KR102584197B1 - Method for manufacturing bellflower fermented product to improve skin condition due to immune hypersensitivity reaction - Google Patents

Abstract

Atopic dermatitis caused by skin immune hypersensitivity is a chronic disease that repeatedly improves and worsens and requires long-term, consistent treatment. However, side effects from the treatments being used as treatments have been reported, so the development of safe materials using natural food materials is necessary. Accordingly, the present invention aims to provide a method for producing fermented bellflower root products that improve skin condition due to immune hypersensitivity reactions through research on improving the antioxidant effect of bellflower root, which is widely used as a food, and the saponin content and activity of bellflower root.
In order to solve the above problem, in the method of manufacturing bellflower fermented product that improves skin condition due to immune hypersensitivity reaction, 27kg of washed and dried bellflower root is extracted at 100℃ for 2 hours using 170L of water to make 150L of final liquid raw material. Bellflower hot water extraction step (S1); and a spawn preparation step (S2) in which Lactobacillus plantarum (CM), used in kimchi fermentation, is subcultured three times as a spawn to check whether the spawn is contaminated. and a seed culture step (S3) of inoculating the seed prepared in the seed preparation step with 1% activated seed in 1.5 L of sterilized Seed Starter (MRS broth) and then culturing the seed for 15 hours; And a main culture step (S4) of inoculating the Lactobacillus plantarum (CM) seed cultured in the seed culture step into the bellflower hot water extract extracted in step S1 and culturing it at 37°C for 15 to 60 hours; And a culture stop step (S5) of stopping the culture when the acidity of the bellflower hot water extract is pH 3.6; and a sterilization step (S6) of sterilizing at 100°C for 20 minutes to prevent the culturing from proceeding; And a drying step (S7) of freeze-drying or spray-drying the sterilized culture solution is provided.
By constructing the invention as described above, the present application provides treatment for skin diseases caused by immune hypersensitivity, such as atopic dermatitis, with natural ingredients derived from nature, thereby providing treatment with fewer side effects or symptom suppression and alleviation even after long-term use. It has the effect of providing natural materials.

Description

Method for manufacturing bellflower fermented product to improve skin condition due to immune hypersensitivity reaction{.}

The present invention relates to a method for producing bioconverted fermented bellflower root. In more detail, it relates to a method of producing fermented bellflower root.

A technology for producing steamed bellflower root is disclosed by mixing grape juice and honey with steamed bellflower root and then fermenting it with Bacillus svtilis fermentation bacteria according to prior art prior to the filing of the present application. This technology was intended to improve the expectorant and antipyretic effects of bellflower root by removing the unique odor and bitter taste of bellflower root and improving medicinal ingredients such as saponin and steroids.

Another prior art relates to a method for producing bellflower fermented broth with enhanced antioxidant activity and alpha-glucosidase inhibitory activity, in which Lactobacillus plantarum is added to a culture broth containing 1 to 10% (w/v) bellflower root. plantarum) strain and fermented at 20 to 40°C for 8 to 24 hours. This is a technology characterized by having an antioxidant effect and an alpha-glucosidase inhibitory effect.

Another prior art is a composition for preventing, improving, or treating atopic dermatitis, which contains bellflower root as a liquid culture medium, inoculated with basidiomycete hyphae, cultured and fermented, and then treated with cellulolytic enzyme as an active ingredient. Related technology has been disclosed.

Registered Patent Publication No. 10-1225868 Public Patent Publication No. 10-2015-0131483 Registered Patent Publication No. 10-2168765

Atopic dermatitis caused by skin immune hypersensitivity is a chronic disease that repeatedly improves and worsens and requires long-term, consistent treatment. However, side effects from the treatments being used as treatments have been reported, so the development of safe materials using natural food materials is necessary. Accordingly, the present invention aims to provide a method for producing fermented bellflower root products that improve skin condition due to immune hypersensitivity reactions through research on improving the antioxidant effect of bellflower root, which is widely used as a food, and the saponin content and activity of bellflower root.

For this purpose, dried bellflower root was fermented using Lactobacillus plantarum , a kimchi lactic acid bacterium, and an analysis method was developed to measure the contents of platycodin D and platycoside E, which are indicators and active ingredients of bellflower root, and the fermented composition was tested using the HaCaT cell line, a human keratinocyte. Through experiments, it was found that fermented bellflower root has the effect of improving immune hypersensitivity.

In order to solve the above problems, the following problem solving means are provided.

