KR20130017431A - An antioxidantive composition of persimmom wine powder using monascus purpureus spp. strain - Google Patents

Abstract

PURPOSE: A functional food composition is provided to have an excellent effect in an antioxidant activity and a cardiac cell protection by using a persimmon wine. CONSTITUTION: A functional food composition for an antioxidant activity and a cardiac cell protection contains a persimmon wine concentrated powder as an active component. A persimmon wine concentrated powder is obtained by sterilizing a persimmon smashing liquid obtained by the smashing of a fallen fruit or a ripe persimmon; inoculating a Monascus purpureus KCCM 60170 strain culture medium against a sterilized persimmon raw material; performing a first fermentation; adding glucose to be 23^o Brix after extracting and filtering; inoculating a Saccharomyces cerevisiae yeast; performing a second fermentation until reaching alcohol content 16%; maturing a tannase or a gelatin; and freezing and drying at -40°C for 48 hours after concentrating at 40°C for 48 hours. A food component is any one formulation among a tablet, syrup, or a capsule. [Reference numerals] (AA) Crushing a raw persimmon material; (BB) Sterilization/K_2S_2O_3 sealing; (CC) Sterilized persimmon raw material pulp; (DD) Inoculating Monascus purpureus; (EE) First fermentation; (FF) Extraction and filtration; (GG) Chaptalization(glucose); (HH) Inoculating yeast; (II) Second fermentation; (JJ) Tanninase(0.2 weight%) or Gelatin(0.3 weight%); (KK) Enzyme treated product; (LL) Maturing; (MM) Concentrating; (NN) Freeze-drying; (OO) Concentrate powder

Description

An antioxidantive composition of persimmom wine powder using Monascus purpureus spp. strain}

The present invention relates to an antioxidant and heart cell protective functional food composition containing a concentrated powder of persimmon wine using red yeast bacteria as an active ingredient.

Recently, the trend of pursuing a health-oriented diet with the improvement of the standard of living of the people has been prominent. As a part of this, there is increasing interest in high-quality foods that prefer natural-oriented foods and safety choices such as well-being foods.

This trend is changing the consumer's choice of liquor from high alcohol to low alcohol consumption, and the consumption of health-related alcoholic beverages is increasing, for example, soju is lowered, makgeolli consumption and wine Income is skyrocketing. In line with this trend, the inventors have developed persimmon wines with unique aromas and flavors using local persimmons. However, the accumulated technology of Gam wine is insufficient and its quality, functional ingredients and health related effects are not widely known to the public. Scientific evaluation was necessary.

Persimmon is a specialty of the Asian region, and it is a fruit that contains unique sweetness only in China, Japan, and Korea. Persimmon is a deciduous broad-leaved arboreous tree growing in villages all over the country. Persimmon trees in Yangsan Nonchon are trees that are at least 30 years old. It is a clean place where the northwest season is well-ventilated, and it is a clean place surrounded by mountains, where dust is not blown away. Due to the natural condition of natural beauty, Yangchon dried persimmon is made by natural sun drying, not artificial drying. Known as dried persimmon, Yangchon Durigam is a nutritious fruit with high sugar content, vitamin C and carotenoids. Especially, tannin, which has a rich taste, is very fruitful because of its excellent antioxidant capacity. The action of tannins is also associated with acetaldehyde, which has the effect of eliminating hangovers. This is why there is a saying that "drink persimmons break quickly." Although it has been neglected to be cultivated by foreign fruits for some time, it is in full bloom again with its unique taste and excellent health function.

Red yeast has long been regarded as the leaven that is beneficial to health. It has been used as a raw material for brewing in China since ancient times, and it is recorded in Chinese ancient medicine. The Chinese emperor's breast was first used as an imperial food and used as a Chinese medicine to improve blood circulation. In the main herb 本草綱目, it helps digestion, revitalizes the blood, strengthens the spleen, regulates the stomach, and revitalizes the woman's blood to cure the disease. 'Dongguk' agrees that the 'Hongguk' is a new country that makes blood flow well, food is digested, and stops dysentery. Hongguk is registered as a dietary supplement by the Food and Drug Administration, and its efficacy has been scientifically proven and sold in the United States. The efficacy of red yeast rice, which has been studied until recently, has been shown to be effective in lowering blood pressure, antibacterial, anti-cancer, antioxidant function, and hypoglycemic activity. Especially, "Monakolin-K" produced by red yeast bacterium has a large effect on lowering blood cholesterol content. It has been shown to be effective. Gam wine developed by the present inventors is a new wine that proved the health functionalities that combine tradition and science by applying the hongguk bacteria to wine production.

