KR20140013339A - Method for producing microorganism lamella construct and composition comprising microorganism lamella construct - Google Patents

Abstract

The present disclosure relates to a producing method of a microorganism lamella structure, which comprises a cell wall degrading step which degrades a cell wall of a microorganism; and a high-pressure emulsion step which emulsifies a microbial cell composition remaining after a cell wall is degraded at a high-pressure, and to a composition comprising a lamella structure derived from a microbial cell composition, and provides a lamellar structure of a liposome which functions as a carrier for an active ingredient, an emulsion, a liquid crystal, etc. and can be variously used in an area of food, cosmetics, and pharmacy by providing a lamellar structure containing various bioactive ingredients from a microorganism. [Reference numerals] (AA) Dexamethasone; (BB) Lactobacillus 0.00016%; (CC) Lactobacillus 0.0008%; (DD) Yeast 0.0004%; (EE) Yeast 0.002%; (FF) Bifidus 0.01%

Description

Method for producing microorganism lamella constructs and composition comprising microorganism lamella constructs {Method for producing microorganism lamella construct and composition comprising microorganism lamella construct}

Disclosed herein is a lamellar structure derived from a microorganism, a method for preparing the same, and a composition comprising the same.

Recently, as the development of food and cosmetics using fermented foods and fermented materials is active, interest in fermented microorganisms is increasing. Natural plant materials or herbal medicines contain various bioactive substances and antioxidants, but they are often unstable and sometimes irritating after extraction, often causing toxicity to the human body. Therefore, in recent years, many attempts have been made to increase the effects of stabilizing natural extracts, reducing toxicity, or converting them into stable derivatives, and as part of this, biological transformation methods using microorganisms or enzymes have been developed. For example, the fermentation technique is an example.

Fermentation is a process in which organic matters derived from plants and animals are decomposed by the enzymatic action of microorganisms, which is a series of processes that are reproduced as essential nutrients of organic life.

Specifically, fermentation refers to a case where it becomes useful to the human body by the decomposition or synthesis process of organic matter by the microorganism. As such, representative fermentation microorganisms that produce substances useful for the human body include Nuruk, yeast, mold, Bacillus subtilis, Lactobacillus, and acetic acid.

In addition, various foods are produced by the fermentation process through the fermentation microorganisms. Such foods obtained by converting the components of the food by the microorganisms are defined as fermented foods, and typical fermented foods include miso, cheonggukjang, red pepper paste, kimchi, salted fish, cheese, yogurt, vinegar, sikhye, and liquor.

On the other hand, food ingested in the human body is decomposed by lactic acid bacteria in the intestine, and is converted into a low-molecular substance that is easily absorbed by human cells. It is absorbed through various actions such as detoxification and decomposition. These results are indicative of the importance of bioconversion by intestinal microorganisms.

Therefore, attempts to maximize the efficacy of natural plant materials and herbal medicines or solve safety problems by microbial fermentation such as lactic acid bacteria or yeast have important meanings in the development of various foods, cosmetics, medicines and the like. In particular, microorganisms that breed in Korean traditional foods, such as kimchi and doenjang, have been reported to have excellent biological activity, and for this reason, many studies have been conducted to use microorganisms separated from traditional fermented foods.

Fermentation has a beneficial effect on the human body as a result of the effects of fermented metabolites and the nutrients of the microorganisms themselves. Yeast or lactic acid bacteria, when ingested as live bacteria, as well as health of the intestines as well as the health of the human body has the effect. In the cosmetics field, fermented products have been used as raw materials, but not many microorganisms are used.

Nutrient nutrients of microorganisms are present in the cell membrane structure, so if the cell membrane structure of the microorganism is properly used, it is possible to develop an excellent raw material that retains the nutrient components of the microbial cells.

Republic of Korea Patent Application No. 10-2004-0024437 Republic of Korea Patent Application No. 10-2000-0002793

An object of one embodiment of the present invention, to provide a lamella structure that functions as a carrier of the active ingredient.

It is an object of another embodiment of the present invention to provide a lamellae structure comprising the nutrient components of a useful microorganism.

