CN114040929A

CN114040929A - Preparation method and application of agar for new agaro-oligosaccharide mixture raw material

Info

Publication number: CN114040929A
Application number: CN202080047657.7A
Authority: CN
Inventors: 李济贤; 金恩妵; 郭我民; 高惠定; 具熙纂
Original assignee: Dynebio Inc
Current assignee: Dynebio Inc
Priority date: 2019-06-04
Filing date: 2020-06-03
Publication date: 2022-02-11
Also published as: KR102214973B1; WO2020246785A2; KR20200139549A; WO2020246785A3; JP2022536097A

Abstract

One embodiment of the present invention provides a method for preparing agar, comprising the steps of extracting lettuce with hot water to prepare an agar-containing solution, cooling and solidifying the agar-containing solution, crushing the agar-containing solution into a predetermined size, and washing the crushed agar-containing solution with purified water to obtain a refined solidified product containing the agar-containing solution. The method for preparing agar according to the present invention is advantageous for commercialization due to its simple process, and is food-grade safe since it does not use a bleaching agent or the like. In addition, the agar prepared according to the method of the present invention has improved sensitivity to decomposition by β -agarase, and thus a neoagaro-oligosaccharide mixture having relatively high contents of neoagarotetraose (neoagarotetraose) and neoagarohexaose (neoagarohexaose) can be prepared.

Description

Preparation method and application of agar for new agaro-oligosaccharide mixture raw material

Technical Field

The present invention relates to a method for preparing agar used as a raw material in preparation of a neoagaro-oligosaccharide mixture, and more particularly, to a method for preparing agar from which a neoagaro-oligosaccharide mixture having a relatively high content of neoagarotetraose (neoagarotetraose) and neoagaro-hexaose (neoagarohexaose) can be prepared by increasing the sensitivity to decomposition by β -agarase, and a method for preparing a neoagaro-oligosaccharide mixture using the same.

Background

Agar (Agar) has been widely used for a long time as a food additive, a pharmaceutical product, a cosmetic product, a livestock feed, an industrial material, etc., and as a representative polysaccharide derived from marine algae, it has a yield of about 2000 to 5000 tons per year in korea, and is one of the relatively abundant aquatic resources. In actual use, only a part of the total yield is simply processed and used as cheap raw materials, and the rest is idle, so that the additional value of the total yield is very low compared with the intrinsic resource yield. Therefore, research for developing new uses and increasing added values of korean abundant agar is highly required.

Agar is mostly composed of polysaccharides except for a small amount of protein, ash and fat, and polysaccharides constituting the agar include neutral polysaccharide agarose (agarose) and acidic polysaccharide agar gum (agaropectin). Agarose is a monomer of agarobiose (agarobiose) in which D-galactose (D-galactose) and 3,6-anhydro-L-galactose (3,6-anhydro-L-galactose) are bound in a β -1,4 form, and has a strong gel formation ability because agarobiose has a linear structure in which α -1,3 binding is repeated. On the other hand, agar gel, like agarose, contains agarobiose as a monomer, but has a weak gelling ability because it contains an acidic group such as a sulfate group (sulfate group).

Wherein, agarose is decomposed into neoagarobiose (neoagarase) by acting on beta-1, 4-conjugated beta-agarase (beta-agarase) and neoagarase (neoagarotetraose), and is finally decomposed into galactose (D-galactose) and 3,6-anhydro-L-galactose (3,6-anhydro-L-galactose) by acting on alpha-1, 3-conjugated alpha-agarase (alpha-agarase). In addition, agarose is broken down into Agarobiose (Agarobiose) by dilute acid or alpha-agarase. In general, a neoagaro-oligosaccharide is an oligosaccharide obtained by hydrolyzing agar or agarose with β -agarase (β -agarase) and binding 2 to 10 monosaccharides such as neoagarobiose, neoagarotetraose, neoagarohexaose (neoagaroohexaose), and neoagarooctaose (neoagarococtaose). The novel agaro-oligosaccharide is an oligosaccharide obtained by hydrolyzing agar or agarose with a dilute acid or α -agarase, and is obtained by binding 2 to 10 monosaccharides such as agarobiose, Agarotetraose (Agarotetraose), Agarohexaose (Agarohexaose), and Agarooctaose (agarococtaose). The agaro-oligosaccharides have 3,6-anhydro-L-galactose (3,6-anhydro-L-galactose) as a non-reducing end, while the agaro-oligosaccharides have D-galactose (D-galactose) as a non-reducing end, and thus show different properties in terms of physiological activities due to such structural differences.

