KR101017594B1 - Method for preparing fermented onion extract with increased quercetin content - Google Patents

Abstract

The present invention relates to a method for producing a fermented onion extract with increased quercetin content, after washing the onion to remove the husk, onion pretreatment step (first step) to grind to a size of 0.5 ~ 10mm; An ethanol extraction step (second step) of mixing 95% concentration of ethanol at a ratio of 1: 5 to 10 with respect to weight of the onion pretreated in the first step and extracting at 50 to 90 ° C. for 3 to 10 hours; A filtration and drying step (third step) of filtering and drying the extract extracted from ethanol in the second step; A buffer mixing step (step 4) of mixing and suspending 100 mM citric acid buffer solution to the dried and filtered products in the third step; In the fourth step is characterized in that the fermentation step (5th step) consisting of inoculating the mesobacteria solution to the suspension mixed with the buffer solution.
Fermented onion extract with increased quercetin content prepared by the above method is fermented onion or onion extract to increase the content of quercetin (quercetinquercetin) in the edible portion of the onion, which has the antioxidant, brain cell protective activity, and anti-inflammatory action of quercetin. To prepare onions or onion extracts with increased various health functionalities, such as anti-cancer action, anti-diabetic, anti-obesity, anti-hypertension, etc., and provides a composition related to foods, health functional foods, natural medicines, etc. containing them can do.

Description

Preparation of fermented onion extract with increased quercetin content {PREPARATION OF FERMENTED ONION CONTAINING INCREASED QUERCETIN CONTENT}

The present invention relates to a method for producing a fermented onion extract with increased quercetin content, and more specifically, an onion pretreatment step of washing and crushing the onions and removing the husk, and mixing 95% concentration of ethanol to the pretreated onions. Ethanol extraction step of extraction, filtration and drying step of filtering and drying the ethanol extracted extract, buffer mixing step of suspending 100 mM citric acid buffer in the filtered and dried product, and a suspension of the buffer mixture It consists of a fermentation step of inoculating the mesobacteria solution.

The present invention fermented onion and onion ethanol extract with microorganisms or enzyme solution obtained from microorganisms to increase the content of quercetin, which is an active ingredient of antioxidant and brain cell protective activity of quercetin, which is low in the edible portion of onion, increasing the content of quercetin It relates to a method for producing increased fermented onions, or fermented onion extracts, and to compositions containing the same, foods, health functional foods, natural medicines, and the like.

Onion is a genus of two years belonging to the family Liliaceae, the diameter of which reaches 10cm in diameter and is spherical or spherical. In September, a large inflorescence grows at the end of the flower garden, and many flowers with sack run in the form of a mountain. In addition, according to the statistics of the Ministry of Food, Agriculture, Forestry and Fisheries (2008 Facility Vegetable Greenhouse Status and Vegetable Production Performance), onions are the top domestic producers of seasoned vegetables (about 1 million tons) and twice the onions (500,000 tons). This is a very important agricultural product.

The ingredients of onions are quercetin glycosides such as quercetin 3-O-glucoside, quercetin 3,4'-diglucoside, and quercetin 4'-glucoside.Sulfides such as propyl allyldisulfide have a spicy taste. It appears to contain a large number of aryl compounds. Quercetin is rarely contained in the edible site, but is contained in a large amount of brown husk (about 8.3 mg per gram of dry matter), and its content is known to be tens to hundreds of times more than the edible site. quercetin) is a compound that has been reported to exhibit various physiological activities such as anti-inflammatory action, brain cell protective activity, anti-cancer action, anti-diabetic, anti-obesity, anti-hypertension.

Fermentation is a phenomenon in which microorganisms decompose or change organic substances with their enzymes to produce unique final products. In a narrow sense, fermentation refers to a process consisting of a complex reaction system in which carbohydrates are anaerobicly decomposed. Alternatively, it may be said that the microorganism secretes various enzymes to oxidize, reduce or decompose and synthesize an organic compound.

