Method for determining monosaccharide composition of an acidic polysaccharide

Abstract

The present application relates to a kind of acid polysaccharide monosaccharide composition determination method.The acid polysaccharide sample is mixed with lysozyme in buffer system and is carried out enzymolysis reaction, after reaction, enzyme is inactivated, and the enzymolysis solution is obtained;Acid liquid is added to the enzymolysis solution, and acid hydrolysis reaction is carried out under heating condition, and the hydrolysis solution containing monosaccharide is obtained, and hydrolysis is completed.The obtained hydrolysis solution is separated and detected, and the monosaccharide composition of the acid polysaccharide sample is determined.Compared with prior art, the present application realizes the complete hydrolysis of enzymolysis oligosaccharide fragment under lower concentration acid and shorter hydrolysis time, so as to realize the complete hydrolysis of polysaccharide chain.Avoid the incomplete hydrolysis of glycosidic bond under the specificity of enzyme hydrolysis and the problem of low hydrolysis rate and monosaccharide easy degradation under extreme acid hydrolysis environment, realize the complete but not excessive hydrolysis of acid polysaccharide, and ensure the accuracy of acid polysaccharide monosaccharide composition analysis.

Description

Technical Field

[0001] This invention relates to the field of monosaccharide composition analysis technology, and in particular to a method for determining the monosaccharide composition of acidic polysaccharides. Background Technology

[0002] Polysaccharides are natural high-molecular polymers composed of monosaccharides linked by glycosidic bonds. Monosaccharide composition, as the smallest structural unit of polysaccharides, is not only the basis of primary structure but also a key factor determining higher-order structures and functional properties. Therefore, accurate determination of monosaccharide composition is crucial for elucidating the higher-order structural features and spatial conformation of polysaccharide chains. Furthermore, it provides a theoretical basis for understanding the mechanisms underlying their diverse functional characteristics at the molecular level. Currently, there are various analytical methods for monosaccharide composition, such as gas chromatography (GC), high-performance liquid chromatography (HPLC), and high-performance anion exchange chromatography (HPAEC). Among these, GC is a commonly used analytical method for monosaccharide composition analysis due to its high sensitivity. However, it requires derivatization of monosaccharides prior to GC analysis, making the operation complex, prone to sample loss and low accuracy, and unable to directly analyze uronic acids. Compared to GC, HPLC can simultaneously analyze neutral monosaccharides and uronic acids, but sample derivatization is still required when using detectors such as ultraviolet (UV), diode array detection (DAD), or fluorescence (FL). In recent years, high-performance anion exchange chromatography combined with pulsed current detector (HPAEC-PAD) has become the mainstream method for monosaccharide composition analysis of polysaccharides. This method can directly and without derivatization separate and quantify neutral monosaccharides and uronic acids, with high sensitivity and low detection limits. However, all of the above instrumental analyses require complete hydrolysis of the polysaccharide sample, and their accuracy fundamentally depends on whether the polysaccharide is completely hydrolyzed in the pretreatment stage. Therefore, achieving complete hydrolysis of polysaccharide samples is a prerequisite for ensuring the accuracy of monosaccharide composition analysis.

[0003] Currently, the commonly used method for polysaccharide hydrolysis is direct acid hydrolysis, such as using trifluoroacetic acid (TFA), sulfuric acid (H2SO4), or hydrochloric acid (HCl) at a certain concentration to hydrolyze the glycosidic bonds of polysaccharides at high temperatures. For example, patent publication number CN112433020A discloses a method and application for detecting the monosaccharide composition based on green brick tea, including the preparation of monosaccharide samples from green brick tea, hydrolyzing the green brick tea polysaccharides with hydrochloric acid followed by pre-column derivatization, and optimizing the hydrochloric acid concentration, hydrolysis temperature, and time during acid hydrolysis. The final acid hydrolysis conditions were determined to be 2M HCl at 90 ℃ for 3 h, with a PMP methanol solution concentration of 0.5M. The monosaccharide composition of neutral green brick tea polysaccharides determined by this method has good accuracy. However, due to the differences in bond energies of different glycosidic bonds, the required hydrolysis reaction conditions for each glycosidic bond are not entirely the same. For example, TFA and HCl are ineffective at breaking the glycosidic bonds between uronic acid residues in acidic polysaccharides; while H2SO4 hydrolysis requires adjusting the acid concentration, hydrolysis temperature, and time to increase the hydrolysis rate, but its effect on acidic polysaccharides (especially pectin-based polysaccharides) still falls short of the requirements for accurate monosaccharide composition analysis. The glycosidic bonds between uronic acids in acidic polysaccharides exhibit high stability under acid hydrolysis, resulting in a slow hydrolysis rate and requiring longer hydrolysis times and stronger, higher concentrations of acid. Furthermore, excessively high acid concentrations and prolonged hydrolysis times may lead to monosaccharide degradation, thus affecting the accuracy of monosaccharide composition analysis.

[0004] Compared to the harsh conditions of acid hydrolysis, enzymatic hydrolysis is a method that uses specific enzymes to selectively cleave glycosidic bonds in polysaccharides under mild conditions. This method can achieve the hydrolysis of specific glycosidic bonds in polysaccharides by selecting different types of enzymes, such as cellulase, pectinase, and glucanase. Furthermore, by controlling the time, temperature, and other conditions during the enzymatic hydrolysis process, mild and controllable hydrolysis of polysaccharide samples can be achieved to reduce the molecular weight of polysaccharides or obtain monosaccharide or oligosaccharide fragments. In addition, enzymatic methods are more environmentally friendly than chemical methods and do not generate byproducts. Patent publication number CN110150668A discloses a method for preparing an oral liquid containing enzymatically hydrolyzed polysaccharides from Inonotus obliquus. After comparing the enzymatic hydrolysis efficiencies of cellulase, α-glucosidase, β-glucanase, and pectinase under fixed enzymatic hydrolysis conditions, β-glucanase, which has the highest hydrolysis efficiency, was selected to achieve specific enzymatic hydrolysis of Inonotus obliquus polysaccharides. By reducing the molecular weight of Inonotus obliquus polysaccharides, modification of the polysaccharides was achieved, improving their antioxidant and other biological activities. However, the application of enzymes in polysaccharide hydrolysis is usually limited by narrow substrate specificity and long incubation times. Furthermore, no single-enzyme system can completely hydrolyze structurally complex acidic polysaccharides. Collapsing enzymes are complex enzymes derived from basidiomycetes, composed of cellulase, kelp polysaccharide enzyme, and xylanase. Studies have shown that they can effectively hydrolyze uronic acid glycosidic bonds in acidic polysaccharides. However, the specificity of enzymatic hydrolysis leads to poor hydrolysis efficiency of collapsing enzymes on some monosaccharides.

[0005] Single acid hydrolysis and enzymatic hydrolysis are insufficient to achieve complete hydrolysis of acidic polysaccharides, leading to difficulties in accurately analyzing their monosaccharide composition and hindering structural elucidation and structure-activity relationship studies. The development of multi-step hydrolysis pretreatment methods may enable the complete hydrolysis of acidic polysaccharides. Patent publication number CN109358154A discloses a two-step acid hydrolysis method for polysaccharides such as carrots, followed by HPAEC-PAD determination of the monosaccharide composition. The method optimized the TFA concentration, hydrolysis temperature, and hydrolysis time in the first step, as well as the hydrolysis temperature and time in the second step. The final hydrolysis process is as follows: in the first step, the polysaccharide is hydrolyzed with 0.1M TFA at 80 ℃ for 1.5 h; in the second step, it is further hydrolyzed with 2M H2SO4 at 100 ℃ for 2 h. The uronic acid content detected in the hydrolyzed polysaccharide obtained by this method is significantly higher than that obtained by the one-step hydrolysis method. However, the total hydrolysis rate of carrot polysaccharide containing 47.24% uronic acid was only 67.30%, while the uronic acid hydrolysis rate was only 58.72%. The total hydrolysis rate and uronic acid hydrolysis rate of tea polysaccharide containing 52.02% uronic acid were 45.31% and 38.87%, respectively; and the total hydrolysis rate and uronic acid hydrolysis rate of okra polysaccharide containing 44.93% uronic acid were 60.25% and 44.25%, respectively. The total hydrolysis rate and uronic acid hydrolysis rate need to be improved to meet the requirements for accurate analysis of its monosaccharide composition. Furthermore, prolonged exposure of acidic polysaccharide samples to an acidic hydrolysis environment, especially a high-acid concentration environment, easily leads to the degradation of neutral monosaccharides into byproducts such as 5-hydroxymethylfurfural and glyceraldehyde, thus affecting the accuracy of monosaccharide analysis.

