KR20080041784A - A method for making special beta-glucan by hydrolyzing crude beta-glucan - Google Patents

Abstract

A method for preparing betaglucan through hydrolysis of natural betaglucan present in oat bran is provided to obtain betaglucan capable of being added to beverage in an amount up to 2% and having an effect of inhibiting cholesterol equivalent to natural betaglucan. A method for preparing betaglucan comprises a step of carrying out hydrolysis of natural betaglucan present in oat bran. The hydrolysis is performed by using cellulase. Particularly, oat bran is treated with 1-3% of cellulase for 2-3 hours. Betaglucan obtained after the treatment using cellulase has a molecular weight of 6.0x10^5-7.0x10^5.

Description

Method for producing beta glucan through hydrolysis of natural beta glucan present in oat bran {A METHOD FOR MAKING SPECIAL BETA-GLUCAN BY HYDROLYZING CRUDE BETA-GLUCAN}

1 is a graph showing the substrate specificity according to the enzyme activity of Example 1.

Figure 2 is a graph showing the molecular weight change of oat bran beta glucan partially hydrolyzed with endo and exo type cellulase of Example 1.

3 is a graph showing the flow curve of the oat bran beta glucan solution partially hydrolyzed with the endo type (3A) and exo type (3B) cellulase of Example 1.

4 is a graph showing the bile acid binding capacity of partially hydrolyzed oat bran beta glucan obtained under various enzymatic hydrolysis conditions of Example 2. FIG.

The present invention relates to a method of producing beta glucan by hydrolyzing natural beta glucan present in oat bran, the viscosity of which is 70% lower than that of natural beta glucan and the inhibitory effect of cholesterol. More specifically, oat beta glucan hydrolyzate having an optimal bile acid binding capacity is selected while minimizing the rate of increase in viscosity when added to liquid, compared to natural beta glucan, and the cholesterol lowering ability through animal experiments is verified. It is possible to add up to%, and the ability to lower cholesterol as compared to natural beta glucan is related to a method for producing beta glucan to develop equivalent beta glucan to contribute to the public health.

Beta-glucan ((1-3), (1-4) -β-D-glucan) is a type of dietary fiber that is not digested and absorbed by the body, and is produced in various forms from yeast, mushrooms, and cereals. In general, beta glucan has a different structure and characteristics depending on the raw material, beta glucan is mainly present in the cell wall, the structure is a straight chain consisting of a variety of β-1,3 and β-1,4 bonds It is a polysaccharide. Such beta glucan is contained in oats and barley in large quantities, and especially contains high oats beta glucan.

Dietary fiber shortens the transit time of the ingested high-fat diet and slows the absorption of fat into the body by reducing the adsorption of bile acids and the dissolution of cholesterol and triglyceride digests. Beta-glucan, a polymer-soluble dietary fiber contained in oats, is a useful physiologically active substance that lowers blood cholesterol and LDL-cholesterol levels, increases HDL-cholesterol levels, inhibits liver accumulation of cholesterol and decreases lipid absorption in the body.

Conventionally, the method for separating beta glucan from grains is generally achieved by pulverizing the grains to powder, separating beta glucans into water by adding hot water, and inactivating beta-glucanase in the grains by applying heat. For example, Korean Patent Laid-Open Publication No. 1999-65617 discloses a fine powder of beta-glucan containing grains of 0.5 mm or less, water is added to the powder to extract an aqueous solution, and the extracted aqueous solution is heated to beta-glu. After deactivation of kanase and coagulation of protein, the reaction was performed by addition of starch dehydrogenase. The solution except for the coagulated material was filtered off, and alcohol or alcohols were added to the filtered solution to obtain solid betaglucan. Disclosed is a method for separating beta glucans of separated grains.