The fermented foods include rice wine made by yeast mold, distilled alcoholic beverages (brandy, rum, whiskey, vodka, gin soju), vinegar made with acetic acid bacteria, wine and beer made with yeast, bread made with yeast bread. There are soybean paste, soy sauce and red pepper paste, kimchi made with lactic acid bacteria, and western cheese, butter and yogurt. Koreans also developed Jang, Kimchi, salted fish, vinegar, Sikhye, and liquor in compliance with the natural environment. There are about 100 kinds of kimchi, 160 kinds of salted seafood, 120 kinds of jang, 10 kinds of vinegar, and 260 kinds of liquor. Fermentation products come out when fermented food extracts, etc. There are cases where there are more specific nutrients than the original food. In addition, soy isoflavones contain about 1.5 ~ 2.5% of dry weight. Among them, glycosides (glycoside, sugar-linked form), daidzin and genistin, account for 60 ~ 70% of the total isoflavones. have. On the other hand, the content of aglycone (glycoside-free form) compound, daidzein and genistein, is known to be about one-third to one-third of each glycoside. In anti-mutation experiments using extracts of various soybean processed foods, the extracts of fermented foods showed much higher antimutagenicity than non-fermented foods, and the degree of anti-mutation increased in proportion to the aglycone content of isoflavones in the foods. Antioxidant activity in conventional meju and doenjang also increases in proportion to the fermentation period, suggesting that the production of antioxidant active substances during fermentation increases. In addition, the inhibitory effect of angiotensin converting enzyme (ACE) activity and immunopotentiation activity have been reported to increase significantly in soybeans produced by traditional fermentation, suggesting the importance of fermentation. It is thought that the most likely reason is that the glycoside is decomposed into the active aglycone form by the hydrolase. In fact, a report from the Ministry of Agriculture has shown that during fermentation, glycosides are converted into non-sugar-forming compounds with increased functionality and bioavailability.

However, there is no production of onion and onion extract with increased health functionalities that can increase the content of quercetin (quercetin) showing a variety of health functional physiological activity through fermentation.

The present invention has been invented to solve the above problems, the object is to increase the content of quercetin (quercetin) is a scientifically edible portion of the onion using edible bacteria, or enzyme liquid derived therefrom It provides a composition related to onion, or onion extract with increased content of quercetin as an active ingredient of antioxidant activity and brain cell protective activity, and foods, health functional foods, and natural products containing the same.

In order to achieve the above, the method of preparing a fermented onion extract with increased quercetin content according to the present invention, after removing the outer skin by washing the onion, onion pre-treatment step of grinding to a size of 0.5 ~ 10mm (first step) Wow; An ethanol extraction step (second step) of mixing 95% concentration of ethanol at a ratio of 1: 5 to 10 with respect to weight of the onion pretreated in the first step and extracting at 50 to 90 ° C. for 3 to 10 hours; A filtration and drying step (third step) of filtering and drying the extract extracted from ethanol in the second step; A buffer mixing step (step 4) of mixing and suspending 100 mM citric acid buffer solution to the dried and filtered products in the third step; In the fourth step is characterized in that the fermentation step (5th step) consisting of inoculating the mesobacteria solution to the suspension mixed with the buffer solution.

In addition, in the fermentation step of the fifth step, the meju bacteria solution,

Bran and 1% glucose solution in a ratio of 1: 1 to 3 by weight and sterilized for 10-30 minutes at 100 ~ 140 ℃ ( Aspergillus kawachii ) inoculated and incubated for 3-7 days at 25 ~ 35 ℃, mixed with 100mM sodium phosphate buffer in a ratio of 1: 1 to 3 by weight to the fermented wheat bran to stand 3 ~ 7 hours at 3 ~ 5 ℃ Then, by centrifugation for 10 to 30 minutes at a speed of 10,000 ~ 12,000rpm characterized in that it was prepared by obtaining a supernatant.

In addition, in the fermentation step of the fifth step, inoculated at a ratio of 1: 1 to 100 by weight of the mesobacteria solution in the suspension of the buffer mixture is characterized in that the fermentation at 10 ~ 50 ℃ for 12 to 36 hours.

According to the fermented onion extract with increased quercetin content according to the present invention, ferment the onion or onion ethanol extract to increase the content of quercetin which is low in the edible portion of onion, antioxidant, brain cell protection of the quercetin Onion or onion extract with increased various health functionalities, such as increased activity, anti-inflammatory action, anti-cancer action, anti-diabetic, anti-obesity, anti-hypertension, etc. can be prepared, foods containing the same, health functional foods, new natural products It has the effect of providing a composition associated with the back.

