KR20150122381A - Quantitaive method of anhydrous galactose in saccharification from red algae - Google Patents

Abstract

The present invention relates to a galactose anhydrous quantitative method and, more specifically, to a method for quantitatively analyzing galactose and galactose anhydrous, both of which have the same retention time in an agarose sugar mixed liquid, by means of using high-performance liquid chromatography (HPLC). According to the present invention, the quantitative method comprises: a step for quantifying galactose in an analyzing specimen comprising galactose and galactose anhydrous by means of Fourier-transform infrared spectroscopy (FT-IR); and a step for quantifying a content of galactose anhydrous by quantifying a total content of both galactose and galactose anhydrous by using HPLC and then deducting the galactose content obtained from the FT-IR.

Description

[0001] The present invention relates to a method for quantifying anhydrous galactose in an alga,

The present invention relates to a method for quantitatively determining anhydrous galactose, and more particularly to a method for quantitatively analyzing galactose and anhydrogalactose having the same retention time in a mixed solution of agarose by high performance liquid chromatography.

Glucose (D-glucose) and galactose (D-galactose), which are the main carbohydrate components of galactan through the glycation reaction, are fermentable sugars which are conventional microorganisms and are easily used for fermentation process. (3,6-anhydro-L-galactose, 3,6-AHG), which is a non-fermentable sugar, is not used as an energy source in living organisms, Has attracted attention as a component of functional cosmetic materials having physiological activities such as blood pressure, anticancer, antihypertensive and whitening effect, and moisturizing effect.

Although anhydrous galactose is a high-value product, the molecular weight and chemical structure of galactose and anhydrous galactose obtained through glycosylation of agarose are very similar, resulting in difficulty in quantification and separation of the two components. Accordingly, studies have been made on a method for independently determining anhydrous galactose in a mixture of anhydrous galactose and galactose.

As an anhydrous galactose and galactose determination method, colorimetric method (Yaphe, W., Arsenault, G. P., 1965 Analytical Biochemistry 13, 143-148), methanolysis (Quemener, B., Lahaye, M., Metro, F., 1995Carbohydrate Research 266, 53-64), mercaptolysis (Hama, Y., Nakagawa, H., Kurosawa, M., Sumi, T., Xia, X., Yamaguchi, K., 1998Analytical Biochemistry265, 42-48.), Amination (Navarro, D. A., Stortz, C. A., 2003Carbohydrate Research 338, 2111-2118], alditol: Jol, C. N., Neiss, T. G., Penninkhof, B., Rudolph, B., De Ruiter, G. A., 1999Analytical Biochemistry 268, 213-222) have been reported. However, these methods have disadvantages that the sample processing is relatively complicated and the accuracy is poor.

Korean Patent Laid-Open Publication No. 10-2011-0072958 discloses a method for separating a crude enzyme solution produced from a marine microorganism Saccharopagus digestans 2-40 with an intracellular enzyme solution and an extracellular enzyme solution, A step of measuring the activity of an enzyme which hydrolyzes an agarose with an enzyme solution to glycosylate and an assay method of simultaneously quantifying galactose and 3,6-anhydro-L-galactose as final decomposition products simultaneously using a gas chromatograph / mass spectrometer .

However, the complexity of the sample preparation step is not improved, and there is a continuing need for a method for easily preparing a sample and accurately and rapidly quantifying the sample.

The present invention provides a method for quantitatively analyzing a sample containing anhydrous galactose and galactose by using high performance liquid chromatography (HPLC), and independently measuring the anhydrous galactose in an aqueous solution state without complicated pretreatment will be.

In order to solve the above-mentioned problems, the present invention provides a method for detecting galactose, comprising the steps of: quantifying galactose in an analytical sample containing anhydrous galactose and galactose using a Fourier transform infrared spectroscopy (FT-IR);

Measuring the total content of anhydrous galactose and galactose using the HPLC of the analytical sample, and subtracting the galactose content obtained from the FT-IR to quantify the anhydrous galactose content.

