KR102048623B1 - A composition comprising acidic polysaccharide in red ginseng marc and method thereof - Google Patents

Abstract

The present invention relates to a method for extracting acidic polysaccharides from red ginseng gourd and a composition comprising the same as an active ingredient, establishing an optimal process for increasing the content of acidic polysaccharides extracted from red ginseng or red ginseng by-products, and extracting acid from red ginseng gourd By preparing a functional composition containing a polysaccharide as an active ingredient, there is an excellent effect of providing a food having excellent antioxidant effects.

Description

A composition containing acidic polysaccharide derived from red ginseng gourd as an active ingredient and a method for preparing the same

The present invention relates to a method for extracting acidic polysaccharide from red ginseng gourd and a composition comprising the same as an active ingredient, and more particularly, to prepare an extract from red ginseng and to optimize the process of extracting acidic polysaccharide from red ginseng gourd discharged as a by-product. It relates to a food composition containing an acidic polysaccharide prepared by the above method as an active ingredient.

Ginseng (Panax ginseng C.A. Meyer) is a traditional medicinal plant that has been used in Asia for a long time and its demand is gradually increasing due to scientific recognition of the pharmacological efficacy of certain bioactive ingredients. Ginseng bioactive substances include ginseng saponins (ginsenosides), polyacetylenes (polyacetylenes), phenolic compounds (alkaloids), alkaloids, acidic polysaccharides, amino acids and inorganic elements, amino sugars, etc. . However, research on ginseng saponins has been mainly conducted and many studies on non-saponin-based substances have not been conducted. However, non-saponin-based chemicals have been found in the non-saponin-based chemicals. The study of ingredients is also increasing.

Among the non-saponin-based compounds of ginseng, the most studied component is acidic polysaccharides. Looking at the polysaccharides isolated until now, among the ginsengs such as red ginseng, white ginseng, and fresh ginseng, blood glucose lowering components from white ginseng such as panaxan AU, etc. Twenty-one species are known, and there is PG 5-1 (protein-containing polysaccharide) which is a bioprotective active substance, and other anti-complement active polysaccharides have been found. The major pharmacological effects of acidic polysaccharides have been reported to have anticancer activity and anticancer activity. In addition, it has been found to have cell division promoting action and anti-complement activity, and has been separated into components that inhibit lipolysis induced by toxohormone-L. In addition, studies on the antitumor effect by oral administration of acidic polysaccharide (RGAP) extracted from red ginseng, alcohol metabolism enzymes and lipid metabolism is activated to improve alcoholic hyperlipidemia.

As interest in red ginseng food continues to increase, the size of the Korean red ginseng market has reached KRW 1 trillion since 2010, and it is ranked first in the sales status of health functional foods by more than 50%. As the consumption of red ginseng increases, red ginseng gourd produced as a by-product is estimated to produce about 8,000 tons annually nationwide, and is partially used as food material and animal feed, but most of it is disposed of as industrial waste.

Red ginseng gourd extracts red ginseng with a solvent such as water or alcohol, and can not extract all of its active ingredients even if the ginseng is repeatedly extracted several times, and only the components dissolved in water or alcohol are extracted and dissolved depending on the extraction solvent. Unsaturated saponins, polyacetylenes, proteins and other active ingredients remain about 20 to 40%. About 65% of these alcohol extract residues are obtained with respect to red ginseng. A significant amount of polysaccharides are not eluted, and acidic polysaccharides of alcohol extract residues have been reported to have high anticancer and immune activity. Red ginseng gourd contains a lot of polysaccharides that have such an immunological activity. Therefore, there is a need for development of new functional materials having better efficacy than conventional red ginseng extracts.

As a method of extracting such acidic polysaccharides, Korean Patent No. 10-0127387 discloses a method of separating acidic polysaccharides from ginseng or alcohol-extracted ginseng foil, but since the method is extracted at a high temperature, the starch content is relatively higher than that of acidic polysaccharides. There was a problem that the high extraction efficiency of the acidic polysaccharides is high. In addition, the Republic of Korea Patent No. 10-0797016 relates to the production technology of ginseng polysaccharides is described that the molecular weight range of ginseng polysaccharides obtained by batch applied to the production technology is about 10 times the difference, the range of the ginseng polysaccharide from the molecular weight 1,100 Because it describes up to 400,000, there was a problem that does not cover the known acidic polysaccharide of ginseng.

Therefore, the present inventors have made an effort to industrially use the red ginseng gourd produced as an extract from the red ginseng and discharged as a by-product, and to solve the above problems, while confirming the conditions for efficiently extracting the acidic polysaccharide from the red ginseng gourd and the present invention. Completed.

KR 10-0127387 B1, October 21, 1997 KR 10-0797016 B1, January 16, 2008

Therefore, the technical problem to be solved in the present invention is to provide a method for extracting the acidic polysaccharide from red ginseng gourd and the acidic polysaccharide prepared by the method.

In addition, another object of the present invention is to provide a functional food composition having an antioxidant effect by mixing the acidic polysaccharide prepared by the above method in a predetermined ratio.

In order to solve the above technical problem, the present invention provides a method for extracting the acidic polysaccharide from red ginseng gourd and the acidic polysaccharide prepared by the method.

In the present invention, the method for extracting the acidic polysaccharide from the red ginseng gourd is dried red ginseng gourd for 12 hours at 60 ℃, pulverized dry red ginseng gourd into 200 mesh, and put the purified water of 10 times in red ginseng gourd , Extracting at 95-98 ℃ for 4 hours, cooling the extract to low temperature and then separating only the supernatant, mixing four times the ethanol to the separated supernatant, separating only the precipitated sediment separately and the separated precipitate Red ginseng acidic polysaccharides may be prepared, including the step of powdering finely after hot air drying.

In the present invention, the composition containing the acidic polysaccharide extracted from red ginseng gourd may exhibit excellent antioxidant effects by excellent DPPH radical scavenging ability, hydroxy radical scavenging ability, superoxide radical scavenging ability, Fe2 + chelation ability.

