KR101637115B1 - Preparation of low molecular weight polysaccharides from red alga - Google Patents

Abstract

According to the present invention, a low molecular weight polysaccharide derived from red algae has a molecular weight of 1,000-10,000 Da, and has excellent antioxidant activities in comparison to a common high molecular weight polysaccharide. A manufacturing method of a low molecular weight polysaccharide derived from Pyropia yezoensis comprises the following steps of: i) removing water soluble proteins by treating a Pyropia yezoensis powder with distilled water; ii) removing lipids by adding 90% ethanol at 150-250°C; iii) obtaining a polysaccharide from residues after adding distilled water at 100-150°C; iv) conducting hydrolysis by injecting amyloglucosidase (AMG) under microwaves having 30-40 W; and v) separating a low molecular weight polysaccharide from the resultant product.

Description

Preparation of low molecular weight polysaccharides from red alga}

The present invention relates to red algae, in particular Pyropia yezoensis ) and its antioxidant effect.

Pyramid , Pyropia yezoensis is a major red algae that can be consumed as food and medicine, and is known in Southeast Asia, including Japan, Taiwan, and China. It contains various components including sulfated polysaccharides, mycosporine-like amino acids, sterols, carotenoids, proteins, essential fatty acids, vitamins and minerals (see Non-Patent Document 1, Non- Patent Document 2).

Of these various functional ingredients, polysaccharides have been used in the food industry for a long time because of their various physiological effects such as antioxidant, anti-cancer, anti-hyperlipemia and anti-fatigue. However, they are poor in solubility and have a very large molecular weight ranging from 300,000 Da to 3,000,000 Da, making it difficult to utilize them because they have difficulties in absorption and denaturation in the cells.

Thus, the present inventors have isolated the low molecular weight polysaccharide of 10 kDa or less from the radiolabeled rice using the microwave assistant rapid enzyme digest system (MAREDS) which provides the microwave easily, promptly and safely.

 K. Kazlowska, H. T. V. Lin, S. H. Chang, G. J. Tsai, Evidence-Based Complementary and Alternative Medicine, 2013, ID 493869: 10.  M. Senevirathne, C. B. Ahn, J. Y. Je, Food Science and Biotechnology, 2010, 19 (6): 1551-1557.

It is an object of the present invention to provide a process for producing a red algae-derived low molecular weight polysaccharide.

Hereinafter, the present invention will be described in detail.

The present invention provides a process for producing a red algae-derived low molecular weight polysaccharide comprising the following steps.

I) treating the red algae powder with distilled water to remove water-soluble proteins;

Ii) removing the water-soluble protein and removing lipid by adding 90% ethanol at 150 to 250 ° C;

Iii) obtaining a polysaccharide in the residue by adding distilled water at 100 to 150 ° C after removing the lipid;

Iv) hydrolyzing the carbohydrate in a molar ratio of 10 to 100: 1 to the polysaccharide in a microwave of 30 to 40W; And

V) separating the low molecular weight polysaccharide having a molecular weight of 1,000 to 10,000 Da from the hydrolyzate.

In the step ii), the lipid is preferably removed by adding the 90% ethanol at 150 to 250 ° C for 1 to 3 hours, but is not limited thereto. If the time for adding the 90% ethanol is less than 1 hour or more than 3 hours, degradation of the sample due to lipid shrinkage or denaturation may occur.

In step (iii), the distilled water is preferably added 3 to 5 times for 10 to 30 minutes, but is not limited thereto. If the time range and the number of times of applying the distilled water exceed the range, denaturation of the polysaccharide may occur.

In the step iv), the hydrolysis is preferably performed for 10 minutes to 2 hours in the hydrolysis efficiency, but is not limited thereto.

The low molecular weight polysaccharide comprises 60 to 90% by weight of glucose as a main constituent sugar, 1 to 30% by weight of galactose and 1 to 30% by weight of mannose, But are not necessarily limited to, all commonly known polysaccharides.

The low molecular weight polysaccharide has an antioxidative effect.

The red algae can be used as long as they contain polysaccharides. However, yezoensis ).