However, the natural beta glucan obtained by such a conventional method is excellent in inhibiting cholesterol when ingested more than 3g per day, but when added to a liquid product, it plays a role of significantly increasing the viscosity of the product and adding 3g to a small amount of liquid product. It is impossible to do this. In order to reduce the molecular weight in order to make a suitable viscosity for such a beverage product using the enzyme method to solve this problem, but the molecular weight is low, the phenomenon of significantly lowering the inhibitory effect of cholesterol occurred.

The present inventors hydrolyzed the natural beta glucan present in oat bran to solve the problems of the prior art as described above, the viscosity is reduced by about 70% than the natural beta glucan can be added to the beverage up to 2% and equivalent cholesterol A method for producing beta glucan having a specific molecular weight showing an inhibitory effect has been found and the present invention has been completed.

Accordingly, an object of the present invention is to provide a method for producing beta glucan having a specific molecular weight that can be added to the beverage product up to 2% and exhibits the same cholesterol inhibitory effect as natural beta glucan.

In order to achieve the above object, the present invention provides a method for producing beta glucan consisting of hydrolyzing the natural beta glucan present in the oat bran.

The present invention will be described in detail as follows.

In the present invention, beta glucan of oat bran was used. In general, oat beta glucan is very viscous and has a sticky property even at low concentrations. Viscosity of beta glucan, which is a kind of dietary fiber, can be changed according to the change of structure. The viscosity can be reduced through hydrolysis of β-1,3 bond of oat bran beta glucan.

In view of the fact that the viscosity of the beta glucan can be reduced through hydrolysis, the optimal enzyme for hydrolyzing oat bran beta glucan was selected using the substrate specificity of the enzyme. As a result, it was confirmed that hydrolyzates having various molecular weights were produced by the treatment of the obtained optimal hydrolase, and the hydrolyzed beta glucan was found to have a viscosity decrease of 70% or more than the natural beta glucan.

That is, in view of the above results, natural beta glucan was not more than 0.6% when added to a beverage because of its high viscosity, but beta glucan having a specific molecular weight was added to the beverage by the hydrolase treatment of the present invention. It could be added up to%

In general, bile acids are synthesized by cholesterol in the liver. It is known that these bile acids and dietary fiber are combined to inhibit the resorption of bile acids in the small intestine, thereby reducing blood cholesterol levels.

In the present invention, the bile acid binding capacity was measured in vitro for the selection of an optimal hydrolyzate having a reduced viscosity than the natural beta glucan and having the best bile acid binding capacity through hydrolysis. As a result, it was confirmed that the hydrolyzate obtained by reaction for 3 hours at 1.6% of enzyme showed the best bile acid binding ability.

Using the selected hydrolyzate, the dietary intake, weight gain and dietary efficiency were measured for 4 weeks through animal experiments, and serum, liver and feces were analyzed to confirm the cholesterol inhibitory effect.

As a result of long-term tissue weight measurement after the high cholesterol diet to which the hydrolyzate obtained by the hydrolysis of the present invention was added, the weight of the intestine most sensitive to lipid accumulation was reduced by 7.96% compared to the control group. Atherosclerotic index (AI) was significantly decreased in LDL and VLDL cholesterol compared to the control group, and the HDL ratio was higher, indicating that the atherosclerosis index was lower. In addition, liver lipid analysis showed that the ratio of total lipid and total cholesterol was lower than that of the control group.

In general, dietary fiber adsorbs bile acids, cholesterol, and toxic substances and is known to increase bile acid excretion in feces. In the present invention, the lipid analysis of feces was performed to study the transition rate and behavior of the ingested lipids to the feces, and as a result, bile acid excretion ability in feces was confirmed to be the same as the in vitro result.

That is, in view of the above results, the hydrolyzed oat bran beta glucan of the present invention has a reduced viscosity compared to natural beta glucan and has an equivalent cholesterol inhibitory effect, so that a small amount of functional units of cholesterol inhibiting functional foods It can be seen that it has various applications to beverage products and products.

