Kit and method for joint detection of glucose, cellobiose and xylose in cellulose hydrolysate

Abstract

This invention discloses a reagent kit and method for the joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate, belonging to the field of biomass conversion and analytical detection technology. The method includes the following steps: setting up three types of detection channels: a glucose oxidase detection channel that specifically responds to the glucose component; a β-glucosidase-glucose oxidase combined detection channel that responds to the total signal of glucose and cellobiose; and a pyranose oxidase detection channel that simultaneously responds to the combined signals of glucose, cellobiose, and xylose. Enzymatic colorimetric reactions are performed on the cellulose hydrolysate sample and the multi-component gradient standard solution in the three types of detection channels, respectively. A ternary linear calibration model is constructed based on the absorbance data of the three types of channels of the multi-component gradient standard solution. This method provides a rapid, low-cost, and highly specific detection method for glucose, cellobiose, and xylose in cellulose biomass hydrolysate, and has practical application value.

Description

Technical Field

[0001] This invention relates to a reagent kit and method for the joint detection of glucose, cellobiose and xylose in cellulose hydrolysate, belonging to the field of biomass conversion, analysis and detection technology. Background Technology

[0002] Lignocellulosic biomass, after pretreatment and hydrolysis, yields hydrolysates containing various sugars. These hydrolysates typically contain glucose derived from cellulose, as well as monosaccharides such as xylose, arabinose, mannose, and galactose derived from hemicellulose, and may also contain oligosaccharide intermediates such as cellobiose. The sugar composition of hydrolysates varies considerably depending on the cellulose raw material and the pretreatment / hydrolysis process conditions. Therefore, accurate analysis of the sugar components in the hydrolysate is crucial for evaluating pretreatment and hydrolysis efficiency, optimizing fermentation processes, and achieving high-value utilization of biomass.

[0003] Currently, chromatographic techniques remain the primary method for analyzing carbohydrate components in cellulose biomass hydrolysates. These include high-performance liquid chromatography (HPLC), high-performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD), gas chromatography (GC), and methods coupled with mass spectrometry. Additionally, there are reports of analysis using nuclear magnetic resonance (NMR), capillary electrophoresis, thin-layer chromatography (TLC), and colorimetric methods such as DNS. Among these, HPLC, especially HPLC-RID, is widely used due to its relatively simple operation and suitability for routine analysis. HPAEC-PAD offers higher sensitivity and better oligosaccharide separation capabilities. GC-MS, LC-MS / MS, and NMR are more suitable for the structural characterization of complex carbohydrates or unknown oligosaccharides.

[0004] Limitations of Existing Technologies: Although existing chromatographic and mass spectrometric methods can accurately analyze the carbohydrate composition in cellulose biomass hydrolysates, they still have the following shortcomings. First, the equipment costs are high, the operation procedures are relatively complex, and the detection cycle is long. For example, although HPLC-RID, HPAEC-PAD, GC-MS, and LC-MS / MS have high sensitivity and resolution, they usually have high requirements for equipment conditions, mobile phase systems, and sample purification or derivatization, which is not conducive to low-cost, rapid, and high-throughput applications. Second, the analysis of oligosaccharide intermediates such as cellobiose remains difficult. Cellobiose is an important intermediate product of cellulose enzymatic hydrolysis, so its quantification is of great significance for evaluating the degree of hydrolysis; however, in existing technologies, cellobiose detection often requires higher resolution chromatographic methods, derivatization, or complex standard systems, making the analytical steps cumbersome, and the poor availability of oligosaccharide standards can easily increase the difficulty of method development and quantitative uncertainty. In addition, existing low-cost rapid analytical methods generally lack sufficient component specificity. For example, while methods for determining total reducing sugars, such as the DNS method, are simple to operate and suitable for initial screening of a large number of samples, they can only reflect the total reducing sugar content and cannot distinguish between different sugar components such as glucose, xylose, and cellobiose. They also cannot meet the need for separate quantification of key components in hydrolysate.

[0005] Therefore, while existing technologies can analyze carbohydrate components in cellulose biomass hydrolysates, they generally suffer from high equipment dependence, cumbersome sample pretreatment, high analysis costs, difficulty in balancing speed and component specificity, and shortcomings in providing convenient and simultaneous analysis of key carbohydrate components such as glucose, cellobiose, and xylose. Especially when applied to process optimization, process monitoring, high-throughput microplate screening, or kit-based applications, existing methods still have room for improvement. Summary of the Invention

[0006] The purpose of this invention is to provide a reagent kit and method for the joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate. This method provides a rapid, low-cost, and highly specific detection method for glucose, cellobiose, and xylose in cellulose biomass hydrolysate, and has practical application value.