1 is a graph comparing the antioxidant effect of the onion extract before and after fermentation by DPPH.
Figure 2 is a graph comparing the neuronal neuroprotective effect of onion extract before and after fermentation by MTT method.
Figure 3 is a graph showing the results of high performance liquid chromatography for onion extract before and after fermentation.
Figure 4 shows the structure of the quercetin compound present in onion and onion extract after fermentation.
Figure 5 is a graph showing the change of the amount of quercetin compound with time when the onion extract fermented with mesobacterial liquid.
Figure 6 is a graph showing the neuronal neuroprotective effect of the compound produced by fermentation.
7 is a graph comparing the quercetin content in the commercially matured onion with the quercetin content in the fermented onion extract according to the present invention.
8 is a graph showing a calibration curve of quercetin, quercetin 3-glucoside, quercetin 4'-glucoside, and quercetin 3,4'-diglucoside in onion extracts before and after fermentation.

Hereinafter, the manufacturing method of the present invention according to the accompanying drawings in detail as follows.

Figure 9 is a flow chart schematically showing a method for producing a fermented onion extract with increased quercetin content according to the present invention.

1. Onion Pretreatment Step (Step 1)

Onion pre-treatment step is to wash the onion to remove the outer skin and then crushed,

After washing the onion to remove the outer skin, it is ground to a size of 0.5 ~ 10mm.

The onion contains quercetin glycoside, quercetin 3-O-glucoside, quercetin 3,4'-diglucoside, quercetin 4'-glucoside, etc., and has a spicy taste of aryl sulfide such as propyl allyldisulfide. It contains a large number of compounds, the quercetin (quercetin) exhibits various physiological activities such as anti-inflammatory action, brain cell protective activity, anti-cancer action, anti-diabetic, anti-obesity, anti-hypertension.

And, after washing the onion to remove the outer husk, crushing to a size of 0.5 ~ 10mm is to allow the ethanol to penetrate smoothly into the onion from which the outer husk is removed.

If, after washing the onion to remove the outer husk, when crushed to less than 0.5mm size is economical because the same yield and activity is obtained when crushing to 0.5 ~ 10mm, when crushing to a size exceeding 10mm It may be difficult for ethanol to penetrate smoothly into onions from which the husks have been removed.

2. Ethanol Extraction Step (Second Step)

Ethanol extraction step is a step of extracting by mixing the ethanol to the onion pretreated in the first step,

The ethanol of 60 ~ 99% concentration in the pre-treated onion in the first step is mixed at a ratio of 1: 5 to 10 by weight to extract for 3 to 10 hours at 50 ~ 90 ℃.

 Here, the ethanol of 60 ~ 99% concentration in the pre-treated onion in the first step is mixed at a ratio of 1: 5 to 10 by weight to extract for 3 to 10 hours at 50 ~ 90 ℃ pre-treatment in the first step This is to ensure that the quercetin in the onions is sufficiently eluted.

In addition, the extraction of the onion pretreated in the first step with ethanol may not only increase safety when ethanol is used for extraction, but also may be suitable for use as an additive for food, medicine, and cosmetics because it is harmless to humans. This is because the cost increase such as strict process control to secure does not occur, so it is economical.

And, if the ethanol of 60 ~ 90% concentration in the pre-treated onion in the first step is mixed at a ratio of less than 1: 5 by weight, quercetin contained in the pre-treated onion in the first step It may not be sufficiently eluted, when mixing the ethanol of 60 ~ 90% concentration in a ratio of more than 1:10 by weight to the onion pretreated in the first step, quercetin contained in the onion pretreated in the first step Using more alcohol than needed to elute quercetin is uneconomical and takes longer to extract.

And, if the onion is pre-treated in the first step 60 to 90% of ethanol in a concentration of 1 to 5 to 10% by weight of the mixture is extracted at less than 3 ℃ 50 ℃, the pre-treatment in the first step Quercetin contained in onions may not be sufficiently eluted, and when extracted at 90 ° C. for more than 10 hours, quercetin contained in onions pretreated in the first step may be sufficiently eluted. It is uneconomical to consume more heating temperature and heating time than necessary.

And preferably, the mixture of 95% ethanol in a ratio of 1: 6 to the weight of the onion pretreated in the first step is to extract for 5 hours at 70 ℃.

Here, the 95% concentration of ethanol in a ratio of 1: 6 to the weight of the onion pretreated in the first step is extracted for 5 hours at 70 ℃ to elute quercetin (quercetin) contained in the optimal onion This is because the alcohol concentration, alcohol content, heating temperature, and heating time were derived through repeated experiments.

