KR20200126207A - Enzymatic lysate of phaeophyceae with enhanced alginate content and method for preparing thereof - Google Patents

Abstract

The present invention relates to an enzymatic lysate of Phaeophyceae with an enhanced content of alginic acid, and a method for preparing the same. According to the method, an extraction yield of alginic acid can be increased and low-molecular-weight alginic acid can be obtained, and thus the method can be effectively used for alginic acid extraction.

Description

Enzymatic lysate of phaeophyceae with enhanced alginate content and method for preparing thereof {Enzymatic lysate of phaeophyceae with enhanced alginate content and method for preparing thereof}

The present invention relates to an enzyme decomposition product of brown algae (phaeophyceae) with an enhanced content of alginic acid and a method for preparing the same, and in more detail, the content of alginic acid is enhanced by treating the hot water extract of kelp with cellulase. It relates to an enzyme digestion product of brown algae.

Seaweed in Korea is a large-scale fishery resource that accounts for approximately 25% of the total aquatic product production. Among them, kelp (sea tangle) is a sea horse of 1.5 to 3.5 m in length and 25 to 40 cm in width. It is a Hanhaeseong plant distributed in the coasts of the cold and subarctic periods. It is known to grow in the areas of Hamgyeongnam-do and Hamgyeongbuk-do.

In addition to various pigments such as carotene, xanthophyll, and chlorophyll, kelp contains a lot of carbohydrates such as manite and laminarin, which are made by carbon assimilation, and alginic acid, a component of the cell wall, and amino acids such as iodine, vitamin B2, and glutamic acid. Contains this. Ingredients vary depending on the type, but are generally about 16% moisture, 7% protein, 1.5% fat, 49% carbohydrates, and 26.5% inorganic salts, and 20% of carbohydrates are fiber, and the rest are polysaccharides such as alginic acid and laminarin. In particular, since it contains a lot of inorganic salts such as iodine, potassium, and calcium, it is a good source of inorganic salts. Laminin, an amino acid in kelp, has the effect of lowering blood pressure.

However, these seaweeds are distributed in a fresh state or are mainly distributed with only simple processing such as small dried products, self-dried products, salted products, and seasoned grilled products. In particular, it is not an exaggeration to say that seaweed, a large-scale resource, is left almost as it is due to the current level of processing or lack of processing technology development, even though the distribution of seaweed is very weak in the fresh state and processing is essential.

These methods have a disadvantage in that the yield of alginic acid extraction is low because they are only simple processing techniques using hot water extraction, simple pasting, and pulverized powder of kelp and seaweed.

Alginic acid is a high molecular polysaccharide with an average molecular weight of 300,000 to 2 million, and has been widely used industrially. However, it has a long dissolving time at room temperature and is not well soluble in alcohol-containing solvents. Low-molecularization studies are being conducted to compensate for the shortcomings while maintaining the function.

As a conventional manufacturing technique for lowering the molecular weight to increase the solubility of alginic acid, there is a partial oxidation method with sodium periodate (NaIO ₄ ) or a high-temperature hydrolysis method using hydrochloric acid. However, the low-molecular alginic acid production method of seaweed polysaccharides by acid requires a large amount of alkali to be discharged by neutralizing and discharging the acid-resistant device and the discarded strong acid due to excessive acid treatment. There are drawbacks.

Therefore, in the present invention, in order to separate alginic acid from kelp, a physical and biological method instead of a conventional chemical method is used to manufacture consumer-friendly food using a safe and eco-friendly method rather than an extraction method using chemicals.

Accordingly, the present inventors found that in extracting alginic acid from kelp, the yield of alginic acid was significantly increased by first performing hot water extraction and then treatment with cellulase, and also low molecular weight alginic acid can be obtained. Confirmed.

Accordingly, it is an object of the present invention to provide an enzyme digestion product of brown algae (phaeophyceae) having an enhanced alginic acid content.

Another object of the present invention is to provide a method for preparing a brown algae enzyme decomposition product having an enhanced alginic acid content.

Another object of the present invention is to provide a food composition with enhanced alginic acid content, prepared by enzymatic treatment of hot water extract of brown algae.

The present invention relates to a brown algae enzyme decomposition product having an enhanced alginic acid content and a method for preparing the same, and the method according to the present invention is to increase the extraction yield of alginic acid by treating the hot water extract of kelp with cellulose, and to obtain alginic acid having a low molecular weight. It can have an effect.

When the present inventors prepared a hot-water extract of kelp, and then treated with cellulose, the content of alginic acid increased, and as a result of measuring the molecular weight of the obtained alginic acid, it was confirmed that the molecular weight was smaller than that of the alginic acid contained in the hot-water extract. .