Accordingly, the present invention provides a method for preparing a food composition by mixing the acidic polysaccharide prepared by the above method in a predetermined ratio and a food composition prepared thereby.

As used herein, the term antioxidant effect refers to a substance that suppresses or attenuates the so-called oxidative action, including hydroxy radicals called superoxide radicals, superoxide radicals, hydrogen peroxide, etc. as active substances or free radicals. As a meaning, it is used by the same meaning as the terms, such as antioxidant composition, antioxidant, antioxidant composition.

According to one preferred embodiment of the present invention, there is provided an antioxidant food composition comprising an acidic polysaccharide as an active ingredient.

The food of the present invention may be a health supplement, a health functional food, a functional food, and the like, but is not limited thereto, and includes adding the compound of the present invention or an acceptable salt thereof to a natural food, a processed food, a general food material, and the like. .

The food composition of the present invention may include acidic polysaccharides alone or may be used with other food or food compositions, and may be suitably used according to conventional methods. The mixed amount of the active ingredient can be suitably determined according to the purpose of use (prevention, improvement or therapeutic treatment).

In general, the acidic polysaccharide of the present invention may be added at 0.1 to 20% by weight, preferably 1 to 10% by weight relative to 100% by weight of the raw material of the food or beverage in the manufacture of the food or beverage.

The effective dose of the acidic polysaccharide of the present invention may be below the above range for long term intake for health and hygiene purposes or for health control purposes, and the amount of the above-mentioned range because the active ingredient has no problem in terms of safety. Can also be used as.

There is no particular limitation on the kind of food. Examples of the food to which the acidic polysaccharide can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea , Drinks, alcoholic beverages and vitamin complexes, and other nutritional supplements, but are not limited to these types of foods.

In one embodiment of the present invention, red ginseng or red ginseng gourd prepared. The red ginseng gourd produced after preparing the red ginseng extract or red ginseng tablets is dried at 60 ° C. for 12 hours. The dried red ginseng gourd is powdered and ground into 200 mesh. 10 times of purified water was added to the prepared red ginseng or red ginseng gourd, and extracted at 95-98 ° C. for 4 hours. After cooling the extract at low temperature, only the supernatant is separated and mixed with ethanol of 4 times the supernatant. Only the sediment is sedimented separately and hot-air dried and finely powdered to produce red ginseng acid polysaccharide. The red ginseng acid polysaccharide, gojija, Schisandra chinensis, ginseng powder into a non-woven fabric and extracted with hot water for 18 hours at a temperature of 85 ℃. After mixing fructose and sodium citrate to the extract, sterilization, sealing, and automatic packaging may produce a functional drink using red ginseng acidic polysaccharide.

In addition, in one embodiment of the present invention, red ginseng or red ginseng gourd prepared. The red ginseng gourd produced after preparing the red ginseng extract or red ginseng tablets is dried at 60 ° C. for 12 hours. The dried red ginseng gourd is powdered and ground into 200 mesh. 10 times of purified water was added to the prepared red ginseng or red ginseng gourd, and extracted at 95-98 ° C. for 4 hours. After cooling the extract at low temperature, only the supernatant is separated and mixed with ethanol of 4 times the supernatant. Only the sediment is sedimented separately and hot-air dried and finely powdered to produce red ginseng acid polysaccharide. Put red ginseng raw material into the extractor, add 10 times purified water and extract hot water at 70-90 ℃ for 12 hours. The red ginseng is mixed with the red ginseng acidic polysaccharide in a predetermined ratio, and the mixture is concentrated to 60% or more solids. After the mixture is sterilized at 85 ° C. or higher for at least 1 hour, the mixture is filled into a packaging container and sealed by closing a lid to prepare a concentrate using red ginseng acidic polysaccharide.

The present invention establishes an optimal process for increasing the content of acidic polysaccharides extracted from red ginseng or red ginseng by-products, and prepares a functional composition containing red ginseng gourd-derived acid polysaccharides as an active ingredient. It works.

Figure 1 shows the results of extracting the acidic polysaccharides by solvent from the red ginseng gourd according to the present invention (A: standard curve of pectin, B: acidic polysaccharide extraction yield by solvent).
Figure 2 shows the results of extracting the acidic polysaccharides by concentration using alcohol from the red ginseng gourd according to the present invention.
3 is a surface reaction analysis result showing the effect of extraction vacuum and time on the amount of crude polysaccharide extraction according to the present invention.
Figure 4 is a surface reaction analysis results showing the effect of extraction vacuum and red ginseng gourd concentration on the amount of crude polysaccharide extraction according to the present invention.
5 is a surface reaction analysis result showing the effect of extraction time and red ginseng gourd concentration on the crude acid polysaccharide extraction amount according to the present invention.
6 is a surface reaction analysis result showing the effect of extraction vacuum and extraction time on the amount of crude polysaccharide extraction according to the present invention.
7 is a surface reaction analysis result showing the effect of extraction vacuum and red ginseng gourd concentration on crude acid polysaccharide extraction yield according to the present invention.
8 is a surface reaction analysis result showing the effect of extraction time and red ginseng gourd concentration on the crude acid polysaccharide extraction yield according to the present invention.
9 is a surface reaction analysis result showing the effect of extraction vacuum and extraction time on the acid production polysaccharide extraction rate according to the present invention.
10 is a surface reaction analysis result showing the effect of extraction vacuum and red ginseng gourd concentration on the crude acid polysaccharide extraction production rate according to the present invention.
11 is a surface reaction analysis results showing the effect of extraction time and red ginseng gourd concentration on the crude acid polysaccharide extraction production rate according to the present invention.
12 is a surface reaction analysis result showing the trend of the crude acid polysaccharide content, yield, production rate according to the vacuum degree, time, concentration of the crude acid polysaccharide according to the present invention.
Figure 13 shows a chromatogram for the elution of the crude acid polysaccharide according to the present invention (A peak: distilled water, B peak: 0.1 M NaCl, C peak: 0.5 M NaCl eluted with chromatogram).
Figure 14 shows the results of a comparative analysis of the DPPH radical scavenging ability of the crude acid polysaccharide according to the present invention (A: distilled water, B: 0.1 M NaCl, C: 0.5 M NaCl).
15 shows the results of FT-IR analysis of the crude acid polysaccharide according to the present invention.
Figure 16 shows the resonance spectrum of ¹ H NMR of the crude acid polysaccharide according to the present invention.
Figure 17 shows the spectrum of ¹³ C NMR of the crude polysaccharide according to the present invention.
Figure 18 shows the antimicrobial activity of the crude acid polysaccharide according to the present invention.
Figure 19 shows the average value for the evaluation of sweetness, sourness, astringent taste, color, aroma, overall preference of the functional beverage containing the crude acid polysaccharide according to the present invention as an active ingredient.
Figure 20 shows the average value for the evaluation on the sweetness, sourness, astringent taste, color, aroma, overall preference of the functional concentrate containing crude acid polysaccharide according to the present invention as an active ingredient.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Experimental Example 1. Extraction of Acidic Polysaccharides from Red Ginseng gourd

