KR20020031561A - Oligosaccahrides from Ginseng and process for preparation thereof - Google Patents

Abstract

PURPOSE: Provided is a method for manufacturing oligosaccharide from ginseng, in particular, from ginseng dregs generated from the manufacturing ginseng extract, by treating polysaccharide hydrolyase. CONSTITUTION: The manufacturing method comprises the steps of: heat-vaporizing or freeze-drying ginseng dregs to remove remaining moisture or solvent therefrom then followed by pulverizing; adding polysaccharide hydrolyase, such as cellulase, hemiplegia, and pectinase, to the ginseng dregs then hydrolyzing the ginseng dregs at 30-60 deg.C for 0.5-6 hours with stirring; heating the above enzyme treated sample at 100 deg.C for 10-30 minutes to inactivate the enzyme then centrifuging it at 5000-10000xg for 10-30 minutes to obtain oligosaccharide from the supernatant; and filtering the supernatant followed by passing it through ultrafiltrator with ultrafiltration membrane of MW 500-5000 then fractioning it according to molecular weight.

Description

Ginseng oligosaccharide and its manufacturing method {Oligosaccahrides from Ginseng and process for preparation etc}

The present invention relates to a ginseng oligosaccharide and a preparation method thereof. More specifically, the present invention relates to a ginseng oligosaccharide prepared from ginseng foil by a polysaccharide hydrolase treatment process and a method for producing the same.

Ginseng (Panax ginseng C. A. Meyer) is a plant native to the Far East of Asia (North 33-48: Korea, North Manchuria, and some parts of the Soviet Union). In particular, ginseng produced in Korea is called Korean ginseng and is well known for its pharmacological activity. Ginseng is manufactured in the form of ginseng, red ginseng, white ginseng, and taeguksam according to the production process.

Ginseng has proven to be very effective in preventing and treating various adult diseases such as arteriosclerosis, hypertension, stress, fatigue recovery, energy enhancement, hypotension, menopausal disorders, diabetes, cancer, and anti-aging (Nutrition , 16 (5), 391-392 (2000); European Journal of Clinical Pharmacology, 55 (8), 567-575 (1999)).

Industrially, ginseng is used to extract the extract using a solvent such as hot water or alcohol. After extracting the extract, the main component of ginseng gourd is mostly made up of dietary fiber material consisting of polysaccharides, so it is expected that various physiological activities are expected.

Oligosaccharides are low-molecular carbohydrate materials that can be divided into two types depending on the type of production. In other words, isomalto-oligosaccharides, fructo-oligosaccharides, galacto-oligosaccharides, and the like, prepared by enzymatic biocon versions of sugars, are used for the hydrolysis of polysaccharides. Inulin oligosaccharides, alginate oligosaccharides, and chitosan oligosaccharides produced by the food industry and nutrition. 3 (1), 18-23 (1998).

The oligosaccharide has a physiological activity as a bifidogenic factor that promotes proliferation of useful intestinal microorganisms, and in the case of oligosaccharides produced by polysaccharide hydrolysates, blood cholesterol lowering action, antibacterial action, immune system activation action, etc. Physiological and pharmacological functionality (Lebensm. Wiss. Technol., 27, 1-9 (1996); Tren. Food Sci. Technol., 7, 353-368 (1996)).

On the other hand, the plant cell wall is composed of polysaccharide components such as cellulose, hemicellulose, pectin, lignin, glycoprotein, and the like. These components, however, do not exist in a free state, but are mostly insoluble and covalently linked through hydrogen bonds, ionic bonds, and hydrophobic bonds (Korean Journal of Nutrition, 23 (2): 358-). 370 (1994).

Generally, acid or alkali solution is used at high temperature as a conventional method for accepting or hydrolyzing polysaccharide components from plant cell walls, but such chemical hydrolysis method has many problems such as generation of large amount of environmental wastewater and corrosion of containers. It is implicated and difficult to apply industrially.

