inoculating the lactic acid bacteria pre-culture solution into the fermentation medium of the aloe polysaccharide extract and fermenting the solution; (d) tyndallizing the aloe-derived lactic acid bacteria fermentation broth; (e) crushing the lactic acid bacteria in the aloe-derived lactic acid bacteria fermentation broth to form a lactic acid bacteria disruption solution; and (f) mixing the tindalized lactic acid bacteria fermented liquid and the lactic acid bacteria disrupted liquid and then drying to prepare a tindalized aloe-derived lactic acid bacteria fermented product.

Separating the lactic acid bacteria from the fresh aloe leaves in step (a) may include separating the lactic acid bacteria from the fresh aloe leaves; Identifying the isolated lactic acid bacteria; And, it may include the step of culturing the isolated and identified lactic acid bacteria in a medium.

Preparation of the lactic acid bacteria pre-culture solution in step (a) is to sterilize the MRS medium for pre-culture, inoculate and shake the lactic acid bacteria to secure the number of lactic acid bacteria of 1 × 10 ¹⁰ to 1 × 10 ¹² CFU / ㎖ or more, and then lactic acid bacteria It may include preparing a pre-culture solution.

Sterilization of the MRS medium may be carried out at 100 to 130 ° C for 10 to 20 minutes, and lactic acid bacteria strains are inoculated and shaken cultured at 37 ° C for preferably 24 to 32 hours, most preferably 28 hours. A pre-culture solution can be prepared.

The lactic acid bacteria may be Lactobacillus gallinarum.

The aloe may be one or more species selected from the group consisting of aloe vera, aloe arborescens, and aloe saponaria.

In step (b), an MRS medium and bird's nest powder are added to the aloe polysaccharide extract obtained from aloe to prepare an aloe polysaccharide extract fermentation medium.

Obtaining the aloe polysaccharide extract in step (b) may include harvesting and pre-treating the aloe; and obtaining a mucilaginous aloe polysaccharide extract from the pretreated aloe.

In the step (b), the pH of the aloe polysaccharide extract may be corrected to pH6 and the concentration adjusted to 10 to 20°Birx before adding the MRS medium and bird's nest powder.

As a health functional food of the aloe family, the daily intake should contain 100 to 200 mg/20g of total polysaccharide content according to the Ministry of Food and Drug Safety, so the aloe polysaccharide extract may have a content of 10% by weight or more.

In the step (c), the fermentation medium of the aloe polysaccharide extract prepared in the step (a) is inoculated with the pre-culture of the lactic acid bacteria prepared in the step (b) and fermented at 37° C. for 68 to 76 hours, so that the lactic acid bacteria in the aloe polysaccharide extract are fermented. prepare a fermentation broth. In step (c), the MRS medium and bird's nest powder added to the aloe polysaccharide extract fermentation medium may be fermented with aloe lactic acid bacteria. Bird's nest powder is fermented by lactic acid bacteria to produce metabolites, that is, swallow's nest powder is fermented by lactic acid bacteria, and sialic acid can be increased in metabolites before fermentation (lactic acid bacteria alone) by not adding bird's nest powder.

In step (c), 1 to 5% by weight of the lactic acid bacteria pre-culture solution may be inoculated and cultured with shaking for 60 to 80 hours to ferment. For example, the lactic acid bacteria pre-culture solution may be inoculated with 1 to 4% by weight, 1 to 3% by weight or 1 to 2% by weight.

After preparing the lactic acid bacteria fermentation broth, the lactic acid bacteria fermentation broth is tindaled in the step (d).

The step of tindalizing the lactic acid bacteria fermentation broth derived from aloe in step (d) may include heating the lactic acid bacteria fermentation broth at 60 to 150° C. for 10 to 60 minutes; And rapid cooling to 40 ° C. or less may include killing live lactic acid bacteria in the lactic acid bacteria fermentation broth.

The tindalization of the aloe-derived lactic acid bacteria fermentation broth is a method of killing live lactic acid bacteria after fermentation in the aloe-derived lactic acid bacteria fermentation broth. It may be to use a method of tindalization by an intermittent sterilization method, which is a method of heating at 60 to 150 ° C. for 10 to 60 minutes and rapidly cooling to 20 to 40 ° C. or below 40 ° C.

In the step (e), the lactic acid bacteria in the lactic acid bacteria fermentation broth prepared in the step (c) are disrupted to form a disrupted lactic acid bacteria solution. The crushing may be centrifuging the lactic acid bacteria fermentation broth to separate the fermentation broth and cells, and then crushing the separated cells, or using an ultrasonicator. The lysate obtained here may be centrifuged once again to remove the cell precipitate, and the supernatant may be obtained as a lactic acid bacteria lysate.

