KR20220126863A - Fermented Medicinal herb method that enhances Anti-acid, antibacterial and allergy suppression by using Lactobacillus bacteria in Bangpungtongseong-san - Google Patents

Abstract

The present invention relates to a method for preparing a herbal fermentation composition and a herbal fermentation composition according thereto and, more specifically, to a method for preparing a herbal fermentation composition, comprising the steps of: preparing Bangpungtongseongsan which is prepared herbal medicine (S1); preparing lactobacillus bacteria (S2); and inoculating the prepared herbal medicine with the lactobacillus bacteria and fermenting the herbal medicine at 30 to 45 ℃ (S3). The present invention may provide an herbal fermentation composition that has various physiologically active functions of herbal medicine, in particular, Bangpungtongseongsan, while further enhancing the antioxidant, antibacterial, and allergy suppression effects of the Bangpungtongseongsan. Accordingly, the herbal fermentation composition according to the present invention is expected to be effective in improving allergies, skin aging, and skin diseases caused by pathogenic microorganisms, so it can be usefully used by being contained in cosmetic compositions, pharmaceutical compositions, food compositions, etc.

Description

Fermented medicinal herb method that enhances Anti-acid, antibacterial and allergy suppression by using Lactobacillus bacteria in Bangpungtongseong-san}

The present invention relates to a method for preparing a fermented oriental herbal composition that can be used in food, cosmetic, and pharmaceutical industries, and to a fermented oriental medicine composition according thereto.

Recently, with the advent of an aging society, a well-being trend is taking root in society, which has amplified interest in natural plant materials and traditional herbal medicines in all fields such as food, cosmetics, and residential environments.

There have been attempts to use natural plant materials as raw materials for a long time in the cosmetic industry, where it is relatively easy to commercialize natural plant materials. And as it began to be commercialized, interest in the human usefulness of natural plant materials and herbal medicines increased.

Natural plant materials or herbal medicines contain various physiologically active substances and antioxidants, so they show various effects such as anti-aging, anti-cancer, anti-inflammatory, and immune function improvement. there are often Therefore, recently, many attempts have been made to stabilize natural extracts, reduce toxicity, or increase the effect by converting them into stable derivatives. and fermentation can be exemplified as a representative method.

Fermentation is a process of producing substances useful in human life by decomposing organic matter using enzymes possessed by microorganisms. Representative fermenting microorganisms that produce substances useful for the human body include lactic acid bacteria and yeast, produced by the process. Also, in addition to producing useful substances during fermentation, lactic acid bacteria involved in fermentation also play a role in immunity and detoxification in the intestine. Furthermore, the ingredients ingested through fermentation by intestinal microbes are decomposed and converted into low-molecular substances that can be easily absorbed by human cells, or are converted from an unstable or inactive form to an active form and absorbed, and these results affect the intestinal microbes. This can be seen as a clear indication of the importance of biotransformation by

Therefore, attempts to maximize the efficacy of natural plant materials and herbal medicines by fermentation of microorganisms such as lactic acid bacteria and yeast or to solve the stabilization problem have an important meaning in the development of various foods, cosmetics, drugs, and the like.

Pungpungtongseongsan is a prescription recorded in the Chinese “Euimyeongdaeron” and the Korean “Donguibogam”, and contains talc, licorice, gypsum, gold, gilgyeong, windbreak, angelica, cheonggung, red peony, rhubarb, ephedra, It is composed of mint, Yeongyo, Mangcho, Hyeonggae, Baekchul, and Gardenia. The windproof acid is useful for excreting and detoxifying self-poisoning substances accumulated through the skin, urinary and digestive organs, etc., and is often used for obese stroke constitution. In addition, it can be applied to obesity, habitual constipation, high blood pressure, chronic nephritis, sole, hemorrhoids, sinusitis, diabetes, and the like. In addition, it is used for stroke, arteriosclerosis, and inflammatory skin diseases, and can be applied to various diseases such as lipid lowering effect, analgesic, anti-inflammatory, antibacterial effect, allergy suppression and immune effect, and liver function enhancing effect.

In the present invention, a method for further enhancing the antioxidant effect, antibacterial effect, and allergy suppression effect of herbal medicine, particularly antiperspirant acid, while having various physiologically active functions, was studied and completed.

An object of the present invention is to provide a method for preparing a fermented oriental medicine composition capable of further enhancing the antioxidant effect, antibacterial effect, and allergy suppression effect of herbal medicines, particularly antiperspirant acid, while having various physiologically active functions.