Insoluble polysaccharide components that make up the plant cell wall can be efficiently solubilized using polysaccharide hydrolase. Ginseng gourd is mostly composed of polysaccharides, so it is possible to produce low molecular weight oligosaccharides when using polysaccharide hydrolysis. The constituent polysaccharides of ginseng are composed of cellulose, hemicellulose, and pectin, so that the hydrolysis is very efficient by using cellulase, hemicellulases, pectinases, etc. It is possible.

In addition, polysaccharide hydrolase such as cellulase, hemicellulase, pectinase and the like can be easily obtained commercially and is easy for industrial application. In particular, since these commercial enzymes are not composed of a single enzyme but are composed of several enzymes, they can be more efficiently used for hydrolysis of plant cell walls composed of complex polysaccharides.

Conventionally, Korean Patent Registration No. 1997-8129 discloses a method for separating acidic polysaccharides from ginseng or alcohol extract ginseng. The present invention is a method for separating high-molecular-weight polysaccharides by separating enzymes by adding enzymes and adding 60% or more alcohol to the final product by precipitating high-molecular weight polysaccharides, while low-molecular-weight sugars are separated by alcohol. The disadvantage is that it cannot be precipitated sufficiently. In addition, the present invention does not specifically specify the specific application characteristics or manufacturing company of the used enzyme, but since the target final product is a polysaccharide of the polymer, it is important to produce a large amount of polysaccharide as much as possible by applying an enzyme having low hydrolytic enzyme activity. It is considered to be an invention based on this.

Therefore, the invention has not been implied at all about the method for producing a low molecular weight oligosaccharide from ginseng foil, and there is no known method for producing a low molecular weight oligosaccharide using ginseng so far.

The present inventors have completed the present invention by efficiently preparing low-molecular-weight ginseng oligosaccharides from ginseng gourd using polysaccharide hydrolase, taking the above points into consideration.

Accordingly, it is an object of the present invention to provide a method for producing low molecular weight oligosaccharides from ginseng by polysaccharide hydrolysis.

Another object of the present invention is to provide a low molecular weight oligosaccharide prepared using polysaccharide hydrolase from ginseng.

The above object of the present invention is to treat the ginseng extract produced as processed by-products during the production of ginseng extract, the polysaccharide hydrolase cellulase, hemicellulase, pectinase alone or a mixture of these enzymes in a weight ratio of ginseng foil 1 It is added at a ratio of 1: 1: 0.001 and reacted at 30 to 60 ° C. for 30 minutes to 6 hours to hydrolyze the polysaccharides present in ginseng foil, and then heat-treated to inactivate the enzyme to obtain a molecular weight of 500 to 5,000. Passed through the ultrafiltration membrane sequentially and then fractionated by molecular weight and dried to achieve ginseng oligosaccharides.

1 is a schematic diagram showing a preferred method for producing ginseng oligosaccharide of the present invention by process.

Figure 2a is a standard curve of gel permeation chromatography prepared using the standard pullulan (laclan) and lactose (lactose).

Figure 2b is a gel permeation chromatography results showing the molecular weight of the ginseng oligosaccharide of the present invention.

3 is a growth curve of Bifidobacterium longum in the medium to which the present invention ginseng oligosaccharide is added.

In the present invention, 500 mL of distilled water was added to 20 g of dried ginseng foil, followed by stirring for 30 minutes. Then, 2 g of Pitraze BR was added thereto, stirred at 50 ° C. for 2 hours, and heated at 100 ° C. for 20 minutes to inactivate the enzyme and centrifuge to obtain a supernatant. Preparing a ginseng oligosaccharide by drying the fraction passed through the ultrafiltration membrane; Measuring molecular weight of the ginseng oligosaccharide using gel permeation chromatography; Measuring the proliferative effect of Bifidobacterium long gum in the MRS broth to which the ginseng oligosaccharide of the present invention is added; Measuring the production yield and molecular weight according to the type of hydrolase when preparing ginseng oligosaccharides; Measuring the yield and molecular weight of the ginseng oligosaccharide when the hydrolase is added to the mixture during production; The ginseng oligosaccharide solution is passed through ultrafiltration membranes having different molecular weights to prepare ginseng oligosaccharides having various molecular weights.