At this time, most of the components constituting the cell precipitate are hydrophobic cell wall components, and the components constituting the supernatant are components constituting lactic acid bacteria cells, which include peptidoglycan called murein and sialic acid In addition, polysaccharides, which are components of cell membranes, intercellular proteins, and various types of intercellular enzymes, are important substances related to immune and brain cell enhancement effects.

When the lactic acid bacteria cells present in the lactic acid bacteria fermentation broth are actually disrupted, all of the cell wall constituents of the lactic acid bacteria and important immune substances in the cells escape, resulting in acquired immunity that can resist potential pathogenic microorganisms along with metabolic components generated during fermentation. immunity), as well as having a stronger immune system.

Finally, in the step (f), the tindalized aloe lactic acid bacteria fermented powder (tindalized aloe-derived lactic acid bacteria fermented product) is prepared by mixing the tindalized lactic acid bacteria fermentation liquid and the lactic acid bacteria disrupted liquid.

The drying may be lyophilization or spray drying.

The present invention relates to a composition for improving brain function (cognitive function or memory ability) containing, as an active ingredient, a novel fermentation product of aloe lactobacillus with increased sialic acid content, and the novel fermentation product with increased sialic acid content of the present invention. silver is not only excellent in inhibiting the activity of acetylcholine degrading enzyme, which inhibits neurotransmission by destroying acetylcholine, but also has excellent ability to restore memory and cognitive ability in animal models with memory impairment induced by scopolamine. It can be usefully used for the prevention or treatment of degenerative cranial nerve diseases such as Alzheimer's dementia, and can also be usefully used as a health functional food composition or pharmaceutical composition for improving or enhancing deteriorated memory, learning ability and cognitive function.

In addition, the present invention provides a food composition for improving brain function comprising, as an active ingredient, a fermented product of aloe-derived lactic acid bacteria using an aloe-derived lactic acid bacterium, Lactobacillus gallina room strain.

The fermented product of lactic acid bacteria derived from aloe may be included in an amount of 5 to 20 parts by weight based on 100 parts by weight of the food composition for improving brain function. For example, 5 to 18 parts by weight, 5 to 16 parts by weight, 5 to 14 parts by weight, 5 to 12 parts by weight, or 5 to 10 parts by weight of the aloe-derived lactic acid bacteria fermented product with respect to 100 parts by weight of the food composition for improving brain function. It may include parts by weight.

In addition, in one embodiment, the present invention provides a pharmaceutical composition for improving brain function comprising, as an active ingredient, a fermented product of aloe-derived lactic acid bacteria using an aloe-derived lactic acid bacterium, Lactobacillus gallina room strain.

The aloe-derived lactic acid bacteria fermented product may contain 40 to 99 parts by weight based on 100 parts by weight of the pharmaceutical composition for improving brain function. For example, 40 to 99 parts by weight, 40 to 80 parts by weight, 40 to 60 parts by weight, 60 to 99 parts by weight, or 80 to 99 parts by weight of the aloe-derived lactic acid bacteria fermented product with respect to 100 parts by weight of the pharmaceutical composition for improving brain function. It may be one containing 99 parts by weight.

Hereinafter, the present invention will be described in more detail through examples and the like according to the present invention, but the scope of the present invention is not limited by the examples presented below.

[Example]

Example 1: Isolation and screening of novel lactic acid bacteria derived from aloe

1 shows a manufacturing process of the fermented tindaled aloe lactobacillus powder of Examples 1 to 6.

Referring to FIG. 1, samples were collected from fresh aloe leaves grown for 3 to 5 years from Kim Jeongmun Aloe Jeju Farm located in Seongsan-eup, Jeju-do, Korea, and lactic acid bacteria isolation and screening tests were consigned for 3 months to the Department of Microbiology at Seoul National University. . Three types of aloe (Vera, Saponaria, Arborescens), diluted solution (0.1% peptone water), and lactobacillus culture medium (BCP-MRS, BS agar) were used as test materials. Divided into the inside, the sharp upper part, and the wide lower part, samples were taken by 30g, and then homogenized with a stomacher with 270mL of the diluted solution. After serial dilution, the homogenized samples were plated on BS medium and BCP-MRS medium, which are selective media for anaerobic lactic acid bacteria isolation, and cultured for 2 to 7 days in a 37 ° C. incubator to separate lactic acid bacteria and complete screening. After the lactic acid bacteria were isolated and screened, they were cultured under anaerobic conditions using an anaerobic jar for mass reproduction of anaerobic bacteria.

As a result of the culture, as shown in FIG. 2, fine colonies appeared in the BCP-MRS medium and BS medium on the 2nd day only in the lactic acid bacteria culture medium and the samples of the Aloe vera sample group, and the culture results until the 7th day were presented as samples. However, no colonies appeared on the 2nd day and 7th day of culture in both the culture media and samples of the lactic acid bacteria in the Aloe saponaria and Aloe aboracense sample groups.