[0007] The method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to the present invention includes the following steps: S1: Three types of detection channels are set up: glucose oxidase detection channel that specifically responds to glucose component, β-glucosidase-glucose oxidase combined detection channel that responds to total signal of glucose and cellobiose, and pyranose oxidase detection channel that responds to comprehensive signal of glucose, cellobiose and xylose components. S2: The cellulose hydrolysate sample to be tested and the multi-component gradient standard solution were subjected to enzymatic colorimetric reactions in three types of detection channels, and the absorbance value of each detection channel was measured. S3: Construct a ternary linear calibration model based on the absorbance data of the three channels of the multi-component gradient standard solution. Substitute the absorbance values ​​of the three channels of the sample to be tested into the ternary linear calibration model to calculate the concentrations of glucose, cellobiose and xylose in the sample to be tested.

[0008] Preferably, the sample pretreatment step is also included: S11: Centrifuge the cellulose hydrolysate to be tested at 8000g for 10min and take the clear upper layer. S12: Filter the obtained clarified liquid through an aqueous filter membrane with a pore size of 0.22μm to remove insoluble impurities; S13: Dilute the filtered sample with 50mM, pH 5.0 phosphate buffer to ensure that the detection signal of the diluted sample falls within the linear response range of the multi-component gradient standard solution.

[0009] Preferably, the reaction step of the β-glucosidase-glucose oxidase coupled detection channel in step S2 is as follows: S21: Add β-glucosidase reagent to the reaction system and incubate at a constant temperature for a certain period of time to completely hydrolyze the cellobiose in the reaction system into glucose. S22: Add horseradish peroxidase reagent, ABTS chromogenic substrate reagent, and glucose oxidase reagent sequentially to the hydrolyzed reaction system, and incubate in the dark for a certain period of time to complete the chromogenic reaction.

[0010] Preferably, the reaction steps for the glucose oxidase detection channel and the pyranose oxidase detection channel in step S2 are as follows: add the sample / standard solution, reaction buffer, horseradish peroxidase reagent, ABTS chromogenic substrate reagent, and the corresponding oxidase reagent to the reaction system in sequence: add glucose oxidase to the glucose oxidase detection channel and add pyranose oxidase to the pyranose oxidase detection channel, and incubate in the dark for a certain period of time to complete the colorimetric reaction.

[0011] Preferably, the ternary linear correction model in step S3 is expressed in the following form: ; Where Y B Y C Y DThe measured absorbance values ​​are for the glucose oxidase detection channel, the β-glucosidase-glucose oxidase combined detection channel, and the pyranose oxidase detection channel, respectively; Glc, Cel, and Xyl represent the concentrations of glucose, cellobiose, and xylose, respectively; b1, b2, b3, c1, c2, c3, d1, d2, d3 are the response coefficients of the three channels to the three sugar components, which together form a 3×3 response coefficient matrix K; a1, a2, and a3 are the intercept values ​​of the linear regression of the three channels, and the coefficient of determination for linear fitting of each channel is: ; Where: n: the total number of multi-component gradient standard solutions; : The measured absorbance value of the i-th standard solution; The absorbance value of the i-th standard solution predicted by the linear regression equation; The average absorbance of all standard solutions in their respective channels; the coefficients of determination for linear fitting of the three types of detection channels must all meet the following requirements. ≥0.99.

[0012] Preferably, the formula for calculating the concentration of the sample to be tested in step S3 is: ; in: Response coefficient matrix The inverse matrix, , , The measured absorbance values ​​of the sample to be tested are in the glucose oxidase detection channel, the β-glucosidase-glucose oxidase combined detection channel, and the pyranose oxidase detection channel, respectively. The concentrations of glucose, cellobiose, and xylose in the sample to be tested can be directly obtained through the above matrix operation.

[0013] The kit for the joint detection of glucose, cellobiose and xylose in cellulose hydrolysate according to the present invention comprises at least the following independently packaged components: β-glucosidase reagent, glucose oxidase reagent, pyranose oxidase reagent, horseradish peroxidase reagent, ABTS chromogenic substrate reagent, reaction buffer, and multi-component mixed standard solution set.

