KR101705482B1 - The Atopic dermatitis treatment lotion or cream containing Ceriporia laceratas - Google Patents

Abstract

The present invention relates to a culturing method for improving the productivity of active ingredients of a Ceriporia lacerata cultured material, and to atopy dermatitis alleviating lotion or cream containing mycelia culture medium exopolysaccharide and betaglucan. Mycelia dried materials, concentrates, exopolysaccharide, and betaglucan cultured by the Ceriporia lacerata is cultured by adjusting rpm for supplying and agitating air at 0-500. The lotion or cream uses Ceriporia lacerata as an active ingredient. A method for producing a Ceriporia lacerata mycelia culture medium and lotion or cream having a high atopy dermatitis alleviating effect by containing an extract thereof and a natural material derived therefrom as an active ingredient are widely provided to people in the world.

Description

The atopic dermatitis treatment lotion or cream containing Ceriporia laceratas,

The present invention relates to a lotion or cream composition for improving atopic dermatitis, which comprises ceropiola lucerata, and more particularly to an extracellular polysaccharide or beta-glucan produced by Ceriporia lacerata. A mycelial culture of three lipolylacerase comprising the extracellular polysaccharide or beta-glucan. A concentrate of mycelium of the above-mentioned extracellular polysaccharide or 3-lipolylacerase comprising beta-glucan. As an active ingredient, an extract of a dried product thereof, or an extract of a culture product of the mycelial culture of the above-mentioned lipopolysaccharide.

Atopic dermatitis is a chronic, highly recurrent inflammatory skin disease in infancy or childhood, accompanied by pruritus, dry skin, and eczema. In infancy, it starts with the face and limbs, but grows and appears as an eczema in the arms and knees. Adults are more likely to develop eczema on the face compared to early childhood, with thickening of the folding skin. Atopic dermatitis is a worldwide increasing trend and its prevalence is reported to be 20% of the population.

As the industrialization progresses, allergic substances increase due to excessive consumption of instant food, changes in concrete, residential space, fine dust, and industrial factors. As a result, a variety of genetic and immunological abnormalities have occurred and various diseases have been induced. Among them, skin diseases including immunological diseases such as atopy are increasing. In particular, atopy is a chronic skin disease accompanied by dry skin and itching.

Patients with atopic dermatitis are treated with steroid ointments and antihistamines in illnesses, clinics and pharmacies. However, side effects such as pigmentation, hair loss, itching, and facial flushing have been reported when steroid medications are used continuously.

It is also known to cause cortical dysfunction, diabetes, gastritis, depression, headache, osteoporosis, and elevated blood pressure. In addition, it has been reported that when steroid ointment is used, serious side effects such as skin infections, steroid acne, steroid dermatitis, and other pituitary adrenal cortical functions are brought about.

Such inflammatory reactions in the skin are caused by infection of microorganisms, excessive secretion of hormones, and excessive secretion of sebum components. The inflammatory response is known to be mediated by prostaglandin E2 produced by the action of cyclooxy or azet-2.

 In order to develop a lotion or cream composition having the effect of improving the atopic dermatitis using a natural substance, various effects of the three lipolylacerases have been studied. It has found that it possesses both a strong prostaglandin E2 inhibitory activity and an antimicrobial activity through anti-inflammatory and antimicrobial activity, and it has been found that it contains at least three kinds of active ingredients such as atopic dermatitis And the present invention has been completed.

Accordingly, it is an object of the present invention to provide a lotion or cream composition to people that has the effect of ameliorating atopic dermatitis.

Conventional techniques for the treatment of atopic diseases developed so far include, for example, Korean Patent Registration No. 10-1528135, which is a pharmaceutical composition for prevention or treatment of atopic dermatitis, which contains, as an active ingredient, an extract of White roots or a pyrogallol compound isolated therefrom Composition for treating atopic dermatitis by extracting pyrogallol material from white beans. Another technology is Korean Patent No. 10-1325094, which is a composition for preventing or treating atopic dermatitis containing extract of mushroom mushroom, which is a method for extracting active ingredient for treating atopy in mushroom mushroom. Another Korean Patent Registration No. 10-1227758 discloses a composition for improving atopic dermatitis containing an extract of Quercus variabilis extracted from a treated oyster oak wood chip as an active ingredient. In addition, a process for producing improved skim-mushroom-derived polysaccharides for mass-producing skimoric acid polysaccharides in high yield, which is disclosed in Korean Patent Laid-Open No. 10-2010-0094868, has also been described in the prior art, This method is useful for mass production of polysaccharide derived from skim mushroom which can be produced at a high yield, but at a low cost. The thus produced skim mushroom polysaccharide is useful for development of cosmetics such as functional soaps, lotions and skins, skin protection, A number of techniques that can be utilized have been described in the prior art, however, as described herein, by improving the medium composition and culture method of the three lipolylaceratra to produce an extracellular polysaccharide or beta-glucan; A mycelial culture of the above-mentioned extracellular polysaccharide or 3-lipolylacerase comprising beta-glucan; The present invention also provides a method for improving the productivity of the extract of mycelial cultures of the above-mentioned Sera lipolactacrata and cultivating it industrially rapidly and in a large amount, and a technology for providing atopy improvement lotion or cream containing Sera lipolactaclora excellent in effect Has not yet been launched anywhere.

The non-patent literature of the present invention also provides a glimpse of a number of related documents. First, functional biomaterials based on natural dye-based biological materials (Park Tae Kyu Trend / Research Report, Konkuk University, 2013) have been made. In addition, this study was conducted based on the characteristics of extracellular polysaccharide production and antidiabetic effect (Kim, Ji - Eun, Ph.D., Keimyung University, 2013) through the liquid cultivation of mycelium of Sella lipolas serratata mushroom. In addition, a method of culturing an extracellular polysaccharide by liquid culture of mycelium of Tremella fuciformis (Cho Eun-Dae Daegu University 2007) was preceded. Antioxidant and anticancer effects of mushroom mycelia fermented by mushroom mycelium (Sangmye Yeong Gyeongsang National University, Korea Traditional Fermented Food Research Institute, 2207) As shown in the paper, various functional materials such as high molecular polysaccharides and beta - Can be utilized as food, cosmetics and functional materials.  In particular, liquid cultivation of mycelium mycelium is economical because it can effectively produce useful substances in a short time by optimizing biotransformation techniques. Therefore, research on cultivation of various mushroom mycelia including edible and medicinal materials, selection of excellent seeds according to production of various functional materials, and optimization technology of bioconversion technology using them are industrially useful.  Accordingly, the inventors of the present invention have developed a liquid culture of the above-mentioned three lipolylacerase and developed a functional substance, thereby being stable without skin irritation through natural substances and having an effect of improving atopic dermatitis containing seripositive liposarate ≪ / RTI >

Although atopic dermatologic diseases cause physical and psychological distress in patients, only temporary remedies with side effects such as steroids and antihistamines are currently available, and alternatives are needed. The present invention provides a lotion or cream for atopic dermatitis which is free from side effects and can infiltrate into the dermis to control inflammatory substances to improve atopic dermatitis.

The present invention improves the culture medium and cultivation method of the cellulolytic enzyme, as described above, to produce an extracellular polysaccharide or beta-glucan. A mycelial culture of three lipolylacerase comprising the extracellular polysaccharide or beta-glucan. A concentrate of mycelium of the above-mentioned extracellular polysaccharide or of three lipolactacera containing beta-glucan. And a method for culturing the same in an industrially rapid and large-scale manner by improving the productivity of the dried product thereof or the extract product of the mycelial culture product of the cellulolyticase. It is an object of the present invention to provide a method for producing a cultured Mycelium lacticera mycelium, an extract of the same and a natural substance derived from the same, as an active ingredient, a bathing agent, a body lotion and an atopic dermatitis improvement lotion or cream composition .