At this time, the ethanol of 60 ~ 99% concentration in the pre-treated onion in the first step is mixed at a ratio of 1 to 5 to 10 by weight, and extracted for 3 to 10 hours at 50 ~ 90 ℃ is called an extract.

3. Filtration and drying step (3rd step)

Filtration and drying step is to filter and dry the ethanol extracted extract in the second step,

After filtering the ethanol extracted extract in the second step, the filtered filtrate is dried.

More specifically, after filtering the extract extracted ethanol in the second step, the filtered filtrate is dried in a rotary pressure reducer.

Here, after filtering the extract extracted ethanol in the second step, drying the filtered filtrate with a rotary pressure reducer is contained in the extract, without losing the activity of the effective components contained in the extract This is to reduce the ethanol content as much as possible.

In addition, the rotary decompression thickener is generally used in the art, and is not limited to the type, and uses a freely set temperature and pressure.

In this case, after filtering the extract extracted ethanol in the second step, the filtered filtrate is called dry matter.

4. Buffer Mixing Step (Step 4)

The buffer mixing step is a step of suspending by mixing the dried and filtered dried in the third step in the buffer,

In the third step, 100 mM citric acid buffer (pH 4.6) is mixed and suspended in the filtered and dried product.

Preferably, the mixture is suspended by mixing 100 mM citric acid buffer (pH 4.6) in a ratio of 1 to 1000 by weight to the dried and filtered dried in the third step.

In addition, the citric acid (citric acid) is a weak organic acid mainly found in the fruit of the tangerine, as a natural preservative, pH adjusters, browning prevention, used to add acidic or sour taste to food or drink.

Here, mixing and suspending 100 mM citric acid buffer (pH 4.6) in a ratio of 1 to 1000 by weight to the dried and filtered dried in the third step is the pH of the dried and filtered dried in the third step This is to maintain the 4 to 5, to further facilitate the fermentation in the following fermentation step.

In addition, when 100 mM citric acid buffer (pH 4.6) is mixed at a ratio of less than 1: 1 by weight to the dried product filtered and dried in the third step, 100 mM citric acid buffer solution in the dried and filtered water in the third step. The mixture may not be evenly mixed to maintain the pH, and may not proceed smoothly in the following fermentation, and 100 mM citric acid buffer (pH 4.6) is added to the weight of the dried 1: filtered and dried in the third step. In the case of mixing with more than 1000, it is uneconomical to use more than necessary to evenly mix 100 mM citric acid buffer in the dried and filtered dried in the third step.

And more preferably, in the dry and filtered in the third step, 100 mM citric acid buffer (pH 4.6) is mixed and suspended in a ratio of 1: 30 to 35 to the weight.

Here, in suspension by mixing 100mM citric acid buffer (pH 4.6) in a ratio of 1: 30 to 35 to the weight of the dried and filtered dried in the third step is the pH of the dried and filtered in the third step It is because the optimal mixing ratio to keep 4 to 5, the smooth fermentation in the following fermentation step was derived through repeated experiments.

In this case, the suspension is obtained by mixing 100 mM citric acid buffer (pH 4.6) in a ratio of 1: 1 to 1000 by weight to the dried and filtered dried in the third step.

5. Fermentation stage (5th stage)

The fermentation step is a step of inoculating the mesobacteria solution to the suspension mixed with the buffer solution in the fourth step,

In the fourth step, the mesococci solution is inoculated into the suspension in which the buffer solution is mixed to ferment.

More specifically, the mesobacteria solution is inoculated at a ratio of 1: 1 to 100 by weight in a suspension in which the buffer is mixed in the fourth step, and is fermented at 10 to 50 ° C. for 12 to 36 hours.

Here, inoculating the mesobacteria solution in a ratio of 1: 1 to 100 to the weight and the fermentation at 10 ~ 50 ℃ 12 ~ 36 hours in the suspension of the buffer mixture is mixed in the fourth step is the buffer solution is mixed in the fourth step The quercetin glycosides contained in the suspension are significantly reduced, and the content of quercetin is drastically increased so that the quercetin has antioxidant, brain cell protective, anti-inflammatory, anti-cancer, anti-diabetic, anti-obesity, and antihypertensive activities. To prepare this enhanced onion extract.