Accordingly, the present invention as a first preferred embodiment (S1) preparing the herbal medicine Bangpungtongseongsan; (S2) preparing Lactobacillus; and (S3) inoculating Lactobacillus into the prepared Bangpungtongseong acid and fermenting it at 30~45℃;

In the method for preparing the oriental fermented composition according to the embodiment, the windbreaktongseong acid in step (S1) may be centrifuged at 1-5°C and 2,000-5,000×g to remove the precipitate.

In the method for preparing the oriental fermented composition according to the embodiment, the windproof acid in step (S1) may be prepared by adjusting the pH to 4-7.

In the method for preparing a fermented herbal composition according to the embodiment, it provides a method for preparing a fermented oriental composition by adding 1-5% (w/v) of sugar to the Bangpungtongseong acid in step (S1).

In the method for preparing the oriental fermentation composition according to the embodiment, the Lactobacillus bacteria in step (S2) are Lactobacillus brevis, Lactobacillus casei, Lactobacillus del Bruechi subspecies bulgaricus (Lactobacillus). Delbrueckii subsp. .

In the method for preparing the oriental fermented composition according to the embodiment, the Lactobacillus in step (S2) may be one in which the number of bacteria is adjusted to 1×10 6 to 1×10 8 CFU/ml.

In the method for preparing the oriental fermentation composition according to the embodiment, the Lactobacillus in step (S2) is 0.8 × 10 6 to 5 × 10 6 CFU / ㎖ Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus casei (Lactobacillus casei), 0.8 Lactobacillus delbrueckii subsp. bultaricus (Lactobacillus delbrueckii subsp.bultaricus) and Lactobacillus plantarum (Lactobacillus plantarum) of × 107 ~ 5 × 107 CFU / ㎖, Lactobacillus permentum of 0.8 × 108 ~ 5 × 108 CFU / ㎖ (Lactobacillus fermentum) and Lactobacillus helveticus (Lactobacillus helveticus) may be included.

In the method for preparing the oriental fermentation composition according to the embodiment, the Lactobacillus in step (S2) is 0.8 × 10 6 to 5 × 10 6 CFU / ㎖ Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus casei (Lactobacillus casei), 0.8 Lactobacillus helveticus and Lactobacillus plantarum at ×107-5×107 CFU/ml, Lactobacillus fermentum and Lactobacillus at 0.8×108-5×108 CFU/ml Delbruecki sub sp. bultaricus (Lactobacillus delbrueckii subsp. bultaricus) may be included.

In the method for preparing the oriental fermented composition according to the embodiment, in step (S3), Lactobacillus brevis, Lactobacillus delbrueckii subsp. bultaricus and Lactobacillus helveticus (Lactobacillus helveticus) ) provides a method for preparing a fermented oriental medicine composition, which is to inoculate Lactobacillus fermentum and Lactobacillus plantarum on the 4th to 5th day after inoculation.

In the method for preparing the oriental fermented composition according to the embodiment, each Lactobacillus may be inoculated after adjusting the number of bacteria to 1×108 CFU/ml.

In the method for preparing the oriental fermented composition according to the embodiment, in step (S3), the Lactobacillus may be inoculated in an amount of 0.1 to 1% (v/v) with respect to the wind-proof acid.

As a second preferred embodiment, the present invention provides a fermented herbal composition that is prepared by the above preparation method and exhibits antibacterial and antiallergic activity.

The herbal fermentation composition according to the embodiment is Pseudomonas aeruginosa (Pseudomonas aeruginosa), Staphylococcus aureus genus. Aureus (Staphylococcus aureus subsp. aureus) and Propionibacterium acnes (Propionibacterium acnes) may exhibit antibacterial activity.

The present invention provides a food composition containing the herbal fermented composition as a third preferred embodiment.

The present invention provides a pharmaceutical composition containing the herbal fermented composition as a fourth preferred embodiment.

As a fifth preferred embodiment, the present invention provides a cosmetic composition containing the fermented herbal composition.

According to the present invention, it is possible to provide a fermented oriental medicine composition that further enhances the antioxidant effect, antibacterial effect, and allergy suppression effect of herbal medicines, particularly antiperspirant acid, while having various physiologically active functions. The oriental fermented composition according to the present invention is expected to be effective in improving allergies, skin aging and skin diseases caused by pathogenic microorganisms.