In order to achieve the above object, the present invention relates to an extracellular polysaccharide or beta-glucan produced by Sera lipolactacera. A mycelial culture of three lipolylacerase comprising the extracellular polysaccharide or beta-glucan. A concentrate of the mycelial culture of the above-mentioned lipopolysaccharide. And a lotion or cream for improving atopic dermatitis containing an extract of the dried product and a culture of the mycelial culture of the aforementioned lipopolysaccharide as an active ingredient.

The present invention also relates to a method for various culturing of cerporolactaceratas, that is to say, during initial and during culture, depending on the pH, the amount of air injected, the oxygen supply during the culture, and the impeller speed at which the culture material can be agitated The present invention discloses a method for maintaining proper DO (Dissolved Oxygen) and uniformly diffusing the medium components, and industrially mass-producing the same according to the optimization, thereby preparing a lotion or cream composition for improving atopic dermatitis.

 The composition of the present invention can be widely used for general use of lotion or cream composition for improving atopic dermatitis through reduction of atopic dermatitis through reduction of skin inflammatory cells.

 An extracellular polysaccharide or beta-glucan produced by three lipolylacerate in the medium composition according to the present invention. A mycelial culture of three lipolylacerase comprising the extracellular polysaccharide or beta-glucan. A concentrate of mycelium of the above-mentioned extracellular polysaccharide or 3-lipolylacerase comprising beta-glucan. A carbon source, a nitrogen source, and an inorganic salt can be optimally optimized for the industrial production of an extract of the dried product thereof or the mycelial culture product of the above-mentioned cellulolytic enzyme.

In addition, the present invention relates to an extracellular polysaccharide or beta-glucan produced by three lipolylaceratases by a culture method. A mycelial culture of three lipolylacerase comprising the extracellular polysaccharide or beta-glucan. A concentrate of mycelium of the above-mentioned extracellular polysaccharide or 3-lipolylacerase comprising beta-glucan. Or by preparing an extract of the dried product thereof or a mycelial culture product of the above-mentioned cellulolytic enzyme.

It is also possible to optimize the method of uniformly diffusing the medium component and maintaining dissolved oxygen in accordance with the impeller speed at which the cultured material can be agitated in order to uniform the initial pH during the culture, the amount of air injected, the oxygen supply during cultivation, . ≪ / RTI >

In addition, nutrients can be added to the culture to improve the amount of mycelium, thereby increasing the amount of dried cellulolytic enzyme and the amount of extracellular polysaccharide and beta-glucan contained in the dried product.

As a method for culturing the mycelium used in the lotion or cream of the present invention, a solid culture method and a liquid culture method can be used. In the solid culture method, the operation for collecting only the mycelium after the cultivation is completed and the culture time is long, The mycelial liquid culture method has a small possibility of contamination compared with the solid culture method and has an advantage that it can be mass-cultured in a relatively compact space in a short time.

In order to produce a large amount of a liquid culture containing mushroom mycelium and useful components in a short period of time, it is required to optimize the medium components and use economical raw materials.

As nutrients for liquid culture of mushrooms, a carbon source derived from by-products such as corn steep liquor, yeast extract and peptone, and an expensive nitrogen source are used.

Most fungi form pellets or filament hyphae during shaking culture, and especially bacillus species form hyphae aggregates of various sizes. Wall growth occurs due to the mycelial aggregate, which makes it difficult to transfer the substance by stirring and aeration, thereby inhibiting mycelial growth and production of the product. Although the air-lift fermenter has been used for some time, the use of a large-scale air-lift fermenter for industrial production is insufficient.

Therefore, cultivation in a fermentation tank is inevitable, and measures for preventing wall growth must be taken. The method of culturing the existing three lipolylaceratra is reported in terms of temperature, pH, CO ₂ concentration, amount of light to be irradiated, and medium component in an air lift type incubator. However, In order to increase the contents of the mycelia, the amount of the dried product, and the extracellular polysaccharide and beta-glucan contained in the cell culture medium, There is no method of culturing using a stirring speed of a stirrer, aeration amount, or the like.

Accordingly, there is a demand for a development method that can improve production using a cultivation method capable of industrial production through optimization of a culturing method of a natural material, such as a cellulolytic enzyme.

Accordingly, the inventors of the present invention have continued intensive studies on the extract of the culture medium of the mycelial L. serrata, and as a result, found that the culture of the L. serrata is improved according to the culture conditions, The inventors of the present invention have completed the present invention by discovering that the extract of the culture medium and the resulting beta-glucan and extracellular polysaccharide have an excellent effect for improving the dermatitis of the lotion or cream composition for improving atopic dermatitis.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the growth of mycelium according to initial pH at the time of culturing three lipriac lactase in one embodiment of the atopic improvement lotion or cream containing the three lipolylacerase of the present invention. FIG.
FIG. 2 is a graph showing productivity of mycelium over time during culturing of three lipolactacerata cultures in one embodiment of lotion or cream for improving atopic sensation containing the present lipolylacerase of the present invention. FIG.
FIG. 3 is a graph showing the results of a culture of seripositive Lactacerata according to the kind of a culture medium in a lotion or cream for improving atopic sensation containing the present lipolylacerase.
Figure 4 shows the results of culturing in a 700 L air culture incubator and an optimal condition of a 5,000 L stirred bioreactor in one embodiment of an atopic recovery lotion or cream comprising the present granola lacerata. Graph.
FIG. 5 is a graph showing the anti-inflammatory activity according to the treatment amount of three lipolactaclata in one embodiment of atopic lotion-containing lotion or cream containing the present lipolylacerase.
FIG. 6 is a graph showing the results of a comparison between the pre-use and the post-treatment of atopic patients using ointment prepared by using three liprias lactacerate concentrates in one embodiment of the atopic improvement lotion or cream containing the three- It is a photograph afterwards.
FIG. 7 is a graph showing the results of a comparison between the pre-use and the post-use of atopy patients using the ointment prepared by using the three lipriac lactacer concentrate in one embodiment of the atopic improvement lotion or cream containing the three lipolylacerase of the present invention for 2 months It is a photograph afterwards.

Hereinafter, embodiments of the present invention will be described in detail. In the following description of the present invention, a detailed description of known functions and configurations incorporated herein will be omitted when it may make the subject matter of the present invention rather unclear. In addition, terms used in this specification are terms used to appropriately express the preferred embodiments of the present invention, which may vary depending on the user, the intention of the operator, or the practice of the field to which the present invention belongs.

Therefore, the definitions of these terms should be based on the contents throughout this specification. Throughout the specification, when an element is referred to as "comprising ", it means that it can include other elements as well, without excluding other elements unless specifically stated otherwise.

The present invention is characterized in that the lotion or cream composition for improving atopic dermatitis as described above contains ceriplora lactaclorata.

The lotion or cream composition for improving atopic dermatitis according to the present invention is characterized in that when cultured in a liquid culture medium of Saporolylaceras due to stirring and aeration change under a dissolved oxygen concentration of a certain concentration or more, the amount of mycelium, Thereby improving the content of the exopolysaccharide and beta-glucan.

 In addition, if the dissolved oxygen amount is not controlled by the stirring speed by controlling the stirring speed and the dissolved oxygen amount is maintained at a predetermined concentration or more, the aeration is controlled in two stages to effectively remove the mycelium from the jar fermenter system in the jar fermenter system. And dried products, extracellular polysaccharides and beta-glucans.

The present invention relates to an extracellular polysaccharide or beta-glucan produced by three lipolylacerase. A mycelial culture of three lipolylacerase comprising the extracellular polysaccharide or beta-glucan. A concentrate of mycelium of the above-mentioned extracellular polysaccharide or 3-lipolylacerase comprising beta-glucan. Or an extract of the mycelial culture of the above-mentioned C. lipolascerata.

Therefore, an extracellular polysaccharide or beta-glucan produced by the above-mentioned lipopolysaccharide. A mycelial culture of three lipolylacerase comprising the extracellular polysaccharide or beta-glucan. A concentrate of mycelium of the above-mentioned extracellular polysaccharide or 3-lipolylacerase comprising beta-glucan. A composition containing the dried extract of the plant or an extract of the mycelial culture of the above-mentioned cellulolytic agent may be commercialized as a lotion or cream composition for improving atopic dermatitis.

