KR20070063112A - Sulfated Polysaccharide Fraction Having Secondary Bile Adsorption Capacity and Its Manufacturing Method - Google Patents

Abstract

The present invention relates to a fraction for adsorbing secondary bile salts from a hydrolyzate obtained by hydrolyzing seaweed with an enzyme.

Description

Sulfonated polysaccharides fraction having binding ability for deoxycholate and preparation method

Figure 1 shows the adsorption reaction of primary bile salts Na-cholate and USF-1, USF-2 for 140 minutes.

Figure 2 shows the adsorption reaction of primary bile salts Na-chenodeoxycholate and USF-1, USF-2 for 140 minutes.

Figure 3 shows the adsorption reaction of secondary bile salts Na-deoxycholate and USF-1, USF-2 for 140 minutes.

Figure 4 shows the adsorption reaction of secondary bile salts Na-deoxycholate and USF-1, USF-2, Cholestyramime for 140 minutes.

There is abundant seaweed resources along the coast of Korea, where the Korean and turbulent flows. There are 218 species of genus 87, 241 genus, and 150 species of genus algae of 25 type, 57 genus 57. These seaweeds have been used for food, fenugreek, medicinal, raw materials of the seaweed industry, livestock feed and fertilizers since ancient times. About 60 to 70 kinds of these seaweeds have been found to have excellent nutritional physiological effects.

Among them, the highest yield is seaweed, and has been used as a postpartum cooked food since ancient times. The development of aquaculture in the early 1970s led to a sharp increase in its production, and in recent years its disposal rate has fallen to less than 10%, leading to an increase in waste.

This is because it is limited to 3 months of harvest season in February ~ April, and is mainly produced as a primary processed product such as dried seaweed and salted seaweed. It is also falling.

There are only a few reports on the use of seaweed as a dietary fiber, the study of carcinogenic nitrosamine production. The use of these low-use seaweed potential resources to increase the value as processed raw materials and to find effective ways to utilize them is expected to greatly contribute to securing food resources and improving the nutrition of the people, as well as increasing fishermen's income.

In this study, we attempted to separate sulfated polysaccharide by chromatography using enzymatic digestion, a new processing technique using seaweed.

Recently, due to the westernization of Koreans' dietary culture, excessive fat intake has been reported that the potential population of hypercholesterolemia reaches 20%. The cholesterol level in Koreans is still lower than that of Westerners, but has recently risen above 10 mg / dl. Therefore, in recent years, the number of hypertension, arteriosclerosis, and heart disease patients is increasing rapidly, and the age of heart disease patients is decreasing.

Cholesterol is an insoluble steroid compound with 27 carbon atoms, which acts as a precursor to progestins, corticosteroids, and sex hormones, and forms bile acids necessary for the absorption of fat or fat-soluble vitamins from the small intestine and is carried by the liver to tissues from the liver. It is also used for the synthesis of ultra low density proteins (VLDL) that are formed.

Too much cholesterol is known to accumulate in blood vessels and cause heart disease. There is no preventive method yet except low cholesterol diet, and drug use is effective as a cholesterol lowering agent, but its use is extremely limited because it causes side effects such as liver dysfunction due to inhibition of cholesterol synthase.

Although cholestyranmine, cholestipol, etc. are used to suppress the incidence of lesions caused by excessive intake of cholesterol, it is known that there are problems in taking the liver as described above. They have been reported to interact with bile acids in the small intestine, contributing to the resynthesis of cholesterol and the release of bile acids.

Bile acids are produced by the oxidation of cholesterol in the liver and secreted into the intestines, which emulsify lipids or fat-soluble vitamins, and excrete only a portion (about 1%) during the intestinal cycle, which is absorbed in the ileum and circulated to the liver. Have At this time, the use of an adsorbent prevents the absorption of bile acids near the ileum, promotes the consumption of cholesterol in the body, and lowers the value of blood cholesterol by blocking and excreting the absorption of cholesterol produced in the urinary tract by the feed back effect. Used. This method was tested by ferric chloride on a rooster by siperstein in 1953, and then, by 1960, Tennent et al. Prepared cholestyramine (a copolymer of syrene and divinylbezene) using animal experiments. It is becoming.

