Method for determining the content of reducing sugars in a hydrophilic deep eutectic solvent system

Abstract

The application belongs to the technical field of quantitative analysis of reducing sugar, and discloses a method for determining the content of reducing sugar in a hydrophilic deep eutectic solvent system, comprising the following steps: preparing 3,5-dinitrosalicylic acid DNS reagent; preparing hydrophilic deep eutectic solvent hi(NA) DES; preparing a reducing sugar RS-hi(NA) DES stock solution; preparing a reducing sugar RS-hi(NA) DES standard solution; preparing a reducing sugar RS-hi(NA) DES test solution or preparing a cellulase hydrolysate based on hi(NA) DES; water-disintegrating the reducing sugar solution RS-hi(NA) DES or alkali-disintegrating the reducing sugar solution RS-hi(NA) DES; water-disintegrating or alkali-disintegrating the hydrophilic deep eutectic solvent hi(NA) DES; and determining the content of reducing sugar in the disintegration solution. The determination method has a wide application range, high measurement accuracy, good sensitivity, good reproducibility, low sample consumption, green safety, and is suitable for all hydrophilic deep eutectic solvent systems.

Description

Technical Field

[0001] This invention belongs to the field of quantitative analysis technology of reducing sugars, and particularly relates to a method for determining the content of reducing sugars in a hydrophilic eutectic solvent system. Background Technology

[0002] Photosynthesis is "the most important chemical reaction on Earth," the largest process on Earth converting solar energy into storable chemical energy, and also the largest process synthesizing organic matter from inorganic matter and releasing oxygen from water. It is no exaggeration to say that photosynthesis created the material foundation and thriving life on Earth today. Inspired by photosynthesis, people are striving to build "artificial photosynthesis" systems, including algal hydrogen production, and "reverse photosynthesis" systems, including biomass refining, to address pressing issues such as resource depletion and environmental pollution. In all these processes, sugars, primarily reducing sugars, are absolutely central; they are both products and raw materials, connecting the entire process of photosynthesis and reverse photosynthesis. Therefore, in-situ determination of reducing sugars is the foundation, focus, and core of research on these processes.

[0003] Reducing sugars are a subclass of sugars, often further subdivided into aldoses and ketoses due to the presence of hemiacetal or ketal groups. The earliest determinations of reducing sugars date back to the mid-1800s and early 1900s. The famous Fehling's reagent, Benedict's reagent, and Torrance's reagent were developed during this period. They are all based on the same principle: the hemiacetal or hemiketal derived from the reducing sugar is first isomerized in alkaline solution to the intermediate enediol, which then undergoes a single-electron reduction reaction with a transition metal complex and is subsequently oxidized to a sugar acid. These reactions usually produce strong colors and can be measured in spectrophotometers or even colorimeters. However, these early reagents could only identify the presence of reducing sugars, not quantify them. Therefore, many methods for the quantitative determination of reducing sugars were subsequently developed. The most widely used methods are the DNS method and the Nelson-Somogyi method, which use 3,5-dinitrosalicylic acid (DNS) and Cu(II) (CuSO4) as oxidizing agents, respectively. Ultimately, this method avoided the use of toxic arsenic compounds AsH2O. 15 Na2O 11 With the adoption of DNS, it gained more users and achieved the widest application.

[0004] Currently, the latest trend in research on both artificial and reverse photosynthesis is the upgrading and transformation of technologies through the introduction of green solvents, especially the latest deep eutectic solvents (DES). DES are homogeneous and ionic solvents synthesized non-covalently through intermolecular hydrogen bonds between binary components (e.g., halide ions and hydrogen donor moieties). Abbott et al. reported the first DES (choline chloride:urea mixture) in 2003. Natural deep eutectic solvents (NADES) are both a subset of DES and an all-natural version of DES, with all their binary or ternary components derived from natural sources, including sugars, sugar alcohols, polyols, amino acids, organic acids, and organic bases. An unknown quantity of NADES solvents actually exist in nature, replacing aqueous and oily liquid phases to form the "third liquid phase" in organisms. Due to its advantages such as being safer (non-toxic), greener, and having better biocompatibility, since its introduction and patenting in 2011, research reports on its application in food, agrochemicals, cosmetics, and pharmaceuticals have shown a trend of doubling year by year. Figure 1 ).

[0005] Taking biomass refining, a representative process of reverse photosynthesis, as an example, recent research has confirmed that incorporating (NA)DES into the refining process of lignocellulose biomass clearly brings more exciting advantages: namely, it is green, sustainable, has high solids content, high lignin removal rate, and integrated bioprocessing (CBP). Related research has received increasing attention over the past five years, mainly focusing on introducing (NA)DES into pretreatment, fractionation, extraction, conversion, and hydrolysis steps, and many reviews have commented on these topics. However, despite these advances, the biggest obstacle to the full application of (NA)DES in biomass refining—the in-situ determination of reducing sugars in pure (NA)DES—remains unresolved. In other words, an accurate, unified, and standardized method has not yet been established to determine the reducing sugar content in anhydrous pure (NA)DES solvent and to apply it to various procedures in biomass refining processes involving reducing sugars and (NA)DES. Figure 2 ).

[0006] Currently, traditional techniques (mainly HPLC and DNS methods) still have the following problems and defects when used to determine the reducing sugar content in eutectic solvents: (1) Over-dilution of (NA)DES solvent (usually 100 times or more) for column safety will result in excessively low sugar concentration or even undetectable sugar concentration; (2) HPLC can only detect soluble sugars and cannot determine their reactivity—that is, reducing power; (3) There is currently no method to determine the reducing sugar content in anhydrous pure (NA)DES solvent in situ. Previous reports have all involved DNS testing in aqueous (NA)DES media, which greatly limits the application range of (NA)DES solvent; (4) In acidic (NA)DES (such as choline chloride: lactic acid) solvents, DNS reagents still fail even when water is added. This is a new phenomenon reported for the first time in this invention. Summary of the Invention

[0007] To address the gaps and shortcomings in existing quantitative analysis techniques for reducing sugars in the field of eutectic solvents, this invention aims to provide a method for in-situ determination of reducing sugar content in anhydrous, pure, hydrophilic eutectic solvent system hi(NA)DES. The method provided by this invention adds two basic steps (water disintegration and pH alkalization) to the traditional DNS standard procedure (referred to as the "DNS method") to adapt to the new characteristics of the hydrophilic eutectic solvent system hi(NA)DES, which is a non-aqueous solvent system that can be switched to an aqueous phase. The former overcomes the difficulties of converting from a non-aqueous phase to an aqueous phase, as well as the problem of decreased measurement accuracy due to the high viscosity of hi(NA)DES. The latter solves the problem of DNS reagent failure caused by the high acidity of some hi(NA)DES. This method has a wide range of applications, high measurement accuracy, good sensitivity, and is applicable to all hydrophilic eutectic solvent systems hi(NA)DES.

[0008] To achieve this objective, the present invention employs the following technical solution: The present invention provides a method for determining the reducing sugar content in a hydrophilic eutectic solvent system hi(NA)DES. The method, the DNS method, involves preparing and synthesizing DNS reagents, a hydrophilic eutectic solvent, a reducing sugar-hydrophilic eutectic solvent stock solution, and a reducing sugar-hydrophilic eutectic solvent standard solution. Water or alkaline solution is used as a phase change agent. The reducing sugar test solution or cellulose enzymatic hydrolysate based on the hydrophilic eutectic solvent is disintegrated by adding water or alkaline solution. The DNS method is used to determine the standard curves of various reducing sugars in hiNADES solvent and to track the enzymatic hydrolysis kinetics curve of microcrystalline cellulose (MCC) in hiNADES.

[0009] Furthermore, the determination method includes the following steps:

[0010] Step 1: Prepare 3,5-dinitrosalicylic acid (DNS) reagent. The purpose of this step is to prepare a colorimetric reagent that can react quantitatively with reducing sugars.

[0011] Step 2: Prepare the hydrophilic eutectic solvent hi(NA)DES. The purpose of this step is to prepare a hydrophilic eutectic solvent that can switch between anhydrous and aqueous states.

[0012] Step 3: Prepare reducing sugar stock solution RS-hi(NA)DES stock The purpose of this step is to create an anhydrous, pure binary system of "reducing sugar - eutectic solvent";

[0013] Step 4: Prepare reducing sugar standard solution RS-hi(NA)DES stand The purpose of this step is to prepare the standard solution needed for plotting the standard curve of reducing sugar concentration;

[0014] Step 5: Prepare the reducing sugar test solution RS-hi(NA)DES based on hi(NA)DES. test The purpose of this step is to simulate or collect a real solution of "reducing sugar-eutectic solvent" at an unknown concentration;

[0015] Step 6: Prepare the cellulose hydrolysate RS-hi(NA)DES based on hi(NA)DES. cellulase The purpose of this step is to hydrolyze cellulose in a "cellulase-eutectic solvent" system to prepare an enzymatic hydrolysis solution mainly composed of "reducing sugar-eutectic solvent".

[0016] Step 7: Disintegrate the reducing sugar solution with water or alkali. The purpose of this step is to use water or alkali as a phase inversion agent to transform the eutectic solution of the reducing sugar into an aqueous solution so that it can undergo an oxidation reaction with the aqueous colorimetric reagent—DNS reagent.

[0017] Step 8: Perform hydrophilic eutectic solvent hi(NA)DES by adding water or alkali. The purpose of this step is to use water or alkali as a phase transformation agent to transform the eutectic phase of hi(NA)DES into the aqueous phase in order to determine the colorimetric blank value of hi(NA)DES itself.

[0018] Step 9: Mix the reducing sugar disintegration solution, the solvent blank disintegration solution, and the DNS reagent. The purpose of this step is to quantify the components involved in the colorimetric reaction. The quantitative parameters include the volume of the reducing sugar disintegration solution, the volume of the solvent blank disintegration solution, the volume of the DNS reagent, and the total volume after mixing.

[0019] Step 10: The mixed solution is subjected to a colorimetric reaction in a boiling water bath. The purpose of this step is to conduct parallel reactions under the same conditions for all mixed solution samples and to maximize the reaction progress in a boiling water bath.

[0020] Step 11: Dilute the colorimetric solution. The purpose of this step is to dilute the solution by a certain ratio to increase the absorbance A of the diluted solution. 540nm It falls within the range of 0.1 to 1.0A (confidence interval);

[0021] Step 12: Measure the absorbance of the colorimetric solution using a UV-Vis spectrophotometer. The purpose of this step is to determine the final absorbance value A of the colorimetric solution. 540nm ;

[0022] Step thirteen, calculate the reducing sugar content. The purpose of this step is to determine the reducing sugar content by measuring the absorbance value A. 540nm A linear standard curve was constructed that corresponds one-to-one with the absolute amount of reducing sugar (mg / mL), and the amount of reducing sugar (mg / mL) in the sample solution was calculated using this standard curve.

[0023] The method for determining reducing sugar content in hydrophilic eutectic solvent systems, implemented through the above steps, namely the "DNS" method, is currently the only accurate, unified, and standardized method for in-situ determination of reducing sugar content in anhydrous pure hi(NA)DES solvent. It overcomes the gaps and deficiencies in existing domestic and international quantitative analysis techniques for reducing sugars in the field of eutectic solvents, expanding the application scope of reducing sugar testing and providing a new analytical tool for the application research of eutectic solvents. The method for determining reducing sugar content in hydrophilic eutectic solvent systems provided by this invention can ultimately be applied to the research and application scenarios of various chemical events related to "hi(NA)DES and reducing sugars".

[0024] In addition to the above technical steps, the present invention uses the following general definitions: ① Eutectic solvent DES is a binary mixture composed of two specific components (one of which is ionic) that remains in the liquid phase at 25°C; ② The concept of natural eutectic solvent NADES mainly involves the following: as a hydrogen bond donor in a binary mixture, the component is mainly derived from any of many naturally sourced substances, which form eutectic solvents through hydrogen bonds with choline chloride or other naturally sourced hydrogen bond acceptor components that are themselves natural products; ③ Hydrophilic eutectic solvent hi(NA)DES includes two solvent systems: hydrophilic eutectic solvent hiDES and hydrophilic natural eutectic solvent hiNADES, and is a subset of the hydrophilic portion of eutectic solvents and natural eutectic solvents.

[0025] Furthermore, the DNS method, DNS' method, and DNS'" method mentioned in this invention are three different methods for determining reducing sugars, and their detailed descriptions and definitions are as follows: ① The DNS method refers to the most widely used method for determining reducing sugars, developed by Miller in 1959 and recommended by the International Union of Pure and Applied Chemistry (IUPAC) and the National Renewable Energy Laboratory (NREL). However, this method is limited to aqueous solvent systems and cannot be applied to the anhydrous eutectic solvent system involved in this patent; ② The DNS' method is an improved method of DNS. The improvement idea is to dissolve the DNS reagent components in a eutectic solvent to prepare a DNS-eutectic solvent reagent, and then use the DNS-eutectic solvent reagent to determine the reducing sugar content. The unlimited miscibility of raw sugar-eutectic solvent solution was used to achieve the subsequent determination of reducing sugar content. Unfortunately, the DNS-eutectic solvent reagent was not successfully prepared. The failure process and reasons are detailed in Table 3. ③ The DNS method is also an improved method of DNS. The improvement idea is different from the DNS' method. In this method, the components and preparation process of DNS reagent remain unchanged. Instead, two basic steps (water disintegration and pH alkalization) are added to the standard procedure of DNS method. This realizes the transformation of various reducing sugar-eutectic solvent solutions to various reducing sugar solutions dissolved in hydrophilic natural eutectic solvents from the non-aqueous phase to the aqueous phase. It also solves the problem of DNS reagent failure caused by excessive acidity of some hydrophilic natural eutectic solvents. See Table 3 for details.

[0026] Furthermore, the preparation of the 3,5-dinitrosalicylic acid (DNS) reagent in step one includes:

[0027] (1) Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to 400mL of distilled water. Heat and stir moderately until completely dissolved. The solution is grass green. Then add 7.0g of NaOH and stir at room temperature until completely dissolved. The solution is orange red. In this step, the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially.

[0028] (2) Add 108g of Rochelle salt (sodium potassium tartrate tetrahydrate), 2.7mL of liquid phenol melted at 50℃ and 2.9g of sodium metabisulfite to the orange-red solution, stir thoroughly and dissolve all the added components;

[0029] (3) After adding the last three components, a large number of suspended particles will appear in the solution. After stirring continuously for more than 1 hour, all the suspended particles will disappear and a clear orange-red solution will be formed. Add distilled water to make up to 500 mL, store in a brown bottle away from light, and use after one week. The shelf life is 6 months.

[0030] Furthermore, the preparation of the hydrophilic eutectic solvent hi(NA)DES in step two includes:

[0031] (1) Solvent synthesis molar ratio: Taking two hi(NA)DES based on choline chloride (ChCl) as examples, one is choline chloride-glycerol (ChCl-Gly) solvent and the other is choline chloride-lactic acid (ChCl-LA) solvent. Both solvents use choline chloride as hydrogen bond acceptor HBA, while the hydrogen bond donors HBD are glycerol (Gly) and lactic acid (LA), respectively; the molar ratio between HBA and HBD in both hi(NA)DES solvents is 1:2.

[0032] (2) Solvent synthesis steps: Add the two components (HBA and HBD) directly into a round-bottom flask at a molar ratio of 1:2, and heat and stir in an oil bath at 60-65°C, avoiding the addition of water as much as possible, until the two components are transformed into a homogeneous transparent liquid. Stop stirring and heating. If it is still liquid when cooled to room temperature, it means that the preparation is successful.

[0033] (3) Solvent components: For the DES system of eutectic solvents, common hydrogen bond acceptors include choline chloride (ChCl) and its analogues (Table 1 lists the synthesis parameters of some hydrophilic eutectic solvents with choline chloride), onium salts, metal salts, metal chlorides, etc., and common hydrogen bond donors include urea and its analogues, sugars, sugar alcohols, polyols (such as glycerol), amino acids, organic acids (such as lactic acid), etc. For the NADES system of natural eutectic solvents, the hydrogen bond acceptors and hydrogen bond donors used must be natural products, usually limited to organic compounds of natural origin produced from primary or secondary metabolic pathways, including but not limited to the types of components constituting DES. For the hi(NA)DES system of hydrophilic eutectic solvents, based on the above solvent components, only components that can make the prepared solvent hydrophilic are selected.

[0034] (4) It should be particularly noted that, since there are numerous solvents that meet the definition of hydrophilic eutectic solvent hi(NA)DES (currently exceeding 100 and continuing to increase in the future), it is impossible to list them all. Therefore, this invention only takes two of them (i.e., ChCl_Gly and ChCl_LA) as examples, which does not mean that the technical solution described in this invention is only applicable to these two solvents. On the contrary, whether now or in the future, any hydrophilic eutectic solvent hi(NA)DES is applicable to the technical solution described in this invention.

