Application of Molecular Rotor in Analyzing the Content and Ratio of Components in α-D-1,4-Glucan Mixtures

Abstract

The present invention discloses the use of molecular rotors in analyzing the content and ratios of components in α-D-1,4-glucan mixtures. The present invention also provides a fluorescence analysis method for analyzing the content and ratios of components in α-D-1,4-glucan mixtures. The method is easy to use and requires no special operator skills. It also provides a simple sample pretreatment method, minimizes solvent consumption, and is environmentally friendly. It requires a small sample amount, down to the microgram level. High-throughput detection is possible, significantly increasing analysis speed. It requires minimal instrumentation and significantly reduces costs. The analytical results are stable and reliable.

Description

Technical Field

[0001] The invention relates to the application of a molecular rotor in analyzing the content and ratio of components of an alpha-D-1,4-glucan mixture. Background Art

[0002] α-D-1,4-glucan is a functional polymer compound with important applications in the food, chemical, and pharmaceutical industries. Common substances containing α-D-1,4-glucan include maltose syrup, maltooligosaccharides, maltodextrin, amylose, and debranched starch. They are typically composed of varying proportions of polysaccharides (with a degree of polymerization greater than 10) and oligosaccharides (with a degree of polymerization less than 10). Numerous studies have shown that the component content and ratios of α-D-1,4-glucan mixtures affect important functional properties such as storage stability, gelation, solubility, sweetness, and reducing properties. Currently, analysis of the component content and ratios of α-D-1,4-glucan mixtures primarily relies on chromatographic techniques such as size exclusion chromatography (SEC) and fluorescence-assisted capillary electrophoresis (FACE). These techniques are expensive, time-consuming, require specialized expertise, and are inefficient. Therefore, a new, sensitive and convenient method for analyzing the component content and ratios of α-D-1,4-glucan mixtures is of great significance.

[0003] At present, the fluorescence behavior of molecular rotors is mainly used to monitor biochemical processes such as changes in the viscosity of the cytoplasm (membrane), protein polymerization, etc. There are no reports on the use of molecular rotors to measure the component content and ratio of α-D-1,4 glucan mixtures. Summary of the Invention

[0004] The present invention aims to provide the use of molecular rotors for analyzing the component contents and ratios of α-D-1,4-glucan mixtures. The present invention first protects the use of molecular rotors for analyzing the component contents and ratios of α-D-1,4-glucan mixtures. By using molecular rotors as fluorescent probes, the fluorescence generated by the molecular rotors can sensitively reflect the spatial constraints of the surrounding microenvironment. Since glucans of different chain lengths in an α-D-1,4-glucan mixture exert varying degrees of constraint on the molecular rotors, the fluorescence properties of the molecular rotors can be used to characterize the component contents and ratios of the α-D-1,4-glucan mixture.

[0005] The present invention also provides a fluorescence analysis method for the component contents and ratios of an α-D-1,4 glucan mixture.

[0006] The fluorescent analysis method for the component contents and ratios of an α-D-1,4-glucan mixture provided by the present invention comprises the following steps:

[0007] (1) dispersing the α-D-1,4-glucan mixture to be tested in a solvent to obtain a dispersion of the glucan mixture to be tested;

[0008] (2) adding a molecular rotor to the above-mentioned glucan mixture dispersion to be tested and dispersing it evenly to obtain a glucan mixture dispersion containing a molecular rotor to be tested;

[0009] (3) measuring characteristic fluorescence emission parameters of the molecular rotor in the dispersion of the dextran mixture containing the molecular rotor to be tested;

[0010] (4) selecting an α-D-1,4-glucan mixture sample with known component contents and ratios, using a solvent with the same components to prepare a standard α-D-1,4-glucan mixture dispersion with the same concentration as the sample to be tested, and adding the same concentration of molecular rotors to disperse uniformly to obtain a standard α-D-1,4-glucan mixture dispersion containing molecular rotors;

[0011] (5) Determine the emission fluorescence characteristic parameters of the molecular rotor in the dispersion of the standard α-D-1,4-glucan mixture containing the molecular rotor, perform single-point calibration based on the obtained characteristic parameter values, or establish a standard curve to determine the polysaccharide and oligosaccharide content and their ratio of the α-D-1,4-glucan mixture to be tested.

[0012] In steps (1) and (4), the solvent is one or more of water, buffer, strong alkaline solution, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, pyridine, acetone, and tetrahydrofuran;

[0013] In the test glucan mixture dispersion and the standard α-D-1,4 glucan mixture dispersion, the concentration of the test glucan mixture may be 0.1%-2% (w / v), specifically 0.5% (w / v) or 1% (w / v);

[0014] In step (4), the method for determining the α-D-1,4 glucan mixture sample with known component contents and ratios thereof may be, but is not limited to, size exclusion chromatography;

[0015] In steps (1) to (5), the molecular rotor is a substance that includes an electron acceptor group, an electron donor group and a conjugated π bond structure, and can generate fluorescence under the blocking effect of the surrounding environment;

[0016] The molecular rotors include but are not limited to julidine derivatives, dialkylaniline derivatives, boron-dipyrromethene derivatives, porphyrin derivatives, etc.;

