KR20230031411A - Method of manufacturing black maekmundong and liquid preparations enhanced steroidal saponin contents and antioxidant activity by fermentation and nine steam and dry - Google Patents

Abstract

The present invention relates to a method for manufacturing Ophiopogon planiscarpus with increased active ingredients of steroid-based saponin, and a method for manufacturing a liquid formulation including the same. More particularly, the present invention relates to: a method for manufacturing original fermented and steamed Ophiopogon planiscarpus with enhanced content of Ophiopogonin, an active ingredient of steroid saponin, and antioxidant activity through five steps of fermentation and aging, and nine steps of steaming and drying Viscum album by using five kinds of natural fruit sugared fermentation liquid; and a method for manufacturing a liquid preparation comprising the same.

Description

Method of manufacturing black maekmundong and liquid preparations enhanced steroidal saponin contents and antioxidant activity by fermentation and nine steam and dry}

The present invention relates to a method for manufacturing black grapefruit with increased active ingredients of steroid-based saponin and a method for manufacturing a liquid formulation including the same, and more particularly, to a five-step fermentation and aging of black grapefruit with five kinds of natural fruit sugar acupuncture fermentation liquid. Method for producing round-shaped fermented and steamed black grapefruit with increased content and antioxidant activity of Ophiopogonin, an active ingredient of steroid saponin, through steaming and drying steps of step 9 and step 9, and a method for manufacturing a liquid formulation including the same will be.

Liriope platyphylla is a perennial evergreen herb belonging to the Liliaceae family.

The height is about 30 to 50 cm, the rhizome is short and thick, and the beard root is thin and long, but some become thick and become tuberous roots.

Leaves are dark green, gathered together at the rhizome, linear, and narrow at the bottom to form a leaf sheath. Flowers bloom in May-June in light purple, and 3-5 flowers are gathered at each node to form raceme inflorescences with a length of 8 to 12 cm.

The fruit is a berry, spherical, and the skin peels off early to expose the black seeds.

When the tuberous root of Macmundong is dried, it becomes translucent light yellow, and it is known to be effective in stopping cough and phlegm, helping the function of the lungs, and boosting energy.

In oriental medicine, it is used as a tonic, expectorant, antitussive, and cardiac medicine.

Macmundong is known to have various functionalities as it contains steroidal saponins such as opiopogonins A, B, C, D, homoisoflavonoids, oligosaccharides and polysaccharides. Anti-inflammatory action, blood sugar lowering effect, brain cell protection action, memory enhancement effect, etc. have been reported.

As such, Maekmundong has various physiological activities, but studies on steroid-based saponins and antioxidants are insignificant.

On the other hand, bellflower containing oleanane saponin contains platycodigenin as an aglycone, platycodins A, C, D, D2, D3, and platycodigenic acid. ) A1~A5, C, polygalacin D, D2, polygalacic acid, and platycodigenic, which account for about 2% of the root.

Bellflower stimulates the gastric mucosa and promotes bronchial secretion, thereby significantly increasing the amount of mucus secretion, thereby exhibiting an expectorant action.

In oriental medicine, it has excellent antitussive, expectorant, analgesic and anti-inflammatory effects, and is widely used for cough and bronchitis as an antitussive and expectorant medicine.

In addition, ginseng containing damaran saponins is mainly composed of Protopanaxadiol and Protopanaxatriol. It is composed of Rb3, Rc, Rd, etc., and Protopanaxatriol is composed of Re, Rf, Rg1, Rg2, Rh1, etc.

As for the pharmacological action of ginseng, it has a strong lymphocyte proliferation action as an immune enhancing action, increases the production of interferon, and remarkably increases the activity of natural killer cells.

As for the action on the cardiovascular system, it lowers heart rate and central venous pressure, normalizes abnormal enzyme activity in ischemic cardiomyocytes, and reduces degenerative changes in cardiomyocytes due to aging.

As for the action on the central nervous system, it stabilizes the mind, stimulates the vomiting center and respiratory center, and shows a central nervous system suppression effect.