[0035] Table 1. Synthesis parameters of some choline chloride hydrophilic eutectic solvents

[0036] name HBA HBD <![CDATA[mol HBA / mol HBD ]]> Synthesis temperature / °C ChCl_Urea choline chloride urea 1:2 60-65 ChCl_LA(1:2) choline chloride lactic acid 1:2 60-65 ChCl_LA(1:5) choline chloride lactic acid 1:5 60-65 ChCl_LA(1:10) choline chloride lactic acid 1:10 60-65 ChCl_LA(1:15) choline chloride lactic acid 1:15 60-65 ChCl_Gly choline chloride glycerin 1:2 60-65 ChCl_MA choline chloride malic acid 1:1 60-65 ChCl_Xyl choline chloride Xylitol 1:1 60-65 ChCl_EG choline chloride Ethylene glycol 1:2 60-65 ChCl_CA choline chloride Citric acid 1:1 60-65 ChCl_AA choline chloride Acetic acid 1:2 60-65 ChCl_OA choline chloride oxalic acid 1:2 60-65

[0037] Furthermore, in step three, the reducing sugar stock solution RS-hi(NA)DES is prepared. stock include:

[0038] (1) 50 mL of prepared hi(NA)DES (such as ChCl_Gly and ChCl_LA) is pre-filled into an Erlenmeyer flask, and 1 g of reducing sugar is weighed and poured into it. The mixture is stirred and dissolved at room temperature for more than 24 h until the solid is completely dissolved and the solution becomes transparent, thus obtaining a series of RS-hi(NA)DES stock solutions with a concentration of 20 mg / mL. The reducing sugar includes glucose, xylose, fructose, galactose, mannose, rhamnose, fucose, cellobiose, maltose and lactose.

[0039] Furthermore, in step four, the reducing sugar standard solution RS-hi(NA)DES is prepared. stand include:

[0040] (1) RS-hi(NA)DES standard solutions are obtained by measuring a certain amount of RS-hi(NA)DES stock solution and diluting it with the corresponding hi(NA)DES solvent in a volumetric flask according to a certain ratio; wherein, the concentration range of the RS-hi(NA)DES standard solution is 0.5–4.0 mg / mL.

[0041] Furthermore, in step five, the preparation of the reducing sugar test solution RS-hi(NA)DES based on hi(NA)DES is described. test include:

[0042] (1) Weigh a certain amount of reducing sugar into an Erlenmeyer flask, pour in a certain volume of hi(NA)DES (such as ChCl_Gly and ChCl_LA), stir and dissolve at room temperature for more than 24 hours. If it is completely dissolved, it can be used directly; if there is some undissolved reducing sugar solid, filter it through an organic membrane under vacuum and collect the liquid part for later use.

[0043] Furthermore, in step six, the preparation of the cellulose hydrolysate RS-hi(NA)DES based on hi(NA)DES is described. cellulase include:

[0044] (1) Weigh 0.5g of cellulose raw material into a 25mL Erlenmeyer flask, and add 10mL of hi(NA)DES solvent; place the Erlenmeyer flask containing the cellulose raw material and hi(NA)DES solvent into a constant temperature water bath shaker, and preheat for 30min at 50-70℃ and 120-160rpm; wherein, the cellulose raw material includes, but is not limited to, microcrystalline cellulose. PH-101, wherein the hi(NA)DES solvent includes, but is not limited to, ChCl_Gly and ChCl_LA;

[0045] (2) After preheating, add cellulase to the conical flask at a loading rate of 60-160 FPU cellulase / g cellulose; react at 50-70℃ and 120-160 rpm for 0-48 hours; wherein the cellulase includes, but is not limited to, NovozymeCelluclast1.5L and NovozymeCtec2.

[0046] (3) Take the enzymatic hydrolysate at different reaction times as RS-hi(NA)DES cellulase When the reaction time is reached, remove the corresponding conical flask, inactivate it in ice water, let it stand until the solid settles at the bottom of the conical flask, then take the supernatant, filter it and use it for later use.

[0047] Furthermore, step seven, which involves the hydrolysis of the reducing sugar solution by adding water or alkali, includes:

[0048] (1) Disintegration with water: Add reducing sugar solution (including RS-hi(NA)DES) to a 10mL graduated cylinder. stand RS-hi(NA)DES test and RS-hi(NA)DES cellulase Add water to the 1 mL mark, then add distilled water to the 4 mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the eutectic phase of RS-hi(NA)DES to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution. This step is applicable to all reducing sugar solutions prepared with non-acidic hi(NA)DES solvent.

[0049] (2) Disintegration with Alkaline Solution: The oxidation reaction between DNS reagent and reducing sugars can only occur under alkaline conditions. When preparing reducing sugar solutions with acidic hi(NA)DES solvent, an alkaline solution is used for disintegration to make the disintegrating solution alkaline. The alkaline solution includes, but is not limited to, NaOH solution. The optimal molar concentration of the alkaline solution is determined by simultaneously meeting the following two criteria: ① The disintegrating solution formed after adding this concentration of alkaline solution can undergo an oxidation reaction with DNS reagent without precipitation, and the colorimetric test tube turns purple-red; ② The absorbance of the disintegrating solution formed after adding this concentration of alkaline solution should not be too high, and the absorbance value should be relatively low compared to other concentrations. This step is applicable to all reducing sugar solutions prepared with acidic hi(NA)DES solvent.

[0050] Furthermore, step eight, involving the hydrophilic eutectic solvent hi(NA)DES, includes either hydrolysis or alkali disintegration.

[0051] (1) Add the hydrophilic eutectic solvent hi(NA)DES to a 10mL graduated cylinder to the 1mL mark. When hi(NA)DES is ChCl_Gly, add distilled water to the 4mL mark. When hi(NA)DES is ChCl_LA, add 2M NaOH solution to the 4mL mark. Shake the graduated cylinder thoroughly to allow the water (or NaOH solution) to impact the non-aqueous phase interface of the sample, causing the eutectic phase of RS-hi(NA)DES to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution. This step is applicable to all hi(NA)DES solvents.

[0052] (2) The hydrophilic eutectic solvent hi(NA)DES applicable to this step is not limited to ChCl_Gly and ChCl_LA. When other hydrophilic eutectic solvent hi(NA)DES is used, either water disintegration or alkali disintegration can be selected, and the selection criteria are the same as in step seven.

[0053] Furthermore, step nine, mixing the reducing sugar disintegrating solution, the solvent blank disintegrating solution, and the DNS reagent, includes:

[0054] (1) Mixing reducing sugar disintegration solution with DNS reagent: Transfer 1.5 mL of reducing sugar disintegration solution (standard solution disintegration solution / test solution disintegration solution / enzyme hydrolysate disintegration solution) into a 10 mL test tube, then add 3 mL of DNS reagent and mix well. The total volume of the solution after mixing is 4.5 mL.

[0055] (2) Mixing the solvent blank disintegration solution with DNS reagent: Transfer 1.5 mL of solvent blank disintegration solution into a 10 mL test tube, then add 3 mL of DNS reagent and mix well. The total volume of the mixed solution is 4.5 mL.

[0056] Furthermore, step ten, which involves conducting a colorimetric reaction of the mixed solution in a boiling water bath, includes:

[0057] (1) Insert all test tubes into the test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the mixed solution in the test tubes. The blank solution and standard / test solution / enzyme digest should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature.

[0058] Furthermore, the diluent for color development in step eleven includes:

[0059] (1) Dilute all test tubes (blank, standard / test / enzymatic digestion) with distilled water. The dilution ratio should be explored based on the actual situation, but the principle is that the A value of the colorimetric solution should be diluted with distilled water. 540nm The dilution ratio is 0.1–1.0A. The dilution ratio of blank test tubes should be consistent with that of standard / test / enzyme digestion test tubes.

[0060] Furthermore, the determination of the absorbance of the colorimetric solution in the UV-Vis spectrophotometer in step twelve includes:

[0061] (1) Pour the diluted colorimetric solution into a glass cuvette, set the wavelength of the UV-Vis spectrophotometer to 540 nm, put the cuvette into the cuvette slot and measure the absorbance value. The colorimetric absorbance value of the standard / test / enzyme digestion tube is the absorbance difference after deducting the solvent blank.

[0062] Furthermore, the calculation of reducing sugar content in step thirteen includes:

[0063] (1) The absolute amount of reducing sugar (mg / mL) relative to A 540nm Plot a linear standard curve. The data of the standard curve should be as close to a straight line as possible, and the correlation coefficient of the fitted line should be in the range of 0.9900-1.0000.

[0064] (2) The reducing sugar content of each test or enzymatic hydrolysis tube was calculated using the linear equation obtained after fitting the standard curve.

[0065] Furthermore, the above thirteen steps can be divided into the following five parts according to their functions: The first part includes steps one and two, which provide the reagents and solvents required for the synthesis of the entire technical solution; the second part includes steps three, four, five, and six, which provide the various reducing sugar solutions RS-hi(NA)DES based on hi(NA)DES that need to be prepared for the entire technical solution, wherein steps three and four respectively prepare the reducing sugar stock solutions RS-hi(NA)DES. stock and reducing sugar standard solution RS-hi(NA)DES stand This was used to plot the glucose absorbance standard curve, while steps five and six were used to prepare the reducing sugar test solution RS-hi(NA)DES. test and cellulose enzymatic hydrolysate RS-hi(NA)DES cellulaseThe first part corresponds to the determination of reducing sugar content in practical application scenarios such as reducing sugar solubility experiments and cellulose enzymatic hydrolysis experiments; the second part includes step seven, which is the necessary phase transition step for successful quantification of reducing sugar content in all hi(NA)DES-based reducing sugar solutions. The solution can be either hydrolyzed or alkali-disintegrated. When the reducing sugar solution is prepared with a non-acidic hi(NA)DES solvent, the hydrolyzed disintegration step is performed; when the reducing sugar solution is prepared with an acidic hi(NA)DES solvent, the alkali-disintegration step is performed. The alkaline disintegration step; Part Four includes step eight, which is a phase transition step performed to eliminate the influence of the absorbance value of the hi(NA)DES solvent itself on the colorimetric value of the reducing sugar. Hi(NA)DES solvent blank disintegration with water and disintegration with alkaline solution can be selected, and the selection criteria are the same as in step seven; Part Five includes steps nine, ten, eleven, twelve, and thirteen, which are used to determine the absorbance values ​​of the reducing sugar disintegration solution and the solvent blank disintegration solution, and finally determine the content of reducing sugar. The functions of steps nine to thirteen are, in order, mixing, color development, dilution, measuring absorbance, and calculating the reducing sugar content.

[0066] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0067] First, addressing the technical problems existing in the prior art and the difficulty in solving them, this paper closely analyzes, in conjunction with the technical solution to be protected by this invention and the results and data obtained during the research and development process, how the technical solution of this invention solves the technical problems, and the inventive technical effects brought about by solving these problems. The specific description is as follows:

[0068] (1) This invention addresses the biggest obstacle to the full application of eutectic solvents in biomass refining: the in-situ determination of reducing sugars in pure anhydrous eutectic solvents. This patent establishes a novel DNS method. This method involves preparing and synthesizing DNS reagents, a hydrophilic eutectic solvent, a reducing sugar stock solution, and a reducing sugar standard solution. Using water or alkali as a phase change agent, the reducing sugar test solution based on the hydrophilic eutectic solvent (hiNADES) or the cellulase hydrolysate (containing reducing sugars) is disintegrated by adding water or alkali. This achieves effective mixing and color development of the reducing sugar disintegration solution and the DNS reagent, avoiding the technical difficulties of the high viscosity of the eutectic solvent affecting the measurement accuracy and precision, and the high acidity of some eutectic solvents causing DNS failure and color development. Furthermore, based on this, the DNS method successfully determines various reducing sugars in hiNADES. The standard curve in the ES solvent was used to track the enzymatic hydrolysis kinetics of microcrystalline cellulose (MCC) in hiNADES, thus successfully extending the DNS method to applications such as the determination of reducing sugar solubility in hiNADES solvent, the determination of cellulase activity (FPU) on filter paper in hiNADES solvent, the determination of endoglucanase (EG) activity (CMCU) in hiNADES solvent, the determination of total reducing sugar in extracts from food raw materials (or food residues) extracted with hi(NA)DES solvent, and the determination of total reducing sugar in pretreatment solutions of lignocellulosic biomass pretreated with hi(NA)DES solvent. Ultimately, the method for determining reducing sugar content in hydrophilic eutectic solvent systems provided by this invention can be widely applied to the research and application of various chemical events related to "hi(NA)DES and reducing sugars".

[0069] (2) The inventive technical effects of this invention:

[0070] The method for determining the reducing sugar content in a hydrophilic eutectic solvent system provided by this invention, namely the "DNS" method, is currently the only method that can be used to determine the reducing sugar content in situ in a pure hiNADES solvent system, thus possessing a certain degree of irreplaceability. The effectiveness and applicability of this method have been fully verified in two anhydrous hiNADES solvents (ChCl_Gly and ChCl_LA) and six application scenarios (see Examples 1 and 2, Application Examples 1-4). It is foreseeable that the "method for determining the reducing sugar content in a hydrophilic eutectic solvent system" provided by this invention will inevitably become a universal and standard method for determining the reducing sugar content in hydrophilic eutectic solvent systems, applicable to all hydrophilic eutectic solvents and all reducing sugars, and applicable to all research scenarios related to chemical events involving "hi(NA)DES and reducing sugars".

[0071] Furthermore, the method for determining the reducing sugar content in hydrophilic eutectic solvent systems provided by this invention has the following significant technical advantages: ① Wide applicability: Applicable to all hydrophilic eutectic solvent systems (both acidic and alkaline) and all reducing sugars; ② High measurement accuracy and sensitivity: Because two essential sample preparation steps—water disintegration and alkali disintegration—are added to the classic DNS method, the two major problems of DNS reagent failure and decreased measurement accuracy due to the high viscosity of hi(NA)DES are solved simultaneously, resulting in high measurement accuracy and sensitivity; ③ Greener and safer: The DNS reagent, solvent (hi(NA)DES), and disintegrant (H2O) used in the entire determination process are all green and pollution-free substances, making the entire testing process more environmentally friendly and safer than other methods; ④ In-situ testing does not damage the sample: This testing method is an in-situ detection method, which does not require separating the reducing sugar from the hydrophilic eutectic solvent before testing, nor does it require... The method first breaks down the hydrophilic eutectic solvent to make it an aqueous solvent, thus distorting the research process (this invention defines an eutectic solvent system with excessive water content as a pseudo-eutectic solvent system), thereby ensuring the broad application prospects of this method; ⑤ Low detection cost: The reagents and solvents used are all synthesized from conventional reagents and low-cost natural products, while the disintegrant is inexpensive and readily available water, and it does not rely on expensive large-scale instruments, thus having a significant cost advantage; ⑥ Extremely rich application scenarios: It can be applied to various chemical events related to "hi(NA)DES and reducing sugars", including the pretreatment, saccharification and fermentation of lignocellulose biomass, cellulase activity determination, carbohydrate conversion, and glycosidic bond synthesis; ⑦ Low dilution ratio: Unlike the excessively high dilution ratio of 50-100 times required for the determination of reducing sugars in eutectic solvents by HPLC, this invention controls the dilution ratio of the solution to a low level of less than 4 times, avoiding the excessive dilution of sugar solutions in the HPLC method for column safety, which results in an undetectable sugar concentration in the diluted solution. In addition, the testing process eliminates the interference of eutectic solvents on the test results, making the results closer to the true values. Moreover, the operation is simple and fast, with high detection sensitivity, good reproducibility, and low sample consumption.

[0072] Ultimately, this invention establishes a universal method for in-situ detection of reducing sugar content in anhydrous pure hi(NA)DES solvent—the "DNS" method. This represents a breakthrough from zero to one and also clears the way for (NA)DES to fully penetrate into two major research fields: (artificial) photosynthesis, which focuses on reducing sugars, and reverse photosynthesis, represented by biomass refining. It can be said that the establishment of this invention represents a breakthrough in the intersection of reducing sugar quantitative analysis technology and green solvent applications, expanding the application scope of reducing sugar testing and providing a new analytical tool for the application research of eutectic solvents.

[0073] Second, considering the technical solution as a whole or from a product perspective, the technical effects and advantages of the technical solution to be protected by this invention are specifically described as follows:

[0074] 1. The detection method is simple and easy to implement, low in cost, high in accuracy, good in sensitivity, strong in reproducibility, and consumes little sample;

[0075] 2. This invention can determine the reducing power, i.e., the reactivity, of carbohydrates, which is precisely what HPLC lacks, and therefore has a certain irreplaceable value.

[0076] 3. In the absence of developing new specialized reagents, the inventors of the patent have tried no less than ten conventional detection methods, and only this invention is feasible. Therefore, in the field of reducing sugar determination in eutectic solvent systems, it has a certain degree of technological advancement and monopoly in the short term.

[0077] 4. It overcomes the interference of low eutectic solvent background values ​​on test results, making the results closer to the true values;

[0078] 5. This method avoids the excessively high dilution ratios of 50-100 times often seen in HPLC methods, controlling the dilution of the sugar solution to a lower level of less than 4 times, thus maximizing the preservation of the original sugar solution concentration. In the method described in this invention, two fundamental steps are added to the traditional DNS method for the first time—hydration disintegration and pH alkalization—to adapt to the new characteristics of the new non-aqueous solvent system that can be switched to aqueous solution—the hydrophilic eutectic solvent system hi(NA)DES. The former overcomes the difficulties of transitioning from a non-aqueous phase to an aqueous phase, and the problem of decreased measurement accuracy due to the high viscosity of hi(NA)DES. The latter solves the problem of DNS reagent failure caused by the high acidity of some hi(NA)DES. The new concept of "disintegration" distinguishes it from the traditional term "dilution," emphasizing the phase transition process from a non-aqueous solvent to an aqueous solvent. Finally, this invention establishes an improved DNS method—the DNS method—which successfully enables in-situ determination of reducing sugars in anhydrous pure hi(NA)DES solvent system (no successful cases have been reported in previous literature). Two implementation schemes of this method are deployed with "the solubility of reducing sugars in hi(NA)DES solvent system" and "the reducing sugar release curve in hi(NA)DES-enzymatic hydrolysis system" as application entry points.