When formulated into a lotion, essence or cream for improving atopic dermatitis, the content of the extracellular polysaccharide or beta-glucan is 0.01 to 10% by weight, preferably 0.05 to 5% by weight based on the total weight of the composition.

At this time, the content of extracellular polysaccharide or beta-glucan is suitably adjusted within the above range according to the content of components contained in the lotion or cream composition for improving atopic dermatitis.

The lotion or cream composition for improving atopic dermatitis of the present invention can be prepared in any formulations conventionally produced in the art, and there is no particular limitation. Acceptable raw materials or additives can be used for the formulation of the atopic dermatitis improvement lotion or cream and any raw material or additive known to be usable in the art for the preparation of the formulation to be prepared can be used have.

The lotion, cream composition for improving atopic dermatitis of the present invention can be prepared in any formulations conventionally produced in the art, and there is no particular limitation. Acceptable raw materials or additives can be used for the formulation of the atopic dermatitis improving cream and cream and any raw materials or additives known to be used in the art in the preparation of the formulations to be manufactured can be used have.

According to one embodiment of the present invention, the content of the extracellular polysaccharide or beta-glucan in the composition of the present invention is preferably 0.01 to 10% by weight or 0.1 to 10% by weight based on the total weight of the total composition, .

The present inventors have determined the optimal medium composition and culture conditions of mycelia in order to establish a liquid culture method for the mass production of the cultured lipase of cereals, which is the core substance of lotion or cream for improving atopic dermatitis.

The present inventors have determined the optimal culture conditions and culture conditions of mycelium in order to establish a liquid culture method for mass production of a cultivar, which is a core substance of lotion and cream for improving atopic dermatitis.

The method for mass production of the mycelium of ceriplora lactaclora according to the present invention comprises the steps of obtaining a mycelium of ceriplora lactaclora for liquid culture from a ceriplora lactacerata in the production method of ceraplora lactaclora, Culturing the mycelium of Serratia in a liquid culture, and culturing the liquid cultured Mycelium lacticera mycelium by using a bioreactor.

(One) Three Li Pori From Rock Serata  For liquid culture Three Li Pori Lacerata  Step of obtaining mycelium.

Sera lipolyacerata mycelia for liquid culture can be obtained from Sera lipolactacera. The cell lipolylacerase was cultured for 7 to 11 days in a PDA (Culture medium 100 plastic culture medium, Difco, Becton Dickinson and Company) on a PDA (Potato dextrose agar) medium.

Then, a PDB (Potato dextrose broth) medium (Difco) 200 was prepared in an Erlenmeyer flask equipped with a 500-baffle, and then one PDA culture was added and cultured for 7-11 days. At this time, the amount of the mycelium to be added to the inoculum is preferably about 0.3% (w / v) based on the amount of the solution to be cultured.

(2) a step of liquid culturing the mycelium of ceriplora lacera.

And then culturing the mycelia of ceriplora lactacerata by inoculating the culture medium for liquid culture with the mycelium of cerifolia lactaclora obtained in the above step (1).

The culture medium for liquid culture can be adjusted to pH 4.06.5 by including carbon source, nitrogen source, vitamin, and inorganic substance in water. Then, the mycelium of ceriplora lactaclora in the pH-adjusted culture medium can be added to the culture liquid at a concentration of 0.1 to 10% relative to the volume of the culture medium, and liquid culture can be performed at 20 to 25 ° C for 7 to 11 days.

 The carbon source may be selected from the group consisting of Maltose, Glucose, Lactose, Starch, Dextrin, Sucrose, Fructose, Galactose, Mannose, ), And oligosaccharide may be used.

 The nitrogen source may be selected from soybean powder, cottonseed powder, amino acid, peptone,

And at least one selected from congestion liquor can be used.

The inorganic material may be selected from the group consisting of potassium monophosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ), calcium carbonate (CaCO ₃ ), calcium chloride (CaCl ₂ ), magnesium sulfate (MgSO ₄ ), sodium chloride (MnSO4) can be used.

In one preferred embodiment of the present invention, the culture medium is prepared by mixing 0.2 ~ 5% glucose, 0.2 ~ 1% peptone, 0.05 to 0.25% potassium monophosphate (KH ₂ PO ₄ ), 0.02 to 0.1% magnesium sulfate (MgSO ₄ ), and water. Preferably, 3 wt% of glucose, 0.5 wt% of peptone, 0.1 wt% of potassium monophosphate (KH ₂ PO ₄ ), and 0.05 wt% of magnesium sulfate (MgSO ₄ ) are preferable.

(3) a liquid culture using a bioreactor Three Li Pori Lacerata  Culturing the mycelium.

The mycelia of the ceripolial lactaceras cultured by the above step (2) may be inoculated into a culture solution in a bioreactor and cultured to produce a culture of ceriplora lactaclora.

The culture medium inside the bioreactor can be adjusted to pH 4.0 to 6.5 by including carbon source, yeast extract, nitrogen source, vitamins and minerals in the purified water.

Then, the liquid cultured Sella lipolactacera mycelium is inoculated into the culture liquid in the bioreactor at a concentration of 0.1 to 10% of the volume of the culture liquid, and then air of 0.02 to 2 vvm (volume of air added to liquid volume per minute) And cultured at a temperature of 20 to 25 DEG C for 7 to 11 days to prepare a culture of ceriplora lactaclora.

In the culture medium inside the bioreactor, the carbon source may be at least one selected from maltose, glucose, lactose, starch, dextrin, sucrose, fructose, galactose and mannose.

In the culture medium inside the bioreactor, the nitrogen source may be selected from soybean powder, cottonseed powder, amino acid, peptone, and CSL.

The inorganic material may be selected from the group consisting of potassium monophosphate (KH ₂ PO ₄ ), potassium diphosphate (K ₂ HPO ₄ ), calcium carbonate (CaCO ₃ ), calcium chloride (CaCl ₂ ), magnesium sulfate (MgSO ₄ ), sodium chloride Manganese (MnSO4) may be used.

The present invention can be used to prepare a culture solution of various constituents for liquid culture of mycelium of ceraria lacera.

Preferred as one embodiment of the medium is 0.2 to 5% glucose, 0.2 ~ 1% of peptone, 0.05 ~ 0.25% one potassium phosphate _{(KH 2 PO 4), 0.02} ~ 0.1% of magnesium sulfate (MgSO ₄₎ and water . ≪ / RTI > Preferably 3% by weight of glucose, 0.5% by weight of peptone, 0.1% by weight of potassium monophosphate (KH ₂ PO ₄ ) and 0.05% by weight of magnesium sulfate (MgSO ₄ ).

In the present invention, a bioreactor for large-scale cultivation of mycelia lacticera mycelium is commonly used in the related field of the present invention, and any bioreactor capable of injecting air can be used. As an example of such a bioreactor in the present invention, any one selected from a circulation type (air bubble type) air bioreactor, a balloon type air bioreactor, a column type air bioremediation bioreactor and an agitated bioreactor can be used .

On the other hand, the present invention is a three-lipolylacerase culture obtained by the above-mentioned method of mass-producing the myelofibrosarcera mycelium. And an extract or a dried product of the culture medium of ceriplora lacerata.

(4) Three Li Pori From the lactase culture  The step of preparing the dried product, the extract, the extracellular polysaccharide or the beta-glucan.

The extracellular polysaccharide or beta-glucan of the present invention can be obtained from a cell culture supernatant.

Preferably, the extracellular polysaccharide or beta-glucan is produced by culturing (a) a cellulolytic enzyme in a liquid medium to produce a mycelial culture, (b) culturing the mycelium of ceriplora lacera, Followed by drying and pulverizing, and (c) extracting the mycelial culture powder with a solvent to obtain an extracellular polysaccharide or beta-glucan from the extract.

In the step (b), the mycelial culture produced in the step (a) may be pulverized by vacuum drying or lyophilization. The drying is preferably carried out at a temperature of 40 DEG C or lower, more specifically, at a temperature of 30 DEG C or lower for 48 to 96 hours in order to prevent the disappearance of the active substance. The drying in the step (b) preferably uses a vacuum freeze dryer rather than a vacuum dryer in which the evaporation temperature is set to be relatively high, so that the change in effective substance content is minimized.