1 is a photograph showing a state in which an oriental fermented composition (Lactobacillus) cultured for 5 days according to a preferred embodiment of the present invention was spread in lactobacilli MRS medium containing BPB and then smeared and cultured for 3 days.
2 is a graph showing the growth curve of Lactobacillus of Example 1 and Example 2 of the present invention, and FIG. 3 is a graph showing the growth curve of Lactobacillus of Example 3 of the present invention.
Figure 4 is a graph showing the change in pH according to the culture period of the oriental fermented composition according to Example 3 of the present invention, Figure 5 is the pH according to the culture period of the oriental fermented composition according to Examples 1 and 2 of the present invention This is a graph showing the change.
6 is a graph showing the DPPH scavenging ability according to the incubation period of each sample according to a preferred embodiment of the present invention, and FIG. 7 is a graph showing the DPPH scavenging ability according to the concentration of BHA as a positive control.
8 is a graph showing the SOD-like activity according to the incubation period of each sample according to a preferred embodiment of the present invention.
9 is a photograph showing the antibacterial activity against Propionibacterium acnes causing acne bacteria of each sample according to a preferred embodiment of the present invention.
10 is a graph showing cytotoxicity according to the concentration of herbal medicine (Pungtongseongsan) targeting RBL-2H3 cell line, and FIG. It is a graph showing the cytotoxicity according to the treatment time of the composition.
12 is a graph showing the effect of inhibiting the release of β-hexosaminidase of the oriental fermentation composition of Example 3 according to the present invention with respect to the RBL-2H3 cell line.
13 is a graph showing the effect of inhibiting the release of β-hexosaminidase on the pH of the oriental fermentation composition of Example 3 according to the present invention for RBL-2H3 cell line.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a method for producing a fermented oriental medicine composition and to a fermented oriental composition according thereto, wherein Lactobacillus bacteria is inoculated and fermented into fenugreek acid, which is an oriental medicine, to have various physiologically active functions of windbreaktongseongsan, while having antioxidant effect, antibacterial effect and To provide a fermented oriental medicine composition that further enhances allergy suppression effect.

To this end, the present invention comprises the steps of (S1) preparing an herbal medicine, Bangpungtongseongsan; (S2) preparing Lactobacillus; and (S3) inoculating Lactobacillus into the prepared Bangpungtongseong acid and fermenting it at 30~45℃;

The herbal medicines in step (S1) (talc, licorice, gypsum, gold, gilgyeong, windbreak, cheongung, angelica, red peony, rhubarb, ephedra, mint, Yeongyo, mangcho, hyeonggae, baekchul and gardenia) are It can also be manufactured by adding or subtracting several medicinal substances within the range that does not significantly deviate from the efficacy. Specific examples include 6.37 g of talc, 4.5 g of licorice, 2.62 g each of gypsum, gold and gilgyeong, 1.68 g each of Bangpung, Cheongung, Angelica, red peony, rhubarb, ephedra, mint, Yeongyo and Mangyo, 1.31 g each of Hyeonggae, Baekchul and Gardenia. It is preferable that it is prepared with herbal medicine consisting of 5 g of ginger.

The windproof acid is preferably used after removing the precipitate by centrifugation at 2000-5000×g at 1-5°C, and the pH is preferably adjusted to 4-7.

In addition, it is preferable to add sugar (especially brown sugar) to the windproof acid in an amount of 1 to 5% (w/v) with respect to the windproof acid in order to increase the growth of microorganisms.

And, in step (S2), Lactobacillus for fermenting the windbreaktongseong acid is prepared. The Lactobacillus bacteria are Lactobacillus brevis (Lactobacillus brevis), Lactobacillus casei (Lactobacillus casei), Lactobacillus del bruecki subspecies Bulgaricus (Lactobacillus delbrueckii subsp. bultaricus), Lactobacillus permentum (Lactobacillus permentum), It is preferable to include two or more selected from the group consisting of Helveticus (Lactobacillus helveticus) and Lactobacillus plantarum (Lactobacillus plantarum). In addition, it is preferable to inoculate the Lactobacillus by adjusting the number of bacteria to 1 × 10 6 ~ 1 × 10 8 CFU / ㎖ for the growth of the bacteria.

In the present invention, it is preferable to complex culture with Lactobacillus. The complex culture in the present invention refers to a case of inoculating and fermenting several species of Lactobacillus with different numbers of bacteria, and a case of inoculating and fermenting several species of Lactobacillus at different times according to growth conditions.