Antioxidants are substances that have the ability to slow down, interfere with, or eliminate oxidative damage. In the human body, there are superoxide dismutase (SOD), glutathione peroxidase, and catalase. The same antioxidant enzymes exist, but these alone are incomplete, so the supply of antioxidants supplied from the outside is necessary to maintain health.

Recently, as interest in the relationship between oxidative stress and aging has increased, many studies have been conducted to use antioxidants for aging delay or life extension. However, the relationship between antioxidants, aging, and longevity is controversial, with studies showing that typical antioxidants such as vitamins A, C, and E actually have little effect on delaying aging or extending lifespan, or have adverse effects depending on the dose. It is continuing. It has been reported that this is because the mechanisms related to aging and longevity are not simply influenced by the effects of antioxidants, but because various internal and external factors work.

The present invention repeats the first fermentation, second fermentation, and aging steps of five types of natural fruit fermented fermentation broth with different types of natural fruit fermented fermentation broth, and repeats the steaming and drying steps nine times to obtain It is intended to develop a liquid formulation that significantly increases the efficacy and antioxidant activity of saponins by increasing the content of steroid saponins and combining black bellflower containing oleanane saponins with black ginseng containing damaran saponins.

As for the efficacy of the macmundong containing steroidal saponin, antipyretic action, antitussive action, expectorant action, anti-inflammatory action, blood sugar lowering effect, brain cell protecting action, memory enhancement effect, etc. have been reported. The efficacy of bellflower is excellent in antitussive, expectorant, analgesic and anti-inflammatory effects, and is used for cough and bronchitis as an antitussive and expectorant. It is widely known for its blood circulation improvement and antioxidant effects.

The present invention is to develop various saponin complex liquid formulations in which the antipyretic and anti-inflammatory action of steroid saponins and the antitussive and expectorant action of oleanane saponins are combined with the immunity enhancing and blood circulation improving effects of damaran saponins.

For efficient product development, first, the 1st fermentation, 2nd fermentation and maturation steps were repeated with five kinds of natural fruit fermented liquids in Maekmundong containing steroid-based saponin, and the steaming and drying steps were repeated 9 times to obtain active ingredients. By combining the manufacturing method of fermented and steamed black maltose with a significantly increased content of phosphorus ophiopogonin, black bellflower containing oleanane saponin, and black ginseng extract containing damaran saponin, the effect of saponin is It is to provide a method for producing a fermented and steamed black maltune complex liquid formulation with increased antioxidant activity.

The present invention includes (1) a pretreatment step of putting wheatgrass in a washing tank, washing it with hot water, and drying it in a hot air dryer;

(2) a first fermentation step of fermenting by adding a natural fruit sweetener fermented liquid to the pre-treated Maekmundong;

(3) a secondary fermentation step of fermenting by injecting the natural fruit sugar syrup fermentation liquid back into the macmundong that has gone through the primary fermentation step;

(4) ripening the maekmundong that has undergone the secondary fermentation process at a constant temperature;

(5) Putting the ripening step into a steamer and steaming it through steam;

(6) drying the maekmundong that has undergone the steaming step; It relates to a method for producing circular fermented steamed wheatgrass comprising a.

In addition, the present invention

Heating and extracting water by adding water to the circular fermented and steamed black coriander, black bellflower concentrate, and black ginseng concentrate;

It relates to a method for producing a fermentation-vaporised black maltune complex liquid preparation comprising the step of filtering the extract and concentrating the filtrate in a vacuum concentrator.