Bile acids are present in the form of salts as anions in vivo. These bile salts include primary bile salts, cholate and chenodeoxycholate, and these primary bile salts produce decyholate as a secondary bile salt by the degradation of intestinal microorganisms.

The present invention relates to a sulfated polysaccharide having excellent adsorptive capacity of secondary bismuth salts by increasing the yield by enzymatic digestion technology using a large-scale domestic seaweed and extracting polysulfated polysaccharide to test the adsorption capacity of bile salts.

An object of the present invention is to provide a sulfated oligosaccharide excellent in enzymatic digestion of wakame seaweed to increase its extraction yield, the sulfated polysaccharide is extracted by molecular weight and then bile salt adsorption.

In order to achieve the above object, the present invention increases the extraction yield by decomposing the seaweed with polysaccharide degrading enzyme. In hot water extraction or solvent extraction, even if purified water is added in the same amount, the extraction yield is less than 5% and the extraction of polysaccharide as a target component is not easy, so it is extracted using polysaccharide enzyme. After extraction, the sulfated polysaccharides are extracted through anion exchange resin and gel chromatography, and the active fractions are separated by testing bile acid adsorption capacity by molecular weight.

Example 1 Enzymatic Degradation of Seaweed

First, the foreign matters of wakame seaweed are sorted and then hot air dried to 7-10% of moisture, and then cut to 10cm in length. In the case of fine powder, the water content of the fermenter is 20 times more than the dry weight of the sample.

10 times purified water is added to 200kg of cut seaweed samples, soaked for 15 minutes, washed, and then drained. In case of excessive washing, sulfuric acid group forming ester bond with dietary fiber and dietary fiber is seriously spilled and discharged by dipping.

Water washing and primary desalted seaweed samples are added to a 5 ton fermentation tank, and 3 tons of purified water (15 times weight W / W of sample) warmed to 80 ° C in advance prior to the addition of Citrate-Phoshate buffer (mixture ratio Citrate 1: Sodium) Phosphate 1.75) is prepared by adding 4 kg of 2% of the dry weight of the sample to pH 5.5 beforehand to prepare a buffer solution. The prepared buffer solution is added to the seaweed sample and stirred for 10 minutes to mix with the ions in the seaweed to stabilize the buffer capacity and to cool the temperature below 60 ° C.

Thereafter, polysaccharide dehydrogenase, which is 2.0% of the sample building amount, is added and decomposed at 57 ° C for 7 hours. At this time, the polysaccharide degrading enzyme is a complex glycolytic enzyme (10,000 CU / g) originated from Aspergillus orija and Trichodermari mari, and its composition ratio is 1: 0.5-1: 0.7%. Can be used. Enzymes are not limited to the items of the manufacturer. As a cellulose unit, an enzyme having a titer of 10,000 unit / g may be added to W / W of the sample weight part.

In order to extract protein components after carbohydrate digestion and remove them by membrane filtration, 1% (W / W) of Pacific Seaweed Protease NP, a complex enzyme as protease, is added and digested for 4 hours under the same conditions. The protease used herein is not limited to the enzyme of the above company, it is possible to use protease derived from the strain of the genus Bacillus. After completion of the enzymatic digestion, the mixture was pressurized under pressure conditions of 1.2 kgf / cm ² at 121 ° C. for 1 to 3 hours to adjust the viscosity and molecular weight. The average molecular weight was 300,000 or less and the final viscosity was adjusted to 15 cP to prepare a wakame enzyme digestion liquid. The digestion solution was continuously centrifuged at 10,000 xg with an ultracentrifuge (Tomoe Industries, Japan), filtered three times with a spark killer filter (10 micro, 1 micro, 0.22 micro) to obtain a filtrate. 2.9 tons of solid content of 5.7% was obtained.

Example  2. Seaweed From enzyme digestion Sulfate  Separation of polysaccharides.

