KR101648130B1 - Method for producing microorganism lamella construct and composition comprising microorganism lamella construct - Google Patents

Abstract

The present disclosure relates to a cell wall degradation step of degrading a cell wall of a microorganism; And a high-pressure emulsification step of emulsifying the microbial cell constituents remaining in the cell walls and decomposing the cell wall, and a composition comprising the microelement cell-derived lamellar structure, wherein the lamellar structure and the lamellar composition having excellent stability are provided And can provide a lamellar structure such as a liposome, emulsion or liquid crystal which functions as a carrier of an active ingredient, and can be used in various fields such as foods, cosmetics and pharmaceuticals by providing a lamellar structure containing various physiologically active ingredients derived from microorganisms .

Description

TECHNICAL FIELD The present invention relates to a method for producing a microorganism lamellar structure and a microorganism lamellar construct,

The present disclosure relates to a lamellar structure derived from a microorganism, a method for producing the same, and a composition containing the same.

Recently, the development of foods and cosmetics using fermented foods and fermented materials has become vigorous, and interest in fermenting microorganisms is increasing. Natural plant material or medicinal herbs contain various physiologically active substances and antioxidant substances, but after extraction, they are unstable and sometimes irritating, and often cause toxicity to human body. Therefore, in recent years, attempts have been made to stabilize natural extracts, reduce toxicity, or convert them into stable derivatives to increase their effectiveness. As a result, biological transformation methods using microorganisms or enzymes have been developed And representative examples of the fermentation technique include fermentation techniques.

Fermentation is a process in which organisms originated from plants and animals are degraded by the enzymatic action of microorganisms, and then they are regenerated as essential nutrients of organic organisms.

Specifically, fermentation refers to a case where the fermentation becomes useful to the human body through decomposition or synthesis of organic substances by microorganisms. As such, typical fermenting microorganisms that produce useful substances for the human body include Mycelia, Yeast, Fungi, Bacillus, Lactobacillus, and Acetic Acid.

In addition, various foods are produced by the fermentation process through the fermenting microorganisms. These foods, which are obtained by converting food ingredients by microorganisms, are called fermented foods. Examples of representative fermented foods include miso, cheonggukjang, kochujang, kimchi, salted fish, cheese, yogurt, vinegar, sikhye, and sake.

On the other hand, the food consumed in the human body is decomposed by the lactic acid bacteria in the intestines and converted into a low-molecular substance which is easily absorbed into human cells, transformed into an active form from an unstable or inactive form, or a human harmful substance contained in the food And is then absorbed through various actions such as detoxification. These results suggest that the intestinal microorganisms are important for bioconversion.

Therefore, attempts to maximize the efficacy of natural plant material and herbal medicine materials by fermentation of microorganisms such as lactic acid bacteria or yeast, or to solve the safety problem, have an important meaning in the development of various foods, cosmetics, medicines and the like. In particular, microorganisms propagating in Korean traditional foods such as kimchi and soybean are reported to have excellent biological activity. Therefore, many studies are being conducted to use microorganisms isolated from traditional fermented foods.

The beneficial effect of fermentation on the human body is the result of the effect of the fermented metabolites and the nutrient content of the microorganism itself. Yeast and lactobacillus can be used to make healthy the intestines when they are ingested as live cells, as well as to make the human body healthy by supplying nutrients. In the field of cosmetics, fermented products have been used as raw materials, but microorganisms themselves are rarely used.

Since the nutrients of microorganisms are present in many membrane structures of cells, it is possible to develop excellent raw materials that have the nutritional components of microbial cells as they are, by appropriately using the cell membrane structure of microorganisms.

Korean Patent Application No. 10-2004-0024437 Korean Patent Application No. 10-2000-0002793

An object of one embodiment of the present invention is to provide a lamellar structure which functions as a carrier of an active ingredient.

It is an object of another embodiment of the present invention to provide a lamellar structure containing useful microbial nutrients.

A method of manufacturing a lamellar structure according to an embodiment of the present invention includes: a cell wall decomposing step of decomposing a cell wall of a microorganism; And a high pressure emulsification step in which the cell wall is decomposed and the remaining microbial cell constituents are emulsified under high pressure.

The microbial lamellar composition according to an embodiment of the present invention may include a lamellar structure derived from microbial cell constituents.

Using the techniques disclosed herein, lamellar structures and lamellar compositions with excellent stability can be provided. In addition, a lamellar structure such as liposome, emulsion or liquid crystal functioning as a carrier of the active ingredient can be provided. In addition, by providing a lamellar structure containing various physiologically active ingredients derived from microorganisms, it can be utilized in various fields such as foods, cosmetics and pharmaceuticals.