[0079] 6. The method developed in this invention not only solves the bottleneck problem arising when (artificial) photosynthesis and reverse photosynthesis research are combined with green solvents—the in-situ determination of reducing sugars in pure (NA)DES—but it is also foreseeable that this method will be widely used in many related fields such as botany, biology, physiology, energy, chemical engineering, food, agrochemicals, cosmetics, and pharmaceuticals, which simultaneously involve "reducing sugars and hydrophilic eutectic solvents."

[0080] 7. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system provided by this invention has important guiding significance for the study of various chemical events related to "hi(NA)DES and reducing sugar". It not only expands the application field of reducing sugar testing, but also provides a new analytical tool for the application research of eutectic solvents.

[0081] 8. The determination method of this invention has a wide range of applications, high measurement accuracy, and good sensitivity, and is suitable for all hydrophilic eutectic solvent systems. The analytical determination method established in this invention is expected to promote the research on chemical events related to "hi(NA)DES and reducing sugars" in a more scientific and rigorous direction.

[0082] Third, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects:

[0083] (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows:

[0084] The technical solution of this invention, once transformed, will generate significant economic benefits and broad commercial prospects, such as: 1. Service revenue from testing, including service fees generated from commissioning testing services provided by the patent holder; 2. Knowledge fees generated from paying the patent holder for authorization to use this invention for various reasons; 3. Consulting service and technical assistance fees paid to the patent holder due to technical needs; 4. Training fees generated from numerous clients requesting instruction on this method as the invention becomes widely used in various fields and application scenarios; 5. The establishment and promotion of this method will inevitably drive the vigorous development of research on chemical events related to "hi(NA)DES and reducing sugars," thereby creating a market demand for DNS reagents, hi(NA)DES solvents, and testing consumables and equipment. If the patent holder uses this patent to engage in the manufacturing and development of reagents, solvents, and testing-related consumables and equipment, they will obtain direct economic benefits.

[0085] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally:

[0086] The technical solution of this invention fills the gap and deficiency of existing quantitative analysis technology for reducing sugars in the field of eutectic solvents at home and abroad—that is, the in-situ determination of reducing sugars in anhydrous pure (NA)DES has not been effectively solved. It establishes an accurate, unified and standardized method for determining the reducing sugar content in anhydrous pure (NA)DES solvent, and can be applied to the study of various chemical events involving "hi(NA)DES and reducing sugars".

[0087] (3) The technical solution of this invention solves a long-standing but unsolved technical problem: the in-situ determination of reducing sugars in anhydrous pure (NA)DES. Simultaneously, this invention successfully develops a universal and standard method for determining the reducing sugar content in a hydrophilic eutectic solvent system. This method adds two basic steps (water disintegration and pH alkalization) to the traditional DNS standard procedure to adapt it to the new characteristics of the new non-aqueous solvent system (hydrophilic eutectic solvent, hi(NA)DES). The former overcomes the difficulties of transitioning from a non-aqueous phase to an aqueous phase, as well as the decrease in measurement accuracy due to the high viscosity of hi(NA)DES. The latter solves the problem of DNS reagent failure caused by the high acidity of some hi(NA)DES. Water here acts as a phase change agent, playing a role in phase disintegration and phase transition in the eutectic solvent system, rather than the dilution effect mentioned in previous reports.

[0088] (4) The technical solution of the present invention overcomes technical bias:

[0089] The technical solution of this invention overcomes two technical biases: Bias 1: Since DNS reagent is an aqueous reagent, the DNS method can only be applied in aqueous eutectic solvent systems. The technical solution of this invention overcomes this bias and successfully implements the determination of reducing sugars based on the DNS method in anhydrous pure eutectic solvent systems; Bias 2: Even when the DNS method is applied to eutectic solvent systems, there is no difference in acidity or alkalinity. The technical solution of this invention discovers the difference in the application of the DNS method in acidic and non-acidic eutectic solvent systems. Acidic eutectic solvent systems are more likely to cause DNS reagent to fail, so it must be disintegrated with an alkaline solution, while non-acidic eutectic solvent systems can be disintegrated simply with water. Attached Figure Description

[0090] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0091] Figure 1 This is a graph showing the annual growth trend of the number of published papers on natural deep eutectic solvents (NADES) using current technologies (data source: Web of Science database);

[0092] Figure 2 This illustrates the central role of reducing sugar determination in (natural) eutectic solvents in the study of (artificial) photosynthesis and reverse photosynthesis, as provided in the embodiments of this invention.

[0093] Figure 3This invention provides a process for upgrading traditional biorefining to "greener" biorefining, as well as the evolution of the traditional method (DNS method) for reducing sugar determination to a new method (DNS "method") that is inevitable due to solvent substitution.

[0094] Figure 4 The physical photographs provided in this embodiment of the invention show the test results of simply cloning the DNS method into the ChCl_LA solvent. The DNS reagent becomes ineffective and does not develop color due to the formation of a large amount of white precipitate.

[0095] Figure 5 This is a flowchart of the method for determining the reducing sugar content in a hydrophilic eutectic solvent system provided in the embodiments of the present invention;

[0096] Figure 6 This is a schematic diagram illustrating the effect of the molar concentration of NaOH solution on the absorbance of the disintegrating solution (hi(NA)DES is ChCl_LA) provided in the embodiments of the present invention;

[0097] Figure 7 This is a schematic diagram of the glucose absorbance standard curve (hi(NA)DES is ChCl_Gly and ChCl_LA) provided in the embodiments of the present invention;

[0098] Figure 8 This is a schematic diagram of the absorbance standard curves of reducing sugars other than glucose in ChCl-Gly solvent, obtained by using the method of the present invention in an embodiment of the present invention.

[0099] Figure 9 This is a schematic diagram of the absorbance standard curves of reducing sugars other than glucose in ChCl_LA solvent, obtained by using the method of the present invention in an embodiment of the present invention.

[0100] Figure 10 This is a schematic diagram of the glucose release kinetic curve (hi(NA)DES is ChCl_Gly) in the enzymatic hydrolysate provided in the embodiments of the present invention;

[0101] Figure 11 This is a semi-logarithmic graph of glucose release from the Cellulase 1.5L-ChCl_Gly system at 50°C, provided in an embodiment of the present invention.

[0102] Figure 12 This is a semi-logarithmic graph of glucose release from the Cellulase 1.5L-ChCl_Gly system at 60°C, provided in an embodiment of the present invention.

[0103] Figure 13 This is a semi-logarithmic graph of glucose release from the Cellulase 1.5L-ChCl_Gly system at 70°C, provided in an embodiment of the present invention.

[0104] Figure 14 This is a semi-logarithmic graph of glucose release from the Ctec2-ChCl_Gly system at 50°C, provided in this embodiment of the invention.

[0105] Figure 15 This is a semi-logarithmic graph of glucose release from the Ctec2-ChCl_Gly system at 60°C, provided in this embodiment of the invention.

[0106] Figure 16 This is a schematic diagram of the enzyme activity of Cellulase 1.5L provided in the embodiments of the present invention at different temperatures in three different solvent systems. Detailed Implementation

[0107] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0108] To address the problems existing in the prior art, the present invention provides a method for determining the reducing sugar content in a hydrophilic eutectic solvent system. The present invention will be described in detail below with reference to the accompanying drawings.

[0109] Sugars (also known as carbohydrates) are the most widely distributed naturally occurring organic compounds on Earth. Their modern definition is a polyhydroxy (two or more) aldehyde or ketone compound. Because they are mainly composed of carbon, hydrogen, and oxygen, they typically have a carbonyl group (C). n (H2O) n The chemical formula of carbohydrates, formally representing a polymerization of carbon and water, is also known as a carbohydrate. Reducing sugars are sugars with reducing properties; they are a subclass of carbohydrate compounds. Their reducing property is manifested in their ability to form aldehyde and ketone groups in alkaline solutions and to be oxidized by suitable oxidizing agents to uronic acids, diacids, etc. Reducing sugars include monosaccharides such as glucose, xylose, fructose, galactose, mannose, rhamnose, and fucose, as well as disaccharides such as cellobiose, maltose, and lactose, and some reducing oligosaccharides. Not all sugars have reducing properties; sugars that cannot form aldehyde and ketone groups in alkaline solutions are not reducing sugars, such as sucrose, trehalose, and most polysaccharides.

[0110] Carbohydrates, including reducing sugars, are produced during photosynthesis, where energy from the sun is converted into chemical energy by combining carbon dioxide with water to form carbohydrates and molecular oxygen (reaction 1). These carbohydrates are then broken down through anaerobic glycolysis (reaction 2), providing energy in the form of adenosine triphosphate (ATP), a phosphorylated, purine-substituted carbohydrate derivative of ribose.

[0111]

[0112] Based on reactions 1 and 2, the reducing properties of reducing sugars play an important role in the life process of organisms, mainly in the following three aspects: (1) providing energy, reducing sugars are oxidized to L-lactic acid during anaerobic glycolysis, and at the same time release 2 units of ATP to provide energy; (2) metabolic balance, the redox process mediated by reducing sugars (such as glycolysis) is an important process for regulating the metabolic balance of the human body; (3) reducing sugars are highly reactive because they contain free aldehyde or ketone groups in their molecules. They are important building blocks and material basis for biological metabolism. There are many derivatives developed from them. Sugar-containing substances alone include polysaccharides, sugar alcohols, sugar acids, ribose, glycoproteins, etc.

[0113] Table 2 lists the main methods for determining sugars and reducing sugars. These methods can be categorized according to their principles into colorimetric methods, physical methods, chromatographic methods, spectroscopic methods, electrochemical methods, and enzymatic methods. Based on the range of sugars measured, i.e., sugar specificity, they can be further divided into reducing sugar determination methods and general sugar determination methods. Among these, only colorimetric methods, based on redox reactions and combined with coloring agents, can capture the reducing groups of sugars and determine their reducing properties; the remaining methods are general sugar determination methods that cannot distinguish between reducing and non-reducing sugars.

[0114] Table 2. Summary of methods for determining carbohydrates (including reducing sugars)

[0115]

[0116] This invention will compare the advantages and disadvantages of various colorimetric methods for the determination of reducing sugars listed in Table 2. The potassium permanganate method, Fehling's reagent method, Benedict's reagent method, Nelson-Somogyi method, and new copper reagent method all rely on the reduction of divalent copper ions to monovalent cuprous ions by reducing sugars. The content of cuprous ions is then identified using different colorimetric reagents, thus indirectly determining the content of reducing sugars. All five methods are indirect methods, which, in addition to requiring numerous reagents and involving cumbersome steps, also generate a large amount of copper salt contaminants. Tollens' silver reagent for determining reducing sugars works by reacting silver ammonia solution with reducing sugars to form a silver mirror. The reaction is sensitive and the phenomenon is obvious, making it arguably the best reagent in the field of reducing sugar detection. However, because Tollens' reagent must be freshly prepared and easily precipitates explosive black silver nitride (Ag3N) and silver fulminate (AgONC) over time, its application is limited. In addition, methods involving potassium ferricyanide and Cu(II)-Cu(I) ions are only suitable for the determination of small amounts of reducing sugars after chromatographic purification. However, experience shows that these methods require a high level of skill, are time-consuming, and are sensitive to small changes in conditions.

[0117] Anthrone reagents are well-suited for standard sugar solutions; however, when used to analyze chromatographically purified sugars, even trace amounts of solvent or developer can render them ineffective. Furthermore, both the phenol-sulfuric acid method and the anthrone method are based on the principle of colorimetric analysis of the dehydration products of sugars. First, reducing sugars are dehydrated to generate 5-hydroxymethylfurfural (5-HMF), and then phenol or anthrone forms a color complex with 5-HMF. Both of these methods are still indirect methods, and their disadvantage lies in the limitation of their reducing sugar detection range, which is affected by the success of the reducing sugar dehydration reaction, as not all reducing sugars can successfully dehydrate to form 5-hydroxymethylfurfural.

[0118] The MBTH (3-methyl-2-benzothiazoline hydrazine hydrochloride) method, originally proposed for aliphatic aldehydes, has been described as a suitable method for sugar detection and quantification. This method involves three stages and analyzes the final product—formaldehyde. The first stage involves the reduction of monosaccharides to the corresponding sugar alcohols using potassium borohydride (KBH4). The sugar alcohols are then treated with periodic acid (HIO4), whose terminal sugar ol diol groups produce formaldehyde. In the final stage, the latter compound reacts with MBTH to form a blue complex with maximum absorption at 635 nm. Neutral sugars (pentoses, hexoses, and deoxysaccharides), amino sugars, uronic acids, sugar alcohols, and some disaccharides (such as maltose) are the major carbohydrate classes that can be analyzed by this method. Because only one compound formed from a sugar is analyzed, there are no issues related to reaction differences between sugars. The advantages of this method are its low detection limit (420–500 nM) and accuracy of less than 10% at the μM level. However, the MBTH method is also relatively time-consuming and laborious because it involves three chemical reactions. Furthermore, all unnecessary reagents need to be eliminated between each step, and the efficiency of each stage is fundamental to obtaining reliable results.

[0119] pHBAH, BCA, and DNS reagents are all reagents that can directly react with reducing sugars to produce a redox reaction and colorimetric result. Literature reports show that pHBAH and BCA reagents can reliably detect different reducing sugars at concentrations below 1 mM with extremely high sensitivity. Compared to the other two reagents, DNS reagent cannot be used for trace or even ultra-trace amounts of reducing sugars and has lower sensitivity. However, the advantages of DNS reagent and its corresponding detection method lie in its low cost, small dosage, minimal equipment requirements, and simple and easy-to-perform steps. It is not only a widely used method for reducing sugar determination but also a method recommended by the International Union of Pure and Applied Chemistry (IUPAC) and the National Renewable Energy Laboratory (NREL). It is also a component of the cellulase filter paper enzyme activity assay. Furthermore, the DNS method is currently the only reported method for reducing sugar determination successfully implemented in non-aqueous solvent systems (ionic liquids, eutectic solvents).

[0120] Technical challenges faced when applying the DNS method to eutectic solvent systems:

[0121] For a long time, the DNS method dominated the determination of reducing sugars in the field of biomass refining, but this was achieved in traditional biomass refining processes using aqueous conventional solvents (citrate buffer, acetate buffer, phosphate buffer, etc.) as the reaction medium. In recent years, as more and more researchers have used green solvents (especially (NA)DES solvent) to replace buffer solvents to carry out greener biomass refining processes, the applicability of the DNS method in new solvent systems has been challenged.

[0122] from Figure 3 It is not difficult to see that the key to upgrading traditional biorefining to "greener" biorefining is to replace traditional aqueous buffer solutions with green solvents such as eutectic solvents. This replacement will inevitably lead to the evolution of the traditional method (DNS method) for reducing sugar determination (DNS "method") to a new method (DNS "method"). However, to develop a new method (DNS "method") for reducing sugar determination adapted to the new solvent system based on the DNS method, the following three main obstacles still need to be overcome (see Table 3 for details): ① DNS reagent can only be prepared with water as its solvent and cannot be prepared with eutectic solvents: because in the exploratory experiments conducted by this invention using ChCl_LA solvent as an example, it was found that the solubility of DNS reagent components in ChCl_LA solvent is low, and some components (Rochelle salt) are completely insoluble, failing to form orange DNS reagent and thus failing to achieve the colorimetric reaction of reducing sugar oxidation; ② The high acidity of some eutectic solvents will cause a large amount of white precipitate to be produced when the DNS reagent is directly mixed with the sugar solution prepared with the eutectic solvent, resulting in failure and no color development. Figure 4 ); ③ Eutectic solvents generally have very high viscosity, which affects the precision and accuracy of reducing sugar determination.

[0123] Table 3. The exploration process of the evolution from the DNS method to the "DNS" method.

[0124]

[0125] Table 3 shows the trial-and-error process of this invention in developing a new method for determining reducing sugars adapted to the new solvent system (hiNADESs). First, this invention directly cloned the original DNS method into ChCl_LA (the DNS method in Table 3), but the DNS reagent failed to develop color and produced a large amount of white precipitate (…). Figure 4The high acidity of ChCl_LA was likely the cause of this failure. Next, this invention attempted to prepare a ChCl_LA-based DNS reagent (DNS@ChCl_LA) by replacing water with ChCl_LA. However, the DNS reagent components had low solubility in the ChCl_LA solvent, and some components (Rochelle salts) were completely insoluble, failing to form an orange DNS reagent. Furthermore, the subsequent oxidation and colorimetric reaction of the reducing sugar could not be achieved (DNS' method in Table 3). After these two failures, this invention shifted its focus from the DNS reagent to the sample preparation process (DNS' method in Table 3). For the first time, two essential steps (water disintegration and pH alkalization) were added to the classic DNS method, leading to successful colorimetric analysis. The former overcame the challenges of the transition of hiNADESs from a non-aqueous to an aqueous phase and the decrease in measurement accuracy due to the high viscosity of hiNADESs. The latter solved the problem of DNS reagent failure caused by the high acidity of some hiNADESs (e.g., ChCl_LA). For detailed standard procedures, see this invention. Figure 5 The flowchart illustrating the method for determining the reducing sugar content in a hydrophilic eutectic solvent system, along with the textual descriptions in the Summary of the Invention and Detailed Embodiments sections, are provided in the document.