In the present invention, Fourier transform infrared spectroscopy (FT-IR) is used as an independent method of quantifying anhydrous galactose and galactose. The Fourier transform infrared spectroscopic analyzer is an apparatus for analyzing the characteristic that the functional groups of organic molecules absorb specific wavelengths in a certain wavelength band of the infrared spectrum. The measurement time is fast and the measurement can be repeated many times in a short time. It is also possible to analyze the sample, and it is characterized not only in qualitative but also in accuracy of quantitative analysis.

In the present invention, when analyzing a sample containing galactose and anhydrous galactose using a Fourier transform infrared spectrometer (FT-IR), it is possible to obtain a mixture of galactose only and pyranose ring absorbs a specific wavelength of a predetermined region, and a single absorption peak appears in the infrared spectroscopic analyzer, so that it can be quantitatively analyzed.

In the present invention, quantitative analysis using a Fourier transform infrared spectrophotometer is performed at 1110 to 1170 cm ^-1 , more preferably at 1120 to 1160 cm ^-1 at which the amount of absorption increases with increasing content of galactose, Lt; ^-1 > cm < ^-1 >.

In the present invention, the quantification may be performed by deriving a calibration curve according to the concentration of galactose, and further introducing a smoothing technique in the quantitation method.

In the practice of the present invention, it is preferable to use the partial least squares method to derive the calibration curve according to the galactose concentration, and the Savitzky-Golay filter method may be used as the smoothing method.

In the present invention, the step of quantifying the total content of anhydrous galactose and galactose by using the above-mentioned HPLC is a method of quantifying the peak appearing at the same position by galactose and anhydrous galactose having the same retention time in high performance liquid chromatography using a calibration curve .

In the present invention, the HPLC calibration curve is not limited but is preferably obtained using a standard method.

In the practice of the present invention, the quantitative analysis of galactose and anhydrous galactose by high performance liquid chromatography was carried out using Agilent Techonolgy 1100 as the instrument, Aminex HPX-87H of BIO RAD, column temperature of 50 ° C, 0.004M sulfuric acid The analysis can be carried out using an aqueous solution, a flow rate of 0.6 ml / min, RID, and all samples are prepared and analyzed in aqueous solution, but are not limited thereto.

In the present invention, the subtraction of the content of galactose in the content of galactose and anhydrous galactose is determined by subtracting the area of the concentration value of galactose determined by a Fourier transform infrared spectrophotometer from the area value indicated by galactose and anhydrogalactose appearing at the same retention time after HPLC analysis And the amount of galactose anhydrous is quantified.

In one embodiment of the present invention, the sugar mixture containing anhydrous galactose is an aqueous solution sample obtained through glycosylation of agarose, but is not limited thereto.

In one aspect of the present invention, galactose and anhydrous galactose samples having different concentrations are weighed using high performance liquid chromatography (HPLC) under the same analytical conditions to derive a standard curve, and using the obtained calibration curve, galactose And anhydrous galactose were measured by HPLC using two concentrations in a mixed sample, thereby obtaining a regression equation between the two concentrations;

Using a Fourier transform infrared spectrometer (FT-IR), a mixture of both substances absorbed a specific wavelength of 1150 cm ^-1 region by galactose-only pyranose ring and showed a single absorption peak in the infrared spectrometer Wherein the calibration curve is derived according to the concentration of galactose, and the smoothing technique is added to increase the accuracy of the calibration curve; And

Measuring the anhydrous galactose content by substituting galactose content in the mixed solution measured by FT-IR in a regression equation derived using the HPLC;

A method for quantifying anhydrous galactose.

In an embodiment of the present invention, for the determination of the anhydrous galactose, a comparative analysis is performed using a gas chromatograph mass spectrometer (GC-MS) as disclosed in Korean Patent Laid-Open Publication No. 10-2011-0072958 For this purpose, the entire disclosure of Korean Patent Publication No. 10-2011-0072958 is incorporated herein by reference.

The invention In one aspect, there is provided a method of measuring the content of galactose in the aqueous solution samples containing galactose and anhydrous galactose, and the aqueous sample Fourier transform analysis using an infrared spectrophotometer (FT-IR), 1150 cm - ^{And a} single peak of ¹ region band is used to quantify galactose.

The quantitative method according to the present invention provides a method for quantitatively analyzing an anhydrous galactose and a galactose-containing sample using high performance liquid chromatography (HPLC), and independently and rapidly quantifying anhydrous galactose in an aqueous solution state without complicated pretreatment do.