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a brown algae enzyme decomposition product having an enhanced alginic acid content, prepared by enzymatic treatment of a brown algae (phaeophyceae) extract.

The brown algae may be selected from the group consisting of kelp, seaweed, green michae and wrinkled rhubarb, and may be, for example, kelp, but is not limited thereto.

The enzyme may be cellulase.

The enzyme digestion product contains alginic acid having a molecular weight of 10 to 600,000, 10 to 400,000, 10 to 200,000, 10 to 100,000, 10 to 80,000, 10 to 10,000, 10 to 1,000 or 10 to 100, for example, 10 to 50 It may be, but is not limited thereto.

Alginic acid aid production worldwide is about 100,000 tons, of which about 30% is used as food additives, but if it is developed as a high-value pharmaceutical ingredient, its value can be doubled. Currently, the unit price of alginic acid is about 10-15 dollars per kg, and when the polymerization degree is lowered and the molecular weight is about 40 kDa, the unit price is about 200 dollars per kg, and alginate oligosaccharide is sold at the level of 600 dollars per kg.

The market of low-molecular alginic acid is used for a variety of purposes, such as the manufacture of beverages, infant formula, animal feed, confectionery and bakery, dairy products, and snacks. In particular, low-molecular sugars used in animal feed are expected to grow at a CAGR of 6.5%. . In addition, low molecular weight sugars in the beverage market are estimated to have the largest share and market value, and are predicted to dominate the global beverage sweetener market in the future.

Low-molecularized alginic acid exhibits the same effect as alginic acid's dietary fiber. It goes down to the intestine and becomes a nutrient source for beneficial bacteria in the intestine, helping to improve the internal environment of the colon, as well as anti-cholesterol and anti-obesity effects. Has the function of.

Therefore, since simple processed products of seaweed have poor price competitiveness, the technology of converting the existing alginic acid extraction yield to a high value-added material through enhancement and low molecular weight is very important.

The'extract' includes a crude solvent extract, a specific solvent-soluble extract (solvent fraction), and a solvent fraction of the crude solvent extract, and the brown algae extract may be in a solution, concentrate or powder state.

The brown algae extract may be a crude extract obtained by extracting brown algae with water and at least one solvent selected from the group consisting of straight chain or branched alcohols having 1 to 4 carbon atoms.

In the case of using a mixture of water and alcohol as a solvent used in the production of crude brown algae extract, 10% or more to less than 100% (v/v), 20% or more to less than 100% (v/v), 30% or more To less than 100% (v/v), more than 40% to less than 100% (v/v), more than 50% to less than 100% (v/v), more than 60% to less than 100% (v/v), 70 % Or more and less than 100% (v/v), 10% or more and less than 90% (v/v), 20% or more and less than 90% (v/v), 30% or more and less than 90% (v/v) , 40% or more and less than 90% (v/v), 50% or more and less than 90% (v/v), 60% or more and less than 90% (v/v) or 70% or more and less than 90% (v/v ) Of a C 1 to C 4 linear or branched alcohol aqueous solution, for example, 80% (v/v) C 1 to C 4 linear or branched alcohol aqueous solution.

In addition, the aqueous alcohol solution may be at least one selected from the group consisting of an aqueous methanol solution, an aqueous ethanol solution, an aqueous propanol solution, and an aqueous butanol solution, and may be, for example, an aqueous ethanol solution, but is not limited thereto.

The brown algae extract according to the present invention may be a solvent fraction obtained by fractionating the crude solvent extract with an additional solvent, for example, using at least one solvent selected from the group consisting of ethyl ether, ethyl acetate, and butanol as the crude solvent extract. It may be a solvent fraction.

For example, the crude solvent extract obtained by extracting the brown algae with at least one solvent selected from the group consisting of water and a straight chain or branched alcohol having 1 to 4 carbon atoms is at least one selected from the group consisting of ethyl ether, ethyl acetate, and butanol. It may be a solvent fraction using a solvent.

One aspect of the present invention relates to a method of preparing a brown algae extract.

The brown algae extract includes a crude solvent extract and a solvent fraction of brown algae, as described above.

The brown algae extract may be a crude extract obtained by extracting at least one selected from the group consisting of leaves, stems and roots of brown algae with at least one solvent selected from the group consisting of water and a straight chain or branched alcohol having 1 to 4 carbon atoms. have.