The extract of the mycelial culture of the present invention can be obtained by culturing the mycelial culture of the above-mentioned mycelium of Cerporaria lactara according to a conventional method known in the art, that is, by using ordinary solvents such as hot water extraction, Heating extraction, ultrasonic extraction, supercritical extraction, and the like.

At this time, each raw organic plant is preferably extracted using an extraction solvent, and then mixed and used.

Specifically, a solvent selected from the group consisting of water, glycols having 1 to 6 carbon atoms, anhydrous or hydrated alcohol having 1 to 4 carbon atoms, acetone, ethyl acetate, chloroform, 1,3-butylene glycol, .

More preferably, 35 to 45% (v / v) 1,3-butylene glycol is extracted using as an extraction solvent. In this case, the amount of the extraction solvent used is 10 to 30 times the volume of the raw material, more preferably 20 times.

The extraction temperature and time may vary depending on the amount of the sample to be extracted. It is preferable that the extraction is performed by heating at 40 to 100 ° C for 3 to 100 hours or at 50 to 70 ° C for 1 to 5 days. Or purification process.

In addition, the extract of the mycelial culture of the present invention may be prepared in powder form by an additional process such as vacuum distillation, freeze-drying or spray-drying.

In step (c), the cultured mycelial body obtained in step (b) is extracted with a solvent, and an extracellular polysaccharide or beta-glucan as an active ingredient of the composition according to the present invention is isolated and prepared.

To obtain an extracellular polysaccharide, 100 mg of distilled water was added to 1 ~ 10 g of dry powder and suspended well. The suspension was centrifuged at 10,000 rpm for 20 minutes, and its purity was 95% or more And the mixture is allowed to stand in a refrigerator at 2 to 6 ° C for 12 to 18 hours.

After centrifugation for 20 minutes at 10,000 rpm, the precipitate is collected and lyophilized to produce a crude extracellular polysaccharide.

Wherein the extracellular polysaccharide comprises from 40 to 60% by weight of sugar, from 30 to 40% by weight of protein, from 40 to 50% by weight of sugar and from 32 to 38% by weight of protein, or from 43 to 47% % Protein, more specifically about 45 wt% sugar and about 34 wt% protein.

The β-glucan is suspended in 10 g of dry powder by adding 50 ml of distilled water, and the pH is adjusted to 10.0 using 20% sodium carbonate at room temperature, followed by allowing to stand for about 10 hours so that the powder sample is sufficiently softened. To this softened sample solution, α-amylase stable at high temperature is added, and the mixture is stirred for 2 hours in a constant temperature water bath at 60 ° C., treated with an enzyme, and cooled to room temperature. This solution is adjusted to pH 5 and then stirred in a constant temperature water bath at 60 ° C for 2 hours with amyloglucosidase.

Then, the pH is adjusted to 4.5 to inactivate the enzyme, and the mixture is stirred in a constant temperature water bath at 5 캜 for 30 minutes and then cooled. The cooled extract is centrifuged (30 min, × 8000 rpm) to remove only the supernatant. The concentrate is concentrated under reduced pressure at a temperature of 40 ° C. Four times as much ethanol is added to the concentrate for 4 hours and centrifuged to obtain a precipitate. The precipitate is dissolved again in water, the pH is adjusted to 4.5, and the enzyme is reprocessed at 58 ° C for 2 hours with Amyloglucosidase. After the treatment, the enzyme is inactivated at 95 ° C for 30 minutes, and then cooled and centrifuged to obtain the supernatant, which is then reprecipitated with ethanol to prepare crude beta-glucan.

Hereinafter, the present invention will be described in detail with reference to Examples and Test Examples.

It should be understood, however, that these examples are provided for illustrative purposes only and are not to be construed as limiting the scope of the present invention.

Three Li Pori Lacerata  Determination of medium and growth conditions in solid culture of mycelium.

The optimal growth medium was determined by measuring mycelial growth and density using various kinds of media described in the following Table 1 in order to determine the medium having the best growth properties of mycelia Lactarata mycelium. Specifically, each kind of medium was sterilized by adjusting pH to 5.5 before sterilization. After culturing the mycelium of C. liparcerata for 10 days in an incubator fixed at 24 ± 2 ℃ for the cultivation, the growth and viability of the mycelium and the diameter of the mycelium of C. lipa ceratata were measured, .

Investigation of Mycelial Growth by Different Media Types. badge PDA YM NU YMG LB MYP MCM Diameter / mm 76 67 54 70 61 71 67 Mycelial density *** ** * *** ** ** *

Mycelial density: excellent (***), good (**), moderate (*)

Place: CL Bio Research Institute.

PDA: Potato 200g, Dextrose 20g, Agar 15g / L

Y M: Yeast extract 3g, Malt extract 3g, Peptone 5g, Dextrose 10g, Agar 15g / L

NU: Peptone 5 g, Beef extract 3 g, Agar 15 g / L

YMG: Yeast extract 4g, Malt extract 10g, Glucose 4g, Agar 15g / L

LB: 10 g of NaCl, 5 g of Yeast extract, 10 g of Tryptone, 15 g / L of Agar

MYP: Malt extract 30 g, Yeast extract 2 g, Peptone 1 g, Agar 15 g / L

MCM: Glucose 20g, Yeast extract 2g , Peptone 3g, MgSO 42 O 0.5 g, K 2 HPO 4 1g, KH 2 PO 4 0.46 g, Agar 20 g / L

As a result, as shown in Table 1, PDA, YMG, and MYP medium showed excellent growth performance. In particular, when the PDA medium was used, the mycelial growth was most excellent.

Three Li Pori Lacerata  Measurement of Growth by Temperature of Mycelium.

In order to measure the growth performance of the mycelium of the cell culture medium, which is the core material of the present invention, using the PDA medium having the best growth characteristics, the culture temperature was adjusted to 18 ° C., 20 ° C., 22 ° C., 24 ° C. , And set at 26 DEG C, and then cultured for 10 days to examine the growth of mycelium according to each temperature.

Investigation of Mycelial Growth by Temperature. Temperature() 18 20 22 24 26 Diameter (mm) 38 64 80 75 70 Mycelial density * ** *** *** ***

Mycelial density: excellent (***), good (**), moderate (*)

Place: CL Bio Research Institute.

As a result, as shown in Table 2, the growth was increased to a temperature of 22 ° C, but decreased at a temperature higher than 22 ° C, and the highest growth temperature was measured at 22 ° C.

Three Li Pori Lacerata  Liquid species culture of mycelium Three Li Pori Lacerata  Investigation of the Growth Properties of Mycelium by Initial pH.

In order to determine the medium having the best growth properties of the mycelial growth medium, the following table 1 shows the amount of mycelium in PDA (Potato dextrose broth) Respectively.

As a result, as shown in FIG. 1, the amount of mycelium almost similar at pH 5 to 6 was exhibited, and the best cell growth was observed at pH 5.5.

Three Li Pori Lacerata  Growth characterization of cultured mycelium over time.

To investigate the completion time of cultivation of mycelia of Cerporaria lacera, cultured mycelium was investigated. For culturing, the mycelia cultured in the PDA solid medium for 12 days were homogenized by a mixer and inoculated into the seed culture broth. The culture conditions were initially incubated at pH 5.5 and 22 ° C and 120 rpm in shaking incubator.

As a result, as shown in Fig. 2, there was almost no cell growth until the third day of cultivation. However, from the fourth day onwards, the cells grew rapidly, and the amount of cells was similar from day 9 to day 12, Respectively. The optimum seed culture time was from 9 to 12 days.

Three Li Pori Lacerata  Production of physiologically active substances by liquid culture of mycelium.