(1) pretreatment of red ginseng gourd

Red ginseng gourd (10%, w / v) was suspended in purified water and centrifuge (Supra R30, Hanil Scientific., Seoul, Korea) was performed at 10,000 rpm x 10 minutes in order to separate solids. In order to separate acidic polysaccharides by solvent (Ethanol, methanol, propanol, acetone, butanol), a solvent was added to the supernatant at a rate of 50% (v / v). Centrifugation was performed to separate the precipitated crude polysaccharide. The amount of crude acid polysaccharide precipitated was measured and dried in a hot air dryer at 50 ° C.

(2) Extraction method of acidic polysaccharide by solvent from red ginseng gourd

In order to quantify the amount of acidic polysaccharide extracted from red ginseng gourd according to the type of solvent, the standard curve was determined using pectin as a representative material and the absorbance was measured by measuring the amount of acidic polysaccharide. The acidic polysaccharide extraction evaluated according to the standard curve is shown in FIG. 1, and the average of 16.64% was obtained when alcohol was used as a solvent. Yields of methanol and propanol were similar at about 10-12%, and acetone and butanol showed no layer separation and no precipitation of acidic polysaccharides.

(3) Extraction of Acidic Polysaccharides by Concentration Using Alcohol

The dried red ginseng gourd was removed impurities using a 425 mesh sieve separator. A 10 ml solution was prepared using red ginseng gourd powder and purified water at a concentration of 0.1 g, 0.5 g, 1.0 g, 1.5 g, and 2.0 g. In order to extract an acidic polysaccharide, it extracted for 2 hours on 40 degreeC and 130 rpm conditions. After extraction, centrifugation (4 ° C., 3000 rpm × 15 minutes) was performed to remove solids. Ethanol was added to the centrifuge supernatant in a ratio of 1: 4 to precipitate crude acid polysaccharide. Centrifugation (4 ° C., 3000 rpm × 15 minutes) was performed for acidic polysaccharide recovery. The acidic polysaccharide slurry solution was then dried at 65 ° C. for 24 hours to powder.

Alcohol was selected as the optimum solvent by comparing the yield of acidic polysaccharide extraction by solvent. This experiment was performed to compare the extraction yield and the amount of crude polysaccharide extraction by varying the concentration of red ginseng. The results are shown in FIG. The yield of acidic polysaccharide increased with increasing red ginseng gourd concentration, but the yield decreased. When the amount of red ginseng gourd sample increased to 1-20%, the amount of acidic polysaccharide gradually increased from 8.49 mg / mL to 14.05 mg / mL, and the yield decreased from 84.95% to 7.03%. The results of this study show that the extraction yield is different depending on the concentration because the reaction kinetics of the polysaccharide components bound to the red ginseng gourd and the solvent are different as the concentration of the red ginseng gourd increases. Therefore, it is necessary to increase the number of extraction of polysaccharides when increasing the amount of red ginseng gourd.

Experimental Example 2. By surface reaction analysis Acidic Polysaccharide Extraction Optimization Process (RSM)

In order to determine the optimal conditions for the acidic polysaccharide extraction process, all experimental designs included a five-run center run with three independent variables: vacuum (temperature), time, and concentration of 370 mmHg, 3 hours, and 10%, respectively. A total of 17 treatment combinations were made. Box-Behnken Design proceeds according to three important procedures: first, the experiment is performed statistically according to the designed experiment, second, the coefficients of model are calculated, and third, the model. Proceed to determining the suitability of the.

In order to facilitate statistical calculation, independent variables are standardized as follows. The three variables were X ₁ (temperature), X ₂ (time) and X ₃ (concentration), respectively. The values of standardization can be obtained by the following formula and the value is Z.

--- (1)

--- (One)

Where X ⁰ is the center value of the normalized value and X is the normalized value. ΔX is the magnitude of the increasing or decreasing value by one unit. The analysis of the experimental results was used as the surface response analysis, and the multiple regression equations representing the optimal process conditions are as follows.

---(2)

---(2)

Here, Y is predicted response and if there are 3 variables as in this experiment, the value of k becomes 3 and ultimately expressed as follows.

---(3)

--- (3)

Statistical analysis of the results confirmed after the experiment was done using Design Expert (Couresy: Stat-ease Inc., Statistics Made Easy, Minneapolis, USA). The values of the independent variables were selected from the results obtained in the preliminary experiments, where X ₁ (vacuum degree) was set to 240 mmHg (-1), 370 mmHg (0) and 500 mmHg (+1), and X ₂ (time) was 2 (1), a 3 (0), 4 (+1), and, X ₃ (concentration) was set to 8% (1), 10% (0), 12% (+1)%.