[0014] Preferably, the pyranose oxidase is derived from *Phanerochaete chrysospora*, serving as the core enzyme component of the pyranose oxidase detection channel. It works in conjunction with the enzyme reagents of the glucose oxidase detection channel and the β-glucosidase-glucose oxidase co-detection channel to achieve the joint detection of the three types of sugars.

[0015] Preferably, the multi-component mixed standard solution group includes six gradient mixed standard solutions, P1 to P6, with the concentration range of each standard solution being: glucose 0~1g / L, cellobiose 0~0.08g / L, and xylose 0~0.08g / L. Among them, P1 is a blank standard solution without the three target sugars, and P2~P6 are mixed standard solutions with increasing concentration gradients of the three target sugars.

[0016] Preferably, the kit is compatible with two detection modes: high-throughput batch detection mode based on 96-well plate microplate reader and single-tube detection mode based on cuvette visible light spectrophotometer.

[0017] Compared with existing technologies, the reagent kit and method for the joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate of the present invention exhibit the following beneficial effects in terms of technical performance and practical application: 1. This invention achieves simultaneous quantification of three key sugar components through joint modeling of the glucose oxidase detection channel (GOX channel), the β-glucosidase-glucose oxidase combined detection channel (β-Glu+GOX channel), and the pyranose oxidase detection channel (POX channel), resulting in higher information content. Compared with methods such as HPLC, HPAEC-PAD, GC-MS, and LC-MS / MS, this invention uses enzymatic colorimetric detection, which can be performed using an ELISA reader or a visible spectrophotometer, requiring less equipment, reducing detection costs, and making it more suitable for routine laboratories and process screening scenarios.

[0018] 2. This invention uses microplates as a carrier, has simple reaction conditions, and directly measures absorbance at 405 nm after color development. It does not require complex separation and long-term detection, and can complete the detection of dozens of samples within half an hour, making it suitable for rapid analysis.

[0019] 3. Cellobiose is an important intermediate product in the enzymatic hydrolysis of cellulose, but existing methods for its detection often rely on high-resolution chromatography or complex standard systems. This invention converts cellobiose into a detectable signal through β-glucosidase pretreatment, significantly reducing the difficulty of detection.

[0020] 4. This invention can simultaneously provide the content of glucose, cellobiose and xylose, thus enabling the determination of whether hydrolysis is sufficient, whether cellobiose has accumulated, and the degradation status of hemicellulose, making it more suitable for process monitoring.

[0021] 5. This invention does not rely on expensive instruments such as high-performance liquid chromatography or mass spectrometry. Through multi-enzyme coupled colorimetric reactions and a three-channel joint calibration model, it can achieve simultaneous quantitative analysis of three sugar components. Compared with existing technologies, this invention has advantages such as low equipment requirements, low detection cost, simple operation, fast detection speed, suitability for high-throughput screening in microplates, more favorable detection of cellobiose intermediates, and ease of kit development and widespread application. It is particularly suitable for monitoring cellulose hydrolysis processes, optimizing biomass conversion processes, and related scientific research and industrial detection scenarios. Attached Figure Description

[0022] Figure 1 This is a flowchart illustrating the method in an embodiment of the present invention; Figure 2 The figures show the screening results of different pyranose oxidases in this embodiment of the invention; in the figure, (a) shows the phylogenetic tree analysis results of the candidate pyranose oxidases and related oxidases, where PCPOX and TVPOX are two candidate pyranose oxidases screened in this embodiment; (b) shows the recombinant expression of PCPOX and TVPOX in the host and the SDS-PAGE analysis results, where M is the protein molecular weight standard, S is the supernatant component, P is the precipitate component, FT is the permeation buffer, W is the washing buffer, and E is the elution buffer; Figure 3 The figures show the substrate response and glucose kinetic analysis results of the candidate pyranose oxidases in the embodiments of the present invention; in the figure, (a) shows the schematic diagram of the response strength of PCPOX and TVPOX to glucose, cellobiose and xylose; (b) shows the Michaelis kinetic fitting curves of PCPOX and TVPOX with glucose as the substrate. Figure 4 The figures show liquid chromatograms of sample 1 and sample 2 in an embodiment of the present invention; in the figures, (a) represents sample 1 and (b) represents sample 2. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0024] Example 1: To verify that the glucose oxidase detection channel (GOX channel), β-glucosidase-glucose oxidase combined detection channel (βGlu+GOX channel), and pyranose oxidase detection channel (POX channel) described in this invention can perform joint quantitative analysis of glucose, cellobiose, and xylose, standard solutions P1-P6 were used to perform detection in the three channels respectively, a three-channel response model was established, and it was used to solve for the inversion of the concentrations of the three sugars in unknown samples.