Excessive amounts of free radicals in the human body cause protein denaturation, oxidation of lipids in the living body, and destruction of DNA. The present invention developed by the present inventors was fermented with a sense of fermentation of hongchonkyun and yeast bacteria, it was confirmed that a large amount of polyphenol components such as tannin, catechin, etc. contained in the persimmon contained in a higher concentration than ordinary wine. The tannin, catechin component is excellent in the antioxidant capacity to remove the free radicals to prevent damage to the cells, as well as provide new functional use.

The present invention has been made in view of the above-mentioned matters, and an object of the present invention is to provide an antioxidant and heart cell protective functional food comprising the persimmon wine of the present invention as an active ingredient.

The above object of the present invention is the primary fermentation step of producing the persimmon wine by inoculating and fermenting the seed culture medium of hongguk spawn in the waste crushed solution; A second fermentation step of adding the yeast strain with glucose after filtration and filtration, and empty culture; Fermentation of persimmon fermented wine by enzymatic treatment; Identifying the properties of the alcoholic fermentation strain; The present invention was achieved through the step of concentrating, lyophilizing and identifying and evaluating antioxidant capacity.

The present invention has a high gallic acid content has an excellent effect of providing an antioxidant functional food composition.

1 is a manufacturing process diagram of the present invention wine.
2 is an HPLC chromatogram of Standards according to the present invention.
Figure 3 is an HPLC chromatogram of the inventive persimmon wine '2-1 (produced February 2009)'.
4 is an HPLC chromatogram of the inventive persimmon wine '2-2 (March 2010 production)'.
5 is an HPLC chromatogram of the inventive persimmon wine inventive product.
6 is an HPLC chromatogram of Standards according to the present invention.
Figure 7 is a Monacolin K related HPLC chromatogram of the present invention Gam wine.
8 is an HPLC chromatogram of the present invention persimmon wine '2-1 (February 2009 production)' product (enzyme treatment).
Figure 9 is an HPLC chromatogram of the inventive persimmon wine '2-2 (March 2010 production)' product (enzyme treatment).
FIG. 10 is an HPLC chromatogram of the inventive persimmon wine 'tracing' product (enzyme treatment).
FIG. 11 is an HPLC chromatogram of the inventive persimmon wine '2-1 (February 2009) gelatin 0.3 wt% processed' product.
12 is an HPLC chromatogram of Standards according to the present invention.
Figure 13 is an HPLC chromatogram of raw raw materials according to the present invention.
Figure 14 is an HPLC chromatogram of the first step of fermentation of ginseng of the present invention (hongguk fermentation).
15 is an HPLC chromatogram of the second step (fermentation) of the persimmon wine fermentation of the present invention.
Figure 16 is the antioxidant activity of the present invention wine (A) and the additional wine powder (B).
17 is an evaluation of free radical removal ability in the heart cells of the present invention wine powder.
18 is a cardioprotective evaluation of the present invention wine.
Figure 19 is an evaluation of the cardiac protective effect of Gallic acid and Monacolin K of the present invention wine powder.

< Example  1> the present invention wine of  Produce

The manufacturing process of the persimmon wine of this invention is shown in FIG.

Monascus perprius KCCM 60170 strain was inoculated in PDB medium and incubated at 30 ° C. for 24 hours to pre-cultivate erythrocytes to 1 × 10 ⁷ cells / mL. 0.3% by weight or less of tannin dehydrogenase or gelatin was added to the persimmon crushed liquid obtained by mashing the fruit or soft berry (soft persimmon), treated at 45 ° C. for 2 hours or 1 hour, and then adjusted to pH 3.6 by adding 0.05% by weight of citric acid. 0.15 g / L of sulfur metasulfite (K ₂ S ₂ O ₅ ) was added thereto to maintain a concentration of sulfuric acid (SO ₂ ) of 60 ppm and sterilized by sealing for 12 hours. The sterilized persimmon pulp was inoculated with 3% by weight of the pre-cultivation of the honggukyun culture medium was fermented primarily at 26 ℃ for 6 days.