Method for producing a lamellar structure according to an embodiment of the present invention, the cell wall decomposition step of decomposing the cell wall of the microorganism; And a high pressure emulsifying step of high pressure emulsifying the remaining microbial cell constituents after the cell wall is decomposed.

The microbial lamella composition according to an embodiment of the present invention may include a lamellae structure derived from a microbial cell construct.

Using the techniques disclosed herein, it is possible to provide lamellar structures and lamella compositions with excellent stability. In addition, a lamellar structure such as a liposome, an emulsion, or a liquid crystal that functions as a carrier of the active ingredient can be provided. In addition, by providing a lamellar structure containing a variety of bioactive components derived from microorganisms, it can be variously used in the food, cosmetics and pharmaceutical fields.

Figure 1 is treated with a lamella composition derived from the amount of secretion of TNF-α, lactobacillus, Saccharomyces and bifidus of the negative control (UV) and dexamethasone treated dexamethason treated nothing after UV irradiation It is a graph which compared the secretion amount of TNF- (alpha) afterwards.

Microbes consist of cell membranes and cell walls. The cell membrane is mainly composed of phospholipids and the cell membrane is composed of a combination of various polymers including a peptidoglycan layer. Microbial cell membranes have a bilayer structure composed mainly of phospholipids. Phospholipid bilayer structure has a self-assembly (self assembly) characteristics it is possible to form a lamellar structure, such as liposomes, emulsions or liquid crystals. Since the biologically active mechanism including various enzymes are located in the cell membrane and cell wall of the microorganism, the lamella composition composed of the microbial cell structure has characteristics that can exhibit various physiological activities.

As used herein, "lamellar structure" means a structure having a multilayer structure of two or more layers. The concept includes the lipid bilayer structure of ordinary cell membranes. Others include, but are not limited to, liposomes, emulsions, or liquid crystal structures.

In the present specification, "high pressure emulsification" is a broad concept that means to emulsify by applying a high pressure. For example, it includes emulsification using a high pressure to enable emulsification of at least 500 bar, at least 600 bar, at least 700 bar, at least 800 bar, at least 900 bar or at least 1000 bar. A device for high pressure emulsification may include a microfluidizer, but is not limited thereto. Microfluidizers, for example, mix lamellar components and pass through cartridge cells or valves (also called interaction chambers) with sub-micro-sized micropores, with the size of the micropores passing therethrough. Is about 50 to 300 micrometers, and lamellar structures such as double lipid membranes are formed by the surfactant due to the large shear stress generated.

As used herein, "liposome" refers to a structure consisting of layers in which phospholipids are suspended in aqueous solution. Liposomes are a kind of carrier, and microcapsulation is possible by inserting drugs, cosmetic raw ingredients, and the like.

As used herein, the term "emulsion" is a general term for a state in which liquid particles are dispersed in colloidal particles or larger particles in a liquid.

As used herein, the term "liquid crystal" refers to a material in which the position occupied by a molecule and the direction of the molecular axis are completely intermediate between solid and liquid, and is also referred to as a mesomorphic phase or mesophase. Liquid crystals behave like liquids in terms of viscosity and fluidity, but they have unique properties, such as physical properties such as scattering and reflection of light.

Method for producing a lamellar structure according to an embodiment of the present invention, the cell wall decomposition step of decomposing the cell wall of the microorganism; And a high pressure emulsifying step of high pressure emulsifying the remaining microbial cell constituents after the cell wall is decomposed.

The cell wall decomposition step may include degrading the cell wall by adding an enzyme to the microorganism. The enzyme may be a cell wall degrading enzyme. For example, enzymes such as Lysozyme or glucanase can be used, but are not limited thereto. The cell wall may form a spheroplast or protoplast form that has been partially or completely degraded to produce a lamellae structure.

In one embodiment of the present invention, the microorganism may be used in the manufacture of food, but the lactic acid bacteria or yeast that has been proven safe, but is not limited thereto. Lactic acid bacteria include Lactobacillus sp. Strain or Bifidobacterium sp. Strain. Yeasts include Saccharomyces sp .