[0006] Techniques combining enzymatic and acidic methods to prepare oligosaccharides have been reported. For example, patent publication number CN101870993A discloses a process for extracting konjac glucomannan oligosaccharides through enzymatic-acid hydrolysis. The hydrolysis is performed in two steps: the first step is enzymatic hydrolysis, where water is added to a reaction vessel, konjac flour is added, and then cellulase and hemicellulase are added for hydrolysis for 3-5 hours at 45-55 °C; the second step is sulfuric acid hydrolysis for 2-4 hours at 70-80 °C. Patent publication number CN102191298A discloses a method for efficiently degrading pectin polysaccharides. Specifically, the first step involves dissolving pectin polysaccharides in a 0.5-1.5 mol / L acid solution and then partially hydrolyzing the solution at 60-100 °C for 3-7 hours; the second step involves enzymatic hydrolysis of the precipitate with pectinase for 10 min-4 h to obtain pectin oligosaccharides. Patent publication number CN110283864A discloses a method for efficiently preparing chitosan oligosaccharides by combining acid degradation and enzymatic degradation. After initial degradation of chitosan with an acid solution, further enzymatic hydrolysis with chitosanase and protease is performed to obtain chitosan oligosaccharides with a weight-average molecular weight of 400-4000. However, the aforementioned existing technologies focus on the random degradation of natural polysaccharides into functional oligosaccharides with certain biological activities through a combination of enzyme and acid treatment, and are mainly applied in fields such as biochemical engineering. However, these processes aim to obtain oligosaccharide products with low degrees of polymerization and do not involve the relevant technical content of completely and quantitatively hydrolyzing polysaccharides into monosaccharides for compositional analysis and structural determination. Therefore, existing methods still lack corresponding technical inspiration for achieving complete and non-destructive hydrolysis of polysaccharides into monosaccharides. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for determining the monosaccharide composition of acidic polysaccharides. This invention, for the first time, combines enzymatic hydrolysis and acid hydrolysis. First, it utilizes the advantage of degrading enzymes to hydrolyze the uronic acid residues in acidic polysaccharides into monosaccharide or oligosaccharide fragments. Then, it utilizes the advantage of acid hydrolysis to achieve complete hydrolysis of the enzymatically hydrolyzed oligosaccharide fragments under lower acid concentrations and shorter hydrolysis times, thereby achieving complete acid hydrolysis of the polysaccharide chain. This invention avoids the problems of incomplete hydrolysis of glycosidic bonds under the specificity of enzymatic hydrolysis and the low hydrolysis rate and easy degradation of monosaccharides under extreme acid hydrolysis conditions. Compared with single acid hydrolysis and enzymatic hydrolysis methods, this method significantly improves the hydrolysis rate of acidic polysaccharides, providing a theoretical and technical basis for the fine structural characterization of acidic polysaccharides. It achieves complete but not excessive hydrolysis of acidic polysaccharides, ensuring the accuracy of monosaccharide composition analysis.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] This invention provides a method for determining the monosaccharide composition of acidic polysaccharides, comprising the following steps:

[0010] S1. Enzymatic hydrolysis: The acidic polysaccharide sample is mixed with the decomposing enzyme in a buffer system to carry out the enzymatic hydrolysis reaction. After the reaction is completed, the enzyme is inactivated to obtain the enzymatic hydrolysate.

[0011] S2, Acid hydrolysis: Add acid to the enzymatic hydrolysate and carry out acid hydrolysis under heating conditions to obtain a hydrolysate containing monosaccharides;

[0012] S3. Separate and analyze the obtained hydrolysate to determine the monosaccharide composition of the acidic polysaccharide sample.

[0013] Further, in step S1, the buffer solution is one of acetate-sodium acetate buffer, citrate-sodium citrate buffer, or phosphate buffer.

[0014] Further, in step S1, the temperature of the enzymatic hydrolysis reaction is 32~38 ℃, more preferably 35 ℃; the reaction time is 24~48 h, more preferably 36 h.

[0015] Further, in step S1, the concentration of the breakdown enzyme in the reaction system is 0.008~0.05 g / mL, preferably 0.02~0.03 g / mL;

[0016] The concentration of the acidic polysaccharide sample in the reaction system is 0.005~0.015 g / mL, preferably 0.015 g / mL.

[0017] Further, in step S1, the mass ratio of the acidic polysaccharide sample to the breakdown enzyme is 0.1~0.625:1, preferably 0.17~0.25:1.

[0018] Further, in step S1, the acidic polysaccharide sample includes one or more of the following: polysaccharides with low uronic acid content, polysaccharides with medium uronic acid content, or polysaccharides with high uronic acid content.

[0019] Furthermore, the low-uronic acid content polysaccharide includes peach gum polysaccharide (PGP), the medium-uronic acid content polysaccharide includes Yunnan olive polysaccharide (PEP), and the high-uronic acid content polysaccharide includes passion fruit pectin (PFP).

[0020] Further, in step S2, the acid solution is one or more of sulfuric acid solution, hydrochloric acid solution, or trifluoroacetic acid solution, wherein the hydrogen ion concentration is 1~4M;

[0021] The acid hydrolysis reaction is carried out at a temperature of 95-105°C, preferably 100°C, for a reaction time of 30-90 minutes. The optimal hydrolysis time differs for polysaccharides with low, medium, or high uronic acid content, and is related to the hydrogen ion concentration. For polysaccharides with low uronic acid content, a further preferred hydrolysis time is 1-4 MH. +Hydrolysis for 60-90 minutes; further optimization of polysaccharides with medium and high uronic acid content (1-4 MH) was performed. + Hydrolyze for 30-90 minutes.

[0022] Further, in step S2, the volume ratio of the enzymatic hydrolysate to the acid solution is 0.1~0.2:1, preferably 0.15:1.

[0023] Further, in step S2, the hydrolysate includes one or more of fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, fructose, glucuronic acid (GlcA), or galacturonic acid (GalA).

[0024] In step S3, the monosaccharide composition that can be determined includes one or more of Fuc, Rha, Ara, Gal, Glc, Man, Xyl, Fru, GlcA, or GalA.

[0025] Furthermore, for polysaccharides with low uronic acid content, the total hydrolysis rate can reach 90.6%~97.4%; for polysaccharides with medium uronic acid content, the total hydrolysis rate can reach 81.1%~86.1%, of which the uronic acid hydrolysis rate can reach 86.4%~98.4%; for polysaccharides with high uronic acid content, the total hydrolysis rate can reach 78.1%~82.8%, of which the uronic acid hydrolysis rate can reach 96.7%~99.2%.

[0026] Furthermore, in step S3, the hydrolysate is separated and detected using high-performance anion exchange chromatography-pulse amperometric detection.

[0027] Furthermore, the chromatographic system of the high-performance anion exchange chromatography-pulse amperometric detection method is equipped with an anion exchange guard column and an analytical column, and uses an aqueous solution of sodium hydroxide and / or sodium acetate as the mobile phase for gradient elution.

[0028] The flow rate for chromatographic analysis was 0.4–0.5 mL / min, the column temperature was 28–35 °C, and the data acquisition time was 50–70 min.

[0029] The technical principle of this invention is to achieve complete but not excessive hydrolysis of acidic polysaccharides through multi-mode synergistic hydrolysis and regulation. First, a breakdown enzyme is used to specifically hydrolyze the glycosidic bonds between uronic acids and some neutral sugar-related glycosidic bonds in the acidic polysaccharide. Second, acid hydrolysis conditions are customized for the hydrolysate based on the uronic acid content in the acidic polysaccharide. This achieves hydrolysis of oligosaccharide fragments in the acidic polysaccharide hydrolysate under low acid concentration and short hydrolysis time, while effectively preventing further dehydration, polymerization, and degradation of the hydrolyzed monosaccharides into byproducts. This achieves a balance between complete polysaccharide hydrolysis and monosaccharide structural integrity in the monosaccharide composition analysis of complex acidic polysaccharides.

[0030] Compared with the prior art, the present invention has the following advantages:

[0031] This invention formally involves enzymatic and acidic hydrolysis steps, but its core objective is to establish a method for monosaccharide composition analysis applicable to complex acidic polysaccharides. The key technical aspect of this invention lies in employing a broad-spectrum hydrolytic enzyme—a complex enzyme—to overcome the limitations of heterogeneity in the structure of complex acidic polysaccharides and the specificity of single enzymes. After depolymerizing large acidic polysaccharides into smaller fragments, the enzyme further achieves complete and thorough hydrolysis of these fragments under low acid concentration and short hydrolysis time conditions, maximizing the protection of monosaccharides from degradation. This effectively resolves the long-standing contradiction between complete hydrolysis and monosaccharide degradation in analytical chemistry. Furthermore, this technique is universally applicable to acidic polysaccharides of unknown structure from different sources.

[0032] The hydrolysis method of this invention significantly improves the total hydrolysis rate of acidic polysaccharides, especially the uronic acid hydrolysis rate, enabling more accurate analysis of the monosaccharide composition of acidic polysaccharides. While traditional concentrated acid high-temperature hydrolysis can effectively break glycosidic bonds associated with neutral monosaccharides, it cannot break the glycosidic bonds between uronic acids in acidic polysaccharides (such as...). α -(1→4)-GalpA) exhibits acid resistance, often leading to incomplete hydrolysis of polysaccharides. Under a two-step acid hydrolysis technique, the highest total hydrolysis rate for acidic polysaccharides with moderate uronic acid content, such as carrot polysaccharide, tea polysaccharide, and okra polysaccharide, is 67.30%, with the highest uronic acid hydrolysis rate being only 58.72%. However, under the hydrolysis method provided by this invention, the total hydrolysis rate and uronic acid hydrolysis rate for acidic polysaccharide with moderate uronic acid content, Yunnan olive polysaccharide, are 86.17% and 98.99%, respectively, representing improvements of over 18.87% and 40.27% compared to the two-step acid hydrolysis method. The advantage of enzyme-mediated hydrolysis lies in its ability to specifically recognize and break glycosidic bonds between uronic acids and some glycosidic bonds associated with neutral monosaccharides. Although its enzymatic hydrolysis effect on glycosidic bonds associated with some neutral monosaccharides, such as fucose and arabinose, is poor, resulting in the cleavage into oligosaccharides, the gentle enzymatic hydrolysis avoids the risk of further degradation of already hydrolyzed monosaccharides. In the process of preparing specific oligosaccharides with a single enzyme system, the enzyme cleavage sites are random, and it is difficult to achieve complete hydrolysis of polysaccharides even with acid hydrolysis. Therefore, single enzymatic hydrolysis and acid hydrolysis have been applied in the preparation of oligosaccharides from different polysaccharide sources.