The fermentation broth was juiced, filtered, and glucose was added so that the sugar content was 23 ^o brix. The fermentation of the yeast Saccharomyces cerevisiae (Wine west, Anchor, Germany) was inoculated with 3% by weight of the total weight, followed by secondary fermentation until the alcohol concentration reached 16%. After tannin dehydrogenase or gelatin was added at less than 0.3% by weight and treated for 2 hours or 1 hour, the cells were aged.

< Experimental Example  1> Alcohol content measurement

100 ml of the present invention was taken and transferred to an Erlenmeyer flask, washed with an equal amount of water, combined with the flask and connected to a distillation and cooling device, and heated to stop the distillation when the distillate became about 80 mL and distilled water up to 100 mL. The display was read using the alcohol system in the essence solution and the alcohol content was corrected to 15 ° C as a Gay-Lussac table.

< Experimental Example  2> soluble solids

A portion of the sample of the present invention was taken and measured by ° Brix at a temperature of 20 ° C using a refractometer (ATAGO Digital Refractometer, Japan).

< Experimental Example  3> pH  Measure

The pH of the present invention wine was measured by dipping directly in the sample using a pH meter (HANNA Instrument, Italy).

< Experimental Example  4> Reducing Sugar

Absorbance of 3-amino-5-nitrosalicylic acid produced by reducing 3,5-dinitrosalicylic acid (DNS) by reducing sugar is measured at 550 nm with a UV / VIS spectrophotometer (Diod-Array) HP 8454. Separately, a calibration curve of a standard solution containing 15 to 300 µg of glucose was prepared, and the reducing equivalent (mg / ml or%) in the sample was obtained.

Analysis results of alcohol, soluble solids, reducing sugars, pH of two wines produced according to the present invention and two different wines produced according to the present invention in order to analyze the physicochemical characteristics of the persimmon wine according to Experimental Examples 1 to 4 above. It is shown in [Table 1]. The alcohol content of all three treatments was about 16%, and the soluble solids and reducing sugars were 10.0 ° Bx and 0.74% in March 2010, respectively. The pH of the 'follow-up' product of the present invention was 4.08, which was somewhat higher than other treatments.

< Experimental Example  5> Main polyphenol composition analysis

Wine samples were analyzed using 0.45 μm membrane filter and analyzed by HPLC (Jasco, Japan). SunFire as an analysis column^TM-C18 (4.6 × 250 mm) was used and analyzed at UV 280 nm using a Jasco MD-2010 Plus detector (Jasco, Japan). The temperature of the oven was 35 ° C. The solvents used were A: 2% acetic acid in Water, B: 0.5% acetic acid in acetonitrile: water = 50:50, 20 μl injection volume and 0.8 ml / min flow rate.

The main polyphenol components of persimmon wine are shown in [Table 2]. Gallic acid was found to occupy most of the polyphenols, and small amounts of catechin and epicatechin were detected. Meanwhile, HPLC chromatograms of polyphenol standards and treatments are shown in FIGS. 2 to 5.

On the other hand, the results of analyzing the monacolin K content of the present invention wine is shown in Figures 6 and 7. The monacolin K content was found to be about 1.3 μg / mL.

< Experimental Example  6> microbiological inspection

Take 1 mL of sample and dilute with 10-fold dilution method using sterile physiological saline, smear it on dry film medium for general bacteria and yeast (Petri film, 3M, USA), incubate at 37 ℃, and count the colony. Represented in mL.

Microbiological tests were performed on four kinds of persimmon wine products (2-1, 2-2, follow-up products 375 mL, 750 mL) and the results are shown in [Table 3]. General bacteria were not detected in all four treatments, and yeast bacteria were detected by 3.3 × 10 ² CFU / mL in 2-2 (March 2010). On the other hand, the physicochemical characterization of the product (750 mL) showed that the alcohol content was 16.7%, the soluble solids was 13.6 ° Bx, the reducing sugar was 3.95%, and the pH was 3.82.

< Example  2> present invention Persimmon  Concentrate and Lyophilize

One liter of the present invention wine was concentrated in an evaporator at 40 ° C. for two days (48 hours) to evaporate the alcohol. After lyophilization at -40 ° C for another two days (48 hours) to form a powder (68 g / liter) form was used as the present invention material. The powder was prepared by mixing with the excipients acceptable in the food industry for various formulations to produce a syrup (Syrup) product, or tableted into tablet products or capsules into edible capsules to be a dietary supplement.