In the high pressure emulsifying step, the microfluidizer treatment may be repeated two or more times, three or more times, or four or more times. If repeated more than two times, the lamellar structure becomes smaller.

The high pressure emulsifying step may include high pressure emulsifying the microbial cell construct with phospholipids. The phospholipid may include one or more phospholipids selected from the group consisting of soybean-derived phospholipids, soybean-derived phospholipids, synthetic phospholipids, hydrogenated soybean-derived phospholipids and partially hydrogenated soybean-derived phospholipids. The phospholipid may be added in an amount of 10 to 50 parts by weight when the weight of the microbial cell construct is 100 parts by weight. If the phospholipid is less than 10 parts by weight, the stabilizing effect by the phospholipids is insignificant, and if it exceeds 50 parts by weight, the content of the microbial cell constituents is lowered and the function by the cell constituents is lowered.

In addition, one embodiment of the present invention provides a food, cosmetic or pharmaceutical composition comprising the microbial lamella composition.

When applying the composition according to one embodiment of the present invention to a pharmaceutical composition, oral administration or parenteral in solid, semi-solid or liquid form by adding a commercially available inorganic or organic carrier including the active ingredient in the lipid lamella composition It can be formulated as a sphere dosage. The oral dosage forms include, for example, tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, powders, fine granules, granules, pellets, and the like, and these formulations include surfactants in addition to the active ingredients. , Diluents such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine, and glidants such as silica, talc, stearic acid and its magnesium or calcium salts and polyethylene glycols. . Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose or polyvinylpyrrolidine, optionally starch, agar, alginic acid or its sodium salt Pharmaceutical additives such as disintegrants, absorbents, colorants, flavors, and sweeteners. The tablets may be prepared by conventional mixing, granulating or coating methods. In addition, the parenteral administration agent may be, for example, formulations such as injections, drops, ointments, lotions, gels, creams, sprays, suspensions, emulsions, suppositories, and patches, but are not limited thereto. no.

Pharmaceutically acceptable doses, ie dosages, of the microbial lamellar compositions will depend on the age, gender, weight of the subject to be treated and the severity of the particular disease or pathology to be treated, the route of administration and the prescriber's judgment. For example, a typical dosage can be 0.01 μg / kg / day to 1000 mg / kg / day and 1 μg / kg / day to 40 mg / kg / day, but the dosage can be seen in any way. It is not intended to limit the scope of the invention.

Microbial lamella composition according to an embodiment of the present invention may be provided included in the food composition. The food composition may be a health food, functional food, and food additive composition. The food composition may include other ingredients that can give a synergistic effect to the main effect within a range that does not impair the main effect of the present invention. For example, additives such as fragrances, pigments, fungicides, antioxidants, preservatives, moisturizers, thickeners, inorganic salts, emulsifiers or synthetic polymer materials may be further included to improve physical properties. In addition, supplementary ingredients such as water soluble vitamins, oil soluble vitamins, polymer peptides, polymer polysaccharides and seaweed extract may be further included. The above components may be mixed and selected without difficulty by those skilled in the art depending on the purpose of formulation or use, and the amount thereof may be selected within a range that does not impair the objects and effects of the present invention. The formulations of the compositions according to the invention may be in various forms, such as solutions, emulsions, viscous mixtures, tablets, powders and the like.

Dosage determination of the active ingredient is within the level of one skilled in the art, and its daily dosage may be, for example, 0.1 μg / kg / day to 5000 mg / kg / day, more specifically 50 μg / kg / day to 500 mg / kg / day, but is not limited thereto and may vary depending on various factors such as age, health condition, complications, etc. of the subject to be administered.

On the other hand, the cosmetic composition according to an embodiment of the present invention is a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation and It may be formulated in a spray or the like, but is not limited thereto. More specifically, softening cream, nourishing cream, lotion, body lotion, nourishing cream, massage cream, moisturizing cream, hand cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, gel, patch, oil in water It may be formulated into a basic cosmetic such as (O / W) type, water-in-oil (O / W) type, and a color cosmetic such as lipstick, makeup base or foundation.