[0126] To address the shortcomings and gaps in existing quantitative analysis techniques for reducing sugars in the field of eutectic solvents, this invention aims to provide a method for in-situ determination of reducing sugar content in anhydrous, pure, hydrophilic eutectic solvent system hi(NA)DES. The method provided by this invention adds two basic steps (water disintegration and pH alkalization) to the traditional DNS standard procedure (referred to as the "DNS method") to adapt to the new characteristics of the hydrophilic eutectic solvent system hi(NA)DES, which is a non-aqueous solvent system that can be switched to an aqueous phase. The former overcomes the difficulties of converting from a non-aqueous phase to an aqueous phase, as well as the problem of decreased measurement accuracy due to the high viscosity of hi(NA)DES. The latter solves the problem of DNS reagent failure caused by the high acidity of some hi(NA)DES. This method has a wide range of applications, high measurement accuracy, good sensitivity, and is applicable to all hydrophilic eutectic solvent systems hi(NA)DES.

[0127] like Figure 5 As shown, the method for determining the reducing sugar content in a hydrophilic eutectic solvent system provided in this embodiment of the invention includes the following steps:

[0128] S101, prepare 3,5-dinitrosalicylic acid DNS reagent, and prepare hydrophilic eutectic solvent hi(NA)DES;

[0129] S102, Preparation of reducing sugar stock solution RS-hi(NA)DES stock Prepare reducing sugar standard solution RS-hi(NA)DES stand;

[0130] S103, Prepare a reducing sugar test solution based on hi(NA)DES: RS-hi(NA)DES test Or cellulose enzymatic hydrolysate RS-hi(NA)DES cellulase ;

[0131] S104 is used for the hydrolysis or alkaline disintegration of reducing sugar solutions.

[0132] S105 is used for the hydrophilic eutectic solvent hi(NA)DES, either by adding water or by adding alkali.

[0133] S106, mix reducing sugar disintegration solution, solvent blank disintegration solution and DNS reagent, then carry out color development reaction in boiling water bath, and dilute the color development solution;

[0134] S107. The absorbance of the colorimetric solution was measured in a UV-Vis spectrophotometer to calculate the reducing sugar content.

[0135] This invention provides a method for determining the reducing sugar content in a hydrophilic eutectic solvent system, the method comprising the following steps:

[0136] (1) Preparation of 3,5-dinitrosalicylic acid (DNS) reagent. Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to about 400mL of distilled water. Heat and stir moderately until completely dissolved. The solution will be grass-green at this point. Then add 7.0g of NaOH to the above solution and stir at room temperature until completely dissolved. The solution will be orange-red at this point. It should be noted that the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially. They should not be added at the same time or in a different order. After that, add 108g of potassium sodium tartrate (Rochelle salt), 2.7mL of phenol (liquid phenol melted at 50℃), and 2.9g of sodium metabisulfite to the orange-red solution. Stir thoroughly and dissolve all the added components. It should be noted that after adding the last three components, a large number of suspended particles may appear in the solution. After stirring continuously for more than 1 hour, all the suspended particles will disappear, and a clear orange-red solution will be formed. Add distilled water to make up to 500 mL, store in a brown bottle away from light, and use after one week. The shelf life is 6 months.

[0137] (2) Preparation of hydrophilic eutectic solvent hi(NA)DES. Two hi(NA)DES based on choline chloride (ChCl) are used as examples: one is ChCl_Gly solvent, and the other is ChCl_LA solvent. Both solvents use choline chloride as the hydrogen bond acceptor (HBA), and the hydrogen bond donors (HBD) are glycerol (Gly) and lactic acid (LA), respectively. The molar ratio of HBA to HBD in both solvents is 1:2. The preparation process involves adding the corresponding mass of choline chloride and glycerol (or lactic acid) in a round-bottom flask at a molar ratio of 1:2, heating and stirring at 60–65°C, avoiding the addition of water as much as possible, until the two components transform into a homogeneous transparent liquid. Stirring and heating are stopped; if the liquid remains after cooling to room temperature, the preparation is successful. In addition to choline chloride, the hydrogen bond acceptor HBA can also be a choline chloride analogue (such as acetylcholine, choline bromide, or acetylcholine). + Hydrogen bond acceptors (HBDs) can be selected from glycerol, lactic acid, urea and its analogues, sugars, sugar alcohols, polyols (such as glycerol), amino acids, organic acids (such as lactic acid), etc., in addition to glycerol and lactic acid. Natural eutectic solvents are a subset of eutectic solvents, characterized by the requirement that both the hydrogen bond acceptor and hydrogen bond donor be natural products, typically limited to organic compounds of natural origin derived from primary or secondary metabolic pathways. It is particularly noteworthy that the hydrophilic eutectic solvent hi(NA)DES encompasses both hydrophilic eutectic solvent hiDES and hydrophilic natural eutectic solvent hiNADES, representing the hydrophilic subset of eutectic solvents and natural eutectic solvents. All hydrophilic eutectic solvents hi(NA)DES are applicable to the technical solutions described in this invention.

[0138] (3) Preparation of reducing sugar stock solution RS-hi(NA)DES stock 50 mL of prepared hi(NA)DES (such as ChCl_Gly and ChCl_LA) is pre-filled into an Erlenmeyer flask. Then, about 1 g of reducing sugar (such as glucose, xylose, fructose, galactose, mannose, rhamnose, fucose, cellobiose, maltose, and lactose) is weighed and added to the flask. The solution is stirred at room temperature for more than 24 hours until the solid is completely dissolved and the solution becomes transparent, resulting in a series of RS-hi(NA)DES stock solutions with a concentration of 20 mg / mL.

[0139] (4) Preparation of reducing sugar standard solution RS-hi(NA)DES stand After step (3) is completed, RS-hi(NA)DES standard solutions (0.5-4.0 mg / L) are obtained by measuring a certain amount of RS-hi(NA)DES stock solution and adding the corresponding hi(NA)DES solvent to a volumetric flask in a certain proportion.

[0140] (5) Preparation of reducing sugar test solution based on hi(NA)DES: RS-hi(NA)DES test Weigh a certain amount of reducing sugar into an Erlenmeyer flask. The dissolution process is the same as in step (3). If all the sugar is dissolved, it can be used directly. If there is some undissolved reducing sugar solid, it can be vacuum filtered through an organic membrane and the liquid portion can be collected for later use.

[0141] (6) Preparation of cellulose hydrolysate RS-hi(NA)DES based on hi(NA)DES cellulase Weigh out 0.5g of cellulose raw material (such as microcrystalline cellulose). Add PH-101 to a 25 mL Erlenmeyer flask, then add 10 mL of hi(NA)DES solvent (ChCl_Gly or ChCl_LA). Place the Erlenmeyer flask containing the cellulose raw material and hi(NA)DES solvent in a constant temperature water bath shaker and preheat for 30 min at 50–70 °C and 120–160 rpm. After preheating, add cellulase (NovozymeCelluclast 1.5 L or NovozymeCtec 2) to the Erlenmeyer flask at a loading rate of 60–160 FPU cellulase / g cellulose. Then, react for 0–48 h at 50–70 °C and 120–160 rpm. Take the enzymatic hydrolysate from different reaction times as RS-hi(NA)DES. cellulase When the reaction time is reached, the corresponding conical flask should be removed, inactivated in ice water, and allowed to stand until the solid settles at the bottom of the conical flask. The supernatant should then be filtered and used for later use.

[0142] (7) Perform hydrolysis or alkali disintegration of the reducing sugar solution. Hydrolysis: Add the reducing sugar solution (including RS-hi(NA)DES) to a 10 mL graduated cylinder. stand RS-hi(NA)DES test and RS-hi(NA)DES cellulaseAdd distilled water to the 4mL mark, and shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the eutectic phase of RS-hi(NA)DES to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution. This step is applicable to all reducing sugar solutions prepared with non-acidic hi(NA)DES solvents. Alkali disintegration: The oxidation reaction between DNS reagent and reducing sugars can only occur under alkaline conditions. When preparing reducing sugar solutions with acidic hi(NA)DES solvent, alkali is used for disintegration to make the disintegrating solution alkaline. The alkali includes, but is not limited to, NaOH solution. The optimal molar concentration of the alkali is determined by simultaneously meeting the following two criteria: ① The disintegrating solution formed after adding this concentration of alkali can undergo an oxidation reaction with the DNS reagent without precipitation, and the colorimetric test tube turns purple-red; ② The absorbance of the disintegrating solution formed after adding this concentration of alkali should not be too high, and the absorbance value should be relatively low compared to other concentrations. This step applies to reducing sugar solutions prepared with all acidic hi(NA)DES solvents.

[0143] (8) Perform hydrophilic eutectic solvent hi(NA)DES disintegration by adding water or alkali. Add the hydrophilic eutectic solvent hi(NA)DES to a 10mL graduated cylinder to the 1mL mark. When hi(NA)DES is ChCl_Gly, add distilled water to the 4mL mark; when hi(NA)DES is ChCl_LA, add 2M NaOH solution to the 4mL mark. Shake the graduated cylinder thoroughly to allow the water (or NaOH solution) to dissolve completely. Figure 6 The impact on the non-aqueous phase interface of the sample causes the eutectic phase of RS-hi(NA)DES to disintegrate until it completely disappears, ultimately forming a transparent and homogeneous aqueous solution. This step is applicable to all hi(NA)DES solvents.

[0144] (9) Mix the reducing sugar disintegration solution, the solvent blank disintegration solution, and the DNS reagent. Mixing the reducing sugar disintegration solution with the DNS reagent: Transfer 1.5 mL of the reducing sugar disintegration solution (standard solution disintegration solution, test solution disintegration solution, enzyme digestion solution disintegration solution) from step (7) into a 10 mL test tube, then add 3 mL of DNS reagent and mix well. The total volume of the mixed solution is 4.5 mL. Mixing the solvent blank disintegration solution with the DNS reagent: Transfer 1.5 mL of the solvent blank disintegration solution from step (8) into a 10 mL test tube, then add 3 mL of DNS reagent and mix well. The total volume of the mixed solution is 4.5 mL.

[0145] (10) Perform the colorimetric reaction in a boiling water bath and dilute the colorimetric solution. Insert all the test tubes from (9) into the test tube rack, then place them in a cylindrical stainless steel mesh basket and boil precisely for 5 minutes in a vigorously boiling water bath. The water level in the boiling water bath should be sufficient to cover the height of the mixed solution in the test tubes. The blank solution and standard solution (or test solution, or enzyme digest) should be boiled together. After boiling, transfer to an ice water bath to cool to room temperature. Then, dilute all the test tubes (blank, standard / test / enzyme digest) that have undergone colorimetric reaction with distilled water. The dilution ratio should be explored according to the actual situation. The principle is that the A of the colorimetric solution after dilution should be... 540nm The dilution ratio is 0.1–1.0A. The dilution ratio of blank test tubes should be consistent with that of standard / test / enzyme digestion test tubes.

[0146] (11) Measure the absorbance of the colorimetric solution in a UV-Vis spectrophotometer. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at a wavelength of 540 nm in a UV-Vis spectrophotometer. Subtract the blank absorbance value of the solvent tube to obtain the colorimetric absorbance value of the standard / test / enzyme digestion tube.

[0147] (12) Calculate the reducing sugar content. Express the absolute amount of reducing sugar (mg / mL) relative to A. 540nm Plot a linear standard curve. The data in the standard curve should be as close to a straight line as possible, and the correlation coefficient of the fitted line should be in the range of 0.9900-1.0000. Calculate the reducing sugar content of each test or enzymatic hydrolysis tube using the linear equation derived from the fitted standard curve.

[0148] Steps (1) and (2) constitute the first part, providing the reagents and solvents required for the synthesis of the entire technical solution; Steps (3), (4), (5), and (6) constitute the second part, providing the various reducing sugar solutions RS-hi(NA)DES based on hi(NA)DES that need to be prepared for the entire technical solution. Steps (3) and (4) respectively prepared the reducing sugar stock solutions RS-hi(NA)DES. stock and reducing sugar standard solution RS-hi(NA)DES stand This was used to plot the glucose absorbance standard curve, while steps (5) and (6) respectively prepared the reducing sugar test solution RS-hi(NA)DES. test and cellulose enzymatic hydrolysate RS-hi(NA)DES cellulaseThe steps (7) and (8) are the third part, which is the phase transition step necessary for the successful quantification of the reducing sugar content in all hi(NA)DES-based reducing sugar solutions. The reducing sugar solution RS-hi(NA)DES can be either hydrolyzed or alkali-disintegrated. When the reducing sugar solution is prepared with a non-acidic hi(NA)DES solvent, the hydrolyzed disintegration step is performed; when the reducing sugar solution is prepared with an acidic hi(NA)DES solvent, the alkali-disintegration step is performed. The fourth part is a phase transition step performed to eliminate the influence of the absorbance value of the hi(NA)DES solvent itself on the colorimetric value of the reducing sugar. Hi(NA)DES solvent blank disintegration with water or disintegration with alkali solution can be selected, and the selection criteria are the same as in step (7). Steps (9), (10), (11) and (12) are the fifth part, which is to determine the absorbance values ​​of the reducing sugar disintegration solution and the solvent blank disintegration solution, and finally determine the content of reducing sugar. The functions of steps (9) to (12) are, respectively, mixing of the analyte with the colorimetric reagent, color development and dilution, absorbance measurement, and calculation of reducing sugar content.

[0149] Example 1

[0150] The solubility of glucose in ChCl-Gly solvent was determined using the DNS method of this invention.

[0151] The specific operating method is as follows:

[0152] (1) Preparation of 3,5-dinitrosalicylic acid DNS reagent:

[0153] Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to approximately 400mL of distilled water. Heat and stir moderately until completely dissolved; the solution will be grass-green at this point. Next, add 7.0g of NaOH to the above solution and stir at room temperature until completely dissolved; the solution will then be orange-red. It is important to note that the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially; they cannot be added simultaneously or in any order. Then, add 108g of potassium sodium tartrate (Rochelle salt), 2.7mL of phenol (liquid phenol dissolved at 50℃), and 2.9g of sodium metabisulfite to the orange-red solution, stirring thoroughly until all components are dissolved. It should be noted that after adding the last three components, a large number of suspended particles may appear in the solution. Continue stirring for at least one hour until all suspended particles disappear, resulting in a clear orange-red solution. Dilute to 500mL with distilled water, store in a brown bottle protected from light, and let stand for one week before use. The shelf life is 6 months.

[0154] (2) Preparation of the hydrophilic eutectic solvent ChCl_Gly:

[0155] Add the corresponding masses of choline chloride and glycerol to a round-bottom flask in a 1:2 molar ratio. Heat and stir at 60–65°C, avoiding the addition of water as much as possible, until both components transform into a homogeneous, transparent liquid. Stop stirring and heating. If the liquid remains after cooling to room temperature, the ChCl_Gly solvent has been successfully prepared.

[0156] (3) Preparation of glucose stock solution (Glucose-ChCl_Gly) stock :

[0157] Pre-fill an Erlenmeyer flask with 50 mL of the prepared ChCl_Gly solvent, then weigh approximately 1 g of glucose and add it to the flask. Stir at room temperature for at least 24 hours until the solid is completely dissolved and the solution becomes transparent, thus obtaining a 20 mg / mL glucose stock solution (Glucose-ChCl_Gly). stock .

[0158] (4) Preparation of glucose standard solution (Glucose-ChCl_Gly) stand :

[0159] After step (3) is completed, the glucose standard solution Glucose-ChCl_Gly stand (0.5-4.0 mg / L) were respectively measured by taking a certain amount of glucose stock solution (Glucose-ChCl_Gly). stock The solution is obtained by diluting the volumetric flask with the appropriate amount of ChCl-Gly solvent according to a certain ratio.

[0160] (5) Glucose standard solution (Glucose-ChCl_Gly) stand Disintegration upon adding water:

[0161] Add glucose standard solutions of different concentrations (Glucose-ChCl_Gly) to a 10 mL graduated cylinder. stand Add distilled water to the 4 mL mark, and shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, thus allowing the Glucose-ChCl_Gly... stand The eutectic phase disintegrates until it disappears completely, eventually forming a transparent and homogeneous aqueous solution—the glucose standard solution disintegration solution.

[0162] (6) Disintegration of ChCl-Gly solvent upon addition of water:

[0163] Add ChCl_Gly solvent to a 10mL graduated cylinder to the 1mL mark, then add distilled water to the 4mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the eutectic phase of ChCl_Gly to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the solvent blank disintegration solution.

[0164] (7) Determination of absorbance of the disintegration solution:

[0165] Take 1.5 mL of the supernatant from both the glucose standard solution disintegration buffer and the solvent blank disintegration buffer, transfer them to 10 mL test tubes, and add 3 mL of DNS reagent. Mix well, resulting in a total solution volume of 4.5 mL. Insert all test tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the mixed solution in the test tubes. The blank solution and standard solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the test tubes (blank and standard) with distilled water. The dilution ratio should be explored based on actual conditions; the principle is that the dilution ratio of the colorimetric solution should be such that the A... 540nm The absorbance should be 0.1–1.0 A. The dilution ratio of the blank test tube and the standard test tube should be consistent. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance value of the blank test tube from the absorbance value of the solvent test tube to obtain the colorimetric absorbance value of the standard test tube.

