KR101088013B1 - Method for producing fermentated citrus unshiu - Google Patents

Abstract

The present invention relates to a method for preparing fermented dermis, and more particularly, to prepare fermented dermis by inoculating subcutaneous Bacillus subtilis and fermenting it to increase the elution amount of hesperidin, which is the main component of dermis, when the Chinese medicine is fermented with dermis. It relates to a fermented dermis manufacturing method that can improve the.

The method for producing fermented dermis according to the present invention is characterized in that fermented dermis is produced by inoculating fermented subtilis of Dermis into the dried dermis of tangerine or a related perennial plant of the same flavor.

Dermis, Bacillus subtilis, Fermentation, Hespyridine, Antioxidant

Description

METHOD FOR PRODUCING FERMENTATED CITRUS UNSHIU}

The present invention relates to a method for preparing fermented dermis, and more particularly, by preparing fermented dermis by inoculating Bacillus subtilis into the dermis and then fermenting dermis by increasing the elution amount of hesperidin which is the main component of dermis to improve antioxidant function. It relates to a method for producing fermented dermis that can be made.

Dermis, which is used as a herbal medicine, refers to the mature skin of tangerine (Citrus unshiu Markovich) or related plant of the same kind in Korea. Meanwhile, in Japan, tangerine (Citrus unshiu Markovich) and persimmon (Citrus reticulata Blanco) are used as the dermis, and tangerine persimmon (Citrus tachibana (Makino) Tanaka) is used as the tangerine skin, and in China, citrus reticulata Blanco and The cultivar is defined as dermis.

The dermis releases clumps and strengthens the spleen to treat only the abdominal swelling, belching, vomiting, nausea, indigestion, flatulence, drowsiness, and diarrhea. Sea water, sputum elimination and diuretic effect, the older the drug is known to increase.

Dermis contains essential oils and hesperidin as main ingredients, and other active ingredients such as narizine and rutin are also contained.

Essential oils, the main components of the dermis, have been reported to have pharmacological effects such as digestive stimulation, digestion promotion, expectoration, anti-ulcer, anti-gastric secretion, cardiac, blood pressure increase, anti-allergy, bile secretion promotion, uterine smooth muscle suppression, and antibacterial action. .

Meanwhile, hesperidin, another main component of dermis, is a pale yellow or light brownish white crystal or crystalline powder, which has little smell and taste, and is combined with disaccharide rutinose and flavanone in flavonoid pigments. It is present in the form of glycosides, hardly soluble in water, ethanol and ether, and can be a caustic agent of canned citrus.

The hesperidin is distributed in citrus pulp and white part 1.5 ~ 3%, is a substance necessary to maintain capillary permeability of vitamin P, inhibits the formation of lipid peroxides, antioxidant effects such as aging delay, anti-inflammatory effect, Capillary protection and anti-cancer effect, lowers cholesterol.

In addition, the hesperidin inhibits the release of histamine from the mammary gland cells, reducing allergy symptoms and fever, and exhibits anti-cancer effects by inhibiting polyamine synthesis.

Ovarian vertebral mice not only reduce serum and liver cholesterol, but also reduce bone loss by reducing the number of osteoclasts.

Other studies have found that hesperidin extracted from citrus fruits has a significant effect on the excretion of cadmium, which is a pollutant through feces, and a significant effect on lowering triglycerides and cholesterol in the blood and a significant synergistic effect of HDL-cholesterol in the body. It was.

Figure 1 shows the molecular formula of hesperidin, soluble hesperidin dissolved in water was found in lemon juice in 1936 as a substance that enhances capillary walls with vitamin C, named vitamin P, which is a mixture of two or more flavonoids in water It is a nutrient enhancer whose main ingredient is 3´-methly-7 (rhamonosyl-2-methylglucosyl) hesperidin, a methylated derivative of hesperidin that is difficult to dissolve.