< Experimental Example  7> Cell Culture

For cell experiments, H9c2 cardiac muscle cells were purchased from the American Type Culture Collection (Manassas, VA). Cells were added to Dulbecco ’s modified Eagle's medium (DMEM) with 10% fetal bovine serum at 37 ° C and 5% CO₂ Cultured under conditions.

< Experimental Example  8> DPPH radical scavenging assay

DPPH radical scavenging ability was measured to confirm the antioxidant capacity of the present invention. Each concentration of the sample and the DPPH solution (5 mg / 100 ml methanol) was mixed in the same amount and then reacted for 20 minutes and the absorbance at 525 nm was investigated.

First, DPPH radical scavenging ability was evaluated to evaluate the antioxidant activity of wine. The antioxidant activity of each follow-up wine itself and the antioxidant activity of the follow-up wine powder are shown in FIG. 16. As shown in FIG. 16A, the follow-up wine itself exhibited radical scavenging activity of 60% at 0.125 μl and almost 100% at 1 μl. It was confirmed that the activity was saturated up to 5 μl. Follow-up wine powder also showed a radical scavenging activity in a concentration-dependent manner, and showed 100% activity at a concentration of 200 μg / ml. As a result, it was confirmed that the follow-up wine had excellent antioxidant activity in vitro.

< Experimental Example  9> Cell viability assay  ( MTT )

Incubate the cells in 96 well plates and process each sample. Two hours before the end of the sample treatment, the absorbance was measured at 570 nm after treatment with 1 mg / ml of MTT dye.

< Experimental Example  10> Reactive oxygen species  ( ROS ) measurement

Incubate the cells in 6 well plates and process each sample. Thirty minutes before the end of the sample treatment, the DCFH-DA dye was treated with 10 μM for 30 minutes, discarded the cell culture medium, and then 1 ml of PBS was added.

< Example  3> invention Persimmon  A bitter taste Reduction  for Tannins  Ingredient control

(1) enzyme treatment

In order to reduce the taste of persimmon in persimmon wine, the main polyphenol component was investigated after the reaction by adding tanninse, a tannin degrading enzyme, and presented in [Table 4].

As can be seen in [Table 4] it was confirmed that the content of the three components treated with gallic acid, catechin, epicatechin after the enzyme treatment was reduced compared to before the enzyme treatment. Therefore, the enzyme treatment method could be an effective way to improve the astringent taste of persimmon wine. However, the high price of tanninase enzyme may be a factor that can affect the use. 8 to 10 show HPLC chromatograms after enzymatic treatment of each treatment.

(2) gelatin treatment method

Since tannin is water-soluble, it binds to proteins and denatures them. Therefore, after a certain amount of gelatin, a derivative of protein derived from the treatment of collagen, a natural protein that constitutes animal skin, tendons, cartilage, etc. with hot water, The change in the polyphenol component was investigated. Gelatin treatment was used in the experiment by adding 0.3% by weight of powdered gelatin to the gamma wine "2-1 (February 2009)" product, stirred for 60 minutes and filtered through a 0.45 ㎛ membrane filter. The results are shown in [Table 5].

As a result of comparing the detected three components of gallic acid, catechin, and epicatechin, it was confirmed that the polyphenol component of persimmon wine was decreased after gelatin treatment. Especially, the gallic acid was rapidly reduced from 157.5 / mL to 84.3 / mL. . Therefore, gelatin treatment method may be a useful alternative to tannin reduction in consideration of problems such as price of enzyme treatment method. Fig. 11 shows the HPLC chromatogram after gelatin treatment of a gamma wine product. Pretreatment of the raw material of the present invention with enzyme treatment or gelatin to remove the astringent taste components will be necessary in the case of the need to prevent constipation as a health functional supplement raw material without significant changes in active ingredients such as gallic acid.

< Experimental Example  11> Gam wine  Changes in Quality Characteristics over Time by Manufacturing Process

To investigate the change in physicochemical properties occurring in the process of producing persimmon wine of the present invention The results of analyzing the chemical properties and main polyphenol components are shown in [Table 6] and [Table 7].

Alcohol content increased as the yeast fermentation stage increased in the fermentation of red yeast rice. Soluble solids, reducing sugars, and pH decreased in the first fermentation stage compared with the year-end, but increased again as the fermentation stage proceeded. It was.