Hereinafter, the configuration of the present invention will be described in detail with reference to the following examples. However, the scope of the present invention is not limited by the description range of an Example.

EXAMPLE

Example  1: selection of optimal enzyme for hydrolysis reaction

Three commercial enzymes from Novozymes A / S (Bagsvaerd, Denmark) (Celluclast 1.5L, Novozyme 188, Viscozyme L) were used to determine the enzyme to be used for oat glucan hydrolysis. Each enzyme activity was compared.

In order to investigate the substrate specificity of the enzyme, 3.5 mL of citric acid buffer solution (pH4.8) and 1.5 mL of enzyme solution (1.67% (v / v)) were added to cellobiose (Cellobiose, 3 mg), and hydrolysis was performed at 50 ° C. At this time, 0.5mL samples were taken at regular intervals to inhibit enzymatic activity, and glucose produced by hydrolysis was colorimetrically determined at 575 nm using dinitrosalicylic acid (3, 5-dinitrosalicylic acid; DNS) (Marcock, 2004). ).

As a result of examining the substrate specificity using the three commercial enzymes is shown in FIG. Viscozyme L has a high initial degradation rate and relatively high glucose production ability, making it difficult to control the random degradation of oat glucan. Therefore, two types of cellulase endo Celluclast 1.5L and exo Novozyme 188, which can control the degree of hydrolysis, were selected and then experimented.

In order to measure the molecular weight change of the hydrolyzate, oat bran betaglucan was hydrolyzed using the two types of cellulases selected above.

Molecular weight changes were investigated using Gel Permeation Chromatogram (GPC, LC 900, JAI, Japan) equipped with JAIGEL-W254 ~ 255 columns. The mobile phase used for the analysis used the RI detector by making distilled water at the rate of 3.5 mL / min. Dextran was used as the molecular weight standard, and the concentration of each sample was 5 mg / mL, and filtered by 0.45 μM syringe filter (Water Co., USA), followed by injection by 1.0 mL.

 The experimental results are shown in FIG. It was confirmed that the molecular weight change according to the hydrolysis by enzyme type decreased with increasing reaction time, but after 60 minutes of reaction, the molecular weight of endo-type Celluclast 1.5L continued to decrease, whereas the degree of hydrolysis in the case of exo-type Novozyme 188 Was reduced so that there was no change in molecular weight (FIG. 2).

The viscosity of the hydrolyzate obtained by treating the two types of cellulase was measured. Viscosity was measured using a plate-plate type (35 mm) viscometer (Rotational Rheostress RS1 Rheometer; Thermo Haake, Germany) with a 3% (w / v) aqueous solution calibrated at 25 ° C. Shear stress was measured while changing the shear rate from 0.1 to 200 / sec, and then the apparent viscosity was shown as the shear stress / shear rate. As a result, endo-type Celluclast 1.5L showed shear thinning in the flow characteristics, while exo-type Novozyme 188 shear thickening was shown (FIGS. 3A and 3B).

According to the above results, in the present experiment, the enzyme has a characteristic of acting on the inner part of the sugar chain to induce partial liberation of the sugar chain, and finally the end-cell Celluclast 1.5L which can produce glucan of various molecular weights with continuous molecular weight reduction is finally selected Proceeded.

Example  2: optimal Bile acid Binding capacity  Having Hydrolyzate  Produce

Changes in bile acid binding capacity according to molecular weight and viscosity decrease by enzymatic hydrolysis of oat glucan were measured.

-Preparation of enzymatic hydrolysates

In the preparation of enzymatic hydrolyzate, two kinds of cellulase were added with varying concentrations of glucose but similar in hydrolysis mechanism and enzymatic activity.