On the other hand, Streptomyces coelicolor A3(2) as an actinomycete has been disclosed (Stanier et al, 1942, J.Bacteriol., Hodgson and Chater,1981, J.Gen.Microbiol.) to produce a β -agarase for decomposing agar or agarose in the form of an extracellular (secreted to the outside of the cell) protein, and this agarase is encoded by the DagA gene or the DagB gene. Of the β -agarases produced by Streptomyces coelicolor A3(2), DagA enzyme mainly decomposes agar or agarose to produce DP4 (neoagarotetraose) and DP6 (neoagarohexaose), and DagB enzyme mainly decomposes agar or agarose to produce DP2 (neoagarobiose). The DagA gene plays an important role in the study of agarase production by actinomycetes. In particular, Streptomyces coelicolor, which is the most widely used strain in the study of the molecular biology of actinomycetes, has been published in 2002 by analyzing the sequence of chromosomal DNA by Sanger centre (Sanger centre) in the UK.

Agar or agarose used as a raw material for preparing a novel agaropectin mixture by using β -agarase can be prepared by various methods. For example, korean laid-open patent publication No. 10-0125214 discloses a method for purifying agar using chitosan, which comprises adding chitosan and ethanol to a 1-5% agar solution, stirring, standing, precipitating agar gel (Agaropectin) present in the agar, precipitating the supernatant with 3 times the amount of ethanol, filtering with a glass filter, and drying under reduced pressure to reduce the content of ash and sulfate groups. Also, korean laid-open patent publication No. 10-0122019 discloses a method for pretreating agar raw grass (sea grass) by preparing agar from other agar raw grass (sea grass) such as Gelidium amansii, hedgerow (Gracilaria verrucosa) and Hypnea charoides through pretreatment, cooking, coagulation, dehydration and drying, wherein in the pretreatment step, the agar raw grass (sea grass) is soaked in a slaked lime or quick lime solution having a concentration of about 0.3 to 1.5% for about 2 to 4 hours at 30 to 60 ℃. Further, Korean patent laid-open publication No. 10-0196679 discloses a method for preparing high quality agar, comprising: adding EDTA salt solution into agar powder, stirring, removing supernatant, and neutralizing; washing and filtering the salt added in the EDTA salt treatment and neutralization step; and a step of adding an organic solvent to the obtained filtration residue to perform dehydration treatment and drying. Also, Korean patent laid-open No. 10-0317579 discloses a method for preparing high purity agarose for electrophoresis, which comprises dissolving agar raw material to 0.1-10% (W/V), adding chitosan or water-soluble chitosan in an amount of 0.1-20% based on the weight of agar, reacting at 50-90 deg.C, adjusting the hydrogen ion concentration of the reaction solution to 9-11 after the reaction is completed, precipitating, filtering or centrifuging the precipitate, and drying the supernatant. Further, Korean patent laid-open publication No. 10-0411481 discloses a method for preparing agar for a microorganism culture medium, it is characterized in that the agar is washed by clear water and dried, and is soaked in water which is 10 times of the agar for one night, then adding sodium hypochlorite of 0.05% liquid volume for bleaching treatment, washing the Eucheuma Gelatinosum with water, treating with 0.2% sulfuric acid solution, washing with water to neutrality, adding 0.02% sodium metaphosphate solution 10 times the amount of Eucheuma Gelatinosum into the Eucheuma Gelatinosum, fixing pH to 6.0, heating for extraction, adding potassium pyrophosphate, adjusting pH to 8.0, adding 2% liquid volume of diatomaceous earth, filtering, standing the filtrate at room temperature for gelation, cutting into 5 × 5 × 300mm size, bleaching with 0.1% hypochlorous acid soda, freezing at-80 deg.C, thawing, dehydrating, and drying, and filtering the agar extract with a fine filter with pore size of 0.2-0.4 μm. Further, korean laid-open patent publication No. 10-0484926 discloses a method for preparing agarose, which comprises adding a chitosan solution to an agar solution, precipitating to remove agar gel, separating the supernatant, and powdering, wherein the method comprises: adding an acidic solution into the supernatant obtained after the agar gel is removed through precipitation to adjust the pH value to 2-4, stirring for 1-6 hours, and adding an alkali for neutralization; and a step of cooling the neutralized agarose aqueous solution at normal temperature, adding 40-70% (v/v) ethanol having a concentration of 1.0 time the total amount of the agarose aqueous solution, separating the precipitate, and recovering the supernatant to obtain a powder. Further, Korean patent laid-open publication No. 10-0759525 discloses a method for preparing agar, which is characterized by comprising: a cleaning step, cleaning the lettuce; cooking, namely putting the raw agar into water, heating and extracting agar-containing agar solution together with steam; a filtering step, filtering the extracted agar solution from the lettuce residues; a solidification step of putting the filtered agar solution into a forming container for solidification, wherein in the solidification step, the depth of the agar solution put into the forming container is set to be the same as the length of the reserved thin agar (strip agar) after 30% of the depth of the agar solution is subtracted from the depth of the agar solution; a vertical cutting step of vertically pressing the agar block solidified in the solidification step on a cutting net to cut the agar to form agar before completion, wherein the step is performed by pressing the solidified agar block on the cutting net by a pressing plate lifted by a cylinder; freezing, dissolving and dehydrating the agar before completion; and a drying step of drying the agar before completion. Further, Korean patent laid-open publication No. 10-1759282 discloses a method for preparing agar from fence using alkali soaking and microwave dehydration process, comprising: a pretreatment step, namely, putting the dried hedgerow into boiled water, cooking the hedgerow, cooling the hedgerow with cold water, washing the hedgerow with flowing water, and drying the hedgerow; an alkaline soaking step, namely soaking the hedgerow treated in the pretreatment step in a sodium hydroxide solution with the concentration of 1-3% as an alkaline solution; an extraction step, heating the alkaline solution for soaking the hedgerow to extract the hedgerow extract; a gel forming step of forming the hedgerow extract into a gel; microwave dehydration step, freezing the gel, thawing with microwave and dehydrating; a drying step of washing and drying the gel dehydrated by the microwave dehydration step, wherein the alkali soaking step is to stir the alkali solution for 30 to 60 minutes, the extraction step is to heat the alkali solution to 100 to 130 ℃ and extract for 30 to 120 minutes, and the gel forming step is to a) filter step of filtering the hedgerow extract to obtain a filtrate; b) a neutralization treatment step of adding an acid to the filtrate to perform neutralization treatment; c) a coagulation step of coagulating the neutralized filtrate to form a gel; the step (b) of dehydrating the gel with microwaves, wherein the gel is frozen at-20 ℃ for 12 hours and microwaves are applied for 30 to 60 minutes. The methods for preparing agar or agarose disclosed in the above prior art have problems that some processes are very complicated and not suitable for commercialization, some use unsuitable compounds and the like in materials for producing health functional foods, and some prepared agar is poor in the decomposition sensitivity to β -agarase and a sufficient amount of a novel agaro-oligosaccharide mixture cannot be obtained.