Experimental results obtained by designing experiments using Box-Behnken Design for three experimental variables, reaction vacuum, time, and concentration, which are the extraction process conditions of acidic polysaccharides, ie, amount (g), yield (%), and production of crude polysaccharides The velocity (Pn) is shown in Table 1 below. The crude acid polysaccharide amount ranges from 62.4 to 135.6 g, the maximum predicted crude polysaccharide amount is 135.6 g, the yield range is 6.24 to 11.52%, the maximum predicted yield is 11.52%, and the production rate ranges from 1.56 to 4.52 Pn. The production rate was measured at 4.52 Pn.

As shown in Table 2 below, the results of the regression analysis of the regression equation for the amount, yield, production rate of the acidic polysaccharide shown in the experimental results. The analysis results of the Quadratic Regression Model indicate the suitability of the model. It was recognized that the amount, yield, and production rate of acidic polysaccharides were affected by reaction vacuum, time, and concentration within 95%.

The model determination coefficient R ² shows the degree of correlation between the observed and predicted values. The acidic polysaccharide content is 0.9877, the yield is 0.9872, and the production rate is 0.9935. %, Yield is 1.28% and production rate is not explained in the 0.65% range.

The lack of fit test test did not show any significance, indicating that the model used in this experiment is very appropriate. Table 3 below shows the regression coefficient of the model, and the acid polysaccharide content, yield, and production rate were significantly affected by three factors, namely, vacuum, time, and concentrations (P <0.05). The results of this experiment show that the influences on the degree of vacuum, time, and concentration are significant in the first, second, and cross product terms, indicating that the factors influence alone or alternately. The regression equations for these are shown in Table 4 below.

(1) Effect of Extraction Vacuum and Time on Crude Acid Polysaccharide Extraction Amount

The optimum surface reaction analysis results of the crude polysaccharide content with vacuum and time are shown in FIG. 3. The maximum value of crude polysaccharide content was obtained when the reaction temperature was 240 mmHg for 3 hours and the minimum value of the crude polysaccharide content was reacted with the vacuum degree 500 mmHg for 4 hours. The degree of vacuum gradually decreased from 240 mmHg to 370 mmHg and gradually increased after 370 mmHg. The time increased steadily from 2 hours to 4 hours, unlike the degree of vacuum. The crude acid polysaccharide extraction contents with vacuum and time ranged from 59.99 g to 114.74 g. Therefore, the optimum acidic polysaccharide extraction vacuum and temperature in the experimental condition region were hardly affected by the extraction time of the experimental region at low vacuum. Therefore, the vacuum degree is related to the extraction temperature, so the breaking point of the vacuum degree of 240 mmHg is about 75 ° C., so the extraction was carried out at 70 ° C. Therefore, the extraction time of the optimum condition extraction vacuum pressure 240 mmHg extraction time was about 98.7 g at 1 hour.

(2) Effect of Extraction Vacuum and Red Ginseng Gum Concentration on Crude Acid Polysaccharide Extraction Amount

The optimum surface reaction analysis results of the crude polysaccharide content according to the vacuum degree and the concentration are shown in FIG. 4. The maximum value of crude polysaccharide content was obtained when the reaction rate was 240 mmHg and 12% concentration, and the minimum value of crude polysaccharide content was obtained when the reaction rate was 500 mmHg and 10% concentration. The degree of vacuum gradually decreased from 240 mmHg to 370 mmHg and gradually increased after 370 mmHg. Concentrations increased steadily from 8% to 12%. The crude polysaccharide content ranged from 69.39 g to 133.51 g depending on the vacuum and concentration. The amount of polysaccharide extracted increased with higher red ginseng gourd concentration. This increase was affected by the extraction vacuum and was less affected by the extraction vacuum when the concentration of red ginseng was less than 9%. Therefore, the lower the vacuum, the greater the amount of extraction.

(3) Effect of Extraction Time and Red Ginseng Gum Concentration on Crude Acid Polysaccharide Extraction Amount

The optimum surface reaction analysis results of the crude polysaccharide content with concentration and time are shown in FIG. 5. The maximum value of crude polysaccharide content was observed when reacted with 12% concentration for 3 hours and the minimum value of crude polysaccharide content was reacted with concentration 10% for 4 hours. Concentration increased steadily from 8% to 12%, and time increased from 2 hours to 3 hours differently from concentration and then gradually decreased until 4 hours later. The crude acid polysaccharide content ranged from 59.85 g to 93.99 g.

(4) Effect of Extraction Vacuum and Extraction Time on Crude Acid Polysaccharide Extraction Amount

The optimum surface reaction analysis results of the degree of vacuum and the yield with time are shown in FIG. 6. The maximum value of crude polysaccharide content was obtained when the reaction temperature was 240 mmHg for 3 hours and the minimum value of the crude polysaccharide content was reacted with the vacuum degree 500 mmHg for 4 hours. The degree of vacuum gradually decreased from 240 mmHg to 370 mmHg and gradually increased after 370 mmHg. The time increased steadily from 2 hours to 4 hours, unlike the degree of vacuum. The yield range with vacuum degree and time ranged from 6.06% to 11.40%.

(5) Effect of Extraction Vacuum and Red Ginseng Extract Concentration on Crude Acid Polysaccharide Extraction Yield

The results of the optimum surface reaction analysis of the yield according to the vacuum degree and the concentration are shown in FIG. 7. The maximum value of the yield was obtained when the reaction temperature was 240 mmHg and 12% concentration, and the minimum value was obtained when the reaction rate was 500 mmHg and 10% concentration. The degree of vacuum gradually decreased from 240 mmHg to 370 mmHg and gradually increased after 370 mmHg. Concentrations decreased slowly from 8% to 12%. Yields ranged from 6.51% to 11.36% depending on vacuum and concentration.