In the present invention, water was added to the dried ginseng powder or ginseng foil, followed by stirring, and then polysaccharide hydrolases, cellulase, hemicellulases, and pectinases were added alone or in combination. At this time, a commercially available enzyme was used as a method for adding a polysaccharide hydrolase.

Commercially used in the present invention is the cellulase (Celluclast, Novo Nordisk, Denmark), hemicellase as Ceremix L (Ceremix L Novo Nordisk, Denmark), Pilatese BR (Filtrase BR Gist-Brocades, Netherlands), Ultraflo L (Novo Nordisk, Denmark) was used as pectinase (Pectinex, Novo Nordisk, Denmark).

After enzymatic reaction, ginseng oligosaccharide fraction was obtained from the supernatant by centrifugation, and then fractionated by molecular weight using ultrafiltration and then lyophilized or hot air dried.

The manufacturing method of ginseng oligosaccharides by the enzymatic method using the polysaccharide hydrolase will be described in detail for each manufacturing process.

Preferred method for preparing ginseng oligosaccharide of the present invention is shown in FIG.

First step: manufacturing ginseng foil

After extracting the extract from the inside of ginseng by adding hot water, solvent such as ethanol or methanol to ginseng products such as red ginseng, white ginseng and taegeuk ginseng, the ginseng foil produced as processed by-products is collected. Water or solvent remaining in the ginseng foil is dried using a heat evaporator or a lyophilizer and then ground to prepare a ginseng foil. Analysis of the sugar component of ginseng meal used in the present invention by the phenol-sulfuric method showed that more than 98% of the ginseng meal is composed of polysaccharides of the sugar component.

Second Process: Polysaccharide Hydrolase Treatment Process

1 to 10% (w / v) of ginseng foil prepared in the first step was added to distilled water, followed by cellulase, hemicellulase, pectinase alone or two kinds of commercial polysaccharide hydrolase by weight ratio. 1: 1 to 1: 0.001 was added to the mixed enzyme solution of the phase, followed by stirring at a temperature of 30 to 60 ° C. for 30 to 480 minutes depending on the reaction conditions of the enzyme. The sample is heated at 100 ° C. for 15 minutes to inactivate the enzyme, and then centrifuged at 5,000 to 10,000 × g for 10 to 30 minutes to obtain a water-soluble ginseng oligosaccharide solution from the supernatant. At this time, the precipitate can be dried and used as dietary fiber.

Third Step: Manufacturing Process of Ginseng Oligosaccharides by Molecular Weight Using Ultrafiltration Membrane

The ginseng oligosaccharide solution prepared in the second step was sequentially passed through an ultrafiltration membrane having a molecular weight of 500 to 5,000 using an ultrafiltrator, followed by drying to prepare a ginseng oligosaccharide for each molecular weight.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Preparation of Ginseng Oligosaccharide of the Present Invention

500 g of distilled water was added to 20 g of dried ginseng foil, followed by stirring for 30 minutes, and then 2.0 g of Pitraze BR (Gist-Brocades, Netherlands) was added thereto, followed by stirring at 50 ° C. for 2 hours. The enzyme was inactivated by heating at 100 ° C. for 20 minutes, centrifuged at 6,000 × g for 10 minutes, and the supernatant was filtered. The yield of ginseng oligosaccharides was determined by the solids content of the supernatant and found to be 56.7%. This result means that the ginseng oligosaccharide component can be successfully separated from ginseng gourd by the treatment of polysaccharide hydrolase. In addition, the oligosaccharide prepared in the above example did not produce a precipitate due to the addition of alcohol showed that it is composed of a low molecular weight oligosaccharide.

Ginseng oligosaccharide of the present invention can be used in beverages, confectionery, bakery and health food.

Experimental Example 1: Measurement of the molecular weight of the ginseng oligosaccharide of the present invention

The molecular weight of ginseng oligosaccharides was measured by gel permeation chromatography. Gel permeation chromatography used for the measurement of molecular weight was made by Waters LC Module I of the United States, the detector was M410-RI with a heating chamber and M2010 Millennium software It was. The column was an ultrahydrogel 120 & 250 column and 0.1M NaNO ₃ as the mobile phase. The rate of mobile phase was 0.8mL / min in the analysis, and pullulan and lactose were used as standards.