If the mesobacterial solution is inoculated at a ratio of less than 1: 1 by weight to the suspension in which the buffer is mixed in the fourth step, the mesobacterial solution is not evenly inoculated in the suspension in which the buffer is mixed in the fourth step. The fermentation may not be performed smoothly. When the mesobacteria solution is inoculated at a ratio of 1: 100 by weight to the suspension in which the buffer is mixed in the fourth step, the suspension is mixed in the buffer in the fourth step. The mesobacterial liquid is inoculated more than necessary so that the fermentation takes longer, but the fermentation may not proceed smoothly.

In addition, inoculating the mesobacterial solution in a ratio of 1: 1 to 100 by weight in a suspension mixed with the buffer solution in the fourth step, and fermentation is less than 12 hours at 10 ° C. It may be difficult to prepare the onion extract, and if the fermentation exceeds 50 ℃, 36 hours, fermentation is not performed smoothly due to the high fermentation temperature, it is difficult to prepare the onion extract with increased quercetin and fermentation time more than necessary As uneconomical.

And, the mesobacterial liquid,

Bran and 1% glucose solution in a ratio of 1: 1 to 3 by weight and sterilized for 10-30 minutes at 100 ~ 140 ℃ ( Aspergillus kawachii ) inoculated and incubated for 3-7 days at 25 ~ 35 ℃, and then mixed with the fermented bran 100mM sodium phosphate buffer (pH 7.0) in a ratio of 1: 1 to 3 by weight to 3 to 5 ℃ 3 After standing for 7 hours, the supernatant was prepared by centrifuging for 10-30 minutes at a speed of 10,000-12,000 rpm.

In addition, in addition to the mesobacterial liquid, one of lactic acid bacteria, chungguk bacteria, and glucosidase, which exhibits similar activities to the mesobacteria, may be used.

And, preferably, inoculated at a ratio of 1: 1 by weight to the mesobacteria solution in the suspension of the buffer mixture in the fourth step, it is fermented at 30 ℃ for 24 hours.

Here, the inoculation of the mesobacteria solution in a ratio of 1: 1 to the weight in the suspension mixed with the buffer in the fourth step, and fermentation at 30 ℃ for 24 hours is contained in the suspension mixed with the buffer in the fourth step This is because the quercetin glycoside content is significantly reduced, and the optimal inoculation ratio, fermentation temperature, and fermentation time for the rapid increase in quercetin content have been derived through repeated experiments.

And, at this time, inoculated at a ratio of 1: 1 to 100 by weight to the suspension mixed with the buffer solution in the fourth step, fermented onions with increased quercetin content that fermented at 10 ~ 50 ℃ for 12 to 36 hours It is called an extract.

In addition, the fermented onions having increased quercetin content may be prepared through the pre-treated onion without the ethanol extraction step, the filtration and the drying step, and the buffer mixing step and the fermentation step.

Fermented onion extract or fermented onion with increased quercetin content prepared by the above method may be used for antioxidant, brain cell protective activity, anti-inflammatory activity, anticancer activity, antidiabetic, anti-obesity, antihypertensive or therapeutic agent in the form of pharmaceutical preparation. When prepared, the active ingredient may be formulated with a pharmaceutically acceptable conventional carrier and / or injectable preparations or injectables, such as tablets, hard or soft capsules, chewing tablets, powders, solutions or suspensions, depending on the purpose of administration. It may be formulated in the form of preparations for parenteral administration such as solutions, suspensions, nasal washes and the like.

And, when the fermented onion extract or fermented onion with increased quercetin content prepared by the above method is formulated into a tablet, capsule, chewing tablet, powder, liquid, suspension or the like for the purpose of oral administration, Arabia Binders such as rubber, corn starch, microcrystalline cellulose or gelatin, excipients such as dicalcium phosphate or lactose, disintegrants such as alginic acid, corn starch or potato starch, glidants such as magnesium stearate, sweeteners such as sucrose or saccharin And flavoring agents such as peppermint, methyl salicylate or fruit flavor, and when the unit dosage form is a capsule, a liquid carrier such as polyethylene glycol or fatty oil may be included in addition to the above components.

Then, the injection of fermented onion extract or fermented onion with increased quercetin content prepared by the above method in the form of a solution or suspension for parenteral administration is administered parenterally, for example, subcutaneously, intravenously, intramuscularly or intraperitoneally. In general, injectable solutions or suspensions are pharmaceutically acceptable, such as water, saline, aqueous dextrose and related sugar solutions, nonvolatile oils, ethanol, glycerin, glycols such as polyethylene glycol, propylene glycol, and the like. It may be prepared by homogeneously mixing an effective amount of the active ingredient in a liquid carrier, and may also contain auxiliary agents such as solubilizers, antibacterial agents, chelating agents, buffers, and preservatives as necessary.