[0025] like Figure 1 As shown, the method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to the present invention includes the following steps: S1: Sample pretreatment: Sample Requirements ① Applicable samples; Cellulose hydrolysate or fermentation broth (requires simple pretreatment); ② Sample pretreatment; The sample to be tested was centrifuged at 8000 g for 10 min; The supernatant was filtered through a 0.22 μm aqueous filter membrane; The filtered sample was appropriately diluted with 50 mM phosphate buffer (pH 5.0) to bring the detection signal into the linear range of the standard solution.

[0026] S2: Three-channel enzymatic colorimetric reaction: Three types of detection channels were set up: the GOX channel, which specifically responds to the glucose component only; the βGlu+GOX channel, which responds to the total signal of glucose and cellobiose; and the POX channel, which responds to the combined signal of glucose, cellobiose, and xylose. Enzymatic colorimetric reactions were performed on the cellulose hydrolysate sample and the multi-component gradient standard solution in the three types of detection channels, and the absorbance value of each detection channel was measured. GOX channel reaction system (total system 200 μL): Add 50 μL of diluted sample / standard solution, 141 μL of reaction buffer, 2 μL of horseradish peroxidase reagent, 5 μL of ABTS chromogenic substrate reagent, and 2 μL of glucose oxidase reagent in sequence. After sealing with sealing film, incubate in a 37℃ constant temperature incubator in the dark for 10 min. After taking it out, place it in an ELISA reader to detect the absorbance value at a wavelength of 405 nm, and record it as YB (standard solution) or YB' (sample to be tested).

[0027] βGlu+GOX channel reaction system (total system 200μL): Add 50μL of diluted sample / standard solution, 139μL of reaction buffer, and 2μL of β-glucosidase reagent sequentially. Pre-incubate at 37℃ for 10min to completely hydrolyze cellobiose into glucose. Then add 2μL of horseradish peroxidase reagent, 5μL of ABTS chromogenic substrate reagent, and 2μL of glucose oxidase reagent. Incubate at 37℃ in the dark for 10min. Detect the absorbance value at 405nm and record it as YC (standard solution) or YC' (sample to be tested).

[0028] POX channel reaction system (total system 200 μL): Add 50 μL of diluted sample / standard solution, 141 μL of reaction buffer, 2 μL of horseradish peroxidase reagent, 5 μL of ABTS chromogenic substrate reagent, and 2 μL of pyranose oxidase reagent in sequence. Incubate at 37°C in the dark for 10 min. Detect the absorbance value at 405 nm and record it as YD (standard solution) or YD' (sample to be tested).

[0029] ① Hole position design: Table 1 Hole Position Design

[0030] Table 2 Composition of each component

[0031] Reaction conditions: 37℃, 10 min, incubation in the dark.

[0032] Detection: Wavelength 405 nm.

[0033] Table 3 Concentrations of glucose, cellobiose, and xylose in the standard solution

[0034] The aforementioned standard solutions P1-P6 were used, and their composition is shown in Table 3.

[0035] The absorbance of each standard solution in the GOX well, β-glucosidase + GOX well and POX well was measured according to the three-well detection system described in the examples. The results are shown in Table 4.

[0036] Table 4. Example absorbance data of standard solutions in three channels (405 nm)

[0037] Among them, the GOX well mainly reflects the glucose signal in the sample; the β-glucosidase + GOX well reflects the glucose signal produced after the hydrolysis of glucose and cellobiose; and the POX well reflects the combined color of glucose, cellobiose and xylose.

[0038] S3: Construction of the ternary linear correction model: A ternary linear calibration model was constructed based on the absorbance data of the three channels of the multi-component gradient standard solution. The absorbance values ​​of the three channels of the sample to be tested were substituted into the ternary linear calibration model to calculate the concentrations of glucose, cellobiose and xylose in the sample to be tested.