(6) Effect of Extraction Time and Red Ginseng Extract Concentration on Crude Acid Polysaccharide Extraction Yield

The results of the optimum surface reaction analysis of the yield with concentration and time are shown in FIG. 8. The maximum value of the yield was obtained when reacted with 8% concentration for 3 hours, and the minimum value was obtained when reacted with 10% concentration for 4 hours. The concentration gradually decreased from 8% to 10% and then gradually increased from 10% to 12%, and the time increased from 2 hours to 4 hours differently from the concentration, and then became constant. The yield range with concentration and time ranged from 6.17% to 9.09%.

(7) Effect of Extraction Vacuum and Extraction Time on Crude Acid Polysaccharide Extraction Production Rate

The results of the optimum surface reaction analysis of the production rate with vacuum degree and time are shown in FIG. 9. The maximum value of the production rate appeared when the reaction rate was 240 mmHg and 3 hours, and the minimum value was produced when the reaction rate was 500 mmHg and 4 hours. The degree of vacuum gradually decreased from 240 mmHg to 370 mmHg, and after 370 mmHg, almost constant results were obtained. Similarly, time showed almost constant results from 2 to 4 hours. The production rate with vacuum and time ranged from 1.49 Pn to 4.19 Pn.

(8) Effect of Extraction Vacuum and Red Ginseng Extract Concentration on Crude Acid Polysaccharide Extraction Production Rate

The results of the optimum surface reaction analysis of the production rate according to the vacuum degree and the concentration are shown in FIG. 10. The maximum production rate was obtained when reacting with a vacuum degree of 240 mmHg and 12% concentration, and the minimum value was obtained when reacting with a vacuum degree of 500 mmHg and 10% concentration. The degree of vacuum gradually decreased from 240 mmHg to 370 mmHg and gradually increased after 370 mmHg. Concentrations increased rapidly from 8% to 12%. The production rate ranged from 2.33 Pn to 4.44 Pn depending on the vacuum and concentration.

(9) Effect of Extraction Time and Red Ginseng Gum Concentration on Crude Acid Polysaccharide Extraction Production Rate

The optimum surface response analysis results of production rate with concentration and time are shown in FIG. 11. The maximum production rate was obtained when reacting with 12% concentration for 3 hours and the minimum value was produced when reacting with 10% concentration for 4 hours. Concentrations increased sharply from 8% to 12%, whereas time decreased rapidly from 2 to 4 hours. The production rate with concentration and time ranged from 1.67 Pn to 3.95 Pn.

(10) Trend of Crude Polysaccharide Content, Yield, and Production Rate According to Vacuum Degree, Time, and Concentration

When extracting red ginseng gourd, the trend of crude polysaccharide content, yield, and production rate according to vacuum degree, time, and concentration is shown in FIG. 12. The trend of crude acidic polysaccharide content shows a sharp decrease with the degree of vacuum and then gradually increases, and gradually increases with time, and then gradually decreases with the concentration. Thus, the acidic polysaccharide content appears to be more affected by the degree of vacuum. The trend of yield shows a rapid decrease with the degree of vacuum and gradually increases from one point of time, gradually increases with time, and then gradually decreases, and gradually decreases with a certain concentration and then increases from a certain point of time. . Therefore, the yield also seems to be most affected by the degree of vacuum. The trend of the production rate shows a tendency to decrease rapidly with the degree of vacuum and then to become constant, to show a tendency to decrease rapidly with time, and to increase gradually with concentration. Therefore, the production rate seems to be affected by time more than vacuum degree, unlike content and yield.

Experimental Example 3. Isolation and Purification of Acidic Polysaccharides Using Ion Exchange Resin and Dialysis

An ion exchange resin was used to separate the antioxidant active acid polysaccharide from the crude acid polysaccharide. The ion exchange resin Dowex® 1 × 2, 200 resin was equilibrated in a column (2.5 × 100 cm), and 1 mL of 10% acidic polysaccharide was injected into the equilibrated resin. The elution sequence of the sample was first eluted with 100 mL of distilled water, followed by 0.1 M NaCl, 0.5 M NaCl and finally 1.0 M NaCl. Fraction samples were collected using an automatic fractionator Retriever 500 (Teledyne ISCO, USA), and eluent was collected in 5 mL in vitro. The polysaccharide of each eluate was measured and the antioxidant activity was measured. 4-14 (distilled water), 17-27 (0.1 M NaCl), and 40-50 (0.5 M NaCl) were collected in a 100 mL round bottom flask. Ten eluents, that is, 50 mL each, were collected in three, and the solvent was blown out with a rotary evaporator. Then, 5 mL of distilled water was added, dissolved in a dialysis membrane, and stirred for about 6 hours for dialysis. The dialyzed solution was frozen at −50 ° C. and then lyophilized with a lyophilizer.

A cation exchange resin was used to separate and purify the polysaccharide showing antioxidant activity from the crude acid polysaccharide. The samples eluting at similar locations were then combined and subjected to dialysis. The samples without salt by dialysis were lyophilized and used in subsequent experiments. Chromatogram for elution is shown in FIG. 13. Peaks of chemically similar polysaccharides appeared in three patterns: A peak shows distilled water, B peak shows 0.1 M NaCl, and C peak shows 0.5 M NaCl.

   In this experiment, the three separated acidic polysaccharides were divided into RAP 1, RAP 2, and RAP 3. In vitro solutions corresponding to RAP 1, RAP 2 and RAP 3 were collected and evaporated with a vacuum concentrator. Concentrated RAP 1, RAP 2, and RAP 3 solutions were desalted using a 10 kDa dialysis membrane.

Experimental Example 4. Measurement of Antioxidant Capacity of Acidic Polysaccharides

(1) DPPH radical scavenging activity measurement

Electron donating ability (EDA) for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging ability was measured using the isolated and purified from crude acid polysaccharide. In other words, a 0.1 mM DPPH solution was prepared, and 0.5 mL, 0.5 mL of distilled water, 0.5 mL of 0.1 M NaCl, 0.5 mL of 0.5 M NaCl, and 0.5 mL of distilled water were mixed in the same amount as the DPPH solution. The reaction was measured for 30 minutes and the absorbance was measured using a UV-1601 spectrophotometer (Shimaszu Co., Australia) at 517 nm, and the absorbance of the control was measured under the same conditions by distilled water. Ascorbic acid was used as a control, and the electron donating ability was measured by the same method as above, and compared with the sample group. Each test was repeated three times. The electron donating ability was calculated using the following formula.