Two enzymes of endo-type Cellucalst 1.5L and exo-type Novoyme188 were diluted 10-fold and 50-fold, respectively, and used for the reaction. The ratio of buffer solution (D.W., pH 4.8), oat bran and enzyme solution was fixed at 4: 4: 1 and then reacted for 0, 10, 30, 60, 90 minutes in a 50 ° C constant temperature water bath. The hydrolyzed oat glucan hydrolysis solution was heat-treated in a 100 ° C. constant temperature water bath for 30 minutes to inactivate the enzyme to prepare a sample.

The molecular weight change of the oat glucan hydrolyzate produced under various enzymatic reaction conditions is shown in Table 1. According to Table 1, as the enzyme concentration increased and the hydrolysis time increased, the average molecular weight of oat glucan decreased from 1,453 x 10 ³ g / mol to 361 x 10 ³ g / mol, and the polydispersity (Mw / Mn) It increased from 1.723 to 8.225. Therefore, it could be seen that hydrolyzate having various molecular weights was produced by the enzyme treatment.

The effect of molecular weight change on the apparent viscosity of oat glucan is shown in Table 1 that the apparent viscosity in the 3% (w / w) aqueous solution of oat glucan and its hydrolyzate decreases with decreasing molecular weight. It showed a tendency. However, the apparent viscosity did not appear similar between hydrolyzates having the same molecular weight produced under different enzymatic reaction conditions. This difference in viscosity is thought to arise from the diversity of the constituent polymers and the reaction between them even though the hydrolyzate produced by the endo cellulase has the same weight average molecular weight.

-Preparation of Oat Glucan Hydrolyzate with Optimal Bile Acid Binding Capacity

Enzymatic hydrolyzate was prepared using a fermenter by adding various concentrations of cellulase to 6.25% (w / v) oat glucan solution. Add 2L of distilled water (pH4.8), each reaction enzyme solution (1.6, 5.0, 10.0% (v / w), dry weight of oat glucan, activate the enzyme at 50rpm for 30 minutes at 50 ℃, and then add 125g of oat bran. The hydrolysis was carried out at 500 rpm for 1 to 5 hours.The finished oat glucan hydrolysis solution was heat treated at 100 ° C. for 30 minutes to inactivate the enzyme, and then left overnight after adding the same amount of ethanol. Under reduced pressure filtration and dried in a 40 ° C. dryer to obtain a sample.

-Measurement of bile acid binding capacity

The bile acid binding capacity was measured by modifying the method of Cammir et al. (Carmir., 1993) and Boyd (Boyd., 1966). Samples were added to 0.01 mg sodium phosphate buffer (pH 7.0) containing 200 μM of taurocholic acid to pH 2.5 mg / mL and hydrated at 37 ° C. for 2 hours, followed by a 0.2 μm syringe filter (Water Co., USA). 0.2 mL of the filtered sample was taken, 1 mL of 70% sulfuric acid was added and reacted for 5 minutes. After adding 0.2 mL of 0.25% furfural, the mixture was left for 1 hour and absorbance at 510 nm was measured to measure bile acid binding ability.

The bile acid binding ability of oat bran beta glucan hydrolyzate according to enzyme treatment was measured, and the results are shown in FIG. 4. It was confirmed that the binding capacity of the enzyme-treated oat bran beta glucan was substantially reduced compared to the 8.45% of the taurocholic acid binding ability of the oat bran beta-glucan without enzyme treatment. However, the hydrolyzate obtained by reaction for 3 hours at 1.6% of enzyme concentration was 8.52%, which was significantly higher than the bile acid binding capacity of other hydrolysates.

The physiological activity of functional dietary fiber is proportional to dietary fiber intake and improves as the concentration of the solution increases (Chen 1984). Therefore, the relative concentration of the aqueous solution of hydrolyzate at the same viscosity was calculated for the selection of the optimum hydrolyzate, and the relative bile acid binding capacity was calculated in consideration of the yield, and the results are shown in Table 2. The hydrolyzate (sample No. 4) obtained by reacting with an enzyme concentration of 1.6% for 3 hours showed the best bile acid binding capacity (12.35%), and the animal experiment was conducted using this.

