KR101750620B1 - Preparation method of osmotic enzyme fermentation product using dendranthema zawadskii, the fermentation product prepared thereby and cosmetic, food or pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention relates to a method for producing a fermented osmotic enzyme using a perennial plant, a fermented product prepared from the fermented product, and a cosmetic, food or pharmaceutical composition containing the fermented product.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a fermented product of osmotic enzyme using a perennial herb, a fermented product prepared from the fermented product, and a cosmetic, food or pharmaceutical composition containing the same. BACKGROUND ART COMPRISING THE SAME}

The present invention relates to a method for producing a fermented osmotic enzyme using a perennial plant, a fermented product prepared from the fermented product, and a cosmetic, food or pharmaceutical composition containing the fermented product.

Dendranthema zawadskii , Chrysanthemum zawadskii ) is a perennial herbaceous plant belonging to the Asteraceae family, and is growing nationwide nationwide. It is known that it is effective for female diseases such as menstrual irregularity, menstrual pain and infertility as well as efficacy for chilling fatigue, cholesterol reduction, removal of vascular waste, inhibition of fat accumulation, bronchial protection, and pain relief.

The varieties showing such various effects can be used as foods, drinks, liquors and the like as they are naturally taken or processed. In order to improve the efficacy of the mouthwashes, the effective powders can be extracted and used. The active ingredient may be extracted by hot water extraction, pressure extraction, solvent extraction, fermentation methods such as alcohol fermentation, lactic acid fermentation, methane fermentation, and the like. Can be prepared.

For example, Korean Patent Laid-Open Publication No. 2005-0058561 discloses a composition comprising a Rhododendron extract extracted using a hot water extraction method.

However, when hot water extraction or pressurized extraction is used, not only the effective ingredient of the substrate is destroyed by high heat and pressure, but also it may be difficult to exhibit the desired effect by being denatured.

In addition, when solvent extraction is used, there is a problem that loss of an active ingredient can be prevented, but it may be harmful to a human body due to a non-polar solvent, an organic solvent, etc. remaining in the extract.

In addition, when fermentation is performed using microorganisms, it is difficult to keep conditions such as temperature, humidity, and oxygen concentration suitable for growing inoculated microorganisms constant, and desired fermentation may not occur due to contamination of other microorganisms not inoculated.

As a method for improving the shortcomings of the extraction method and the fermentation method, a direct method can be used. In the current method, a high concentration of sugar is added to elute an active ingredient together with moisture in a substrate by an osmotic pressure phenomenon. Although the active ingredient can be easily obtained while reducing the loss of the active ingredient by heat or pressure by the direct method, the immediate method does not require a long time to obtain the extract compared with the extraction method and the fermentation method, but also depends on the state and kind of the microorganism Repeatedly it may be difficult to obtain a uniform extract.

In order to solve such a problem, a method of fermenting microorganisms by inoculating the extract extracted by the conventional method is also known. However, there remains a need for a method for rapidly and uniformly obtaining an extract containing an active ingredient in a substrate.

Korean Patent Publication No. 2005-0058561

In order to solve the above problems, the present invention provides a process for producing an osmotic enzyme fermented product capable of uniformly extracting an effective ingredient of the herbicide without heat loss, a fermented product prepared from the osmotic enzyme, and a cosmetic, food or pharmaceutical composition containing the fermented product The purpose is to provide.

The present invention relates to (a) a mixture comprising (i) Saccharomyces cerevisiae as a substrate and (ii) sugar as a substrate and (iii) a lactobacillus fermentum as a lactic acid bacterium Lt; RTI ID = 0.0 > 50 C < / RTI > to produce a primary fermentation product; (b) removing the solid content from the primary fermentation product and then performing secondary fermentation at 20 to 50 ° C to produce a secondary fermentation product; And (c) removing the solid content from the secondary fermentation and then aging the fermented product at 0 to 10 < 0 > C.