However, since hesperidin, an important active ingredient of dermis, is insoluble in water, most of it is not used because it is insoluble in water when it is infused with water, and only part of it is converted into water-soluble hesperidin and used as soluble vitamin P [Methyl Hesperidin]. The rest is abandoned.

There is a patent (patent registration No. 0736901) which extracts and liquefies dermis into specific sugars according to the prior art, but there is still a problem that hesperidin, which is a main ingredient of dermis, does not leak well when treating herbal medicine or extracting with food ingredients.

The dermis used for Chinese medicine is old (陳), and the longer the tangerine is stored, the more fragrant it is and the better the medicinal effect is. The old righteous people put it on a sack under the eaves and matured it for 3 years. It was a product. Not only the actual clinic but also various experimental analyzes confirmed that old tangerine is excellent. Knowing the basics of aging fermentation, it can be seen that it is the wisdom of ancestors who applied natural fermentation according to the characteristics of products.

Therefore, the present invention solves the problems of the prior art, such as the use of fermentation science to apply this in a modern scientific method based on the wisdom of the ancient ancestors, when hesperidin, which is the main ingredient of the dermis, does not flow well when using the fermentation science. In order to improve the antioxidant function by increasing the elution amount of hesperidin, which is the main component of the dermis when the Chinese herbal medicine is fermented by preparing the fermented dermis by inoculating the subtilis Bacillus subtilis into the dermis, the object of the present invention can be improved. It is to provide a method for producing fermented dermis.

The fermented dermis manufacturing method according to the present invention to achieve the above object is characterized in that the fermented dermis by inoculating the mature peel of tangerine or coriander related plants of the fortune inoculated with D. subtilis to the dermis.

In addition, the method for producing fermented dermis according to the present invention is characterized in that the Bacillus subtilis is cultured in a liquid medium containing rice bran and soybean powder as main components.

In addition, in the method for producing fermented dermis according to the present invention, the Bacillus subtilis is incubated for 24 to 48 hours at a temperature of 37 to 42 ℃ in the liquid medium, the dermis is 1 to 5 hours at a temperature of 80 to 120 ℃ After the sterilization by heat sterilization, the Bacillus subtilis is inoculated, it is fermented for 1 to 5 days in a fermentation chamber maintaining a temperature of 37 ~ 42 ℃ characterized in that it is dried at a temperature of 55 ℃ in a dryer.

Fermented dermis manufacturing method according to the present invention having the configuration as described above to increase the elution amount of hesperidin which is the main component of the dermis when the Chinese medicine as the dermis to enhance the antioxidant function to improve the efficacy of the purifier system, fatigue recovery and budding It further enhances the effect on metabolism and has the effect of broadening the application of dermis to the herbal medicine prescription.

Although it is said that it is good to have long-lasting dermis in the herb, etc., but in modern times, natural ripening fermentation of dermis for a long time is a reality that cannot be produced due to various bacteria contamination and decay. There is no situation.

The mukjin dermis (mukjin dermis) is believed to have been fermented during the storage for a long period of time, especially by Bacillus subtilis, which is widely distributed in the natural world in summer, and breeds in general hay or skin.

Therefore, the present invention has been completed based on the experiment of artificially liquid culture of Bacillus subtilis, and fermentation of the Bacillus subtilis into the normal dermis.

The actual dermis contains about 4% of hesperidin, but the extract of hesperidin was extracted by extracting it in water at 100 ° C for 3 hours as if it were a Chinese medicine. By not melting, we could confirm the reality of being thrown away as drug residue.

The present invention aims to extract a large amount of hesperidin from the dermis through the fermentation of Bacillus subtilis as a water-soluble vitamin P, and to extract more amounts of other active ingredients, thereby enhancing the antioxidant power.

[Table 1] below is the result of the comparative analysis between normal dermis and mukjin dermis.

Table 1 shows that hesperidin effluent and antioxidant activity (S.O.D. activity, DPPH activity) are better than murine dermis.