[0039] Each standard solution, P1–P6, was analyzed using three channels: Table 5 Three-channel detection

[0040] Constructing the data matrix: ; Corresponding concentration matrix: ; Establish three sets of equations and perform regressions on the three channels respectively:

[0041] , , These represent the GOX channel, βGlu+GOX channel, and POX channel, respectively.

[0042] Matrix representation:

[0043] in: ; K: Response coefficient matrix 5.0; : Intercept vector; Coefficient calculation:

[0044] (3) Coefficient of determination Calculation: Calculate separately for each channel : ; : Actual measured absorbance; : Signals predicted by regression models; : The average value of the standard solution signal in this channel; The closer the calculated result is to 1, the better the fit. Generally, this is required. ≥0.99.

[0045] Using glucose concentration, cellobiose concentration, and xylose concentration as independent variables, and three-channel absorbance as the dependent variable, linear regression models were established respectively, yielding the following results:

[0046] in, , and The absorbance values ​​represent the absorbance of the GOX well, β-glucosidase + GOX well, and POX well, respectively; Glc, Cel, and Xyl represent the concentrations of glucose, cellobiose, and xylose in the sample, respectively, all in g / L.

[0047] This can be further written in matrix form: ; That is, the response coefficient matrix K is: ; The intercept vector a is: ; S4: Model fitting results: Calculate the coefficient of determination for each of the three channels. The results are as follows: GOX Channel: ; β-glucosidase + GOX channel: 0.99875; POX channel: 0.99951; The results show that all three-channel models have good linear fit.

[0048] In one implementation, the coefficient of determination of the three-channel regression model All are not less than 0.99.

[0049] Calculation of sample concentration: Solve for the inverse matrix K of the response coefficient matrix. -1 Substitute the measured absorbance of the three channels of the sample into the formula: ; The concentrations of the three sugars in the diluted sample can then be obtained, and multiplied by the dilution factor to obtain the actual concentrations in the original hydrolysate. In this example, the detection results of the hydrolysate to be tested were glucose 8.23 ​​g / L, cellobiose 0.47 g / L, and xylose 0.62 g / L, with a relative error of less than 2.5% compared with the HPLC detection results, verifying the accuracy of the detection method.

[0050] Example 2: The kit described in this invention for the combined detection of glucose, cellobiose, and xylose in cellulose hydrolysate as described in Example 1 contains at least the following individually packaged components: β-glucosidase reagent, glucose oxidase reagent, pyranose oxidase reagent, horseradish peroxidase reagent, ABTS chromogenic substrate reagent, reaction buffer, and multi-component mixed standard solution set.

[0051] This kit is used for the quantitative determination of glucose (Glc), cellobiose (Cel), and xylose (Xyl) in cellulose biomass hydrolysates. It is suitable for monitoring the cellulose hydrolysis process and optimizing biomass conversion processes. High-throughput screening can be performed using a microplate reader (microplate system), or low-cost detection can be performed using a visible light spectrophotometer.

[0052] Simultaneous quantification of three sugars was achieved through multi-enzyme coupling reaction and three-well signal joint modeling.

[0053] ① GOX channel (glucose signal): Glucose oxidase (GOX) specifically catalyzes the production of H2O2 from glucose, which then reacts with the chromogenic substrate under the action of HRP to generate an absorbance signal.

[0054] ②β-glucosidase (βGlu) + GOX channel (glucose + cellobiose): βGlu hydrolyzes cellobiose into glucose (1Cel→2Glc), and then GOX detects the total glucose signal.

[0055] ③POX channel (comprehensive sugar signal): Pyranose oxidase (POX) responds to glucose, xylose and cellobiose, producing a comprehensive signal.

[0056] The following system of linear equations was established based on the absorbance of the three channels: ; After establishing the model through standard solution calibration, the concentrations of the three sugars in the unknown sample can be solved.

[0057] Table 6. Reagent Kit Components

[0058] The kit is compatible with two detection modes: high-throughput batch detection mode based on 96-well plate microplate reader and single-tube detection mode based on cuvette visible light spectrophotometer.

[0059] Example 3: Screening and application verification of different pyranose oxidases To screen for pyranose oxidases suitable for the combined detection system of this invention, two candidate enzymes from *Phanerochaete chrysosporium* and *Trametes versicolor* were selected and named PCPOX and TVPOX, respectively. Expression verification and substrate response analysis were performed using protein expression methods commonly used in the prior art.