In the method of manufacturing bellflower fermented product for improving skin condition due to immune hypersensitivity reaction,

A bellflower hot water extraction step (S1) in which 27 kg of washed and dried bellflower root is extracted at 100°C for 2 hours using 170 L of water to obtain a final liquid raw material of 150 L; and

A spawn preparation step (S2) in which Lactobacillus plantarum (CM), used in kimchi fermentation, is subcultured three times as a spawn to check whether the spawn is contaminated; and

A seed culture step (S3) of inoculating the seed prepared in the seed preparation step with 1% activated seed in 1.5 L of sterilized Seed Starter (MRS broth) and then culturing for 15 hours; and

A main culture step (S4) in which the Lactobacillus plantarum (CM) seed cultured in the seed culture step is inoculated into the bellflower hot water extract extracted in step S1 and cultured at 37° C. for 15 to 60 hours; and

Cultivation stop step (S5) of stopping the culture when the acidity of the bellflower hot water extract is pH 3.6; and

A sterilization step (S6) of sterilizing at 100°C for 20 minutes to prevent the culturing from proceeding; and

A method for producing fermented bellflower root product for improving skin condition due to immune hypersensitivity reaction is provided, comprising a drying step (S7) of freeze-drying or spray-drying the sterilized culture solution.

In addition, an ointment containing bellflower root fermentation product for improving skin condition due to immune hypersensitivity reaction is provided, wherein the fermented bellflower root product for improving skin condition due to immune hypersensitivity reaction is mixed with propolis water extract and applied to the skin.

In addition, it provides a fermented bellflower root composition for improving skin condition due to immune hypersensitivity reaction, which is characterized by mixing the bellflower fermented product for improving skin condition due to immune hypersensitivity reaction and thistle extract at a weight ratio of 1 to 0.1 to 10.

In addition, the method of manufacturing balloon flower fermentation to improve skin condition due to immune hypersensitivity reaction is

Bellflower hot water extraction step (SA1) of extraction at 100°C for 2 hours using dried bellflower water after washing; and

A starter preparation step (SA2) using Lactobacillus plantarum (CM), which is used in kimchi fermentation, as a starter; and

A seed culture step (SA3) in which the seed prepared in the seed preparation step is inoculated into a sterilized Seed Starter (MRS broth) and then incubated for 15 hours; and

A main culture step (SA4) in which the Lactobacillus plantarum (CM) seed cultured in the seed culture step is inoculated into the bellflower hot water extract extracted in step S1 and cultured at 37° C. for 15 to 60 hours; and

Cultivation stop step (S5) of stopping the culture when the acidity of the bellflower hot water extract is pH 3.6; and

A sterilization step (SA6) of sterilizing the culture to prevent it from proceeding; and

A method for producing fermented bellflower root product for improving skin condition due to immune hypersensitivity reaction is provided, comprising a drying step (SA7) of freeze-drying or spray-drying the sterilized culture solution.

In addition, the bellflower hot water extraction step involves extracting 27kg of dried bellflower after washing at 100°C for 2 hours using 170L of water to make a final liquid raw material of 150L. Manufacturing bellflower fermented product that improves skin condition due to immune hypersensitivity reaction. Provides a method.

In addition, it provides a method of manufacturing bellflower fermented product for improving skin condition due to immune hypersensitivity reaction, characterized in that the dried bellflower fermented product in the drying step is 12.15 kg.

In addition, an ointment containing bellflower root fermentation product for improving skin condition due to immune hypersensitivity reaction is provided, wherein the bellflower root fermentation product for improving skin condition due to immune hypersensitivity reaction is mixed with propolis water extract and applied to the skin.

In addition, it provides a fermented bellflower root composition for improving skin condition due to immune hypersensitivity reaction, which is characterized by mixing the bellflower fermented product for improving skin condition due to immune hypersensitivity reaction and thistle extract at a weight ratio of 1 to 0.1 to 10.

By constructing the invention as described above, the present application provides an ingredient that treats or alleviates skin diseases caused by immune hypersensitivity, such as atopic dermatitis, with natural ingredients derived from nature, with fewer side effects even when used for a long time. It has an effect.

On the one hand, a composition effective against immune hypersensitivity reactions was provided by mixing two or more ingredients with complementary acidity that are effective against immune hypersensitivity reactions.

Figure 1 is a diagram illustrating the strain activation steps of the present invention
Figure 2 is a diagram illustrating the culture process steps of the present invention
Figure 3 is a graph of the culture results of the fermented bellflower extract of the present invention
Figure 4 shows the experimental results of indicator substance analysis of the fermented bellflower extract according to the bioconversion of the present invention.
Figure 5 is a bar graph of cell viability change for evaluating cell toxicity according to treatment of fermented bellflower extract of the present invention.
Figure 6 is a bar graph showing changes in expression of inflammatory biomarkers according to treatment with the fermented bellflower extract of the present invention.
Figure 7 shows the bellflower fermented product manufacturing process of the present invention

□ Risk of immune hypersensitivity reaction

A hypersensitivity reaction refers to an immune response that occurs excessively against a specific antigen and causes inappropriate damage, such as tissue damage. Hypersensitivity reactions are classified into types 1 to 4, and type 1 hypersensitivity reactions are well known as allergies. Currently, allergy is defined in a more limited sense as ‘a disease caused by the immune system’s response to various antigens.’ Allergies are hypersensitivity reactions, one of several harmful immune reactions that can cause tissue damage and serious diseases.