The present invention measures and compares the content of opioponin among steroidal saponins contained in dried and fermented fermented black grapefruit,

Measure and compare the DPPH radical scavenging ability of dried and fermented black malts,

After extracting fermented vaporized black maltose with several organic solvents, the DPPH radical scavenging ability in the fractions was measured and compared,

Using Caenorhabditis elegans , the antioxidant enzyme (SOD, Catalase) activity of fermented and steamed black grapes was measured and compared,

Using Caenorhabditis elegans to measure and compare the inhibitory activity of intracellular reactive oxygen species (ROS) accumulation of fermented and distilled black grapes,

Measurement and comparison of oxidative stress resistance of fermented steamed black grapefruits using Caenorhabditis nematodes,

The intensity of SOD-3::GFP fluorescence expression in the transgenic nematodes of fermented nematodes was measured and compared using Caenorhabditis elegans.

The present invention goes through 5 stages of primary fermentation, secondary fermentation and maturation using five kinds of natural fruits such as fig, pear, kiwi, plum, and peach, etc. Through the steaming and drying steps, it is possible to produce a circular fermented and steamed black maltongdong with significantly increased content of Ophiopogonin, an active ingredient of steroid saponin.

In addition, the present invention extracts a complex liquid preparation from the original fermented and steamed black coriander, black bellflower concentrate containing oleanane saponin, and black ginseng concentrate containing damaran saponin, thereby significantly increasing the complex efficacy of saponins. It is possible to provide a method for preparing a complex liquid formulation of gupo black malaria.

1 shows a method for producing fermentation-aged gujeunggupo black maltmundong of the present invention.
Figure 2 shows the peak of the steroid-based saponin opiopogonin of dried rhubarb.
Figure 3 shows the peak of the steroid-based saponin opiopogonin of fermented and steamed black malaria.
4 shows the results of experiments on the effect of dried and fermented black malts on DPPH radical scavenging ability.
Figure 5 is the result of the experiment on the effect on the DPPH radical scavenging ability of several organic solvent-extracted fractions of fermented and evaporated black malt.
Figure 6 is the result of the experiment on the effect of fermented steamed black grapefruit on the increase in the activity of SOD, an antioxidant enzyme in Caenorhabditis elegans.
Figure 7 is an experimental result confirming the increase in the activity of catalase, an antioxidant enzyme in Caenorhabditis nematodes of fermented and evaporated black grapes.
8 is an experimental result of the effect on the accumulation of ROS in C.
Figure 9 is the result of experiments on the effect of fermented steamed black grapefruit on oxidative stress resistance of Caenorhabditis nematodes.
10 is a result of observing the expression intensity of fluorescence after culturing a sample of CF1553 including SOD-3::GFP as a nematode transformed with fermented and evaporated black vein extract in a culture medium prepared for each concentration.
11 and 12 are the manufacturing process of fermentation-aged Gujeung Gupo Black Maltmundong and the manufacturing process of the liquid composite preparation.

Hereinafter, a preferred implementation process of the present invention will be described with reference to the drawings. However, the scope of the present invention is not limited to the description or drawing of the following implementation process. That is, terms used in this specification are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly dictates otherwise. In addition, terms such as "include" or "having" described in this specification are intended to designate that there is a feature, number, step, operation, component, part, or combination thereof described in the specification, but one or the other It should be understood that the above does not preclude the possibility of the presence or addition of other features, numbers, steps, operations, components, parts, or combinations thereof.

1 shows a method for producing circular fermented steamed black maltose of the present invention.

Referring to FIG. 1 , the method for manufacturing black malt bean sprouts of the present invention includes a pretreatment step, a primary fermentation step, a secondary fermentation step, a maturation step, a steaming step, and a drying step.

The method for manufacturing circular fermented steamed black maltongdong of the present invention is five kinds of natural fruit fermented ripening liquids. The first fermentation, the second fermentation, and the aging step may be repeated 4 or more 6 times, respectively, and preferably 5 times.

Subsequently, the steaming and drying steps may be repeated 8 or more 10 times, preferably 9 times.

The (1) pretreatment step is a step of putting the wheatgrass in a washing machine, washing it with hot water, and then putting it in a hot air dryer and drying it.

For example, in the (1) pretreatment step, after putting the root of the rhubarb in a washing machine and washing it three times with hot water at 40 to 60 ° C, transferring it to a hot air dryer and drying it at 50 to 70 ° C for 4 to 6 hours, a cold air dryer It may include a step of drying with cold air at 10 to 20 ° C. for 12 to 24 hours after putting in.