[0060] Figure 2Phylogenetic analysis in (a) showed that both PCPOX and TVPOX belong to the fungal pyranose oxidase family, but are located in different branches. The two candidate enzymes were heterologously expressed and purified using *Escherichia coli* BL21, as shown in the figure. Figure 2 The results in (b) show that both PCPOX and TVPOX yielded clear bands of the target protein, indicating that both enzymes can achieve effective expression and purification.

[0061] Figure 2 (a) Phylogenetic tree analysis results of candidate pyranose oxidases and related oxidases, where PCPOX and TVPOX are two candidate pyranose oxidases screened in this embodiment; Figure 2 (b) Recombinant expression of PCPOX and TVPOX in the host and SDS-PAGE analysis results, where M is the protein molecular weight standard, S is the supernatant, P is the precipitate, FT is the permeation buffer, W is the washing buffer, and E is the elution buffer.

[0062] Further comparisons were made of the substrate response characteristics of the two candidate enzymes. Figure 3 The results in (a) indicate that both PCPOX and TVPOX exhibit strong responses to glucose; PCPOX also shows a significant response to xylose and some response to cellobiose, while TVPOX shows relatively weaker responses to both cellobiose and xylose. Kinetic analysis was performed using glucose as a substrate. Figure 3 The results in (b) show that both PCPOX and TVPOX conform to the Michaelis kinetics, and their overall catalytic efficiencies are similar. PCPOX's apparent Vmax is slightly higher than that of TVPOX, indicating that its maximum reaction rate is slightly higher.

[0063] Based on the above results, in the specific embodiments of this invention, the two screened enzyme proteins (PCPOX and TVPOX) both have a sequence length of approximately 600 amino acids. Although the alignment results show that the sequence similarity between the two proteins is only about 39.1%, experiments show that they both retain good pyranose oxidase catalytic activity and can effectively achieve a comprehensive response to target sugars. PCPOX has a wider substrate response range and a slightly higher maximum reaction rate. According to actual detection requirements, *Phanerochaete chrysosporium* was selected as the enzyme component of the POX channel, and it was used in combination with the GOX channel and β-glucosidase + GOX channel to achieve the joint detection of glucose, cellobiose, and xylose.

[0064] Example 4: Simulated Sample Recovery Validation Example To verify the quantitative accuracy and repeatability of the combined detection method of the present invention for mixed samples of known concentrations, three sets of simulated mixed samples were artificially prepared, denoted as G1, G2, and G3. Specifically, G1 contained 0.30 g / L glucose (Glc), 0.02 g / L cellobiose (Cel), and 0.01 g / L xylose; G2 contained 0.60 g / L glucose, 0.05 g / L cellobiose, and 0.03 g / L xylose; and G3 contained 0.90 g / L glucose, 0.03 g / L cellobiose, and 0.07 g / L xylose. Each simulated sample was measured three times in parallel according to the detection steps of the kit of the present invention.

[0065] Among them, the absorbance YB of the GOX well measured three times for sample G1 was 0.2028, 0.2029, and 0.2032, respectively; the absorbance Yc of the βGlu+ GOX well was 0.2391, 0.2392, and 0.2391, respectively; and the absorbance YD of the POX well was 0.2202, 0.2205, and 0.2202, respectively. The YB of sample G2 measured three times was 0.3735, 0.3734, and 0.2032, respectively. The values ​​of Yc for sample G3 were 0.3725, 0.4610, 0.4607, and 0.4606, respectively, and the values ​​of YD were 0.4201, 0.4200, and 0.4205, respectively. The values ​​of YB for sample G3 were 0.5439, 0.5432, and 0.5429, respectively, the values ​​of Yc were 0.5987, 0.5981, and 0.5984, respectively, and the values ​​of YD were 0.6368, 0.6366, and 0.6367, respectively.

[0066] Substitute the above three-channel absorbance data into the regression model established in Example 1:

[0067] The measured concentrations of glucose, cellobiose, and xylose in each simulated sample were calculated, and the average value, recovery rate, and relative standard deviation (RSD) were further calculated. The recovery rate was calculated using the following formula: ; The relative standard deviation (RSD) is calculated using the following formula: ; Table 7. Determination results, recoveries, and relative standard deviations of each component in the simulated samples.