Table 1

Dermis Hesperidin
(%) SOD activity
(%) DPPH active
(%) ACE
(%) pH General dermis 0.51 35.9 24.0 33 4.18 Mukjinpi 0.59 62.6 24.2 32 4.09

Here, pH value is the value measured after leaching 10g of dermis in 100cc of purified water for 3 hours.

When explaining the SOD Super Oxide Dismutase, free radicals in the metabolism of organic matter are the active oxygen, mainly superoxide or superoxide anion, hydrogen peroxide and hydroxyl radical. It is called oxygen poison because it causes various detoxification. Their free radicals can cause mutations in DNA, leading to cancer, and heart damage to many organs and blood vessel tissues. Preventing cell and connective tissue damage caused by free radicals is the group of enzymes such as superoxide dismutase, glutathione peroxidase (glutathione peroxidase GSH-PX, GPX), catalase and β-carotene, α. Vitamin group, such as carotene, vitamin C, E, folic acid (folic acid) and minerals such as Selenium.

If these enzymes and nutrients are deficient, cancer, genetic disease, or peroxidation progresses to adult disease, and furthermore, it is susceptible to infectious diseases due to a decrease in immunity. I can speak.

In other words, SOD is an antioxidant enzyme that removes harmful oxygen produced in the body as a physiologically active enzyme present in the organs and blood of humans and animals. It is the most powerful and large amount of superoxide radicals produced in the body. It is a very strong enzyme that inhibits activity. Therefore, ingesting a substance that helps S.O.D. or acts like S.O.D. can protect the body from the effects of oxidation, restore fatigue, and prevent aging.

In other words, if active oxygen occurs in the body, it is harmless to SOD enzyme or similar substance (β-carotene, α-carotene, vitamin C, E, folic acid), Selenium, etc. Mineral group) acts to break down superoxide radicals into hydrogen peroxide and oxygen to weaken its oxidizing power, and Catalase finally decomposes hydrogen peroxide into water and oxygen to protect cells from oxidation.

In addition, when explaining DPPH free radical scavenging ability, DPPH (1,1-diphenyl-2-picryl hydrazyl, Sigma, D9132-1G) radical scavenging ability is a water-soluble substance with chemically stabilized free radicals, When it encounters a substance, radicals (DPPHs) disappear and change color, giving off electrons. The antioxidant effects of extracts, beverages and oils, and pure phenolic compounds with various antioxidants can be analyzed.

Therefore, in order to analyze the antioxidant component of the dermis extract, a DPPH quenching experiment was conducted to determine the electron donating ability.

Hereinafter, a method for measuring hesperidin, S.O.D and DPPH free radical scavenging ability in an experiment for the present invention will be described.

First, look at how hesperidin is measured.

The extract used in measuring the hesperidin was used by concentrating 3 hours of hot water extraction at 100 ° C in consideration of the actual Chinese medicine.

That is, 10 g of the sample was subjected to agitation with purified water for 3 hours, concentrated, and extracted with methanol (10 g / 100 mL) again to measure the concentrate by HPLC.

Table 2 below shows the HPLC operating conditions.

[Table 2]

ANIONS column  ODS column Column temperature  35 ℃ Mobile phase  Methanol-water mixture (40:60) flux  1.0 mL / min Inj.Volume  20 uL Detector  Ultraviolet absorption spectrophotometer (SPD-20A, Simadzu, Japan)

Second, S.O.D. If you look at how to measure,

S.O.D was measured using Fluka's SOD determination kit (19160-1KT-F).

Dermis samples were made horizontally and weighed exactly 10 g and extracted three times with distilled water.

The extract was filtered and collected in one place to prevent the inhibition of the polymer material, filtered with Ultra Filter (SK, UF10-106), and collected only low molecular weight substances with molecular weight of 10,000 or less, and concentrated in a rotary vacuum concentrator to pH 8.0 with sodium phosphate buffer. Finally it was applied.

The extract was aliquoted and stored frozen and thawed slowly in the refrigerating chamber one day before the experiment, and diluted to 1.0g / 100ml with distilled water immediately before use.