[0033] In acidic polysaccharides, the glycosidic bonds of some neutral monosaccharides are incompletely hydrolyzed by decomposition enzymes, requiring further acid hydrolysis to achieve complete hydrolysis. However, neutral monosaccharides undergo dehydration reactions under strong acid and high temperature catalysis, degrading into byproducts, such as pentoses degrading into furfural and hexoses degrading into 5-hydroxymethylfurfural. If hydrolysis continues for a prolonged period under these conditions, these byproducts will further degrade into organic acids such as formic acid. In addition, hydroxyl intermediates such as furfural may react with undegraded monosaccharides to form humicin, irreversibly consuming the degraded monosaccharides and causing browning of the solution. Therefore, adjusting the acid hydrolysis conditions from high acid concentration and long hydrolysis time to low acid concentration and short hydrolysis time is necessary to avoid the formation of byproducts from monosaccharide degradation. The hydrolysis method of this invention significantly reduces the intensity of acid hydrolysis conditions by breaking glycosidic bonds between uronic acids and some neutral sugar-related glycosidic bonds using a breakdown enzyme. Acid hydrolysis conditions are customized based on the uronic acid content in the acidic polysaccharide, achieving complete hydrolysis of oligosaccharide fragments by the breakdown enzyme under low acid concentration and short hydrolysis time, without causing degradation of already hydrolyzed monosaccharides. This invention achieves complete but not excessive hydrolysis of acidic polysaccharides, ensuring the accuracy of monosaccharide composition analysis.

[0034] Meanwhile, the analytical method for determining monosaccharide composition in this invention is high-performance anion exchange chromatography combined with a pulsed current detector. Compared with gas chromatography, it can directly and without derivatization separate neutral monosaccharides and uronic acids. Compared with high-performance liquid chromatography using ultraviolet (UV), diode array detection (DAD), or fluorescence (FL) as detectors, it also does not require complex derivatization operations, has high sensitivity and low detection limit, and can conveniently achieve accurate analysis of monosaccharide composition in polysaccharide hydrolysates. Attached Figure Description

[0035] Figure 1 HPAEC-PAD chromatograms of 10 monosaccharide standards;

[0036] Figure 2 The uronic acid hydrolysis rate and total hydrolysis rate of PEP were determined by enzymatic hydrolysis with a decomposing enzyme at different temperatures for 36 h.

[0037] Figure 3 The uronic acid hydrolysis rate and total hydrolysis rate of PEP were determined by enzymatic hydrolysis with a decomposing enzyme at 35 °C for different times.

[0038] Figure 4 The uronic acid hydrolysis rate and total hydrolysis rate of PEP were obtained by enzymatic hydrolysis at 35 °C for 36 h with different concentrations of decomposing enzyme (i.e., different sample-to-decomposing enzyme mass ratios).

[0039] Figure 5 The total hydrolysis rate of acidic polysaccharide hydrolysate at different temperatures;

[0040] Figure 6 The total hydrolysis rate of acidic polysaccharides is denoted as ... Detailed Implementation

[0041] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments. All other embodiments obtained by those skilled in the art based on the given embodiments without creative effort are within the scope of protection of this application.

[0042] Unless otherwise specified, the reagents, methods, instruments, and equipment used in this invention are conventional in the art. Fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, fructose, glucuronic acid, galacturonic acid (all chromatographic grade), and degrading enzymes were obtained from Sigma-Aldrich, USA; m-hydroxybiphenyl and dialysis bags (8000~14000 Da) were from Shanghai Yuanye Biotechnology Co., Ltd.; sodium acetate (electrochemical grade) and sodium hydroxide (chromatographic grade) were from Thermo-Fisher, USA. Peach gum, Yunnan olive, and purple passion fruit were all purchased from the local Shanghai market.

[0043] Peach gum polysaccharide (PGP) extraction: Weigh 15 g of raw peach gum into a beaker, add 1500 mL of ultrapure water, allow to swell overnight at room temperature, and extract at 100 ℃ for 6 h. After cooling to room temperature, centrifuge, concentrate the supernatant to 200 mL, dialyze to ultrapure water for 3 days, and freeze-dry to obtain PGP.

[0044] Extraction of Yunnan olive polysaccharide (PEP): 150 g of Yunnan olives were pitted, dried at 55 ℃, pulverized, and soaked overnight in 300 mL of 80% ethanol to remove alcohol-soluble impurities. The powder was then dried at 55 ℃ to obtain Yunnan olive fruit powder. 15 g of the Yunnan olive fruit powder was weighed into a beaker, and 375 mL of ultrapure water was added. Extraction was carried out at 94 ℃ for 3.5 h. The supernatant was concentrated to 40 mL, and 4 times its volume of anhydrous ethanol was added for precipitation overnight. The precipitate was dissolved in an appropriate amount of ultrapure water, dialyzed in ultrapure water for 3 days, and then freeze-dried to obtain PEP.

[0045] Passion fruit pectin (PFP) extraction: 150 g of passion fruit peel was boiled in boiling water for 20 min to inactivate enzymes, dried at 55 ℃, pulverized, and soaked in 300 mL of 80% ethanol overnight to remove alcohol-soluble impurities. The powder was then dried at 55 ℃ to obtain passion fruit powder. 15 g of passion fruit powder was weighed into a beaker, and 450 mL of ultrapure water was added for extraction at 80 ℃ for 1 h. The supernatant after centrifugation was concentrated to 70 mL, and 4 times its volume of anhydrous ethanol was added for precipitation overnight. The precipitate was dissolved in an appropriate amount of ultrapure water, dialyzed in ultrapure water for 3 days, and then lyophilized to obtain PFP. The uronic acid content and neutral sugar content of the acidic polysaccharide were determined colorimetrically. This provides guidance for selecting conditions for further acid hydrolysis of polysaccharides in the enzymatic hydrolysate during the analysis of monosaccharide composition of other acidic polysaccharides besides the three mentioned above. Using galacturonic acid as a standard, the uronic acid content of polysaccharide samples was determined by the m-hydroxybiphenyl method. The steps were as follows: Add 5 mL of sodium tetraborate / concentrated sulfuric acid solution to 1 mL of standard / sample solution in an ice-water bath, mix well, and then boil in a water bath for 10 min; after cooling to room temperature, add 0.1 mL of m-hydroxybiphenyl solution, react at room temperature for 20 min, and then measure the absorbance at 525 nm using a UV spectrophotometer. Using glucose as a standard, the neutral sugar content of polysaccharide samples was determined by the phenol-sulfuric acid method. The specific steps were as follows: Add 0.5 mL of 5% phenol solution to 1 mL of standard / sample solution and mix well; add 5 mL of concentrated sulfuric acid solution in an ice-water bath and mix well, react at room temperature for 30 min, and then measure the absorbance at 490 nm using a UV spectrophotometer. In the following examples, the polysaccharide with low uronic acid content was peach gum polysaccharide (PGP); the polysaccharide with medium uronic acid content was Yunnan olive polysaccharide (PEP); and the polysaccharide with high uronic acid content was passion fruit pectin (PFP). The uronic acid content determined by the m-hydroxybiphenyl method was 7.86%, 41.77%, and 71.77%, respectively. The neutral sugar content determined by the phenol-sulfuric acid method was 91.66%, 48.86%, and 33.01%, respectively.

[0046] Ten monosaccharide standard solutions: Using ultrapure water as the solvent, ten monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, fructose, glucuronic acid, and galacturonic acid) were diluted to standard solutions with monosaccharide concentrations of 0.00025 mg / mL, 0.0005 mg / mL, 0.001 mg / mL, 0.002 mg / mL, 0.004 mg / mL, 0.008 mg / mL, 0.01 mg / mL, and 0.02 mg / mL. The solutions were then filtered through a 0.22 μm aqueous filter membrane to obtain calibration curves for sample qualitative and quantitative analysis. All measurements were performed in triplicate. The HPAEC-PAD chromatograms of the ten monosaccharide standards are shown below. Figure 1As shown in Table 1, the linear equations for different monosaccharides are presented. The linear calibration curves between the mass concentration (mg / mL) and peak area (nC·min) of each monosaccharide all exceed 0.99, indicating that the analytical method is reliable.

[0047] Table 1. Linear equations for different monosaccharides

[0048]

[0049] The procedure for analyzing monosaccharide composition using HPAEC-PAD is as follows: High-performance anion exchange chromatography with a tandem pulsed amperometric detector (HPAEC-PAD, Dionex ICS-5000) is employed. + (USA) This system analyzes the monosaccharide composition of polysaccharides. It is equipped with a Dionex CarboPac... TM PA20 protective pillars (3 mm × 30 mm) and Dionex CarboPac TM A PA20 analytical column (3 mm × 150 mm) was used. The mobile phase consisted of H₂O (A), 25 mmol / L NaOH solution (B), 1 mol / L NaOAc solution (C), and 200 mmol / L NaOH solution (D), with gradient elution. The flow rate was set to 0.5 mL / min, the column temperature to 30 °C, and the data acquisition time to 60 min.

[0050] Example 1

[0051] The lyase was dissolved in acetate-sodium acetate buffer to obtain a 0.075 g / mL lyase solution. 3 mg of PEP sample was accurately weighed into a sample vial, and 200 μL of lyase solution and 400 μL of ultrapure water were added sequentially. This resulted in a PEP concentration of 0.005 g / mL and a lyase concentration of 0.025 g / mL, meaning the mass ratio of acidic polysaccharide to lyase in the enzymatic hydrolysis system was 0.2:1. The PEP-lyase mixture was thoroughly mixed at room temperature and incubated at 32 °C for 36 h. The enzyme was then inactivated by heating to 100 °C, cooled, and centrifuged. 70 μL of the cooled PEP hydrolysate was diluted with ultrapure water, filtered through a 0.22 μm aqueous filter, and analyzed for monosaccharide composition using HPAEC-PAD. The remaining PEP hydrolysate was further hydrolyzed with sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution, resulting in a hydrolysate to acid solution volume ratio of 0.15. The sulfuric acid concentration was fixed at 1 M, the hydrolysis temperature was 100 °C, and the hydrolysis time was 90 min. After cooling, the PEP hydrolysate was diluted with ultrapure water, filtered through a 0.22 μm aqueous filter membrane, and analyzed for monosaccharide composition using HPAEC-PAD.

[0052] Example 2

[0053] Most of the contents were the same as in Example 1, except that the PEP-disruption enzyme mixture was incubated at 35 °C.