In particular, DP4 and DP6 are reported to have better improving, anticancer and immunity enhancing effects than DP2 on metabolic diseases such as obesity, diabetes, hyperlipidemia and the like in beta-agarase reaction products of agar or agarose. Therefore, it is desired to develop a method for preparing safe edible agar or agarose which can prepare a new agaro-oligosaccharide mixture having a high content of DP4 and DP 6.

Disclosure of Invention

Problems to be solved by the invention

The invention is completed under the background of the prior art, and aims to provide a preparation method of agar which is simple in process, beneficial to commercialization and safe in food grade, can improve the decomposition sensitivity to beta-agarase, and can prepare a new agaro-oligosaccharide mixture with relatively high contents of new agaro tetrasaccharide and new agaro hexaose. In addition, another object of the present invention is to provide a method for preparing a novel agaro-oligosaccharide mixture using the method for preparing agar.

Means for solving the problems

The present inventors have come to study and develop a method for preparing agar that is simple in process and safe in food grade, since commercially available agar products are not suitable as raw materials for preparing functional food materials for health care such as a novel agaro-oligosaccharide mixture because of the use of a bleaching agent or the like in the preparation process. The present inventors have confirmed that when agar-containing solutions are prepared from lettuce (i.e., raw agar) by hot water extraction and immediately reacted with β -agarase to prepare a neoagaro-oligosaccharide mixture, the final reaction product has relatively low contents of DP4 (neoagarotetraose) and DP6 (neoagarohexaose), although it is simple in process and food-grade safe, so that the process is further improved. As a result, it was confirmed that when an agar-containing solution was prepared from lettuce by hot water extraction, cooled to solidify and then pulverized to a certain size, washed with purified water and then dissolved again to prepare a refined agar-containing solution, and this was reacted with β -agarase to prepare a novel agaro-oligosaccharide mixture, the contents of DP4 and DP6 in the final reaction product were significantly increased, thereby completing the present invention.

In order to achieve the above object, one embodiment of the present invention provides a method for preparing agar, comprising: adding lithocarpus capsulifera and water into an extraction tank, extracting with hot water, and separating hot water extract from residues to obtain an agar-containing solution; transferring the agar-containing solution into a stirring tank, carrying out primary cooling while stirring to cool the agar-containing solution to 50-55 ℃, then stopping stirring, and carrying out secondary cooling in a standing state to cool the agar-containing solution to 10-25 ℃ to obtain a solidified substance containing the agar solution; and (c) crushing the coagulated product of the agar-containing solution in the stirring tank to a size of 0.5 to 15cm, and then washing the crushed product with water (water) to obtain a refined coagulated product of the agar-containing solution.