As the polyphenol component progressed from the wine fermentation stage, gallic acid increased and catechin, epicatechin, gallocatechin, gallocatechin gallate and catechin gallate decreased. Tannin in persimmon fruit is a polymer of proanthocyanidin whose basic structure is catechin and gallocatechin, etc., and it is thought that gallic acid has ester bond in its side chain. Therefore, the content of catechin components is decreased due to the natural desorption phenomena during fermentation, and the increase of gallic acid by catechin decomposition is estimated.

12 to 15 show HPLC chromatograms for the year-round and first and second fermentation wines.

< Experimental Example  12> present invention Persimmon  Free radicals in cell models Removability  evaluation

Heart disease is caused by various causes and is one of the important diseases that threaten human survival. Among the pathogens of many heart diseases, free radicals are known to cause damage to cells due to cardiac ischemia / reperfusion. Accordingly, based on the results of in vitro antioxidant activity of the wine of the present invention, the cardiac cell model was used to evaluate the efficacy of intracellular free radicals (ROS) removal. H9c2 cardiac muscle cells were pretreated with follow-up wine powder concentration-dependently (10-800 μg / ml) for 1 hour and then H₂O₂ 500 μM was treated for 4 hours to induce the production of ROS. At this time, the green fluorescence was confirmed by fluorescence microscopy after 30 minutes of DCFH-DA dye treatment with 10 μM.₂O₂(0) cells showed green fluorescence when treated, and the follow-up wine powder was H-dependent.₂O₂ It was found that ROS induced by can be removed (FIG. 17). As a result, the present invention was able to confirm that the present invention has excellent antioxidant activity in cell model as well as in vitro.

< Experimental Example  13> present invention Follow-up wine H ₂ O ₂ Induced Cardiac apoptosis  Protective effect

Next, using the heart cell model, the follow-up wine was H₂O₂ The following experiments were conducted to evaluate whether there is protection in induced apoptosis. H9c2 cardiac muscle cells were pretreated with follow-up wine powder concentration-dependently (10-800 μg / ml) for 1 hour and then H₂O₂ 500 μM was treated for 4 hours to induce apoptosis. At this time, 2 hours before the end of the experiment, MTT dye was treated with 1 mg / ml, and the absorbance was measured.₂O₂Treatment with (0) showed that cell death was induced. In the follow-up wine powder, concentrations from 100 μg / ml to significantly 800 μg / ml were observed.₂O₂ It was found that the cell death induced by the cells was suppressed (FIGS. 18A and B). As a result, the follow-up wine was found to have excellent antioxidant activity and cytoprotective effect in the cell model.

< Experimental Example  14> present invention Follow-up wine major compounds of Cardiac cell  Protective efficacy evaluation

Next, the main components of the present invention wine were analyzed, and among them, Gallic acid and Monacolin K were evaluated to play an important role in the cardioprotective effect of the wine. H9c2 cardiomyocytes were treated with gallic acid or Monacolin K in concentration-dependent (10-200 μM) for 1 hour before H₂O₂ 500 μM was treated for 4 hours to induce cell death. At this time, 2 hours before the end of the experiment, MTT dye was treated with 1 mg / ml, and the absorbance was measured. As a result, the concentration of gallic acid and gallic acid in Monacolin K of the persimmon wine powder of the present invention₂O₂ It was found that the cell death induced by the cells was suppressed (FIG. 19). As a result, it could be inferred that gallic acid acts as an important component of the protective effect of the present invention.

The present invention is a very useful invention in the food processing industry because it has an excellent effect of providing an antioxidant functional food composition and a cardioprotective functional food by recycling waste shredded persimmon or soft waste (pulp).

Claims (3)

After sterilization of the crushed persimmon crushed solution of the fruit or new year, inoculated with the pre-culture of the Monascus perprius KCCM 60170 strain to the sterilized raw materials, and then fermented to 23 ^o Brix after juice and filtration Second fermentation was inoculated with yeast inoculation to Mises cerevisiae yeast until the alcohol content was reached 16%, and it was obtained by treating and aging tannin degrading enzyme or gelatin, concentrating for 48 hours at 40 ℃, and lyophilizing at -40 ℃ for 48 hours. Fermented persimmon wine powder of red yeast bacteria and yeast in the yeast.
Antioxidant and cardioprotective functional food composition comprising the persimmon wine powder of claim 1 as an active ingredient.
3. The antioxidant and cardioprotective functional food composition of claim 2, wherein the food composition is in the form of a tablet, syrup or capsule.

Images