DPPH radical scavenging activity (%) = [(B-A) / B × 100]

A: absorbance at sample addition, B: absorbance at sample addition

In this experiment, DPPH radical scavenging ability was calculated with peaks in three patterns. DPPH radical scavenging activity is the antioxidant activity of phenolic acids, flavonoids and other phenolic compounds. The greater the reducing power of these substances, the greater the DPPH radical scavenging activity.

As shown in FIG. 14, the comparative analysis of DPPH radical scavenging ability of the three fractions showed 67.70% in distilled water (A), 19.48% in 0.1 M NaCl (B), and 32.29% in 0.5 M NaCl (C). . In this experiment, the radical scavenging activity ranged from 19.48 to 67.70%, and the highest predicted polysaccharide eluted DPPH radical scavenging activity among the three peaks was 67.70%. The highest antioxidant activity was measured in distilled water (A). From the results of this experiment, the separated A using the eluent as distilled water showed the highest activity of DPPH, and the acidic polysaccharide structural analysis sample was measured using A.

(2) hydroxy radical scavenging ability

Scavenging activity against hydroxyl radicals was determined by modifying Avellar's method (Avellar et al 2004). Standard preparation was mixed with 10 mg Ascorbic acid in 10 ml deionized water (DIW). As a control, 0.1 M sodium phosphate buffer (pH 7.4) is used, and samples are prepared at 10,000 ppm and 20,000 ppm. The radical reaction was mixed with 300 ul sample solution, 300 ul 3 mM 1,10-phenanthroline, 300 ul 3 mM FeSO _4, and 300 ul 0.01% hydrogen peroxid, followed by shaking culture at 37 ° C. for 1 hour, followed by absorbance at 536 nm using a spectrophotometer. Was measured. Hydroxyl radical scavenging activity was calculated by the following formula comparing the absorbance of the sample to the control.

Hydroxyl radical scavenging activity (%) = {[(ΔA / min) b- (ΔA / min) s] / (ΔA / min) b} × 100

b: control, s: sample, ΔA / min: absorbance after reaction-absorbance before reaction

(3) superoxide radical scavenging ability

Digestive activity against superoxide was determined by modifying Xie's method (Xie et al 2008). As a control, 50 mM TrisHCl buffer (pH8.3) solution was used. Reaction was carried out by adding 500 ug of peptide to 500 μl 50 mM TrisHCl buffer (pH8.3) solution prepared using a micro tube and then mixing 400 μl 1.5 mM pyrogallol solution for 4 minutes at room temperature followed by spectrophotometer. Absorbance at 420 nm was measured. Digestive activity against superoxide was calculated by the following equation comparing the absorbance of the sample to the control.

Superoxide scavenging activity (%) = {[(ΔA / min) b− (ΔA / min) s] / (ΔA / min) b} × 100.

b: control. s: sample, ΔA / min: absorbance after reaction-absorbance before reaction

(4) Fe2 + chelation ability

Iron chelation ability was measured according to the method of Le et al. (2007). The extracted sample was dissolved in distilled water at a constant concentration (0-1%) and mixed with 500 μL, 100 μL FeCl ₂ (0.6 mM), and 900 μL methanol. After reacting at room temperature for 5 minutes, 100 μL ferrozine (5 mM) was added to the mixture and reacted at room temperature for 10 minutes. Absorbance was measured at 562 nm, and ethylenediaminetetraacetic acid (EDTA) was measured under the same conditions as the sample.

Iron chelation activity (%) = (1A / B) × 100

A: absorbance at sample addition, B: absorbance at sample addition

(5) red ginseng gourd acid polysaccharide in vitro Antioxidant experiment

Acidic polysaccharides derived from red ginseng gourd showed different DPPH scavenging ability, superoxide radical scavenging ability, hydroxy radical scavenging ability, and metal chelation ability, depending on the characteristics of the structure. DPPH scavenging ability and hydroxy radical scavenging ability were different, but the trends were similar, and superoxide radical scavenging ability and metal chelation ability were similar but showed similar tendency. These reasons can be attributed to the difference in the size and structure of the molecular weight constituting the acidic polysaccharide. To further compare the antioxidant inhibitory capacity of the hydrolysates, IC _{50 was} evaluated for each hydrolyzate.

As shown in Table 5 below, the overall antioxidant inhibition was significantly different depending on the acidic polysaccharide isolated. RAP 1 showed high inhibition in order of metal chelation ability, DPPH scavenging ability, superoxide radical scavenging ability, and hydroxy radical scavenging ability. Inhibition concentrations of metal chelation ability, DPPH scavenging ability, superoxide radical scavenging ability, and hydroxy radical scavenging ability were 4.08, 6.10, 7.80 and 55.7 mg / ml, respectively. RAP 2 showed high inhibitory ability in the order of metal chelation ability, superoxide radical scavenging ability, hydroxy radical scavenging ability and DPPH scavenging ability. Metal chelation ability, superoxide radical scavenging activity, hydroxy radical scavenging activity and DPPH scavenging inhibitory concentrations were 1.19, 7.20, 9.15 and 15.8 mg / ml, respectively. RAP 3 showed high inhibition ability in the order of metal chelation ability, superoxide radical scavenging ability, hydroxy radical scavenging ability, DPPH scavenging ability. Metal chelation ability, superoxide radical scavenging activity, hydroxy radical scavenging activity and DPPH scavenging inhibitory concentrations were 3.20, 5.68, 13.40 and 55.7 mg / ml, respectively.