Distilled water was added instead of the sample as a control, and the absorbance was measured at 450 nm by adding a buffer instead of enzyme, and the average was measured three times each.

S.O.D. Activity inhibition rate was calculated by the following [Equation 1].

[Equation 1]

Where C is the absorbance of the control, Cb is the absorbance of the control blank, S is the absorbance of the sample, and Sb is the absorbance of the blank sample.

Thirdly, how to measure DPPH free radical scavenging ability,

In the experiment of the present invention, the method described by Blosi et al. Was used with a slight modification. In other words, the sample was prepared by extracting with 95% ethanol, and immediately after the experiment was added DPPH solution diluted to 0.4 mM and reacted for 15 minutes at room temperature, the absorbance was measured at 520 nm using a UV spectrophotometer.

In the control group, ethanol was added instead of the sample, and the average was measured twice.

DPPH radical scavenging ability was calculated by Equation 2 below.

&Quot; (2) "

Where A 0 is the absorbance of the control and A 1 is the absorbance of the sample.

In the above experiments on the method of measuring hesperidin, SOD and DPPH free radical scavenging activity in the experiment for the present invention, the following will be described in more detail with respect to the fermentation dermal preparation method according to the present invention with an embodiment of the present invention. Shall be.

The fermented bacteria used in the experiments of the present invention were cultivated by the Korean spawn association and inoculated into the sterilized dermis of the liquid cultured various kinds of Bacillus subtillis, and the fermented dermis, the non-fermented dermis, stored in nature Hesperidin outflow and antioxidant activity (SOD activity, DPPH activity) were examined when mukjin dermis (mukjin dermis) was put into Chinese medicine. Hesperidin effluent was confirmed to be an improved product and enhanced antioxidant power to reach the present invention.

In the experiment of the present invention, the first strain screening was extensively tested for Bacillus subtilis currently used in fermentation of Cheonggukjang, natto fermentation in Japan, soybean fermentation product, and liquid enzyme production, but among them, high temperature strains have good effect of short-term fermentation. It was judged that it would be convenient to use in actual fermentation because it had little rejection in smell or taste and was selected as high temperature strain first.

Secondary strain screening was performed by selecting four high temperature Bacillus subtilises as shown in Table 3 below, which can be distributed to the Korean spawn association.

[Table 3]

Bacillus subtilis BACTERIA One KCCM: 12149 IFO: 14132 New Sale 2 KCCM: 12259 ATCC: 15841 New Sale 3 KCCM: 40938 ATCC: 23059 New Sale 4 KCCM: 70078 ATCC: 33608 New Sale

The above-mentioned Bacillus subtilis were sold by the Korean spawn association and cultivated with liquid shake culture with Nutrient Broth medium (hereinafter referred to as NB medium) in a face flask and inoculated into 121 ° C 30 minutes autoclaved dermis and incubated at 37 ° C for 2 days, followed by drying. It was made into the prototype.

Prototypes were extracted with 100 ° C. for 3 hours and then analyzed for the outflow of S. pyridine, SOD activity, and DPPH activity. The results are shown in Table 4 below.

Table 4

Strain name Hesperidin content (%) S.O.D. activation(%) DPPH activity (%) 1.KCCM 12149 0.55 54.1 25.1 2.KCCM 12259 0.64 61.8 33.6 3.KCCM 40938 0.57 53.5 24.3 4.KCCM 70078 0.58 41.3 26.3 Raw material dermis (fresh dermis) 0.54 35.9 24.0

In general, the results of the KCCM 12259 showed excellent results.

According to the present invention, the method for preparing the dermis according to the present invention inoculates fermented supernatant after inoculating the dried dermis with the tangerine or the related perennial plant of the related plant, and the fermented dermis is prepared. Incubated in, the Bacillus subtilis incubated for 24 to 48 hours at a temperature of 37 ~ 42 ℃ in the liquid medium, the dermis is inoculated with the Bacillus subtilis after heat sterilization for 1 to 5 hours at a temperature of 80 ~ 120 ℃ , After fermentation in a fermentation chamber for maintaining the temperature of 37 ~ 42 ℃ for 1 to 5 days and dried at a temperature of 55 ℃ in a dryer, the experiment as follows <Example 1>, <Example 2> The effect can be confirmed through.