In order to achieve the above object, one embodiment of the present invention provides a method for preparing a novel agaro-oligosaccharide mixture, comprising: adding lithocarpus capsulifera and water into an extraction tank, extracting with hot water, and separating hot water extract from residues to obtain an agar-containing solution; transferring the agar-containing solution into a stirring tank, carrying out primary cooling while stirring to cool the agar-containing solution to 50-55 ℃, then stopping stirring, and carrying out secondary cooling in a standing state to cool the agar-containing solution to 10-25 ℃ to obtain a solidified substance containing the agar solution; crushing the coagula containing the agar solution to 0.5-15 cm in the stirring tank, and then washing with water to obtain a refined coagula containing the agar solution; and (d) heating and dissolving the refined agar solution-containing solidified product, stirring, cooling to 35-50 ℃, adding beta-agarase, and performing an enzymatic reaction to obtain the new agaro-oligosaccharide.

Effects of the invention

The preparation method of agar according to the present invention is advantageous for commercialization due to its simple process, and is food-grade safe since it does not use a bleaching agent or the like. In addition, the agar prepared by the method improves the decomposition sensitivity to beta-agarase, so that a new agaro-oligosaccharide mixture with relatively high contents of new agaro tetrasaccharide and new agaro hexaose can be prepared.

Drawings

FIG. 1 is a diagram showing the properties of the purified agar-containing solution prepared in preparation example 1 and the agar-containing solution prepared in comparative preparation example 1 in a liquid state, a coagulated state and a freeze-dried state.

Detailed Description

The present invention will be described in detail below.

One aspect of the present invention relates to a method for preparing agar, which is simple in process, safe in food grade, and excellent in the decomposition sensitivity to β -agarase. A method for preparing agar according to one embodiment of the present invention comprises: adding raw gelidium amansii (unprocessed gelidium amansii) and water into an extraction tank, extracting with hot water, and separating hot water extract from residues to obtain an agar-containing solution; transferring the agar-containing solution into a stirring tank, carrying out primary cooling while stirring to cool the agar-containing solution to 50-55 ℃, then stopping stirring, and carrying out secondary cooling in a standing state to cool the agar-containing solution to 10-25 ℃ to obtain a solidified substance containing the agar solution; and (c) crushing the solidified product containing the agar solution to 0.5-15 cm in the stirring tank, and then washing with water to obtain a refined solidified product containing the agar solution.

In one example of the method for preparing agar according to the present invention, the step (a) is a step of obtaining an agar-containing solution having a high temperature from lettuce by hot water extraction, filtration or the like in an extraction tank. The lettuce put into the extraction tank in the step (a) is preferably lettuce which has been subjected to pretreatment such as desalting and drying. The dry weight of the lettuce charged into the extraction tank in the step (a) is not limited to a large amount, but in consideration of smooth transportation after hot water extraction, smooth solidification after cooling, and the like, it is preferable to add 1 to 5kg per 100L of water (extraction solvent), and more preferably 2 to 3kg per 100L of water. In the step (a), the hot water extraction temperature is preferably 80 to 110 ℃, more preferably 85 to 105 ℃ in view of extraction efficiency, economy, and the like. In the step (a), the hot water extraction time is preferably 1 to 10 hours, more preferably 2 to 8 hours, in view of extraction efficiency, economy, and the like. In the step (a), the method of separating the hot water extract from the lettuce residue after the hot water extraction is not particularly limited, and examples thereof include a method of putting the lettuce in an extraction bag and extracting with hot water and then discharging only the extract, and a method of filtering with a filter cloth or a filter net (or a filter sieve).

In one embodiment of the method for preparing agar according to the present invention, the step (b) is a step of obtaining a solidified product of an agar-containing solution from the agar-containing solution at a high temperature by adjusting conditions such as cooling and stirring in a stirring tank. And (c) cooling the agar-containing solution moved to the stirring tank in the step (b) to 50-55 ℃ while stirring. In the step (b), if the agar-containing solution is once cooled to a temperature of 55 ℃ or higher or 50 ℃ or lower and then stirring is stopped, the coagulation of the agar-containing solution may not be smooth enough. In the step (b), the time for which the agar-containing solution is allowed to stand after once cooling and stopping stirring is preferably 15 to 30 hours, more preferably 18 to 25 hours, in view of smooth solidification and properties of the solidified product. In the step (b), the agar-containing solution is cooled to 10 to 25 ℃ by secondary cooling, preferably 12 to 22 ℃ in a state where stirring and standing are stopped.

