KR101599588B1 - Methods for producing and separation of 3,6-anhydro?l-galactose from red algae by biological treatment - Google Patents

Abstract

According to the present invention, galactose and 5-hydro-methylpurfural can be easily removed through metabolism by culturing a mixed strain using a sugar mixture produced by hydrolyzing red algae and agarose as a substrate . As a result, the anhydrous galactose can be separated and purified more easily by using the centrifugal separator and the high performance liquid chromatography, and high-purity anhydrous galactose can be mass-produced efficiently.

Description

METHODS FOR PRODUCING AND SEPARATION OF 3,6-ANHYDRO? L-GALACTOSE FROM RED ALGAE BY BIOLOGICAL TREATMENT BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for producing 3,6-

More particularly, the present invention relates to a method for efficiently mass-separating and purifying anhydrous galactose from red algae and agarose, and more particularly, to a method for efficiently purifying anhydrous galactose by adjusting the conditions of hydrolysis using an acid as a catalyst, And a method for efficiently mass-producing anhydrous galactose remaining in a culture solution by selectively decomposing only galactose and 5-hydroxymethyl furfural contained in the saccharide mixture through a biological fermentation process using a mixed strain will be.

The content of red algae varies depending on the region and the time and type of red algae. The composition of Gelidium Amaansii , the representative species of red algae, is composed of 75.5% of carbohydrates, 18.5% of protein, 0.6% of lipids, 5.4%.

Agar, which forms most of the mugwort, is composed of agarose (about 70%), a polysaccharide having a linear molecular structure, and agaropectin (about 30%), a branched polysaccharide .

Agarose is a disaccharide in which β-D-galactose and α-L-3,6 anhydrogalactose are alternately formed by α-1,3 bonds and β-1,4 bonds. agarobiose, which is a linear polymer,

Agaropectin is essentially an agarose unit, but some galactose is transversely bound to one another through ionic bonds between sulfate groups and sulfate ions. The ratio of galactose to anhydrous galactose in the agar component is known to be 0.44: 0.56 (Duckworth, M. and W. Yaphe (1971) Carbohydrate Research 16, 435-445).

Galactose and anhydrous galactose can be obtained by an acid hydrolysis process or an enzyme saccharification process of red algae, most of which are known to be obtained in the form of a mixture of galactose and anhydrous galactose.

In the acid hydrolysis method, agarose is reacted more sensitively under acidic conditions due to anhydrous galactose having a large ring strain due to its chemical structure, unlike cellulose, and the connection between agarose is not C1 -C3, which is advantageous in that the conversion reaction can proceed even under acidic conditions, which are much milder than cellulose, which is a starting material derived from woody biomass. The mechanism of the saccharification reaction of agarose is as follows. First, hydrogen ions bind to oxygen atoms in agarose molecules and are activated as cations. Then, electrons in the agarose molecules move and reaction starts. At this time, the chemical bonds in the molecule are simultaneously broken or regenerated due to the movement of electrons, resulting in a large number of intermediates having an equilibrium relationship in the reaction solution.

The acid hydrolysis process is preferred to the acid hydrolysis process in the mass production of anhydrous galactose because the acid hydrolysis method is inexpensive compared to the enzyme saccharification method, the reaction time is short, and the reaction process is simple. However, the problem of the acid hydrolysis method of agarose caused by the ring deformation of galactose anhydrous is that 5-hydroxymethylfurfural (5-HMF), which inhibits fermentation as galactose anhydrous is very unstable in acid ), Which makes it difficult to use the fermentation process as a post-process.

The enzyme saccharification has a disadvantage in that the reaction time is longer and the process cost is higher than that of the saccharification method using acid hydrolysis and the reaction is more complicated than the acid hydrolysis method, . As an example of an enzymatic saccharification method of agarose, Korean Patent Laid-Open No. 10-2011-0072958 discloses a saccharification method in which crude enzyme solution is fractionated from Saccharophagus digaltone 2-40 to decompose agarose to produce anhydrogalactose .

Recently, it has been found that anhydrous galactose can be used as a high-priced substance used in functional cosmetic materials having known antifibrinolytic activity, anticancer activity, antihypertensive activity, etc. and having a whitening effect and moisturizing effect, Methods for separating anhydrous galactose from a mixture of monomers obtained by decomposition have been developed.

Korean Patent Publication No. 2010-0116932 discloses a method for separating and purifying anhydrous galactose from a mixture of galactose and anhydrous galactose using circulating aliquot chromatography. However, this method requires a time-consuming period of 4 or more cycles to separate anhydrous galactose from the galactose peak. When analyzing the sample, the separation of galactose is completely performed by the chromatography technique alone There is a problem in that adducts generated in the glycation reaction using an acid remain.

