A rapid-detection continuous degradation reactor for polysaccharides

Abstract

This invention discloses a rapidly detectable continuous degradation reactor for polysaccharides. The reactor uses metal-supported two-dimensional nanoporous carbon derived from metal-organic frameworks (MOFs) as a heterogeneous catalyst. By placing this catalyst in a temperature-controlled reactor with partitioned chambers, rapid recovery of oligosaccharides and controlled continuous degradation of various polysaccharides are achieved. The automated collection of the degraded oligosaccharide solution integrates the degradation reactor with LC-MS / MS, enabling rapid and continuous detection of polysaccharide structures.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide degradation and mass spectrometry structure detection, and in particular to a continuous polysaccharide degradation reactor capable of rapid detection. Background Technology

[0002] Polysaccharides are a class of high-molecular-weight carbohydrates composed of monosaccharide units linked by glycosidic bonds, possessing complex structures and diverse biological functions. Unlike well-studied proteins and nucleic acids, characterizing the chemical structure of polysaccharides remains quite challenging due to their diversity and complexity. In recent years, combinations of chemical methods and instrumental analytical techniques have been widely used for polysaccharide structural characterization, such as high-performance gel permeation chromatography (HPGPC), high-performance anion exchange chromatography (HPAEC), gas chromatography-mass spectrometry methylation (GC-MS), and nuclear magnetic resonance (NMR). However, these analytical processes are typically very time-consuming and require large sample volumes. Mass spectrometry (MS) has been applied to determine the structure of polysaccharides, accurately characterizing oligosaccharide structures, including bonding patterns, degree of polymerization (DP), and molecular weight. Polysaccharide mass spectrometry structural detection mainly includes methods such as MALDI-TOF-MS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometry) and ESI-TOF-MS (electrospray ionization time-of-flight mass spectrometry). Electrospray ionization (ESI) is a soft ionization technique widely used in carbohydrate analysis. ESI-MS generates mass spectra primarily consisting of single-charged ions, which are further broken down into fragments, providing information on the monosaccharide, glycosidic bond types, and sequences of polysaccharides. This allows for rapid inference of the composition and structure of carbohydrates. ESI-MS offers significant advantages such as low sample consumption, simple sample preparation, suitability for continuous ionization of liquid samples, and high-throughput determination. It is also suitable for online separation of complex polysaccharide mixtures when coupled with liquid chromatography (e.g., HPLC-ESI-MS), and it is easier to achieve tandem mass spectrometry (MS / MS) for glycosidic bond sequence resolution. However, ESI-MS typically requires a series of pretreatment processes before detection, including polysaccharide extraction, purification, and degradation. The degradation process directly affects the effectiveness in detecting polysaccharide fragment structures. Despite continuous exploration and varying degrees of success in polysaccharide degradation technologies based on physical methods such as ultrasound and microwaves, as well as chemical methods such as acid hydrolysis, Fenton reaction, and enzymatic methods, ESI-MS has achieved remarkable results. The high cost and incomplete degradation of physical degradation methods in ESI-MS detection, the environmental and structural loss issues of chemical methods, and the high specificity and lack of specific enzymes in enzymatic methods, along with the cumbersome degradation steps that may introduce errors, all complicate the rapid and consistent degradation of structurally complex and heterogeneous polysaccharides. These challenges limit the high throughput and accuracy of ESI-MS in polysaccharide structure detection. Developing standardized, green, safe, and efficient polysaccharide degradation strategies that preserve key structural features while facilitating rapid and reliable structural and functional analysis and food applications remains a significant need for advancing polysaccharide research. Summary of the Invention

[0003] The purpose of this invention is to address the problems of insufficient polysaccharide degradation efficiency and cumbersome operation in the pretreatment process of existing polysaccharide mass spectrometry detection technology. A continuous polysaccharide degradation reactor has been constructed. Compared with the traditional pretreatment process of polysaccharide mass spectrometry detection technology, this reactor realizes rapid, efficient and heterogeneous degradation of polysaccharides. The reactor can achieve high-throughput rapid preparation of different oligosaccharides, and the prepared oligosaccharides do not contain metals.