According to one example of the method for preparing agar of the present invention, the step (c) is a step of washing the coagulated product of the agar-containing solution with clean water such as purified water to remove impurities such as agar and the like which adversely affect the enzymatic reaction of β -agarase, in order to increase the yield of neoagarotetraose and neoagarohexaose produced by the enzymatic reaction of β -agarase. In the step (c), the coagulated product containing the agar solution is pulverized to a size of 0.5 to 15cm, preferably 1 to 10cm, more preferably 1 to 5cm, before being washed with purified water. In the step (c), when the agar solution-containing coagulation product is crushed to a size of less than 0.5cm, when the agar solution-containing coagulation product is washed by injecting purified water into a stirring tank and discharging the purified water to the outside of the stirring tank, which will be described later, there are many problems such as the agar solution-containing coagulation product leaking out through a discharge pipe provided with a filter screen, the agar recovery rate or the agarose recovery rate being lowered, or the filter screen being clogged so that the agar solution-containing coagulation product cannot be washed continuously. In the step (c), the amount of water used for washing the coagulate of the agar-containing solution is preferably 2000 to 10000L, more preferably 3000 to 80000L, based on 1kg of the dry weight of the agar-containing solution used in the hot water extraction, in consideration of the purification level of the coagulate. In the step (c), in order to continuously perform the process of washing the congelation containing the agar solution, the congelation containing the agar solution may be washed by supplying water to the stirring tank after the congelation containing the agar solution is crushed in the stirring tank, and the water for washing may be discharged to the outside of the stirring tank through a discharge pipe provided with a filter mesh. In addition, in the step (c), in order to further remove impurities and factors that adversely affect the enzymatic reaction of β -agarase, it is more preferable that the method comprises washing the coagulated product of the agar-containing solution by supplying water to the inside of the agitation tank and discharging water to the outside of the agitation tank, transferring the coagulated product of the agar-containing solution and the water that has not been discharged from the inside of the agitation tank to a sieve (sieve), and removing the water that has not been discharged by vibration. In the step (c), when the process of washing the coagulate of the agar-containing solution is continuously performed, the flow rate of water supplied to the agitation tank for washing the coagulate of the agar-containing solution is preferably 3 to 8L/min, more preferably 4 to 6L/min, and the supply time of water is preferably 12 to 20 hours, more preferably 14 to 18 hours, based on 1kg of the dry weight of the agar used in the hot water extraction, in consideration of the agar recovery rate, the agarose content in the purified agar, or the agarose recovery rate.

An aspect of the present invention relates to a method for producing a neoagaro-oligosaccharide mixture having a relatively high content of neoagaro-tetrasaccharide and neoagaro-hexaose, and a method for producing a neoagaro-oligosaccharide mixture according to an embodiment of the present invention uses the aforementioned method for producing agar. A method for producing a novel agaro-oligosaccharide mixture according to one embodiment of the present invention comprises: injecting the lettuce and water into an extraction tank, extracting with hot water, and separating hot water extract from residues to obtain an agar-containing solution; transferring the agar-containing solution into a stirring tank, carrying out primary cooling while stirring to cool the agar-containing solution to 50-55 ℃, then stopping stirring, and cooling the agar-containing solution to 10-25 ℃ in a standing state to obtain a solidified substance of the agar-containing solution; and a step (c) of pulverizing the coagulated product of the agar-containing solution in the stirring tank to a size of 0.5 to 15cm, and then washing the pulverized product with water to obtain a purified coagulated product of the agar-containing solution; and (d) heating and dissolving the refined agar solution-containing solidified product, stirring, cooling to 35-50 ℃, and adding beta-agarase to perform an enzymatic reaction to produce new agaro-oligosaccharides.

In the method for producing a novel agar oligosaccharide mixture according to one embodiment of the present invention, steps (a) to (c) correspond to the method for producing agar described above, and therefore, detailed descriptions of the technical features are omitted.

In the method for producing a novel agaro-oligosaccharide mixture according to one embodiment of the present invention, the step (d) is a step of reacting agarose in a solidified product of the purified agar-containing solution with β -agarase to produce novel agaro-oligosaccharides. In the step (d), the refined agar-containing solution is heated to dissolve again, and the heating temperature of the refined agar-containing solution is preferably 80 to 110 ℃, more preferably 85 to 105 ℃. In the step (d), the β -agarase to be used for converting agarose in the solidified product of the purified agar-containing solution into a novel agaropectin may be selected from a variety of known β -agarases.