[0068] As shown in Table 7, the method of this invention can achieve quantitative detection of glucose, cellobiose, and xylose in simulated samples. The average values ​​of glucose and cellobiose were basically consistent with the theoretical values, with a recovery rate close to 100% and a low RSD, indicating that this invention has good accuracy and repeatability for glucose and cellobiose. The recovery rates of xylose in samples G2 and G3 were 100.88% and 100.61%, respectively, showing good repeatability. Under the low concentration conditions of G1, the xylose recovery rate was 95.51%, with a relatively high RSD, indicating that the measurement fluctuation increases slightly when the xylose concentration is low, but it is still within an acceptable range overall. In summary, the three-channel combined detection method established in this invention can simultaneously quantify glucose, cellobiose, and xylose, with good accuracy and repeatability.

[0069] Example 5: Detection of Real Cellulose Hydrolysate To verify the applicability and quantitative accuracy of the glucose / cellobiose / xylose combined detection kit of this invention in real cellulose hydrolysate samples, two real cellulose hydrolysate samples were selected, designated as Sample 1 and Sample 2, for detection and analysis. Sample pretreatment was performed according to the conditions described in the instructions: the sample was centrifuged at 8000 g for 10 min, and the supernatant was filtered through a 0.22 μm aqueous filter membrane. If necessary, appropriate dilution with 50 mM phosphate buffer (pH 5.0) was performed to ensure the detection signal fell within the linear range of the standard curve.

[0070] Cellobiose, glucose, and xylose in samples 1 and 2 were compared and detected using liquid chromatography (HPLC). After enzymatic digestion, the reaction solution was centrifuged at 8000×g for 10 min, and the supernatant was filtered through a 0.22 μm filter before analysis. Sugar content was determined using HPLC with an Aminex HPX-87P column and a suitable guard column. A refractive index detector (RID) was used. The mobile phase was HPLC-grade ultrapure water (filtered and degassed at 0.2 μm), the flow rate was 0.6 mL / min, the column temperature was 80 ℃, the injection volume was 10 μL, and the acquisition time was 20 min. Glucose content was quantified using the external standard method; standards and samples were analyzed under the same conditions. The HPLC chromatograms of samples 1 and 2 are shown below. Figure 4 .

[0071] Table 8 Comparison of detection results of enzymatic reagent kit and HPLC method for real hydrolysate samples

[0072] The kit of this invention was used to perform a three-channel enzymatic assay on the two real samples. Specifically, the reaction was carried out in the GOX well, the β-glucosidase + GOX well, and the POX well, respectively, and the absorbance was measured at 405 nm. The obtained three-channel absorbance data were then substituted into the three-channel regression model established in Example 1 to calculate the concentrations of glucose, cellobiose, and xylose in the sample.

[0073] Comparison of the enzymatic detection results and the liquid chromatography detection results of this invention shows that the determination results of the three target sugar components in real cellulose hydrolysate by the two methods are generally consistent. Using the liquid chromatography results as a reference, the relative deviations of cellobiose, glucose, and xylose in sample 1 are approximately 0.98%, 0.01%, and 3.80%, respectively; the relative deviations of cellobiose, glucose, and xylose in sample 2 are approximately 0.79%, 1.29%, and 1.48%, respectively. The relative deviations of each component are all less than 5%, indicating that the three-channel combined detection method established in this invention has good quantitative accuracy in real cellulose hydrolysate samples.

[0074] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for the joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate, characterized in that, Includes the following steps: S1: Three types of detection channels are set up: glucose oxidase detection channel that specifically responds to glucose component, β-glucosidase-glucose oxidase combined detection channel that responds to total signal of glucose and cellobiose, and pyranose oxidase detection channel that responds to comprehensive signal of glucose, cellobiose and xylose components. S2: The cellulose hydrolysate sample to be tested and the multi-component gradient standard solution were subjected to enzymatic colorimetric reactions in three types of detection channels, and the absorbance value of each detection channel was measured. S3: Construct a ternary linear calibration model based on the absorbance data of the three channels of the multi-component gradient standard solution. Substitute the absorbance values ​​of the three channels of the sample to be tested into the ternary linear calibration model to calculate the concentrations of glucose, cellobiose and xylose in the sample to be tested.