[0166] (8) Plot the standard curve of glucose absorbance:

[0167] A scatter plot was created with the concentration of the glucose standard solution (mg / mL) on the x-axis and the absorbance at 540 nm (after subtracting the solvent blank) on the y-axis. A linear fit was then applied to the scatter plot, and the fitted curve passed through the point (0, 0). The resulting glucose absorbance standard curve is shown below. Figure 7 As shown.

[0168] Glucose standard solution (Glucose-ChCl_Gly) stand The relationship between the concentration of glucose and the absorbance at 540 nm is given by the function w = A / 0.06192, where w is the glucose concentration in mg / mL and A is the colorimetric absorbance obtained by subtracting the absorbance of the solvent from the absorbance of the disintegrated glucose standard solution at 540 nm.

[0169] (9) Preparation of saturated glucose solution (Glucose-ChCl-Gly) saturated :

[0170] Pre-fill a conical flask with 50 mL of the prepared ChCl_Gly solvent, then weigh approximately 20 g of glucose and add it to the flask. Stir at room temperature for at least 24 hours until the solid content in the solution no longer decreases, indicating a saturated solution has been formed. If the solid is completely dissolved, add another 20 g of glucose and stir for 24 hours, repeating this process until no more solid remains in the solution, at which point the saturated solution is ready. Undissolved glucose solids are then filtered through an organic membrane under vacuum to react with the glucose-ChCl_Gly saturated solution. saturated Separation.

[0171] (10) Glucose-ChCl-Glycine saturated solution saturated Disintegration upon adding water:

[0172] Add a saturated glucose solution (Glucose-ChCl_Gly) to a 10 mL graduated cylinder. saturated Add distilled water to the 1 mL mark, then add distilled water to the 4 mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the non-aqueous phase to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the glucose saturated solution disintegration solution.

[0173] (11) Determination of absorbance of glucose saturated solution disintegration:

[0174] Take 1.5 mL of the supernatant from both the glucose saturated solution disintegration buffer and the solvent blank disintegration buffer, transfer them to 10 mL test tubes, and add 3 mL of DNS reagent. Mix well, resulting in a total solution volume of 4.5 mL. Insert all test tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the mixed solution in the test tubes. The blank solution and standard solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the test tubes (blank and saturated) with distilled water. The dilution ratio should be explored based on actual conditions; the principle is that the colorimetric solution should be diluted to a concentration equal to the required concentration of A. 540nm The absorbance should be 0.1–1.0 A. The dilution ratio of the blank test tube and the saturated test tube should be consistent. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance value of the blank test tube from the absorbance value of the solvent test tube to obtain the colorimetric absorbance value of the saturated test tube.

[0175] The formula for calculating the glucose concentration in a saturated glucose solution is w = A / 0.06192. Where w is the glucose concentration in mg / mL, and A is the absorbance value obtained by subtracting the absorbance value of the solvent disintegration from the absorbance value of the saturated solution at 540 nm.

[0176] (12) Calculation of the solubility of glucose in ChCl-Gly solvent:

[0177] Converting the concentration of a saturated glucose solution (mg / mL) to g / 100g gives the solubility of glucose in ChCl-Gly solvent at room temperature.

[0178] (13) Although this embodiment takes the determination of the solubility of glucose in ChCl-Gly solvent as an example, it can be further extended to determine the solubility of any reducing sugar in any hydrophilic eutectic solvent. The reducing sugar can be selected from glucose, xylose, fructose, galactose, mannose, rhamnose, fucose, cellobiose, maltose and lactose. The hydrophilic eutectic solvent can be selected from the set of hydrophilic solvents in the eutectic solvent family and the natural eutectic solvent family.

[0179] Example 2

[0180] The enzymatic hydrolysis curve of cellulose in ChCl-Gly solvent was determined using the DNS method of this invention.

[0181] The specific operating method is as follows:

[0182] (1) Preparation of 3,5-dinitrosalicylic acid DNS reagent:

[0183] Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to approximately 400mL of distilled water. Heat and stir moderately until completely dissolved; the solution will be grass-green at this point. Next, add 7.0g of NaOH to the above solution and stir at room temperature until completely dissolved; the solution will then be orange-red. It is important to note that the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially; they cannot be added simultaneously or in any order. Then, add 108g of potassium sodium tartrate (Rochelle salt), 2.7mL of phenol (liquid phenol dissolved at 50℃), and 2.9g of sodium metabisulfite to the orange-red solution, stirring thoroughly until all components are dissolved. It should be noted that after adding the last three components, a large number of suspended particles may appear in the solution. Continue stirring for at least one hour until all suspended particles disappear, resulting in a clear orange-red solution. Dilute to 500mL with distilled water, store in a brown bottle protected from light, and let stand for one week before use. The shelf life is 6 months.

[0184] (2) Preparation of the hydrophilic eutectic solvent ChCl_Gly:

[0185] Add the corresponding masses of choline chloride and glycerol to a round-bottom flask in a 1:2 molar ratio. Heat and stir at 60–65°C, avoiding the addition of water as much as possible, until both components transform into a homogeneous, transparent liquid. Stop stirring and heating. If the liquid remains after cooling to room temperature, the ChCl_Gly solvent has been successfully prepared.

[0186] (3) Preparation of glucose stock solution (Glucose-ChCl_Gly) stock :

[0187] Pre-fill an Erlenmeyer flask with 50 mL of the prepared ChCl_Gly solvent, then weigh approximately 1 g of glucose and add it to the flask. Stir at room temperature for at least 24 hours until the solid is completely dissolved and the solution becomes transparent, thus obtaining a 20 mg / mL glucose stock solution (Glucose-ChCl_Gly). stock .

[0188] (4) Preparation of glucose standard solution (Glucose-ChCl_Gly) stand :

[0189] After step (3) is completed, the glucose standard solution Glucose-ChCl_Gly stand (0.5-4.0 mg / L) were respectively measured by taking a certain amount of glucose stock solution (Glucose-ChCl_Gly). stock The solution is obtained by diluting the volumetric flask with the appropriate amount of ChCl-Gly solvent according to a certain ratio.

[0190] (5) Glucose standard solution (Glucose-ChCl_Gly) stand Disintegration upon adding water:

[0191] Add glucose standard solutions of different concentrations (Glucose-ChCl_Gly) to a 10 mL graduated cylinder. stand Add distilled water to the 1 mL mark, then add distilled water to the 4 mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the eutectic phase of Glucose-ChCl_Gly to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the glucose standard solution disintegration solution.

[0192] (6) Disintegration of ChCl-Gly solvent upon addition of water:

[0193] Add ChCl_Gly solvent to a 10mL graduated cylinder to the 1mL mark, then add distilled water to the 4mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the eutectic phase of ChCl_Gly to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the solvent blank disintegration solution.

[0194] (7) Determination of absorbance of the disintegration solution:

[0195] Take 1.5 mL of the supernatant from both the glucose standard solution disintegration buffer and the solvent blank disintegration buffer, transfer them to 10 mL test tubes, and add 3 mL of DNS reagent. Mix well, resulting in a total solution volume of 4.5 mL. Insert all test tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the mixed solution in the test tubes. The blank solution and standard solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the test tubes (blank and standard) with distilled water. The dilution ratio should be explored based on actual conditions; the principle is that the colorimetric solution should be diluted to a concentration equal to the required concentration of A. 540nm The absorbance should be 0.1–1.0 A. The dilution ratio of the blank test tube and the standard test tube should be consistent. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance value of the blank test tube from the absorbance value of the solvent test tube to obtain the colorimetric absorbance value of the standard test tube.

[0196] (8) Plot the standard curve of glucose absorbance:

[0197] A scatter plot was created with the concentration of the glucose standard solution (mg / mL) on the x-axis and the absorbance at 540 nm (after subtracting the solvent blank) on the y-axis. A linear fit was then applied to the scatter plot, and the fitted curve passed through the point (0, 0). The resulting glucose absorbance standard curve is shown below. Figure 7 As shown.

[0198] Glucose standard solution (Glucose-ChCl_Gly) stand The relationship between the concentration of glucose and the absorbance at 540 nm is given by the function w = A / 0.06192, where w is the glucose concentration in mg / mL and A is the colorimetric absorbance obtained by subtracting the absorbance of the solvent from the absorbance of the glucose standard solution disintegration at 540 nm.

[0199] (9) Preparation of cellulose hydrolysate based on ChCl_Gly solvent: Glucose-ChCl_Gly cellulase :

[0200] Weigh approximately 0.5g of microcrystalline cellulose ( Add PH-101 to a 25 mL Erlenmeyer flask, then add 10 mL of ChCl_Gly solvent. Preheat the flask in a constant temperature water bath at 50–70 °C and 120–160 rpm for 30 minutes. Afterward, add cellulase (PH-101) to the Erlenmeyer flask at a loading rate of 60–160 FPU cellulase / g cellulose. 1.5L and CTec2 (Novozymes, filter paper enzyme activities were 130.45 FPU and 271.3 FPU, respectively) was reacted at 50–70°C and 120–160 rpm for 0–48 h. Enzyme hydrolysates from different reaction times were taken as Glucose-ChCl_Gly cellulase When the reaction time is reached, remove the corresponding conical flask, inactivate it in ice water, let it stand until the solid settles at the bottom of the conical flask, then take the supernatant, filter it and use it for later use.

[0201] (10) Cellulose hydrolysate Glucose-ChCl_Gly cellulase Disintegration upon adding water:

[0202] Add the cellulase hydrolysate Glucose-ChCl_Gly to a 10mL graduated cylinder. cellulase Add distilled water to the 1 mL mark, then add distilled water to the 4 mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the non-aqueous phase to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the cellulase hydrolysate disintegration solution.

[0203] (11) Determination of absorbance of cellulase hydrolysate disintegration:

[0204] Take 1.5 mL of the supernatant from both the cellulase hydrolysate and the solvent blank hydrolysate, transfer them to 10 mL test tubes, add 3 mL of DNS reagent, and mix well. The total volume of the mixed solution is 4.5 mL. Insert all test tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the height of the mixed solution in the test tubes. The blank solution and the hydrolysate solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the test tubes (blank and hydrolysate) with distilled water. The dilution ratio should be explored based on actual conditions; the principle is that the A value of the colorimetric solution should be diluted with distilled water. 540nm The absorbance should be 0.1–1.0 A. The dilution ratio of the blank test tube and the enzyme digestion test tube should be consistent. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance value of the blank test tube from the absorbance value of the solvent test tube to obtain the colorimetric absorbance value of the enzyme digestion test tube.

[0205] The formula for calculating the glucose concentration in the cellulose enzymatic hydrolysis solution is w = A / 0.06192. Where w is the glucose concentration in mg / mL, and A is the absorbance value of the disintegrating solution obtained by subtracting the absorbance value of the solvent disintegrating solution from the absorbance value of the disintegrating solution at 540 nm.

[0206] (12) Plot the enzymatic hydrolysis curve of cellulose in ChCl-Gly solvent.

[0207] Enzymatic hydrolysis was timed at 1h, 3h, 6h, 12h, 24h, 36h, and 48h, and the corresponding cellulose hydrolysis solutions were collected. The glucose concentration in the cellulose hydrolysis solution at the above hydrolysis times was obtained according to steps (9) to (11), and the corresponding glucose release kinetic curves were obtained (see...). Figure 10 ).

[0208] The applicant declares that this invention illustrates the method for testing the reducing sugar content in a hydrophilic eutectic solvent system through the above embodiments, but this invention is not limited to the above steps, i.e., it does not mean that this invention must rely on the above steps to be implemented. Those skilled in the art should understand that any improvements to this invention, equivalent substitutions of the raw materials used in this invention, additions of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this invention.

[0209] To demonstrate the inventiveness and technical value of the technical solution of this invention, this section provides specific product or related technology application examples of the technical solution claimed.

[0210] Application Example 1

[0211] The filter paper enzyme activity of cellulase in ChCl-Gly solvent was determined using the DNS method of this invention.

[0212] The specific operating method is as follows:

[0213] (1) Preparation of 3,5-dinitrosalicylic acid DNS reagent:

[0214] Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to approximately 400mL of distilled water. Heat and stir moderately until completely dissolved; the solution will be grass-green at this point. Next, add 7.0g of NaOH to the above solution and stir at room temperature until completely dissolved; the solution will then be orange-red. It is important to note that the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially; they cannot be added simultaneously or in any order. Then, add 108g of potassium sodium tartrate (Rochelle salt), 2.7mL of phenol (liquid phenol melted at 50℃), and 2.9g of sodium metabisulfite to the orange-red solution, stirring thoroughly until all components are dissolved. It should be noted that after adding the last three components, a large number of suspended particles may appear in the solution. Continue stirring for at least one hour until all suspended particles disappear, resulting in a clear orange-red solution. Dilute to 500mL with distilled water, store in a brown bottle protected from light, and let stand for one week before use. The shelf life is 6 months.

[0215] (2) Preparation of the hydrophilic eutectic solvent ChCl_Gly:

[0216] Add the corresponding masses of choline chloride and glycerol to a round-bottom flask in a 1:2 molar ratio. Heat and stir at 60–65°C, avoiding the addition of water as much as possible, until both components transform into a homogeneous, transparent liquid. Stop stirring and heating. If the liquid remains after cooling to room temperature, the ChCl_Gly solvent has been successfully prepared.

[0217] (3) Preparation of glucose stock solution (Glucose-ChCl_Gly) stock (10mg / mL):

[0218] Pre-fill a conical flask with 50 mL of the prepared ChCl_Gly solvent, then weigh approximately 0.5 g of glucose and add it to the flask. Stir at room temperature for at least 24 hours until the solid is completely dissolved and the solution becomes clear, thus obtaining a 10 mg / mL glucose stock solution (Glucose-ChCl_Gly). stock .

[0219] (4) Prepare the substrate for enzyme activity assay – Whatman No. 1 filter paper strips:

[0220] Cut Whatman No.1 filter paper into strips of 1.0 cm × 6.0 cm. Accurately weigh the cut filter paper strips on a balance of 0.001%, and keep the weight of all cut filter paper strips within the range of 50 ± 0.5 mg.

[0221] (5) Prepare blank and control test tubes:

[0222] The blank and control test tubes include three different types of test tubes: reagent blank, enzyme control, and substrate control. The reagent blank is a test tube containing only 1.5 mL of ChCl_Gly solvent; the enzyme control is a test tube containing both 1.0 mL of ChCl_Gly solvent and 0.5 mL of enzyme diluted sample (a separate control is prepared for each diluted sample tested); and the substrate control is a test tube containing both 1.5 mL of ChCl_Gly solvent and a filter paper strip.

[0223] (6) Prepare glucose standard test tubes:

[0224] Take four test tubes and add 1 mL of the glucose stock solution (10 mg / mL) prepared in step (3) to each. Then, add 0.5 mL, 1.0 mL, 2.0 mL, and 4.0 mL of ChCl_Gly solvent to each test tube in turn, and mix well to obtain glucose standard diluted solutions of 3.35 mg / 0.5 mL, 2.5 mg / 0.5 mL, 1.65 mg / 0.5 mL, and 1.0 mg / 0.5 mL in turn. Take four new test tubes and add 0.5 mL of the above glucose standard diluted solution to each test tube, then add 1.0 mL of ChCl_Gly solvent and mix well to prepare glucose standard test tubes.

[0225] (7) Prepare enzyme assay tubes:

[0226] Take 1 mL of each of the two cellulases (Celluclast 1.5L or Ctec2) and dilute them in 19 mL of ChCl_Gly solvent to prepare a 1:20 working enzyme solution. Then, dilute the 1:20 working enzyme solutions of the two different cellulases a second time according to the dilution sequence of Celluclast 1.5L shown in Table 4 and the dilution sequence of CTec2 shown in Table 5 to obtain the corresponding enzyme concentrations (dimensionless). Take 5 test tubes and 5 filter paper strips, fold the filter paper strips horizontally and place them into each test tube. Add 1.0 mL of ChCl_Gly solvent to the test tubes, and the solvent should be used to saturate the filter paper strips as much as possible. Seal the 5 test tubes and place them in a constant temperature water bath shaker at 50-70℃ and 120-160 rpm for 30 minutes for preheating. After preheating, take out 5 test tubes and add 0.5 mL of the diluted enzyme solution (numbered 1-5 in Table 4 / 1-5 in Table 5) to each of the 5 test tubes, mix well, and you will have 5 enzyme assay test tubes. It is important to note that each cellulase must be diluted at least twice. One dilution will release slightly more than 2.0 mg of glucose (absolute amount), while the other will release slightly less than 2.0 mg of glucose. The target values ​​for these two dilutions are 2.1 and 1.9 mg of glucose, respectively. Due to the nature of the enzyme, these targets may be difficult to achieve, therefore additional dilutions are necessary.

[0227] (8) Perform parallel reactions on all test tubes:

[0228] Place a total of 12 test tubes (3 blank and control tubes, 4 glucose standard tubes, and 5 enzyme assay tubes) into a shaker and shake for 60 minutes at 50–70°C and 120–160 rpm.