For example, the β -agarase is preferably a β -agarase DagA derived from Streptomyces coelicolor (Streptomyces coelicolor) in view of the kind and content ratio of the new agaro-oligosaccharide mixture. The present invention refers to examples disclosed in the specification of Korean patent laid-open No. 10-1632262, regarding the amino acid sequence and production method of DagA as described above. In the step (d), the amount of β -agarase to be added is not particularly limited, but is preferably 10 to 200 units/mL, more preferably 75 to 175 units/mL, in view of the efficiency of producing the novel agaro-oligosaccharide. The 1 Unit (Unit) used to show the activity of the above β -agarase was defined as follows: agarose was dissolved in 50mM PBS (pH 7) at a concentration of 0.2% (w/v) and 3.9mL was reacted at 40 ℃ for 5 minutes (reaction solution 4mL), DNS reagent (dinitrosalicylic acid 6.5g, 2M NaOH 325mL and propylene glycol 45mL were dissolved in 1L distilled water) was added in an amount equivalent to the reaction solution, the mixture was boiled for 10 minutes and cooled, and when absorbance was measured at 540nm, the amount of enzyme that produced an absorbance of 0.001 at 540nm was measured. In the step (d), the enzyme reaction temperature is not particularly limited as long as the activity of the β -agarase is in a suitable level or more without causing re-coagulation of the agar-containing solution purified under stirring, and is preferably 35 to 50 ℃ and more preferably 38 to 45 ℃ in view of the efficiency of production of new agaro-oligosaccharides. In the step (d), the enzyme reaction time is not particularly limited, but is preferably 10 to 24 hours, and more preferably 12 to 20 hours, in view of the efficiency of production of the novel agaro-oligosaccharide and the economy. In the step (d), the neoagaro-oligosaccharides produced by the enzymatic reaction are mainly neoagarotetraose and neoagarohexaose. For example, in the step (d), the final product obtained by the enzymatic reaction preferably contains 30 to 40% by weight of neoagarotetraose and 18 to 26% by weight of neoagarohexaose, based on the total weight of the solid component. In addition, in the step (d), the total content of the neoagarotetraose and the neoagarohexaose in the final product obtained by the enzymatic reaction is 50 to 65% by weight based on the total weight of the solid components.

Hereinafter, the present invention will be described in more detail by examples. However, the following examples are only for clearly illustrating the technical features of the present invention and do not limit the scope of the present invention.

1. Experimental raw materials and measuring method

(1) Production and titer of beta-agarase (titer)

The β -agarase used in the following examples is Streptomyces coelicolor (DagA) enzyme produced by Daneyobo, Inc., of the applicant. The specific production method of Streptomyces coelicolor DagA enzyme is the same as that of example 1 disclosed in the specification of Korean patent laid-open publication No. 10-1632262.

The activity of β -agarase was measured by the reducing sugar assay (DNS method). Specifically, the method for measuring β -agarase activity and the definition of 1U (Unit) are the same as those of example 2 disclosed in the specification of Korean patent laid-open No. 10-1632262.

(2) Determination of the Agarose (Agarose) content

The agarose content of the sample was measured by a known DMSO (Dimethyl sulfoxide) reaction method (refer to Journal of the Korean Fisheres Society v.18no.1,1985, pp.37-43).

(3) Determination of content of new agaro-oligosaccharide

The content of the new agaro-oligosaccharides in the sample is determined by HPLC.

2. Preparation of agar and preparation of new agar oligosaccharide mixture by using agar

Preparation example 1

Every 1kg of desalted and dried lettuce was put into extraction bags, and 20 extraction bags containing lettuce were prepared (equivalent to 20kg of lettuce in total). Then, 20 prepared extraction bags are placed in an extraction tank, 500L of purified water is injected for soaking, the temperature is kept at about 40-50 ℃ for about 10 minutes, and then the injected purified water is removed. Then, 800L of purified water was injected into the extraction tank, steam was supplied to the jacket outside the extraction tank, and hot water extraction was performed at about 95. + -. 5 ℃ for about 4 hours. After the hot water extraction is completed, a discharge pipe connected to the extraction tank is subjected to a pressure reduction treatment to separate the hot water extract from the residue to obtain an agar-containing solution, which is then transferred to a stirring tank through a sterilized valve pipe. Thereafter, cooling water was supplied to the outer jacket of the stirring tank while keeping the rotation speed of the impeller of the stirring tank at 35rpm, the agar-containing solution was cooled, and when the temperature of the agar-containing solution reached about 55 ℃, the operation of the impeller was stopped, and the stirring tank was allowed to stand for about 20 hours in a state where cooling water was supplied only to the outer jacket of the stirring tank, thereby solidifying the agar-containing solution. Then, the agar solution-containing coagulated product in the agitation tank is pulverized to a size of about 1 to 3cm, purified water is supplied to the interior of the agitation tank through an external pipe at a flow rate of about 100L/min for about 16 hours, the agar solution-containing coagulated product is washed, and the purified water for washing is discharged into the agitation tank through a discharge pipe equipped with a filter screen. Then, the coagulated product of the agar-containing solution washed in the stirring tank and the purified water not discharged were transferred to a vibrating screen, and the purified water not discharged from the washed agar-containing solution was removed by vibration to obtain a purified coagulated product of the agar-containing solution. Thereafter, the refined agar-containing solution was dissolved in the solidified product of the refined agar-containing solution by placing the solidified product of the refined agar-containing solution in a stirring tank, supplying steam to the outer jacket of the stirring tank to raise the temperature inside the stirring tank to 95. + -. 5 ℃ and then holding the temperature for about 1 hour.