The present invention also relates to a process for the production of (a) a process for the production of (i) a herbicide as a substrate, (ii) sugar, and (iii) Saccharomyces cerevisiae cerevisiae) and the momentum spread Lactobacillus (Lactobacillus fermentum) primary fermentation in a mixture mixture of 20 to 50 ℃ as the lactic acid bacteria to prepare a first fermented product; (b-1) removing the solid content from the primary fermentation product and sterilizing the product at 100 to 140 캜; (b-2) followed by the addition in my process three Levy Jia as Saccharomyces (Saccharomyces cerevisiae) and Lactobacillus buffer momentum (Lactobacillus fermentum) as a lactic acid bacteria as yeast and (v) per (iv) in the first fermented product of the sterile Secondary fermentation at 20 to 50 캜 to prepare a secondary fermentation product; And (c) removing the solid content from the secondary fermentation and then aging the fermented product at 0 to 10 < 0 > C.

Also, there is provided an osmotic enzyme fermented product prepared by the above-mentioned method.

Further, there is provided a cosmetic, food or pharmaceutical composition comprising the osmotic enzyme fermented product as an active ingredient.

According to the production method of the present invention, the osmotic enzyme fermented product containing the effective ingredient of the herbicide can be prepared by extracting the active ingredient of the herbicide without loss of the active ingredient by heat. Such an osmotic enzyme fermented product has excellent effects such as anti-inflammation, anti-allergy, skin regeneration, and can be used as a cosmetic, food or pharmaceutical composition.

1 is a schematic view of a method of producing an osmotic fermentation product according to an embodiment of the present invention.
FIG. 2 is a schematic view of a method for producing an osmotic fermentation product according to another embodiment of the present invention.
3 and 4 are graphs showing cell stability according to Experimental Example 1 of the present invention.
5 and 6 are graphs showing anti-inflammatory effects according to Experimental Example 2 of the present invention.
7 is a graph showing the anti-allergy effect according to Experimental Example 3 of the present invention.
FIGS. 8 and 9 are graphs showing skin regeneration effects according to Experimental Example 4 of the present invention. FIG.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, a method for preparing a fermented osmotic enzyme using a perennial plant according to the present invention, a fermented product prepared from the fermented product, and a cosmetic, food or pharmaceutical composition containing the fermented product will be described in detail with reference to the accompanying drawings. However, these descriptions are provided only to illustrate the present invention, and the scope of the present invention is not limited by these exemplary explanations.

<Osmotic enzyme Fermented  Manufacturing method>

The method for producing the osmotic enzyme fermented product according to an embodiment of the present invention is characterized in that (a) (i) is used as a substrate, (ii) sugar, and (iii) saccharomyces cerevisiae as yeast Preparing a first fermentation product by firstly fermenting a mixture of Saccharomyces cerevisiae and Lactobacillus fermentum as a lactic acid bacterium at 20 to 50 ° C; (b) removing the solid content from the primary fermentation product and then performing secondary fermentation at 20 to 50 ° C to produce a secondary fermentation product; And (c) aging at 0 to 10 &lt; 0 &gt; C after removing the solid content from the secondary fermentation product.

Hereinafter, the manufacturing method will be described separately for each step.

(a) a primary fermentation step

The step (a) is a step of firstly fermenting a mixture of a substrate (peritoneal), sugar and a microorganism (yeast, lactic acid bacteria) at 20 to 50 ° C to prepare a first fermented product.

The substrate was Dendranthema zawadskii ), and the kind, origin and form thereof are not particularly limited. It can use outposts including leaves, flowers, stems, roots, and the like. At this time, the substrate may be pulverized to a predetermined size or may be in the form of a juice using a presser to easily extract the active ingredient.

As the saccharide, there can be used any of conventional saccharides known in the art without limitation, and at least one selected from the group consisting of white sugar, sulfur sugar and raw sugar (non-saccharified sugar) can be used. As the sugar, it is preferable to use a yellow sugar or a raw sugar, and it is more preferable to use a raw sugar.