[0004] Based on this reactor setup, the present invention further integrates HPLC-ESI-TOF-MS / MS; the HPLC-ESI-TOF-MS / MS consists of a Waters ACQUITY UPLC with a BEH amide column (1.7 μm, 1.2 × 150 mm) and is connected to a SYNAPT XS mass spectrometer; the integration of HPLC-ESI-TOF-MS / MS enables real-time monitoring of oligosaccharide quality and timely monitoring of the polysaccharide structure in the product during the reaction process.

[0005] Specifically, the rapidly detectable continuous degradation reactor for polysaccharides provided by the present invention includes the following parts: (1) Nanoporous carbon catalytic material; the nanoporous carbon catalytic material is obtained by carbonization of Cu-MOF; (2) A degradation reactor device with built-in nanoporous carbon; the degradation reactor device includes two inlet tubes and a reaction chamber; the reaction chamber has an inlet and an outlet equipped with a filter membrane; the nanoporous carbon catalyst material is located inside the reaction chamber; the two inlet tubes are respectively connected to the inlet to input polysaccharide solution and hydrogen peroxide solution into the degradation reactor; (3) Reaction quenching device; the reaction quenching device is automatically injected with ammonia water; the reaction quenching device is connected to the sample outlet through a hose; the concentration of ammonia water is 0.1M.

[0006] (4) HPLC-ESI-TOF-MS / MS system; consisting of a Waters ACQUITY UPLC with a BEH amide column (1.7 μm, 1.2 × 150 mm) connected to a SYNAPT XS mass spectrometer; (4) Peristaltic pump; the peristaltic pump uses pressure to transport the liquid in the degradation reactor to the reaction quenching device, and then to the HPLC-ESI-TOF-MS / MS system; the two ends of the peristaltic pump are connected to the reaction quenching device and the HPLC-ESI-TOF-MS / MS system respectively.

[0007] Further, the preparation method of the nanoporous carbon catalytic material is as follows: 1,3,5-tris(4-carboxyphenyl)benzene (BTB) and copper(II) nitrate trihydrate (Cu(NO3)2·3H2O) are continuously stirred and completely dissolved at room temperature. The mixed solvent system is transferred to a stainless steel autoclave for reaction. After reacting at 80 °C for 1 hour, the precursor material is collected by centrifugation. The precursor material is carbonized under a nitrogen atmosphere with programmed temperature increase. Finally, the carbonized material is etched in nitric acid solution and washed until neutral to obtain the nanoporous carbon catalytic material (Cu-BTB@C / A).

[0008] Furthermore, the pore size of the filter membrane described in step 2 is 0.45 μm.

[0009] Furthermore, the HPLC-ESI-TOF-MS / MS system consisted of a Waters ACQUITY UPLC with a BEH amide column (1.7 μm, 1.2 × 150 mm) connected to a SYNAPT XS mass spectrometer.

[0010] Furthermore, the flow rate of the peristaltic pump is 0.1-10 mL / min.

[0011] Furthermore, the bottom of the reaction chamber is an automatic temperature control platform, which can heat and cool the reactor.

[0012] The advantages of this invention are: it solves the shortcomings of traditional polysaccharide mass spectrometry detection technology, such as harsh pretreatment conditions, complex process flow, easy destruction of oligosaccharide structure, and difficulty in achieving high-throughput sequencing. By integrating a polysaccharide degradation reactor and LC-MS / MS, it is possible to analyze polysaccharide structure within 120 minutes (including 60 minutes of degradation and 60 minutes of analysis), realize real-time monitoring of oligosaccharide quality, and provide a rapid reaction platform for high-throughput sequencing and rapid continuous large-scale preparation of polysaccharides. Attached Figure Description

[0013] Figure 1 This is a schematic diagram of the internal structure of the degradation reactor; Figure 2 This is a schematic diagram of a traditional polysaccharide mass spectrometry detection method combined with a degradation reactor for mass spectrometry detection. Figure 3 Chromatographic elution profiles of different polysaccharide standards degraded in the degradation reactor; Figure 4 The chromatographic elution diagram shows the polysaccharide degradation in the degradation reactor. Figure 5 This is a schematic diagram of a traditional polysaccharide mass spectrometry detection method combined with a degradation reactor for mass spectrometry detection. Figure 6 Annotate the base peak chromatograms of oligosaccharides from xylan degradation in homogeneous and degradation reactors; Figure 7 A schematic diagram of the detectable oligosaccharide degree of polymerization distribution in a homogeneous and degradation reactor for xylan degradation. Detailed Implementation

[0014] The following examples are provided to further illustrate the present invention and are intended to explain the invention, not to limit its scope. Unless otherwise specified, all figures are expressed in parts by weight and weight percentages.