The (2) primary fermentation step is a step of fermenting by adding a natural fruit sugar syrup fermentation liquid to the pretreated Maekmundong.

The natural fruit sugar syrup fermentation liquid is a fruit enzyme liquid prepared by fermenting after adding sugar to any one fruit of fig fruit, pear, kiwi, plum, and peach.

In the present invention, a natural fruit fermented fermented liquid made from any one of the fig fruit, pear, kiwi, plum, and peach can be used, preferably three or more natural fruit fermented liquids, preferably four or more. , More preferably, all five natural fruit fermented liquids can be used.

For example, the fermented fig fermented liquid is mixed evenly by adding 10 to 14 kg of refined sugar to 8 to 12 kg of figs, and then placed in a traditional pottery, sealed, and exposed to direct sunlight. Fermented for more than 3 months in a cool and well-ventilated place After that, filter through cotton, take the filtrate, mature it in a storage tank at 20~25℃ for more than 3 months, filter it through cotton again, and store it in a glass container.

For example, the pear fermentation ripening liquid is mixed evenly by adding 10 to 14 kg of brown sugar to 8 to 12 kg of pears, and then placed in a traditional pottery, sealed, and exposed to direct sunlight. Fermented for more than 3 months in a cool and well-ventilated place After that, filter through cotton, take the filtrate, mature it in a storage tank at 20~25℃ for more than 3 months, filter it through cotton again, and store it in a glass container.

For example, the kiwi fermented liquid is mixed evenly by adding 10 to 14 kg of refined sugar to 8 to 12 kg of kiwi, and then placed in a traditional pottery, sealed, and exposed to direct sunlight. Fermented for more than 3 months in a cool and well-ventilated place After that, filter through cotton, take the filtrate, mature it in a storage tank at 20~25℃ for more than 3 months, filter it through cotton again, and store it in a glass container.

For example, the plum fermentation broth is mixed evenly by adding 10 to 14 kg of brown sugar to 8 to 12 kg of plums, and then sealed in a traditional pottery, and then exposed to direct sunlight. Fermented for more than 3 months in a cool and well-ventilated place After that, filter through cotton, take the filtrate, mature it in a storage tank at 20~25℃ for more than 3 months, filter it through cotton again, and store it in a glass container.

For example, the peach fermented ripening liquid is mixed evenly by adding 10 to 14 kg of refined sugar to 8 to 12 kg of dog peaches, and then placed in a traditional earthenware pot and sealed, and then stored in a cool and well-ventilated place without direct sunlight for 3 months. After over-fermentation, filter through cotton, take the filtrate, mature in a storage tank at 20-25 ° C for more than 3 months, filter again through cotton and store in a glass container.

The (2) primary fermentation step is a step of fermenting by adding a natural fruit sugar syrup fermentation broth to the pretreated walnut.

The (3) secondary fermentation step is a step of injecting and fermenting the fermented liquid from the natural fruit sugar needles again to the macmundong that has passed through the primary fermentation step.

The (4) aging step is a step of aging the maekmundong that has undergone the secondary fermentation process at a constant temperature.

In the present invention, (2) primary fermentation, (3) secondary fermentation, and (4) aging may be performed using the prepared natural fruit syrup fermentation broth.

More specifically, the (2) primary fermentation step to (4) aging step

(a) adding or spraying any one of the natural fruit sugar needle fermented liquids to the maltberry to perform the (2) primary fermentation step, (3) secondary fermentation step, and (4) aging step;

(b) Adding or spraying the remaining one of the natural fruit sugar needle fermentation liquid to the wheatgrass that has gone through the step (a), the (2) primary fermentation step, (3) secondary fermentation step, and (4) aging step The step of proceeding, and

Among the natural fruit sugar needle fermented liquids, up to the last matured liquid is sequentially added to or sprayed to Maekmundong that has undergone the previous (2) 1st fermentation step to (4) ripening step, so that the above (2) 1st fermentation step, (3) 2 It may include a step of proceeding with a tea fermentation step and (4) aging step.