Experimental Example 5. Quantitative and structural analysis of acidic polysaccharides

(1) Acidic Polysaccharide Quantitative Analysis

Acidic polysaccharides contained in the red ginseng alcohol extract were measured by the carbazole sulfuric acid method, which is commonly used for the determination of pectin, and carbazole was used as a control. 10 mL of distilled water was added to the dried powder to dissolve well. 1 mL of the solution dissolved in a 20 mL Falcon tube was taken and 9 mL of distilled water was added. 0.5 mL of the diluted solution and 0.25 mL of carbazole were added, mixed, and 3 mL of sulfuric acid was added thereto. The dissolved solution was reacted for 5 minutes in an 85 ° C. water bath. After the reaction, the mixture was cooled for 15 minutes at room temperature. Absorbance at 525 nm was measured using a spectrophotometer.

(2) Acid Polysaccharide Structure Analysis

FT-IR used a surface reflection infrared spectrometer. Sample preparation was made by mixing 1.5 mg (100: 1) of sample with 150 mg of KBr, grinding it in a mortar, and making KBr pellets and fixing them on an ATR apparatus. Absorbance was measured by VERTEX 80v FT-IR (Bruker, USA). NMR measured both ¹H NMR (400 MHz) and ¹³C NMR spectra (100 MHz). Sample preparation was prepared by dissolving a 30 mg sample in 0.6 mL of CDCl₃ solvent. ¹ H NMR (400 MHz) and ¹³ C NMR spectra (100 MHz) were measured with a JNM-AL400 NMR spectrometer (Jeol, Japan).

(3) acidic polysaccharide structure analysis

The FT-IR was used to analyze the specific functional groups of the sugars constituting the acidic polysaccharides by combining the elution amount corresponding to the peak with the highest antioxidant activity.

As shown in Fig. 15, the analysis shows that the chromatogram shows a typical OH stretching vibration of carbohydrates at 3294 cm ⁻¹ and saturated CH at 2932 cm ⁻¹ , with a relatively strong absorption peak of C═O at 1018 cm ⁻¹ . At 1149 cm ^-1 , COC and glycosidic bonds showed a typical polysaccharide absorption pattern. Two strong absorption 1149, 932 cm ^-1 appeared on pyrano seed peak, 1,100 - two peaks on furanyl no seed of 1,010 cm ^-1 range appeared in 1,636 cm ^-1 to the strongest peak. In addition, the absorption bands of the carboxyl groups at about 1,636 cm ⁻¹ (asymmetric COO stretching vibration) and 1,343 cm ⁻¹ (symmetrical COO stretching vibration) were most prominent in the FT-IR spectrum. Therefore, the results of this study indicate that the sample with antioxidant function consists of glycosidic bonds with residues such as L-arabinise, D-galactose, L-rhamnose, D-galacturonic acid, D-gluconic acid and D-galactosyl. This is consistent with previous studies. Subsequently, NMR was used to analyze the chemical bond structure of the sample acidic polysaccharide.

NMR data is shown in Figure 16 to measure the dialysis using distilled water as the eluent when the acidic polysaccharide is separated. Resonance spectra of the ¹H NMR of the acidic polysaccharide were observed in the 8.46-10.45 ppm region. At 8.46- 9.34 ppm, H-2, H-3, H-4, H-5 and H-6 and hemiacetal H-1 were observed as multiplats. And at 9.83 ppm, it was found that it is a typical (1 → 6) chain-extending anomeric peak. Bonds linked by (1 → 3) appeared at 10.45 ppm.

As shown in FIG. 17, the spectra of ¹³C NMR of the acidic polysaccharide were observed at 94.84-134.00 ppm. The peaks of C-2, C-3, C-4 and C-5 were found around 105-110 ppm and anomeric C-6 peaks at 94.84 ppm. The peaks of C-1 and C-6 show approximately the same intensity, indicating that there is a (1 → 6) glycosidic bond.

Experimental Example 6. Determination of antimicrobial activity of acidic polysaccharides

In this experiment, the antimicrobial experiment of acidic polysaccharide was performed by the disk diffusion method. In this method, agar containing a certain concentration of a sample on agar plate inoculated with the test bacteria is incubated, and the sample is diffused at a constant speed on the medium to make a gradient of a certain concentration. Thus, inoculated test bacteria will only grow where the concentration of the sample in the medium does not inhibit proliferation. Therefore, a growth inhibition zone is formed around the disk, and its size is proportional to the sample concentration and the sensitivity of the test organism. Therefore, it is possible to qualitatively determine the effect of a product or raw material having an antimicrobial effect by observing the presence or absence of the development low zone.

In order to prepare a TSA agar medium, a certain amount was put in the flask, the flask was capped tightly with aluminum foil, and steam autoclave sterilized (121 ° C., 1.5 psi, 15 min) in an autoclave. After completion of sterilization, the flask was removed from the autoclave and cooled while stirring at room temperature. When the medium cooled to about 45-50 ° C., the medium was poured into a petri-dish in a clean-bench and hardened.

Sample solutions Acidic polysaccharides were prepared by concentration, sterilized in autoclave (121 ℃, 1.5psi, 15min) wrapped in aluminum foil using a disk with a diameter of about 8 mm, and sufficiently wet after drying in an oven. The test strain was Eschrichia coli and diluted after incubation so that the number of bacteria in the suspension was 10 ^5-6 cfu / plate.

100 μl each of the test bacteria suspension prepared in the prepared agar medium was injected into TSA, and the bacteria solution was evenly applied using a sterile swab. Covered with a lid of Petri-dish, dried for about 3 to 5 minutes and placed on the surface of the prepared disk medium, and pressed lightly. The bacteria-inoculated agar medium was confirmed whether the growth zone was formed after 24 hours incubation at 30 ℃.