The solvent is 10% or more to less than 100% (v/v), 20% or more to 100% (v/v), 30% or more to 100% (v/v), 40% or more to 100% (v /v) less than, 50% or more to less than 100% (v/v), 60% or more to 100% (v/v), 70% or more to less than 100% (v/v), 10% or more to 90% Less than (v/v), more than 20% and less than 90% (v/v), more than 30% and less than 90% (v/v), more than 40% and less than 90% (v/v), more than 50% Less than 90% (v/v), 60% or more to 90% (v/v) or 70% or more to 90% (v/v) straight chain or branched alcohol aqueous solution having 1 to 4 carbon atoms, for example, It may be an 80% (v/v) C 1 to C 4 linear or branched aqueous alcohol solution.

The manufacturing process of the brown algae extract according to the present invention will be described in more detail as follows: After cutting the brown algae and washing with water to remove constrictions, extraction at room temperature with an extraction solvent of about 10 to 50 times the weight of the brown algae do. After extraction, filter and collect the filtrate. The extraction temperature is not particularly limited, but is preferably 15 to 110, preferably 20 to 90.

The extraction process may be repeated once or several times, and in one preferred example of the present invention, a method of re-extraction after the first extraction may be employed. This is the case of mass production of herbal extracts, even if the herbal medicine itself is effectively filtered. This is to prevent the loss because the moisture content is high, and the extraction efficiency is lowered only by the first extraction. In addition, as a result of verifying the extraction efficiency of each step, it was found that about 80 to 90% of the total extraction amount was extracted by the secondary extraction.

In an example of the present invention, when the extraction process is repeated twice, the obtained residue is extracted under reflux with an extraction solvent, about 5 to 15 times by volume, preferably 8 to 12 times by volume. After extraction, it is filtered, combined with the filtrate obtained previously, and concentrated under reduced pressure to prepare a brown algae extract. As described above, extraction efficiency can be increased by mixing the second extraction and the filtrate obtained after each extraction, but the extract of the present invention is not limited to the number of extractions.

If the amount of the solvent used in the manufacture of the brown algae extract is too small, stirring becomes difficult and the solubility of the extract decreases, resulting in lower extraction efficiency, and if too large, the amount of solvent used in the next purification step increases, which is not economical. Since handling problems may occur, the amount of the solvent is preferably within the above range.

The filtered extract thus obtained is about 10 to 30 times by weight, preferably 15 to 25 times by weight, more preferably about 10 to 30 times by weight of the total amount of the concentrate in order to adjust the content of the remaining lower alcohol to be suitable for use as a pharmaceutical ingredient. It can be prepared as a powdery brown algae extract by azeotropic concentration 1 to 5 times, preferably 2 to 3 times with 20 times the weight of water, and homogeneously suspending by adding the same amount of water again, followed by lyophilization.

The extraction method used in the present invention may be any commonly used method, for example, cold sedimentation, hot water extraction, ultrasonic extraction, or reflux cooling extraction method, but is not limited thereto.

Another aspect of the present invention is a method for producing a brown algae enzyme decomposition product with an enhanced alginic acid content comprising the following steps:

Hot water extraction step of preparing a hot water extract of brown algae; And

Enzymatic treatment step of reacting by adding an enzyme to the hot water extract.

The brown algae may be selected from the group consisting of kelp, seaweed, green michae and wrinkled rhubarb, and may be, for example, kelp, but is not limited thereto.

The hot water extraction step may be performed under a temperature condition of 90 to 120°C or 90 to 110°C, for example, 95 to 105°C, but is not limited thereto.

The hot water extraction step may be performed for 1 to 8 hours, 2 to 8 hours, or 4 to 8 hours, for example, 5 to 7 hours, but is not limited thereto.

The enzyme treatment step may be performed under a temperature condition of 60 to 90°C, 70 to 90°C, 60 to 85°C, or 70 to 85°C, for example, 75 to 85°C.

The enzyme treatment step may be performed for 1 to 10 hours, 3 to 10 hours, or 5 to 10 hours, for example, may be performed for 5 to 8 hours. There was no significant change in viscosity after 6 hours in the enzyme treatment time, and the change was also very insignificant.

The enzyme may be cellulase.

The molecular weight of the alginic acid may be 10 to 600,000, 10 to 400,000, 10 to 200,000, 10 to 100,000, 10 to 80,000, 10 to 10,000, 10 to 1,000 or 10 to 100, for example, 10 to 50, but limited thereto. It does not become.

Another aspect of the present invention is a food composition with improved alginic acid content, prepared by enzymatic treatment of hot water extract of brown algae.

The brown algae may be selected from the group consisting of kelp, seaweed, green michae and wrinkled rhubarb, and may be, for example, kelp, but is not limited thereto.

The enzyme may be cellulase.