As a result of the experiment, the weight molecular weight of the ginseng oligosaccharide prepared in Example 1 as shown in FIGS. 2A and 2B was 1,050.

Experimental Example 2: Intestinal microbial proliferation effect of the ginseng oligosaccharide of the present invention

In this experiment, Bifidobacterium longum isolated from Korean feces was used as intestinal microorganism (Korean J. Dairy Sci., 11 (1), 16-25, 1989). Bifidobacterium longgum was used in the experiment after increasing the activity by third passage in anaerobic conditions in 20ml MRS broth having the composition shown in Table 1 below. At this time, the medium containing no oligosaccharide was used as a control. Inoculate Bifidobacterium longgum to the medium to which the ginseng oligosaccharide was added and the medium to which the ginseng oligosaccharide was added, while incubating in an incubator at 37 ° C., the culture medium was sampled at an interval of 2 hours to measure absorbance at 660 nm and to measure growth curve. Created.

As a result, as shown in FIG. 3, when the ginseng oligosaccharide prepared in Example 1 was added to the medium, the absorbance after 20 hours of cultivation was 2.95, and the absorbance of the control group was 0.22. That is, when the present invention ginseng oligosaccharides were added, the growth of Bifidobacterium long gum was much better than that of the control group which was not added.

Preparation of MRS broth Medium for Cultivation of Bifidobacterium Longgum Furtherance content Peptone 20 g Meat extract 10 g Yeast extract 1 mL Tween 80 2 g Ammonium citrate 5 g Sodium Acetate 0.1 g Magnesium Sulfate (MgSO4, 7H2O) 0.05 g Manganese Sulfate (MnSO4 · 5H2O) 2 g Agar 15 g Ginseng Oligosaccharide 20 g [Note] 1L of total volume of medium, pH 6.0

Examples 2 to 6: Effects of Kinases on the Production of Ginseng Oligosaccharides

The effect of the type of hydrolase on the production of ginseng oligosaccharides was investigated. 500 g of distilled water was added to 10 g of dried ginseng foil and stirred for 30 minutes, followed by Celluclast (Novo Nordisk, Denmark), Ultraflo L (Novo Nordisk, Denmark), Ceremix (Novo Nordisk, Denmark), Pectinex (Novo Nordisk, Denmark). ), FiltraseBR (Gist-Brocades, Netherlands) was added to each 0.1g each ginseng oligosaccharides were prepared according to the conditions shown in Table 2. Production yield and molecular weight of the prepared water-soluble ginseng oligosaccharides were confirmed by the same method used in Example 1 and Experimental Example 1, respectively.

As a result, as shown in Table 2, the production yield was 19.6∼43.1% and the molecular weight was 1,180∼1,820, depending on the type of hydrolase, and the oligosaccharide yield was obtained when hemicellulase, Filtrase BR, was used. The highest yield was 43.1%, and the yield of oligosaccharide was 40.7% even when pectinase was used. This means that it is possible to produce ginseng oligosaccharides having various yields and molecular weights depending on the type of hydrolase.

Production of Ginseng Oligosaccharides According to the Types of Hydrolase Example Yeo (brand name) Reaction temperature Reaction time Reaction pH Oligosaccharide yield (%) Molecular Weight Example 2 Celluclast 60 ℃ 1 hours 5.0 20.5 1,820 Example 3 Ceremix 30 ℃ 1 hours 6.5 38.9 1,530 Example 4 Filtrase 50 ℃ 1 hours 6.0 43.1 1,250 Example 5 Pectinex 50 ℃ 1 hours 5.0 40.7 1,180 Example 6 Ultraflo 50 ℃ 1 hours 6.0 20.3 1,470

Examples 7-10: Mixing Effect of Hydrolase in Preparation of Ginseng Oligosaccharides

Changes in yield and molecular weight of ginseng oligosaccharides were investigated when the enzymes used in Examples 2 to 6 were mixed in the same amount by weight or two and three at the same conditions as in Examples 2 to 6.