[0015] Unless otherwise specified, the raw materials used in this invention are all conventional commercially available products; unless otherwise specified, the methods used in this invention are all conventional methods in the field.

[0016] The embodiments of the present invention will be further described below with reference to several examples.

[0017] It should be understood that the described embodiments are merely some, not all, of the embodiments in this application. All other embodiments obtained by those skilled in the art based on the embodiments in this application without inventive effort are within the scope of protection of this application.

[0018] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms “a,” “the,” and “the” used in the embodiments of this application and the appended claims are also intended to include the plural forms unless the context clearly indicates otherwise.

[0019] Example 1 Material Preparation A homogeneous reaction solution was prepared by mixing dimethylformamide (DMF), methanol (MeOH), and deionized water. Subsequently, 1,3,5-tris(4-carboxyphenyl)benzene (BTB) and copper(II) nitrate trihydrate (Cu(NO3)2·3H2O) were completely dissolved in the mixed solvent system under continuous stirring at room temperature for 60 minutes, with concentrations of 0.10 mM and 0.05 mM for BTB and Cu(NO3)2·3H2O, respectively. The resulting solution was then transferred to a stainless steel autoclave for reaction. After cooling to ambient temperature, pale blue Cu-BTB crystals were collected by centrifugation, washed, and vacuum dried to obtain the purified MOF precursor. The precursor material was heated at 1100 °C for 1 hour to produce a carbonized material. The carbonized material was then treated with nitric acid solution under reflux at 80 °C for 4 hours. The resulting suspension was repeatedly centrifuged and washed with ultrapure water to finally obtain the nanoporous carbon product (Cu-BTB@C / A).

[0020] Example 2: This embodiment provides, as follows: Figure 1 The rapidly detectable continuous degradation reactor for polysaccharides shown includes: The degradation reactor device includes two inlet tubes and a reaction chamber 2; the reaction chamber has an inlet 1 and an outlet 3 equipped with a filter membrane; the reaction chamber 2 is loaded with the nanoporous carbon catalytic material prepared in Example 1; the two inlet tubes are respectively connected to the inlet 1 to input polysaccharide solution and hydrogen peroxide solution into the degradation reactor; The reaction quenching device 4 is automatically injected with ammonia water; the reaction quenching device is connected to the sample outlet 3 via a hose; the concentration of ammonia water is 0.1M.

[0021] Peristaltic pump 5; The peristaltic pump uses pressure to transport the liquid in the degradation reactor to the reaction quenching device, and then to the HPLC-ESI-TOF-MS / MS system; The two ends of the peristaltic pump are connected to the reaction quenching device and the HPLC-ESI-TOF-MS / MS system, respectively.

[0022] HPLC-ESI-TOF-MS / MS system 6; consisting of a Waters ACQUITY UPLC with a BEH amide column (1.7 μm, 1.2 × 150 mm), connected to a SYNAPT XS mass spectrometer; Peristaltic pump; the peristaltic pump uses pressure to deliver liquid from the degradation reactor to the reaction quenching device, and then to the HPLC-ESI-TOF-MS / MS system; the two ends of the peristaltic pump are connected to the reaction quenching device and the HPLC-ESI-TOF-MS / MS system, respectively.

[0023] The ability of the degradation reactor to catalyze the continuous degradation of polysaccharides was evaluated using xylan and other polysaccharide standards (5 mg / mL).

[0024] The implementation process is as follows: The first polysaccharide solution (5 mg / mL) and 10 mM H2O2 were injected into the reaction chamber through two injection tubes to carry out the degradation reaction at 60 °C. After the reaction was completed, the first reaction solution was formed; the first reaction solution was squeezed out of the filter membrane by a peristaltic pump.