Thereafter, while maintaining the impeller rotation speed of the agitation tank at 25rpm, cooling water was supplied to the outer jacket of the agitation tank, the temperature of the refined agar-containing solution was lowered to about 43 ℃, β -agarase was added in an amount corresponding to a titer of about 125U/mL, and an enzymatic reaction was carried out for about 16 hours to obtain a solution containing a reaction product. After completion of the enzyme reaction, steam was supplied to the jacket outside the stirring tank, and the temperature inside the stirring tank was raised to 95. + -. 5 ℃ and then kept for about 1 hour to inactivate the β -agarase. Thereafter, the steam supplied to the outer jacket of the stirring tank was cut off, cooling water was injected, the temperature inside the stirring tank was lowered to 65. + -. 5 ℃ and concentrated under reduced pressure, and then the concentrated solution containing the reaction product was obtained by filtration through a 1 μm filter. Then, the reaction product-containing concentrate was freeze-dried and pulverized to obtain a reaction product-containing powder.

Comparative preparation example 1

Every 1kg of desalted and dried lettuce was put into extraction bags, and 20 extraction bags containing lettuce were prepared (equivalent to 20kg of lettuce in total). Then, 20 extraction bags are placed into an extraction tank, 500L of purified water is injected for soaking, and the injected purified water is removed after the temperature is maintained at about 40-50 ℃ for about 10 minutes. Then, 800L of purified water was injected into the extraction tank, steam was supplied to the jacket outside the extraction tank, and hot water extraction was performed at about 95. + -. 5 ℃ for about 4 hours. After the hot water extraction is completed, a discharge pipe connected to the extraction tank is depressurized to separate the hot water extract from the residue to obtain an agar-containing solution, which is then transferred to the inside of the agitation tank through a valve pipe for sterilization.

Thereafter, while keeping the impeller rotation speed of the agitation tank at 25rpm, cooling water was supplied to the outer jacket of the agitation tank, the temperature of the agar-containing solution was lowered to about 43 ℃, β -agarase was added thereto in an amount corresponding to a titer of about 125U/mL, and an enzymatic reaction was carried out for about 16 hours to obtain a solution containing a reaction product. After completion of the enzyme reaction, steam was supplied to the jacket outside the stirring tank, and the temperature inside the stirring tank was raised to 95. + -. 5 ℃ and then kept for about 1 hour to inactivate the β -agarase. Thereafter, the steam supplied to the outer jacket of the stirring tank was cut off, cooling water was injected, the temperature inside the stirring tank was lowered to 65. + -. 5 ℃ and concentrated under reduced pressure, and then the concentrated solution containing the reaction product was obtained by filtration through a 1 μm filter. Then, the reaction product-containing concentrate was freeze-dried and pulverized to obtain a reaction product-containing powder.

3. Characterization according to Agar (Agar) preparation method

(1) Analysis of agar Properties

The solidified product of the agar-containing solution purified in preparation example 1 was dissolved into a purified agar-containing solution, and then a sample was taken, followed by solidification and freeze-drying to solidify it. In addition, the agar-containing solution of comparative preparation example 1 was transferred to the inside of a stirring tank, and then sampled, coagulated and freeze-dried to solidify it. FIG. 1 is a diagram showing the properties of the purified agar-containing solution prepared in preparation example 1 and the agar-containing solution prepared in comparative preparation example 1 in a liquid state, a coagulated state and a freeze-dried state. In FIG. 1, "extract agar" corresponds to the agar-containing solution prepared in comparative preparation example 1, and "water-washed agar" corresponds to the purified agar-containing solution prepared in preparation example 1. As shown in FIG. 1, the refined agar-containing solution prepared in preparation example 1 had almost no impurities in appearance as compared with the agar-containing solution prepared in comparative preparation example 1.

(2) Quantitative analysis of agar recovery and the like

The solidified product of the agar-containing solution purified in preparation example 1 was dissolved into a purified agar-containing solution, and then a sample was taken, followed by solidification and freeze-drying to solidify it. The weight of the solidified sample was defined as the weight of agar, and the agarose content in the solidified sample was measured. In addition, the agar-containing solution of comparative preparation example 1 was transferred to the inside of a stirring tank, and then, a sample was taken, and solidification was carried out by coagulation and freeze-drying. Thereafter, the weight of the agar and the agarose content were determined in the same manner. Table 1 below shows the recovery rate of agar with respect to the weight of lettuce, the recovery rate of agarose with respect to the weight of lettuce, and the content of agarose in the prepared agar according to the preparation method of agar. The values in table 1 below are average values determined after 5 times of total implementation by changing only the relative scale in the agar preparation of preparation example 1 and comparative preparation example 1.

TABLE 1

As shown in Table 1, the method of preparation example 1 showed relatively low recovery rate of agarose compared to the method of comparative preparation example 1, but the content of agarose, which is a substrate for β -agarase, was significantly increased, and thus, there was no significant difference in recovery rate of agarose as a result.