2. The method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to claim 1, characterized in that, It also includes the following pretreatment steps for the sample to be tested: S11: Centrifuge the cellulose hydrolysate to be tested at 8000g for 10min and take the clear upper layer. S12: Filter the obtained clarified liquid through an aqueous filter membrane with a pore size of 0.22μm to remove insoluble impurities; S13: Dilute the filtered sample with 50mM, pH 5.0 phosphate buffer to ensure that the detection signal of the diluted sample falls within the linear response range of the multi-component gradient standard solution.

3. The method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to claim 1, characterized in that, The pyranose oxidase in the pyranose oxidase detection channel is derived from the pyranose oxidase of Phanerochaete chrysospora. As the core enzyme component of the pyranose oxidase detection channel, it works in conjunction with the enzyme reagents of the glucose oxidase detection channel and the β-glucosidase-glucose oxidase co-detection channel to achieve the joint detection of the three types of sugars.

4. The method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to claim 1, characterized in that, The reaction steps for the β-glucosidase-glucose oxidase coupled detection channel in step S2 are as follows: S21: Add β-glucosidase reagent to the reaction system and incubate at a constant temperature for a certain period of time to completely hydrolyze the cellobiose in the reaction system into glucose. S22: Add horseradish peroxidase reagent, ABTS chromogenic substrate reagent, and glucose oxidase reagent sequentially to the hydrolyzed reaction system, and incubate in the dark for a certain period of time to complete the chromogenic reaction.

5. The method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to claim 1, characterized in that: The reaction steps for the glucose oxidase detection channel and the pyranose oxidase detection channel in step S2 are as follows: Add the sample / standard solution, reaction buffer, horseradish peroxidase reagent, ABTS chromogenic substrate reagent, and the corresponding oxidase reagent to the reaction system in sequence: add glucose oxidase to the glucose oxidase detection channel and add pyranose oxidase to the pyranose oxidase detection channel, and incubate in the dark for a certain period of time to complete the colorimetric reaction.

6. The method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to claim 1, characterized in that: The ternary linear correction model in step S3 is expressed as follows: ； Where Y B Y C Y D The measured absorbance values ​​are for the glucose oxidase detection channel, the β-glucosidase-glucose oxidase combined detection channel, and the pyranose oxidase detection channel, respectively; Glc, Cel, and Xyl represent the concentrations of glucose, cellobiose, and xylose, respectively; b1, b2, b3, c1, c2, c3, d1, d2, d3 are the response coefficients of the three channels to the three sugar components, which together form a 3×3 response coefficient matrix K; a1, a2, and a3 are the intercept values ​​of the linear regression of the three channels, and the coefficient of determination for linear fitting of each channel is: ； Where: n: the total number of multi-component gradient standard solutions; : The measured absorbance value of the i-th standard solution; The absorbance value of the i-th standard solution predicted by the linear regression equation; The average absorbance of all standard solutions in their respective channels; the coefficients of determination for linear fitting of the three types of detection channels must all meet the following requirements. ≥0.

99.

7. The method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to claim 6, characterized in that: The formula for determining the concentration of the sample to be tested in step S3 is as follows: ； in: Response coefficient matrix The inverse matrix, , , These are the measured absorbance values ​​of the sample under test in the glucose oxidase detection channel, the β-glucosidase-glucose oxidase combined detection channel, and the pyranose oxidase detection channel, respectively. The concentrations of glucose, cellobiose, and xylose in the sample under test can be directly obtained through the above matrix operation.

8. A kit for implementing the method for joint detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to any one of claims 1 to 7, characterized in that, The kit contains at least the following individually packaged components: β-glucosidase reagent, glucose oxidase reagent, pyranose oxidase reagent, horseradish peroxidase reagent, ABTS chromogenic substrate reagent, reaction buffer, and multi-component mixed standard solution set.

9. The kit for the combined detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to claim 8, characterized in that, The multi-component mixed standard solution group contains six gradient mixed standard solutions, P1 to P6, with the following concentration ranges: glucose 0-1 g / L, cellobiose 0-0.08 g / L, and xylose 0-0.08 g / L. Among them, P1 is a blank standard solution without the three target sugars, and P2 to P6 are mixed standard solutions with increasing concentration gradients of the three target sugars.

10. The kit for the combined detection of glucose, cellobiose, and xylose in cellulose hydrolysate according to claim 8, characterized in that, The kit is compatible with two detection modes: high-throughput batch detection mode based on 96-well plate microplate reader and single-tube detection mode based on cuvette visible light spectrophotometer.

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