The microwave can be generated using a microwave assistant rapid enzyme digest system (MAREDS), but not limited thereto, any microwave generating machine can be used.

MAREDS refers to a water bath in which a tube or the like can be placed in a chamber through which microwaves are radiated so that the composition or enzyme in the tube is activated by the microwave to perform the action of the enzyme on the composition (for example, Hydrolysis, etc.).

The carbohydrate hydrolase may be any one or more selected from the group consisting of Viscozyme, Celluclast, Termarmyl, amyloglucosidase (AMG), and Ultraflo But is not limited thereto.

In one embodiment according to the present invention, the molecular weight of the low molecular weight polysaccharide and the high molecular weight polysaccharide isolated from the hydrolyzate of amyloglucosidase (AMG), which is a carbohydrate hydrolase, was evaluated as 2755 kDa And 782 Da in the case of low molecular weight polysaccharides. As a result of the monosaccharide analysis, it was confirmed that the high molecular weight polysaccharide accounted for more than 90% of the galactose. In the case of the low molecular weight polysaccharide, the glucose accounted for 64.50% and the galactose accounted for 27.25%.

As a result of the antioxidant activities of the low molecular weight polysaccharide and the high molecular weight polysaccharide isolated from the hydrolyzate of amyloglucosidase (AMG), which is a carbohydrate hydrolase, in one embodiment according to the present invention, an alkyl radical and hydrogen peroxide (H ₂ O ₂ ) IC ₅₀ value of lower molecular weight polysaccharides on, it was confirmed that each of 114.36 g / ml and 13 g / ml as shown, 181.38g / ml and 81g / ml of high indicating an extremely high antioxidant effect than the molecular weight polysaccharide antioxidant activity.

Polysaccharides having different molecular weights exhibit differentiated effects on the antioxidative effect. In particular, the red algae-derived low molecular weight polysaccharides according to the present invention are superior in antioxidant activity to general high molecular weight polysaccharides.

FIG. 1 shows the results of a microwave assistant rapid enzyme digest system ( MAREDS ) (A) and Pyropia (B) of the step of obtaining a low molecular weight polysaccharide and a high molecular weight polysaccharide from pseudomonas sp . yezoensis .
Figure 2 is a plot of the degree of hydrolysis (DH) according to the hydrolysis time under microwave; Amyloglucosidase (AMG), a carbohydrate hydrolase, is administered at a ratio of 10: 1 to the substrate.
Figure 3 is a diagram for a monosaccharide chromatogram; (A), unfractionated polysaccharide (glucoamylase (AMG) hydrolyzate), (B) amyloglucosidase (AMG) hydrolyzate derived high molecular weight polysaccharide (AMG-HMWP), (C) glucoamylase (AMG-LMWP) derived from amyloglucosidase (AMG) hydrolyzate, (D) Pyropia yezoensis . * 1: Galactose, 2: Glucose, 3: Mannose.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Sample preparation of red algae

Pyropia (red flounder) yezoensis ) were collected from the Wando coast of Korea, washed twice in fresh water, and stored frozen at -20 ° C until use. The frozen sample was lyophilized and pulverized in a pulverizer. The dried sample powder was refrigerated until used. The composition of the dried sample powder was analyzed according to the Association of Official Agricultural Chemists (AOAC) method (Washington, DUSA, 1990). The moisture content was analyzed in a drying oven at 105 DEG C for 24 hours and the quench was analyzed according to calcination in an oven at 550 DEG C. [ Crude protein content was analyzed by Kjeldahl method (Kjeltec ^TM 2300, Foss Co. Ltd., Denmark) and crude lipid content by Sohxlet method (Sohxlet system 1046, TacatorAB, Sweden).

The results of the composition analysis of dried P. yezeonsis are shown in Table 1. The content of polysaccharide in dried samples was 44.6 ± 2.3%, and protein was the second most abundant. Red algae such as Pyropia sp. Are high in protein and fibrin content, so protein and carbohydrate use values are considered to be very high.