Preferred specific examples of Lactobacillus bacteria for complex culture in the present invention are 0.8 × 10 6 ~ 5 × 10 6 CFU / ㎖ Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus casei (Lactobacillus casei), 0.8 × 107 ~ 5 × 107 CFU Lactobacillus delbruecki subsp. bultaricus (Lactobacillus delbrueckii subsp.bultaricus) and Lactobacillus plantarum (Lactobacillus plantarum) at /ml, Lactobacillus fermentum (Lactobacillus fermentum) and lactobacillus fermentum at 0.8 x 108-5 x 108 CFU/ml Bacillus helveticus (Lactobacillus helveticus).

Or 0.8 × 106 ~ 5 × 106 CFU / ㎖ of Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus casei (Lactobacillus casei), 0.8 × 107 ~ 5 × 107 CFU / ㎖ of Lactobacillus helveticus (Lactobacillus helveticus) and Lactobacillus Plantarum (Lactobacillus plantarum), Lactobacillus fermentum (Lactobacillus fermentum) and Lactobacillus delbrueckii subsp. bultaricus (Lactobacillus delbrueckii subsp.

And, in step (S3), the prepared Lactobacillus bacteria are inoculated into the prepared windproof acid and fermented at 30-45°C. At this time, it is preferable to inoculate the Lactobacillus in an amount of 0.1 to 1% (v/v) with respect to the windproof acid.

Another preferred example for the complex culture of the present invention, Lactobacillus brevis (Lactobacillus brevis), Lactobacillus delbrueckii subsp. bultaricus (Lactobacillus delbrueckii subsp. bultaricus) and Lactobacillus helveticus (Lactobacillus helveticus) was inoculated into the windproof acid, and on the 4th to 5th day from the inoculation day, the number of bacteria was adjusted to 1×108 CFU/ml, respectively Lactobacillus fermentum and Lactobacillus planta There is a method of fermenting rum (Lactobacillus plantarum) by inoculating 0.1 to 0.3% (v/v) of each.

The oriental fermented composition of the present invention prepared as described above has better antioxidant activity than the herbal medicine before fermentation (Bacillus subtilis) PCI 219, Pseudomonas aeruginosa (Pseudomonas aeruginosa) KCTC 2004, Staphylococcus aureus genus. Aureus (Staphylococcus aureus subsp. aureus) KCTC 1916 showed antibacterial activity and, in particular, showed antibacterial activity against Propionibacterium acnes KCTC 3314, which causes acne. Antibacterial activity against Propionibacterium acnes KCTC 3314 was not present in the antibacterial acid before fermentation. In addition, it exhibited an allergy inhibitory effect close to 60%. Therefore, the fermented herbal composition according to the present invention is expected to be effective in alleviating allergies, skin aging and skin diseases caused by pathogenic microorganisms.

Hereinafter, the configuration of the present invention will be described in more detail through examples, but the scope of the present invention is not limited to the following examples.

Example

Herbal medicine selection and manufacturing

As shown in Table 1 below, talc 6.37g, licorice 4.5g, gypsum, gold and gilgyeong each 2.62g, windbreak, cheongung, angelica, red peony, rhubarb, ephedra, mint, yeonkyo and mangcho 1.68g each, hyeonggae, baekchul and gardenia Bangpungtongseongsan, a herbal medicine containing 1.31g and 5g of ginger, respectively, was commissioned by an oriental clinic in Yangnyeong-si, Daegu.

Then, the prepared windproof acid was centrifuged at 4 ℃, 4,000 × g for 10 minutes to remove the precipitate, the pH was adjusted to 6.3, and sterilized at 121 ℃ for 15 minutes.

Preparation of Lactobacillus

Lactobacillus was selected as a useful microorganism for fermenting the prepared antiperspirant acid, and as shown in Table 2 below, it was purchased from the Korea Research Institute of Bioscience and Biotechnology Genetic Resource Center (KCTC) and Lactobacilli MRS medium (proteose peptone No. 3 10g). , beef extract 10g, yeast extract 5g, dextrose 20g, Polyoxyethylene sorbitan monooleate 1g, ammonium citrate 2g, maganessium sulfate 0.1g, manganesse selfate 0.05g, dipotassium phosphate 2g, sodium acetate 5g/L, pH 6.5, Difco Co.) After culturing, the cells were placed in a stock solution containing 20% glycerol and stored at -70°C, and subculture was performed before use in the experiment.

Lactobacillus inoculation and fermentation

After preparing a Lactobacillus stock solution by varying the number of bacteria as shown in Table 3 below, 0.1% (v/v) of each was inoculated in the prepared windproof acid.