Accordingly, there is a continuing need for effective mass production of anhydrous galactose in the glycosylation step of decomposing red algae.

On the other hand, the glycosylation process of red algae (eg, persimmon leaves, cucurbit, etc.) is largely divided into chemical methods of hydrolyzing an aqueous solution such as dilute sulfuric acid and hydrochloric acid and agarase as an enzyme capable of degrading polysaccharides such as agarose And the enzymatic saccharification method for decomposing.

A problem to be solved by the present invention is to provide a method for efficiently mass-producing anhydrous galactose in the saccharification process of red algae or agarose.

Another object of the present invention is to provide a method for effectively separating anhydrous galactose from a glycoside of red algae or agarose obtained through acid hydrolysis.

Another object of the present invention is to provide a method for mass production of anhydrous galactose by effectively removing galactose from a glycoside of red algae or agarose obtained through acid hydrolysis.

Another object of the present invention is to provide a method for mass production of anhydrous galactose by removing 5-hydroxymethyl furfural from a glycoside of red algae or agarose obtained through acid hydrolysis.

A further object of the present invention is to provide a method of separating anhydrous galactose from a glycosylated red alga or agarose obtained through acid hydrolysis and purifying it again with high purity.

In order to solve the above problems,

Decomposing algae or agarose to produce a sugar mixture comprising galactose and anhydrogalactose;

Decomposing the galactose through culturing the mixed strain using the sugar mixture as a substrate; And

And separating and separating anhydrous galactose.

In the present invention, it is preferable that the saccharide mixture containing galactose and anhydrous galactose be saccharified through acid hydrolysis reaction of red algae and / or agarose to facilitate mass production.

In the present invention, in the saccharification process using the acid as a catalyst, the sulfuric acid aqueous solution is catalyzed at a temperature of 100 to 140 ° C. for 10 minutes or less, preferably, It is preferable to use 0.1 N sulfuric acid aqueous solution as a catalyst at a temperature of 140 캜 for 10 minutes.

In the present invention, the above-mentioned mixed strain is a mixture of two or more strains capable of metabolizing galactose. Preferably, it is preferably capable of metabolizing 5-hydroxymethylpurfural produced by over-cleaving agarose together with galactose Do.

In the practice of the present invention, the mixed strains can be obtained by separating from seaweed seaweed, and specifically, Vibrio parahaemolyticus , Vibrio furnissii strain, Klebsiella ornithinolytica , Klebsiella pneumonia , Citrobacter murliniae , Citrobacter sedilakii , Citrobacter braakii , Enterobacter ludwigii , Proteus mirabilis strain, Shigella flexneri strain, and preferably all of them.

In the practice of the present invention, it is possible to add minimum salts or the like to the mixed culture medium for the purpose of assisting the growth of mixed strains, but it is preferable to use only the mixture of the saccharides in consideration of easiness in purification.

In one embodiment of the present invention, the mixed strains are cultured for 12 hours to a maximum of 60 hours using galactose present in the glycosylated hydrolyzate of agarose as a carbon source.

In the present invention, in the step of purifying and separating anhydrous galactose, the cultured medium is separated into a mixed culture and a supernatant using a centrifuge, and anhydrous galactose is separated from the supernatant.

In the practice of the present invention, the centrifugation can be performed at 4000 rpm for 10 minutes. The supernatant obtained after the centrifugation is separated and purified from other components present in the supernatant by collecting only anhydrous galactose using high performance liquid chromatography .

In a preferred embodiment of the present invention, the separation and purification process using the high performance liquid chromatography uses water as a mobile phase (flow rate) of 0.3 ml / min. In order to confirm the purity of the purified anhydrous galactose, it can be finally analyzed using GC-MS as disclosed in Korean Patent Laid-Open Publication No. 10-2011-0072958, The entire contents of the publication 10-2011-0072958 are incorporated herein by reference.

In one aspect, the present invention provides a method for selectively removing galactose using a strain that metabolizes galactose in a sugar mixture comprising galactose and anhydrogalactose.

The term " selectively removed " is understood to mean that the metabolic rate for galactose is higher than that for anhydrous galactose, and preferably galactose can be degraded, but anhydrous galactose is understood to mean that it is difficult to substantially decompose .

In another aspect of the present invention, red algae or agarose is treated with 0.1 N sulfuric acid aqueous solution as a catalyst so as to inhibit the formation of 5-hydroxymethyl furfural in the saccharification process using acid, Which is characterized in that it decomposes.