In the case of using all the five natural fruit fermented liquids prepared above, for example, using fig fermented liquid, (2) 1st fermentation step, (3) 2nd fermentation step, and (4) ripening step are performed. (1st time), and then, by adding or spraying pear fermented liquid to the fermented and aged fig fermented liquid, the (2) primary fermentation step, (3) secondary fermentation step and (4) aging Proceed through the steps (2nd time).

In addition, by adding or spraying kiwi fermented liquid to the fermented and aged macmundong for the second time, the (2) first fermentation step, (3) second fermentation step, and (4) aging step are performed (third time), and So, by adding or spraying plum fermented liquid to the fermented and aged Maekmundong for the 3rd time, the (2) 1st fermentation step, (3) 2nd fermentation step and (4) aging step are performed (4th time), 4 The (2) 1st fermentation step, (3) 2nd fermentation step and (4) aging step are performed by adding or spraying dog peach fermented liquid to the fermented and aged Maekmundong (5th time),

The first fermentation step is a step of fermenting at a constant temperature after immersing and infiltrating fermented fig fermented liquid, pear fermented matured liquid, kiwi fermented matured liquid, plum fermented matured liquid and dog peach fermented matured liquid in sequence.

For example, in the first fermentation step, 10 kg of the pre-treated Mammundong is spread out on 5 trays, and 2L of a natural fruit fermented liquid (eg, fig fermented liquid) is added to each tray, and the temperature is 20 ~ 35 ~ 45 ℃. It can be fermented for 24 hours (by soaking and infiltrating the aging liquid).

In addition, for the secondary fermentation, 1L of the fermented fermentation liquid (fig fermentation liquid) is sprayed on Maekmundong after the primary fermentation, and fermented at 35~45℃ for 20~24 hours.

Singgi ripening step is a step of storing the first and second fermentation processes at a certain temperature (eg, 15 ~ 25 ℃) for 48 hours.

The steaming step is a step of steaming the maekmundong that has passed through the aging step by putting it in a steamer and passing steam therethrough.

The first fermentation, the second fermentation, and the maturation step are put into a pressurized steam passage device and steamed for 1 to 2 hours with steam at 100 to 110 ° C., the steam passing in the temperature range is 12 to 22p. s.i, and preferably a pressure of 14.65 p.s.i (100° C.) to 20.76 p.s.i (110° C.) is required.

In the steaming step, if the temperature is lower than the temperature range, the steaming of the brown rice is impossible, and if the temperature is higher than the temperature, the active ingredient of the brown rice may be decomposed.

The drying step is a step of gradually drying the wheat moondong that has gone through the steaming step.

In the drying step, the steamed wheatgrass is put in a hot air dryer and dried first by passing hot air at 60 to 80 ° C for 18 to 24 hours, and then secondarily dried by passing cold air at 10 to 20 ° C for 6 to 8 hours. do.

The present invention can maintain the original form of black maltongdong as it is by repeating the steaming step and the drying step 9 times to gujeunggupo.

Unlike the present invention, when the steamed black grapefruit is quickly dried at a high temperature of 115 ° C. or higher, the original shape of the black grapefruit is not maintained, the shape is distorted, and the active ingredient may be decomposed.

In the present invention, the primary fermentation, secondary fermentation and aging steps may be repeated 5 times, and the steaming and drying steps may be repeated 9 times.

In the present invention, after 5 times of primary fermentation, 2nd fermentation and maturation, the content of active ingredients increases rapidly through repeated 9 times of steaming and drying, and when repeated 9 times, the maximum efficiency of active ingredients is obtained. You can get it.

When repeated more than 10 times, the content of the active ingredient no longer increases, but rather decomposition can be found.