The three lipolylacerases were incubated for 10 days in PDA, medium (100 plastic culture dishes, Difco, Becton Dickinson and Company). Then, 200 ml of PDB and Difco (Difco) were prepared in an Erlenmeyer flask with a 500-ml baffle. One PDA culture was added and cultured for 10 days with shaking. And ₂ L of liquid culture containing 3 wt% of glucose, 0.5 wt% of peptone, 0.1 wt% of potassium monophosphate (KH ₂ PO ₄ ), 0.05 wt% of magnesium sulfate (MgSO ₄ ), 0.2 wt% of silicone foam, The medium was sterilized in a 5 L incubator, 200 mL of the PDB culture strain to be used as an inoculum was inoculated in a state cooled to 25 캜, and incubated at 25 캜 for 7 to 11 days with stirring at a stirring speed of 300 rpm and air at 0.5 vvm. Liquid culture was carried out to prepare a culture of mycelium of ceriplora lactaclorata.

Three Li Pori Lacerata  Preparation of Dried Powder.

The cultured Mycelium lacticera mycelium prepared in Example 5 was lyophilized at a temperature of 35 ° C or lower for 48 hours using a vacuum freeze dryer to obtain dry granules of the cultured mycelium lacticera mycelium .

Three Li Pori Lacerata  Preparation of extracellular polysaccharide (EPS) from cultures.

100 ml of distilled water was added to 2 g of the dried powder prepared in Example 6, stirred well, and centrifuged (10,000 rpm, 20 minutes). Ethanol equivalent to three times its amount was added to the supernatant, 2 to 8 占 폚) for 16 hours. The precipitate was taken out from the abovementioned lyophilizate and lyophilized to prepare an extracellular polysaccharide from the cultured mycelium lacticera mycelium.

Three Li Pori Lacerata  Preparation of beta-glucan from cultures.

10 g of the dry powder prepared in Example 6 was suspended in 50 ml of distilled water, and the pH was adjusted to 10.0 using 20% sodium carbonate at room temperature. After 10 hours, the powder sample was sufficiently softened Respectively. To this softened sample solution, α-amylase stable at high temperature was added and the mixture was stirred for 2 hours in a constant temperature water bath at 60 ° C., treated with enzyme, and cooled to room temperature. This solution was adjusted to pH 5 and then stirred with a amyloglucosidase in a constant temperature water bath at 60 ° C for 2 hours. Then, the pH was adjusted to 4.5 to inactivate the enzyme, followed by stirring in a constant temperature water bath at 95 캜 for 30 minutes, followed by cooling.

The cooled extract was centrifuged (30 min x 8000 rpm) to remove only the supernatant, and then concentrated under reduced pressure at a temperature of 40 ° C. To this concentrate, 4-fold amount of ethanol was added and allowed to stand for about 4 hours, followed by centrifugation to obtain a precipitate. The precipitate was dissolved in water again, the pH was adjusted to 4.5, and the enzyme was reprocessed with amyloglucosidase at 58 DEG C for 2 hours. After the treatment, the enzyme was inactivated at 95 ° C for 30 minutes, then cooled, centrifuged, and the supernatant was taken out and reprecipitated with ethanol to prepare crude beta-glucan.

Depending on the type of bioreactor Three Li Pori Lacerata  Mycelial culture.

Since mycelial cultivation using the above-described bioreactor differs in the amount of mycelial growth depending on the type of the bioreactor, in order to select an appropriate incubator type for the cultivation of the mycelium of the three lipolakarerata mycelium, The amount of growth, the amount of dried matter, the extracellular polysaccharide contained in the dried material and the content of beta glucan were compared. The culture conditions were as follows: 0.5 vvm bubble column type, 300 rpm and 0.5 vvm stirring type, and the incubation temperature was 25 ° C. To measure mycelial growth, cultures were centrifuged at 8000 rpm for 10 minutes, washed twice with distilled water and centrifuged twice. After centrifuging, the precipitated mycelia were dried at 105 ° C. and then the amount of dried mycelium (DCW, dry cell weight) was measured. In addition, extracellular polysaccharide and beta glucan were measured by the contents of the dried product, respectively.

Mycelium, dried matter, extracellular polysaccharide, and beta-glucan produced in each incubator are shown in FIG. As shown in FIG. 3, the amount of mycelium and dry matter was the highest in the agitated type, but the contents of extracellular polysaccharide and beta-glucan contained in the dried matter were similar in the column type and agitated type of the air floating type.

Therefore, an extracellular polysaccharide and beta-glucan are produced in an agitating type incubator which is similar to the column type and the agitation type of the air floating type, but the amount of the mycelium and the amount of the dried material is high.

Stirring type  In a bioreactor Three Li Pori Lacerata  Culture optimization.

The medium composition and culture conditions of Example 5 were such that the concentrations of the mycelium of ceriplora lactacerus, the concentration of the powder, and the content of extracellular polysaccharide and beta-glucan contained in the dried material, Respectively.

The agitation speed of the incubator was maintained at 0, 50, 100, 200, 300, 400 and 500 rpm from the beginning of culture to the completion of cultivation.

according to rpm Three Li Pori Lacerata  Mycelial culture. rpm A steady state of growth
(Work) Amount of mycelium
(g / L) Amount of dried matter
(g / L) Extracellular polysaccharide content
(mg / g Powder) Beta-glucan content
(mg / g Powder) 0 4 4.7 15.9 2.42 14.1 50 5 6.7 26.5 3.78 20.1 100 7 7.9 29.9 4.55 25.5 200 9 8.8 34.2 5.01 29.2 300 10 8.5 31.0 4.97 28.6 400 8 8.3 30.7 4.95 28.7 500 5 3.9 12.8 1.72 11.4

Place: Lab. Of Food and Nutrition, Keimyung University (2015.09)

As a result, as shown in Table 3, the concentration of the mycelium of cerolaria lacera, the concentration of the dried material, and the content of extracellular polysaccharide and beta-glucan contained in the dried matter were low at a low rpm of 100 or less, To 400 was somewhat higher.

However, it decreased significantly at 500 rpm. In terms of the growth retardation period, the low rpm was faster, but the mycelial concentration, the concentration of the dried material, and the content of extracellular polysaccharide and beta - glucan contained in the dried matter were higher than the incubation period. However, at the rpm of 200 to 400, the point of stagnation of growth was more than 8 days, but the efficiency was lower than that of low rpm considering culture period. Therefore, it was found that the cultivation of Sera lipolyceratata was changed by the initial rpm control. In addition, it was estimated that when the initial agitation was not performed, or starting from a low rpm and then gradually increased, the culturing performance would be improved.

Culture by air supply (Aeration).

The medium composition and culture conditions of Example 5 were the same as in Example 1 except that the concentrations of the mycelium of ceriplora lactacerus, the concentration of the powder, and the extracellular polysaccharide and beta-glucan contained in the dried matter, And the content thereof was measured. The agitation speed of the incubator was rpm 200, which was irradiated under the most favorable condition. The air supply was maintained at 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2 vvm from the beginning to the completion.

Depending on air supply Three Li Pori Lacerata  Mycelial culture. air
flow
(vvm) Growth
Stagnation (days) Amount of mycelium
(g / L) Amount of dried matter
(g / L) Extracellular polysaccharide content
(mg / g Powder) Beta-glucan content
(mg / g Powder) 0 2 1.9 5.4 1.12 5.2 0.01 8 8.3 29.7 4.78 27.5 0.02 10 8.6 30.8 4.89 28.8 0.05 10 8.5 31.1 5.02 29.1 0.1 10 8.5 30.9 5.01 29.0 0.2 10 8.6 31.4 5.10 30.1 0.5 10 8.4 30.6 4.98 29.4 One 9 8.4 30.8 5.01 28.1 2 3 3.7 8.9 1.89 9.5

Place of Experiment: Laboratory of Food and Nutrition, Keimyung University

As a result, as shown in Table 4, when air was not supplied, the culture was not performed. As the air supply from 0.01 vvm to 1 vvm was increased, the culture results showed no significant difference. However, at 2 vvm, the concentration of myceliallacerase mycelium, the concentration of the dried material, and the extracellular polysaccharide The content of beta-glucan was significantly decreased. Therefore, it was found that the air supply amount from 0.01 vvm to 1 vvm was advantageous for the culture in the agitated bioreactor.