Fig. 1 shows the results of treatment of the lamellar composition derived from each of the negative control (UV) treated with UV and the secretion amount of TNF- [alpha] of the positive control (dexamethason) treated with dexamethasone, the lactobacillus, the saccharomyces and the bifidus, Of TNF- [alpha]. ≪ / RTI >

Microorganisms are composed of cell membranes and cell walls. The cell membrane consists mainly of phospholipids and the cell membrane consists of a combination of various polymers including a peptidoglycan layer. The cell membranes of microorganisms have a bilayer structure composed mainly of phospholipids. The phospholipid bilayer structure has a self-assembly property, and it is possible to form a lamellar structure such as a liposome, an emulsion or a liquid crystal. Since the cell membranes and cell walls of the microorganisms contain physiologically active agents including various enzymes, the lamellar composition composed of the microbial cell structure has various physiological activities.

As used herein, the term " lamellar structure "means a structure having a multilayer structure of two or more layers. It is a concept that includes the lipid bilayer structure of normal cell membranes. But are not limited to, liposomes, emulsions, or liquid crystal structures.

In the present specification, the term " high-pressure emulsification "is a broad concept meaning that high pressure is applied to emulsify. For example, by 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 around 1000 bar. A device for high pressure emulsification may include, but is not limited to, a microfluidizer. Microfluidic devices, for example, mix the components of a lamellar structure and pass through a cartridge cell or valve (also referred to as an interaction chamber) having micro-pore micro-pores, where the size of the micro- Is about 50 to 300 micrometers, and a lamellar structure such as a double lipid film formed by a surfactant is formed by the generated large shear stress.

As used herein, "liposome" means a structure in which a phospholipid is suspended in an aqueous solution to form a layer. Liposomes can be microcapsulated by inserting a drug or a cosmetic ingredient as a carrier.

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

As used herein, the term " liquid crystal "refers to a material in which the position of a molecule and the direction of a molecular axis is a complete intermediate state between a solid and a 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 the properties of crystallinity in the properties such as light scattering and reflection.

A method of manufacturing a lamellar structure according to an embodiment of the present invention includes: a cell wall decomposing step of decomposing a cell wall of a microorganism; And a high pressure emulsification step in which the cell wall is decomposed and the remaining microbial cell constituents are emulsified under high pressure.

The step of degrading the cell wall may include decomposing the cell wall by adding an enzyme to the microorganism. Enzymes can be cell wall degrading enzymes. For example, enzymes such as Lysozyme or glucanase may be used, but are not limited thereto. The cell walls can be made into partially or fully degraded sphaeroplast or protoplast forms and then lamellar structures.

In one embodiment of the present invention, microorganisms may be used, but not limited to, lactic acid bacteria or yeast that have been used for food production and have been proven to be safe. Lactic acid bacteria include Lactobacillus sp. Strain or Bifidobacterium sp. Strain. Examples of the yeast include a strain of Saccharomyces sp .

The high-pressure emulsification step may be repeated two or more times, three times, or four or more times with a microfluidizer treatment. Repeating more than 2 times reduces the size of the lamellar structure.

The high pressure emulsification step may comprise high pressure emulsification of the microbial cell construct with a phospholipid. 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 component is 100 parts by weight. If the amount of the phospholipid is less than 10 parts by weight, the effect of stabilization by the phospholipid is insignificant. If the amount of the phospholipid exceeds 50 parts by weight, the content of the microbial cell components is decreased and the function of the cell constituent is deteriorated.

Also, one embodiment of the present invention provides a food, cosmetic or pharmaceutical composition characterized by containing the microbial lamellar composition.

When a composition according to an embodiment of the present invention is applied to a pharmaceutical composition, an inorganic or organic carrier, including an active ingredient, is added to the lipid lamellar composition to prepare an oral or semi-solid preparation in the form of solid, semi-solid or liquid Formulation may be made with the former administration agent. Examples of the oral administration agent include tablets, pills, hard and soft capsules, liquids, suspensions, emulsions, syrups, powders, powders, granules, granules and pellets, , Diluents (such as lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (such as silica, talc, stearic acid and magnesium or calcium salts thereof and polyethylene glycols) . Tablets may also contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose or polyvinylpyrrolidine, optionally mixed with starch, agar, alginic acid or its sodium salt Such as disintegrants, absorbents, coloring agents, flavoring agents, and sweetening agents. The tablets may be prepared by conventional mixing, granulating or coating methods. The parenteral administration may be, for example, an injection, a drip, an ointment, a lotion, a gel, a cream, a spray, a suspension, an emulsion, a suppository, no.