According to the present invention, galactose and 5-hydro- methylpurfural can be easily removed through metabolism by culturing a mixed strain using a sugar mixture produced by hydrolyzing red algae and agarose as a substrate . As a result, the anhydrous galactose can be separated and purified more easily using a centrifugal separator and high-performance liquid chromatography, and high-purity anhydrous galactose can be efficiently mass-produced.

Fig. 1 shows the results of quantitative determination of galactose, anhydrous galactose and 5-hydroxymethyl furfural produced when a red algae (cicadas) was subjected to heat treatment at 140 ° C using 0.1 N aqueous sulfuric acid solution.
FIG. 2 shows the results of quantitative determination of galactose, anhydrous galactose and 5-hydroxymethylfurfural produced when agarose was subjected to heat treatment at 140 ° C. using 0.1 N sulfuric acid aqueous solution.
FIG. 3 shows the results of gas chromatography / mass spectrometry of the saccharified solution A) cicada B) agarose.
FIG. 4 shows the growth curve and metabolic process of the mixed strains using the alginate alginate as a substrate.
FIG. 5 shows the growth curve and metabolic process of mixed strains using an agarose saccharified solution as a substrate.
FIG. 6 shows the result of performing the culture after the mixed strain was cultured for 60 hours in A) gas chromatography / mass spectrometry B) high performance liquid chromatography.
FIG. 7 shows the results of separating anhydrous galactose by performing preparative liquid chromatography according to an embodiment of the present invention. A) Gas Chromatography / Mass Spectrometry B) High Performance Liquid Chromatography Analysis
Figure 8 is a quantification straight line obtained by performing high performance liquid chromatography at various concentrations of anhydrous galactose and galactose in an independent state.

Hereinafter, the present invention will be described in detail with reference to examples.

The embodiments described below are for illustrative purposes only and are not intended to limit the scope of the present invention.

Reference Example 1: Experimental preparation and analysis method

(D-galactose, Aldrich, G0750) and anhydrous galactose (3 mg / ml) were used as the reference materials for the quantitation. , 6-Anhydrogalactose, Viol Systems, 28251-55-0) was used.

As a method of analysis, a sample in which a galactose material and an anhydrous galactose material were mixed was carried out by a gas chromatography / mass spectrometry (GC-MS) method with reference to the disclosure in Korean Patent Laid-open No. 10-2011-0072958, When analyzed by chromatography, the retention times of two peaks are similar due to the similar molecular weight and molecular structure of galactose and anhydrous galactose, and overlapping peaks are formed. Therefore, only for samples in which the two materials are not mixed, (Agilent Technologies 1100 series). The columns were Aminex HPX-87H Ion Exclusion Column (BIORAD Inc., USA), 0.004 M aqueous sulfuric acid solution was used as the mobile phase, 0.6 ml / min, and the column temperature was quantitatively analyzed at 50 ° C. In the case of preparative liquid chromatography, the mobile phase under the above conditions was carried out by changing only the water and the flow rate.

Example 1 Production of Anhydrous Galactose from Red Algae and Agarose by Saccharification

In order to obtain anhydrous galactose from red algae and agarose, the saccharification reaction was carried out through a chemical hydrolysis method as follows. In order to maximize the chemical treatment effect, experiments were carried out with the aim of finding the optimal conditions of the glycation reaction by varying the concentration of the sulfuric acid aqueous solution used as the acid catalyst, the temperature of the heat treatment condition and the reaction time. And the amount of 5-hydroxymethyl furfural produced as a mixed strain inhibiting substance was compared with each other to find an optimum condition of the glycation reaction condition in which the overdispersion of anhydrous galactose is minimized.

Specifically, 100 ml of 1% solution of dried cod (red algae) and agarose was added to 250 ml of Erlenmeyer flask, and the acid catalyst conditions were adjusted to the concentrations of 0.001 N, 0.01 N, 0.1 N, 0.2 N and 0.5 N aqueous sulfuric acid . The reaction was carried out at 100 ° C., 120 ° C. and 140 ° C. for 5, 10, 15 and 20 minutes under the temperature and time conditions for applying heat.

After the reaction was completed, the glycation temperature was lowered to room temperature, and the pH of the solution was adjusted to 7 by using 5N NaOH. The resulting solution was subjected to high performance liquid chromatography and gas chromatography / RTI > was quantitated using the < RTI ID = 0.0 > The main components of the saccharification solution were galactose, anhydrous galactose and 5-hydroxymethyl furfural, and galactose and anhydrous galactose were mixed. Quantitative analysis of the two components was carried out by gas chromatography / mass spectrometry, and 5-hydroxymethylpurine Fuller was quantitated using high performance liquid chromatography.