As the pharmaceutically acceptable carrier, any adjuvant may be used that is pharmaceutically pure, substantially non-toxic, and which does not inhibit the action of the active ingredient.

In addition, the daily dose of the prophylactic or therapeutic agent according to the present invention may be changed depending on various factors such as the severity, complications, weight, age, sex, etc. of the target subject to be administered, but in general, extracts 1 to 1,000 mg. / Kg, preferably 10 to 500 mg / kg, more preferably 10 to 100 mg / kg can be administered orally 1-3 times a day.

In addition, the fermented onion extract or fermented onion with increased quercetin content produced by the above method is a natural extract, in addition to the pharmaceutical preparations described above, beverage products such as soft drinks, mineral water, alcoholic beverages, or chewing gum or caramel products , Candy, ice cream, confectionary, or the like, or may be incorporated into health supplements, including vitamins and minerals, or food additives, to produce food or food supplements.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only intended to specifically illustrate the present invention, it is obvious to those skilled in the art that the scope of the present invention is not limited. That is, simple modifications or changes of the present invention can be easily carried out by those skilled in the art, and all such modifications or changes can be seen to be included in the scope of the present invention.

Example 1 Fermented Onion Extract Prepared According to the Present Invention

Onion is a commercially available yellow onion to remove the crust, which is not edible, and then crushed the peeled onion to 5mm size, 5.96kg in 95% ethanol 36L extracted for 5 hours at 70 ℃ After filtering, the filtrate was dried with a rotary pressure reducer to obtain 399.95 g of dried product. 3.24 g of this was dispersed in 100 ml of 100 mM citric acid buffer (pH 4.6), and then the same volume of mesobacteria was added, followed by incubation at 30 ° C. for 24 hours to obtain fermented onion extract.

Preparation of the bacteria solution is Meju, 10g wheat bran and 1% glucose solution, followed by kneading the 10㎖ a packed and sterilized by 15 minutes at 121 ℃ 100㎖ Erlenmeyer flasks, bags Aspergillus (Aspergillus kawachii) inoculation and five days incubation at 30 ℃ It was. 30 ml of 100 mM sodium phosphate buffer (pH 7.0) was added to the fermented bran mass, and the mixture was left at 4 ° C. for 5 hours, and the supernatant centrifuged at 12,000 rpm for 15 minutes was used as a fermentation broth.

Example 2 Preparation of Hard Capsule

As shown in Table 1 below, according to the method for preparing a conventional capsules, the above-mentioned ingredients were combined in a prescribed amount, and then filled into hard gelatin capsules of appropriate size so as to have a blend of 33 mg per capsule, thereby preparing a desired capsule.

10-fold concentrate of the mixed extract of Example 1 10 mg D-sorbitol 10 mg Lactose 5 mg Colloidal silicon dioxide 5 mg Magnesium stearate 3 mg Total amount  33 mg

Example 3: Preparation of Tablets

After mixing the ingredients in Table 2 in the specified amount, to prepare a tablet according to the conventional tablet manufacturing method.

10-fold concentrate of the mixed extract of Example 1 10 mg D-sorbitol 10 mg Lactose 5 mg Colloidal silicon dioxide 5 mg Magnesium stearate 3 mg Total amount  33 mg

Example 4 Preparation of Soft Capsule

After blending the ingredients of Table 3 in the specified amount, to prepare a soft capsule according to the conventional soft capsule preparation method.

10-fold concentrate of the mixed extract of Example 1 10 mg Soybean oil 100 mg                  palm oil 76 mg                  Total amount 186 mg

Example 5 Preparation of Injection

For the injection of one vial (10 μs) the above ingredients were prepared according to conventional injection preparation methods.

10-fold concentrate of the mixed extract of Example 1 10 mg Tocopherol 172 mg Selenium 30 mg Distilled water for injection Quantity PH regulator Quantity

Experiment 1: Determination of Antioxidant Activity by DPPH Method

When Example 1 (fermented onion extract prepared according to the present invention) is treated, it is an experiment to evaluate the antioxidant activity by measuring the degree of reduction of a radical substance called DPPH (1,1-Diphenyl-2-picrylhydrazyl). DPPH is dark purple, but the more reduction, the more colorless experiment. 10 μl of Example dissolved in DMSO and 190 μl of 150 μM DPPH solution dissolved in ethanol were mixed in a 96-well plate to block light at room temperature and reacted for 30 minutes. This is taken as the absorbance (OD) value measured at 517 nm. The control was the OD value of the DMSO solution of DPPH, and the radical scavenging ability was calculated by the following equation.