In addition, as a result of taking FE-SEM (field emission scanning electron microscope; SIGMA, Cal Zeiss, UK) at the Joint Institute of Agriculture and Life Sciences of Seoul National University, as shown in FIG. 3, it showed typical rod shape morphological characteristics of lactic acid bacteria.

Example 2: 16S rRNA sequence analysis and identification of novel lactic acid bacteria derived from aloe

For the genetic identification of the new aloe-derived lactic acid bacteria obtained from Example 1, the identification was requested to Macrogen Co., Ltd., an organization specializing in microbial identification, and was found to have 16S rRNA with the base sequence shown in FIGS. 4 and 5 and Table 1 below. When the 16S rRNA base sequence was compared with the base sequence registered in the GenBank database using the BLAST program, 99% homology on the base sequence with the known Lactobacillus gallinarum as shown in FIG. 6 It was confirmed to have, and it was determined to be Lactobacillus gallina room taxonomically based on the nucleotide sequence of 16S rRNA.

Therefore, the name of the strain was set as 'KJM-ALOE2112' in the sense of Lactobacillus gallinarum derived from Kim Jung-moon's aloe, and this strain was submitted to the Microbial Resource Center of the Korea Research Institute of Bioscience and Biotechnology as shown in Figure 7 on November 15, 2021 After the microbiological safety deposit (number: SD1458) on the date, the KJM-ALOE2112 strain as shown in Figure 8 from KRIBB (Korea Research Institute of Bioscience and Biotechnology) was granted accession number KCTC 14824BP as a patent deposit on December 15, 2021.

[Table 1]

Example 3: Acquisition of aloe polysaccharide extract

Fresh aloe leaves grown for 3 to 5 years are collected from Kim Jeong-moon Aloe Jeju Farm, washed 3 times with purified water in KGMP facility, peeled, and then subjected to the grinding step of the obtained aloe, and U-tech patented method is applied to obtain a viscous polysaccharide The extract was obtained (item declaration and manufacturing number: 1992063113512, other processed products).

In the present invention, about 98% or more of aloe is water, and most of the solid part is composed of fibers or polysaccharides, so it is preferable to use powdered aloe for storage. A polysaccharide extract of the phase was used. As a liquefaction method, the green gel obtained after removing the bark of the aloe leaf was extracted using a U-tech patented method, and anthraquinone-based compounds and other pigments were removed using an activated carbon column to obtain an aloe polysaccharide extract.

As a health functional food of the aloe family, the daily intake should contain 100 to 200mg/20g of total polysaccharide content according to the Ministry of Food and Drug Safety, so it is preferable to use an aloe polysaccharide extract having a content of 10% by weight or more.

Example 4: Preparation of new lactic acid bacteria pre-culture solution derived from aloe

The aloe-derived lactic acid bacteria used in the present invention was registered as a new strain as Lactobacillus gallinarum KJM-ALOE2112, KCTC 14824BP.

The aloe-derived lactic acid bacteria pre-culture medium of the present invention is MRS medium (1.00% Pancreatic digest of gelatin, 0.80% Beef extract, 2.00% Dextrose, 0.20% Dipotassium phosphate, 0.10% Polysorbate 80, 0.50% Sodium acetate, 0.20% Ammonium citrate, 0.02% % Magnesium sulfate, 0.01% Manganese sulfate, 95.18% Water) was prepared, sterilized at 121 ° C for 15 minutes, cooled, and Lactobacillus gallina room strain was inoculated into the medium and cultured with shaking at 37 ° C and 90 rpm for 28 hours to obtain 1 × After securing the number of lactic acid bacteria of 10 ¹⁰ to 1 × 10 ¹² CFU / ㎖ or more, a lactic acid bacteria pre-culture solution was obtained.

Example 5: Preparation of aloe-derived lactic acid bacteria fermentation broth

The fermentation broth of aloe-derived lactic acid bacteria used in the present invention was prepared as follows. First, the pH of the 95.18% aloe polysaccharide extract obtained in Example 3 was corrected to 6.0, and then the concentration was adjusted to 10 to 20° Brix. MRS medium components (1.00% Pancreatic digest of gelatin, 0.80% Beef extract, 2.00% Dextrose, 0.20% Dipotassium phosphate, 0.10% Polysorbate 80, 0.50% Sodium acetate, 0.20% Ammonium citrate, 0.02% Magnesium sulfate, 0.01 % Manganese sulfate) and 1.0 to 10.0% of bird's nest powder were added and autoclaved at 121°C for 15 minutes. The fermentation medium thus prepared was cooled, and 1.0 to 5.0% of the pre-culture of the aloe-derived lactic acid bacteria obtained in Example 4 was inoculated and cultured with shaking at 37° C. and 90 rpm for 68 to 76 hours to obtain a fermentation broth of the aloe-derived lactic acid bacteria.