Example  3: Cholesterol in Laboratory Rats Inhibition  Experiment

About 50 male rats of about 100 ± 10 g 4 weeks of age were fed from central experimental animals, and three were raised in stainless cages. Adapted to provide a pellet-type lab-chow for the first week, then divided into four groups by randomized complete block design. The experimental diet composition is based on the AIN-76 dietary composition, divided into the normal diet group and 1% high cholesterol diet group, and then the high cholesterol diet group again oat glucan diet group (OB) and optimal hydrolyzate diet group (HG) as shown in Table 3. Were bred for 4 weeks. Experimental diet was prepared by pelleting to be the same energy level in all groups except the normal control, refrigerated and supplied freely with water. The temperature of the feeding room was maintained at 24 ± 2 ℃ and the contrast was adjusted at 12 hour intervals. Cholesterol inhibitory effect was confirmed by measuring dietary intake, weight gain and dietary efficiency during 4 weeks and analyzing serum, liver, adipose tissue and feces.

-Measurement of dietary intake, weight gain and dietary efficiency

Dietary intake and body weight were measured and recorded every other day at a given time, and the food efficiency ratio (FER) was calculated by dividing the total weight gain by the dietary intake over the same period.

-Collection of serum, liver, adipose tissue and feces

After 4 weeks of breeding, the animals were anesthetized with dry ice, an abdominal incision was made, blood was collected from the heart, left at room temperature for 2 hours, and centrifuged at 3,000 rpm for 20 minutes to separate serum. Immediately after blood collection, organ tissues (liver, kidney, epididymis) were removed, washed with 0.9% saline, and then drained and weighed. Collected serum and individual organs were stored at -70 ℃ until sample analysis.

Feces were collected once a week during the breeding period and the day before sacrifice, dried, weighed dry weight and stored at -70 ° C.

Serum, liver and fecal lipid analysis

Total lipid (TL), triglyceride (TG), total cholesterol (TC), and high density lipoprotein (HDL) cholesterol in serum were analyzed by an automated blood analyzer (Olympus AU400, Tokyo, Japan). ). Low density lipoprotein (LDL) cholesterol and very low density lipoprotein (VLDL) cholesterol were calculated by the Friedewald formula (Friedewald et al., 1986), Atherogenic index was calculated by Fiordaliso (Fiordaliso et al., 1995).

Total lipids in liver and feces were extracted according to the method of Folk (Folch., 1956), and TG and TC concentrations were analyzed using analytical quantitative kits (AM 157S-3, AM202-K, Asan Pharmaceutical Co., Seoul, Korea). It was measured by. Fecal bile acid was extracted by modifying the method of Uchida (Uchida., 1977) and then quantified using a bile acid measurement kit (Wako chemical Co., Japan).

① organ tissue weight

Organ weights were measured to measure lipid accumulation in the organs and lipid metabolism in the liver using the experimental mice, and the measured organ weights are shown in Table 4. Liver weight, which is the most sensitive to lipid accumulation by high cholesterol diet, was decreased by 4.66% and 7.96%, respectively, due to the addition of oat bran betaglucan and optimal hydrolyzate to inhibit the accumulation of liver lipids. It is reported that dietary fiber inhibits fat absorption or active ingredient decomposes and burns body fat to inhibit triglyceride synthesis in liver (Yoshioka et al., 1994) and water-soluble dietary fiber psyllium, gum (gums), pectin (pectin), etc. in the group was given a decrease in liver weight, showing a hypocholesterolemic effect is consistent with the results of Anederon et al. (1994).

② Lipid analysis in serum

Table 5 shows the results of analyzing the Atherosclerosis Index (AI), which is used to determine the cholesterol content of serum and the risk of cardiovascular disease. Total cholesterol was not significantly different from the control group, but LDL and VLDL cholesterol were significantly decreased in HG group. In addition, the HDL-cholesterol / total cholesterol ratio (HTR) of all the experimental groups was higher than that of the control group, resulting in a low arteriosclerosis index (AI).