The pharmaceutically acceptable dose, i.e., the dosage, of the microbial lamellar composition will vary depending on the age, sex, body weight of the subject to be treated and the severity of the particular disease or condition to be treated, the route of administration and the judgment of the prescriber. For example, a typical dosage may be from 0.01 μg / kg / day to 1000 mg / kg / day and may be from 1 μg / kg / day to 40 mg / kg / day, And are not intended to limit the scope of the invention.

The microbial lamellar composition according to an embodiment of the present invention may be provided in a food composition. The food composition may be a health food, a functional food, and a food additive composition. The food composition may contain other components which can give rise to a synergistic effect on the main effect to the extent that the main effect of the present invention is not impaired. For example, additives such as perfume, coloring agent, bactericide, antioxidant, preservative, moisturizing agent, thickening agent, inorganic salt, emulsifier or synthetic polymer substance may be further added to improve physical properties. In addition, it may further contain auxiliary components such as water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymeric polysaccharides and seaweed extract. 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 present invention may be in various forms such as solutions, emulsions, viscous mixtures, tablets, powders, and the like.

The dosage of the active ingredient is within the level of those skilled in the art and its daily dose is, for example, from 0.1 μg / kg / day to 5000 mg / kg / day, more specifically from 50 μg / kg / day, but it is not limited thereto, and may be varied depending on various factors such as the age, health condition, and complications of the subject to be administered.

Meanwhile, the cosmetic composition according to an embodiment of the present invention may be used as a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing oil, a powder foundation, an emulsion foundation, Spray, or the like, but is not limited thereto. More specifically, the present invention relates to a cosmetic composition, which comprises at least one of a softening agent, a nutritional lotion, a lotion, a body lotion, a nutritional cream, a massage cream, a moisturizing cream, a hand cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, (O / w) type, water-in-oil type (O / W) type, lipstick, makeup base or foundation.

The 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. Examples of the compounding ingredients that may be added include organic solvents such as a preservative component, a moisturizer, an emollient, a surfactant, an organic and inorganic pigment, an organic powder, an ultraviolet absorbent, a preservative, a bactericide, an antioxidant, a plant extract, a pH adjuster, A blood circulation promoting substance, a cooling agent, an antiperspirant agent, and purified water. In addition, the components to be added in addition to the above components are not limited thereto, and any of the above components may be compounded within a range that does not impair the objects and effects of the present invention.

Hereinafter, the present invention will be described in further detail with reference to the preferred embodiments of the present invention. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not limited thereto.

[Example 1] Production of microbial lamellar structure

Microorganisms of the genus Lactobacillus ( Lactobacillus plantarum , KCTC 3103) and microorganisms of Bifidobacterium ( Bifidobacterium longum, KCTC 3420), each emal-S (MRS, Difco) in a culture, and saccharose in MRS yeast (Saccharomyces cerevisiae, KCTC 7904) were cultured in a 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 and suspended in 10 mM Tris-EDTA buffer. Lysozyme, a cell wall lytic enzyme, was treated with lactic acid bacteria and glucanase was treated with yeast to dissolve the cell wall. Enzyme treatment was carried out by adding 1 ml of an enzyme solution having a concentration of 10 mg / ml to 10 ml of the culture solution and reacting at 37 ° C for 2 hours. The cell wall-lysed cells were recovered 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 enzyme-free microorganism was repeated three times at 1000 bar with a high pressure emulsifier (Microfluidizer Processor M-110EHI, Microfluidics Corporation) to prepare a microbial lamellar composition. The solution prepared from the microbial lamellar composition was filtered with a membrane having a pore size of 1.2 mu m.

Table 1 shows the change in the size of the microbial lamellar composition with the number of times of processing the microfluidizer.

Strain name Number of high-pressure emulsifier treatment 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 Sakaromisses 0 pass > 3000 Sakaromisses 1 pass 1177 Sakaromisses 2 pass 676 Sakaromisses 3 pass 423

As shown in the above table, a lamellar structure of 600-700 nm was obtained in all strains in two repeated treatments, and a lamellar structure of 400 nm in all strains was obtained in three repeated treatments.

[Experimental Example 1] Stability test of microbial lamellar composition

The stability of each microbial lamellar composition prepared through the treatment of the microfluoridizer three times in Example 1 was tested.

The stability of the microbial lamella composition was measured at room temperature, 4 ℃, and for 3 months at 40.

The results are shown in Table 2 below.

As shown in the above table, it can be seen that a very stable state is maintained for three months at all temperatures.