Various oligosaccharides containing sulfate groups, such as sulfated fucan, sulfated galactan, sulfated fucogalactan, etc., are combined with dietary fiber in alginic acid of algae and carrageenan of red algae, and have anticoagulant, blood pressure lowering, and antitumor activity. It is known that research on its physiological activity has been actively conducted in Japan and Russia recently.

In the present invention, by obtaining the sulfated oligosaccharides from the enzymatic digestion solution to invent a fraction having a bile acid adsorption capacity to provide a simple and high-yield food material.

2.9 tons of seaweed digestion filtrate was added to a stirring tank equipped with a side outlet for each level, and 23.2 kg of calcium chloride was added at 8% of the total weight, and the mixture was stirred at 65 rpm for 15 minutes to gradually produce a polymer alginate aggregate. To precipitate the polymer calcium alginate salt. Obtained by draining the supernatant of the precipitation site through the water pipe. The liquid amount was 2.3 tons at this time. Dion SA10AP (strong base anion exchange resin, Samyangsa), which was regenerated with 0.01 N HCl and NaOH, was charged into an ion exchange resin tower (30 cm x 200 cm in diameter), and the filtrate from which the polymer alginic acid was removed was adsorbed. Re-elution with saline was collected by desorption. Two times the amount of purified water was added to the collection solution, followed by simultaneous desalination, amino acid removal and concentration using an ultrafilter (excluding 5,000 Daltons of molecular weight), followed by dilution of 3 times of deionized water. It is added with diluent to facilitate column chromatography elution. In this case, when the viscosity of the concentrate is 40 cP or more, deionized water may be further added so as to be 15 cP or less. The diluted sample solution was prepared using a Moduline Purificatin system (14 cm in diameter x 160 cm in height, Millipore Inc.) filled with Toyopearl HW-65s using 25 mM Tris-HCl (pH 7.0) and 1: 1 (v / v) eluent. Elution was performed by 50 ml. These fractions were combined according to the type of peaks measured by the phenol sulfate method using fucose as a standard, and lyophilized to obtain a seaweed sulfated polysaccharide sample. Two fractions were obtained from the sample, and the UNDARIA SULFHATED FRACTION was reduced to USF, and each was referred to as USF-1 fraction (average molecular weight 120,000) and USF-2 fraction (average molecular weight 40,000).

The following experiment was conducted to measure the degree of sulfated polysaccharide sulfate of each fraction.

General ingredients were tested according to the Food Code, and the content of sugar content was tested by the phenol-sulfuric acid method of Real Code, but the standard product was Fucos. Composition analysis was performed on the basis of calibration curves obtained by using HPLC as a standard.

     General Composition of USF Fractions sample moisture protein Fat carbohydrate Ash USF-1 6.5 2.8 1.1 65.6 24 USF-2 7.1 4.1 1.5 67.3 20

As a result, the amount of protein adsorbed and removed by ultrafiltration and the amount removed by ultrafiltration at 20% or more of seaweed dry weight during the enzymatic digestion process after the first enzymatic digestion and the second protein enzymatic digestion and the polymer alginate removal process. Accounted for.

The content of heavy sulfate groups in seaweed polysaccharides has recently become a new field of development. Indeed, even in export markets such as carrageenan, those with high sulfuric acid content also sell high. In addition, polysaccharides containing sulfate groups must contain at least 25% in order to obtain product qualification in export markets such as Japan.

Therefore, the sulfuric acid group content was measured by the composition of the sulfuric acid group and constituent sugar of the fraction of this invention.

        Uronic acid, sulfuric acid content, and composition of each fraction. sample Uronic acid Sulfate Composition per Composition (%) FUCOSE GALACTOSE GLUCOSE XYLOSE RIBOSE USF-1 8.34 28.7 50.2 12.7 1.5 6.9 1.8 USF-2 3.45 27.1 45.8 13.2 0.8 5.6 0.9

In the above results, uronic acid content is a basic structure of alginic acid, the smaller the content, the lower the viscosity, and a measure of purity of polysaccharides except alginic acid. In addition, the composition of constituent sugar shows the composition ratio of each component when the peak per each component totals 100. It was high in fucose and next in galactose. In particular, the sulfuric acid content is more than 25%. That is, in the above results, each fraction represented sulfated polysaccharide containing 25% or more sulfate groups.