In the above, in Example 3, the inoculation time was changed, and the stock solution was prepared at 1×108 CFU/ml, and then Lb. brevis KCTC 3498, Lb. delbrueckii subsp. bulgaricus KCTC 3635 and Lb. Helveticus KCTC 3545 and Lb.fermentum KCTC 3112 and Lb. plantarum KCTC 3108 was inoculated.

As described above, the herbal medicines of Examples 1, 2 and 3 inoculated with Lactobacillus were fermented for 7 days to prepare the oriental fermented composition of the present invention. 1 is a photograph showing the results of plating the oriental fermented composition (Lactobacillus) of Example 3 cultured for 5 days in Lactobacillus MRS medium containing BPB after 100 μl dispensing. Here, A is cultured only with windproof acid, B is cultured by adding 1% (w/v) of brown sugar to windproof acid, and C is cultured with sugar (brown sugar) added to windproof acid with 5% ( w/v) was added and cultured.

In addition, FIG. 2 is a graph showing the growth curve of Lactobacillus of Example 1 and Example 2 by comparison, and FIG. 3 is a graph showing the growth curve of Lactobacillus of Example 3. As shown in FIG. 3 , it can be seen that the Lactobacillus bacteria of Example 3 grew similarly to the initial stage of fermentation even in the latter stage of fermentation.

Experimental example

pH measurement

The growth degree of the test strains in the oriental medicine fermented composition (fermented herbal medicine) was confirmed after inoculating the bacteria in Bangpungtongseongsan, taking 100 μl of each culture day and smearing it on Lactobacilli MRS agar medium, and measuring the number of viable cells. Whether or not the test strain produced acid was confirmed by inoculating the test strain on Pungongtongseong acid, taking the culture solution for each culture day, centrifuging it at 1,000 × g for 10 minutes, and measuring the pH of the supernatant.

In Examples 1, 2 and 3, the acid production of the strain on the first day of culture was more excellent when cultured in a medium containing 5% sugar added to Bangpungtongseongsan. The acid production of the strain in Example 1 was the highest at pH 3.49 when cultured for 7 days by adding 1% sugar, and the acid production of the strain in Example 2 was pH 3.52 when cultured for 7 days by adding 5% sugar. was the highest In Example 1 and Example 2, when cultured with herbal medicine alone, acid production was similar at pH 4.84 and pH 4.78, respectively (FIG. 5), and when cultured with herbal medicine alone in Example 3, acid production was excellent at pH 3.87 (FIG. 4) .

antioxidant activity

electron donating ability

The electron donating ability was performed by modifying the method of Blois (Blois 1985). DPPH was dissolved in methanol before the start of the experiment and used to be 0.3 mM. The DPPH solution and the sample (or fermented herbal medicine) diluted with sterile water were mixed 1:1 and reacted at room temperature for 30 minutes, and then absorbance was measured at 517 nm. The negative control was measured by adding methanol instead of the sample, and the positive control was measured with BHA, which is known as a conventional antioxidant. The electron donating ability was expressed as a percentage as follows by calculating the absorbance of the sample-added group and the negative control group.

Among the methods for measuring antioxidant activity, DPPH radical scavenging ability is relatively simple and can be measured in large quantities, and is a method highly correlated with actual antioxidant activity. It is a relatively stable compound among substances having DPPH radicals, and is colored purple in ethanol or methanol. However, when it comes into contact with substances with antioxidant activity, the antioxidant substance removes the radicals of DPPH and is decolorized. The electron donating action is used as a measure of suppressing lipid oxidation by donating electrons to active radicals, and is also used as a measure of the action of inhibiting aging caused by active radicals in the human body.

As a result of examining the electron donating ability with the scavenging ability of DPPH, as shown in FIG. 6, when diluted 100-fold, the herbal medicine (non-fermented Bangpungtongseong acid) was 31.7%, and all of the fermented oriental medicine compositions of the present invention were 70% or more. . When the Lactobacillus inoculation time was changed and cultured with herbal medicine alone (Example 3), the DPPH scavenging ability was the highest at 94.43% on the 7th day of culture ( FIGS. 6 and 3A ), and as shown in FIG. 7 , 0.5 of BHA, a positive control, was It was shown to have DPPH scavenging activity corresponding to mM.