[Experimental Example 2] Evaluation of skin effect of microbial lamellar composition

Human keratinocyte (ATCC CRL-2404) was 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) And incubated at 37 [deg.] C in an incubator containing 5% carbon dioxide. The obtained keratinocytes were dispensed into 6-well plates at 3 × 10 ⁵ per well and cultured for 24 hours under cell culture conditions. After culturing, the cells were irradiated with ultraviolet rays in an amount of 25 mJ / cm 2, and the microbial lamella composition prepared in Example 1 was added and further cultured for 24 hours. The culture supernatant was collected and the secretion amount of TNF-α, an inflammatory cytokine, was measured. Dexamethasone 1 Um was used as a positive control. The results are shown in Fig.

Figure 1 shows the result of treatment of the negative control (UV) with no treatment after UV irradiation and the result of the positive control (dexamethason) treated with dexamethasone and the lamellar composition derived from Lactobacillus. As a result, the lamellar composition derived from bifidus The results of treatment, and the results of treatment of the lamellar composition derived from yeast.

As a result of the experiment, the microbial lamellar composition showed an excellent anti-inflammatory effect by suppressing the increase of secretion of TNF-α by the stimulation of ultraviolet rays. This seems to be due to the physiologically active substance of the microorganism.

Hereinafter, a formulation example of a composition according to an embodiment of the present invention will be described.

[Formulation Example 1]

Lactobacillus lamellar composition of Example 1 5.00

Maintaining 75

Sodium hydroxide 5

Fragrance 10

Purified water balance

(Unit: wt%)

[Formulation Example 2] A lotion-type preparation

The bifidal lamellar composition of Example 1 3.00

L-ascorbic acid-2-phosphate magnesium 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 balance

(Unit: wt%)

[Formulation Example 3] Cream-type preparation

The saccharomyces lamellar composition of Example 1 1.00

Polyethylene glycol monostearate 2.00

Self emulsifying monostearate glycerin 5.00

Cetyl alcohol 4.00

Squalane 6.00

Tri-2-ethylhexane glyceryl 6.00

Sphingo glycolipid 1.00

1.3-butylene glycol 7.00

Purified water balance

(Unit: wt%)

[Formulation Example 4]

Lactobacillus lamellar composition of Example 1 5.00

Polyvinyl alcohol 13.00

L-ascorbic acid-2-phosphate magnesium 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 balance

(Unit: wt%)

[Formulation Example 5] Cosmetic liquid preparations

The bifidal lamellar 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 balance

(Unit: wt%)

Claims (13)

An effective component delivery carrier composition,
Wherein the cell wall comprises a lamellar structure formed from a high pressure emulsion derived from a partially or fully degraded microbial cell construct,
The microorganism is at least one microorganism selected from the group consisting of a Lactobacillus sp. Strain, a Bifidobacterium sp. Strain, and a Saccharomyces sp. Strain,
Lt; RTI ID = 0.0 > lamellar < / RTI > structure. delete The method according to claim 1,
Wherein the high pressure emulsion is roughened more than once with a microfluidizer treatment. The method according to claim 1,
Wherein the high pressure emulsion is a high pressure emulsion of a mixture comprising the microbial cell construct and a phospholipid. 5. The method of claim 4,
Wherein the phospholipid is in an amount of 10 to 50 parts by weight when the weight of the microbial cell component is 100 parts by weight. Comprising as an active ingredient a microbial-derived lamellar composition formed from a high pressure emulsion derived from a microbial cell construct in which the cell wall is partially or fully degraded,
Wherein the microorganism is at least one microorganism selected from the group consisting of a Lactobacillus sp. Strain, a Bifidobacterium sp. Strain, and a Saccharomyces sp. Strain. delete The method according to claim 6,
Wherein the high-pressure emulsion is subjected to microfluidizer treatment twice or more. The method according to claim 6,
Wherein the high pressure emulsion is a high pressure emulsion of a mixture comprising the microbial cell construct and a phospholipid. 10. The method of claim 9,
Wherein the phospholipid is in an amount of 10 to 50 parts by weight when the weight of the microbial cell component is 100 parts by weight. 7. A method for producing a carrier composition according to any one of claims 1 to 5,
A cell wall degradation step of decomposing the cell wall of the microorganism; And
A high pressure emulsification step in which the cell wall emulsifies the microbial cell constituents, partially or fully degraded, under high pressure. 10. A method for producing a anti-inflammatory composition according to any one of claims 6 to 9,
A cell wall degradation step of decomposing the cell wall of the microorganism; And
A high pressure emulsification step in which the cell wall emulsifies the microbial cell constituents, partially or fully degraded, under high pressure. delete

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