In the present invention, the method for producing a fermented and evaporated black maltune complex liquid formulation is firstly, the steps of putting the circular fermented steamed black maltongdong, gujeunggupo black bellflower, and gujeunggupo black ginseng into a herbal medicine extraction device, adding purified water to heat extraction, filtering the extract, and obtaining a filtrate It includes the step of putting in a vacuum concentrator and concentrating.

The weight ratio of the prototype fermented and steamed black maltongdong, gujeunggupo black bellflower, and gujeunggupo black ginseng is 1: 1 to 2; It may be 0.5 to 1.

For example, 8-12kg of fermented, fermented black malt, 12-18kg of Gujeunggupo black bellflower, and 6-8kg of Gujeunggupo black ginseng are put into an extraction container, heat-extracted at 90-100°C for 4-6 hours, and then filtered through cotton. there is.

Preferably, 10 kg of round fermented steamed black maltongdong, 15 kg of Gujeunggupo black bellflower, and 7kg of Gujeunggupo black ginseng are put into an extraction container, heated and extracted, and filtered through cotton.

The filtrate is taken and put in a vacuum concentrator and concentrated under reduced pressure at 60 to 80 ° C. for 4 to 6 hours to obtain a fermented vaporized black malt brown complex concentrate (40 to 60 brix).

After mixing fructooligosaccharide and black garlic concentrate with the fermented and evaporated black maltberry complex concentrate, 15g of each is filled into a polyethylene liquid stick and sealed to prepare a liquid formulation.

Experimental Example 1. Mcmundong analysis

1) Sample pretreatment process

Crush the fermented and steamed black grapefruit, take 1g of the powder, put it in a 15mL test tube, add 10mL of methanol (99.99%), put it in a sonicator, and pass ultrasonic waves for 10 minutes.

After passing ultrasonic waves, 5mL of the sample was taken in a centrifuge tube, centrifuged at 13,000 rpm for 10 minutes, and 1mL of the supernatant was taken to prepare a fermentation sample solution.

A control solution was obtained by operating the same method as the conjunctivitis (Comparative Example 1), which had not been separately fermented and thickened.

2) Analyzer

Device name: Mass spectrometer (MS QQQ Mass Spectrometer model 6410B, Agilent Technologies Co., USA)

Column: Phenomenex Kinetex 2.6um C8 100A 50x2.1mm

3) Analysis conditions

Mobile solvent A: 0.1% formic acid (distilled water)

Moving solvent B: 0.1% formic acid (acetonitrile)

Maintenance time: 20 minutes

4) Analysis result

A sample obtained by fermenting and distillation of dry grapefruit was pulverized, and 1 g of powder was added to 10 mL of methanol, passed through ultrasonic waves, and centrifuged. Mass Spectrometer), then A solvent (0.1% formic acid (distilled water)) and B solvent (0.1% formic acid (acetonitrile)) were programmed at an appropriate ratio for 20 minutes to compare the areas of the peaks that appeared before and after fermentation. was compared.

Table 1 shows the component areas and content ratios of opiopogonins A, B, C, and D, which are the active ingredients of Examples (fermented steamed black coriander) and Comparative Example (general corticosteroids).

sample
ingredient dried maekmundong Fermentation and expansion black malt mundong peak area µg/g peak area µg/g Opiopogonin A ND - ND Opiopogonin B ND - 6.1 4.78 Opiopogonin C ND - ND Opiopogonin D 24.32 19.07 100.0 78.41

Referring to Table 1, among the opiopogonin components of dried rhizomes, A, B, and C were not detected, and opiopogonin D was 19.07 μg/g, whereas opiopogonin B was 4.78 μg/g in fermented and fermented black malt buds. A small amount was detected, and opiopogonin D was found to be 78.41 μg/g, which was more than 4 times amplified compared to the dry rhizomatous sinus.