[0229] (9) Disintegrate all test tubes with water:

[0230] After the reaction is complete, remove all test tubes, add 4.5 mL of distilled water to each tube, and shake each tube 70 times to carry out the disintegration reaction until the solution in the test tube becomes a clear and homogeneous aqueous solution.

[0231] (10) Determination of absorbance values ​​for all test tube disintegration solutions:

[0232] Take 1.5 mL of the supernatant from each of the blank and control tubes, glucose standard tubes, and enzyme assay tubes, and transfer them to a 10 mL tube. Add 3 mL of DNS reagent and mix well, resulting in a total solution volume of 4.5 mL. Insert all tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to completely cover the mixed solution in the tubes. The blank, control, glucose standard, and enzyme assay solutions should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the colorimetric tubes (blank, control, glucose standard, and enzyme assay) with distilled water. The dilution ratio should be explored based on actual conditions; the principle is that the colorimetric solution should be diluted to a concentration equal to the A value of the previous solution. 540nm The absorbance should be 0.1–1.0 A. The dilution ratio should be consistent across all test tubes. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance of the enzyme control from the absorbance value to obtain the colorimetric absorbance of the enzyme test tube.

[0233] (11) Calculate the enzyme activity FPU on filter paper

[0234] The absolute glucose concentration (mg / 0.5mL) was used to determine the concentration of glucose in A. 540nm Plot a linear glucose standard curve. The data on the standard curve should be as close as possible to the calculated fitted straight line, and the correlation coefficient of the straight line should be in the range of 0.9900-1.0000. Verify this standard curve using a standard solution (CVS) with a known glucose concentration. If the solution falls exactly on the curve, the standard curve is correct.

[0235] Using this standard curve, the absorbance of the sample tube (after subtracting the enzyme control) is converted into the absolute amount of glucose (= the number of milligrams of glucose produced during the reaction).

[0236] By plotting the relationship between glucose release and enzyme concentration on semi-logarithmic graph paper, the enzyme concentration required to release exactly 2.0 mg of glucose can be estimated.

[0237] Convert the dilution used to the enzyme concentration using the following formula:

[0238]

[0239] Calculate the FPU using the following formula:

[0240]

[0241] Note: To find the required enzyme concentration, take two data points that are very close to 2.0 mg (one slightly more and one slightly less), connect the two points and draw a straight line. Find the enzyme concentration that corresponds to the precise release of 2.0 mg on this line.

[0242] Because FPU analysis is non-linear, using the universal International Units of Enzyme Activity (IU / mL) is incorrect, as this unit is based on the initial rate, i.e., a linear reaction, where the product is produced at the same rate every minute of the reaction. Therefore, the unit should be changed to units / mL.

[0243] Derivation of FPU vitality calculation:

[0244] The unit of FPU is based on International Units (IU).

[0245] Substrate conversion of 1 IU = 1 μmol / min

[0246] =2.0 μmol / min of glucose (reducing sugar is glucose) is formed in the hydrolysis reaction.

[0247] =0.18 mg / min, the product is glucose

[0248] At this critical dilution, the absolute release of glucose in the FPU assay is 2.0 mg.

[0249] 2.0 mg glucose = 2.0 / 0.18 × 0.5 × 60 μmol / (min × mL) = 0.37 μmol / (min × mL)

[0250] Therefore, the amount of enzyme that releases 2.0 mg of glucose in the FPU reaction (=critical enzyme concentration) is estimated to contain 0.37 activity units.

[0251] (12) Detailed examples and data:

[0252] 12.1 Steps for calculating the enzyme activity of filter paper used for saccharified cellulase

[0253] Same as step (11) of application example 1.

[0254] 12.2 Dilute the working enzyme solution according to the specified ratio.

[0255] As shown in Tables 4 and 5, the 1:20 working enzyme solutions used in the tables below were all diluted in ChCl_Gly solvent at a ratio of 1:20 as described in step (7) of Application Example 1. The dilution ratios and enzyme concentrations after dilution for the two enzymes (Celluclast 1.5L and Ctec2) are as follows:

[0256] Table 4. Dilution sequences of Celluclast 1.5L

[0257]

[0258]

[0259] Table 5. Dilution sequences of CTec2

[0260]

[0261] The term "enzyme concentration" is used to indicate the proportion of the original enzyme solution in the diluted enzyme solution (added to the assay mixture). For example, a 1:20 enzyme working stock solution diluted at a ratio of 1:10 has an "enzyme concentration" of 0.005.

[0262] 12.3 Quantitative Analysis of Sugar Released by Cellulase in ChCl_Gly Solvent

[0263] As shown in Tables 6 and 7.

[0264] Table 6. Sugar release amount of Cellulase 1.5 in ChCl-Gly solvent

[0265]

[0266]

[0267] Table 7 Sugar release amount of CTec2 in ChCl_Gly solvent

[0268]

[0269] 12.4 Calculation of filter paper enzyme activity (FPU) of cellulase in ChCl-Gly solvent

[0270] Using the amount of sugar released from each enzyme tube as the x-axis and the ratio of enzyme dilution as the y-axis (semi-logarithmic coordinate), plot the graph using Origin software to obtain the dilution ratio at which 2.0 mg of sugar is released. Then, substitute the values ​​into the formula in 12.1 to calculate the activity.

[0271] Figure 11 The figure shows a semi-logarithmic graph of glucose release from the Cellulase 1.5L-ChCl_Gly system at 50℃:

[0272] At an enzyme concentration of 0.0121, exactly 2.0 mg of glucose can be released.

[0273] according to Conclusion:

[0274] FPU = 0.37 / 0.0121 = 30.58

[0275] Figure 12 The figure shows a semi-logarithmic graph of glucose release from the Cellulase 1.5L-ChCl_Gly system at 60℃:

[0276] At an enzyme concentration of 0.015, exactly 2.0 mg of glucose can be released.

[0277] according to Conclusion:

[0278] FPU = 0.37 / 0.015 = 24.67

[0279] Figure 13 The figure shows a semi-logarithmic graph of glucose release from the Cellulase 1.5L-ChCl_Gly system at 70℃:

[0280] At an enzyme concentration of 0.0081, exactly 2.0 mg of glucose can be released.

[0281] according to Conclusion:

[0282] FPU = 0.37 / 0.0081 = 45.68

[0283] Figure 14 The figure shows a semi-logarithmic plot of glucose release from the Ctec2-ChCl_Gly system at 50℃:

[0284] At an enzyme concentration of 0.00253, exactly 2.0 mg of glucose can be released.

[0285] according to Conclusion:

[0286] FPU = 0.37 / 0.00253 = 146.24

[0287] Figure 15 The figure shows a semi-logarithmic plot of glucose release from the Ctec2-ChCl_Gly system at 60℃:

[0288] At an enzyme concentration of 0.0021, exactly 2.0 mg of glucose can be released.

[0289] according to Conclusion:

[0290] FPU = 0.37 / 0.0021 = 176.19

[0291] As can be seen from the glucose release data in Table 7, Ctec 2 could not be diluted to 2.0 mg of sugar in ChCl_Gly solvent at 70℃, and therefore did not have activity. Thus, it can be concluded that the Ctec 2-ChCl_Gly system was inactivated at 70℃.

[0292] Application Example 2

[0293] The activity of endoglucanase (EG) in ChCl-Gly solvent was determined using the DNS method of this invention.

[0294] The specific operating method is as follows:

[0295] (1) Preparation of 3,5-dinitrosalicylic acid DNS reagent:

[0296] Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to approximately 400mL of distilled water. Heat and stir moderately until completely dissolved; the solution will be grass-green at this point. Next, add 7.0g of NaOH to the above solution and stir at room temperature until completely dissolved; the solution will then be orange-red. It is important to note that the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially; they cannot be added simultaneously or in any order. Then, add 108g of potassium sodium tartrate (Rochelle salt), 2.7mL of phenol (liquid phenol melted at 50℃), and 2.9g of sodium metabisulfite to the orange-red solution, stirring thoroughly until all components are dissolved. It should be noted that after adding the last three components, a large number of suspended particles may appear in the solution. Continue stirring for at least one hour until all suspended particles disappear, resulting in a clear orange-red solution. Dilute to 500mL with distilled water, store in a brown bottle protected from light, and let stand for one week before use. The shelf life is 6 months.

[0297] (2) Preparation of the hydrophilic eutectic solvent ChCl_Gly:

[0298] Add the corresponding masses of choline chloride and glycerol to a round-bottom flask in a 1:2 molar ratio. Heat and stir at 60–65°C, avoiding the addition of water as much as possible, until both components transform into a homogeneous, transparent liquid. Stop stirring and heating. If the liquid remains after cooling to room temperature, the ChCl_Gly solvent has been successfully prepared.

[0299] (3) Preparation of glucose stock solution (Glucose-ChCl_Gly) stock (2mg / mL):

[0300] Pre-fill a conical flask with 50 mL of the prepared ChCl_Gly solvent, then weigh approximately 0.1 g of glucose and add it to the flask. Stir at room temperature for at least 24 hours until the solid is completely dissolved and the solution becomes transparent, thus obtaining a glucose stock solution with a concentration of 2 mg / mL, namely, Glucose-ChCl_Gly. stock .

[0301] (4) Preparation of CMC substrate solution for enzyme activity assay: 2% CMC-ChCl_Gly solution:

[0302] 250 mL of the prepared ChCl_Gly solvent was pre-filled into an Erlenmeyer flask, and then about 5 mg of CMC (carboxymethyl cellulose, degree of substitution = 0.7) solid powder was weighed and poured into it. The mixture was stirred at room temperature for more than 24 hours until the solid was completely dissolved and the solution became transparent, thus obtaining a 2% (w / v) CMC-ChCl_Gly solution.

[0303] (5) Prepare the spectral zero-sum enzyme control test tube:

[0304] The spectral zero tube is a tube containing only 1.5 mL of CMC substrate solution (2% CMC-ChCl_Gly solution), while the enzyme control tube is a tube containing both 1.0 mL of ChCl_Gly solvent and 0.5 mL of enzyme diluted sample.

[0305] (6) Prepare glucose standard test tubes:

[0306] Take four test tubes and add 1 mL of the glucose stock solution (2 mg / mL) prepared in step (3) to each. Then, add 0 mL (undiluted), 0.5 mL, 1.0 mL, and 3.0 mL of ChCl_Gly solvent to each test tube in turn, and mix well to obtain glucose standard diluted solutions of 1.0 mg / 0.5 mL, 0.67 mg / 0.5 mL, 0.5 mg / 0.5 mL, and 0.25 mg / 0.5 mL in turn. Take four new test tubes and add 0.5 mL of the above glucose standard diluted solution to each test tube, then add 1.0 mL of ChCl_Gly solvent and mix well to prepare glucose standard test tubes.

[0307] (7) Prepare enzyme assay tubes:

[0308] Take 3-5 test tubes and add 1.0 mL of CMC substrate solution to each tube. Seal the test tubes and place them in a constant temperature water bath shaker at 50-70°C and 120-160 rpm for 30 minutes. Add 0.5 mL of enzyme dilution in ChCl-Gly solvent to each test tube. Each enzyme sample must be diluted at least twice at different ratios. Under the reaction conditions, the glucose release (reducing sugar is glucose) of one enzyme dilution should be greater than 0.5 mg (absolute amount), and the glucose release of another enzyme dilution should be less than 0.5 mg. Mix the above solution components thoroughly to obtain the enzyme assay test tubes.

[0309] (8) Perform parallel reactions on all test tubes:

[0310] Place the spectral zero test tube, enzyme control test tube, glucose standard test tube, and enzyme assay test tube together in a shaker and shake for 60 minutes at 50–70°C and 120–160 rpm.

[0311] (9) Disintegrate all test tubes with water:

[0312] After the reaction is complete, remove all test tubes, add 4.5 mL of distilled water to each tube, and shake each tube 70 times to carry out the disintegration reaction until the solution in the test tube becomes a clear and homogeneous aqueous solution.

[0313] (10) Determination of absorbance values ​​for all test tube disintegration solutions:

[0314] Take 1.5 mL of the supernatant from each of the spectral zero, enzyme control, glucose standard, and enzyme assay tubes, and transfer them to a 10 mL tube. Add 3 mL of DNS reagent and mix well, resulting in a total solution volume of 4.5 mL. Insert all tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the mixed solution in each tube. The blank solution and standard solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the tubes that have undergone color development (spectral zero, enzyme control, glucose standard, enzyme assay) with distilled water. The dilution ratio should be explored based on actual conditions; the principle is that the color development solution should be diluted to a concentration equal to the A value of the standard solution. 540nm The absorbance should be 0.1–1.0 A. The dilution ratio should be consistent across all test tubes. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance of the enzyme control from the absorbance value to obtain the colorimetric absorbance of the enzyme test tube.

[0315] (11) Calculate the enzyme activity CMCU of endoglucanase (EG).

[0316] The absolute glucose concentration (mg / 0.5mL) was used to determine the concentration of glucose in A. 540nmPlot a linear glucose standard curve.

[0317] Using this standard curve, the absorbance of the sample tube (after subtracting the enzyme control) is converted into the absolute amount of glucose (= the number of milligrams of glucose produced during the reaction).

[0318] Convert the dilution used to enzyme concentration.

[0319]

[0320] By plotting the relationship between glucose release and enzyme concentration on semi-logarithmic graph paper, the enzyme concentration that releases exactly 0.5 mg of glucose can be estimated.

[0321] Calculate CMCU using the following formula:

[0322]

[0323] Note: To find the required enzyme concentration, take two data points that are very close to 0.5 mg (one slightly more and one slightly less), connect the two points and draw a straight line. Find the enzyme concentration that corresponds to the precise release of 0.5 mg on this line.

[0324] Because CMCU analysis is non-linear, using the universal International Units of Enzyme Activity (IU / mL) is incorrect, as this unit is based on the initial rate, i.e., a linear reaction, where the product is produced at the same rate every minute of the reaction. Therefore, the unit should be changed to units / mL.

[0325] Derivation of CMCU vitality calculation:

[0326] The units of CMCU are based on International Units (IU), and its calculation is similar to that of FPU vitality.

[0327] 1 IU = 1 μmol / min releases hydrolysis products

[0328] =0.18 mg / min when the product is glucose

[0329] The critical glucose threshold in the CMC test is 0.5 mg.

[0330] 0.5 mg glucose = 0.5 / 0.18 μmol

[0331] This amount of glucose, 0.5 mg, is produced within 30 minutes, and also during the CMC reaction:

[0332]

[0333] Therefore, the amount of enzyme that releases 0.5 mg of glucose in the CMC reaction (=critical enzyme concentration, ml·ml) -1 The estimated activity value is 0.185 IU, and:

[0334]

[0335] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

[0336] Application Example 3

[0337] Determination of total reducing sugars in extracts of food raw materials (or food residues) by using the DNS method of this invention—taking the extraction of Houttuynia cordata by ChCl-Gly solvent as an example.

[0338] The specific operating method is as follows:

[0339] (1) Preparation of 3,5-dinitrosalicylic acid DNS reagent:

[0340] Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to approximately 400mL of distilled water. Heat and stir moderately until completely dissolved; the solution will be grass-green at this point. Next, add 7.0g of NaOH to the above solution and stir at room temperature until completely dissolved; the solution will then be orange-red. It is important to note that the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially; they cannot be added simultaneously or in any order. Then, add 108g of potassium sodium tartrate (Rochelle salt), 2.7mL of phenol (liquid phenol dissolved at 50℃), and 2.9g of sodium metabisulfite to the orange-red solution, stirring thoroughly until all components are dissolved. It should be noted that after adding the last three components, a large number of suspended particles may appear in the solution. Continue stirring for at least one hour until all suspended particles disappear, resulting in a clear orange-red solution. Dilute to 500mL with distilled water, store in a brown bottle protected from light, and let stand for one week before use. The shelf life is 6 months.

[0341] (2) Preparation of the hydrophilic eutectic solvent ChCl_Gly:

[0342] Add the corresponding masses of choline chloride and glycerol to a round-bottom flask in a 1:2 molar ratio. Heat and stir at 60–65°C, avoiding the addition of water as much as possible, until both components transform into a homogeneous, transparent liquid. Stop stirring and heating. If the liquid remains after cooling to room temperature, the ChCl_Gly solvent has been successfully prepared.

[0343] (3) Preparation of glucose stock solution (Glucose-ChCl_Gly) stock :

[0344] Pre-fill an Erlenmeyer flask with 50 mL of the prepared ChCl_Gly solvent, then weigh approximately 1 g of glucose and add it to the flask. Stir at room temperature for at least 24 hours until the solid is completely dissolved and the solution becomes transparent, thus obtaining a 20 mg / mL glucose stock solution (Glucose-ChCl_Gly). stock .

[0345] (4) Preparation of glucose standard solution (Glucose-ChCl_Gly) stand :

[0346] After step (3) is completed, the glucose standard solution Glucose-ChCl_Gly stand (0.5-4.0 mg / L) were respectively measured by taking a certain amount of glucose stock solution (Glucose-ChCl_Gly). stock The solution is obtained by diluting the volumetric flask with the appropriate amount of ChCl-Gly solvent according to a certain ratio.