Radial Removability (%) =

The results are shown in FIG.

As shown in FIG. 1, it can be seen that the histogram after the fermentation on the right side shows stronger radical scavenging ability as the concentration on the x-axis increases as compared to before the fermentation on the left side. The concentration required to exhibit about 20% of inhibitory activity was 100 ppm before fermentation, and the same effect was observed at 10 ppm after fermentation. Based on this, the antioxidant effect was increased by about 10 times.

Experiment 2: Measurement of Brain Cell Protective Activity by MTT Method

Samples were treated on HT22 cells, a mouse hippocampus-derived neuronal cell line, and then tested for cell viability by inducing oxidative stress with glutamate to assay neuronal cell death inhibitory effects. This experiment is based on the conversion of water-soluble MTT (3-4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide) into a purple water-insoluble substance called formazan by the action of mitochondrial redox enzymes in living cells. It is known to be suitable for evaluating cell viability by using. Each hole of the 24-well plate was inoculated to have a cell number of 3 × 10 ⁴ per ml to incubate for 24 hours and the material was treated. After 12 hours, 10 mM glutamate was treated to induce oxidative stress, and after 12 hours, MTT solution (0.5 mg / ml) was treated for 4 hours. The MTT solution in the well plate was removed and the resulting formazan was dissolved using dimethylsulfoxide (DMSO). After shaking well for more than 1 hour, the absorbance (optical density, OD) was measured at 575 nm, and cell viability was calculated as a% value for the control using the following equation.

×100
Cell survival rate (%) =

× 100

Figure 2 is a comparison of the brain cell protection effect of onion extract before and after fermentation, the cell survival rate of 82% before fermentation (left bar graph) at a concentration of 25 ppm 102% after fermentation (right bar graph) It shows that the cell survival rate of about 20% increased the neuronal neuroprotective effect by fermentation.

Experiment 3: Compound Changes of Onion Extract Before and After Fermentation

Onion extracts before and after fermentation are evaporated to dryness and then methanol is added and centrifuged. Only the supernatant, which is a methanol soluble portion, was evaporated to dryness, and methanol was added to 100,000 ppm to dry matter, and then passed through a membrane of 0.45 μm, 30 μl of which was used as a sample for HPLC analysis. The column uses Hyper Hyper BDS C18 (5μ, 250 × 4.6 mm) from Thermo Hypersil-Keystone, and the mobile phase uses 99% deionized distilled water + 1% acetic acid (A) and 99% acetonitrile + 1% (B). The solvent was transferred at a rate of 0.8 ml per minute. The detector uses a photodiode array (PDA) that can analyze multiple wavelengths simultaneously. Solvent gradient is from 0% (B) → 100% (B) for 40 minutes, then hold 100% (B) for 5 minutes, then return to 100% (B) → 0% (B) again for the last 5 minutes. Programmed. The injection volume of the sample was 20 μl and the chromatogram showed 368 nm, the maximum absorption wavelength of quercetin.

In conclusion, as shown in FIG. 3, as a result of HPLC analysis, the peaks of the two peaks increased instead of the two peaks as shown by the arrows after the fermentation (below), compared to before (top). Were quercetin and quercetin 3-glucoside.

Experiment 4: Determination of Optimal Fermentation Time

As shown in FIG. 4 and FIG. 5, the change according to the fermentation time was observed. As a result, the chromatogram before the reaction showed a retention time of 20 minutes [Compound (1), 2.65 ± 0.23 mg / g _ext ] and 23 minutes [Compound (2 ), The peak of 0.86 ± 0.05 mg / g _ext ] was the largest (A), but the amount of these compounds gradually decreased with fermentation time, respectively, 0.80 ± 0.19 mg / g _ext And 0.03 ± 0.05 mg / g _ext and reduced to [(B) ~ (G), (I)], and 22 minutes [Compound (3)] and 0.00 ± 0.01 mg / g, which were 0.00 ± 0.01 mg / g _ext before fermentation. 26 minutes was _ext [compound 4] amount to a significant increase of the [(B) ~ (g), (H) after the fermentation 24 hours compared to pre fermentation quercetin 3-glucoside (3) is 0.56 ± 0.02 mg / g _{With ext} , quercetin (4) increased rapidly to 0.61 ± 0.10 mg / g _ext .