Experimental Example 2. Measurement of DPPH radical scavenging ability

1) Experiment method

Dried and fermented wheatgrass were extracted with water, and freeze-dried samples were prepared for each concentration using ethanol as a solvent in a 96-well plate, and 0.2 mM 1,1-diphenyll-2-picrylhydrazyl ( 1,1-diphenyl-2-picrylhydrazyl, DPPH) ethanol solution was added in a certain amount. After shaking for 10 seconds, the mixture was allowed to stand at 25 ^° C. for 30 minutes, and absorbance was measured at 517 nm using a microplate reader. The control drug was measured using vitamin C (L-ascorbic acid), and the measurement was repeated three times for each sample.

2) Experimental results

Referring to FIG. 4, the DPH radical scavenging effect was more than twice as strong in the fermentation-vaporised black maltsong compared to the dried malts.

Referring to FIG. 5, among the fractions of fermented and distilled black grapefruit, the methylene chloride fraction and the ethyl acetate fraction showed strong activity in a concentration-dependent manner, and the ethyl acetate fraction showed slightly better results.

Experimental Example 3. Effect of increasing antioxidant enzyme (SOD, Catalase) activity in the body of nematodes

1) Experiment method

N2 nematodes of the same growth stage as the water extract of fermented steamed black grapefruit were cultured on a plate containing the composition at each concentration (0 μM, 250 μM, 500 μM).

For SOD activity, nematodes were collected on the 4th day after becoming adults, washed three times with M9 buffer, and then ground and used to measure enzyme activity.

First, a reaction mixture [1.6 mM xanthine and 0.48 mM nitroblue tetrazolium (NBT) 0.49 mL] was prepared using 10 mM phosphate buffer (pH 8.0) as a solvent, and then pre-prepared for 5 minutes at 37 ° C with 10 μL of sample. cultured.

Thereafter, 1 mL (0.05 U/mL) of xanthine oxidase was added and incubated at 37° C. for another 20 minutes, and then the reaction was stopped with 69 mM SDS and absorbance was measured at 570 nm.

Catalase activity was determined by the Aebi method (Method Enzymol. 105: 121-126), followed by reacting 50 μL of the sample with 25 mM H ₂ O ₂ for 3 minutes and measuring the absorbance at 240 nm.

2) Experimental results

Referring to Figures 6 and 7, the activity of SOD was measured using superoxide anion generated during the enzymatic reaction of Xanthine oxidase. As a result, the group administered with the ethyl acetate fraction of C. made it On the other hand, catalase activity was found to increase by 128%.

Experimental Example 4. Reactive oxygen species (ROS) accumulation inhibitory activity in nematode cells

1) Experiment method

N2 nematodes of the same growth stage of the fermented and fermented extracts of Zephyr vulgaris were cultured on a plate containing the composition at each concentration (0 μM, 250 μM, 500 μM).

Reactive oxygen species (ROS) in nematode cells were measured using 2',7'-dichlorodihydro fluorescein diacetate (H2DCF-DA). On the 4th day after adulthood, they were placed in M9 buffer containing 50 μM zuglon and exposed for 2 hours, and then transferred to 50 μL M9 buffer in a 96-well plate in groups of 5. Finally, after adding 50 μL of 25 μM H2DCF-DA, the absorbance was measured at 485 nm for excitation and 535 nm for emission.

2) Experimental results

Referring to FIG. 8, in the fermentation-enhanced black rhododendron extract, ROS accumulation in the 250 μM treatment group was reduced by 6.71% (p <0.05) and ROS accumulation in the 500 μM treatment group was reduced by 14.67% (p <0.01), indicating that ROS accumulation in nematode cells was reduced. It was found to be significantly suppressed.

Experimental Example 5. Measurement of oxidative stress resistance increasing efficacy

1) Experiment method

Nematodes of the same growth stage were cultured on the plate for each concentration of the fermented and steamed black lentil extract (EDZ 250, 500 μg/mL). Resistance to oxidative stress was evaluated by slightly modifying the existing method. On the 7th day after becoming an adult, the nematodes were temporarily transferred to a 96-well plate containing M9 buffer containing 1 mM juglone and the survival rate was measured over time. Confirmed.