In one aspect, the present invention provides a method for rapidly determining the content of galactose in a mixture of galactose and anhydrogalactose using a Fourier transform infrared spectrometer (FT-IR).

FIGS. 1 and 2 are graphs showing the quantification of galactose and anhydrogalactose by an area value according to concentration using high performance liquid chromatography (HPLC).
FIG. 3 is a graph showing an absorption spectrum of water, anhydrous galactose solution, and a range of 4000 to 700 cm ^-1 area of a galactose solution sample using an infrared spectrometer.
4 is a graph showing the absorption spectrum of the entire region of the galactose solution sample at various concentrations.
5 is a graph showing a calibration curve of galactose using an infrared spectrometer.
FIG. 6 is a graph showing a calibration curve of galactose, which is obtained by increasing the accuracy of quantitative analysis using the Savizky-Golay method using a smoothing technique in the calibration curve of FIG.
Fig. 7 is a flowchart showing steps of the measuring method of the present invention.

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.

Sample Preparation

In this experiment, agarose (Aldrich, A0701) was used as a substrate, and standard materials used for quantitative analysis were galactose (D-galactose, Aldrich, G0750) and anhydrogalactose Systems, 28251-55-0).

Specifically, 100 ml of a 1% agarose solution was added to 250 ml Erlenmeyer flask, and the concentration of 0.1 N, 0.2 N, and 0.5 N aqueous sulfuric acid solution The acid catalyst conditions were suitably determined at 0.1 N. In addition, the reaction was carried out at 140 ° C for 10 minutes under the conditions of temperature and time to apply heat, thereby forming a saccharified solution of agarose (a mixture of anhydrous galactose and galactose).

Example 1: Determination of galactose and anhydrous galactose using high performance liquid chromatography (HPLC) and derivation of regression equation

For the determination of galactose and anhydrous galactose by high performance liquid chromatography, the following conditions were used.

- High Performance Liquid Chromatography (HPLC) System: Agilent Technology 1100 series

Analysis Column: Aminex HPX-87H Ion Exclusion Column (Manufacturer: BIORAD Inc., USA)

- Flow rate and mobile phase: 0.6 ml / min, 0.004 M aqueous sulfuric acid solution

- Column temperature: 50 ° C

- Refractive Index

The concentration of galactose and anhydrogalactose to be analyzed under the above conditions may be in the range of 0.1 g / L to 2 g / L, and more specifically, in the case of galactose, four points of 0.1, 0.5, Was determined by using the standard method using four points of anhydrous galactose of only 0.1 0.3 0.7 0.9 g / L. In case of galactose and anhydrous galactose, both peaks were observed at the same retention time of 9.587 minutes on HPLC. Therefore, it was confirmed that analysis was difficult when the two components were mixed. FIGS. 1 and 2 are graphs of galactose and anhydrogalactose quantitated by area values according to their concentrations using high performance liquid chromatography (HPLC). Q.s. curves R ² correlation coefficient value by the formula of y = 148.92x of galactose eoteonaen using standard methods (Standard method) is exhibited to 0.9933, a suitable amount curve y = 34.893x, a correlation coefficient R ² is values of anhydrous galactose 0.9995.

D-galactose (mg / L) 3,6-AHG (mg / L) Estimated value (area) Experimental value (area) Accuracy 200 150 35017.95
(29784 + 5233.95) 33845 96.7% 300 200 51654.6
(44676 + 6978.6) 49984 96.8% 500 450 90161.85
(74460 + 15701.85) 88124 97.7%

As shown in Table 1, when calculating the area values in the mixed solution predicted using the quantitative curves of galactose and anhydrogalactose as shown by the standard method, the accuracy with the actual measured values was found to be as follows: galactose was 200 mg / L, When the anhydrous galactose was 150 mg / L, the concentration was 96.7%. When the galactose was 300 mg / L and the anhydrous galactose was 200 mg / L, the concentration was 96.8%, while the galactose was 500 mg / L and the anhydrous galactose was 450 mg / L, it was 97.7%. The average accuracy was 97.06%.