According to Hick et al. (Hick et al., 1995), the cholesterol lowering ability of oat bran reduces the risk of atherosclerosis with the increase of HDL-cholesterol. In the present study, the addition of oat bran beta glucan (OG) and hydrolyzate (HG) in high cholesterol diet rats induced lower serum cholesterol, increased HDL-cholesterol ratio and decreased atherosclerosis index. Therefore, oat bran beta glucan and its hydrolyzate may be useful for the prevention of vascular diseases such as atherosclerosis.

③ Geological analysis of liver

Table 6 shows the total lipid, total cholesterol, and triglyceride contents of the liver that play a central role in lipid metabolism. Total lipids and total cholesterol were significantly lower in both OG and HG supplemented groups than in the control group. Although there was no significant difference between the experimental groups, it was confirmed that the HG group was somewhat lower. The triglyceride concentration was 25.34 mg / g in the control group, and the OG and HG-added groups were 17 mg / g and 17.73 mg / g, respectively, which were reduced by 28.3% and 30% compared to the control group. It is believed that the amount accumulated in the liver is lowered due to the low concentration of fat.

④ lipid analysis of feces

The daily dry weight, total lipid, total cholesterol and triglyceride content of feces were measured to study the rate of transition and the mode of transition of ingested lipids to feces. The results are shown in Table 7. The dry weight of daily feces was significantly higher in all experimental groups than in the control group. Total lipids, total cholesterol and triglycerides were all significantly increased in the OG group, while fecal bile excretion was significantly increased in the HG group, which was the same as in vitro results.

From the above results, it was confirmed that the addition of oat bran beta glucan and its hydrolyzate to the high cholesterol diet increased the serum and liver cholesterol lowering effect and fecal bile acid excretion in rats. In other words, oat bran beta glucan and the optimal hydrolyzate of the bile acid binding ability of the cholesterol in the liver due to the lack of bile acid in the liver showed a decrease in cholesterol and serum. In addition, this resulted in an increase in the amount of fecal cholesterol and bile acid excretion ultimately ingested beta glucan and its hydrolyzate lowered cholesterol in the body.

That is, when natural beta glucan is treated with 0.1% to 3% of cellulase for 2 to 3 hours, it is possible to prepare hydrolyzed beta glucan having a molecular weight of 6.0 × 10 ⁵ to 7.0 × 10 ⁵ , but if the enzyme is more than 3% The decomposition rate of hydrolyzed beta glucan is too fast to make the above molecular weight, and if the enzyme is less than 0.1%, the decomposition rate is too slow, making it difficult to manufacture a product industrially. Therefore, it was confirmed that the above condition range is necessary to prepare beta glucan having the molecular weight.

Beta glucan of the present invention has a viscosity of more than 70% lower than natural beta glucan, and the cholesterol inhibitory effect is equal, so that a large amount can be added to a small amount of beverage products and other products, so a small amount of beverage products and other products among cholesterol-lowering functional foods Applicable to

Claims (6)

A method for producing beta glucan, characterized by hydrolyzing natural beta glucan present in oat bran. The method of claim 1, The hydrolysis is carried out using a cellulase, characterized in that Method for preparing beta glucan. The method of claim 2, Hydrolysis using the cellulase is characterized in that the oat bran is treated with a cellulase at a concentration of 1 to 3% for 2 to 3 hours Method for preparing beta glucan. The method of claim 3, The molecular weight of the beta glucan obtained by the cellulase treatment is characterized in that the 6.0 × 10 ⁵ to 7.0 × 10 ⁵ Method for preparing beta glucan. Beta-glucan prepared according to any one of claims 1 to 4. A beverage comprising beta glucan prepared according to any one of claims 1 to 4.

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