In conclusion, the content of quercetin was maximum after 16 ~ 24 hours after fermentation.

Experiment 5: Cerebral Neuronal Cell Protective Effects of Compounds Increased by Fermentation

To investigate whether the neuroprotective activity of onion extract increased by fermentation was actually related to the compound increased by fermentation, the neuroprotective activity of quercetin and quercetin 3-glucoside was measured. In general, the activity increases with increasing concentration, so the degree of protective activity was evaluated while gradually increasing the concentration.

As a result, as shown in FIG. 6, quercetin 3-glucoside showed little activity, but when quercetin was treated at a concentration of 5 μM or more, almost 100% of the survival rate without treatment with cytotoxic substances was nearly 100%. Survival rate showed better protection than estrogen (ES) or trolox (TRX) used as a positive control. On the other hand, when 50μM was treated, the cell viability was lower than the concentration below that, because the concentration of the sample was too high may show cytotoxicity.

In conclusion, it can be seen that the protective effect of fermented onion was increased by quercetin increased by fermentation.

Experiment 6: Comparison of Quercetin Content in Commercially Aged Onions with Quercetin Content in Fermented Onions by the Present Invention

The content of quercetin in fermented onions of the present invention and the quercetin content in commercially matured onions were compared by HPLC. HPLC conditions are the same as those used for quercetin quantification. Three commercial onion fermentation solutions were obtained, each weighed by evaporation to dryness, weighed, dissolved in methanol, concentrated, and concentrated to 100,000 ppm after adding methanol.

As a result, as shown in Fig. 7, the quercetin content per 1g of dry matter was 24.2 ㎍ for commercial A company, 2.4 ㎍ for B company, 0.2 ㎍ for Company C product, the quercetin content (0.61 mg) of fermented onions according to the present invention At least 25 times as much as 3,000 times more.

Therefore, fermenting onions or onion extracts can not only obtain a substance with an increased content of quercetin, but also provide a composition with enhanced antioxidant and neuronal cell protective effects. In addition, quercetin is known to have antioxidant, brain cell protective activity, anti-inflammatory action, anti-cancer action, anti-diabetic, anti-obesity, anti-hypertension, and the like, it is possible to produce onion and onion extract with increased various health functionalities.

Claims (4)

After washing the onion to remove the outer skin, onion pre-treatment step of grinding to a size of 0.5 ~ 10mm (first step);
Ethanol extraction step of extracting the ethanol of the 95% concentration in the ratio of 1: 1 to 5 to 10 by weight in the first step to extract for 3 to 10 hours at 50 ~ 90 ° C (second step);
A filtration and drying step of filtering and drying the extract extracted from ethanol in the second step (third step);
A buffer mixing step (fourth step) of mixing and suspending 100 mM citric acid buffer in the dried and filtered filtered in the third step; And
Method of producing a fermented onion extract with increased quercetin content, characterized in that the fermentation step (the fifth step) to inoculate and ferment the mesobacteria solution to the suspension of the buffer mixture in the fourth step.
The method of claim 1,
In the fermentation step of the fifth step,
Bran and 1% glucose solution in a ratio of 1: 1 to 3 by weight and sterilized for 10-30 minutes at 100 ~ 140 ℃ ( Aspergillus kawachii ) inoculated and incubated for 3-7 days at 25 ~ 35 ℃, mixed with 100mM sodium phosphate buffer in a ratio of 1: 1 to 3 by weight to the fermented wheat bran to stand 3 ~ 7 hours at 3 ~ 5 ℃ Then, the method of producing a fermented onion extract with increased quercetin content, characterized in that it was produced by centrifugation for 10-30 minutes at a speed of 10,000 ~ 12,000rpm to obtain a supernatant.
The method of claim 1,
In the fermentation step of the fifth step, the quercetin content is increased by inoculating a suspension of the mesobacteria solution in a ratio of 1: 1 to 100 by weight to fermentation at 10 to 50 ° C. for 12 to 36 hours. Fermented onion extract preparation method.
Composition for the protection of antioxidant and cerebral nerve cell, comprising a fermented onion extract with an increased content of ulcetin prepared by the method of any one of claims 1 to 3.

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