Fig. 1 is a graph showing the results of a gelatinization reaction of red algae and agarose in 0.1 N sulfuric acid aqueous solution at 140 < 0 > It is a graph that quantifies Fural.

As a result of comparing the anhydrogalactose saccharification rate according to the concentration of the aqueous sulfuric acid solution, it is preferably 0.1 N to 0.2 N, and most preferably 0.1 N. As the concentration increased under the condition of 0.1N or lower, the glycation rate tended to increase, but there was no significant difference in the glycation rate.

The rate of glycation was greatly changed by the heat treatment time and temperature condition than the concentration of sulfuric acid aqueous solution. The saccharification reaction was most effective at 100 ℃, 120 ℃ and 140 ℃, and the anhydrous galactose yield was 3.49% (g (g anhydrous galactose / g dried croaker) x 100%), and anhydrous galactose was found to be the highest in the yield of 39.67% ((g anhydrous galactose / g agarose) X 100%) in agarose.

In the case of treatment at 140 ℃ for more than 10 minutes, the glycation rate was decreased in both substrates. The production of 5-hydroxymethylfurfural was affected by the concentration of acid catalyst solution used in the glycation reaction, , The higher the number, the higher the increase.

In addition, it was confirmed that 5-hydroxymethyl furfural production was not produced in the pure substance galactose glycosylation reaction but only when anhydrous galactose was used as a substrate. That is, it was confirmed that the glycation reaction conditions did not affect the conversion of 5-hydroxymethylfurfural into galactose.

FIG. 3 shows the results of gas chromatography / mass spectrometry of the saccharified solution A) cicada B) agarose. The retention time of anhydrous galactose was 34.778 min, and it was confirmed that galactose was analyzed as two peaks at 45.328 minutes and 46.394 minutes in anomer state of alpha and beta forms. The remaining fine peaks not mentioned above are the base peaks used in the sample preparation step.

Example 2: Galactose metabolism through culturing of mixed strains

The mixed strain was cultured using the glycated solution obtained in Example 1 as a substrate to metabolize galactose from the culture solution to easily separate anhydrous galactose. As a mixed strain used for culturing, a mixed strain which can metabolize 5-hydroxymethyl furfural, which is a toxic substance as well as galactose, at a specific concentration is recommended.

Specifically, the mixed strains used in the above culture were separated from kelp, which was undergoing end-of-summer recording, and the strains were cultured in the presence of Vibrio parahaemolyticus ( Vibrio furnissii strain), Klebsiella ornithinolytica ( Klebsiella pneumonia ), Citrobacter murliniae , Citrobacter sedlakii , Citrobacter braakii , Enterobacter ludwigii , Proteus mirabilis strain, and Shigella flexneri strain were mixed and cultured.

The above-mentioned mixed strains were precultured in LB (Luria Bertani) medium, and the mixed strains used were mixed with glycerol 30% and stored at -70 ° C in a cryogenic freezer. In the experiment using the saccharified liquid as a substrate, 5% (v / v) of a mixed strain pre-cultured in LB medium for 18 hours was inoculated and cultured in a shaking incubator at 25 ° C. and 200 rpm for about 3 days to 5 days . In addition, M9 minimum salts may be added to the culture to help grow bacteria, but this procedure was carried out without adding minimal medium for better purification.

FIG. 4 shows the growth curve and metabolic process of the mixed strains using the alginate red ginseng solution as a substrate, and FIG. 5 shows the growth curve and metabolic process of the mixed strains using the agarose alginate as a substrate. After 60 hours of incubation, the bacterial growth was reduced and galactose degradation was completely observed. In addition, it was confirmed that the used mixed strains can be metabolized to 5-hydroxymethyl furfural, so that they are preferably used in the anhydrous galactose separation and purification process. Since there was no change in the anhydrous anhydrous galactose concentration and the anhydrous galactose concentration after completion of the culture of the mixed strain, it was confirmed that the mixed strains did not have an ability to metabolize anhydrogalactose.

FIG. 6 shows the result of gas chromatography / mass spectrometry of the culture solution after culturing the mixed strain for 60 hours. As a result, it was confirmed that the galactose peaks (45.328 and 46.394 min) shown in FIG. 3 were removed through the culturing of the mixed strains. 2-Amino-2-deoxy-D -galactopyranose was generated at 37.694 min.

Example 3: Separation of anhydrous galactose in a mixed culture medium

High performance liquid chromatography was performed to separate anhydrous galactose from the culture product produced through the above-mentioned mixed strain culture. First, the culture broth was centrifuged (4000 rpm, 10 minutes) to separate the mixed strains from the culture broth. The supernatant portion was separated and purified by chromatography using anhydrous galactose alone.