Culture according to pressure.

The culture medium and the culture conditions of Example 5 were used to measure the content of the mycelium of ceriplora lactaceras, the amount of the dried material, and the content of extracellular polysaccharide and beta-glucan contained in the dried matter according to the initial internal pressure. The agitation speed of the incubator was set to rpm 200, which was the most favorable condition in the above embodiment. The air supply amount was 0.2 vvm, which is the most favorable condition in the above embodiment, and the internal pressure was 0.05, 0.1, 0.2, 0.5, cm < ^{2 &} gt ;.

Pressure-dependent Three Li Pori Lacerata  Mycelial culture. Pressure
(kg / cm ² ) Growth
Congestion
(Work) Amount of mycelium
(g / L) Amount of dried matter
(g / L) Extracellular polysaccharide content
(mg / g Powder) Beta-glucan content
(mg / g Powder) 0.05 10 8.5 28.9 4.95 28.1 0.1 10 8.6 28.5 4.88 29.1 0.2 10 8.5 29.1 4.89 28.7 0.5 10 8.4 30.1 4.91 29.2 One 10 8.5 29.5 4.94 29.4 1.5 10 8.6 29.9 4.92 29.3

Place of Experiment: Laboratory of Food and Nutrition, Keimyung University

As a result, as shown in Table 5, even when the internal pressure of the agitated bioreactor was increased or decreased, there was almost no influence on the culture. Therefore, it was found that the pressure resistance was negligible in culturing mycelia lactase.

Culture by dissolved oxygen control.

As shown in the above Examples and the like, it was found that the effect of rpm was higher than that of the air supply or the internal pressure when the agitated bioreactor was cultivated, which is an important factor in the cultivation of the ceriplora lactaclor.

Therefore, there are shear stresses (mycelial shear stress) and dissolved oxygen content in the culture medium, which is represented by the influence of rpm.

In terms of the shear stress, the concentration of mycelium, the concentration of the dried material, and the concentration of the cells contained in the dried matter in the culture of the three lipolylaceratta depending on the amount of dissolved oxygen, which are presumed to have a large influence on the mycelium, The contents of exopolysaccharide and beta - glucan were investigated.

The initial culture conditions were rpm 50, air supply 0.2 vvm, internal pressure 0.5 kg / cm ² , and the dissolved oxygen levels were 0, 5, 10, 20, 30, 40, 50 and 70%. As the culture progressed, the rpm was firstly controlled from the time when the target dissolved oxygen amount was reached, and when the target dissolved oxygen amount was not maintained, the air supply amount and the internal pressure were controlled to be constant.

Depending on the amount of dissolved oxygen Three Li Pori Lacerata  Mycelial culture. DO
(%) A steady state of growth
(Work) Amount of mycelium
(g / L) Amount of dried matter
(g / L) Extracellular polysaccharide content
(mg / g Powder) Beta-glucan content
(mg / g Powder) No control 4 4.3 12.6 2.13 13.3 5 10 12.6 39.1 6.91 40.2 10 11 12.7 40.2 7.15 39.7 20 12 15.2 45.3 8.22 46.1 30 11 13.6 41.1 7.45 42.3 40 11 13.2 41.3 7.27 40.9 50 9 8.9 30.2 5.23 31.1 70 7 6.3 25.4 4.11 23.2

Place: Lab. Of Food and Nutrition, Keimyung University (2015.09)

As a result, as shown in Table 6, the amount of dissolved oxygen was as small as 40% in terms of the growth of the cells, but there was no significant difference. However, the amount of mycelium, the amount of the dried material and the extracellular polysaccharide And the content of beta-glucan were remarkably increased.

The amount of extracellular polysaccharide and β-glucan contained in the dried matter was 8.22 mg / g Powder, and the amount of β-glucan was 12.2 mg / L. And irradiated with 46.1 mg / g Powder.

Cultivation was improved by more than 50% compared to the case without consideration of dissolved oxygen.

Therefore, it was found that it is advantageous to control the rpm, the air supply, and the internal pressure to maintain a certain concentration of dissolved oxygen in the cultivation of ceraplora lucerata.

Cultivation by addition of nutrients during culture.

In addition, when the mycelium is cultured using a bioreactor, the nitrogen source and the carbon source, which affect the growth of the cell and the productivity of the useful substance, are added in order to increase the productivity of the useful substance.

As a result, the addition of nitrogen source was advantageous for cell growth, but the content of extracellular polysaccharide and beta - glucan contained in the dried material was decreased.

Therefore, the addition of the carbon source, which is a method of increasing the productivity of the useful substance, was performed. According to the conditions described in the above examples, the initial conditions and the composition of the medium and the amount of dissolved oxygen retained during the culture were set to 20% Respectively.

Cultivation according to the kind of carbon source added.

Glucose, fructose, mannose, sucrose, maltose and starch were added to the culture medium for 3 days at 5 days. The starch was 20% and the remaining carbon was 70%.

Depending on the type of carbon source added during the culture Three Li Pori Lacerata  Mycelial culture. Carbon source Amount of mycelium
(g / L) Amount of dried matter
(g / L) Extracellular polysaccharide content
(mg / g Powder) Beta-glucan content
(mg / g Powder) glucose
(Glucose) 17.7 52.1 9.12 50.9 fruit sugar
(Fructose) 16.4 51.9 8.30 50.7 Mannoose
(Mannose) 18.1 55.2 9.25 54.8 Sugar
(Sucrose) 18.0 57.1 9.51 59.2 Flesh
(Maltose) 16.7 49.7 8.66 47.3 Starch
(Starch) 15.2 45.8 8.18 44.9

Place of Experiment: Laboratory of Food and Nutrition, Keimyung University

As a result, as shown in Table 7 above, when the carbon source was added as a whole, the cultivation performance was improved by culturing Serrachia lacera. Especially, the addition of mannose or sugar increased the amount of mycelial lipid peroxidase, beta - glucan, and extracellular polysaccharide contained in the dried matter.

Therefore, when the carbon source was added during the culture, the productivity of the mycelium and various useful materials was improved. Especially, when mannose and sugar were added, the productivity was improved.

Cultural characterization by sugar concentration maintained during culture.

As shown in the results of the above examples, the addition of a carbon source to the sugar added during the culture improved the culturing ability. Therefore, in order to investigate the optimum sugar concentration to be added, 0.1, 0.2, 0.5, 1, 2, 3, 4, 5% relative to the culture volume was added to the culture solution for 5 days.

Depending on the carbon source concentration added during the culture Three Li Pori Lacerata  Mycelial culture. Addition concentration
(%) Amount of mycelium
(g / L) Amount of dried matter
(g / L) Extracellular polysaccharide content
(mg / g Powder) Beta-glucan content
(mg / g Powder) 0 14.7 44.2 8.16 45.1 0.1 15.4 47.6 8.91 48.2 0.2 15.9 48.2 9.11 47.9 0.5 16.7 50.7 9.23 51.5 One 17.6 53.2 9.39 58.7 2 18.2 59.2 9.78 61.3 3 17.9 57.4 9.56 60.1 5 15.1 48.1 8.92 50.8

Place of Experiment: Laboratory of Food and Nutrition, Keimyung University

As a result, as shown in the above Table 8, the addition of sugar did not increase the cultivation, but the concentration of added sugar increased up to 2%, but decreased at higher concentrations.

Therefore, when 2% of sugar was added, the amount of mycelial lipase, amount of dried matter, and extracellular polysaccharide and beta - glucan content contained in the dried material were increased.

Cultivability according to size of incubator.

The size of the agitated bioreactor performed in the above example is 5 L, which is not suitable for industrial use. In order to carry out large-scale cultivation of Sella lipolactacera for industrial production in the future, cultivation studies according to the size of the incubator are required.