□ The need to develop materials to improve skin conditions caused by immune hypersensitivity reactions

Atopic dermatitis is a chronic, incurable inflammatory skin disease characterized by itching, skin redness, lichenification, and skin infection. The cause can be divided into congenital factors (genetic factors) and acquired factors. Genetic factors include genetic mutation of filaggrin, and acquired factors include stimulation by various triggers and immunological factors. These factors and damage to the skin barrier occur through complex interactions.

The above-mentioned atopic dermatitis is characterized by an immune hypersensitivity reaction of the skin, and is a disease that appears more commonly in industrially developed and westernized countries. It has a prevalence of 10-20% in children and 1-3% in adults, and has a similar prevalence in Korea. It represents. Genetic predisposition plays a very important role in atopic dermatitis, with a family history in about 70-80% of cases, but environmental factors are also known to have a strong influence. Typical symptoms include dry, itchy skin and rashes on the face, inside the elbows, behind the knees, and hands and feet.

Atopic dermatitis is a chronic disease with diverse causes and mechanisms that repeatedly improves and worsens, requiring long-term, consistent treatment. Due to the nature of the disease, which repeatedly recurs, it often becomes severe and incurable even after adulthood.

According to data released by the National Health Insurance Corporation in 2016, children under the age of 12 account for nearly half (48.6%) of the 933,000 atopic dermatitis patients based on the population covered by health insurance in Korea in 2015. Atopic dermatitis is a series of allergic immune diseases characterized by severe itching and chronic skin rashes. It is the most common form of infantile eczema, commonly called fever, and is a disease that occurs in 10-20% of the total population not only domestically but also internationally. Atopic disease often disappears naturally with age, but it often gets worse in 30-80% of patients, especially due to the increase in air pollution due to the effects of industrialization and urbanization, changes in food, clothing, and shelter such as the increase in imported agricultural products and westernization of life. As a result, atopic dermatitis disease is gradually increasing due to new factors.

According to data monitor data, more than 40 million people in the world's seven largest markets, including the United States, Japan, and France, are suffering from atopic dermatitis. By country, atopic dermatitis appears to occur at the highest rate in the United States, at 64 out of 1,000 people.

It has been analyzed that atopic dermatitis occurs.

As a result of analyzing the age-specific incidence rate of atopic dermatitis patients in 7 countries around the world, a high incidence rate of about 21% was found in children aged 0 to 4 years.

Limitations and side effects of atopic dermatitis treatments Currently used treatments for atopic dermatitis are non-specific immunosuppressants such as steroids and tacrolimus and antihistamines. Topical steroids are the most commonly used drugs, but if used continuously, side effects such as skin atrophy, swelling lines, and damage to the skin barrier function occur. Therefore, there is a need for the development of new treatments that are antigen-specific and capable of fundamentally treating the disease. Currently, atopic dermatitis treatment is based on skin moisturization, removal of aggravating factors, topical steroids, and patient and family education. Depending on the severity of symptoms, antihistamines, phototherapy, topical immune response agents, and immunosuppressants are used. However, for a long time, non-specific treatment has been used. Treatments cause many side effects, so there is an urgent need to develop new atopic dermatitis treatments that are more effective and have fewer side effects.

Bellflower (Platycodon grandiflorum), which is used as the material of the present invention, is a plant of the Campanula family that grows widely in mountainous regions of Korea, Japan, and China, and the root part is mainly used. Among them, the bellflower root used as an herb is mainly 1-2 years old, and the bellflower root used for medicinal purposes is mainly 3 years old or older (Kim et al., 2007; Lim, 1971; Sung and Seo, 1998). The main effects of bellflower root include lowering blood sugar (Seo et al., 2004), improving cholesterol metabolism (Seo et al., 2006; Zhao et al., 2004), anti-obesity (Byen, 2003), and anti-inflammatory (Choi et al. ., 2001), increased immune response (Choi et al., 2001), antioxidant effect (Jang et al., 2011; Kim et al., 1986), improvement of atopic dermatitis (Kim et al., 2011; Kim et al. , 2012), etc. studies have been reported. More than 30 types of saponins, such as platycodin A, C, D, and polygalacin D, contained in bellflower root, are known as active ingredients of bellflower root with this functionality. The saponin of bellflower root has a structure of oleanane triterpene as an aglycone and is used in each reactor. It combines one or more sugars (Konish et al., 1976; Li et al., 2010; Tada et al., 1975). According to a study by Yan et al. (2012), the saponin content of bellflower root from Korea and China was examined and the content of platycoside E and platycodin D was found to be the highest. According to a study by Ha et al. (2006), among the saponin components contained in bellflower root, platycoside E, platycodin D and polygalacin D showed high contents.

The present invention relates to a technology for producing a fermented product by fermenting the bellflower root described above.

For this purpose, conditions for extraction and fermentation of bellflower root according to bioconversion (fermentation) are being set.