2) Experimental results

Referring to FIG. 9, the effect of fermented vaporized ethyl acetate fraction on the survival rate of nematodes induced by oxidative stress with juglone was that the maximum survival time of the control nematode was 25 hours, but the ethyl acetate fraction 500 μg / mL concentration increased the survival time to 30 hours. The average survival time of the control group was 11.5±0.7 hours, but the 500 μg/mL concentration treatment group extended the average survival time to 16.6±0.9 hours by 44.3%.

Experimental Example 6. Measurement of SOD-3::GFP fluorescence in transgenic nematodes

1) Experiment method

CF1553 samples containing SOD-3::GFP as nematodes transformed from the fermented and steamed black rhombus extract were cultured in a medium prepared for each concentration. It was used on the 4th day after becoming an adult. The nematodes were anesthetized with sodium azide (4%), and GFP expression was observed under a fluorescence stereomicroscope (Olympus, Japan). In order to quantify and analyze the expression intensity, photographs were taken using a microscope and analyzed using ImageJ software, and all experiments were repeated three times.

2) Experimental results

Referring to FIG. 10, in order to determine whether the protein for resisting oxidative stress in the nematodes of the fermented and distilled ethyl acetate fraction was tested using the transformed nematode CF1553 containing SOD-3, CF1553 transformation In the nematodes treated with 500 μg/mL of fermented nematode ethyl acetate fraction, the expression rate of SOD-3::GFP was 11.7% higher than that of the untreated nematodes.

In the above description, various embodiments of the present invention have been presented and described, but the present invention is not necessarily limited thereto. It will be readily apparent that branch substitutions, modifications and alterations are possible.

Claims (6)

(1) a pretreatment step of putting the wheatgrass in a washing tank, washing it with hot water, and drying it in a hot air dryer;
(2) a first fermentation step of fermenting by adding a natural fruit sweetener fermented liquid to the pre-treated Maekmundong;
(3) a secondary fermentation step of fermenting by injecting the natural fruit sugar syrup fermentation liquid back into the macmundong that has gone through the primary fermentation step;
(4) ripening the maekmundong that has undergone the secondary fermentation process at a constant temperature;
(5) Putting the ripening step into a steamer and steaming it through steam;
(6) drying the maekmundong that has undergone the steaming step; A method for producing circular fermented steamed black maltose comprising a. The method of claim 1, wherein the natural fruit syrup fermented liquid is
A method for producing circular fermented steamed black maltose, characterized in that it is a fruit enzyme liquid prepared by adding sugar to any one of fig fruit, pear, kiwi, plum and peach and then fermenting it. The method of claim 2, wherein the (2) primary fermentation step to (4) aging step
(a) performing the (2) primary fermentation step, (3) secondary fermentation step, and (4) ripening step by adding or spraying any one of the natural fruit sugar needle fermentation liquid to the maltberry;
(b) Adding or spraying the remaining one of the natural fruit sugar needle fermentation liquid to the wheatgrass that has gone through the step (a), the (2) primary fermentation step, (3) secondary fermentation step, and (4) aging step to proceed; and
Among the natural fruit sugar needle fermented liquids, up to the last matured liquid is sequentially added or sprayed to Maekmundong that has undergone step (b) to proceed with the (2) 1st fermentation step, (3) 2nd fermentation step and (4) ripening step A method for producing circular fermented vaporized black maltose, characterized in that it comprises the step of. The method of claim 1, wherein the steaming step is a step of steaming for 1!2 hours with steam having a temperature of 100 to 110 ° C and a pressure in the range of 12 to 22 psi. The method according to claim 1, wherein the steaming step (5) and the drying step (6) are repeated nine times. Heat-extracting by adding water to the circular fermented and steamed black maltose, black bellflower concentrate, and black ginseng concentrate prepared according to any one of claims 1 to 5;
A method for producing a fermentation-vaporized black maltose complex liquid preparation comprising the step of filtering the extract and concentrating the filtrate in a vacuum concentrator.

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