[0017] In the dispersion of the dextran mixture containing molecular rotors to be tested and the dispersion of the standard α-D-1,4-dextran mixture containing molecular rotors, the concentration of the molecular rotors is greater than 1 μM, specifically 10 μM;

[0018] In steps (3) and (5), the characteristic parameters of the emitted fluorescence are one or more of fluorescence intensity and quantum yield;

[0019] In step (5), the single-point calibration operation is as follows: using the emission fluorescence characteristic parameters of a single standard α-D-1,4-glucan mixture dispersion and the emission fluorescence characteristic parameters of a blank dispersion as references, a standard working curve is established to determine the component contents and ratios of the α-D-1,4-glucan mixture to be tested;

[0020] The operation of establishing the standard curve is as follows: preparing two or more standard α-D-1,4-glucan mixture dispersions with known component contents and ratios, determining points on the standard working curve based on their contents and emission fluorescence spectral characteristics, and determining the component contents and ratios of the α-D-1,4-glucan mixture to be tested based on the standard working curve.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The method of the present invention is easy to operate and does not require special skills from the operator; the sample pretreatment method is simple, solvent consumption is low, and it is relatively environmentally friendly; the required sample amount is small, which can be as low as microgram level; high-throughput detection can be achieved, and the analysis speed is greatly improved; the instrument requirements are low, and the cost is significantly reduced; and the analysis results are stable and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the standard working curve made for Example 1 of the present invention.

[0024] Figure 2 This is the standard working curve made for Example 2 of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0026] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0027] The present invention provides application of molecular rotors in the content and ratio of components of an α-D-1,4-glucan mixture.

[0028] The present invention also provides a fluorescence analysis method for the content of components of an α-D-1,4-glucan mixture and their ratios.

[0029] The fluorescent analysis method for the content and ratio of components of an α-D-1,4-glucan mixture provided by the present invention comprises the following steps:

[0030] (1) dispersing the α-D-1,4-glucan mixture to be tested in a solvent to obtain a dispersion of the glucan mixture to be tested;

[0031] (2) adding a molecular rotor to the above-mentioned glucan mixture dispersion to be tested and dispersing it evenly to obtain a glucan mixture dispersion containing a molecular rotor to be tested;

[0032] (3) measuring characteristic fluorescence emission parameters of the molecular rotor in the dispersion of the dextran mixture containing the molecular rotor to be tested;

[0033] (4) selecting an α-D-1,4-glucan mixture sample with known component contents and ratios, using a solvent with the same components to prepare a standard α-D-1,4-glucan mixture dispersion with the same concentration as the sample to be tested, and adding the same concentration of molecular rotors to disperse uniformly to obtain a standard α-D-1,4-glucan mixture dispersion containing molecular rotors;

[0034] (5) Determine the emission fluorescence characteristic parameters of the molecular rotor in the dispersion of the standard α-D-1,4-glucan mixture containing the molecular rotor, perform single-point calibration based on the obtained characteristic parameter values, or establish a standard curve to determine the polysaccharide and oligosaccharide content and their ratio of the α-D-1,4-glucan mixture to be tested.

[0035] Example 1: Application of polysaccharide and oligosaccharide contents and ratios of glucan mixture 1 to be tested

[0036] (1) Dispersing the glucan mixture 1 to be tested in pure water at a concentration of 0.5% (w / v) to obtain a dispersion of the α-D-1,4-glucan mixture to be tested;

[0037] (2) adding 9-(2-carboxy-2-cyanovinyl)juracil to the dispersion of the α-D-1,4-glucan mixture to be tested, wherein the concentration of 9-(2-carboxy-2-cyanovinyl)juracil in the test solution is 10 μM, and heating under shaking in a boiling water bath for 30 minutes to disperse uniformly;

[0038] (3) Add the solution to the ELISA plate and measure the fluorescence intensity using a ELISA reader (excitation wavelength 420 nm, emission wavelength 490 nm);

[0039] (4) Standard α-D-1,4-glucan mixture samples 1 and 2 (oligosaccharide contents of 27.37 ± 0.12% and 35.04 ± 0.06%, respectively) with known polysaccharide and oligosaccharide component contents and ratios were prepared using the same solvent composition to prepare a standard α-D-1,4-glucan mixture dispersion with the same concentration as the sample being tested, and the same concentration of 9-(2-carboxy-2-cyanovinyl)julidine was added and uniformly dispersed;

[0040] (5) Determine the emission fluorescence spectrum characteristic parameters (fluorescence intensity) of 9-(2-carboxy-2-cyanovinyl) julodidine in the dispersion of the standard α-D-1,4-glucan mixture, and use the data to establish a standard working curve to determine the polysaccharide and oligosaccharide content and their ratio of the glucan mixture 1 to be tested, such as Figure 1 shown.

[0041] The oligosaccharide content in the glucan mixture 1 was measured three times by this method and the result was 32.79±0.68%. The oligosaccharide content in the glucan mixture 1 was measured three times by high performance size exclusion chromatography-differential refractive index detection and the result was 32.57±0.06%. The two-tailed p value calculated by t-test was 0.61, indicating no significant difference.