Protein (%) Carbohydates (%) Lipid (%) Moisture (%) Ash (%) Calories (kcal / 100g) P. yezoensis 41.7 ± 1.7 44.6 ± 2.3 1.8 ± 0.12 7.4 ± 1.7 5.3 ± 0.1 351.4 ± 12.7

< Example  2> Obtain a polysaccharide from a red algae sample

(Food Research International, 2012, 49: 364-372.) And Takahashi et al. (Journal of agricultural and food chemistry, 2000, 48 (7): 2721-48), to prepare a pure polysaccharide. 2725.) (Figure 1B). The water-soluble protein was removed from 50 g of the dried powder at 20 ° C using distilled water, and then 90% ethanol at 200 ° C was added thereto for 2 hours to remove lipids. After lipid removal, 2 g of polysaccharide was obtained from the residue obtained by adding distilled water at 120 캜 for 15 minutes three times.

The composition of crude protein and polysaccharide of P. yezeonsis was analyzed in the same manner as in FIG. 1B, and the results are shown in Table 2. The polysaccharide showed a high sugar content of 91.71 ± 5.07%, while a low sulfate content of 5.41%. Porphyran, a sulfated polysaccharide, is a major component of Pyropia sp. And has been reported to have a sulfate content of about 8% in P. yezeonsis (Takahashi et al., Journal of agricultural and food chemistry, 2000 , 48 (7): 2721-2725., Yu et al., Carbohydate Polymers, 2015, 117: 650-656.) This is similar to the experimental results for crude polysaccharide.

Sugar (%) Protein (%) Sulfate (%) Proteins fraction 11.2 ± 0.43 89.03 + - 7.35 - Polysaccharides fraction 91.71 + 5.07 8.7 ± 3.3 5.41 ± 0.02

< Example  3> polysaccharide hydrolysis and Low molecular weight  Obtain polysaccharide

The polysaccharide may be a carbohydrate hydrolase such as Viscozyme (Novozyme Co., Denmark), Celluclast (Novozyme Co., Denmark), Termarmyl (Novozyme Co., Denmark), Glucoamylase (microwave assistant (AMI)) under the treatment of amyloglucosidase (AMG) (Novozyme Co., Denmark), and Ultraflo (Novozyme Co., Denmark) (10: 1 and 100: Rapid enzyme digest system (REDS ^TM , ASTA INC., USA) (Fig. 1A). Low molecular weight polysaccharides (LMWP) of 10 kDa or less and high molecular weight polysaccharides (HMWP) of 10 kDa or more are prepared with a 10 kDa molecular weight cut-off (MWCO) ultrafilteration membrane (BIONEER, USA) AccuPrep® plasmid extraction kit. The obtained low molecular weight polysaccharides (LMWP) was 504 mg, and the yield was 25.2%.

The sugar content was analyzed using Dubois et al.'S phenol-sulfuric acid colorimetric method (Anal. Chem, 1954, 28 (3): 350-356.). Degree of hydrolysis (DH) of the polysaccharides derived from the enzymatic hydrolyzate was measured by Pyropia yezoensis ) was calculated as the total sugar content of the low molecular weight polysaccharide after filtration of the polysaccharide hydrolyzate against the total sugar content of the crude polysaccharide. The formula for this is as follows.

Table 3 shows that 40 W of MAREDS at various hydrolysis conditions including different ratios of enzyme to substrate (10: 1 and 100: 1) and different hydrolysis times (10 min, 30 min, 1 hr and 2 hr respectively) The degree of hydrolysis (DH) of the polysaccharide hydrolyzate used was shown. The degree of hydrolysis of amyloglucosidase (AMG) increased with the hydrolysis time (10 minutes to 2 hours), while other enzymes did not affect the hydrolysis of the polysaccharide.