□ Bellflower extraction and fermentation conditions setting according to bioconversion

(1) Bellflower root extract manufacturing method

- Extract 27kg of washed and dried bellflower root using 170L of water at 100℃ for 2 hours, resulting in final liquid.

Extract the raw material to make 150L.

- When 150L of the extracted liquid raw material is freeze-dried, the extract solution, which has a recovery rate of 45% compared to the final pharmaceutical raw material and 7% compared to the extracted liquid, is used as the raw material.

(2) Spawn activation and seed starter preparation

The present invention uses Lactobacillus plantarum (CM), which is used in kimchi fermentation, as a starter. Once a production plan is established, a seed activation plan is established, and then the strain is inoculated and culture is started to activate the strain. After inoculating the activated strain, the strain is checked under a microscope to ensure that it is not contaminated, and then subculture is performed for three generations. Figure 1 shows this process.

- Activation of Lactobacillus plantarum (CM) spawn is done by subculturing in MRS broth medium for up to 3 generations after inoculation with the first ampoule.

- Add 1% activated Lactobacillus plantarum seed to 1.5L of sterilized Seed Starter (MRS broth), and incubate for 15 hours.

(3) Production manufacturing fermentation process

Using the activated spawn, fermented bellflower root is produced through the process of Figure 2.

The seed is inoculated into the produced medium and the main culture is performed. After the culture, heat treatment is performed to prevent further fermentation, the fermented product is recovered, and the freeze-dried fermented product is pulverized to produce bellflower fermented product. The sterilization process of the culture medium and the main culture process are shown in Tables 1 and 2 below.

- Culture medium sterilization process

work method apparatus / time caution note Culture tanks and lines should be sufficiently steam sterilized.
High pressure steam sterilizer/
15 minutes Weigh the raw materials for the medium that fit the production plan, insert them into the main culture tank, and mix well by stirring.
Raw materials, weighing scale / 1~2 hours Measure the medium according to its content and be careful not to mix in other influents. After high-pressure steam sterilization at 121°C for 15 to 20 minutes, lower the temperature to 37°C while stirring at 40 to 60 RPM and sufficiently replace with N ₂ .
High pressure steam sterilizer/cooler/
N ₂ gas/2~3 hours When sterilizing the medium, carefully control the temperature, pressure, and time to prevent the medium from burning, and allow sufficient time for CO ₂ replacement. If there is a problem with the machine, report it to the manager in charge and take action.

- Main culture process

work method Equipment / Liquid / Time caution note Seed starter with no contamination in sterilized medium
Inoculate at 1%.
Incubator/Seed starter
/10 minutes When inoculating, check the temperature and pH appropriate for the strain before inoculating. After inoculation, culture for 15 hours at 37°C and pH 3.5 to 6, check temperature and pH every 2 hours, sample and perform a speculum, and record the culture status.
Sampling tube, microscope/10 minutes If any abnormalities occur during cultivation, report them to the manager in charge and record them in the production management form. If there are no abnormalities until the end of the culture, end the culture and place at 100℃.
After sterilizing for 15 minutes, cool to 50℃.
Incubator/1~2 hours When treating sterilization, carefully control temperature, pressure, and time to prevent the medium from burning.
Adjust it. The cooled culture medium is recovered through a recovery line. incubator/min Make sure the recovery line is sterilized and perform work with caution against inflow of external contamination. . Freeze the recovered culture medium
Freeze-dried for 4 days in the dry season.
After that, retrieve the original horse and
After grinding, store in a double-sealed container to prevent moisture from entering. Freeze dryer, double sealed container/min When recovering or moving to the freeze dryer, be careful of external contamination. The final raw material is tested for contamination and functionality.
Conduct an inspection,
Report any abnormalities to the manager in charge and record them in the production management record.

(4) Production manufacturing fermentation process culture results

- As a result of measuring the number of viable bacteria and pH change in the fermented bellflower root extract, the number of viable cells is shown in Table 3 and Figure 3. It was confirmed that the number of viable bacteria in the fermented bellflower root extract significantly increased with fermentation time, and pH decreased as fermentation time increased.

Time(hr) 1batch pH cfu/ml brix 0 4.9 1.50E+07 8.2 2 4.89 6.09E+07 8.2 4 4.64 1.49E+08 8.2 6 4.19 6.40E+08 8.1 8 3.89 7.50E+08 8.1 10 3.8 8.00E+08 8.1 12 3.72 9.10E+08 8 14 3.66 1.07E+09 8 15 3.64 1.17E+09 8

(5) Production manufacturing drying process

- The fermented bellflower root extract was freeze-dried for 4 days using a freeze dryer under the conditions shown in Table 4, and a powder containing 45% of the raw material and 7% of the dried dosage was produced.