Figure 8 is a quantification straight line obtained by performing high performance liquid chromatography at various concentrations of anhydrous galactose and galactose in an independent state. Generally, when galactose and anhydrous galactose are mixed together, since they have similar molecular weights and structures, they have the same retention time value in high performance liquid chromatography. However, in the case where galactose is not present as shown in FIGS. 4 and 5 , It was confirmed that anhydrous galactose can be quantitatively and fractionally separated by high performance liquid chromatography.

(Agilent Technology 1100 series) was used for the analysis using the high performance liquid chromatography. Aminex HPX-87H Ion Exclusion Column (BIORAD Inc., USA) was used as a column. A 0.004 M aqueous sulfuric acid solution was used as the mobile phase, and the flow rate was quantified at 0.6 ml / min and the column temperature at 50 ° C.

6 shows the result of performing a high performance liquid chromatography on the culture solution after culturing the mixed strain for 60 hours. Dodecanoic acid, which is one of the glycation reaction products produced during the glycation reaction using the aqueous sulfuric acid solution, appeared at 8.082 min and the amino sugar 2-Amino-2-deoxy-D-galactopyranose produced from the culture product appeared at 10.221 min Respectively. The anhydrous galactose appeared at 9.589 minutes between these two peaks, and the fractionation process for purifying anhydrous galactose was carried out for purifying from two peaks before and after anhydrous galactose.

As a condition for the preparative liquid chromatography, water was used as a mobile phase as the same column as the high performance liquid chromatography as described above, and the injection volume was 500 μl (1000 ppm), flow rate ) Can be variously performed to increase the purity of anhydrous galactose from 1 ml / min to 0.3 ml / min, 0.2 ml / min and a minimum of 0.1 ml / min, preferably from 0.3 ml / min to 0.6 ml / min . ≪ / RTI > Refractive index detector was used. For mass production of anhydrous galactose, the flow rate, injection amount, and column conditions can be changed.

FIG. 7 shows the results of separating anhydrous galactose by performing preparative liquid chromatography according to an embodiment of the present invention. Through preparative liquid chromatography, the saccharification and culture products shown in Fig. 6 were removed to confirm loading. Thus, it was confirmed that the purity of anhydrous galactose can be finally obtained as 99% or more of pure substance through the fractionation process.

Claims (16)

A method for producing anhydrous galactose using red algae or agarose,
Decomposing red algae or agarose to produce a sugar mixture comprising galactose and anhydrogalactose;
Culturing a mixed strain isolated from kelp using the sugar mixture as a substrate to decompose the galactose; And
And separating and separating the undegraded anhydrous galactose,
Wherein said mixed strain comprises Vibrio parahaemolyticus , Vibrio furnissii strain, Klebsiella ornithinolytica , Klebsiella pneumonia , Citrobacter murliniae , Citrobacter sedilakii , Citrobacter braakii , Enterobacter ludwigii , Proteus mirabilis strain, Shigella flexneri strain. The method of claim 1, wherein the sugar mixture is produced through an acid-catalyzed hydrolysis reaction. 3. The method according to claim 2, wherein the hydrolysis reaction is heat-treated using an aqueous sulfuric acid solution. 4. The method according to claim 3, wherein the sulfuric acid aqueous solution is a 0.1 N sulfuric acid aqueous solution. 4. The method according to claim 3, wherein the heat treatment is performed at 100 to 140 DEG C for 5 to 10 minutes. delete delete The method of claim 1, wherein the mixed strain metabolizes 5-hydroxymethyl furfural. 2. The method according to claim 1, wherein the mixed culture medium comprises only the sugar mixture. The method according to claim 1 or 2, wherein the mixed strains are cultured for 12 to 60 hours using galactose as a carbon source. The method according to claim 1, wherein the culture broth is centrifuged to purify anhydrous galactose in the supernatant. 12. The method according to claim 11, wherein the supernatant is fractionated with high performance liquid chromatography to obtain anhydrous galactose. Galactose is selectively removed using a mixed strain isolated from kelp selectively metabolizing galactose in a sugar mixture containing galactose and anhydrous galactose,
Wherein said mixed strain is composed of Vibrio parahaemolyticus , Vibrio furnissii strain, Klebsiella ornithinolytica , Klebsiella pneumonia , Citrobacter murliniae , Citrobacter sedilakii , Citrobacter braakii , Enterobacter ludwigii , Proteus mirabilis strain, Shigella flexneri strain. delete 14. The method according to claim 13, wherein the mixed strain metabolizes 5-hydroxymethyl furfural together. delete

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