SOD-like activity

SOD-like activity was performed according to Marklund's method (Marklund and Marklund 1974). To 0.2 ml of each sample solution, 2.6 ml of Tris-HCl buffer (50 mM Tris, 10 mM EDTA, pH 8.5) and 0.2 ml of 7.2 mM pyrrogallol (Sigma Co.) were added and reacted at 25° C. for 10 minutes, followed by 1.0 N HCl. 0.1 ml was added to stop the reaction, and the amount of oxidized pyrrogallol in the reaction solution was measured at 420 nm. The SOD-like activity was expressed as the absorbance reduction rate of the experimental group and the control group of the sample solution.

The SOD-like activity of the herbal medicine was confirmed to be 36.34% as shown in FIG. 8 . The SOD-like activity of the complex-cultured herbal medicine (oriental medicine fermented composition of the present invention) was higher than that of the herbal medicine, and was higher when sugar was added at 1% and 5%. The oriental fermented composition (Example 3) cultured at different inoculation times of Lactobacillus had increased SOD-like activity according to the number of culture days (Figs. 8, 3C).

Antibacterial activity measurement

The antibacterial activity of herbal medicines and herbal fermented compositions was measured by agar diffusion method. The black strain is a Gram-positive bacterium, Bacillus subtilis PCI 219, genus Staphylococcus aureus. Aureus (Staphylococcusaureus subsp. Lium (Salmonella typhimurium) KCTC 2504 was used. In addition, yeast Candida albicans (Candida albicans), Cryptococcus neoformans (Cryptococcus neoformans) was used, the mold Aspergillus niger (Aspergillus niger), Aspergillus duck material (Aspergillus oryzae) was used. .

Bacteria were LB medium (peptone 10 g, yeast extract 5 g, sodium chloride 5 g/ℓ, pH 6.8), nutrient medium (beef extract 3 g, peptone 5 g/ℓ, pH 6.8, Difco Co.) and Reinforced Clostridial ( RC) subcultured in medium (tryptose 10 g, beef extract 10 g, yeast extract 3 g, dextrose 5 g, sodium chloride 5 g, soluble starch 1 g, cysteine hydrochloride 0.5 g, sodium acetate 3 g/ℓ, pH 7.6) The yeast was subcultured in YM medium (yeast extract 3 g, malt extract 3 g, dextrose 10 g, peptone 5 g/ℓ, pH 7.6) and stored at 4°C for use. The mold was subcultured in PDA medium (potato starch 4 g, dextrose 20 g/ℓ, pH 5.6, Difco Co.) to prepare a spore suspension, and then stored at -20°C for use.

For antibacterial activity, the culture medium was centrifuged at 4°C and 1,000 × g for 10 minutes, and then 20 μl of the supernatant was added to a paper disc (=6 mm, Whatman Co.), dried, and placed on a plate medium containing the assay strain. Bacteria were cultured at 37° C. for 1 day, and yeast and mold were cultured at 28° C. for 2 days and 4 days, respectively, and the resulting clear zone was judged by the presence and size of the clear zone.

As a result of examining the antibacterial activity, the herbal medicine (non-fermented antibacterial acid) did not have antibacterial activity on all test strains, and the oriental medicine fermented composition (complex cultured antibacterial acid) of the present invention was Bacillus subtilis PCI 219 , Pseudomonas aeruginosa KCTC 2004, Staphylococcus aureus genus. It showed antibacterial activity against Staphylococcus aureus subsp. aureus KCTC 1916, and showed antibacterial activity against Propionibacterium acnes KCTC 3314, which causes acne (Table 4, FIG. 9).

A: Herbal medicine (antibacterial acid), B: Herbal medicine + 1% sugar, C: Herbal medicine + 5% sugar

Cytotoxicity check

For cytotoxicity, MTT colorimetric reduction assay indicating mitochondrial dehydrogenase activity was performed, and the effect of the assay material on cell survival was measured according to the method reported by Mosmann (Mosmann 1983). The RBL-2H3 cell line, which is an indicator cell, was seeded in a 96-well plate at a density of 1×105 cells per well and cultured for 24 hours. After cell culture, 0.5 mg/ml of MTT reagent (Generay Biotech Co.) was added to each well of 10 μl and cultured for 4 hours, the remaining MTT reagent and medium were removed, DMSO and EtOH were mixed 1:1, and 100 μl After each aliquot, the precipitate was sufficiently dissolved. Cytotoxicity was confirmed after measuring the absorbance based on the measurement wavelength of 540 nm using an ELISA reader.