[0347] (5) Glucose standard solution (Glucose-ChCl_Gly) stand Disintegration upon adding water:

[0348] Add glucose standard solutions of different concentrations (Glucose-ChCl_Gly) to a 10 mL graduated cylinder. stand Add distilled water to the 4 mL mark, and shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, thus allowing the Glucose-ChCl_Gly... stand The eutectic phase disintegrates until it disappears completely, eventually forming a transparent and homogeneous aqueous solution—the glucose standard solution disintegration solution.

[0349] (6) Disintegration of ChCl-Gly solvent upon addition of water:

[0350] Add ChCl_Gly solvent to a 10mL graduated cylinder to the 1mL mark, then add distilled water to the 4mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the eutectic phase of ChCl_Gly to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the solvent blank disintegration solution.

[0351] (7) Determination of absorbance of the disintegration solution:

[0352] Take 1.5 mL of the supernatant from both the glucose standard solution disintegration buffer and the solvent blank disintegration buffer, transfer them to 10 mL test tubes, and add 3 mL of DNS reagent. Mix well, resulting in a total solution volume of 4.5 mL. Insert all test tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the mixed solution in the test tubes. The blank solution and standard solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the test tubes (blank and standard) with distilled water. The dilution ratio should be explored based on actual conditions; the principle is that the dilution ratio of the colorimetric solution should be such that the A... 540nm The absorbance should be 0.1–1.0 A. The dilution ratio of the blank test tube and the standard test tube should be consistent. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance value of the blank test tube from the absorbance value of the solvent test tube to obtain the colorimetric absorbance value of the standard test tube.

[0353] (8) Plot the standard curve of glucose absorbance:

[0354] A scatter plot was created with the concentration of the glucose standard solution (mg / mL) on the x-axis and the absorbance at 540 nm (after subtracting the solvent blank) on the y-axis. A linear fit was then applied to the scatter plot, and the fitted curve passed through the point (0, 0). The resulting glucose absorbance standard curve is shown below. Figure 7 As shown.

[0355] Glucose standard solution (Glucose-ChCl_Gly) stand The relationship between the concentration of glucose and the absorbance at 540 nm is given by the function w = A / 0.06192, where w is the glucose concentration in mg / mL and A is the colorimetric absorbance obtained by subtracting the absorbance of the solvent from the absorbance of the glucose standard solution disintegration at 540 nm.

[0356] (9) Preparation of ChCl-Gly solvent extract of Houttuynia cordata: Houttuynia cordata-ChCl-Gly extracted :

[0357] Wash and air-dry (or oven-dry, or freeze-dry) the Houttuynia cordata sample beforehand, grind it into powder in a grinder, and pass it through a 75-mesh sieve to separate the coarse and fine powders. Place the Houttuynia cordata powder (or coarse powder) and ChCl_Gly solvent in a 50℃ oven for 24 hours to ensure drying. Add the dried Houttuynia cordata powder (or coarse powder) and ChCl_Gly solvent at a solid-liquid ratio of 1:10 (S:L) to a 25mL pressure-resistant colorimetric tube, place a heat-resistant stir bar in the tube, and react in an oil bath at 90–120℃ for 2–6 hours under magnetic stirring. After the reaction is complete, immediately place the tube in an ice-water bath to terminate the reaction, and then separate the solid and liquid components by vacuum filtration, collecting the liquid portion, which is the ChCl_Gly solvent extract of the Houttuynia cordata powder.

[0358] (10) ChCl-Gly solvent extract of Houttuynia cordata Houttuynia cordata-ChCl-Gly extracted Disintegration upon adding water:

[0359] Add the ChCl-Gly solvent extract of Houttuynia cordata to a 10mL graduated cylinder. extracted Add distilled water to the 1mL mark, then add distilled water to the 4mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the non-aqueous phase to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the disintegration solution of the ChCl_Gly solvent extract of Houttuynia cordata.

[0360] (11) Determination of absorbance of disintegrating solution of ChCl-Gly solvent extract of Houttuynia cordata:

[0361] Take 1.5 mL of the supernatant from the disintegration buffer of the ChCl-Gly solvent extract and the solvent blank disintegration buffer of Houttuynia cordata, respectively, and transfer them to 10 mL test tubes. Add 3 mL of DNS reagent and mix well. The total volume of the mixed solution is 4.5 mL. Insert all test tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level in the bath is sufficient to cover the height of the mixed solution in the test tubes. The blank solution and standard solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the test tubes (blank and saturated) with distilled water. The dilution ratio should be explored based on the actual situation, but the principle is that the A value of the colorimetric solution should be diluted with distilled water. 540nm The absorbance should be 0.1–1.0 A. The dilution ratio of the blank test tube and the saturated test tube should be consistent. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance value of the blank test tube from the absorbance value of the solvent test tube to obtain the colorimetric absorbance value of the extract test tube.

[0362] (12) Quantitative calculation of total reducing sugar in the ChCl-Gly solvent extract of Houttuynia cordata:

[0363] The formula for calculating the total reducing sugar concentration in the ChCl-Gly solvent extract of Houttuynia cordata is w = A / 0.06192. Where w is the total reducing sugar concentration in mg / mL; A is the absorbance value obtained by subtracting the absorbance value of the solvent-disintegrated solution from the absorbance value of the disintegrated ChCl-Gly solvent extract of Houttuynia cordata at 540 nm.

[0364] The formula for calculating the total reducing sugar mass is W. TRS =w×v=A / 0.06192×10. Where W TRS The total reducing sugar mass is expressed in grams; w and A are as described above; v is the total volume of the ChCl-Gly solvent, which is 10 mL in this example.

[0365] The formula for calculating the extraction rate of total reducing sugars is:

[0366]

[0367] Where Y TRS Total reducing sugar extraction rate, %; W TRS Total reducing sugar mass, in grams; W S The dry weight of the Houttuynia cordata powder sample is in grams; C C The relative content of cellulose in the Houttuynia cordata powder sample, by mass ratio, %; C H , where is the relative content of hemicellulose in the Houttuynia cordata powder sample, by mass ratio, %; 0.88 is the dehydration correction factor when converting the measured pentose (pentose, such as xylose) concentration to hemicellulose concentration; 0.90 is the dehydration correction factor when converting the measured hexose (hexose, such as glucose) concentration to cellulose concentration.

[0368] (13) Although this embodiment uses the determination of total reducing sugar in the ChCl-Gly solvent extract of Houttuynia cordata as an example, it can be further extended to determine the concentration, mass, and extraction rate of total reducing sugar in the extract of any food raw material (or food residue) extracted with any hydrophilic eutectic solvent. This example is an example of food raw material. In addition to Houttuynia cordata listed in this example, it can also be various plant-based food raw materials such as onion, white fungus, ginger, garlic, Sichuan pepper, and avocado. Food residue can be soybean residue, coffee grounds, Sichuan pepper seeds, etc. The hydrophilic eutectic solvent can be selected from the set of hydrophilic solvents in the eutectic solvent family and the natural eutectic solvent family.

[0369] Application Example 4

[0370] Determination of total reducing sugars in pretreated solutions of lignocellulosic biomass pretreated with hi(NA)DES solvent using the DNS method of this invention—taking rapeseed straw pretreated with ChCl-Gly solvent as an example.

[0371] The specific operating method is as follows:

[0372] (1) Preparation of 3,5-dinitrosalicylic acid DNS reagent:

[0373] Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to approximately 400mL of distilled water. Heat and stir moderately until completely dissolved; the solution will be grass-green at this point. Next, add 7.0g of NaOH to the above solution and stir at room temperature until completely dissolved; the solution will then be orange-red. It is important to note that the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially; they cannot be added simultaneously or in any order. Then, add 108g of potassium sodium tartrate (Rochelle salt), 2.7mL of phenol (liquid phenol dissolved at 50℃), and 2.9g of sodium metabisulfite to the orange-red solution, stirring thoroughly until all components are dissolved. It should be noted that after adding the last three components, a large number of suspended particles may appear in the solution. Continue stirring for at least one hour until all suspended particles disappear, resulting in a clear orange-red solution. Dilute to 500mL with distilled water, store in a brown bottle protected from light, and let stand for one week before use. The shelf life is 6 months.

[0374] (2) Preparation of the hydrophilic eutectic solvent ChCl_Gly:

[0375] Add the corresponding masses of choline chloride and glycerol to a round-bottom flask in a 1:2 molar ratio. Heat and stir at 60–65°C, avoiding the addition of water as much as possible, until both components transform into a homogeneous, transparent liquid. Stop stirring and heating. If the liquid remains after cooling to room temperature, the ChCl_Gly solvent has been successfully prepared.

[0376] (3) Preparation of glucose stock solution (Glucose-ChCl_Gly) stock :

[0377] Pre-fill an Erlenmeyer flask with 50 mL of the prepared ChCl_Gly solvent, then weigh approximately 1 g of glucose and add it to the flask. Stir at room temperature for at least 24 hours until the solid is completely dissolved and the solution becomes transparent, thus obtaining a 20 mg / mL glucose stock solution (Glucose-ChCl_Gly). stock .

[0378] (4) Preparation of glucose standard solution (Glucose-ChCl_Gly) stand :

[0379] After step (3) is completed, the glucose standard solution Glucose-ChCl_Gly stand (0.5-4.0 mg / L) were respectively measured by taking a certain amount of glucose stock solution (Glucose-ChCl_Gly). stock The solution is obtained by diluting the volumetric flask with the appropriate amount of ChCl-Gly solvent according to a certain ratio.

[0380] (5) Glucose standard solution (Glucose-ChCl_Gly) stand Disintegration upon adding water:

[0381] Add glucose standard solutions of different concentrations (Glucose-ChCl_Gly) to a 10 mL graduated cylinder. stand Add distilled water to the 4 mL mark, and shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, thus allowing the Glucose-ChCl_Gly... stand The eutectic phase disintegrates until it disappears completely, eventually forming a transparent and homogeneous aqueous solution—the glucose standard solution disintegration solution.

[0382] (6) Disintegration of ChCl-Gly solvent upon addition of water:

[0383] Add ChCl_Gly solvent to a 10mL graduated cylinder to the 1mL mark, then add distilled water to the 4mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the eutectic phase of ChCl_Gly to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the solvent blank disintegration solution.

[0384] (7) Determination of absorbance of the disintegration solution:

[0385] Take 1.5 mL of the supernatant from both the glucose standard solution disintegration buffer and the solvent blank disintegration buffer, transfer them to 10 mL test tubes, and add 3 mL of DNS reagent. Mix well, resulting in a total solution volume of 4.5 mL. Insert all test tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the mixed solution in the test tubes. The blank solution and standard solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the test tubes (blank and standard) with distilled water. The dilution ratio should be explored based on actual conditions; the principle is that the dilution ratio of the colorimetric solution should be such that the A... 540nm The absorbance should be 0.1–1.0 A. The dilution ratio of the blank test tube and the standard test tube should be consistent. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance value of the blank test tube from the absorbance value of the solvent test tube to obtain the colorimetric absorbance value of the standard test tube.

[0386] (8) Plot the standard curve of glucose absorbance:

[0387] A scatter plot was created with the concentration of the glucose standard solution (mg / mL) on the x-axis and the absorbance at 540 nm (after subtracting the solvent blank) on the y-axis. A linear fit was then applied to the scatter plot, and the fitted curve passed through the point (0, 0). The resulting glucose absorbance standard curve is shown below. Figure 7 As shown.

[0388] Glucose standard solution (Glucose-ChCl_Gly) stand The relationship between the concentration of glucose and the absorbance at 540 nm is given by the function w = A / 0.06192, where w is the glucose concentration in mg / mL and A is the colorimetric absorbance obtained by subtracting the absorbance of the solvent from the absorbance of the glucose standard solution disintegration at 540 nm.

[0389] (9) Preparation of ChCl-Gly solvent pretreatment solution of rapeseed straw: rapeseed straw-ChCl-Gly pretreated :

[0390] Wash and air-dry (or oven-dry or freeze-dry) the rapeseed straw sample beforehand, grind it into powder in a grinder, and pass it through a 75-mesh sieve to separate the coarse and fine powders. Place the rapeseed straw fine powder (or coarse powder) and ChCl_Gly solvent in a 50℃ oven for 24 hours to ensure drying. Add the dried rapeseed straw fine powder (or coarse powder) and ChCl_Gly solvent at a solid-liquid ratio of 1–4:20 (S:L) (i.e., 1–4 g of rapeseed straw powder to 20 mL of ChCl_Gly solvent) into a 50 mL pressure-resistant screw-top blue-capped bottle, place a high-temperature resistant stir bar inside, and react in an oil bath at 90–120℃ for 2–6 hours under magnetic stirring. After the reaction is complete, immediately place the bottle in an ice-water bath to terminate the reaction, and then separate the solid and liquid components by vacuum filtration, collecting the liquid portion, which is the ChCl_Gly solvent pretreated solution of the rapeseed straw powder.

[0391] (10) Pretreatment solution of rapeseed straw with ChCl-Gly solvent: rapeseed straw-ChCl-Gly pretreated Disintegration upon adding water:

[0392] Add rapeseed straw ChCl_Gly solvent pretreatment solution to a 10mL graduated cylinder. pretreated Add distilled water to the 1 mL mark, then add distilled water to the 4 mL mark. Shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, causing the non-aqueous phase to disintegrate until it completely disappears, finally forming a transparent and homogeneous aqueous solution—the disintegration solution of the rapeseed straw ChCl_Gly solvent pretreatment solution.

[0393] (11) Determination of absorbance of disintegrating solution of rapeseed straw pretreated with ChCl-Gly solvent:

[0394] Take 1.5 mL of the supernatant from the ChCl-Gly solvent pretreatment solution disintegration buffer and the solvent blank disintegration buffer, respectively, and transfer them to 10 mL test tubes. Add 3 mL of DNS reagent and mix well, resulting in a total solution volume of 4.5 mL. Insert all test tubes into a test tube rack, then place them in a cylindrical stainless steel mesh basket. Boil precisely for 5 minutes in a vigorously boiling water bath, ensuring the water level is sufficient to cover the mixed solution in the test tubes. The blank solution and standard solution should be boiled together. After boiling, transfer to an ice-water bath to cool to room temperature. Then, dilute all the test tubes (blank and saturated) with distilled water. The dilution ratio should be explored based on actual conditions, but the principle is that the dilution ratio of the colorimetric solution should be such that the A... 540nm The absorbance should be 0.1–1.0 A. The dilution ratio of the blank test tube and the saturated test tube should be consistent. Pour the diluted colorimetric solution into a glass cuvette and measure the absorbance at 540 nm using a UV-Vis spectrophotometer. Subtract the absorbance value of the blank test tube from the absorbance value of the solvent test tube to obtain the colorimetric absorbance value of the pretreatment solution test tube.

[0395] (12) Quantitative calculation of total reducing sugar in the ChCl-Gly solvent pretreated solution of rapeseed straw:

[0396] The formula for calculating the total reducing sugar concentration in the ChCl_Gly solvent pretreatment solution of rapeseed straw is w = A / 0.06192. Where w is the total reducing sugar concentration in mg / mL; A is the absorbance value obtained by subtracting the absorbance value of the solvent-disintegrated solution from the absorbance value of the disintegrated solution of the ChCl_Gly solvent pretreatment solution of rapeseed straw at 540 nm.

[0397] The formula for calculating the total reducing sugar mass is W. TRS =w×v=A / 0.06192×10. Where W TRS The total reducing sugar mass is expressed in grams; w and A are as described above; v is the total volume of the ChCl-Gly solvent, which is 10 mL in this example.

[0398] The formula for calculating the extraction rate of total reducing sugars is:

[0399]

[0400] Where Y TRS Total reducing sugar extraction rate, %; W TRS Total reducing sugar mass, in grams; W S The dry weight of the rapeseed straw powder sample is in grams; C C The relative content of cellulose in the rapeseed straw powder sample, by mass ratio, %; C H1. Relative content of hemicellulose in rapeseed straw powder sample, mass ratio, %; 0.88 is the dehydration correction factor when converting the measured pentose (pentose, such as xylose) concentration to hemicellulose concentration; 0.90 is the dehydration correction factor when converting the measured hexose (hexose, such as glucose) concentration to cellulose concentration.

[0401] (13) Although this embodiment uses the determination of total reducing sugar in the ChCl-Gly solvent pretreatment solution of rapeseed straw as an example, it can be further extended to determine the total reducing sugar concentration, mass, and extraction rate in the pretreatment solution of any lignocellulosic biomass raw material pretreated with any hydrophilic eutectic solvent. In addition to rapeseed straw selected in this example, it can also be various agricultural and forestry lignocellulosic biomass raw materials such as corn straw, rice straw, reeds, sugarcane bagasse, and citrus peel residue. The hydrophilic eutectic solvent can be selected from the set of hydrophilic solvents in the eutectic solvent family and the natural eutectic solvent family.

[0402] The embodiments of the present invention have achieved some positive results during the research and development or use process, and have indeed great advantages compared with the prior art. The following content describes the experimental process with data, charts and other information.

[0403] (1) The positive effects and significant advantages achieved by the embodiments of the present invention during the research and development process are illustrated below with examples in conjunction with Embodiment 2 and Application Embodiment 1:

[0404] Due to the successful implementation of Example 2, the inventors of this invention, for the first time, fully tracked the enzymatic saccharification kinetics of microcrystalline cellulose in an anhydrous, pure hiNADES solvent system, such as... Figure 10 As shown in the figure. Previous literature reports mostly describe the determination of enzymatic saccharification kinetics curves using the DNS method in an aqueous pseudo-DES solvent system, or the determination of sugar concentration by diluting the reaction solution 50-100 times with water and then using HPLC. However, the reducing power of the sugar cannot be determined.