[0054] Example 3

[0055] Most of the contents were the same as in Example 1, except that the PEP-disruption enzyme mixture was incubated at 38 °C.

[0056] Example 4

[0057] Most of the contents were the same as in Example 2, except that the PEP-decomposition enzyme mixture was incubated for 24 h.

[0058] Example 5

[0059] Most of the contents were the same as in Example 4, except that the PEP-decomposition enzyme mixture was incubated for 48 h.

[0060] Example 6

[0061] The lyase was dissolved in acetate-sodium acetate buffer to obtain a 0.024 g / mL lyase solution. 3 mg of PEP sample was accurately weighed into a sample vial, and 200 μL of lyase solution and 400 μL of ultrapure water were added sequentially. This resulted in a PEP concentration of 0.005 g / mL and a lyase concentration of 0.008 g / mL in the enzymatic hydrolysis system, meaning the mass ratio of acidic polysaccharide to lyase in the hydrolysis system was 0.625:1. The PEP-lyase mixture was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The enzyme was then inactivated by heating to 100 °C, cooled, and centrifuged. 70 μL of the cooled PEP hydrolysate was diluted with ultrapure water, filtered through a 0.22 μm aqueous filter, and analyzed for monosaccharide composition using HPAEC-PAD. The remaining PEP hydrolysate was further hydrolyzed with sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution, resulting in a hydrolysate to acid solution volume ratio of 0.15. The sulfuric acid concentration was fixed at 1 M, the hydrolysis temperature was 100 °C, and the hydrolysis time was 90 min. After cooling, the PEP hydrolysate was diluted with ultrapure water, filtered through a 0.22 μm aqueous filter membrane, and analyzed for monosaccharide composition using HPAEC-PAD.

[0062] Example 7

[0063] The process is largely the same as in Example 6, except that the catalytic enzyme is dissolved in sodium acetate buffer to obtain a 0.03 g / mL catalytic enzyme solution, which means that the concentration of the catalytic enzyme in the enzymatic hydrolysis system is 0.01 g / mL, and the mass ratio of acidic polysaccharide to catalytic enzyme in the enzymatic hydrolysis system is 0.5.

[0064] Example 8

[0065] The process is largely the same as in Example 6, except that the catalytic enzyme is dissolved in sodium acetate buffer to obtain a 0.06 g / mL catalytic enzyme solution, which means that the concentration of the catalytic enzyme in the enzymatic hydrolysis system is 0.02 g / mL, and the mass ratio of acidic polysaccharide to catalytic enzyme in the enzymatic hydrolysis system is 0.25.

[0066] Example 9

[0067] The process is largely the same as in Example 6, except that the catalytic enzyme is dissolved in sodium acetate buffer to obtain a 0.09 g / mL catalytic enzyme solution, which means that the concentration of the catalytic enzyme in the enzymatic hydrolysis system is 0.03 g / mL, and the mass ratio of acidic polysaccharide to catalytic enzyme in the enzymatic hydrolysis system is 0.17.

[0068] Example 10

[0069] The process is largely the same as in Example 6, except that the catalytic enzyme is dissolved in sodium acetate buffer to obtain a 0.15 g / mL catalytic enzyme solution, which means that the concentration of the catalytic enzyme in the enzymatic hydrolysis system is 0.05 g / mL, and the mass ratio of acidic polysaccharide to catalytic enzyme in the enzymatic hydrolysis system is 0.1.

[0070] Example 11

[0071] The polysaccharide-polysaccharide hydrolysate was dissolved in an acetate-sodium acetate buffer solution to obtain a 0.75 g / mL hydrolysate solution. 3 mg of PGP sample was accurately weighed into a sample vial, and 200 μL of the hydrolysate solution and 400 μL of ultrapure water were added sequentially. The concentration of PGP in the hydrolysis system was 0.005 g / mL, and the concentration of the hydrolysate was 0.025 g / mL, i.e., the mass ratio of acidic polysaccharide to hydrolysate in the hydrolysis system was 0.2:1. The PGP-hydrolysate mixture was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. Then, the mixture was heated to 100 °C to inactivate the enzyme, cooled, and centrifuged. The PGP hydrolysate was further hydrolyzed with sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution, i.e., the volume ratio of hydrolysate to acid solution was 0.15. The sulfuric acid concentration was fixed at 1 M, the hydrolysis temperature was 100 °C, and the hydrolysis time was 90 min. The PGP hydrolysate was diluted with ultrapure water after cooling, filtered through a 0.22 μm aqueous filter membrane, and then analyzed for monosaccharide composition using HPAEC-PAD.

[0072] Example 12

[0073] The process was largely the same as in Example 11, except that 9 mg of PGP sample was weighed into a sample vial, the concentration of PGP in the enzymatic hydrolysis system was 0.015 g / mL, and the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 0.6:1.

[0074] Example 13

[0075] The polysaccharide-polysaccharide hydrolysate (PPH) was dissolved in an acetate-sodium acetate buffer solution to obtain a 0.75 g / mL PEP solution. 3 mg of PEP sample was accurately weighed into a sample vial, and 200 μL of PEP solution and 400 μL of ultrapure water were added sequentially. The concentration of PEP in the hydrolysis system was 0.005 g / mL, and the concentration of PEP-polysaccharide hydrolysate was 0.025 g / mL, meaning the mass ratio of acidic polysaccharide to PEP-polysaccharide hydrolysate was 0.2:1. The PEP-polysaccharide hydrolysate was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The mixture was then heated to 100 °C to inactivate the enzyme, cooled, and centrifuged. The PEP hydrolysate was further hydrolyzed with sulfuric acid. 300 μL of the polysaccharide hydrolysate was obtained, and 2000 μL of sulfuric acid solution was added to it, resulting in a hydrolysate-to-acid solution volume ratio of 0.15. The sulfuric acid concentration was fixed at 1 M, the hydrolysis temperature was 100 °C, and the hydrolysis time was 90 min. The PEP hydrolysate was cooled, diluted with ultrapure water, filtered through a 0.22 μm aqueous filter membrane, and analyzed for monosaccharide composition using HPAEC-PAD.

[0076] Example 14

[0077] The process was largely the same as in Example 13, except that 9 mg of PEP sample was weighed into a sample bottle and the concentration of PEP in the enzymatic hydrolysis system was 0.015 g / mL, which means that the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 0.6:1.

[0078] Example 15

[0079] The polysaccharide-polysaccharide hydrolysate (PPHH) was dissolved in an acetate-sodium acetate buffer solution to obtain a 0.75 g / mL PPHH solution. 3 mg of PFP sample was accurately weighed into a sample vial, and 200 μL of PPHH solution and 400 μL of ultrapure water were added sequentially. The concentration of PFP in the hydrolysis system was 0.005 g / mL, and the concentration of PPHH was 0.025 g / mL, meaning the mass ratio of acidic polysaccharide to PPHH in the hydrolysis system was 0.2:1. The PFP-PPHH mixture was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The mixture was then heated to 100 °C to inactivate the enzyme, cooled, and centrifuged. The PFP hydrolysate was further hydrolyzed with sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution, resulting in a hydrolysate-to-acid solution volume ratio of 0.15. The sulfuric acid concentration was fixed at 1 M, the hydrolysis temperature was 100 °C, and the hydrolysis time was 90 min. The PFP hydrolysate was diluted with ultrapure water after cooling, filtered through a 0.22 μm aqueous filter membrane, and analyzed for monosaccharide composition using HPAEC-PAD.

[0080] Example 16

[0081] The process was largely the same as in Example 15, except that 9 mg of PFP sample was weighed into a sample vial and the concentration of PFP in the enzymatic hydrolysis system was 0.015 g / mL, which means that the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 0.6:1.

[0082] Example 17

[0083] The polysaccharide-polysaccharide hydrolysate was dissolved in acetate-sodium acetate buffer to obtain a 0.75 g / mL polysaccharide-polysaccharide hydrolysate solution. 9 mg of PGP was accurately weighed into a sample vial, and 200 μL of the polysaccharide-polysaccharide hydrolysate solution and 400 μL of ultrapure water were added sequentially. This resulted in a PGP concentration of 0.015 g / mL and a polysaccharide-polysaccharide-polysaccharide hydrolysate concentration of 0.025 g / mL, meaning the mass ratio of acidic polysaccharide to polysaccharide-polysaccharide hydrolysate in the hydrolysis system was 0.6:1. The PGP-polysaccharide-polysaccharide hydrolysate was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The hydrolysate was then heated to 100 °C to inactivate the enzyme, cooled, and centrifuged. Further hydrolysis of PGP was performed using sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution at 95 °C. The PGP hydrolysate was hydrolyzed for 90 min at 0.5 M sulfuric acid concentration, with a hydrolysate-to-acid solution volume ratio of 0.15. The PGP hydrolysate was diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition was analyzed by HPAEC-PAD.

[0084] Example 18

[0085] Most of them are the same as in Example 17, except that the acid hydrolysis temperature is 100°C.

[0086] Example 19

[0087] Most of the components are the same as in Example 17, except that the acid hydrolysis temperature is 105°C.

[0088] Example 20

[0089] It is almost identical to Example 17, except that the acidic polysaccharide sample weighed is PEP.

[0090] Example 21

[0091] It is almost identical to Example 18, except that the acidic polysaccharide sample weighed is PEP.

[0092] Example 22

[0093] It is almost identical to Example 19, except that the acidic polysaccharide sample weighed is PEP.

[0094] Example 23

[0095] Most of the samples were the same as in Example 17, except that the acidic polysaccharide sample weighed was PFP.

[0096] Example 24

[0097] It is almost identical to Example 18, except that the acidic polysaccharide sample weighed is PFP.

[0098] Example 25

[0099] It is almost identical to Example 19, except that the acidic polysaccharide sample weighed is PFP.