Cosmetic composition according to an embodiment of the present invention may include a composition selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract. Other components that may be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, Blood circulation promoting substances, cooling agents, restriction agents, purified water and the like. In addition, the compounding component which may be added other than this is not limited to this, Moreover, any of the above components can be mix | blended within the range which does not impair the objective and effect of this invention.

Hereinafter, the present invention will be described in more detail through preferred embodiments of the present invention, but the following examples and the like are for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation of Microbial Lamellar Structures

Lactobacillus genus microorganisms (Lactobacillus plantarum , KCTC 3103) and Bifidobacterium longum , KCTC 3420) were cultured in MRS and Difco, respectively, and Saccharomyces cerevisiae , KCTC 7904) were cultured in yeast culture medium (Yeast Extract 3g / L, Malto extract 3g / L, Peptone 5g / L, Dextrose 10g / L). The cultured microbial cells were centrifuged at 8,000 rpm for 10 minutes to remove the supernatant and the cells were recovered. The recovered cells were washed with distilled water, suspended in 10 mM Tris-EDTA buffer, and then the cell wall lysis enzyme Lysozyme was treated with lactic acid bacteria and glucanase was treated with yeast to lyse the cell walls. In the enzyme treatment, 1 ml of an enzyme solution of 10 mg / ml concentration was added to 10 ml of the culture solution and reacted at 37 ° C. for 2 hours. Cells in which cell walls were lysed were collected by centrifugation at 5,000 rpm for 20 minutes, and then the supernatant was discarded and washed three times with distilled water to completely remove the remaining enzyme.

The microorganisms from which the enzyme was removed were repeatedly treated at 1000 bar three times using a high pressure emulsifier (Microfluidizer Processor M-110EHI, Microfluidics Corporation) to prepare a microbial lamella composition. The solution from which the microbial lamella composition was prepared was filtered through a 1.2 μm pore size membrane.

Table 1 shows the change in the size of the microbial lamella composition according to the number of times the microfluidizer is treated.

Strain name High Pressure Emulsifier Process size (nm) Lactobacillus 0 pass 1612 Lactobacillus 1 pass 1071 Lactobacillus 2 pass 775 Lactobacillus 3 pass 475 Bifidus 0 pass 2008 Bifidus 1 pass 1064 Bifidus 2 pass 668 Bifidus 3 pass 448 Saccharomyces 0 pass > 3000 Saccharomyces 1 pass 1177 Saccharomyces 2 pass 676 Saccharomyces 3 pass 423

As shown in the above table, two repeated treatments yielded lamellar structures of 600-700 nm in all strains, and three repeated treatments obtained lamellar structures of about 400 nm in all strains.

Experimental Example 1 Stability Experiment of Microbial Lamela Composition

Stability was tested for each microbial lamella composition prepared through three times of microfluidizer treatment in Example 1.

Stability was observed in the size change of the microbial lamella composition for 3 months at room temperature, 4 ℃, 40.

The results are shown in Table 2 below.

As shown in the table above, it can be seen that the state remains very stable up to 3 months at all temperatures.

Experimental Example 2 Evaluation of Skin Efficacy of Microbial Lamela Composition

Human keratinocytes (ATCC CRL-2404) were placed in DMEM (Dulbecco's Modified Eagle's Medium) medium containing penicillin (100 IU / mL), streptomycin (100 g / mL) and 10% FBS (fetal bovine serum). Culture was maintained in an incubator containing 5% carbon dioxide while maintaining 37 ℃. The obtained keratinocytes were dispensed into 6-well plates at 3 X 10 ⁵ per well and incubated for 24 hours under cell culture conditions. After culturing, the cells were irradiated with an amount of 25 mJ / cm 2 of ultraviolet rays, and further cultured for 24 hours after the addition of each microbial lamella composition prepared in Example 1 above. The culture supernatant was collected to measure the secretion amount of TNF-α, an inflammatory cytokine. Dexamethasone 1 Um was used as a positive control. The results are shown in Fig.

Figure 1 shows the result of the negative control (UV) that did not process anything after UV irradiation (dexamethason) and the result of the positive control treated with dexamethasone, the lamellar composition derived from bifidus, as a result of treating the lamellar composition derived from Lactobacillus The result of processing and the result of processing the lamellar composition derived from yeast are shown.