Saccharomyces cerevisiae as a yeast and Lactobacillus fermentum as a lactic acid bacterium are all used as the microorganisms to be inoculated, and yeast fermentation and lactic acid fermentation can proceed simultaneously. Saccharomyces cerevisiae MAB Y1 (accession number: KCTC 11386BP) and Saccharomyces cerevisiae (KCTC 7904) can be used as the yeast, and saccharomyces cerevisiae (KCTC 7904) It is preferable to use Saccharomyces cerevisiae MAB Y1 (KCTC 11386BP). Lactobacillus fermentum Miev L1106 (accession number: KCTC 12082BP) and Lactobacillus fermentum (KCTC 3112) can be used as the above-mentioned lactic acid bacteria, preferably Lactobacillus fermentum , Miev L1106 (KCTC 12082BP) can be used. The microorganisms may be mixed in a ratio of yeast: lactic acid bacterium = 1: 0.5 to 2 based on the weight ratio, and the yeast and lactic acid bacteria are preferably mixed in a ratio of 1: 1.

When the substrate, the sugar and the microorganism are mixed, it is possible to extract the active ingredient of the substrate to a maximum extent by controlling the mixing ratio or the amount used. The mixing ratio of the substrate to the saccharide may be 1: 0.5 to 2, preferably 1: 1 to 1: 1, based on the weight ratio. The amount of the microorganism to be used may be 1 to 10% by weight, preferably 3 to 5% by weight based on the total weight of the mixture of the substrate and the saccharide.

A mixture of such a substrate, a sugar and a microorganism is prepared by primary fermentation in an incubator having appropriate temperature and aerobic conditions, and a primary fermentation product in which the active ingredient is eluted together with the moisture of the substrate.

The primary fermentation temperature is not particularly limited, but it may be 20 to 50 캜, preferably 25 to 45 캜.

At this time, when the mixture is fermented under anaerobic conditions in which oxygen is intercepted, alcohol is generated upon saccharification by the yeast to inhibit the enzyme activity of the microorganism, so that fermentation proceeds under aerobic conditions in which oxygen is supplied. For example, the nonwoven fabric is used to prevent the anaerobic condition by blocking the inlet of the fermentation vessel to only pass oxygen.

During the primary fermentation, the pH of the primary fermentation product is measured at regular intervals to check for contamination, and the bubbles accumulated in the upper layer of the liquid phase are removed. In addition, the amount of enzyme produced by the protein assay method such as BCA assay or Bradford assay is estimated. Specifically, the protein amount is quantitated in a supernatant containing no inoculated microorganism or cells. The amount of protein in the supernatant is quantitated to exclude the protein content of the microorganism, and the amount of enzyme is estimated by the protein amount.

The pH of the primary fermentation product is preferably 3 to 6, and if it is out of the above range, anaerobic fermentation by yeast may occur or contamination may be caused by external microorganisms other than the inoculated microorganisms. When the protein amount of the primary fermentation product is 400 to 1000 ug / ml, the enzyme is sufficiently generated, and the primary fermentation is terminated. At this time, the primary fermentation period is not particularly limited, but it may be fermented for 4 to 10 days.

(b) a second fermentation step

(b) is a step of removing the solid content from the primary fermentation product and then performing secondary fermentation at 20 to 50 ° C to produce a secondary fermentation product.

The solid content is removed from the primary fermentation product prepared in the step (a) for the secondary fermentation. At this time, the primary fermentation product is removed from the solid matter using a fine mesh, and the primary fermentation solution from which the solid content has been removed is used for the secondary fermentation.

The primary fermentation liquid is subjected to secondary fermentation under the same temperature and aerobic conditions as the primary fermentation in the step (a), whereby the active ingredient of the substrate contained in the primary fermentation broth is reacted with the microorganism enzyme to produce a secondary fermentation product.

During the secondary fermentation, the pH of the secondary fermentation product is measured at regular intervals to confirm whether or not the product is contaminated, and the reduction equivalents converted through the invertase, a saccharide degrading enzyme of the microorganism, are measured. The pH of the primary fermentation product is preferably 3 to 6, and if it is out of the above range, anaerobic fermentation by yeast may occur or contamination may be caused by external microorganisms other than the inoculated microorganisms. When the reducing sugar amount of the secondary fermentation product is 3.0 to 10.0 mg / ml, the activity of the enzyme is active and the secondary fermentation is terminated. At this time, the secondary fermentation period is not particularly limited, but it may be fermented for 4 to 23 days.

(c) Aging step

(c) is a step of removing the solid content from the secondary fermentation and aging at 0 to 10 &lt; 0 &gt; C.