Therefore, culturing was carried out at 5, 10, 30, 100, 500, and 5,000 L according to the results of the above examples. The initial culture condition was rpm 0, air supply 0.2 vvm, internal pressure 0.5 kg / cm ² , and the rpm was firstly increased to maintain the dissolved oxygen content 20%, and then when the rpm reached the maximum value, The internal pressure was raised to adjust. On the 5th day of fermentation, sugar was added to a concentration of 2% and cultured for 12 days.

Depending on incubator size Three Li Pori Lacerata  Mycelial culture. Size of incubator
(L) Amount of mycelium
(g / L) Amount of dried matter
(g / L) Extracellular polysaccharide content
(mg / g Powder) Beta-glucan content
(mg / g Powder) 5 18.3 59.5 9.53 60.2 10 18.5 60.1 9.51 59.3 30 17.9 60.3 9.47 60.7 100 18.2 58.9 9.60 60.7 500 18.5 60.5 9.52 61.5 5,000 18.4 60.7 9.61 61.7

Place: Gyeongbuk Bio Industry Research Institute

As a result, as shown in Table 9, there were no significant differences in the amount of mycelium, the amount of dried matter, and the content of extracellular polysaccharide and beta-glucan contained in the cellulolytic enzyme, depending on the size of the incubator.

Therefore, it was found that the size of the incubator was not influenced by the cultivation of.

Comparison of existing and optimized cultures.

In Korean Patent No. 10-1031605, production was carried out using an air-part column type 700 L incubator. Experiments were performed in the present invention to compare optimized culture conditions in an agitated incubator.

Experimental conditions were as follows: The conditions for the air column 700L were 3 vvm and 25 ℃ in the patented condition, and the initial condition of 25 ℃, 0 rpm, air supply 0.5 vvm and internal pressure 0.5 kg / cm ^{2 in} the 5,000 L agitator The amount of dissolved oxygen was maintained at 20% or more during the culture and 2% of sugar was added to the tea for 5 days.

As a result, as shown in FIG. 4, the amount of mycelium, the amount of dried matter, and the content of extracellular polysaccharide and beta-glucan contained in the dried cultivar were increased by more than 100% Especially the amount of mycelium increased to 150%.

Therefore, it can be seen that compared to the air lifting type, the maintenance of the dissolved oxygen amount by the regulation of rpm and the cultivation performance in the agitation type bioreactor by the addition of the carbon source during the culture are improved.

Three Li Pori Rock Serrat  Antibacterial and antifungal test.

The antimicrobial activity and antifungal activity of the dried product of Example 2 were investigated by adding the dried product to the cell lysate containing the test strain as a method for the antibacterial and antifungal test of Sellapora lacerata. 4.0 g of the sample prepared in Example 6 was dissolved in 96 g of purified water and used as a preparation. The test strain used for the antibacterial activity was Staphylococcus aureus subsp . aureus ATCC 6538. The test strains used for antifungal activity were Candida albican s ATCC 11006 and Trichophyton rubrum ATCC 28188.

For the test method, the test strain used for the antimicrobial activity is cultured in a nutrient broth at 37 ° C for 24 hours, and the cultured cells are diluted with physiological saline to prepare a test strain dilution (1 × 10 ⁷ CFU / ml). 0.5 ml of the preparation solution of Example 2 was added to 0.5 ml of the test strain dilution. After 1, 5, and 10 minutes, 0.2 ml of the suspension was plated on Nutrient agar plate medium and incubated at 37 ° C for 48 hours. The colonies were counted and the bactericidal activity was observed.

The test strain used for antifungal activity was Candida The growth of albican s ATCC 11006 is cultured in YPD liquid medium at 25 ° C for 48 hours, and the cultured cells are diluted with physiological saline to make diluted test solution (1 × 10 ⁷ CFU / ml). 0.5 ml of the preparation solution of Example 3 was added to 0.5 ml of the test strain dilution. After 1 minute, 5 minutes, and 10 minutes, 0.2 ml of the suspension was plated on a YPD agar plate medium and incubated at 25 ° C for 48 hours. The colonies were counted and the bactericidal activity was observed. As a test strain used for antifungal activity , Trichophyton The growth of rubrum ATCC 28188 was incubated in Sabourauds liquid medium at 25 ° C for 72 hours, suspended in 5 ml, and centrifuged (3,500 rpm) to collect spores. The recovered spores were mixed with sterilized distilled water and Tween 20 to prepare 0.5 ml of the spore solution adjusted to have a concentration of about 1 x 10 ⁶ cells / ml, 0.5 ml of the preparation solution of Example 2, After 10 minutes, 0.2 ml of the suspension was plated on a Sabourauds agar plate medium and cultured at 25 ° C for 96 hours. The colonies were counted and the bactericidal activity was observed. Saline was used as a control. The results are shown in Tables 12 and 13 to 14 below.

Results of antibacterial test. Results of Antimicrobial Activity / Strain Number of cells (CFU / ml) 1 minute 5 minutes 10 minutes Saline solution 7.5 10 ⁶ 3.5 10 ⁶ 2.8 10 ⁶ Staphylococcus aureus subsp . aureus ATCC 6538 1.5 10 ⁵ 2.7 10 ³ Below 100

Place: CL Bio Research Institute.

Antifungal activity test results on yeast. Antifungal activity test results on yeast / strain Number of cells (CFU / ml) 1 minute 5 minutes 10 minutes Saline solution 7.5 10 ⁶ 3.5 10 ⁶ 2.8 10 ⁶ Candida albican s ATCC 11006 2.5 10 ⁵ 3.4 10 ² Below 100

Place: CL Bio Research Institute.

Antifungal activity test results against mold. Antifungal activity test results against fungi / strain Number of cells (CFU / ml) 1 minute 5 minutes 10 minutes Saline solution 5.2 10 ⁵ 5.1 10 ⁵ 4.9 10 ⁵ Trichophyton rubrum ATCC 28188 1.5 10 ⁵ 2.7 10 ⁴ 2.1 10 ²

Place: CL Bio Research Institute.

As shown in Tables 12 and 13 to 14, in the control group using saline, the number of cells was constant except for a slight decrease. In the case of adding the preparation of Example 3, Respectively. Therefore, it can be seen that the effect of antibacterial and antifungal agents is exhibited in the case of Sellapora lacera.

Three Li Pori Rock Serratia  Anti-inflammatory activity test for Korean.

The anti-inflammatory effect of the above-mentioned culture medium, concentrate, dried product, its beta-glucan, and extracellular polysaccharide was measured. The synthesis of prostaglandin E2, an inflammation mediator, was induced by adding phorbol 12-myristate 13-acetate, which is an inflammatory substance, to human U937 cell line, , And at the same time, the amount of biosynthesis of prostaglandin E2 was measured after 4 hours after the addition of the culture medium, the concentrate, the dried product, the extract of betaglucan and the extracellular polysaccharide to the concentration, 6, the biosynthesis of prostaglandin E2 was inhibited by increasing the concentration of cerporolactaceratta.

Atopy improvement efficacy experiment.

To examine the effect on atopic dermatitis, a lotion or cream containing 1 to 4% of at least one of the culture medium, concentrate, dried product, extracellular polysaccharide and beta-glucan obtained in Example 3 was prepared. Other compositions were prepared as follows but are not limited thereto.

Atopic dermatitis was diagnosed according to the criteria of atopy. A total of 10 patients with atopic dermatitis were evaluated. Clinical efficacy was evaluated by using it twice a day in the morning and evening for 1 month.

<Lotion>

 The above-mentioned emulsifiable concentrate of the present invention can be obtained by mixing the above-prepared three lipolactaceratas, cetostearyl alcohol, self emulsifying monostearic acid, dimethicone, monostearic acid sorbitan, herbal oil, herbal extract, stearic acid, mineral oil, polyethylene glycol monostearate, glycerin, - Butyleneglycol, carbomer, triethanolamine were heated and injected and emulsified. After the emulsification was completed, the mixture was thermally cooled to 50 DEG C with stirring using an agitator, and a small amount of fragrance was added thereto. The mixture was cooled to 25 DEG C and then aged.