[0042] Example 2: Application of polysaccharide and oligosaccharide contents and ratios of glucan mixture 2 to be tested

[0043] (1) dispersing the glucan mixture 2 to be tested at 1% (w / v) in a 10% volume concentration dimethyl sulfoxide aqueous solution to obtain a dispersion of the α-D-1,4-glucan mixture to be tested;

[0044] (2) adding 9-(2-carboxy-2-cyanovinyl)juracil to the dispersion of the α-D-1,4-glucan mixture to be tested, wherein the concentration of 9-(2-carboxy-2-cyanovinyl)juracil in the test solution is 10 μM, and heating under shaking in a boiling water bath for 30 minutes to disperse uniformly;

[0045] (3) Add the solution to a cuvette and measure the fluorescence intensity using a fluorescence spectrometer (excitation wavelength 420 nm, emission wavelength 490 nm);

[0046] (4) Standard α-D-1,4-glucan mixture samples 1 and 2 (oligosaccharide contents of 27.37 ± 0.12% and 35.04 ± 0.06%, respectively) with known polysaccharide and oligosaccharide component contents and ratios were prepared using the same solvent composition to prepare a standard α-D-1,4-glucan mixture dispersion with the same concentration as the sample being tested, and the same concentration of 9-(2-carboxy-2-cyanovinyl)julidine was added and uniformly dispersed;

[0047] (5) Determine the emission fluorescence spectrum characteristic parameters (fluorescence intensity) of 9-(2-carboxy-2-cyanovinyl) julodidine in the dispersion of the standard α-D-1,4-glucan mixture, and use the data to establish a standard working curve to determine the polysaccharide and oligosaccharide content and their ratio of the glucan mixture 2 to be tested, such as Figure 2 shown.

[0048] The oligosaccharide content of the glucan mixture 2 was determined three times by this method and the result was 30.24±0.44%. The oligosaccharide content of the glucan mixture 2 was determined three times by high performance size exclusion chromatography-differential refractive index detection and the result was 30.06±0.05%. The two-tailed p value calculated by t-test was 0.52, indicating no significant difference.

[0049] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using conventional techniques known in the art.

Claims

1. Application of molecular rotors in analyzing the content and ratio of components in α-D-1,4-glucan mixtures; the molecular rotors are substances that include electron acceptor groups, electron donor groups, and conjugated π-bond structures and produce fluorescence under the retardation of the surrounding environment.

2. A fluorescence analysis method for the content and ratio of components in an α-D-1,4-glucan mixture, comprising the following steps: (1) dispersing the α-D-1,4-glucan mixture to be tested in a solvent to obtain a dispersion of the glucan mixture to be tested; (2) adding the molecular rotor described in claim 1 to the above-mentioned dispersion of the dextran mixture to be tested, and dispersing it evenly to obtain a dispersion of the dextran mixture containing the molecular rotor to be tested; (3) measuring characteristic fluorescence emission parameters of the molecular rotor in the dispersion of the dextran mixture containing the molecular rotor to be tested; (4) Select an α-D-1,4-glucan mixture sample with known component contents and ratios, use a solvent with the same components to prepare a standard α-D-1,4-glucan mixture dispersion with the same concentration as the sample to be tested, and add the same concentration of molecular rotors to disperse evenly to obtain a standard α-D-1,4-glucan mixture dispersion containing molecular rotors; (5) Determine the emission fluorescence characteristic parameters of the molecular rotor in the dispersion of the standard α-D-1,4-glucan mixture containing the molecular rotor, perform single-point calibration based on the obtained characteristic parameter values, or establish a standard curve to determine the content and ratio of the components of the α-D-1,4-glucan mixture to be tested.

3. The method according to claim 2, wherein: In steps (1) and (4), the solvent is one or more of water, buffer solution, strong alkaline solution, N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, pyridine, acetone, and tetrahydrofuran.

4. The method according to claim 2, wherein: The molecular rotor is selected from the group consisting of julidine derivatives, dialkylaniline derivatives, boron-dipyrromethene derivatives, and porphyrin derivatives; The concentration of the molecular rotor in the molecular rotor-containing dextran mixture dispersion to be tested and the molecular rotor-containing standard α-D-1,4-dextran mixture dispersion to be tested is 1 μM or more.

5. The method according to claim 2, wherein: In steps (3) and (5), the characteristic parameters of the emitted fluorescence are one or more of fluorescence intensity and quantum yield.

6. The method according to claim 2, wherein: In step (5), the single-point calibration operation is as follows: using the emission fluorescence characteristic parameters in a single standard α-D-1,4-glucan mixture dispersion and the emission fluorescence characteristic parameters of a blank dispersion as references, a standard working curve is established to determine the content of the components of the α-D-1,4-glucan mixture to be tested and their ratios.

7. The method according to claim 2, wherein: The operation of establishing the standard curve is: preparing a dispersion of an α-D-1,4-glucan mixture with two or more known component contents and ratios, determining points on the standard working curve based on the content and emission fluorescence spectral characteristics, and determining the component contents and ratios of the α-D-1,4-glucan mixture to be tested based on the standard working curve.

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