The food composition contains alginic acid having a molecular weight of 10 to 600,000, 10 to 400,000, 10 to 200,000, 10 to 100,000, 10 to 80,000, 10 to 10,000, 10 to 1,000 or 10 to 100, for example, 10 to 50 It may be, but is not limited thereto.

When the food composition of the present invention is used as a food additive, the food composition may be added as it is or may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. In general, in the manufacture of food or beverage, the health functional food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less based on the raw material.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, Alcoholic beverages and vitamin complexes, and all foods in the usual sense are included.

The beverage may contain various flavoring agents or natural carbohydrates as additional ingredients. As the natural carbohydrates described above, monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, synthetic sweeteners such as saccharin and aspartame may be used. . The proportion of the natural carbohydrate can be appropriately determined by the choice of a person skilled in the art.

In addition to the above, the food composition of the present invention includes various nutrients, vitamins, electrolytes, flavoring agents, colorants, pectic acids and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols , Carbonated beverages used in carbonated beverages, and the like. In addition, the health functional food composition of the present invention may contain pulp for the manufacture of natural fruit juice, fruit juice beverage and vegetable beverage. These components may be used independently or in combination. The proportion of these additives can also be appropriately selected by a person skilled in the art.

The present invention relates to an enzyme decomposition product of brown algae (phaeophyceae) with an enhanced content of alginic acid and a method for preparing the same.According to the method, the extraction yield of alginic acid can be increased and alginic acid having a low molecular weight can be obtained. It can be used for alginic acid extraction.

1 is a graph showing the amount of reducing sugar produced by cellulase treatment after hot water extraction of kelp.
2 is a result of a viscosity check experiment according to a single treatment for hot water extraction of kelp and a cellulase treatment after hot water extraction.
3 is a molecular weight gel chromatography analysis result of a single treatment of hot water extraction of kelp.
4 is a result of molecular weight gel chromatography analysis by cellulose treatment after hot water extraction of kelp.

Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)% for solids/solids, (weight/volume)% for solids/liquids, and Liquid/liquid is (vol/vol)%.

Example 1: Confirmation of changes in reducing sugar production in kelp treated with cellulose

The cell wall of kelp is composed of cellulose, and cellulose is a polymer composed of β-D-glucose bonded to β-glucoside, so it is decomposed into β-D-glucose when treated with cellulase. . Therefore, since the content of reducing sugar increases, the content of reducing sugar was measured to determine whether the cell wall of kelp was degraded by cellulase treatment.

The experiment for measuring the reducing sugar content was carried out as follows. 15 kg of 10% (w/v) kelp powder was added to 150 L distilled water and stirred, followed by hot water extraction at 100°C for 6 hours, and then the temperature was lowered to 37°C.

Thereafter, cellulose was dissolved in a buffer (Citrate-Phosphate buffer, pH 7.4) to 1% (v/v), treated with the hot water extract, and samples were obtained under the conditions of 30, 60, 90, 120, 150, 180 minutes. The free reducing sugar was measured.

The measured reducing sugar was quantified by 3,5-dinitrosalicylic acid (DNS) method. The reaction was stopped by adding 3 mL of a DNS reagent to the kelp hot water extract sample obtained by time, and then reacted in a constant temperature water bath at 100° C. for 5 minutes to develop color, and the content of reducing sugar was confirmed by measuring the absorbance at 540 nm. In addition, in order to check whether alginic acid changes due to cellulose, the content of alginic acid was confirmed by measuring absorbance at 240 nm in commercial alginic acid samples treated with cellulose under the same conditions.

As can be seen in Fig. 1 and Table 1, it was confirmed that reducing sugar increased as the cell wall of kelp was destroyed by cellulose, and that cellulose did not affect alginic acid itself.

Time (minutes) 0 30 60 90 120 150 180 Hot water extraction + cellulase treatment sample 0.552 0.943 1.083 1.257 1.321 1.263 1.385 Commercial alginic acid + cellulase treatment sample 0.384 0.335 0.413 0.428 0.433 0.341 0.409

What can be seen from the measurement of reducing sugar is the result of measurement of β-D-glucose produced by the decomposition of the kelp cell wall. As a result, the alginic acid attached between the kelp cell walls is eluted as the cell wall collapses, so the degree of degradation of the kelp cell wall can be confirmed from the reducing sugar value. The increase in reducing sugar can be used as an indicator of alginic acid elution due to the destruction of the kelp cell wall.

Example 2: Confirmation of viscosity change of extract by cellulase treatment

The viscosity was measured to check whether the viscosity of the extract was increased by the cellulose treatment.