[0025] After cleaning the reaction chamber with water, the second polysaccharide solution and H2O2 are injected, and the reaction process is repeated.

[0026] When polysaccharide solution and hydrogen peroxide are simultaneously introduced into the reactor, a catalytic reaction occurs, generating bubbles that cause the nanoporous carbon to circulate repeatedly in the reaction solution, thus achieving relatively uniform catalytic degradation. Compared to the traditional Fenton degradation method, this polysaccharide degradation reactor can achieve continuous degradation of polysaccharides and rapid separation of metal-free oligosaccharides. Figure 2The degradation reactor's ability to degrade polysaccharides was demonstrated by its degradation effect on 12 polysaccharides, including pectin, mannan, galactan, arabinogalactan, arabinoxylan, galactomannan, rhamnuronic acid, xylan, β-glucan, glucomannan, laminarin, and rhamnose-galacturonic acid I.

[0027] For comparative analysis, a control experiment was conducted under the same conditions using 0.1 mM CuSO4 solution and 10 mM H2O2. The reactor degradation liquid (200 μL) was then collected, the reaction was immediately quenched by adding 5 μL of ammonia, and the solution was adjusted to neutral. After filtration through a 0.22 μm membrane, the molecular weight distribution of the degraded polysaccharides was analyzed by size exclusion chromatography (Superdex™ peptide 10 / 300 GL and Ultradex 250-120).

[0028] 50% acetonitrile was automatically added to the oligosaccharide solution obtained from the degradation reactor, and the structure was characterized using an HPLC-TOF-MS / MS system consisting of a Waters ACQUITY UPLC with a BEH amide column (1.7 μm, 1.2 × 150 mm) connected to a SYNAPT XS mass spectrometer. Chromatographic separation was achieved using a binary gradient system: solvent a (20% acetonitrile, containing 10 mM ammonium formate and 0.2% formic acid) and solvent B (80% acetonitrile, containing 10% ammonium formate and 0.2% formic acid) at a flow rate of 0.25 mL / min and a temperature of 45 °C. The gradient program increased from 100% to 70% over 0–35 min, then decreased to 20% over 50 min (hold for 55 min), and then reequilibrated to 100% over 56 min (hold for 60 min), with an injection volume of 2 μL. Mass spectrometry analysis was performed in negative ion mode (m / z 100-1500) with the following optimized parameters: 55 psi for nebulizer gas, 35 psi for curtain gas, 550 °C for ion source temperature, and -4500 V for ion spray voltage. For fragmentation analysis, the instrument was operated in MSE mode with a declustering potential of 100 V, a collision energy of 10 V for the first scan, and a CID energy of 40 ± 20 eV for the second scan. Mass calibration was performed using a CDS pump prior to analysis to maintain mass accuracy within ± 2 ppm. All data were processed using the Waters MassLynx (v4.2) and UNIFI (v4.2) software platforms.

[0029] like Figure 3As shown, most peaks were observed in the chromatographic elution profiles of all degraded polysaccharides within a retention time interval exceeding 36 minutes, indicating that these polysaccharides had been degraded into oligosaccharides with a DP of 2-13. Furthermore, the reactor maintained high activity after more than 50 consecutive polysaccharide degradation cycles, demonstrating a significantly improved degradation efficiency compared to homogeneous catalytic reactions. Figure 4 This indicates that the degradation reactor is a versatile polysaccharide degrader with high degradation efficiency and easy separation of oligosaccharides, which helps to rapidly and completely degrade most polysaccharides in a continuous oligosaccharide production system.

[0030] Unlike traditional, cumbersome methods for detecting polysaccharide structures, this system seamlessly integrates a degradation reactor with LC-MS / MS. The degraded oligosaccharide solution is further introduced through a pipeline into a reaction quencher to remove unreacted hydrogen peroxide. The oligosaccharide solution from the quencher is then collected and injected into the LC-MS / MS for detection. Figure 5 This system eliminates the need for partitioning experiments, reducing experimental errors. The reaction conditions are mild, and there are no metal residues. This method enables the analysis of polysaccharide structures within 120 minutes (including 60 minutes of degradation and 60 minutes of analysis), which is of great significance for polysaccharide structure detection and instrument protection.