Degree of hydrolysis (%)
Viscozyme Celluclast AMG Termarmyl Ultraflo 10: 1 100: 1 10: 1 100: 1 10: 1 100: 1 10: 1 100: 1 10: 1 100: 1 10 min 12.11 0.62 4.51 1.51 8.65 6.42 4.90 5.37 2.84 2.90 30 min 14.78 2.98 3.55 1.01 15.78 7.73 3.54 5.86 3.83 3.59 1 h 13.57 2.70 6.56 1.16 13.96 7.66 2.32 5.07 2.97 2.17 2 h 13.05 1.59 5.73 1.16 25.02 9.00 5.30 4.80 3.19 0.98

In addition, the hydrolysis of glucoamylase in MAREDS showed a higher degree of hydrolysis than only treatment with glucoamylase or treatment with microwave (Fig. 2). The hydrolyzate of glucoamylase in MAREDS reached the optimal hydrolysis state at 2 hours and did not show a significant increase in the degree of hydrolysis at more than 2 hours. This means that glucoamylase accelerates the hydrolysis of polysaccharides in MAREDS for a short time of 2 hours.

< Example  4> Antioxidant efficacy evaluation of polysaccharide

The alkyl radical scavenging activity was analyzed by the method described by Hiramoto et al. (Free radical research communications, 1993, 19 (5): 323-332). Experimental conditions of an electron spin resonance (ESR) spectrometer (JES-FA machine, JEOL, Tokyo, Japan) were as follows; For example, for alkyl, magnetic field 336.5 ± 5 mT, power 1 mW, modulation frequency 100 kHz, amplitude 1 × 1000, modulation width 0.2 mT, sweep width 10 mT, sweep time 30 sec, time constant 0.03 sec.

The hydrogen peroxide (H ₂ O ₂ ) scavenging activity was analyzed according to the method of Heo et al. (Bioresource Technology, 2005, 96: 1613-1623.). 100 μl each of 0.1 M phosphate buffer solution (pH 5.0), amyloglucosidase (AMG) hydrolyzate, AMG hydrolyzate-derived low molecular weight polysaccharide (AMG-LMWP) and high molecular weight polysaccharide (AMG-HMWP) Lt; / RTI &gt; 20 [mu] l of hydrogen peroxide was added to the mixture and incubated at 37 [deg.] C for 5 minutes. After incubation, 30 μl of 1.25 mM ABTS (2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)) and peroxidase (1 unit / ml) were added and incubated at 37 ° C for 10 minutes. Absorbance was measured at 405 nm using an enzyme-linked immunosorbent assay (ELISA).

The antioxidative effect of hydrolyzate of amyloglucosidase (AMG) was found to increase with the treatment time of glucoamylase (Table 4). In a previous study, polysaccharides derived from P. yezoensis were hydrolyzed with macrophage-stimulated beta-agarase (Nomura et al., Japan Society for Bioscience, Biotechnology, and Biochemistry, 1998, 62 (6) And enhanced the functionality of polysaccharides derived from P. yezoensis through enzymatic hydrolysis. In the present invention, glucoamylase hydrolyzate treated with MAREDS for 2 hours was selected and tested.

The low molecular weight polysaccharide (AMG-LMWP) isolated from glucoamylase hydrolyzate showed strong antioxidative effects against alkyl radical and hydrogen peroxide compared to high molecular weight polysaccharide (AMG-HMWP) (Table 4, Table 5). These results indicate that the molecular weight of the polysaccharide is related to the antioxidative effect. To date, the biological effects of low molecular weight polysaccharides derived from P. yezoensis have not been determined.

IC ₅₀ values (mg / ml) Alkyl radical H ₂ O ₂ AMG 10 min (10: 1) 0.542 ± 0.002 > 0.180 AMG 30 min (10: 1) 0.482 + 0.018 0.175 + - 0.000 AMG 1 h (10: 1) 0.341 ± 0.008 0.125 ± 0.002 AMG 2 h (10: 1) 0.196 + 0.001 0.096 ± 0.002

IC ₅₀ values (mg / ml) Alkyl radical H ₂ O ₂ AMG-HMWP (2 h, 10: 1) 181.38 + 0.328 81 ± 0.150 AMG-LMWPs (2 h, 10: 1) 114.36 ± 3.476 13 ± 0.000

< Example  5> Analysis of monosaccharides on polysaccharides derived from red algae