[0100] Example 26

[0101] The polysaccharide-polysaccharide hydrolysate was dissolved in acetate-sodium acetate buffer to obtain a 0.75 g / mL polysaccharide-polysaccharide hydrolysate solution. 9 mg of PGP was accurately weighed into a sample vial, and 200 μL of the polysaccharide-polysaccharide hydrolysate solution and 400 μL of ultrapure water were added sequentially. This resulted in a PGP concentration of 0.015 g / mL and a polysaccharide-polysaccharide-polysaccharide hydrolysate concentration of 0.025 g / mL, meaning the mass ratio of acidic polysaccharide to polysaccharide-polysaccharide hydrolysate was 0.6:1. The PGP-polysaccharide-polysaccharide hydrolysate was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The mixture was then heated to 100 °C to inactivate the enzyme, cooled, and centrifuged. Further hydrolysis of PGP was performed using sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution, resulting in a hydrolysis ratio of hydrolysate to acid solution of 0.15. The hydrolysis temperature was 100 °C, and the PGP hydrolysate was hydrolyzed for 60 min at 0.5 M sulfuric acid concentration. The PGP hydrolysate was diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition was analyzed by HPAEC-PAD.

[0102] Example 27

[0103] Most of the steps are the same as in Example 26, except that the acid hydrolysis time is adjusted to 90 min.

[0104] Example 28

[0105] Most of the results are the same as in Example 26, except that the acid hydrolysis time is adjusted to 30 min.

[0106] Example 29

[0107] Most of the components are the same as in Example 26, except that the 0.5 M sulfuric acid is changed to 1 M sulfuric acid.

[0108] Example 30

[0109] Most of the components are the same as in Example 27, except that the 0.5 M sulfuric acid is changed to 1 M sulfuric acid.

[0110] Example 31

[0111] Most of the components are the same as in Example 28, except that the 0.5 M sulfuric acid is changed to 1 M sulfuric acid.

[0112] Example 32

[0113] Most of the components are the same as in Example 26, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0114] Example 33

[0115] Most of the components are the same as in Example 27, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0116] Example 34

[0117] Most of the components are the same as in Example 28, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0118] Example 35

[0119] The polysaccharide-polysaccharide lyase was dissolved in acetate-sodium acetate buffer to obtain a 0.75 g / mL polysaccharide-polysaccharide lyase solution. 9 mg of PEP was accurately weighed into a sample vial, and 200 μL of the polysaccharide-polysaccharide lyase solution and 400 μL of ultrapure water were added sequentially. This resulted in a PEP concentration of 0.015 g / mL and a polysaccharide-polysaccharide lyase concentration of 0.025 g / mL, meaning the mass ratio of acidic polysaccharide to polysaccharide-polysaccharide lyase in the enzymatic hydrolysis system was 0.6:1. The PEP-polysaccharide-polysaccharide mixture was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The mixture was then heated to 100 °C to inactivate the enzyme, cooled, and centrifuged. Further hydrolysis of PEP was performed using sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution, resulting in a hydrolysis ratio of hydrolysate to acid solution of 0.15. The hydrolysis temperature was 100 °C, and the PEP hydrolysate was hydrolyzed for 60 min at 0.5 M sulfuric acid concentration. The PEP hydrolysate was diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition was analyzed by HPAEC-PAD.

[0120] Example 36

[0121] Most of the results are the same as in Example 35, except that the acid hydrolysis time is adjusted to 90 min.

[0122] Example 37

[0123] Most of the results are the same as in Example 35, except that the acid hydrolysis time is adjusted to 30 min.

[0124] Example 38

[0125] Most of the components are the same as in Example 35, except that the 0.5 M sulfuric acid is changed to 1 M sulfuric acid.

[0126] Example 39

[0127] It is largely the same as Example 36, except that the 0.5 M sulfuric acid is changed to 1 M sulfuric acid.

[0128] Example 40

[0129] Most of the components are the same as in Example 37, except that the 0.5 M sulfuric acid is changed to 1 M sulfuric acid.

[0130] Example 41

[0131] Most of the components are the same as in Example 35, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0132] Example 42

[0133] Most of the components are the same as in Example 36, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0134] Example 43

[0135] Most of the components are the same as in Example 37, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0136] Example 44

[0137] The polysaccharide-polysaccharide hydrolysate (PFP) was dissolved in an acetate-sodium acetate buffer solution to obtain a 0.75 g / mL PFP solution. 9 mg of PFP was accurately weighed into a sample vial, and 200 μL of PFP solution and 400 μL of ultrapure water were added sequentially. The concentration of PFP in the hydrolysis system was 0.015 g / mL, and the concentration of PFP was 0.025 g / mL, meaning the mass ratio of acidic polysaccharide to PFP in the hydrolysis system was 0.6:1. The PFP-PFP mixture was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The mixture was then heated to 100 °C to inactivate the enzyme, cooled, and centrifuged. Further hydrolysis of PFP was performed using sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution, resulting in a hydrolysis volume ratio of 0.15 to acid. The hydrolysis temperature was 100 °C, and the PFP hydrolysate was hydrolyzed for 60 min at 0.5 M sulfuric acid concentration. The PFP hydrolysate was diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition was analyzed by HPAEC-PAD.

[0138] Example 45

[0139] Most of the results are the same as in Example 44, except that the acid hydrolysis time is adjusted to 90 min.

[0140] Example 46

[0141] Most of the results are the same as in Example 44, except that the acid hydrolysis time is adjusted to 30 min.

[0142] Example 47

[0143] Most of the components are the same as in Example 44, except that the 0.5 M sulfuric acid is changed to 1 M sulfuric acid.

[0144] Example 48

[0145] It is largely the same as Example 45, except that the 0.5 M sulfuric acid was changed to 1 M sulfuric acid.

[0146] Example 49

[0147] Most of the components are the same as in Example 46, except that the 0.5 M sulfuric acid is changed to 1 M sulfuric acid.

[0148] Example 50

[0149] Most of the components are the same as in Example 44, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0150] Example 51

[0151] Most of the components are the same as in Example 45, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0152] Example 52

[0153] Most of the components are the same as in Example 46, except that the 0.5 M sulfuric acid is changed to 2 M sulfuric acid.

[0154] Example 53

[0155] The polysaccharide-polysaccharide hydrolysate was dissolved in acetate-sodium acetate buffer to obtain a 0.75 g / mL polysaccharide-polysaccharide hydrolysate solution. 9 mg of PGP was accurately weighed into a sample vial, and 200 μL of the polysaccharide-polysaccharide hydrolysate solution and 400 μL of ultrapure water were added sequentially. This resulted in a PGP concentration of 0.015 g / mL and a polysaccharide-polysaccharide-polysaccharide hydrolysate concentration of 0.025 g / mL, meaning the mass ratio of acidic polysaccharide to polysaccharide-polysaccharide hydrolysate was 0.6:1. The PGP-polysaccharide-polysaccharide hydrolysate was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The mixture was then heated to 100 °C to inactivate the enzyme, cooled, and centrifuged. Further hydrolysis of PGP was performed using sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 3000 μL of sulfuric acid solution, resulting in a hydrolysis ratio of hydrolysate to acid solution of 0.1. The hydrolysis temperature was 100 °C, and the PGP hydrolysate was hydrolyzed at 2 M sulfuric acid for 90 min. The PGP hydrolysate was diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition was analyzed by HPAEC-PAD.

[0156] Example 54

[0157] The process is largely the same as in Example 53, except that 300 μL of polysaccharide hydrolysate is taken and 2000 μL of sulfuric acid solution is added to it, that is, the volume ratio of hydrolysate to acid solution is 0.15.

[0158] Example 55

[0159] The process is largely the same as in Example 53, except that 300 μL of polysaccharide hydrolysate is taken and 1500 μL of sulfuric acid solution is added to it, that is, the volume ratio of hydrolysate to acid solution is 0.2.

[0160] Example 56

[0161] It is largely the same as Example 53, except that PGP is changed to PEP.

[0162] Example 57

[0163] It is largely the same as Example 54, except that PGP is changed to PEP.

[0164] Example 58

[0165] It is largely the same as Example 55, except that PGP is changed to PEP.

[0166] Example 59

[0167] It is largely the same as Example 53, except that PGP is changed to PFP.

[0168] Example 60

[0169] It is largely the same as Example 54, except that PGP is changed to PFP.

[0170] Example 61

[0171] It is largely the same as Example 55, except that PGP is changed to PFP.

[0172] Example 62

[0173] The polysaccharide-polysaccharide hydrolysate was dissolved in acetate-sodium acetate buffer to obtain a 0.75 g / mL hydrolysate solution. 9 mg of PGP was accurately weighed into a sample vial, and 200 μL of the hydrolysate solution and 400 μL of ultrapure water were added sequentially. The concentration of PGP in the hydrolysis system was 0.015 g / mL, and the concentration of the hydrolysate was 0.025 g / mL, i.e., the mass ratio of acidic polysaccharide to hydrolysate in the hydrolysis system was 0.6:1. The PGP-hydrolysate mixture was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The enzyme was then inactivated by heating to 100 °C, cooled, and centrifuged. Further hydrolysis of PGP was performed using sulfuric acid. 300 μL of the polysaccharide hydrolysate was added to 2000 μL of sulfuric acid solution, i.e., the volume ratio of hydrolysate to acid solution was 0.15. The hydrolysis temperature was 100 °C, and the PGP hydrolysate was hydrolyzed at 2 M sulfuric acid for 90 min. The PGP hydrolysate was diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition was analyzed by HPAEC-PAD. Six independent analyses of the entire process, from hydrolysis to instrumental detection, were performed on this sample within one day.