The results of confirming the cytotoxicity of the herbal medicine (non-fermented Bangpungtongseongsan) for the RBL-2H3 cell line are shown in FIG. 10 . The treatment time of the herbal medicine was constant and only the amount of the herbal medicine added was checked for cytotoxicity. As a result, it was confirmed that there was no cytotoxicity even when the herbal medicine was added at a high concentration (300 μl/ml).

When confirming the cytotoxicity of the oriental medicine fermentation composition, the treatment concentration was 10 μl/ml, and the treatment time was 30 minutes, 1 hour, 3 hours, 6 hours and 12 hours. As shown in FIG. 11 , there was no cytotoxicity when the fermented herbal medicines were treated in cell lines for 30 minutes, 1 hour, and 3 hours, but the cells were affected after 6 hours.

Effect of inhibiting release of β-hexosaminidase

For the measurement of β-hexosaminidase (Yamada et al. 2007), the RBL-2H3 cell line was prepared in D?MEM medium containing 10% FBS and 1% antibiotics and 5% at 37°C. After culturing in saturated humidity air conditions including CO2 and reaching 80 to 90% confluency, the number of cells is adjusted to 5 × 105 cells/ml, aliquoted into a 24-well plate, and then IgE is added at 0.5 μg/ml. and cultured for 24 hours. After incubation, the medium was removed and washed twice with 500 μl Siraganian buffer (119 mM NaCl, 5 mM KCl, 0.4 mM MgCl2, 25 mM PIPES, 40 mM NaOH, pH 7.2), followed by 5.6 mM glucose, 1 mM 160 μl of Siraganian buffer containing CaCl2 and 0.1% BSA (Sigma Co.) was added and incubated for 10 minutes. Cells sensitized with IgE (Sigma Co.) were treated with samples by concentration and cultured for a certain period of time, and then antigen (DNP-KLH, Alpha diagnostic Intl Inc.) was added at 1 μg/ml and reacted for 10 minutes. After putting the reaction mixture on ice for 10 minutes to complete the reaction, the reaction mixture was centrifuged at 500 × g for 10 minutes to separate the supernatant. 25 μl each of the supernatant and substrate (1 mM nitrophenyl-N-acetyl-β-D-glucosaminide, Sigma Co.) was added to a 96-well plate, incubated at 37° C. for 1 hour, and then 200 μl of 0.1 M Na2CO3/NaHCO3 was added. to stop the reaction and measured at 405 nm with an ELISA reader. The inhibition of the release of β-hexosaminidase in the RBL-2H3 cell line was calculated as follows.

Control: Addition of antibody and antigen, no sample;

Treated: addition of antibodies, antigens and samples;

Blank: Add sample and substrate only;

Spontaneous: No antibody, antigen and sample added

Among the methods for measuring the allergy inhibitory effect of a test substance, the method of measuring the amount of histamine released from mast cells sensitized to IgE tends to show large deviations because the concentration of histamine in mast cells is low and complex steps must be performed. . Recently, a method for measuring β-hexosaminidase, which is present in cells at high concentrations and released together with chemical mediators during degranulation, is widely used (Choi 2002).

As a result of measuring the release inhibitory effect of β-hexosaminidase in Example 3 having excellent antioxidant and antibacterial activity using this method, as shown in FIG. 12 , the allergy inhibitory effect did not increase according to the number of culture days. and was similar. For those cultured only with herbal medicine (Pungbangseongsan), 56.81% on the 5th day of culture, 53.93% for herbal medicine with 1% brown sugar added on the 3rd day, and Chinese medicine with 1% brown sugar in culture 3 It was as high as 52.41% on the first day.

The effect of pH on the release of β-hexosaminidase was not significant as shown in FIG. 13 . The effect of inhibiting the release of β-hexosaminidase of the herbal medicine having a pH of 5.4 was 68%, and the effect of inhibiting the release of β-hexosaminidase of the fermented herbal medicine was 57%.

statistical analysis

In the above experimental example, statistical analysis was performed using SigmaStat for analysis of variance, and the mean-to-mean significance test of the test section was performed at the 5% level using a multiple test method according to the SNK (Student-Newman-Keuls) test.

As described above, the present invention uses Pungbangseongsan and Lactobacillus. Specifically, various kinds of lactobacilli are inoculated with different numbers of bacteria or inoculated with herbal medicine (Pungtongseongsan) at different times and then fermented through a complex culture method. Bacillus bacteria were allowed to grow well. For this reason, the present invention has a variety of physiologically active functions of the antiperspirant acid, and has better antioxidant activity than the herbal medicine before fermentation (Bacillus subtilis) PCI 219, Pseudomonas aeruginosa (Pseudomonas aeruginosa) KCTC 2004, Staphylococcus aureus genus. Aureus (Staphylococcus aureus subsp. aureus) KCTC 1916 showed antibacterial activity and, in particular, showed antibacterial activity against Propionibacterium acnes KCTC 3314, which causes acne. In addition, it exhibited an allergy inhibitory effect close to 60%. Accordingly, the present invention is a very useful invention in the cosmetic industry, pharmaceutical industry, food industry, and the like.