[0405] Due to the successful implementation of Example 1, the inventors of this invention have for the first time achieved the determination of filter paper enzyme activity of commercial cellulase in an anhydrous, pure hiNADES solvent system, such as... Figure 16As shown, the inventors of this invention first measured the enzyme activity of cellulase 1.5L in two different hiNADES solvents, ChCl_Gly and ChCl_LA, at three different temperatures: 50°C, 60°C, and 70°C. The results showed that the two hiNADES solvents, ChCl_Gly and ChCl_LA, endowed Cellulase 1.5L with good thermostability, allowing it to remain active at all three temperatures. However, in citrate buffer, Cellulase 1.5L exhibited inactivation. This result is consistent with the previous conclusions of Zhao et al., who suggested that ChCl-based DES can improve the thermostability of proteases and lipases in biocatalytic transformations. On the other hand, in ChCl_Gly solvent, higher temperatures resulted in higher Cellulase 1.5L enzyme activity, with the highest activity observed at 70°C in ChCl_Gly solvent.

[0406] Supplementary explanation regarding the application of this patent in the process of commercialization / industrialization.

[0407] This patent can be commercialized in the following six ways:

[0408] (1) Synthesis and production of reagents and consumables - chemical output: In conjunction with the DNS method provided in this patent, the patent purchaser or institution can produce and synthesize standardized reagents, solvents and consumables for profit. The standardized reagents and solvents include DNS reagents, various types of phase change agents - water and alkaline solutions of various concentrations (including 0.00 to 4.00 mol / L NaOH / KOH / NH4OH solutions, etc.), various hiNADES solvents, and various reducing sugar standard solutions (concentration 0.01 to 10.00 mg / mL) based on different hiNADES solvents. The standardized consumables include conventional consumables such as beakers, graduated cylinders, volumetric flasks, graduated test tubes, pipettes, glass rods, magnetic stir bar, conical flasks, and glass cuvettes (which can be sold individually or in sets), as well as newly developed special consumables such as disintegration tubes.

[0409] (2) Establishment of a miniaturized standard laboratory – testing site output: Based on the samples, reagents, consumables, equipment, processes, and data processing methods described in the DNS method provided in this patent, a miniaturized standard laboratory shall be constructed. The standard laboratory shall have the following infrastructure: not less than 40m² 2The laboratory should include a suitable experimental area, fire alarm system, independent floor drains and drainage, fume hood, experimental workbench with water tank, reagent and consumable cabinet, oven, refrigerator, and computer. It should also be equipped with the following specialized equipment: UV-Vis spectrophotometer, constant temperature water bath / oil bath device, magnetic stirring and heating device, vacuum pump, and suction filtration device. Furthermore, it should be equipped with the following consumables: beakers, graduated cylinders, volumetric flasks, graduated test tubes, pipettes, glass rods, magnetic stir bar, conical flasks, glass cuvettes, Soxhlet extractors, Whatman filter paper, Buchner funnel filtration kits, and solvent filter kits. The completed standard laboratory can apply for certification from authoritative testing institutions and can undertake long-term testing tasks related to the determination of reducing sugars in hydrophilic eutectic solvents, issuing authoritative certified test reports.

[0410] (3) Development of large / micro / portable specialized equipment – ​​Equipment output: Based on the steps and characteristics of the DNS method provided in this patent, develop a specialized device for the determination of reducing sugar content in hydrophilic eutectic solvents, namely a high-throughput reducing sugar content analyzer. This device should be equipped with at least the following four modules: 1. A six-channel pump input module, capable of inputting DNS reagent, hiNADES solvent, reducing sugar-hiNADES standard solution, reducing sugar-hiNADES test solution, phase change agent, and distilled water via separate channels; 2. A disintegration sample preparation module, utilizing digital microfluidic technology to disintegrate the pumped phase change agent and the pumped hiNADES solvent / reducing sugar-hiNADES standard solution / reducing sugar-hiNADES test solution into a solid state. The instrument comprises four main components: 1. **Preparation of Disintegrating Solutions for Various Reducing Sugar Samples:** 1. **Preparation of Disintegrating Solutions:** A fixed-ratio mixing and phase transition process is performed to prepare disintegrating solutions for various reducing sugar samples. 2. **Reaction Color Development Module:** Utilizing digital microfluidic technology, the pumped DNS reagent is mixed with the disintegrating solution prepared in the disintegration module at a fixed ratio, followed by heating and timing to achieve full color development. 3. **Absorbance Testing Module:** Using digital microfluidic technology, distilled water is mixed with the color-developing solution in the reaction color development module at a fixed ratio to dilute the solution before reading. The diluted solution is then automatically added to a 96-well plate, and the detection wavelength is set to 540 nm for high-throughput reading of absorbance data from the 96-well plate. This high-throughput reducing sugar content analyzer is available for sale to research institutes, universities, government agencies, and enterprises that have long been engaged in related testing work.

[0411] (4) Development of a dedicated data processing system – software output: Based on the relevant steps and specific requirements of the data acquisition and processing of the “DNS” method provided in this patent, develop digital products such as codes, programs, and apps specifically for the determination of reducing sugar content in hydrophilic eutectic solvents. The above-mentioned digital products can be sold to research institutes, universities, government agencies, enterprises and other units that have long been engaged in related determination work.

[0412] (5) Building a professional testing team and related institutions - talent and service output: Combining the DNS method provided by this patent, the patent purchaser or institution can form a professional service team to provide paid services such as technical consultation, standardization training and business outsourcing to research institutes, universities, government agencies, enterprises and institutions that have been engaged in related testing work for a long time.

[0413] (6) Development of customized engineering process packages - Module output: Combining the DNS method provided in this patent, the patent purchaser or organization can customize engineering process packages for factories and enterprises, and integrate them into existing workshops or sections to match relevant pipelines, pipelines, materials and equipment, so as to meet the growing demand of factories and enterprises for real-time determination of reducing sugar content in hydrophilic eutectic solvents.

[0414] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for determining the reducing sugar content in a hydrophilic eutectic solvent system, characterized in that, The DNS'' method involves preparing and synthesizing DNS reagents, a hydrophilic eutectic solvent, a reducing sugar-hydrophilic eutectic solvent stock solution, and a reducing sugar-hydrophilic eutectic solvent standard solution. Water or an alkaline solution is used as a phase change agent. The reducing sugar test solution or cellulase hydrolysate based on the hydrophilic eutectic solvent is disintegrated by adding water or an alkaline solution. The DNS'' method is used to determine the levels of various reducing sugars. hi The standard curve in NADES solvent was used to track the performance of microcrystalline cellulose MCC. hi Enzymatic hydrolysis kinetics curves in NADES.

2. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 1, characterized in that, The method for determining the reducing sugar content in the hydrophilic eutectic solvent system includes the following steps: Step 1: Prepare 3,5-dinitrosalicylic acid (DNS) reagent; Step 2: Prepare a hydrophilic eutectic solvent hi (NA)DES; Step 3: Prepare reducing sugar stock solution RS- hi (NA)DES stock ; Step 4: Prepare reducing sugar standard solution RS- hi (NA)DES stand ; Step 5, prepare based on hi (NA)DES reducing sugar test solution RS- hi (NA)DES test ; Step 6, prepare based on hi (NA)DES cellulose hydrolysate RS- hi (NA)DES cellulase ; Step 7: Disintegrate the reducing sugar solution with water or with alkali. Step 8: Use a hydrophilic eutectic solvent hi (NA)DES disintegrates upon addition of water or alkali; Step 9: Mix the reducing sugar disintegration solution, the solvent blank disintegration solution, and the DNS reagent; Step 10: Perform a colorimetric reaction in the mixed solution in a boiling water bath; Step 11: Dilute the colorimetric solution; Step 12: Measure the absorbance of the colorimetric solution in a UV-Vis spectrophotometer; Step thirteen: Calculate the reducing sugar content.

3. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 2, characterized in that, The preparation of the 3,5-dinitrosalicylic acid (DNS) reagent in step one includes: (1) Weigh 3.75g of 3,5-dinitrosalicylic acid and add it to 400mL of distilled water. Heat and stir moderately until completely dissolved. The solution is grass green. Then add 7.0g of NaOH and stir at room temperature until completely dissolved. The solution is orange red. In this step, the 3,5-dinitrosalicylic acid and NaOH solid must be added in batches and sequentially. (2) Add 108g of Rochelle salt and 2.7mL of 50 to the orange-red solution. o Melt liquid phenol and 2.9g sodium metabisulfite at temperature C, stir thoroughly and dissolve all added components; (3) After adding the last three components, suspended particles will appear in the solution. After stirring continuously for more than 1 hour, all suspended particles will disappear and a clear orange-red solution will be formed. Add distilled water to make up to 500 mL, store in a brown bottle away from light, and use after one week. The shelf life is 6 months. The preparation of the hydrophilic eutectic solvent in step two hi (NA)DES includes: (1) Solvent synthesis molar ratio: using two choline chloride-based ChCl solutions. hi Taking (NA)DES as an example, one solvent is choline chloride-glycerol (ChCl-Gly), and the other is choline chloride-lactic acid (ChCl-LA). Both solvents use choline chloride as the hydrogen bond acceptor (HBA), while the hydrogen bond donors (HBD) are glycerol (Gly) and lactate (LA), respectively. hi The molar ratio of (NA)DES solvent HBA to HBD is 1:2; (2) Solvent synthesis step: The two components HBA and HBD are added directly to a round-bottom flask at a molar ratio of 1:2, and then heated in an oil bath at 60-65°C. o Heat and stir at C until the two components turn into a homogeneous and transparent liquid. Stop stirring and heating. If it is still a liquid when cooled to room temperature, the preparation is successful. (3) Solvent components: For the DES system, a eutectic solvent, common hydrogen bond acceptors include choline chloride (ChCl) and its analogues, onium salts, metal salts, and metal chlorides; for the NADES system, a natural eutectic solvent, the hydrogen bond acceptors and hydrogen bond donors used are organic compounds of natural origin produced from primary or secondary metabolic pathways; for hydrophilic eutectic solvents... hi The (NA)DES system selects components that enable the prepared solvent to be hydrophilic, based on the solvent component molecules.

4. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 2, characterized in that, Step three involves preparing the reducing sugar stock solution RS- hi (NA)DES stock include: 50 mL of the prepared solution was pre-filled into an Erlenmeyer flask. hi (NA)DES, then weigh 1g of reducing sugar and add it to the solution. Stir at room temperature for more than 24 hours until the solid is completely dissolved and the solution becomes transparent, yielding a series of RS- compounds with a concentration of 20mg / mL. hi (NA)DES stock solution; wherein the reducing sugars include glucose, xylose, fructose, galactose, mannose, rhamnose, fucose, cellobiose, maltose and lactose; Step four involves preparing the reducing sugar standard solution RS- hi (NA)DES stand include: RS- hi (NA)DES standard solution was obtained by measuring a certain amount of RS- hi (NA)DES stock solution is added to the volumetric flask in a certain proportion. hi The solution was obtained by diluting with (NA)DES solvent; wherein, the RS- hi The concentration range of (NA)DES standard solutions is 0.5 – 4.0 mg / mL.

5. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 2, characterized in that, The preparation in step five is based on hi (NA)DES reducing sugar test solution RS- hi (NA)DES test Includes: Weighing a certain mass of reducing sugar into an Erlenmeyer flask, and pouring in a certain volume of... hi (NA)DES should be stirred and dissolved at room temperature for more than 24 hours. If it is completely dissolved, it can be used directly. If there is some undissolved reducing sugar solid, it should be vacuum filtered through an organic membrane and the liquid portion collected for later use.

6. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 2, characterized in that, The preparation in step six is ​​based on hi (NA)DES cellulose hydrolysate RS- hi (NA)DES cellulase include: (1) Weigh 0.5g of cellulose raw material into a 25mL Erlenmeyer flask, and then add 10mL of cellulose raw material. hi (NA)DES solvent; containing cellulose raw materials and hi The conical flask containing (NA)DES solvent was placed in a constant temperature water bath shaker at 50-70°C. o C. Preheat at 120~160 rpm for 30 minutes; wherein, the cellulose raw material includes microcrystalline cellulose Avicel® PH-101. hi (NA)DES solvents include ChCl_Gly and ChCl_LA; (2) After preheating, add cellulase to the conical flask at a loading rate of 60~160 FPU cellulase / g cellulose; at 50~70 o C. The reaction is carried out at a speed of 120-160 rpm for 0-48 hours; wherein the cellulase includes NovozymeCelluclast 1.5L and Novozyme Ctec2; (3) Take the enzyme hydrolysate at different reaction times as RS- hi (NA)DES cellulase When the reaction time is reached, remove the corresponding conical flask, inactivate it in ice water, let it stand until the solid settles at the bottom of the conical flask, then take the supernatant, filter it and use it for later use.

7. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 2, characterized in that, Step seven, which involves the hydrolysis or alkaline disintegration of the reducing sugar solution, includes: (1) Disintegration with water: Add reducing sugar solution to a 10mL graduated cylinder to the 1mL mark, including RS- hi (NA)DES stand RS- hi (NA)DES test and RS- hi (NA)DES cellulase Add distilled water to the 4mL mark, and shake the graduated cylinder thoroughly to allow the water to impact the non-aqueous phase interface of the sample, thus allowing RS- hi The eutectic phase of (NA)DES disintegrates until it disappears completely, eventually forming a transparent and homogeneous aqueous solution. (2) Disintegration upon addition of alkaline solution: The oxidation reaction of DNS reagent with reducing sugars can only occur under alkaline conditions; when acidic solutions are used... hi When preparing reducing sugar solutions with (NA)DES solvent, an alkaline solution is used for disintegration to make the disintegrating solution alkaline. The alkaline solution includes NaOH solution. The optimal molar concentration of the alkaline solution is determined by simultaneously meeting the following two criteria: ① After adding the alkaline solution of this concentration, the disintegrating solution reacts with DNS reagent in an oxidation reaction without precipitation, and the colorimetric test tube turns purple-red; ② The absorbance of the disintegrating solution after adding the alkaline solution of this concentration should not be too high, and the absorbance value should be relatively low compared to other concentrations. The step eight involves using a hydrophilic eutectic solvent. hi The disintegration of (NA)DES by adding water or alkali includes adding a hydrophilic eutectic solvent to a 10 mL graduated cylinder. hi (NA)DES to the 1mL mark, when hi When (NA)DES is ChCl_Gly, add distilled water to the 4mL mark. hi When (NA)DES is ChCl_LA, add 2M NaOH solution to the 4mL mark; shake the graduated cylinder thoroughly to allow the water or NaOH solution to impact the non-aqueous phase interface of the sample, so that RS- hi The eutectic phase of (NA)DES disintegrates until it disappears completely, eventually forming a transparent and homogeneous aqueous solution.

8. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 2, characterized in that, Step nine, which involves mixing the reducing sugar disintegrating solution, the solvent blank disintegrating solution, and the DNS reagent, includes: (1) Mixing reducing sugar disintegration solution with DNS reagent: Transfer 1.5 mL of reducing sugar disintegration solution into a 10 mL test tube, add 3 mL of DNS reagent and mix well; the total volume of the mixed solution is 4.5 mL. (2) Mixing the solvent blank disintegration solution with DNS reagent: Transfer 1.5 mL of solvent blank disintegration solution into a 10 mL test tube, add 3 mL of DNS reagent and mix well. The total volume of the solution after mixing is 4.5 mL. The step ten, which involves performing a colorimetric reaction of the mixed solution in a boiling water bath, includes: inserting all test tubes into a test tube rack, then placing them into a cylindrical stainless steel mesh basket, and boiling them precisely for 5 minutes in a boiling water bath; the blank solution and the standard solution / test solution / enzyme digest should be boiled together; after boiling, transfer them to an ice water bath to cool to room temperature.

9. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 2, characterized in that, The dilution of the colorimetric solution in step eleven includes: diluting all the test tubes that have completed color development with distilled water. The dilution ratio should be explored based on the actual situation, but the principle is that the colorimetric solution should be diluted to A after dilution. 540nm The dilution ratio should be consistent between the blank test tube and the standard / test / enzyme digestion test tube, ranging from 0.1 to 1.0A. The step twelve, measuring the absorbance of the colorimetric solution in a UV-Vis spectrophotometer, includes: pouring the diluted colorimetric solution into a glass cuvette, setting the wavelength of the UV-Vis spectrophotometer to 540 nm, placing the cuvette in the cuvette holder and measuring the absorbance value. The colorimetric absorbance value of the standard / test / enzyme digestion tube is the absorbance difference after deducting the solvent blank.

10. The method for determining the reducing sugar content in a hydrophilic eutectic solvent system as described in claim 2, characterized in that, The calculation of reducing sugar content in step thirteen includes: (1) The absolute amount of reducing sugar relative to A 540nm Plot a linear standard curve. The data of the standard curve should be close to a straight line, and the correlation coefficient of the fitted straight line should be in the range of 0.9900-1.0000. (2) The reducing sugar content of each test or enzymatic hydrolysis tube is calculated using the linear equation obtained after fitting the standard curve.

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