The solid content is removed from the secondary fermentation product prepared in step (b) for aging. At this time, the secondary fermentation product is removed from the solid matter using a fine mesh, and the secondary fermentation solution in which the solid content is removed is aged.

These secondary fermentation broths are kept at low temperature and matured, so that the components contained in the secondary fermentation broth can interact with each other while stopping the fermentation by the microorganisms, thereby producing a final osmotic enzyme fermentation product. The aging temperature is preferably 0 to 10 ° C.

2, the method of producing the osmotic fermented product according to another embodiment of the present invention comprises the steps of (a) (i) preparing a mixture of (ii) a saccharose as a substrate, (ii) Saccharomyces cerevisiae) and the momentum spread Lactobacillus (Lactobacillus fermentum) primary fermentation in a mixture mixture of 20 to 50 ℃ as the lactic acid bacteria to prepare a first fermented product; (b-1) removing the solid content from the primary fermentation product and sterilizing the product at 100 to 140 캜; (b-2) followed by the addition in my process three Levy Jia as Saccharomyces (Saccharomyces cerevisiae) and Lactobacillus buffer momentum (Lactobacillus fermentum) as a lactic acid bacteria as yeast and (v) per (iv) in the first fermented product of the sterile Secondary fermentation at 20 to 50 캜 to prepare a secondary fermentation product; And (c) aging at 0 to 10 &lt; 0 &gt; C after removing the solid content from the secondary fermentation product.

Hereinafter, the manufacturing method will be described separately for each process step as follows.

(a) a primary fermentation step

The step (a) is a step of firstly fermenting a mixture of a substrate (peritoneal), sugar and a microorganism (yeast, lactic acid bacteria) at 20 to 50 ° C to prepare a first fermented product.

The step (a) is the same as the step (a) of the method for preparing the osmotic fermentation product according to the example of the present invention, and the first fermentation product containing the active ingredient of the substrate and the enzyme of the microorganism is prepared.

(b-1) Sterilization step

The step (b-1) is a step of removing the solid content from the primary fermentation product and sterilizing the product at 100 to 140 ° C.

The primary fermentation product prepared in the step (a) is sterilized by removing the solid content using a mesh and then sterilizing the remaining primary fermentation broth. The sterilization conditions are not particularly limited, but the temperature may be 100 to 140 캜, and the time may be 5 to 30 minutes. Thus, secondary fermentation can be carried out by re-inoculating microorganisms with 20 to 50% of the primary fermentation substance to the substrate. At this time, the dead microorganism contained can be supplied with the protein source during the secondary fermentation.

(b-2) Second fermentation step

The step (b-2) is a step of adding a saccharide and a microorganism (yeast, lactic acid bacteria) to the sterilized primary fermentation product and then performing a secondary fermentation at 20 to 50 ° C to produce a secondary fermentation product.

The primary fermented product sterilized in the step (b-1) is added with sugars and microorganisms for secondary fermentation.

The sugars and microorganisms are the same as the sugars and microorganisms used in step (a) above.

When the primary fermentation product, the sugar and the microorganism are mixed, the mixing ratio or amount of the primary fermentation product, sugar, and microorganism may be adjusted to improve the reaction between the active ingredient contained in the primary fermentation product and the enzyme produced from the microorganism. The mixing ratio of the primary fermentation product to the saccharide may be 1: 0.5 to 2, preferably 1: 1 to 1: 1, based on the weight ratio. The amount of the microorganism to be used may be 1 to 10% by weight, preferably 3 to 5% by weight, based on the total weight of the mixture of the primary fermentation product and the saccharide.

The mixture of the sterilized primary fermentation product, saccharide and microorganism is subjected to secondary fermentation under the same temperature and aerobic condition as the primary fermentation in the step (a), whereby the added microorganism is mixed with the active ingredient In addition, a dead fermented microorganism is used as a protein source to produce a secondary fermentation product by an enzymatic reaction.

(c) Aging step

(c) is a step of removing the solid content from the secondary fermentation and aging at 0 to 10 &lt; 0 &gt; C.

The step (c) is the same as the step (c) of the method for preparing the osmotic enzyme fermented product according to the example of the present invention, and the final osmotic enzyme fermented product is prepared.