Claims (16)

(S1) preparing the herbal medicine, Bangpungtongseongsan;
(S2) preparing Lactobacillus; and
(S3) inoculating Lactobacillus into the prepared windbreak and fermenting acid and fermenting at 30~45℃; The method of claim 1, wherein (S1) in the step of the windbreaktongseongsan
A method for producing a fermented oriental medicine composition in which the precipitate is removed by centrifugation at 1-5°C and 2,000-5,000×g. According to claim 1, (S1) in step (S1) is the oriental fermented composition preparation method by adjusting the pH to 4-7. The method of claim 1, wherein 1 to 5% (w/v) of sugar is added to the Bangpungtongseong acid in step (S1). According to claim 1, (S2) Lactobacillus bacteria in step Lactobacillus brevis (Lactobacillus brevis), Lactobacillus casei (Lactobacillus casei), Lactobacillus del Bruecki sub sp. bultaricus (Lactobacillus delbrueckii subsp. bultaricus) Lactobacillus fermentum (Lactobacillus fermentum), Lactobacillus helveticus (Lactobacillus helveticus), Lactobacillus plantarum (Lactobacillus plantarum) at least two kinds selected from the group consisting of oriental fermentation composition manufacturing method. The method according to claim 1, wherein the number of Lactobacillus bacteria in step (S2) is adjusted to 1×10 6 to 1×108 CFU/ml. According to claim 1, (S2) Lactobacillus in step 0.8 × 106 ~ 5 × 106 CFU / ㎖ Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus casei (Lactobacillus casei), 0.8 × 107 ~ 5 × 107 CFU /ml of Lactobacillus delbrueckii subsp. bultaricus (Lactobacillus delbrueckii subsp. bultaricus) and Lactobacillus plantarum (Lactobacillus plantarum), 0.8 × 108-5 × 108 CFU / ml of Lactobacillus fermentum (Lactobacillus fermentum) and lactobacillus fermentum Bacillus helveticus (Lactobacillus helveticus) will contain the herbal fermentation composition manufacturing method. According to claim 1, (S2) Lactobacillus in step 0.8 × 106 ~ 5 × 106 CFU / ㎖ Lactobacillus brevis (Lactobacillus brevis) and Lactobacillus casei (Lactobacillus casei), 0.8 × 107 ~ 5 × 107 CFU Lactobacillus helveticus (Lactobacillus helveticus) and Lactobacillus plantarum (Lactobacillus plantarum) of / ml, Lactobacillus fermentum (Lactobacillus fermentum) and Lactobacillus delbru Eki subspecies of 0.8 × 108 ~ 5 × 108 CFU / ㎖ Cus (Lactobacillus delbrueckii subsp. bultaricus), the oriental fermentation composition manufacturing method comprising. The method of claim 1, wherein in step (S3)
After inoculation with Lactobacillus brevis, Lactobacillus delbrueckii subsp. bultaricus and Lactobacillus helveticus, 4-5 days from the inoculation day Fermentum (Lactobacillus fermentum) and Lactobacillus plantarum (Lactobacillus plantarum) to inoculate the oriental fermentation composition manufacturing method. The method of claim 9, wherein each Lactobacillus is inoculated after adjusting the number of bacteria to 1×108 CFU/ml. The method of claim 1, wherein in step (S3), Lactobacillus is inoculated in an amount of 0.1 to 1% (v/v) with respect to the windbreak-tongseong acid. According to any one of claims 1 to 11, prepared according to any one of the methods, the oriental fermented composition exhibits antibacterial and anti-allergic activity. According to claim 12, Pseudomonas aeruginosa (Pseudomonas aeruginosa), Staphylococcus aureus genus. Aureus (Staphylococcus aureus subsp. aureus) and Propionibacterium acnes (Propionibacterium acnes) against the oriental fermented composition showing antibacterial activity. A food composition comprising the herbal fermented composition of claim 12 . A pharmaceutical composition comprising the herbal fermented composition of claim 12. A cosmetic composition comprising the fermented herbal composition of claim 12.

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