In the case of hot water extraction samples, hot water extraction was performed in the same manner as in Example 1, and samples were sampled at 1 hour intervals. The viscosity was measured (Spinder 06, 100 rpm condition) using a viscometer (Vicometer, BrookField) from each sample taken.

In the case of the cellulose-treated sample, the cellulose reaction was performed on the extract subjected to hot water extraction for 6 hours in the same manner as in Example 1, and the samples were sampled at 1 hour intervals, the same as the viscosity of the hot water extraction sample was measured. The viscosity was measured by the method.

As can be seen in Fig. 2 and Table 2, in the case of the hot water extraction sample, it was confirmed that the viscosity of the sample obtained by the hot water extraction for 6 hours showed the highest value of 20 cP. In the cellulose-treated sample, it was confirmed that the viscosity of the sample in which cellulose was reacted for 6 hours showed the highest value of 400 cP.

Time (hours) 0 One 2 3 4 5 6 Hot water extraction sample (cP) 0 0 10 10 10 20 20 Cellulase treatment sample (cP) 20 40 80 120 160 240 400

Since it was confirmed that the viscosity increased by 20 times when the cellulose was treated compared to the single hot water extraction treatment, it was confirmed that the content of alginic acid in the kelp extract was increased due to the cellulose treatment through this.

Example 3: Confirmation of molecular weight of alginic acid whose content is enhanced by cellulose treatment

Gel chromatography analysis was performed to confirm the molecular weight of alginic acid produced by hot water extraction of kelp and cellulose treatment. The hot-water extract sample was sampled after hot-water extraction for 6 hours, and the cellulose-treated sample was sampled after hot-water extraction for 6 hours and then treated with cellulose for 12 hours. Samples were sequentially filtered using 11.00, 5.00, 3.00 and 0.45 um filters after sampling, and then lyophilized.

20 mg of the lyophilized powder was diluted in a buffer (phosphate buffer, pH 7.0) to a concentration of 0.25 to 1.0% (w/v), and gel permeation chromatography (GPC) analysis was performed using an HPLC (HPLC-YL9100) analyzer. (Analytical columns: Shodex SB-804 HQ and SB-802.5 HQ OHPak, 55° C., Flow volume: 0.6 mL/min, Injection volume: 50 uL).

As can be seen in FIG. 3, alginic acid polymerized with a molecular weight of 780,245 was extracted from the hot water extraction sample.

On the other hand, as can be seen in Figure 4, it was confirmed that relatively low molecular weight alginic acid was extracted from the cellulose-treated sample compared to the single treatment of hot water extraction with a molecular weight of 492,047, 83,988, 244, 22.833, 24.868, 29.710.

Claims (16)

Brown algae enzyme decomposition product with enhanced alginic acid content, prepared by enzymatic treatment of phaeophyceae extract. According to claim 1, wherein the brown algae is selected from the group consisting of kelp, seaweed, green michae and wrinkled rhubarb, brown algae enzyme digestion with enhanced alginic acid content. According to claim 1, wherein the enzyme is cellulose (cellulase), the brown algae enzyme decomposition product with enhanced alginic acid content. The brown algae enzyme digestion product of claim 1, wherein the enzyme digestion product contains alginic acid having a molecular weight of 10 to 600,000. A method for producing a brown algae enzyme decomposition product with an enhanced alginic acid content comprising the following steps:
Hot water extraction step of preparing hot water extract of brown algae (Phaeophyceae); And
Enzymatic treatment step of reacting by adding an enzyme to the hot water extract. The method of claim 5, wherein the brown algae is selected from the group consisting of kelp, seaweed, green michae and wrinkled rhubarb. The method of claim 5, wherein the hot water extraction step is performed under a temperature condition of 90 to 120°C. The method of claim 5, wherein the hot water extraction step is performed for 1 to 8 hours. The method of claim 5, wherein the enzyme treatment step is performed under a temperature condition of 60 to 90°C. The method of claim 5, wherein the enzyme treatment step is performed for 1 to 10 hours. The method of claim 5, wherein the enzyme is cellulase. The method of claim 5, wherein the alginic acid has a molecular weight of 10 to 600,000, wherein the alginic acid content is enhanced. A food composition with enhanced alginic acid content, prepared by enzymatic treatment in hot water extract of brown algae (Phaeophyceae). The food composition of claim 13, wherein the brown algae is selected from the group consisting of kelp, seaweed, green michae and wrinkled rhubarb. 14. The food composition of claim 13, wherein the enzyme is cellulase. The food composition according to claim 13, wherein the food composition contains alginic acid having a molecular weight of 10 to 600,000.

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