[0174] Example 63

[0175] The lyase was dissolved in acetate-sodium acetate buffer to obtain a 0.75 g / mL lyase solution. 9 mg of PEP was accurately weighed into a sample vial, and 200 μL of the lyase solution and 400 μL of ultrapure water were added sequentially. The concentration of PEP in the enzymatic hydrolysis system was 0.015 g / mL, and the concentration of the lyase was 0.025 g / mL, i.e., the mass ratio of acidic polysaccharide to lyase in the enzymatic hydrolysis system was 0.6:1. The PEP-lyase mixture was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The enzyme was then inactivated by heating to 100 °C, cooled, and centrifuged. PEP was further hydrolyzed using sulfuric acid, with a volume ratio of enzymatic hydrolysate to acid solution of 0.15 and a hydrolysis temperature of 100 °C. The PEP hydrolysate was hydrolyzed for 60 min at a concentration of 0.5 M sulfuric acid. The PEP hydrolysate was diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition was analyzed by HPAEC-PAD. The sample was independently analyzed six times within one day, from hydrolysis to instrument detection.

[0176] Example 64

[0177] The lyase was dissolved in acetate-sodium acetate buffer to obtain a 0.75 g / mL lyase solution. 9 mg of PFP was accurately weighed into a sample vial, and 200 μL of the lyase solution and 400 μL of ultrapure water were added sequentially. This resulted in a PFP concentration of 0.015 g / mL and a lyase concentration of 0.025 g / mL in the enzymatic hydrolysis system, meaning the mass ratio of acidic polysaccharide to lyase in the hydrolysis system was 0.6:1. The PFP-lyase mixture was thoroughly mixed at room temperature and incubated at 35 °C for 36 h. The enzyme was then inactivated by heating to 100 °C, cooled, and centrifuged. Further hydrolysis of PFP was performed using sulfuric acid, with a hydrolysis solution to acid volume ratio of 0.15 and a hydrolysis temperature of 100 °C. The PFP hydrolysate was hydrolyzed for 60 min in 0.5 M sulfuric acid. The PFP hydrolysate was diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The monosaccharide composition was analyzed by HPAEC-PAD. Six independent analyses of the entire process from hydrolysis to instrument detection were performed on the sample within one day.

[0178] Comparative Example 1

[0179] Most of the contents were the same as in Example 1, except that the PEP-disruption enzyme mixture was incubated at 30 °C.

[0180] Comparative Example 2

[0181] Most of the contents were the same as in Example 1, except that the PEP-disruption enzyme mixture was incubated at 40 °C.

[0182] Comparative Example 3

[0183] Most of the contents were the same as in Example 2, except that the PEP-decomposition enzyme mixture was incubated for 12 h.

[0184] Comparative Example 4

[0185] Most of the contents were the same as in Example 2, except that the PEP-decomposition enzyme mixture was incubated for 72 h.

[0186] Comparative Example 5

[0187] The process is largely the same as in Example 6, except that the catalytic enzyme is dissolved in sodium acetate buffer to obtain a 0.0075 g / mL catalytic enzyme solution, which means that the concentration of the catalytic enzyme in the enzymatic hydrolysis system is 0.0025 g / mL, and the mass ratio of acidic polysaccharide to catalytic enzyme in the enzymatic hydrolysis system is 2.0.

[0188] Comparative Example 6

[0189] The process is largely the same as in Example 6, except that the catalytic enzyme is dissolved in sodium acetate buffer to obtain a 0.015 g / mL catalytic enzyme solution, which means that the concentration of the catalytic enzyme in the enzymatic hydrolysis system is 0.005 g / mL, and the mass ratio of acidic polysaccharide to catalytic enzyme in the enzymatic hydrolysis system is 1.

[0190] Comparative Example 7

[0191] The process was largely the same as in Example 11, except that 30 mg of PGP sample was weighed into a sample vial, the concentration of PGP in the enzymatic hydrolysis system was 0.050 g / mL, and the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 2.0.

[0192] Comparative Example 8

[0193] The process was largely the same as in Example 11, except that 60 mg of PGP sample was weighed into a sample vial, the concentration of PGP in the enzymatic hydrolysis system was 0.100 g / mL, and the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 4.0.

[0194] Comparative Example 9

[0195] The process was largely the same as in Example 13, except that 30 mg of PEP sample was weighed into a sample vial and the concentration of PEP in the enzymatic hydrolysis system was 0.050 g / mL, which means that the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 2.0.

[0196] Comparative Example 10

[0197] The process was largely the same as in Example 13, except that 60 mg of PEP sample was weighed into a sample vial, the concentration of PEP in the enzymatic hydrolysis system was 0.100 g / mL, and the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 4.0.

[0198] Comparative Example 11

[0199] The process was largely the same as in Example 15, except that 30 mg of PFP sample was weighed into a sample vial, the concentration of PFP in the enzymatic hydrolysis system was 0.050 g / mL, and the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 2.0.

[0200] Comparative Example 12

[0201] The process was largely the same as in Example 15, except that 60 mg of PFP sample was weighed into a sample vial, the concentration of PFP in the enzymatic hydrolysis system was 0.100 g / mL, and the mass ratio of acidic polysaccharide to degrading enzyme in the enzymatic hydrolysis system was 4.0.

[0202] Comparative Example 13

[0203] Most of the components are the same as in Example 17, except that the acid hydrolysis temperature is 90°C.

[0204] Comparative Example 14

[0205] Most of the components are the same as in Example 17, except that the acid hydrolysis temperature is 110 °C.

[0206] Comparative Example 15

[0207] Most of the components are the same as in Example 20, except that the acid hydrolysis temperature is 90 °C.

[0208] Comparative Example 16

[0209] Most of the components are the same as in Example 20, except that the acid hydrolysis temperature is 110 °C.

[0210] Comparative Example 17

[0211] It is almost identical to Example 23, except that the acid hydrolysis temperature is 90 °C.

[0212] Comparative Example 18

[0213] It is almost identical to Example 23, except that the acid hydrolysis temperature is 110 °C.

[0214] Comparative Example 19

[0215] Most of the components are the same as in Example 53, except that the volume ratio of the enzymatic hydrolysate to the acid solution is 0.05.

[0216] Comparative Example 20

[0217] Most of the components are the same as in Example 53, except that the volume ratio of the enzymatic hydrolysate to the acid solution is 0.25.

[0218] Comparative Example 21

[0219] The majority of them are the same as Comparative Example 19, except that PGP is changed to PEP.

[0220] Comparative Example 22

[0221] The majority of the data is the same as Comparative Example 20, except that PGP is changed to PEP.

[0222] Comparative Example 23

[0223] The majority of the data is the same as Comparative Example 19, except that PGP is changed to PFP.

[0224] Comparative Example 24

[0225] The majority of the data is the same as Comparative Example 20, except that PGP is changed to PFP.

[0226] Comparative Example 25

[0227] Accurately weigh 9 mg of PGP, and add 0.5 mL of 12M H2SO4 sequentially under ice-water bath conditions. After stirring at room temperature for 30 min, add 2.5 mL of ultrapure water to dilute the H2SO4 to a final concentration of 2M. Hydrolyze at 105 °C for 2 h, then cool to room temperature. Each polysaccharide hydrolysate is diluted with ultrapure water and filtered through a 0.22 μm aqueous filter membrane. The filtrates are then subjected to HPAEC-PAD analysis. The 12M H2SO4 is used for the initial depolymerization of PGP, which allows for more thorough high-temperature hydrolysis in the next step of adjusting the final concentration of sulfuric acid to 2M. The concentration of sulfuric acid in the sample hydrolysis stage is 2M.

[0228] Comparative Example 26

[0229] The majority of the data is the same as Comparative Example 25, except that PGP is changed to PEP.

[0230] Comparative Example 27

[0231] Most of them are the same as Comparative Example 25, except that PGP is changed to PFP.

[0232] Comparative Example 28

[0233] Accurately weigh 9 mg of PGP, and add 3 mL of 2M TFA sequentially under ice-water bath conditions. Hydrolyze at 105 °C for 3 h. After drying each polysaccharide hydrolysate with nitrogen, dilute with ultrapure water, filter the hydrolysate through a 0.22 μm aqueous filter membrane, and then perform HPAEC-PAD analysis.

[0234] Comparative Example 29

[0235] The results are almost identical to Comparative Example 28, except that PGP is changed to PEP.

[0236] Comparative Example 30

[0237] The results are almost identical to Comparative Example 28, except that PGP is changed to PFP.

[0238] Comparative Example 31

[0239] The polysaccharide lyase was dissolved in acetate-sodium acetate buffer to obtain a 0.75 g / mL lyase solution. 9 mg of PGP was accurately weighed and added sequentially to 200 μL of the lyase solution and 400 μL of ultrapure water. This resulted in a polysaccharide concentration of 0.015 g / mL and a lyase concentration of 0.025 g / mL in the enzymatic digestion system. The mixture was incubated at 35 °C for 36 h. After inactivating the enzymes, each polysaccharide digest was heated at 100 °C for 20 min and then centrifuged. The polysaccharide digest supernatant was diluted with ultrapure water to a specific concentration and filtered through a 0.22 μm aqueous filter membrane. The filtrate was then subjected to HPAEC-PAD analysis.

[0240] Comparative Example 32

[0241] The majority of the samples are the same as Comparative Example 31, except that PGP is changed to PEP.

[0242] Comparative Example 33

[0243] Most of them are the same as Comparative Example 31, except that PGP is changed to PFP.

[0244] Performance data analysis:

[0245] I. Optimization of enzymatic hydrolysis conditions in enzyme-acid hydrolysis

[0246] (1) Optimization of enzymatic hydrolysis temperature

[0247] Compared with Examples 1, 2, and 3 and Comparative Examples 1 and 2, the temperatures during enzymatic hydrolysis of PEP in the enzymatic hydrolysis system were 32, 35, 38, 30, and 40 °C, respectively. The experimental results are as follows: Figure 2 As shown, when PEP was enzymatically hydrolyzed at 35 °C for 36 h, the uronic acid hydrolysis rate and total hydrolysis rate in the hydrolysate reached their highest values, at 88.89% and 65.02%, respectively. At hydrolysis temperatures of 30 °C and 40 °C, the uronic acid hydrolysis rate and total hydrolysis rate were lower, below 67% and 43%, respectively. This may be because the enzyme's enzymatic reaction rate was low at excessively low temperatures, while excessively high temperatures may have caused irreversible conformational changes in the enzyme, leading to the destruction of its active sites. Therefore, the hydrolysis efficiency is best when the hydrolysis temperature is between 32 and 38 °C.