As a result, as shown in Table 3, the production yield of ginseng oligosaccharides was low as 20.5% when Celluclast alone was used in Example 2, but the production yield was large when other enzymes were mixed. Increasingly, the production yield of oligosaccharide was 50.4% when the reaction was added with Celluclast, Filtrase, and Pectinex.

Preparation of Ginseng Oligosaccharides by Mixing Hydrolytic Enzymes Example Yeo (brand name) Reaction temperature Reaction time Reaction pH Oligosaccharide yield (%) Molecular Weight Example 7 Celluclast + Ceremix 40 ℃ 1 hours 6.0 38.5 1,670 Example 8 Celluclast + Filtrase 50 ℃ 1 hours 6.0 48.6 1,320 Example 9 Celluclast + Pectinex 50 ℃ 1 hours 6.0 47.2 1,120 Example 10 Celluclast + Ultraflo 50 ℃ 1 hours 6.0 33.6 1,420 Example 11 Celluclast + Filtrase + Pectinex 50 ℃ 1 hours 6.0 50.4 980

Examples 12-16: Molecular Weight Fraction of Ginseng Oligosaccharides Using Ultrafiltration

The ginseng oligosaccharide solution prepared in Examples 2 to 6 was sequentially passed through an ultrafiltration membrane having a molecular weight of 3000, 1000, and 500 using an ultrafilter (Amicon, USA) and then lyophilized. As shown in Table 3, the yield of oligosaccharide fraction by molecular weight of ginseng oligosaccharide was 10% or less when the molecular weight was 3,000 or more, and the oligosaccharide production yield was 15-30% when the molecular weight was 1,000 to 3,000, and the molecular weight was 500 to In the case of 1,000, the yield of oligosaccharide was 40-50%, and the yield of the molecular weight 500 or less was 18-20%. Therefore, it could be seen that the use of ultrafiltration according to the type of enzyme applied to prepare ginseng oligosaccharides with various molecular weight distributions.

Production of Ginseng Oligosaccharides with Various Molecular Weight Distributions Using Ultrafiltration Molecular weight fraction Yield (%) Example 12 Example 13 Example 14 Example 15 Example 16 > 3,000 9.4 10.6 8.9 8.1 7.4 1,000 to 3,000 30.9 28.5 15.6 20.7 25.3 500-1,000 39.1 40.7 52.1 53.4 47.6 <500 20.6 20.2 23.4 17.8 19.7

As described above, the ginseng oligosaccharide of the present invention and the method for preparing the same have an effect of easily preparing ginseng oligosaccharides having intestinal bacterial growth activity from ginseng meal by using a polysaccharide hydrolase and an ultrafilter. Since ginseng oligosaccharides with various molecular weight distributions are produced by the type of enzyme and ultrafiltration method applied during production, they are very useful inventions in the food processing industry.

Claims (2)

(a) extracting ginseng extract (extract) and collecting ginseng foil produced as a processed by-product and drying and grinding the water or solvent remaining in the ginseng foil using a heat evaporator or a lyophilizer to prepare a ginseng foil ; (b) the ginseng foil of step (a) was treated with cellulase, hemicellulase, and pectinase as polysaccharide hydrolase alone or a mixture of these enzymes was added at a weight ratio of 1: 1 to 1: 0.001. And then hydrolyzing the polysaccharide in ginseng foil by stirring at 30 to 60 ° C. for 30 minutes to 6 hours; (c) heating the enzyme treatment sample of step (b) at 100 ° C. for 10 to 30 minutes to inactivate the enzyme, and then centrifuging at 5,000 to 10,000 × g for 10 to 30 minutes to obtain a water-soluble ginseng oligosaccharide solution from the supernatant. step; (d) filtering the supernatant obtained after centrifugation in step (c) and then sequentially passing through an ultrafiltration membrane having a molecular weight of 500 to 5,000 using an ultrafilter, and then fractionating by molecular weight to prepare ginseng oligosaccharides. Ginseng oligosaccharide manufacturing method characterized in that. A ginseng oligosaccharide prepared by the method according to claim 1, which is a low molecular polysaccharide having a molecular weight of 500 to 5,000.