Example 1

Bacteria cultured strain of Bacillus subtilis KCCM-10082 in a Nutrient Broth sieve in vitro medium with 37-42 ° C 24-48 hours shake culture with liquid medium of the following [Table 5] separately at 20-50% 10-80% mixed inoculation in the heat sterilized dermis at 80-120 ° C. for 1-5 hours, incubated for 1-5 days in a fermentation chamber maintained at 37-42 ° C. in a perforated box, and dried at 55 ° C. in a dryer to obtain a product.

[Table 5]

Rice bran 10% Soybean Powder 2% Sodium Phosphate [Na ₂ HPO ₄ ] 2% Ammonium diphosphate [(NH ₄ ) ₂ HPO ₄ ] 0.5% Sodium carbonate [Na ₂ CO ₃ ] 0.5% Defoamer (soybean oil) 0.15% glucose 2% pH 7.0 to 7.4

* Glucose is mixed when inoculated after sterilization

Under the above conditions, the flow rate of S. pyridine, SOD activity and DPPH activity between mukjin dermis and fermented dermis according to the present invention were analyzed and the results are shown in Table 6 below.

Table 6

Analysis Content Hesperidin
(%) SOD activity
(%) DPPH active
(%) Mukjinpi 0.59 62.6 23.2 Fermented dermis 0.84 71.8 43.6

That is, looking at the above results, it is confirmed that the fermented dermis prepared by the method for preparing the fermented dermis according to the present invention is superior in the amount of hesperidin outflow and the antioxidant power (S.O.D. activity, DPPH activity) than mukjin dermis.

Example 2

Storage NB solid test tubes shake cultured at 37-42 ° C 24-48 hours with NB liquid medium and transplanted with the liquid medium of [Table 5] in culture tank to expand and culture 20-50% 80-120 ° C for 1-5 hours The mixture was inoculated into sterilized dermis, put in a perforated box, cultured in a fermentation chamber maintained at 37-42 ° C. for 1-5 days, and dried at 55 ° C. in a dryer to obtain a product.

The effluent, SOD and DPPH activity of the bulk fermented dermis prepared by the method for preparing fermented dermis according to the present invention under the above conditions are shown in Table 7 below.

Table 7

Analysis Content Hesperidin
(%) SOD activity
(%) DPPH active
(%) Bulk fermented dermis 1.1 75.0 44.8

In other words, the results of the fermented dermis manufacturing method according to the present invention to increase the elution amount of hesperidin which is the main component of the dermis when the herbal medicine as the dermis as described above to improve the antioxidant function to enhance the efficacy of the purifier system In addition, the effect on the recovery of fatigue and metabolism can be further enhanced, and the application range of the dermis can be broadened.

The method for preparing fermented dermis described above is only one embodiment for carrying out the present invention, and should not be construed as limiting the technical idea of the present invention. The scope of protection of the present invention is defined only by the matters set forth in the claims below, and the embodiments which have been improved and changed without departing from the gist of the present invention will be apparent to those skilled in the art. It will be said to belong to the protection scope of the present invention.

1 is a molecular formula of hesperidin, a major component of the dermis

Claims (3)

Fermented dermis is prepared by inoculating Bacillus subtilis into the dermis, which is a mature rind of citrus fruits. The Bacillus subtilis, incubated for 24 to 48 hours at a temperature of 37 ~ 42 ℃ in a liquid medium containing rice bran and soybean powder components, The dermis is heat sterilized for 1 to 5 hours at a temperature of 80 ~ 120 ℃ after inoculation of the Bacillus subtilis, fermented for 1 to 5 days in a fermentation chamber maintaining a temperature of 37 ~ 42 ℃ 55 ℃ in a dryer A method for producing fermented dermis, which is dried at a temperature. delete delete

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