Conditions for freeze-drying fermented bellflower root extract date hour shelf temperature trap temperature degree of vacuum 2020-11-30 10:20:00 AM -7.6 5:20:00 PM -45 8:10:00 PM -44 -69 51 2020-12-01 8:30:00 AM -21.1 -79 30 1:30:00 PM -8 -77.9 32 5:40:00 PM 1.8 -77.3 27 2020-12-02 8:30:00 AM 34.3 -79.6 22 3:40:00 PM 45 -81.4 20 5:30:00 PM 45.2 -82.1 17 2020-12-03 9:20:00 AM 45 -82.4 17 3:40:00 PM 45 -80.4 17 2020-12-04 9:20:00 AM 40.1 -81.2 17

Another problem solved by the invention of this application lies in the method of analyzing indicator substances of fermented bellflower extract. During the development of the bellflower fermented product according to the invention of the present application, it is also a problem of the invention of the present application to develop this analysis method without an analysis method of an indicator substance that has the effect of suppressing immune hypersensitivity of bellflower through fermentation.

□ Development and verification of indicator material analysis method of fermented bellflower extract following bioconversion

(1) Setting the analysis conditions for indicator components (STD) of fermented bellflower root

- HPLC analysis of platycoside E and platycodin D contained in fermented bellflower root was performed by simultaneously analyzing two different standard substances under the analysis conditions shown in Table 5. The equipment used for analysis was Waters 2695 Separation Module HPLC system and Waters 996 Photodiode Array Detector (Waters, Milford, Ma, USA). The analysis conditions are as shown in Table 5, and the analysis column was Capcell pak C ₁₈ (4.6 mm × 250 mm, 5.0 μm, Shiseido, Tokyo, Japan) was used.

Table 5. Platycoside E and platycodin D analysis conditions using HPLC Instrument Conditions Column Capcell Pak C ₁₈ UG12, (5.0 μm, 4.6 × 250 mm) Column temp. 30℃ Mobile phase
(Gradient) Time (min) A ¹⁾ (%) B ²⁾ (%) 0 82 18 15 75 25 30 70 30 35 82 18 40 82 18 Detector Waters 996 Photodiode Array Detector (210 nm) Flow rate 1.0mL/min Injection volume 20 μL Run time 40min ¹⁾ Distilled water (DW)
²⁾ Acetoniltrile

(2) Preparation of standard solution of indicator components (STD) of fermented bellflower root

- After taking 10 mg each of the standard substances Platycoside E and platycodin D, using a 10 mL volumetric flask, the concentration was adjusted to 1,000 μg/mL with 18% acetonitrile up to the mark, which was used as a stock solution. Working solution was used by using the prepared stock solution and diluting it with 18% acetonitrile to 0.78125, 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, and 200 μg/mL. 10 mg each of platycoside E and platycodin D standard substances were taken, and the concentration was adjusted to 1,000 μg/mL in a 10 mL volumetric flask with 18% acetonitrile up to the mark, which was used as a stock solution. The working solution was diluted with 18% acetonitrile to 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μg/mL using the prepared stock solution. The test solution was prepared by weighing 40 mL of the freeze-dried sample, adding 18% acetonitrile to a concentration of 4,000 μg/mL using a 10 mL volumetric flask, and filtering through a 0.45 μm pore size PET syringe filter (Whatman, USA). It was used as a test solution.

(3) Validation of analytical method

- The effectiveness of the analysis method was verified using linearity, precision, accuracy, limits of detection (LOD), and limits of quantification (LOQ). .

(4) Experiment results

- Check linearity

Linearity refers to the ability to obtain a linear measurement value proportional to the amount of the analyte within a certain range of the sample. Depending on the analysis results, the y-axis represents the peak area and the x-axis represents the concentration of the standard solution (μg/mL). Linearity was confirmed with the R2 value of the calibration curve prepared as . Platycoside E and platycodin D standard solutions were stepwise diluted to concentrations of 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, and 200 μg/mL and analyzed by HPLC. As a result, the standard calibration curves for platycoside E and platycodin D were y, respectively. =2830.2x + 84.333, y=3545.5x + 23.544, and the correlation coefficient (R ² ) values were 0.9999 for platycoside E and 0.9997 for platycodin D, showing excellent linearity. The experimental result graph is shown in Figure 4. Figure 4(A) is the platycoside E measurement result, and Figure 4(B) is the platycodin D.

- Precision and accuracy

Precision refers to the proximity between each measurement value obtained through continuous analysis of a uniform sample, and accuracy refers to the degree to which the measurement value is close to a known true value or standard value. The standard solution was added to the fermented bellflower extract of known concentration at low concentration (1.5625 μg/mL), medium concentration (12.5 μg/mL), and high concentration (100 μg/mL), and then analyzed by HPLC. Precision was measured for each measurement. The degree of dispersion of the results was checked and evaluated, and accuracy was evaluated by measuring the recovery rate. The precision results were confirmed by relative standard deviation (RSD), and the precision of platycoside E and platycodin D was 0.58-2.91% and 1.21-3.59% in intra-day precision, respectively, and 0.4-4.77% and 0.9-4.23% in intra-day precision, which is 5%. The following excellent precision was shown. The accuracy results were expressed by measuring the recovery rate, and as shown in Table 2, the daily accuracy of platycoside E and platycodin D was 94.81-110.62%, 94.02-115.73%, and the intra-day accuracy was 94.63-104.35%, 94.11-122.82%, showing excellent accuracy. indicated. The experimental results are shown in Table 6.