[0031] The practicality of the degradation reactor was further verified by LC-MS / MS results. Characteristic spectra of oligosaccharides with parental structures were obtained using collision-induced ionization (CID) LC-MS / MS and compared with reported oligosaccharide reference libraries to obtain polysaccharide structural information. Prominent peaks were labeled with DP and monosaccharide components (e.g., hexose [hexagonal], pentose [pentagonal], hexuronic acid [hexagonal], 4-O-methylglucuronic acid). LC-MS / MS results of oligosaccharides generated from xylan degradation showed fewer spectral peaks in homogeneous Fenton degradation reactions due to incomplete degradation and signal interference from metal ions. In contrast, the oligosaccharides provided by the degradation reactor could obtain oligosaccharides with DPs of 2–11 from precise mass and exhibited more characteristic basic peaks (…). Figure 6 The measurable particle size distribution of the oligosaccharide products indicates that xylan produced 11 structurally unique oligosaccharides through degradation in the degradation reactor, and isomers also exist. Figure 7 This degradation reactor simplifies the analysis of complex polysaccharide mixtures, and the reaction is easily scalable and can be extended to a wider range of polysaccharides, significantly advancing research in the field of polysaccharides.

[0032] The above embodiments describe in detail the structure, features, and effects of the present invention. The above description is only a preferred embodiment of the present invention. Any changes made in accordance with the concept of the present invention, or equivalent embodiments modified to have equivalent changes, shall still fall within the scope of protection of the present invention if they do not exceed the scope covered by the specification.

Claims

1. A rapidly detectable continuous degradation reactor for polysaccharides, characterized in that, Includes the following parts: (1) Nanoporous carbon catalytic material; the nanoporous carbon catalytic material is obtained by carbonization of Cu-MOF precursor; (2) A degradation reactor device with built-in nanoporous carbon; the degradation reactor device includes two inlet tubes and a reaction chamber; the reaction chamber has an inlet and an outlet equipped with a filter membrane; the nanoporous carbon catalyst material is located inside the reaction chamber; the two inlet tubes are respectively connected to the inlet to input polysaccharide solution and hydrogen peroxide solution into the degradation reactor; (3) Reaction quenching device; the reaction quenching device is automatically injected with ammonia water; the reaction quenching device is connected to the sample outlet through a hose; (4) HPLC-ESI-TOF-MS / MS system; consisting of a Waters ACQUITY UPLC with a BEH amide column (1.7 μm, 1.2 × 150 mm) connected to a SYNAPT XS mass spectrometer; (4) Peristaltic pump; the peristaltic pump uses pressure to transport the liquid in the degradation reactor to the reaction quenching device, and then to the HPLC-ESI-TOF-MS / MS injection system; the two ends of the peristaltic pump are connected to the reaction quenching device and the HPLC-ESI-TOF-MS / MS injection system, respectively.

2. The polysaccharide continuous degradation reactor according to claim 1, characterized in that, The preparation method of the nanoporous carbon catalytic material is as follows: 1,3,5-tris(4-carboxyphenyl)benzene (BTB) and copper(II) nitrate trihydrate (Cu(NO3)2·3H2O) are continuously stirred and completely dissolved at room temperature. The mixed solvent system is transferred to a stainless steel autoclave for reaction. After reacting at 80 °C for 1 hour, the Cu-MOF precursor is obtained by centrifugation. The Cu-MOF precursor is carbonized under a nitrogen atmosphere. Finally, the carbonized material is etched in a nitric acid solution and washed until neutral to obtain the nanoporous carbon catalytic material.

3. The polysaccharide continuous degradation reactor according to claim 1, characterized in that, The pore size of the filter membrane described in step 2 is 0.45 μm.

4. The polysaccharide continuous degradation reactor according to claim 1, characterized in that, The flow rate of the peristaltic pump is 0.1-10 mL / min.

5. The polysaccharide continuous degradation reactor according to claim 1, characterized in that, It also includes an automatic temperature control platform for heating and cooling the reactor.

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