As described above, the method for producing the osmotic enzyme fermented product of the present invention can rapidly and uniformly extract the active ingredient of the herbicide without loss.

<Osmotic enzyme Fermentation product >

The osmotic fermented product produced by the method of the present invention contains the active ingredient of the herbicide, and when used, it can exhibit the effect of the herbicide.

<Osmotic enzyme The fermented product  Included Cosmetics , Food, pharmaceutical composition>

The osmotic enzyme fermented product according to the present invention can be used as a cosmetic, food or pharmaceutical composition containing an osmotic enzyme fermented product of the genus Orthoptera as an effective ingredient derived from an organism and safe for human body. At this time, the compositions may be effective for anti-inflammation, anti-allergy, skin regeneration by increasing immunity and regenerative function.

Specifically, the cosmetic composition containing the osmotic enzyme fermented product may be used in the form of a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, an oil, a powder, an aerosol, Additives usually added may be added. The cosmetic composition may be used as a cosmetic composition for hair such as a shampoo, a rinse, a tonic, a hair conditioner and a hair essence or may be used as a cosmetic composition for a face or wrist, such as a body shower, a body lotion, a body oil, a body mist, a foundation, a cleanser, have.

In addition, the food composition containing the osmotic enzyme fermented product may be used in the form of various foods, beverages, gums, tea, vitamin complex, functional beverage, health supplement and the like. .

In addition, the pharmaceutical composition containing the osmotic enzyme fermented product can be used in the form of an oral preparation, a granule, a tablet, a capsule, a suspension, an emulsion, a syrup or an aerosol, an external preparation, a suppository, Additives usually added during manufacture may be added.

Hereinafter, the present invention will be described concretely with reference to Examples. However, the following Examples are intended to illustrate one embodiment of the present invention, but the scope of the present invention is not limited by the following Examples.

[ Example  1] Neurotoxin osmotic enzyme Fermentation product

As a substrate, it was washed with running water and then water was completely removed, and then cut into 2 cm in length, length and height. The prepared substrate and sugar were mixed in a weight ratio of 1: 1. Yeast and lactic acid bacteria were inoculated into the mixture in an amount of 5% by weight based on the total weight of the mixture of substrate and sugar. The raw materials and the contents used are shown in Table 1 below.

mixture content temperament Guryeocho (Outpost) 50 wt% Party Natural sugar (raw sugar) 50 wt% leaven MAKY1 (KCTC 11386BP) in Saccharomyces cerevisiae 5 wt% based on substrate and sugar mixture Lactobacillus Lactobacillus fermentum MieVL1106 (KCTC 12082BP) 5 wt% based on substrate and sugar mixture

The mixture was placed in a fermentation vessel, and the inlet of the fermentation vessel was blocked with a nonwoven fabric to barely pass oxygen, followed by primary fermentation in a 30 ° C incubator. The first fermentation start time was regarded as a sample on the 0th day, and the primary fermentation product was sampled at intervals of 24 hours to measure the pH, and the amount of enzyme was measured by the protein determination method (BCA assay). Bubbles formed on the surface of the primary fermentation product during the primary fermentation were removed. Approximately 7 days after confirming that the amount of enzyme in the first fermentation product was 800 ug / ml, the solid content of the first fermentation product was removed using a fine mesh (300 mesh).

The primary fermentation broth in which the solid content was removed was placed in a sterilized new fermentation vessel. The inlet of the fermentation vessel was closed with a nonwoven fabric so as to pass only oxygen, followed by secondary fermentation in an incubator at 30 ° C. The second fermentation was started at the 0th day, and the second fermentation product was sampled at 24 hour intervals. The pH was measured, and the invertase activity was measured as the reducing equivalent. Bubbles formed on the surface of the secondary fermentation product during the secondary fermentation were removed. Approximately 7 days after confirming that the reducing sugar amount of the secondary fermentation product was 4 mg / ml, the solid content of the secondary fermentation product was removed using a fine mesh (300 mesh).

The secondary fermentation broth in which the solid content was removed was placed in a sterilized new container and aged at 4 캜 to prepare a fermentation product of the herbicidal osmotic enzyme.