Example  3 Bile acid Adsorption capacity  exam

Preparation of bile salts: Sodium cholate, sodium chenodeoxycholate, sodium Deoxycholate was purchased from Wako pure chemical Inc. (Osaka, Japan).

Quantification of bile salts was determined using the method of Chan, H j, et al. (Korean J. Food Sci, Technol, 56, 348 (1994), bile acid solution 0.1M phosphate buffer solution containing 0.1% sodium azide pH7.0 Each bile acid contained 1-15 mM) 0.5 ml of 45% sulfuric acid and 0.5 ml of 0.3% furfural solution were added and reacted at 65 ° C. for 30 minutes, and then the absorbance at 680 nm was measured using a spectrophotometer.

     Adsorption test of bile acids and samples: Measured according to the method of Suzuki T et al. (Fisheries Sci. 1996; 62: 454-461). 20 ml of each bile salt of 2.5 mM in each fraction was added to a centrifuge tube, reacted for 2 hours at 37 ° C. for 10 minutes at 13,000 × g, filtered, and the amount of bile salt in the filtrate was measured. Distilled water was added instead of each fraction as a control, and the amount of bile acid adsorption was calculated by subtracting the control from the experiment.

For each experiment, a comparative experiment was conducted using cholestyramine, a commercial preparation with bile salt adsorption capacity.

1 to 3 show the results of experiments with adsorption of each fraction with cholate, chenodeoxycholate and deoxycholate while maintaining the incubation time up to 140 minutes. In all cases, adsorption capacity was expressed from the starting point of adsorption with bile acid and maintained for 140 minutes. However, over time, there was no specific alleviation, indicating that the adsorption was initially completed. The concentration for adsorption is expressed in umol / g. As a result, the adsorption capacity for primary bile salts was inadequate, but the adsorption capacity for secondary bile salts was less effective than cholestyramine, which is commonly used for medicinal purposes. 4 is a diagram showing that the USF-1 fraction of the second bile salt, sodium deoxycholate, has the adsorption capacity after the cholestylamine, and the adsorption capacity reaches 78% of the control. Therefore, USF-1 fraction of 120,000 molecular weight contains 28.7% of sulfate group, and it is a fraction of sulfated polysaccharide with high content of fucose and galactose in constituent sugar ratio.

Secondary bile salts were fractionated with deoxycholate adsorption capacity by a new process using seaweed, a large-scale production resource. This fraction, which is a sulfated polysaccharide and oligomer as a food material, can be industrialized as a food material involved in preventing endogenous cholesterol accumulation due to excessive cholesterol intake. In addition, it is possible to overcome lactation lowered by removing the polymer alginic acid in the secondary process by increasing the solids yield by decomposing seaweed into enzymes. The present invention relates to a sulfated polysaccharide fraction which causes a feed back effect of adsorbing and releasing secondary bile acids by using seaweed and reusing cholesterol in vivo to produce a deficiency of bile acids. It is expected to contribute to securing income for fishermen by expanding the utilization rate, avoiding simple processing, and securing a role as a food material that can be expected to grow in the Japanese and European markets where demand for sulfated polysaccharide is high.

Claims (2)

(a) degrading brown algae with polysaccharides; (b) removing the alginic acid from the degradation product obtained in step (a); And (c) chromatographically treating the degraded product from which the alginic acid has been removed, and having an average molecular weight of 120,000 and adsorbing capacity for secondary bile salts. (a) degrading brown algae with polysaccharides; (b) removing the alginic acid from the degradation product obtained in step (a); And (C) a method for producing a sulfated polysaccharide fraction comprising the step of purifying the digested product from which the alginic acid has been removed, having an average molecular weight of 120,000, and having an adsorptive capacity to secondary bile salts.

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