Example 6: Production of fermented powder after tindalization of aloe-derived lactic acid bacteria fermented liquid and cell disruption

The tindalization of the fermentation broth of aloe-derived lactic acid bacteria of the present invention is a method for killing live lactic acid bacteria after fermentation in the fermentation broth obtained in Example 5. Any known method for tindalization of the fermentation broth can be used, but in the present invention, aloe A method of tindalization by intermittent sterilization, which is a method of heating the derived lactic acid bacteria fermentation broth at 60 to 150 ° C. for 10 to 60 minutes and rapidly cooling to 20 to 40 ° C. or below 20 ° C., was used.

In addition, in the method of disrupting lactic acid bacteria in the aloe-derived lactic acid bacteria fermentation broth obtained from Example 5 of the present invention, when the total number of lactic acid bacteria reaches 5.0 to 8.0 × 10 ⁹ CFU/mL or more, the fermentation broth is continuously centrifuged at 8,000 rpm for 30 minutes (Continuous Centrifuge, J-075AT, MDM Co., Ltd, Korea) to separate fermentation broth and cells after centrifugation, and then dilute the separated cells with 5 to 10 times sterile water to obtain 40% amplitude, pulse On 20 sec, off 10 sec, 15 ℃, 25 minutes (Vibracell, VCX2500, SONICS, USA) conditions were crushed. The lysate obtained here was centrifuged again in the same way as above to remove cell debris, and the supernatant was obtained as the final lactic acid bacteria lysate, which was then combined with the aloe fermented liquid to prepare a fermented aloe lactic acid bacteria powder. At this time, most of the components constituting the cell sediment are hydrophobic cell wall components, and the components constituting the supernatant are components constituting lactic acid bacteria cells, which include peptidoglycan called murein, sialic acid, and Cell membrane components such as polysaccharide, intercellular protein, and various types of intercellular enzymes were important substances related to the immune and brain cell enhancement effects.

In addition, 0.01 to 0.1% of the weight of the mixture of the tindal aloe-derived lactic acid bacteria fermented liquid and the lactic acid bacteria crushed mixture was concentrated under reduced pressure and freeze-dried or spray-dried using an inclusion agent such as maltodextrin, The dried powder was used as a final sample of the fermented lactic acid bacteria derived from tindaled aloe.

Example 7: Sialic acid component analysis before and after fermentation of aloe-derived lactic acid bacteria

In order to investigate the component change before and after fermentation of the fermented tindalhwa aloe-derived lactic acid bacteria powder prepared by the methods of Examples 1 to 6, the content of sialic acid was measured. As the sample before fermentation, the sample in Example 5 to which bird's nest powder was not added was used. The measurement of the content of the sialic acid component is the result of requesting HPLC-FLD (1200, Agilent Technologies, USA) instrumental analysis by the manual method of a 100 μM sialic acid fluorescence labeling kit (Sialic acid fluorescence labeling Kit, Takara, Japan) to KOTITI Testing & Research Institute. , as shown in FIG. 9, it was confirmed that the total sialic acid content of the fermented product of aloe-derived lactic acid bacteria was generally higher than that of the non-fermented product. This is because the content of sialic acid among fermentation metabolites in the fermented tindaled aloe-derived lactic acid bacteria powder prepared according to the above example was increased by the addition of bird's nest powder.

The sample pretreatment method is to add 0.5 mL of 0.05N hydrochloric acid solution to the powder before and after fermentation, heat it at 80 ° C for 1 hour, and then use it with distilled water and mix reagent 1 (DMB solution), reagent 2 (coupling solution), and distilled water in a 1:5: A mixed solution was prepared at a ratio of 4, treated with 0.2 mL of each sample and reacted at 50 ° C. for 2 hours and 30 minutes under light-shielding conditions, followed by cooling to perform instrumental analysis. HPLC-FLD instrumental analysis conditions are column (CAPCELL PAK, 4.6 x 250mm, Particle size 5μm), wavelength (excitation 373nm, emission 448nm), flow rate 0.9ml/min, injection volume 10μl, oven temperature 35℃, mobile phase solvent (methanol) : Acetonitrile : Purified water = 7 : 8 : 85 (v / v / v), run time was analyzed under the condition of 40 minutes.