[ Comparative Example  1] Heat number  extract

After washing with running water, the water was completely removed and then cut into 2 cm in length, length and height. The prepared rhizomes and water were mixed in a ratio of 1: 1 and heated at 100 ° C for 1 hour. Subsequently, the solid content was removed with a fine mesh (300 mesh) to prepare a hot water extract of Rhizoma root.

[ Comparative Example  2] Heat number  extraction Fermentation product

5% by weight of the same yeast and 5% by weight of lactic acid bacterium as in Example 1 were inoculated to the extract of Rhododendron japonicum prepared in Comparative Example 1 and then fermented at room temperature for 72 hours. Then, the solid content was removed with a fine mesh (300 mesh) to prepare a fermentation product of the hydrothermal extract.

[ Experimental Example  1] Confirm cell stability

(1) Confirmation of survival rate of macrophages

RAW 264.7 cells (1 × 10 ⁵ cells / ml), a macrophage, were cultured for 18 hours, and the osmotic enzyme fermentations of Example 1 were simultaneously treated at a concentration of 1 μg / ml and cultured for 48 hours. Then, 3- (4,5-dimethylthiazole) -2,5-diphenyltetrazolyl bromide (MTT, Sigma) was added to a concentration of 1 mg / ml and further cultured for 4 hours. After removing the culture medium, 100 μl of dimethylsulfoxide (DMSO, Sigma) or 0.04N HCl / isopropanol was added to dissolve the formazan precipitate produced by the reduction of MTT, and absorbance at 570 nm was measured using an absorbance meter Were measured.

The cell viability was expressed as a percentage by comparing the absorbance value when the osmotic enzyme fermented product of Example 1 was treated at each concentration with the absorbance value when the osmotic enzyme fermented product was not treated (control group) Respectively.

As shown in FIG. 3, when the osmotic fermentation product of Example 1 was treated at 0.1 to 2.5%, the survival rate of macrophages was maintained at 95% or more. On the other hand, when the osmotic enzyme fermented product of Example 1 was treated at 5 to 25%, it was toxic to macrophages and the cell survival rate was reduced to less than 25%.

(2) Confirmation of survival rate of mast cells

Except that RBL-2H3 cells were used instead of macrophages, and 0.05% trypsin-EDTA solution was treated to dissolve the overcorrection caused by cell proliferation and the cells were suspended and subcultured Absorbance was measured in the same manner as in (1) of Experimental Example 1, and the results are shown in FIG.

As shown in Fig. 4, when the osmotic enzyme fermented product of Example 1 was treated, it did not show toxicity to mast cells.

[ Experimental Example  2] Identification of anti-inflammatory effect

(1) NO production inhibition confirmation

RAW 264.7 cells were cultured in 96-well plates at 1 × 10 ⁴ cells / well for 24 hours. The culture was carried out at 37 ° C and 5% CO 2 using high glucose DMEM (Lonza, USA) containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)

The cell culture broth was treated with 0.1% of an anti-inflammatory drug, indomethacin, or the osmotic enzyme fermented product of Example 1 was treated at a concentration of 5% CO 2 for 1 hour at a concentration of 1 μg / mL LPS was treated and further cultured for 18 hours.

To 50 μl of the cell culture medium, 50 μl of a Griess reagent (Sigma chemical Co., USA) containing 1% sulfanilamide, 5% phosphoric acid and 0.1% naphthylethylethylene diamine was added Respectively. After incubation for 10 min, the absorbance was measured at 540 nm using an absorbance meter (TECAN / infinite M200, Switzerland). The absorbance value when the indomethacin or the osmotic enzyme fermentation product of Example 1 was treated at each concentration was compared with the absorbance value obtained when only LPS was treated, and the degree of NO production was expressed as a percentage. The results are shown in FIG. 5 .

As shown in FIG. 5, when the osmotic fermentation product of Example 1 was treated, NO production was suppressed as compared with the case where only LPS was treated. In addition, when the osmotic enzyme fermented product of Example 1 was treated at 0.5 to 25%, NO production was suppressed as compared with the case of treatment with indomethacin.