Table 6. Platycoside E and platycodin D analysis conditions using HPLC Analyzes Concentration (μg/mL) Mean ± SD (μg/mL) RSD (%) Recovery (%) Fermented
Platycodon
grandiflorum
extracts Platycoside E Intra-day 1.5625 1.63 ± 0.08 4.77 104.35 12.5 12.06 ± 0.39 3.20 96.44 100.0 94.63 ± 0.38 0.40 94.63 Inter-day 1.5625 1.73 ± 0.05 2.91 110.62 12.5 11.97 ± 0.24 2.00 95.79 100.0 94.81 ± 0.55 0.58 94.81 Platycodin D Intra-day 1.5625 1.92 ± 0.08 4.23 122.82 12.5 12.54 ± 0.40 3.22 100.35 100.0 94.11 ± 0.85 0.90 94.11 Inter-day 1.5625 1.81 ± 0.03 1.58 115.73 12.5 12.27 ± 0.44 3.59 98.15 100.0 94.02 ± 1.13 1.21 94.02

- Detection limit and quantification limit

The detection limit refers to the minimum amount of an analyte that can be detected at a given confidence level, and the quantification limit refers to the minimum amount of an analyte that can be quantitatively analyzed with a certain level of reliability. As a result of analyzing the detection and quantification limits, the detection limits of platycoside E and platycodin D were measured to be 0.22 μg/mL and 0.31 μg/mL, respectively, and the quantification limits were measured to be 0.68 μg/mL and 0.93 μg/mL. Considering the above results, it was shown that simultaneous and quantitative analysis of platycoside E and platycodin D of fermented bellflower extract was possible using HPLC. Table 7 shows the detection limit and quantitation limit test results.

Table 7. Detection limit and quantitation limit of fermented bellflower extract Analyzes Range
(μg/mL) Slope Intercept Correlation
coefficient (R ² ) LOD
(μg/mL) LOQ
(μg/mL) Platycoside E 0.78-200 3018.48 204.90 0.9999 0.22 0.68 Platycodin D 0.78-200 3928.47 366.36 0.9997 0.31 0.93

□ Analysis of indicator substances of bioconverted fermented bellflower extract

(1) Analysis of platycoside E and platycodin D contents of fermented bellflower extract

- As a result of analyzing the contents of platycoside E and platycodin D in fermented bellflower extract using an established analysis method, it was found that platycoside E was contained at a level of 2.29±0.08 mg/dry weight g and platycodin D was 2.09±0.37 mg/dry weight g. was analyzed. Table 8 shows the experimental results.

Table 8. Indicator substance content of fermented bellflower root extract Sample (mg/dry weight g) Platycoside E Platycodin D Non-fermented Platycodon grandiflorum extract 1.09 ± 0.04 1.99 ± 0.05 Fermented Platycodon grandiflorum extract 2.29 ± 0.04 2.09 ± 0.08

□ Efficacy evaluation of bioconverted fermented bellflower extract

(1) Human Keratinocyte (HaCaT) cytotoxicity evaluation of fermented bellflower extract

- Cytotoxicity evaluation of bellflower root and fermented bellflower root was measured using XTT assay, and silymarin was used as a positive control. All samples containing Silymarin at a concentration of 12.5 μg/mL and bellflower root and fermented bellflower root at a concentration of 200 μg/mL did not show cytotoxicity. In addition, it induces an inflammatory response in HaCaT cells. Even when TNF-α and INF-γ were treated simultaneously, no cytotoxicity was observed, and no changes in cell morphology were observed through microscopic observation in all experiments.

Figure 5 is a graph of cytotoxicity evaluation of bellflower root and fermented bellflower root.

(2) Immune hypersensitivity reaction improvement effect of fermented bellflower extract using HaCaT cells

- HaCaT cells, an epidermal cell, were used to measure the effect of fermented bellflower root on improving skin immune hypersensitivity. As a positive control group, silymarin was used, and the skin inflammatory reaction was treated with TNF-α and INF-γ to induce inflammation. IL-6, IL-1β, MDC, and TARC were selected as biomarkers to determine abnormalities in skin immune regulation function and were measured using enzyme-linked immunosorbent assay (ELISA). The expression of all biomarkers increased due to treatment with TNF-α and INF-γ, but decreased in bellflower root and fermented bellflower root extract. In particular, bioconverted fermented bellflower root induced a significant decrease in immune function regulating biomarkers compared to bellflower root. Figure 6 shows the results of measuring the effect of fermented bellflower root on improving skin immune hypersensitivity. Here, NP is Non-fermented Platycodon grandiflorum extract, which is bellflower extract, and FP is Fermented Platycodon grandiflorum extract, which is a fermented product of bellflower root. In the graphs below, the indications are the same.