[0248] (2) Optimization of enzymatic hydrolysis time

[0249] Compared with Examples 2, 4, and 5 and Comparative Examples 3 and 4, the enzymatic hydrolysis times of PEP in the breakdown enzyme hydrolysis system were 36, 24, 48, 12, and 72 h, respectively. The experimental results are as follows: Figure 3As shown, when PEP was enzymatically hydrolyzed at 35 °C for 36 h, both the uronic acid hydrolysis rate and the total hydrolysis rate in the hydrolysate reached their highest levels. The lowest hydrolysis rates for both uronic acid and total hydrolysis were observed at 12 h, indicating incomplete enzymatic reactions due to the short reaction time. At 72 h, the reaction time was too long, potentially leading to slow inactivation or self-degradation of the enzyme protein. Furthermore, it increased the risk of microbial contamination of the sample solution, resulting in interfering substances. Therefore, a hydrolysis time of 24–48 h yielded the best hydrolysis efficiency.

[0250] (3) Optimization of the amount of breakdown enzyme added and the mass ratio of acidic polysaccharide to breakdown enzyme

[0251] Comparative Examples 6, 7, 8, 9, 10 and Comparative Examples 5, 6, the mass concentrations of the decomposing enzyme in the PEP enzymatic hydrolysis system were 0.008, 0.01, 0.02, 0.03, 0.05, 0.0025, and 0.005 g / mL, respectively. That is, when the mass ratio of acidic polysaccharide sample to decomposing enzyme in the enzymatic hydrolysis system was 0.625, 0.5, 0.25, 0.17, 0.1, 2.0, and 1.0, respectively, the experimental results are as follows: Figure 4 As shown, when the concentration of the lyase in the enzymatic hydrolysis system is 0.02–0.03 g / mL, i.e., the mass ratio of acidic polysaccharide sample to lyase in the enzymatic hydrolysis system is 0.17–0.25, the uronic acid hydrolysis rate and total hydrolysis rate in the PEP enzymatic hydrolysate are high. However, when the concentration of the lyase in the enzymatic hydrolysis system is 0.0025–0.005 g / mL, and the mass ratio of acidic polysaccharide sample to lyase in the enzymatic hydrolysis system is 1–2, the low hydrolysis rate of PEP is due to the relatively high content of the acidic polysaccharide substrate compared to the lyase content, resulting in incomplete hydrolysis. Therefore, when the concentration of the lyase in the enzymatic hydrolysis system is 0.008–0.05 g / mL, i.e., the mass ratio of acidic polysaccharide sample to lyase in the enzymatic hydrolysis system is 0.1–0.625, the uronic acid hydrolysis rate and total hydrolysis rate in PEP enzymatic hydrolysis are relatively high.

[0252] II. Optimization of Polysaccharide Addition in Enzyme-Acid Hydrolysis

[0253] (1) Optimization of PGP addition amount

[0254] Comparing Examples 11 and 12 with Comparative Examples 7 and 8, the concentrations of PGP in the enzymatic hydrolysis system were 0.005, 0.015, 0.050, and 0.100 g / mL, respectively. The experimental results are shown in Table 2. The total hydrolysis rate was highest (90.63%) when the concentration of PGP in the enzymatic hydrolysis system was 0.015 g / mL; the hydrolysis rate was low when the concentration was between 0.050 and 0.100 g / mL. Therefore, for enzymatic-acid hydrolysis of the low-uronic acid content polysaccharide PGP, a polysaccharide concentration of 0.005–0.015 g / mL in the enzymatic hydrolysis system yields the best hydrolysis rate.

[0255] Table 2. Changes in hydrolysis rate under different PGP addition amounts in the enzyme-acid hydrolysis method.

[0256]

[0257] Note: "-" indicates that the component is not present.

[0258] (2) Optimization of PEP addition amount

[0259] Comparing Examples 13 and 14 with Comparative Examples 9 and 10, the concentrations of PEP in the enzymatic hydrolysis system were 0.005, 0.015, 0.050, and 0.100 g / mL, respectively. The experimental results are shown in Table 3. When the concentration of PEP in the enzymatic hydrolysis system was 0.015 g / mL, the total hydrolysis rate was the highest (81.15%), which was significantly better than the hydrolysis effect at other polysaccharide concentrations. When the concentration was between 0.050 and 0.100 g / mL, the total hydrolysis rate was less than 70%, and the effect was not good. Similarly, for the enzymatic-acid hydrolysis of PEP, a polysaccharide with moderate uronic acid content, the hydrolysis rate was better when the polysaccharide concentration in the enzymatic hydrolysis system was between 0.005 and 0.015 g / mL.

[0260] Table 3. Changes in hydrolysis rate under different PEP addition amounts in the enzyme-acid hydrolysis method.

[0261]

[0262] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0263] (3) Optimization of PFP addition amount

[0264] Comparing Examples 15 and 16 with Comparative Examples 11 and 12, the concentrations of PFP in the enzymatic hydrolysis system were 0.005, 0.015, 0.050, and 0.100 g / mL, respectively. The experimental results are shown in Table 4. When the concentration of PFP in the enzymatic hydrolysis system was 0.015 g / mL, the total hydrolysis rate was the highest (75.15%), which was significantly better than the hydrolysis effect at other polysaccharide concentrations. When the concentration was between 0.050 and 0.100 g / mL, the total hydrolysis rate was less than 55%, indicating poor performance. Similarly, for the enzymatic-acid hydrolysis of the high-uronic acid content polysaccharide PFP, the hydrolysis rate was better when the polysaccharide concentration in the enzymatic hydrolysis system was between 0.005 and 0.015 g / mL.

[0265] Table 4. Changes in hydrolysis rate under different PFP addition amounts in the enzyme-acid hydrolysis method.

[0266]

[0267] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0268] III. Optimization of Sulfuric Acid Hydrolysis Conditions in Enzyme-Acid Hydrolysis

[0269] (1) Optimization of acid hydrolysis temperature

[0270] Comparative Examples 17-25 and Comparative Examples 13-18, PGP, PEP, and PFP enzyme hydrolysates were further acid-hydrolyzed at 95, 100, 105, 90, and 110 °C. The experimental results are as follows: Figure 5 As shown, the PGP, PEP, and PFP hydrolysates all achieved the highest total hydrolysis rates (93.76%, 83.34%, and 77.67%, respectively) upon further acid hydrolysis at 100 °C. The total hydrolysis rates of each acidic polysaccharide hydrolysate were higher when the acid hydrolysis temperature was between 95 and 105 °C. However, the total hydrolysis rates were lower when the acid hydrolysis temperatures were 90 and 110 °C. Therefore, further acid hydrolysis at 95–105 °C yielded the best hydrolysis rates for each acidic polysaccharide hydrolysate.

[0271] (2) Optimization of sulfuric acid hydrolysis conditions for enzyme-acid hydrolysis of PGP

[0272] Comparative Examples 26-34: PGP enzymatic hydrolysates were hydrolyzed at sulfuric acid concentrations of 0.5 M, 1 M, and 2 M for 30 min, 60 min, and 90 min, respectively. Table 5 shows the experimental results: when the sulfuric acid concentration in the acid hydrolysis step of the enzyme-acid hydrolysis method remained constant, the hydrolysis rate of PGP significantly increased with increasing hydrolysis time. This indicates that complete hydrolysis of PGP requires a relatively long hydrolysis time. Furthermore, uronic acid in PGP could be hydrolyzed at different sulfuric acid concentrations (0.5–2 M) and different hydrolysis times (30–90 min), but the hydrolysis effect was better at 90 min, allowing for the determination of uronic acid content. The hydrolysis effect was poor at 30 min. When the sulfuric acid concentration was 2 M and the hydrolysis time was 90 min, the uronic acid hydrolysis rate and total hydrolysis rate of the low-uronic acid content polysaccharide PGP were the highest, at 119.6% and 97.41%, respectively. The uronic acid hydrolysis rate of PGP exceeded 100%. This may be because the standard used in the colorimetric determination of its uronic acid content using the m-hydroxybiphenyl method was only galacturonic acid, which failed to accurately quantify the glucuronic acid and galacturonic acid content in PGP. Therefore, the optimal acid hydrolysis conditions for enzymatic-acid hydrolysis of polysaccharides with low uronic acid content are 100 °C and 0.5–2 M H₂SO₄ for 60–90 min.

[0273] Table 5. Optimization of acid hydrolysis conditions for enzyme-acid hydrolysis of polysaccharide PGP with low uronic acid content.

[0274]

[0275] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0276] (3) Optimization of the sulfuric acid hydrolysis conditions for PEP by enzyme-acid hydrolysis

[0277] Comparative Examples 35-43 showed that PEP enzymatic hydrolysates were hydrolyzed at 0.5 M, 1 M, and 2 M sulfuric acid concentrations for 30 min, 60 min, and 90 min, respectively. Table 6 shows that the enzyme-acid method for hydrolyzing PEP, followed by hydrolysis with 0.5-2 M sulfuric acid for 30-90 min, effectively hydrolyzed trace amounts of Fuc and Rha in PEP. When the sulfuric acid concentration was 0.5 M and the hydrolysis time was 60 min, the uronic acid hydrolysis rate and total hydrolysis rate of PEP with moderate uronic acid content were the highest, at 98.99% and 86.17%, respectively. The optimal acid hydrolysis conditions for enzymatic-acid hydrolysis of polysaccharides with moderate uronic acid content were 100 °C and 0.5-2 M H₂SO₄ for 30-90 min.

[0278] Table 6. Optimization of acid hydrolysis conditions for PEP, a polysaccharide with moderate uronic acid content, via enzyme-acid hydrolysis.