Experimental results show that the microbial lamella composition exhibits an excellent anti-inflammatory effect by inhibiting increased secretion of TNF-α by stimulation of ultraviolet rays. This seems to be due to the bioactive material possessed by the microorganisms.

Hereinafter, an example of the formulation of the composition according to an embodiment of the present invention.

Formulation Example 1 Soap-Type Formulation

Lactobacillus lamella composition of Example 1 5.00

Upkeep 75

Sodium hydroxide 5

Spices 10

Purified water level

(Unit: wt%)

Formulation Example 2 Lotion Formulation

Bifidus lamella composition of Example 1 3.00

L-ascorbic acid-2-magnesium phosphate salt 1.00

Water Soluble Collagen (1% Aqueous Solution) 1.00

Sodium Citrate 0.10

Citric Acid 0.05

Licorice Extract 0.20

1,3-butylene glycol 3.00

Purified water level

(Unit: wt%)

Formulation Example 3 Cream-Type Formulation

Saccharomyces lamella composition 1.00 of Example 1

Polyethylene Glycol Monostearate 2.00

Self-emulsifying glycerin monostearate 5.00

Cetyl Alcohol 4.00

Squalene 6.00

Tri2-ethylhexaneglyceryl 6.00

Sphingolipids 1.00

1.3-butylene glycol 7.00

Purified water level

(Unit: wt%)

Formulation Example 4 Packed Formulations

Lactobacillus lamella composition of Example 1 5.00

Polyvinyl Alcohol 13.00

L-ascorbic acid-2-magnesium phosphate salt 1.00

Lauroylhydroxyproline 1.00

Water Soluble Collagen (1% Aqueous Solution) 2.00

1,3-butylene glycol 3.00

Ethanol 5.00

Purified water level

(Unit: wt%)

Formulation Example 5 Cosmetic Liquid Formulation

Bifidus lamella composition of Example 1 2.00

Hydroxyethylene Cellulose (2% Aqueous Solution) 12.00

Xanthan gum (2% aqueous solution) 2.00

1,3-butylene glycol 6.00

Dark Glycerin 4.00

Sodium Hyaluronate (1% aqueous solution) 5.00

Purified water level

(Unit: wt%)

Claims (13)

A cell wall decomposing step of decomposing the cell wall of the microorganism; And
Method for producing a lamella structure comprising a high pressure emulsification step of high pressure emulsification of the remaining microbial cell constituents after cell wall is decomposed. The method of claim 1,
The microorganism is at least one microorganism selected from the group consisting of Lactobacillus sp. , Bifidobacterium sp. And Saccharomyces sp . The method of claim 1,
The high pressure emulsifying step is to repeat the microfluidizer treatment two or more times, a method for producing a lamella structure. The method of claim 1,
The high pressure emulsifying step includes the high pressure emulsification of the microbial cell construct with phospholipids. 5. The method of claim 4,
Wherein the phospholipid is added in an amount of 10 to 50 parts by weight when the weight of the microbial cell construct is 100 parts by weight. A microbial lamella composition comprising a lamellae structure derived from a microbial cell construct. 7. The microbial lamella composition of claim 6, wherein the microbial cell construct is a construct from which the cell wall of the microorganism is degraded. The method according to claim 6,
The microorganism is at least one microorganism selected from the group consisting of Lactobacillus sp. , Bifidobacterium sp. And Saccharomyces sp. Strain, microbial lamella composition. The method according to claim 6,
Wherein said lamellae structure is derived from a mixture of microbial cell constructs and phospholipids. 10. The method of claim 9,
The phospholipid is 10 to 50 parts by weight, when the weight of the microbial cell constituent is 100 parts by weight, microbial lamellae composition. The method according to claim 6,
The lamellar structure is prepared by the method for producing a lamellar structure according to any one of claims 1 to 5, microbial lamella composition. An anti-inflammatory composition comprising the microbial lamella composition according to any one of claims 6 to 10. An anti-inflammatory composition comprising the microbial lamella composition according to claim 11.

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