Figure 7 shows a production process diagram of bellflower fermented product for improving skin condition due to immune hypersensitivity according to the invention of the present application.

The process of activating the strain from the previously described starter, providing a production medium using bellflower root extract, performing main culture, heat treatment, recovering the fermented product, and freeze-drying are shown as a whole.

To prepare the fermented bellflower root of the present invention, bellflower root was extracted with hot water at 100°C for 2 hours, sterilized at 121°C for 20 minutes, and then inoculated and fermented.

For this fermentation condition, 1% by weight of the strain is inoculated and fermented at 37°C for 15 to 16 hours. Fermentation ends when the acidity of the fermented broth reaches pH 3.6.

To prevent further fermentation, when the fermentation broth reaches the end of fermentation, it is sterilized at 100°C for 20 minutes. The fermentation broth produced in this way is freeze-dried or spray-dried and, if necessary, pulverized to produce the final fermentation product.

The fermented product produced in this way can be added and used as various functional foods, drugs, or applied ingredients.

In the present invention, in order to further increase the effect of improving skin condition due to immune hypersensitivity reaction of fermented bellflower root, it can be used by mixing Korean thistle extract obtained by freeze-dried Korean thistle hot water extract at an approximate ratio of 1 to 0.1 to 10.

The mixing ratio of the fermented bellflower root and Korean thistle extract is characterized in that the acidity of the mixture is adjusted to a concentration of pH 4.5 ~ 5.5. If there is a lot of skin inflammation, the mixing ratio is adjusted to close to pH 4.5, otherwise, close to pH 5.5. It is characterized by

The Korean thistle extract is an alkaline ingredient, and when used by mixing with the bellflower fermented product, it neutralizes the acidity of the bellflower fermented product, and the atopic dermatitis alleviating function of the Korean thistle extract has a synergistic effect, resulting in an improved immune hypersensitivity reaction suppressing effect. .

This is based on the invention of Patent Publication No. 10-2010-0079586, which clarifies that the thistle extract can be used as an anti-atopic dermatitis composition. This invention shows that thistle has an inhibitory effect on IL-4, IL-13, etc. Therefore, if the fermented bellflower root product of the present application is used in combination with the function of inducing a decrease in immune function regulating biomarkers such as IL-6 IL-1Beta MDC TARC as shown in Figure 6, anti-atopy, that is, immune hypersensitivity reaction It can be of great help in improving skin diseases caused by In addition, if the cause of the immune response is found depending on the patient's condition and the mixing ratio of the fermented bellflower root and thistle extract is varied, it is expected that there will be a greater therapeutic or suppressive effect. Although the contents of the above-mentioned published patent publication No. 10-2010-0079586 are not actually described, the number of the above-mentioned published patent publication is replaced by the description of all contents.

The extraction method of thistle (gondre) is as follows. After sorting the biological thistle, washing it with water and steaming it in a steaming container at 90℃ for 40 minutes. After removing the steamed thistle from the steaming container, it is moved to a well-ventilated place and dried so that the moisture content is 10% by weight. 100 g of the dried thistle and 2,000 ml of alcohol (ethanol) were placed in an extraction container, ultrasonic waves were applied for 5 minutes, and heat was applied at 1.5 atm and 65° C. for 4 hours. The extracted thistle extract is freeze-dried, concentrated, and powdered to obtain 25 g of powdered thistle extract.

The structure of the invention, which produces the above-mentioned effects, is as follows. In the method of manufacturing bellflower fermented product for improving skin condition due to immune hypersensitivity reaction,

A bellflower hot water extraction step (S1) in which 27 kg of washed and dried bellflower root is extracted at 100°C for 2 hours using 170 L of water to obtain a final liquid raw material of 150 L; and

A spawn preparation step (S2) in which Lactobacillus plantarum (CM), used in kimchi fermentation, is subcultured three times as a spawn to check whether the spawn is contaminated; and

A seed culture step (S3) of inoculating the seed prepared in the seed preparation step with 1% activated seed in 1.5 L of sterilized Seed Starter (MRS broth) and then culturing for 15 hours; and

A main culture step (S4) in which the Lactobacillus plantarum (CM) seed cultured in the seed culture step is inoculated into the bellflower hot water extract extracted in step S1 and cultured at 37° C. for 15 to 60 hours; and

Cultivation stop step (S5) of stopping the culture when the acidity of the bellflower hot water extract is pH 3.6; and

A sterilization step (S6) of sterilizing at 100°C for 20 minutes to prevent the culturing from proceeding; and

A method for producing fermented bellflower root product for improving skin condition due to immune hypersensitivity reaction is provided, comprising a drying step (S7) of freeze-drying or spray-drying the sterilized culture solution.

In addition, an ointment containing bellflower root fermentation product for improving skin condition due to immune hypersensitivity reaction is provided, wherein the fermented bellflower root product for improving skin condition due to immune hypersensitivity reaction is mixed with propolis water extract and applied to the skin.