(2) NO generation inhibition confirmation

Except that the osmotic enzyme fermented product of Example 1 was treated with concentration of 2.5% of the osmotic enzyme fermented product of Example 1, 1% of the herbicidal hot-water extract of Comparative Example 1, and 1% of the herbicidal hot-water extract of Comparative Example 2 Absorbance was measured in the same manner as in (1) of Experimental Example 2, and the results are shown in FIG.

As shown in FIG. 6, when the osmotic fermentation product of Example 1 was treated, NO production was inhibited as compared with the case where only LPS was treated.

In addition, when the osmotic enzyme fermented product of Example 1 was treated, it was found that the inhibitory effect of NO production was superior to that of the indomethacin, the herbicidal hot-water extract (Comparative Example 1) and the herbicidal hot-water extract fermented product (Comparative Example 2).

[ Experimental Example  3] Anti-allergy  Check the effect

In order to confirm the degranulation phenomenon of mast cells that cause early allergic reaction, β-hexosaminidase secreted during degranulation was measured.

RBL-2H3 cells were inoculated into 48 wells of microplates (DMEM) containing 1% penicillin-streptomycin and 10% FBS at 37 ° C and 5% CO ₂ at 5 × ⁵ cells / Time. Subsequently, the cells were sensitized with anti-DNP IgE (0.5 ug / ml) and cultured for 12 hours. The cells were washed twice with Siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl ₂ , 25 mM PIPES, 40 mM NaOH, pH 7.2), and 5.6 mM glucose, 1 mM CaCl ₂ and 0.1% BSA After the addition of the Shiraganian buffer, the osmotic enzyme fermentation product of Example 1 was treated for each concentration and cultured for 1 hour. The cells were treated with DNP-BSA (20 ug / ml) for 1 hour, and then allowed to stand in ice water for 10 minutes to complete the reaction. 20 μl of the supernatant was transferred to 96 well microplates and 20 μl of substrate buffer (1 mM 4-p-nitrophenyl-N-acetyl-bD-glucosaminide, 0.05 M sodium citrate, pH 4.5) And cultured at 37 ° C for 30 minutes. After completion of the reaction, 200 μl of a stop solution (0.1 M Na ₂ CO ₃ / NaHCO ₃ ) was added and the absorbance was measured at 405 nm using an absorbance meter (TECAN / infinite M200, Switzerland). The absorbance value when the osmotic enzyme fermented product of Example 1 was treated at each concentration was compared with the absorbance value when only anti-DNP IgE and DNP-BSA were treated, and the degree of hexosaminidase secretion was expressed as a percentage, The results are shown in Fig.

As shown in FIG. 7, when the osmotic enzyme fermented product of Example 1 was treated at different concentrations, the secretion of hexosaminidase was inhibited as the treatment concentration was increased.

[ Experimental Example  4] Confirm skin regeneration effect

(1) Confirmation of cell viability by UV irradiation

Microtiter plates (24 well) fibroblasts of human dermis cells in a (human dermal fibroblast, NHDF) ( Lonza, USA) to 5 × 10 ⁴ 1% by cell / well penicillin-medium supplemented with a low concentration of glucose that contains the streptomycin and 10% FBS (low glucose DMEM) for 24 hours at 37 ° C and 5% CO ₂ . The osmotic enzyme fermented product of Example 1 was treated for each concentration, cultured for 4 hours, washed twice with PBS, and irradiated with 100 mJ / cm ² UV-B. The osmotic enzyme fermented product was treated at different concentrations and cultured for 48 hours. Then, 3- (4,5-dimethylthiazole) -2,5-diphenyltetrazolyl bromide (MTT, Sigma) was added to a concentration of 1 mg / ml and further cultured for 4 hours. After removing the culture medium, 100 μl of dimethylsulfoxide (DMSO, Sigma) or 0.04N HCl / isopropanol was added to dissolve the formazan precipitate produced by the reduction of MTT, and absorbance at 570 nm was measured using an absorbance meter Were measured. The absorbance value when the osmotic enzyme fermentation product of Example 1 was treated was compared with the absorbance value when the UV was not irradiated (control group) to show the cell survival rate as a percentage, and the results are shown in FIG.

As shown in Fig. 8, when the osmotic enzyme fermented product of Example 1 was treated with UV light, it was found that there was little or no cell damage by UV irradiation.