Example 8: Evaluation of in vitro brain nerve cell proliferation using MTT assay

The lactic acid bacteria fermented powder derived from tindalhwa aloe prepared by the method of Examples 1 to 6 was tested by MTT (3-(4,5-dimethylthiazol-2yl)-2 ,5-diphenyl-2Htetrazolium bromide) analysis was performed, and through this, the protective effect on brain neurons according to scopolamine treatment was confirmed. The samples prepared in Example 6 were treated with SH-SY5Y cells, a human neuroblastoma cell line treated with scopolamine, which induces cranial nerve disease. As a result, as shown at the bottom of FIG. 10, the normal control group on the left had reduced cell viability due to scopolamine treatment, whereas each production lot of the present invention. When different samples were treated, cell viability was similar to that of the positive control group (Donepezil 1.0 to 10 μM). In particular, SA0, SA1, and SA4 among the samples significantly increased cell viability compared to SA2 and SA3 within the concentration range of 1.0 to 100 μl. It was confirmed that the effect of protecting nerve cells was excellent.

Example 9: Evaluation of in vitro antioxidant activity using DPPH assay

Antioxidant activity of the lactic acid bacteria fermented powder derived from tindalhwa aloe prepared by the methods of Examples 1 to 6 was measured at the Department of Oriental Science, Kyung Hee University, as in the experimental method shown at the top of FIG. 11 . Antioxidant activity tests were performed on each production lot. DPPH scavenging activity was measured for each sample, and 0.2 mg/ml of ascorbic acid (Sigma-Aldrich, USA) known as an antioxidant was used as a positive control. Each sample and ascorbic acid were mixed with 80 μl and 200 μl of 0.1 mM DPPH, respectively, left at 25 ° C for 30 minutes, and then the absorbance was measured at 530 nm to analyze the antioxidant level. At this time, each sample was analyzed by varying the concentration as shown in FIG. 11 . As a result of the analysis, as shown in FIG. 11 and Table 2 below, the antioxidant activity of each sample was found to increase in a concentration-dependent manner, but the SA1 sample had a low DPPH IC ₅₀ value and high antioxidant activity and cell protection according to Example 8 The effect was excellent, and it could be confirmed as a sample suitable for this example.

[Table 2]

Example 10: Evaluation of total glutathione in vitro antioxidant activity

As for the total intracellular glutathione antioxidant activity test, the SA1 sample in Example 9 had a low DPPH IC ₅₀ value, high antioxidant activity, and excellent cell protection effect according to Example 8, confirming that the sample was suitable for this example. In this example, glutathione reductase was prepared by diluting the buffer solution 1× to a concentration of 6 units/mL and filling it on ice, and was diluted 5-fold with the buffer solution immediately before adding to the sample. Standard solutions of oxidized glutathione (hereinafter abbreviated as "GSH") and reduced glutathione (hereinafter abbreviated as "GSSG") were diluted to 2μM, 5μM, 10μM, 20μM, 30μM, 40μM by making 1mM GSH and 1mM GSSG solutions. and used it. Extraction of cell line samples for quantification of total glutathione and treatment and non-treatment of SA1 samples were performed using SH-SY5Y cells, a human neuroblastoma cell line, provided by the Department of Oriental Science, Kyung Hee University.

In order to accurately measure GSSG, it is necessary to inactivate GSH with an appropriate reagent. In the present invention, N-ethylmaleimide (hereinafter abbreviated as “NEM”) was used as a GSH inactivator. For fractionation, 100 μl of 250 mM NEM was added so that the final concentration of NEM was 50 mM, and 210 μl of 2M KHCO ₃ solution was added to neutralize the mixture, followed by reaction at room temperature for 30 minutes. In order to accurately quantify GSH and GSSG, the completion of the NEM reaction is key. As a result of HPLC using an NH2 column, no GSH was observed in the NEM-treated fraction, confirming that the reaction was complete (FIG. 12).

SA1 sample treatment For quantification of total glutathione in SH-SY5Y cell line samples, 20 mg of the cell mass harvested by centrifugation after culturing the cells was homogenized in 500 μl of 5% PCA solution, treated with NEM, and described in each step below. While reacting according to one glutathione quantification method, the slope of the section in which absorbance increases in a straight line was obtained, and compared with the standard curve of total glutathione and GSSG, total glutathione in the cell line sample (from the total glutathione standard curve in FIG. 13 (a)) and GSSG ( from the GSSG standard curve in Fig. 13(b)) was obtained.