In order to substitute the appropriate curve for each component in the mixture of galactose and anhydrous galactose, the accuracy of the mixture can be increased by inputting a correction factor of 1.03 in the area indicated by HPLC.

Specifically, the HPLC area of the mixed solution to which the values of the appropriate curves of the two components are substituted can be estimated as follows,

[Equation 1]

갈락토오스의 농도 + 34.893

무수갈락토오스의 농도)

1.03
Mixed liquid area (Area) = (148.92

Concentration of galactose + 34.893

Concentration of anhydrous galactose)

1.03

[Formula 2] Concentration of anhydrous galactose

갈락토오스의 농도)

1/34.893
AHG concentration = (Area of mixed liquid by HPLC / 1.03 - 148.92

Galactose concentration)

1 / 34.893

Example 2: Determination of galactose by Fourier transform infrared spectroscopy (FT-IR)

Galactose alone among galactose and anhydrogalactose coexisting in the saccharified solution of agarose absorbs a specific wavelength of 1150 cm ^-1 region by the pyranose ring and a single absorption peak appears in the infrared spectrometer , And the degree of absorption varies depending on the concentration. That is, various concentrations of galactose were prepared to independently determine only galactose in the agarose sugar mixture using FT-IR, and a calibration curve was derived.

Specifically, the infrared spectrophotometer was Nicolet 6700 (Thermo Scientific, USA). The analyzed samples were prepared by dissolving galactose in an aqueous solution at a concentration of 0.1 g / L to 5.0 g / L, 0.2, 0.3, 0.4, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.5, 1.6, 1.8, 2.1, 2.4, 2.5, 2.7, 2.8, 3.0, 3.6, A total of 26 samples were measured with a Fourier transform infrared spectroscopy (FT-IR).

And OMNIC software that provides algorithms for calibration and verification of assays and quantitation. FIG. 3 is a graph showing the absorption spectrum of a water, anhydrous galactose solution, and a galactose solution sample in the range of 4000 to 700 cm ^-1 area using the infrared spectroscopic analyzer. In the case of water, it is difficult to quantify the solid samples in the aqueous solution state due to the wide absorption peaks such as the absorption peak due to OH stretching vibration at 3500 ~ 3300 cm ^-1 region and the scissors at 1600 cm ^-1 region. This can be. In this result, it can be seen that only the absorption of a specific wavelength of 1150 cm ^-1 region by galactose-only pyranose ring in the aqueous solution of galactose appears, and thus an absorption peak appears.

FIG. 4 is a graph showing the absorption spectrum of the entire region of the galactose solution sample at various concentrations, with the information that only the galactose has a specific peak change in the aqueous solution state. In the whole region, the regions showing different transmittance according to the concentration are largely 5 regions (2390-2280 cm ^-1 , 1820-1700 cm ^-1 , 1390-1340 cm ^-1 , 1250-1180 cm ^-1 , 1150-1110 cm ^-1 ).

Absorption region (cm ^-1 ) 2390-2280 1820-1700 1390-1340 1250-1180 1150-1110 ↑
(high)

% Transmittance
(transmittance)

(low)
↓ 5 0.9 0.9 0.9 water 0.8 2.1 2.1 2.1 0.1 0.1 2.4 water water 0.2 0.9 water 2.4 2.4 0.3 One 1.2 1.2 1.2 0.4 water 0.4 0.4 0.4 0.5 0.5 4.5 One 0.5 0.8 0.4 One 1.4 0.7 0.7 0.2 1.4 0.5 One 0.9 0.3 1.8 4.5 1.5 One 4.5 0.5 2 1.4 1.2 1.8 0.7 1.8 2 1.4 2.4 1.6 0.7 1.8 1.5 2.1 0.8 1.6 1.6 1.6 3 0.3 0.8 0.8 1.7 2.7 3 0.3 0.3 1.8 1.2 0.2 0.2 0.2 2 1.6 5 0.1 2.7 2.4 2 0.1 3 3 3 0.7 3.6 3.6 3.6 3.6 1.4 2.7 2.7 0.1 4.5 1.5 1.5 1.5 1.5 5

Table 2 shows low to high galactose concentrations from high to low transmittance spectra. As the concentration of galactose increases with water, it is confirmed that the range of the region that can be confirmed that the transmittance to the wavelength of the following region decreases (= the galactose concentration increases as the galactose concentration increases) is 1150-1110 cm ^-1 I could.