In this experiment, the results of the antimicrobial experiment on the isolated and purified acidic polysaccharide are shown in FIG. 18. For further verification, purified acidic polysaccharides were sent to Mokwon National University Ecological Resource Institute for analysis. The purified acidic polysaccharide RAP A and the purified acidic polysaccharide RAP B showed antimicrobial activity in laboratory results, whereas the results of the Ecological Resources Research Institute showed that only purified acidic polysaccharide RAP A showed antimicrobial efficacy. This difference seems to be due to the concentration of the sample used in the experiment. However, isolated acidic polysaccharides have been shown to have antioxidant and antimicrobial activity. Therefore, the prospect of manufacturing functional food using raw materials produced by the process developed in the present invention is expected to be high.

Example 1 Functional Beverage Production Method Using Acidic Polysaccharides

(1) Preparation and blending ratio of functional drinks using acidic polysaccharides

In preparing functional drinks using acidic polysaccharides, the red ginseng gourd provided by the company was separated by 425mesh, and the fine red ginseng gourd was put in distilled water and extracted in a vacuum extraction concentrator. The precipitate was removed and the supernatant concentrated in the same vacuum extraction concentrator. Centrifugation was performed to separate only the supernatant.

The recovery of the acidic polysaccharide was carried out in a ratio of supernatant: ethanol = 1: 4, and after 1 hour, the precipitate was separated and hot air dried at 60 ° C. for 24 hours. Hot air dry acidic polysaccharide was ground finely and powdered. Ginseng to be used for beverage preparation was washed clean and hot air dried 72 hours. The dried ginseng was ground finely using a blender. Finely ground ginseng powder was filtered with 200 and 425 mesh and collected separately for beverage preparation. Wolfberry and Schisandra chinensis were used for preparing the beverage as it was dried.

The blending ratio of the liquid teas is shown in Table 1 below. Basically, the ratio of fructose, sodium citrate, wolfberry and schizandra fruits was fixed and the ratio of acidic polysaccharides was prepared.

Compounding ratio (% by weight) APB1 APB2 APB3 APB4 Red Ginseng Acid Polysaccharide 0.5 1.5 3 5 Dry wolfberry fruit 0.5 0.5 0.5 0.5 Dried schizandra 0.5 0.5 0.5 0.5 Ginseng Powder 1.25 0.25 0.25 0.25 fruit sugar 5 5 5 5 Sodium citrate 0.1 0.1 0.1 0.1 Purified water 92.15 92.15 90.65 88.65 total 100 100 100 100

(2) Sensory Evaluation of Functional Drinks Using Red Ginseng Extract Acid Polysaccharides

The sensory test of the prototype was performed on five panel and five points at the lowest score of 1 panel of 20 Food and Biomedical Sciences students for the color, taste, aroma, and overall acceptability. The analysis was carried out. The basic characteristics of the liquid tea should have its own color and flavor and be free of odors. The final product must be sterilized. In this sensory evaluation, the basic rules were conducted to minimize the biases of the evaluators. Samples of the sensory evaluation APB1, APB2, APB3, APB4 were put into 50 ml test subject tea in about 100 ml cup and measured for sweetness, sourness, astringent taste, color, aroma, and total acceptability. The overall sensory evaluation results are shown in Table 2 below, and the average values for the evaluation of sweetness, sourness, astringent taste, color, aroma, and total acceptability are shown in FIG. 19. As a result of the evaluation, APB 2 had the highest preference.

Example 2 Preparation of Functional Concentrate Using Acidic Polysaccharides

(1) Preparation and Formulation Table of Functional Concentrate Using Acidic Polysaccharides

The red ginseng solution was supplied by Geumsan Eunhye Red Ginseng, which is the association of agricultural associations. The raw materials and ingredients are 100% of red ginseng (domestic) (70mg / g of red ginseng components, more than 60% of solid content) and the proportion of red ginseng root is 70% and red ginseng 30%. The blending ratio of the concentrates is shown in Table 3 below. Red ginseng solution was prepared by varying the ratio of acidic polysaccharide and red ginseng concentrate.

Compounding ratio (% by weight) APC1 APC2 APC3 Red Ginseng Acid Polysaccharide 5 One 3 Red Ginseng Concentrate 95 99 97 total 100 100 100

(2) Concentration Sensory Evaluation of Red Ginseng Pak Acid Polysaccharide

The sensory test of the prototype was performed on five panel and five points at the lowest score of 1 panel of 20 Food and Biomedical Sciences students for the color, taste, aroma, and overall acceptability. The analysis was carried out. The basic character of the concentrate should be inherent in color and flavor and free of odors and odors. The final product must be sterilized. In this sensory evaluation, the basic rules were conducted to minimize the bias of evaluators. Samples of APC1, APC2, and APC3, which are subjects of sensory evaluation, were placed in a 100 ml cup of 50 ml test subject tea and measured for sweetness, sourness, astringent taste, color, aroma, and total acceptability. The overall sensory evaluation results are shown in Table 4 below, and the average values for the evaluation of sweetness, sourness, astringent taste, chromaticity, aroma, and total acceptability are shown in FIG. 20. According to the evaluation results, APC 2 was the most preferred in overall preference.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (2)

A beverage composition further comprising a dry goji fruit, dried Schisandra chinensis and ginseng powder to the acidic polysaccharide powder, characterized in that it is prepared in the following steps, containing as an active ingredient.
(a) drying the red ginseng gourd for 12 hours at 60 ° C., then grinding the dried red ginseng gourd into 425 mesh and removing impurities using a 425 mesh sieve separator;
(b) 10 times of purified water was added to the pulverized red ginseng gourd powder, and extracted for 4 hours at a temperature of 95-98 ° C. to prepare a red ginseng gourd solution of 12% concentration. Extracting at 130 rpm for 2 hours;
(c) precipitating crude polysaccharide by centrifugation at 4 ° C. and 3000 rpm for 15 minutes to remove solids after the extraction and then adding ethanol in a ratio of 1: 4 to the centrifuge supernatant; And
(d) centrifuging at 4 ° C. and 3000 rpm for 15 minutes to recover the acidic polysaccharide after the precipitation, and then drying the acidic polysaccharide slurry solution at 65 ° C. for 24 hours to powder it delete

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