As a result, after measuring the absorbance of total glutathione in the 20-fold diluted SH-SY5Y cell line sample, the amount of change in spectral absorbance in the section where the absorbance increases linearly was measured to be 74.49. From this, to obtain the amount of total glutathione in the cell line sample As a result of converting the amount of total glutathione corresponding to the change in spectral absorbance using the total glutathione standard curve of FIG. 13 (a), a value of 467.45 nmole/ml was obtained. In addition, GSSG also measured absorbance in the same manner as above to obtain a change in spectral absorbance of 28.49, and as a result of converting the amount of GSSG corresponding to the slope value from the GSSG standard curve in FIG. 13 (b), 9.63 nmole/ml sample got the value of

On the other hand, in the SH-SY5Y cell line for quantification of total glutathione, SA1 untreated, after measuring the absorbance of total glutathione in the cell line sample diluted 20 times in the same way as above, the change in spectral absorbance in the section where the absorbance increases in a straight line was 21.31 , and the amount of total glutathione corresponding to the change in spectral absorbance was converted using the total glutathione standard curve of FIG. 13 (a) to obtain the amount of total glutathione in the cell line sample. got the value of In addition, GSSG also measured absorbance in the same manner as above to obtain a change in spectral absorbance of 4.89, and as a result of converting the amount of GSSG corresponding to the slope value from the GSSG standard curve in FIG. 13(b), 1.37 nmole/ml sample got the value of

In conclusion, the concentration of total glutathione in the SH-SY5Y cell line sample was 6 to 7 times higher than that of the SA1 treated group, and the antioxidant activity of the SH-SY5Y cell line administered with the SA1 sample was 6 to 7 times higher. could confirm that

Example 11: Evaluation of total sialic acid in vitro cell uptake

In the experiment of total sialic acid influx into cells, the SA1 sample in Example 9 had high IC50 antioxidant activity and excellent cell proliferation and other effects according to Examples 8 to 10, confirming that the sample was suitable for this example. In this example, SH-SY5Y cells, a human neuroblastoma cell line, were used to extract cell line samples for quantification of total sialic acid, and treatment and non-treatment of SA1 samples were provided by the Department of Oriental Science, Kyung Hee University.

Treatment and non-treatment of the SA1 sample were pretreated as follows for HPLC quantitative analysis.

1) To 100 μl of the provided SH-SY5Y cells, 5 μl (1250 units) of neuraminidase and 150 μl of buffer were added and placed in a water bath to proceed with the reaction (37° C., 16 h).

2) Ethanol at -40 to -42°C was added and centrifuged (14,000 rpm, 4°C, 20 minutes) to remove glycoproteins from which sialic acid was separated.

3) Salts in the sample were removed and sialic acid was concentrated through solid phase extraction using a PGC (porous graphitic carbon) cartridge.

Sialic acid obtained from the above pretreatment was quantitatively analyzed using HPLC. In more detail, an HPLC system consisting of an autosampler (maintained at 4 ° C), an ultra-high pressure pump and a column oven was used for diluted sialic acid, a PGC column (100 × 2.1 mm, 3 μm) was used, and a chromatographic method was used. Separation was performed according to optimized separation conditions. After loading the sample, the sialic acid eluting concentration gradient solution was flowed at a rate of 0.3 ml per minute. The gradient solutions are (A) 96.9% water, 0.1% formic acid (v/v) sap, 3.0% acetonitrile and (B) 9.9% water, 90.0% acetonitrile, 0.1% formic acid (v/v) sap. , respectively, treated by time/concentration below. That is, 0% B, 0-0.5 min; 0-10% B, 0.5-6min; 10-100% B, 6-8 min. Finally, the analytical column was equilibrated with 0% B for 3 minutes.

In this Example, in order to analyze the NeuGc content by applying to the pre-treated samples for the Donepezil (Donepezil 1.0mg/kg) treated cell control group, SA1 cell treated group, and SA1 cell untreated group, FIG. 14a is donepezil through acid treatment This is the result of analysis by selectively releasing sialic acid in the treated cell control group, removing interfering substances through PGC SPE, and then injecting the purified sialic acid into an HPLC-FLD system through a fluorescence induction process. As a result, NeuAc and NeuGc contained in the control cells treated with donepezil could be simultaneously quantified, and it was confirmed that NeuAc and NeuGc were present at levels of 22.4 μg/g and 22.4 μg/g in the cell sample. 14b is a chromatogram obtained by applying the same method to the SA1 cell-treated group and the SA1 cell-untreated group of the present invention. The blue chromatogram is the result of analysis applied to the SA1 cell untreated group, and the red chromatogram is the result of applying the SA1 cell treatment group. As a result, it was confirmed that NeuGc and NeuAc were present in the SA1 cell treated group at about 10 times higher concentrations than the SA1 cell untreated group.