In addition, it provides a fermented bellflower root composition for improving skin condition due to immune hypersensitivity reaction, which is characterized by mixing the bellflower fermented product for improving skin condition due to immune hypersensitivity reaction and thistle extract at a weight ratio of 1 to 0.1 to 10.

In addition, the method of manufacturing balloon flower fermentation to improve skin condition due to immune hypersensitivity reaction is

Bellflower hot water extraction step (SA1) of extraction at 100°C for 2 hours using dried bellflower water after washing; and

A starter preparation step (SA2) using Lactobacillus plantarum (CM), which is used in kimchi fermentation, as a starter; and

A seed culture step (SA3) in which the seed prepared in the seed preparation step is inoculated into a sterilized Seed Starter (MRS broth) and then incubated for 15 hours; and

A main culture step (SA4) in which the Lactobacillus plantarum (CM) seed cultured in the seed culture step is inoculated into the bellflower hot water extract extracted in step S1 and cultured at 37° C. for 15 to 60 hours; and

Cultivation stop step (S5) of stopping the culture when the acidity of the bellflower hot water extract is pH 3.6; and

A sterilization step (SA6) of sterilizing the culture to prevent it from proceeding; and

A method for producing fermented bellflower root product for improving skin condition due to immune hypersensitivity reaction is provided, comprising a drying step (SA7) of freeze-drying or spray-drying the sterilized culture solution.

In addition, the bellflower hot water extraction step involves extracting 27kg of dried bellflower after washing at 100°C for 2 hours using 170L of water to make a final liquid raw material of 150L. Manufacturing bellflower fermented product that improves skin condition due to immune hypersensitivity reaction. Provides a method.

In addition, it provides a method of manufacturing bellflower fermented product for improving skin condition due to immune hypersensitivity reaction, characterized in that the dried bellflower fermented product in the drying step is 12.15 kg.

In addition, an ointment containing bellflower root fermentation product for improving skin condition due to immune hypersensitivity reaction is provided, wherein the bellflower root fermentation product for improving skin condition due to immune hypersensitivity reaction is mixed with propolis water extract and applied to the skin.

In addition, it provides a fermented bellflower root composition for improving skin condition due to immune hypersensitivity reaction, which is characterized by mixing the bellflower fermented product for improving skin condition due to immune hypersensitivity reaction and thistle extract at a weight ratio of 1 to 0.1 to 10.

Claims (10)

In the method of manufacturing bellflower fermented product for improving skin condition due to immune hypersensitivity reaction,
A bellflower hot water extraction step (S1) in which 27 kg of washed and dried bellflower root is extracted at 100°C for 2 hours using 170 L of water to obtain a final liquid raw material of 150 L; and
A spawn preparation step (S2) in which Lactobacillus plantarum (CM), used in kimchi fermentation, is subcultured three times as a spawn to check whether the spawn is contaminated; and
A seed culture step (S3) of inoculating the seed prepared in the seed preparation step with 1% activated seed in 1.5 L of sterilized Seed Starter (MRS broth) and then culturing for 15 hours; and
A main culture step (S4) in which the Lactobacillus plantarum (CM) seed cultured in the seed culture step is inoculated into the bellflower hot water extract extracted in step S1 and cultured at 37° C. for 15 to 60 hours; and
Cultivation stop step (S5) of stopping the culture when the acidity of the bellflower hot water extract is pH 3.6; and
A sterilization step (S6) of sterilizing at 100°C for 20 minutes to prevent the culturing from proceeding; and
A method for producing fermented bellflower root product for improving skin condition due to immune hypersensitivity reaction, comprising a drying step (S7) of freeze-drying or spray-drying the sterilized culture solution. delete delete delete The method for producing fermented bellflower root to improve skin condition due to immune hypersensitivity is
Bellflower hot water extraction step (SA1) of extraction at 100°C for 2 hours using dried bellflower water after washing; and
A starter preparation step (SA2) using Lactobacillus plantarum (CM), which is used in kimchi fermentation, as a starter; and
A seed culture step (SA3) in which the seed prepared in the seed preparation step is inoculated into a sterilized Seed Starter (MRS broth) and then incubated for 15 hours; and
A main culture step (SA4) in which the Lactobacillus plantarum (CM) seed cultured in the seed culture step is inoculated into the bellflower hot water extract extracted in the SA1 step and cultured at 37° C. for 15 to 60 hours; and
A culture stop step (SA5) of stopping the culture when the acidity of the bellflower hot water extract is pH 3.6; and
A sterilization step (SA6) of sterilizing the culture to prevent it from proceeding; and
It includes a drying step (SA7) of freeze-drying or spray-drying the sterilized culture solution,
In the bellflower hot water extraction step, 27kg of washed and dried bellflower is extracted at 100°C for 2 hours using 170L of water to obtain a final liquid raw material of 150L,
A method of manufacturing bellflower fermented product for improving skin condition due to immune hypersensitivity reaction, characterized in that the dried bellflower fermented product in the drying step is 12.15 kg.
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