4. Content analysis of Neoagarotetraose and Neoagarohexaose according to the preparation method of Neoagaroogosaccharide mixture

The content of neoagaro-oligosaccharide in the reaction product-containing powders finally obtained in preparation example 1 and comparative preparation example 1 was analyzed, and the results thereof are shown in table 2 below. The values in table 2 below are average values determined after a total of 5 runs, with only the relative dimensions in the preparation of the new agaro-oligosaccharide mixtures of preparation 1 and comparative preparation 1 being changed.

TABLE 2

DP 4: neoagarotetraose

DP 6: neoagarohexaose

As shown in table 2 above, the reaction product-containing powder finally obtained in preparation example 1 was very high in the content of neoagarotetraose and the content of neoagarohexaose compared to the reaction product-containing powder finally obtained in comparative preparation example 1. In addition, in the reaction product-containing powders finally obtained in production example 1 and comparative production example 1, the content of neoagaro-oligosaccharides having different degrees of polymerization was 0.1% or less except for neoagarotetraose and neoagarohexaose, and the remaining solid components were mainly present in the form of agarose, agarose gel, and polysaccharides having a degree of polymerization of more than 10.

The present invention has been described above by way of examples, and it is needless to say that various modifications can be made without departing from the scope and spirit of the present invention. Therefore, it should be understood that the scope of the present invention encompasses all embodiments falling within the scope of the claims of the present invention.

Claims

1. A method for preparing agar, which is characterized by comprising the following steps:

adding lithocarpus capsulifera and water into an extraction tank, extracting with hot water, and separating hot water extract from residues to obtain an agar-containing solution;

transferring the agar-containing solution into a stirring tank, carrying out primary cooling while stirring to cool the agar-containing solution to 50-55 ℃, then stopping stirring, and carrying out secondary cooling in a standing state to cool the agar-containing solution to 10-25 ℃ to obtain a solidified substance containing the agar solution; and

and (c) crushing the coagulum containing the agar solution to a size of 0.5-15 cm in the stirring tank, and then washing with water to obtain a refined coagulum containing the agar solution.

2. The method for preparing agar according to claim 1, wherein,

the dry weight of the lettuce added into the extraction tank is 1-5 kg per 100L of water.

3. The method for preparing agar according to claim 1, wherein,

the hot water extraction temperature is 80-110 ℃, and the hot water extraction time is 1-10 hours.

4. The method for preparing agar according to claim 1, wherein,

and the agar-containing solution is cooled once, then the stirring is stopped, and the standing time is 15-30 hours.

5. The method for preparing agar according to claim 1, wherein,

the amount of water used for washing the coagula containing the agar solution is 2000-10000L based on 1kg of dry weight of the agar used in hot water extraction.

6. The method for preparing agar according to claim 1, wherein,

in the step (c), the agar solution-containing coagulate is pulverized in the stirring tank, and then water is supplied into the stirring tank to wash the agar solution-containing coagulate, and the water for washing is discharged to the outside of the stirring tank through a discharge pipe provided with a filter screen.

7. The method for preparing agar according to claim 6, wherein,

the flow rate of water supplied to the stirring tank for washing the agar solution-containing coagulate is 3 to 8L/min based on 1kg of the dry weight of the agar used in the hot water extraction, and the supply time is 12 to 20 hours.

8. The method for preparing agar according to claim 6, wherein,

in the step (c), the coagulum of the agar-containing solution is washed by supplying water to the stirring tank and discharging water to the outside of the stirring tank, and then the coagulum of the agar-containing solution and the undischarged water inside the stirring tank are transferred onto a sieve and the undischarged water is removed by vibration.

9. A method for preparing a new agaro-oligosaccharide mixture comprises the following steps:

injecting the lettuce and water into an extraction tank, extracting with hot water, and separating hot water extract from residues to obtain an agar-containing solution;

transferring the agar-containing solution into a stirring tank, carrying out primary cooling while stirring to cool the agar-containing solution to 50-55 ℃, then stopping stirring, and carrying out secondary cooling in a standing state to cool the agar-containing solution to 10-25 ℃ to obtain a solidified substance containing the agar solution;

crushing the coagula containing the agar solution to 0.5-15 cm in the stirring tank, and then washing with water to obtain a refined coagula containing the agar solution; and

and (d) heating and dissolving the refined solidified product containing the agar solution, stirring the solution, cooling the solution to 35-50 ℃, and adding beta-agarase to perform an enzymatic reaction to generate new agaro-oligosaccharide.

10. A process for the preparation of a mixture of neoagaro-oligosaccharides as claimed in claim 9, characterized in that,

the heating temperature of the refined solidified product containing the agar solution is 80-110 ℃, and the generated new agar oligosaccharides are new agar tetrasaccharide and new agar hexasaccharide.