Example 12: In vivo efficacy evaluation for AChE activity

In the in vivo efficacy test for AChE activity, the SA1 sample in Example 9 had high antioxidant activity and excellent cell proliferation and other effects according to Examples 8 to 11, and it was confirmed that the sample was suitable for this example. In this example, comparison of efficacy on AChE activity at the Department of Oriental Science, Kyung Hee University using Donepezil (Donepezil 1.0mg/kg) treated cell control group and SA1 (200 mg/kg) and sialic acid (50 mg/kg) as experimental groups was performed as follows: It was carried out as A 5-week-old male ICR mouse (Orient Bio, Korea) was purchased as an experimental animal and maintained for a week of adaptation. In order to compare the in vivo AChE activity inhibitory effect, control cells treated with donepezil and SA1 and sialic acid were orally administered once to each mouse (three mice in each group). 6 hours after administration of each substance, the hippocampus was separated and then homogenized using 1 M phosphate buffer (pH 8.0) to obtain a homogenate. The obtained homogenate was measured for AChE activity in vivo using Amplex red assay kit (Molecular probe, USA). As a result, as shown in FIG. 15, the control group treated with donepezil and the group treated with sialic acid showed little AChE activity inhibition effect, whereas the SA1 treatment group showed a relatively high AChE activity inhibition effect of about 71.5%.

Example 13: Evaluation of spatial memory recovery through Morris water maze test

In this example, the SA1 sample in Example 9 has high antioxidant activity and excellent effects such as cell proliferation according to Examples 8 to 11, as well as excellent in vivo efficacy for AChE activity according to Example 12, A suitable sample was identified. Therefore, in this example, SA0 to SA4 were used as the experimental group and the scopolamine-only treated group as the control group, and the spatial memory recovery effect through the Morris water maze test was performed at the Department of Oriental Science, Kyung Hee University as follows. The animals used in this experiment were 10-week-old BLAB/C male mice with an average body weight of 26 g, and 3 animals were placed in one breeding case, and constant temperature (23 ± 2 ° C) and constant humidity (50-55%) were maintained. Feed and water were supplied autonomously while rearing in the same laboratory environment, alternating day and night at 12-hour intervals. After allowing these mice to undergo an environmental adaptation period for one week, each sample was orally administered twice a day at 100 mg/kg, and the control group was orally administered with the same amount of distilled water. After learning the water maze for 5 days, the platform was removed, and the degree of spatial memory was measured according to how accurately the mice remembered the location of the platform. did 2 mg/kg of scopolamine was administered IP (intraperitoneal) 30 minutes before memory capacity measurement. As a result, compared to the control group not treated with anything, in the group treated with scopolamine alone, not only did the mice try numerous directions to find the location of the platform due to memory impairment, but also the length of the movement line was complicated and the retention time (54 seconds) was shorter. increased. On the other hand, in the sample (SA0 to SA4) treatment group of the present invention, it was found that the number of direction changes and the length of the copper wire were slightly decreased, and the retention time was also reduced. Moreover, when the SA1 sample of Example 9 was orally administered among the samples of the present invention, it was confirmed that the short-term memory ability damaged by scopolamine was remarkably recovered compared to SA0, SA2, SA3, and SA4. Similarly, in the results of long-term memory analysis, it was found that the long-term memory ability damaged by scopolamine was remarkably restored when SA1 of the present invention was orally administered among the samples (FIG. 16).

Example 14: In vivo efficacy evaluation for Aβ (amyloid-β) activity

In this example, the SA1 sample from Example 9 has high antioxidant activity and excellent effects such as cell proliferation according to Examples 8 to 11, as well as in vivo efficacy on AChE activity and spatial memory ability according to Examples 12 to 13. It was excellent and could be confirmed as a sample suitable for this example. Therefore, using the SA1 treatment group of Example 9 as the experimental group and the untreated group as the control group, a histological examination of the hippocampus was performed in a rat model with amyloid beta protein-induced dementia at the Department of Oriental Science, Kyung Hee University as follows. After the behavioral examination, rats were anesthetized with diethyl ester, and then the abdominal cavity was opened and saline was perfused through the heart. Then, the brain was removed, fixed in 10% neutral buffered formalin, and the degree of degeneration of neurons in the hippocampus was observed through hematoxylin & eosin staining. As a result, in the group treated with amyloid beta protein in the ventricle, as shown in FIG. 15, degeneration of memory-related pyramidal cells in the dentate gyrus of the hippocampus was observed compared to the group not treated with anything. In the group administered SA1 at a concentration of 200 mg/kg for 4 weeks, the degree of damage was weak.

Example 15: Preparation of food or pharmaceutical composition for improved brain function

1. Food composition for improving brain function

The following components were mixed with water in the tindal aloe-derived lactic acid bacteria fermented powder (SA1) prepared in Example 9 to prepare a beverage in the usual way, but after setting the total to 100 ml, sterilizing and cooling at 95 ° C. for 15 seconds to make a drink was manufactured.

SA1 powder ------------------- 10.0g

Citric acid ---------------------- 1.0g

Gum arabic ------------------ 1.0g

Sugar ------------------------ 5.0g

2. Pharmaceutical composition for improving brain function

Tablets, capsules, powders and injections were prepared according to the following compositions, respectively.