Calibration curves are obtained by using PLS (Partial Least Squares) quantification method using TQ software used for quantitative analysis among OMNIC software, which is the basic software of Nicolet Product, and precisely using first derivatives of peaks of 1147 ~ 1133 cm ^-1 region When using the quantitative method, the accuracy of the quantitative analysis was improved by using the Savizky-Golay method (number of samples is 17, number of polynomial is 4) provided by the program among the smoothing methods.

FIG. 5 is a graph showing a calibration curve of galactose using an infrared spectrometer. FIG. 6 is a graph showing the calibration curve of galactose obtained by increasing the accuracy of quantitative analysis using the Savizky-Golay method by smoothing in the calibration curve of FIG. Fig. The following two calibration curves show that the correlation coefficient is improved when the smoothing technique is used (0.9945) than when it is not used (0.9796).

Example 3: Determination of anhydrous galactose by HPLC and FT-IR

When the experiment was carried out in the same manner as in Example 2, only the galactose concentration in the mixed solution became quantifiable, and based on the [Formula 1] obtained in Example 1, the galactose concentration corresponding to the HPLC area value of the mixed solution was substituted The concentration of anhydrous galactose can be quantified. [Equation 2]

As a method for comparing the concentration of two components in a mixed solution, it is preferable to determine the concentration of anhydrous galactose and galactose in a mixed solution to a known value, or to use a GC-MS to finally determine the concentration of anhydrous galactose and galactose as disclosed in Korean Patent Publication No. 10-2011-0072958 .

Claims (12)

Quantifying galactose in an analytical sample containing anhydrous galactose and galactose using a Fourier transform infrared spectrometer (FT-IR); And
Determining the total content of anhydrous galactose and galactose using HPLC, and subtracting the galactose content obtained from the FT-IR to quantify the anhydrous galactose content. The method according to claim 1, wherein the determination of galactose using FT-IR is performed using an absorption peak of a pyranose ring of galactose. 3. The method according to claim 1 or 2, wherein the quantification of galactose using the FT-IR quantifies a peak in the range of 1110 to 1170 cm ^-1 . 4. The method of claim 3, wherein the galactose quantitation is substantially at 1150 cm <" ^{1 &} gt ;. The method according to claim 1, wherein the determination of galactose is performed by preparing a calibration curve according to the concentration of galactose and introducing a smoothing technique. 6. The method of claim 5, wherein the calibration curve is generated using a least squares method. 6. The method of claim 5, wherein the smoothing method uses a Savitzky-Golay filter method. The method according to claim 1, wherein the subtraction of the galactose content is performed by removing an area value of the concentration value of galactose determined through the Fourier transform infrared spectroscopic analyzer at an area value indicated by galactose and anhydrogalactose after HPLC analysis Way. The method according to claim 1, wherein the sample is a sample obtained in a saccharification step of agarose. The method according to claim 1, wherein the sample is an aqueous solution sample. Galactose and anhydrous galactose samples having different concentrations were weighed using high performance liquid chromatography (HPLC) under the same analytical conditions, and a standard curve was derived. Using the obtained calibration curve, galactose and anhydrogalactose were mixed at a predetermined concentration Deriving a regression equation between the two concentrations by measuring two concentrations in the mixed sample using HPLC;
Using a Fourier transform infrared spectrometer (FT-IR), a mixture of both substances absorbed a specific wavelength of 1150 cm ^-1 region by galactose-only pyranose ring and showed a single absorption peak in the infrared spectrometer Wherein the calibration curve is derived according to the concentration of galactose, and the smoothing technique is added to increase the accuracy of the calibration curve; And
Measuring the anhydrous galactose content by substituting galactose content in the mixed solution measured by FT-IR in a regression equation derived using the HPLC;
≪ / RTI > In the method for measuring the content of galactose in an aqueous solution sample containing galactose and anhydrous galactose, the aqueous solution sample is analyzed using a Fourier transform infrared spectrophotometer (FT-IR), and a single peak of 1150 cm ^-1 region band is used And then quantitatively determining the amount of galactose.

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