(Monosaccharide) on the low molecular weight polysaccharides (AMG-LMWP) derived from glucoamylase hydrolyzate-derived high molecular weight polysaccharides (AMG-HMWP) and glucoamylase hydrolyzate ) Composition is shown in Fig. The low molecular weight polysaccharide (AMG-LMWP) showed a monosaccharide content of 64.50% glucose, 27.25% galactose, and 8.25% mannose. The unfractionated polysaccharide (glucoamylase (AMG) hydrolyzate) and the high molecular weight polysaccharide (AMG-HMWP) have higher galactose content (93.60% and 92.28%, respectively) than the low molecular weight polysaccharide (AMG- Respectively. In addition, the low molecular weight polysaccharide (AMG-LMWP) exhibited a low sulfate content of less than 1%, which was lower than the sulfate content of the polysaccharide (5.41%, Table 2). Microwaves caused desulfation of sulfated polysaccharides without significant molecular weight loss (Navarro et al., Carbohydrate Polymers, 2007, 69 (4): 742-747).

Porphyran, a sulfated polysaccharide associated with agarose consisting of 1,4-linked 3,6-anhydro- _L- galactose and 1,3-linked _D -galactose residues, is a major cell wall constituent of the red algae _{D -galactose, 3,6-anhydro- L -galactose} , consists of _{6- O -methyl- D -galactose (Hatada et} al., Journal of Agricultural and Food chemistry, 2006, 54 (26): 9895-9900., Jiang et al., The Journal of biochemistry, 2012, 151 (1): 65-74.), Antioxidant, anti-cancer, anti-hyperlipidemia, (4): 377-382., Qian et al., International Journal of Biological Macromolecules, 2014 &quot;, International Journal of Biological &lt; RTI ID = , 68: 48-49., Inoue et al., Biotechnology, and Biochemistry, 2009, 73 (2): 447-449.).

However, in the present invention, the desulfate polysaccharide showed remarkably enhanced antioxidative effect. It was thought that the biologic effect was affected by the degradation and monosaccharide composition of the polysaccharide in which enzymatic hydrolysis by microwave treatment occurred. Amyloglucosidase (AMG) hydrolyzed 1,4-α and 1,6-α linkages in liquefied starches. During hydrolysis, glucose was stepwise removed from the non-reducing end of the substrate molecule. The hydrolysis rate is affected not only by the chain length but also by the type of bonding as 1,4-α bond is hydrolyzed faster than 1,6-α bond, and maltotriose and maltose are longer (Bock et al., Carbohydrate Research, 1985, 145 (1): 135-140). &Lt; / RTI &gt; Accordingly, the invention is a glucosidase amylase P. yezoensis Suggesting hydrolysis of the polysaccharide by decomposing the 1,4-linked 3,6-anhydro- _L- galactose of the derived polysaccharide.

All measurements for the experiments according to the invention were repeated three times and all values were expressed as standard error. The results were analyzed by ANOVA and a Turkey test was conducted to analyze the difference. The p <0.05 value was considered to represent statistical significance.

Claims (6)

I) treating Pyropia yezoensis powder with distilled water to remove water-soluble proteins;
Ii) removing the water-soluble protein and removing lipid by adding 90% ethanol at 150 to 250 ° C;
Iii) obtaining a polysaccharide in the residue by adding distilled water at 100 to 150 ° C after removing the lipid;
Iv) hydrolyzing amyloglucosidase (AMG), which is a carbohydrate hydrolase, in a weight ratio of 10 to 100: 1 with respect to the polysaccharide in a microwave of 30 to 40 W; And
V) separating the low molecular weight polysaccharide having a molecular weight of 1,000 to 10,000 Da from the hydrolyzate;
( Pyropia yezoensis ) -containing low molecular weight polysaccharide,
The low molecular weight polysaccharide may include 64 to 65% by weight of glucose as a main constituent sugar, 27 to 28% by weight of galactose and 8 to 9% by weight of mannose, ( Pyropia yezoensis ) -containing low molecular weight polysaccharide. delete The method of claim 1, wherein the polysaccharide is a radiation pattern Kim (Pyropia yezoensis) method derived from a low molecular weight polysaccharide, characterized in that it has an antioxidant effect. delete 2. The method according to claim 1, wherein the microwave is produced using a microwave assistant rapid enzyme digest system ( MAREDS ). delete

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