1) Refining

500.0 mg of lactic acid bacteria fermented powder (SA1) derived from tindalhwa aloe prepared in Example 9

Lactose ------------------------------------------------- 500.0mg

Talc ------------------------------------------------- -- 5.0 mg

Magnesium Stearate ---------------------------------- 1.0 mg

2) Capsules

500.0 mg of lactic acid bacteria fermented powder (SA1) derived from tindalhwa aloe prepared in Example 9

Starch 1500 --------------------------------------------- 10.0 mg

Magnesium Stearate ----------------------------------- 100.0 mg

3) powder

500.0 mg of lactic acid bacteria fermented powder (SA1) derived from tindalhwa aloe prepared in Example 9

Lactose ------------------------------------------------- 100.0 mg

Talc ------------------------------------------------- -- 5.0 mg

4) Injectables

50.0 mg of lactic acid bacteria fermented powder (SA1) derived from tindalhwa aloe prepared in Example 9

Antioxidants -------------------------------------------- 1.0 mg

Tween 80 ----------------------------------------------- 1.0 mg

Distilled water for injection ------------------------------------ 2.0 ml or less

Claims (12)

(a) preparing a pre-culture solution of lactic acid bacteria by inoculating lactic acid bacteria isolated from fresh aloe leaves into an MRS medium;
(b) preparing an aloe polysaccharide extract fermentation medium by adding MRS medium and bird's nest powder to the aloe polysaccharide extract obtained from aloe;
(c) preparing an aloe-derived lactic acid bacteria fermentation broth by inoculating the lactic acid bacteria pre-culture solution into the fermentation medium of the aloe polysaccharide extract and fermenting the solution;
(d) tyndallizing the aloe-derived lactic acid bacteria fermentation broth;
(e) crushing the lactic acid bacteria in the aloe-derived lactic acid bacteria fermentation broth to form a lactic acid bacteria disruption solution; and,
(f) A method for producing a composition for improving brain function comprising mixing the tindalized lactic acid bacteria fermented liquid and the lactic acid bacteria disrupted liquid and then drying to prepare a tindalized lactic acid bacteria fermented product derived from aloe.
According to claim 1,
Separating lactic acid bacteria from fresh aloe leaves in step (a),
Separating lactic acid bacteria from fresh aloe leaves;
Identifying the isolated lactic acid bacteria; and,
Method for producing a composition for improving brain function, comprising the step of culturing the isolated and identified lactic acid bacteria in a medium.
According to claim 1,
Obtaining the aloe polysaccharide extract in step (b),
Harvesting and pre-treating aloe; and
A method for preparing a composition for improving brain function, comprising the step of obtaining a mucilaginous aloe polysaccharide extract from the pretreated aloe.
According to claim 1,
In the step (b), the pH of the aloe polysaccharide extract is corrected to pH6 and the concentration is adjusted to 10 to 20 ° Birx.
According to claim 1,
In step (c), 1 to 5% by weight of the pre-cultured lactic acid bacteria is inoculated and cultured with shaking for 60 to 80 hours to ferment.
According to claim 1,
The step of tindalizing the aloe-derived lactic acid bacteria fermentation broth in step (d),
Heating the lactic acid bacteria fermentation broth at 60 to 150 ° C. for 10 to 60 minutes; and
A method for producing a composition for improving brain function, comprising rapidly cooling to 40 ° C. or less to kill live lactic acid bacteria in the lactic acid bacteria fermentation broth.
According to claim 1,
The method for producing a composition for improving brain function, characterized in that the lactic acid bacteria is Lactobacillus gallinarum.
According to claim 1,
The method for preparing a composition for improving brain function, characterized in that the aloe is at least one species selected from the group consisting of aloe vera, aloe arborescens and aloe saponaria.
A food composition for improving brain function comprising, as an active ingredient, a fermented product of aloe-derived lactic acid bacteria using an aloe-derived lactic acid bacterium, Lactobacillus gallina room strain.
According to claim 9,
The food composition for improving brain function, characterized in that it contains 5 to 20 parts by weight of the fermented product of the aloe-derived lactic acid bacteria with respect to 100 parts by weight of the food composition for improving brain function.
A pharmaceutical composition for improving brain function comprising, as an active ingredient, a fermented product of aloe-derived lactic acid bacteria using an aloe-derived lactic acid bacterium Lactobacillus gallina room strain.
According to claim 11,
The pharmaceutical composition for improving brain function, characterized in that it comprises 40 to 99 parts by weight of the fermented product of the aloe-derived lactic acid bacteria with respect to 100 parts by weight of the pharmaceutical composition for improving brain function.