Clinical efficacy evaluation of lotion for improvement of atopy.
Contains by substance
Atopy lotion
skin
Itching
Moisture power
Skin inflammation Culture After 1 month + + + Two months later ++ + + Concentrate After 1 month ++ ++ ++ Two months later +++ +++ +++ Building After 1 month + + + Two months later ++ ++ ++ Extracellular polysaccharide After 1 month + + + Two months later + + + Beta Glucan After 1 month + + + Two months later ++ ++ +

(+++: very good, ++: good, +: improvement effect, -: no change)

<Cream>

The above prepared triglycerol lactase, carboxy polymer, butylene glycol, glycerin and purified water, stearic acid, herbal oil, herbal extract, glyceryl monostearate and capric triglyceride were heated and injected and emulsified. After completion of the emulsification, the mixture was cooled to 35 DEG C with stirring using an agitator, cooled to 25 DEG C and then aged.

Clinical efficacy evaluation of atopy cream.
Contains by substance
Atopic Cream
Itchy skin
Moisture power
Skin inflammation Culture After 1 month + + + Two months later + + + Concentrate After 1 month ++ ++ ++ Two months later +++ ++ +++ Building After 1 month ++ + ++ Two months later +++ +++ +++ Extracellular polysaccharide After 1 month + + + Two months later + + + Beta Glucan After 1 month + + + Two months later ++ + +

(+++: very good, ++: good, +: improvement effect, -: no change)

It can be seen from Tables 15, 16 and 17 that when the lotion or cream for improving atopic dermatitis is used, it is possible to provide a product having a high effect on improvement of skin inflammation and itching according to the concentration.

Specific clinical cases.

1) Age, sex and occupation: teenagers, female students

Period of use: The concentrate prepared in the Example was applied twice a day to the atopic dermatitis site for 2 months. As shown in FIG. 6, the clinical subject began to show effects 14 days after the application to the atopic dermatitis site, and 80% or more of the atopic skin inflammation decreased after 2 months.

2) Age, sex and occupation: 40s, male, office worker

Period of use: The concentrate prepared in the Example was applied twice a day to the atopic dermatitis site for 2 months. As shown in FIG. 7, the clinical subjects began to show effects on the atopic dermatitis area 10 days after the application, and after 2 months, 80% or more of atopic skin inflammation decreased.

The present invention relates to a method for the treatment of atopic dermatitis in a lotion or cream for improving atopic dermatitis, which comprises culturing a three-lipolylacerase culture, a dried product and a concentrate thereof, an extracellular polysaccharide derived from three lipolylaceratara, And the like.

In addition, the above-mentioned cultivar Lacerata culturing method includes a step of culturing a solid culture medium and a liquid seed culture medium followed by a large volume culture, and a culture method using cultured three-lipoa lactaclora will use an agitated bioreactor.

And the culture, the dried product or concentrate thereof, and the extracellular polysaccharide and the beta-glucan derived from the cellulolytic enzyme are contained in the lotion or cream. The extracellular polysaccharide and beta-glucan derived from the mycelial body cultured with the above-mentioned cultivar lucerata and the cellulolytic enzyme, and the beta-glucan are adjusted to 0-500 rpm for air supply and agitation in the culture step.

In addition, the amount of air supplied was controlled to 0.01 to 10 vvm in the step of culturing the mycelial body cultured from the cultured mycelium and the extracellular polysaccharide and beta-glucan derived from the cell lipolactacera, 1.5 kg / cm ² , and the carbon source is added during the culturing in the main culturing step. When the carbon source is added in the culturing step, 0.1 to 10% of carbon source is added per liter of the culture liquid.

In addition, the lotion or cream for improving atopic dermatitis has a constitution comprising at least one or at least one of the mycelial culture, extract, concentrate and dried product of the above-mentioned Sellapora lactacrata.

In addition, a lotion or cream for improving atopy, which comprises the above-mentioned granulocyte lucerata as an active ingredient, is a cultured lipolylacerase culture liquid cultured. Concentrate of culture of S. lipolytica. The dried product of the culture of Sellapora lacera. Thereby increasing the content of extracellular polysaccharide or beta-glucan contained in the dried product of the above-mentioned culture lipase.

The present invention also provides a lotion or cream for improving atopic dermatitis, which comprises 0.01 to 10% by weight, based on the total weight of the composition, of at least one or at least one of the mycelial culture, extract, concentrate and dried product of the above- By weight, preferably 0.05 to 5% by weight, based on the total weight of the composition. And at least one or at least one of the mycelial cultures and concentrates of the above-mentioned granulocyte lucerata, wherein the lotion or cream for improving atopic dermatitis is contained in an amount of 1 to 50% by weight based on the total weight of the composition, Wherein the lotion or cream for improving atopic dermatitis comprises 0.1 to 20% by weight based on the total weight of the composition, and at least one of extracellular polysaccharide or beta - Glucan composition, characterized in that it contains 0.05 to 5% by weight, based on the total weight of the lotion or cream composition for improving atopic dermatitis, of one or more of the following ingredients: Atopic improvement lotions or creams are preferred examples.

In addition, the lotion or cream has an effect of improving atopic dermatitis that alleviates antibacterial, antifungal, anti-inflammatory and itching, and is useful for the treatment of the above-mentioned cultivar laceratta culture, its dried or concentrated product, and cells derived from the cell lipolactacera In the culture for preparing the polysaccharide and beta-glucan, the carbon source is at least one selected from among maltose glucose, lactose, starch, dextrin, sucrose, fructose, galactose, mannose and sugar, and the nitrogen source is soybean powder, cottonseed powder, amino acid, , And a cone-shaped liquor.

The present invention relates to a method of producing an atopic dermatitis suppressant containing three lipolylaceratases, which is superior to general atopic dermatologic agents currently marketed in the market, has excellent effects, has few side effects, and has a high effect on skin improvement. Could know.

The present invention may be embodied in many other specific forms without departing from the spirit or essential characteristics of the invention.

The inventors of the present invention have found that the use of ceripolial lactacerate as an active substance useful for improving skin inflammation and itching can be achieved by incorporating it into a lotion or cream for improving atopic dermatitis, which is a cleaning product.

The inventors of the present invention have found that the use of the three lipolylacerase can be used as an effective substance for improving skin inflammation and itching by containing the lipolytic enzyme in a lotion or cream for improving atopic dermatitis.

Claims (15)

In the lotion or cream for improving atopic dermatitis, the lotion or cream is produced by using the mycelium of Seripolya lacera as an active ingredient,
A step of obtaining a three-lipolylacerase mycelium for liquid culture from the three lipolylacerase, a step of liquid culture of the myelofibrosarcera mycelia, a step of culturing the mycelia of the cultured three-lipolylacerase by a bioreactor And culturing the cells.
The culture medium of the above-mentioned cultivar L. serrata is cultivated in an agitation type bioreactor and the culture medium is adjusted to 0 to 500 rpm. An extracellular polysaccharide and beta-glucan as an active ingredient of the lotion or cream. The method according to claim 1,
The lotion or cream for improving atopy which comprises the above-mentioned granulocyte lucerata as an active ingredient is cultured in an agitating type incubator of 100 L or more in the main cultivation step of mycelium lacticera mycelium, and the carbon source during culturing is carbon source 0.1 to 10% by weight of the composition. The method according to claim 2,
The carbon source to be added in the culture in the stirring type incubator is at least one selected from maltose glucose, lactose, starch, dextrin, sucrose, fructose, galactose, mannose and sugar, and the nitrogen source is soybean powder, cottonseed powder, amino acid, Wherein the at least one selected from the group consisting of Staphylococcus aureus and Strip Liquor is added in an amount of 0.1 to 5% based on the total weight of the culture solution on the 5th day of the culture. The method according to claim 1,
Wherein the atopic dermatitis improving lotion or cream comprises 0.1 to 20 wt% of the total weight of the composition, and the atopic dermatitis improving lotion or the atopic dermatitis improving lotion is at least one of extracts and dried extracts of the mycelium culture of the above- Wherein the lotion or cream has an effect of antifungals against yeasts and fungi against Gram-negative bacteria and Gram-positive bacteria, and a lotion or cream for improving atopy.

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