(2) Oxidative  Confirm cell viability by stress

The absorbance was measured in the same manner as in (1) of Experimental Example 4, except that 200 uM hydrogen peroxide (H ₂ O ₂ ) was used instead of UV irradiation, and the result is shown in FIG.

As shown in FIG. 9, when the osmotic enzyme fermented product of Example 1 was treated with hydrogen peroxide, it was found that there was little or no cell damage caused by hydrogen peroxide treatment.

Korea Biotechnology Research Institute KCTC11386BP 20080903 Korea Biotechnology Research Institute KCTC12082BP 20111122

Claims (15)

(a) a mixture of (i)
(ii) Saccharomyces cerevisiae as a yeast and ( Lactobacillus fermentum ) as a lactic acid bacterium are firstly fermented at a temperature of 20 to 50 ° C at a time, Preparing a fermented product;
(b) removing the solid content from the primary fermentation product and then performing secondary fermentation at 20 to 50 ° C to produce a secondary fermentation product; And
(c) removing the solid matter from the secondary fermentation and aging at 0 to 10 &lt; 0 &gt; C
The present invention relates to a method for producing an osmotic enzyme fermented product,
Wherein the yeast and the lactic acid bacteria are mixed at a weight ratio of 1: 0.5 to 2 during the primary fermentation,
Wherein the primary fermentation is terminated when the protein amount of the primary fermentation product is 400 to 1000 ug / ml. (a) a mixture of (i)
(ii) saccharomyces cerevisiae as a yeast and Lactobacillus fermentum as a lactic acid bacterium are subjected to primary fermentation at 20 to 50 ° C at a time, Producing water;
(b-1) removing the solid content from the primary fermentation product and sterilizing the product at 100 to 140 캜;
(iv) saccharomyces cerevisiae as a yeast and lactobacillus fermentum as a lactic acid bacterium were added to the sterilized primary fermentation product (b-2) Secondary fermentation at 20 to 50 캜 to prepare a secondary fermentation product; And
(c) removing the solid matter from the secondary fermentation and aging at 0 to 10 &lt; 0 &gt; C
The present invention relates to a method for producing an osmotic enzyme fermented product,
Wherein the yeast and the lactic acid bacteria are mixed at a weight ratio of 1: 0.5 to 2 during the primary fermentation,
Wherein the primary fermentation is terminated when the protein amount of the primary fermentation product is 400 to 1000 ug / ml. 3. The method according to claim 1 or 2,
Wherein the saccharide is at least one selected from the group consisting of white sugar, sulfur sugar, and raw sugar. 3. The method according to claim 1 or 2,
The yeast is in my process to the three Levy Jia Saccharomyces (Saccharomyces A method for producing an osmotic enzyme fermented product using a. 3. The method according to claim 1 or 2,
The lactic acid bacteria are Lactobacillus momentum spread (Lactobacillus fermentum) Miev L1106 osmotic enzyme fermentation process for producing water with a gujeolcho (KCTC 12082BP). delete 3. The method according to claim 1 or 2,
Wherein the mixing ratio of the substrate and the saccharide in the step (a) is in the range of 1: 0.5 to 2 based on the weight ratio. 3. The method according to claim 1 or 2,
Wherein the amount of yeast and lactic acid bacteria used in step (a) is 1 to 10% by weight based on the total weight of the mixture of the substrate and the saccharide, respectively. 3. The method of claim 2,
Wherein the mixing ratio of the primary fermentation product and the saccharide in the step (b-2) is 1: 0.5 to 2 based on the weight ratio of the primary fermentation product: saccharide fermentation product. 3. The method of claim 2,
Wherein the yeast and the lactic acid bacteria are used in the step (b-2) in an amount of 1 to 10% by weight based on the total weight of the mixture of the primary fermentation product and the sugar, respectively. 3. The method according to claim 1 or 2,
Wherein the primary fermentation and the secondary fermentation proceed under aerobic conditions. An osmotic enzyme fermentation product using a transgenic plant produced by the method of claim 1 or 2. A cosmetic composition comprising the fermented product of claim 12 as an active ingredient. A food composition comprising the fermented product of claim 12 as an active ingredient. delete

Images