[0279]

[0280] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0281] (4) Optimization of sulfuric acid hydrolysis conditions for enzyme-acid hydrolysis of PFP

[0282] Comparative Examples 44-52, PFP enzymatic hydrolysates were hydrolyzed at sulfuric acid concentrations of 0.5 M, 1 M, and 2 M for 30 min, 60 min, and 90 min, respectively. The experimental results, as shown in Table 7, indicate that for enzymatic-acid hydrolysis of PFP, the highest uronic acid hydrolysis rate and total hydrolysis rate (99.26% and 82.81%, respectively) were achieved when the sulfuric acid concentration was 1 M and the hydrolysis time was 60 min. The optimal acid hydrolysis conditions for high uronic acid content polysaccharides were hydrolysis at 100 °C with 0.5–2 M H₂SO₄ for 30–90 min.

[0283] Table 7 Optimization of acid hydrolysis conditions for enzyme-acid hydrolysis of polysaccharide PFP with high uronic acid content

[0284]

[0285] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0286] (5) Optimization of the volume ratio of sample enzymatic hydrolysate to acid solution in enzyme-acid hydrolysis

[0287] Comparative Examples 53-61 and Comparative Examples 19-24 showed that the volume ratios of PGP, PEP, and PFP enzyme hydrolysates to acid solution were 0.05, 0.1, 0.15, 0.2, and 0.25, respectively. The experimental results are as follows: Figure 6 As shown, the total hydrolysis rates of the three acidic polysaccharides PGP, PEP, and PFP reached their highest values ​​(97.41%, 86.17%, and 82.81%, respectively) when the volume ratio of the enzymatic hydrolysate to the acid solution was 0.15. The hydrolysis effect was better when the volume ratio of the enzymatic hydrolysate to the acid solution was between 0.1 and 0.2.

[0288] IV. Comparison of the effects of single hydrolysis method on acidic polysaccharides

[0289] (1) Hydrolysis of sulfuric acid alone

[0290] Comparative Examples 25-27 showed the effects of single sulfuric acid hydrolysis on PGP, PEP, and PFP. Under sulfuric acid hydrolysis, the uronic acid hydrolysis rates of PGP, PEP, and PFP were 58.40%, 54.92%, and 59.66%, respectively, with total hydrolysis rates of 86.03%, 66.71%, and 50.72%, respectively. This indicates that sulfuric acid hydrolysis is more effective at hydrolyzing neutral monosaccharides in acidic polysaccharides. However, the higher the uronic acid content in the polysaccharide sample, the worse the overall hydrolysis effect of single sulfuric acid hydrolysis. This may be attributed to the poor effectiveness of sulfuric acid hydrolysis in hydrolyzing uronic acids in acidic polysaccharides.

[0291] (2) Hydrolysis of trifluoroacetic acid

[0292] Comparative Examples 28-30 involved the hydrolysis of PGP, PEP, and PFP by trifluoroacetic acid alone. After hydrolysis with trifluoroacetic acid, the uronic acid hydrolysis rates of PGP, PEP, and PFP were 39.19%, 18.79%, and 11.41%, respectively, with total hydrolysis rates of 84.99%, 52.12%, and 14.96%, respectively. Clearly, trifluoroacetic acid alone is ineffective at hydrolyzing acidic polysaccharides.

[0293] (3) Enzymatic hydrolysis by a single breakdown enzyme

[0294] Comparative Examples 31-33 involved the hydrolysis of PGP, PEP, and PFP using a single catalytic enzyme. The catalytic enzyme was ineffective in hydrolyzing PGP; the uronic acid hydrolysis rates for PEP and PFP were 88.89% and 91.11%, respectively, with total hydrolysis rates of 65.02% and 68.39%, respectively. While the overall hydrolysis efficiency of the catalytic enzyme in acidic polysaccharide samples needs improvement, it exhibits a unique advantage in the hydrolysis of uronic acids.

[0295] (4) Comparison of hydrolysis effects between enzyme-acid hydrolysis and single hydrolysis

[0296] <1> Low uronic acid content polysaccharide

[0297] The hydrolysis effects of enzymatic-acid hydrolysis (Example 33, 2 M, 90 min) and three single hydrolysis methods—sulfuric acid hydrolysis (Comparative Example 25), trifluoroacetic acid hydrolysis (Comparative Example 28), and catabolite hydrolysis (Comparative Example 31)—on polysaccharides with low uronic acid (PGP) content are shown in Table 8. Among the single hydrolysis methods, the total hydrolysis rates of the two single acid hydrolysis methods for PGP with low uronic acid content were not significantly different, but the uronic acid hydrolysis rates were 58.40% and 39.19%, respectively. The incomplete hydrolysis of PGP with low uronic acid content under single hydrolysis pretreatment resulted in lower measured monosaccharide composition results than the actual values.

[0298] The total hydrolysis rate of low-uronic acid polysaccharides achieved by enzyme-acid hydrolysis (Example 33, 2 M, 90 min) reached 97.41%, with a uronic acid hydrolysis rate of 119.59%. Under this pre-hydrolysis treatment method, the uronic acid and total hydrolysis rates in the sample were significantly improved compared to the three single hydrolysis methods, with increases of over 61.19% and 11.38% respectively compared to single acid hydrolysis. Therefore, the enzyme-acid hydrolysis method provided by this invention has a better hydrolysis effect on acidic polysaccharides with low uronic acid content.

[0299] Table 8. Comparison of single hydrolysis and enzymatic-acid hydrolysis effects of polysaccharide PGP with low uronic acid content

[0300]

[0301] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0302] <2> Polysaccharides with moderate uronic acid content

[0303] The differences in the hydrolysis effects of three single hydrolysis methods—sulfuric acid hydrolysis (Comparative Example 26), trifluoroacetic acid hydrolysis (Comparative Example 29), cytokinase hydrolysis (Comparative Example 32), and enzyme-acid hydrolysis (Example 35, 0.5 M, 60 min)—on polysaccharides with moderate uronic acid content (PEP) are shown in Table 9. The total hydrolysis rate of PEP with moderate uronic acid content was not significantly different under sulfuric acid hydrolysis and cytokinase hydrolysis, but the uronic acid hydrolysis rate was only 54.92% under sulfuric acid hydrolysis; cytokinase hydrolyzed 88.89% of uronic acid, but its effect on some neutral monosaccharides in PEP was poor. The total hydrolysis rate of PEP hydrolyzed by trifluoroacetic acid was only 52.12%, and the uronic acid hydrolysis rate was only 18.79%, indicating poor hydrolysis performance. Under the enzymatic-acid hydrolysis method (Example 35, 0.5 M, 60 min), the total hydrolysis rate of polysaccharides containing moderate uronic acid reached 86.17%, and the uronic acid hydrolysis rate reached 98.99%. The total hydrolysis rate and uronic acid hydrolysis rate of the polysaccharides were increased by more than 19.46% and 44.07% respectively compared to the results of single acid hydrolysis. Therefore, the enzymatic-acid hydrolysis method used in this invention is still superior to the three single hydrolysis methods in the application of acidic polysaccharides containing moderate uronic acid.

[0304] Table 9 Comparison of single hydrolysis and enzymatic-acid hydrolysis effects of polysaccharide PEP with medium uronic acid content.

[0305]

[0306] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0307] <3> High uronic acid content polysaccharides

[0308] The differences in hydrolysis effects of three single hydrolysis methods—sulfuric acid hydrolysis (Comparative Example 27), trifluoroacetic acid hydrolysis (Comparative Example 30), lyase hydrolysis (Comparative Example 33), and enzyme-acid hydrolysis (Example 47, 1 M, 60 min)—on polysaccharides with high uronic acid content are compared, and the results are shown in Table 10. Although sulfuric acid hydrolysis is more effective than trifluoroacetic acid hydrolysis, the uronic acid hydrolysis rate and total hydrolysis rate of PFP hydrolyzed by sulfuric acid are only 59.66% and 50.72%, respectively, which cannot accurately analyze the monosaccharide composition of the polysaccharide. Compared with single acid hydrolysis, the total hydrolysis rate of PFP hydrolyzed by lyase reaches 68.39%, mainly because this pretreatment method has a good effect on hydrolyzing uronic acid residues in polysaccharides with high uronic acid content, and the uronic acid hydrolysis rate in the polysaccharide hydrolysate reaches 91.11%.

[0309] Under the enzymatic-acid hydrolysis method (Example 47, 1 M, 60 min), the total hydrolysis rate of polysaccharides with high uronic acid content reached 82.81%, and the uronic acid hydrolysis rate reached 99.26%. Compared with the two single acid hydrolysis methods, the total hydrolysis rate and uronic acid hydrolysis rate of polysaccharides were increased by more than 32.09% and 39.60%, respectively. Therefore, the enzymatic-acid hydrolysis method provided by this invention has a better effect on hydrolyzing polysaccharides with high uronic acid content.

[0310] Table 10 Comparison of the effects of single hydrolysis and enzymatic-acid hydrolysis on polysaccharide PFP with high uronic acid content

[0311]

[0312] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0313] The enzyme-acid hydrolysis method provided by this invention avoids the problems of low acid hydrolysis rate and monosaccharide degradation caused by high-concentration acids and excessively long hydrolysis times for polysaccharides. It has a wide range of applications for acidic polysaccharides, and its hydrolysis effect on polysaccharides with low, medium, and high uronic acid content is better than that of single hydrolysis methods. It is simple to operate and has high uronic acid hydrolysis and total hydrolysis rates. The total hydrolysis rate of low-uronic acid polysaccharides by the enzyme-acid method can reach 97.41%, the uronic acid hydrolysis rate and total hydrolysis rate for polysaccharides with medium uronic acid content can reach 98.99% and 86.17%, respectively, and the uronic acid hydrolysis rate and total hydrolysis rate for polysaccharides with high uronic acid content can reach 99.26% and 82.81%, respectively. Especially for polysaccharides with high uronic acid content, the total hydrolysis effect and uronic acid hydrolysis effect of the enzyme-acid method are improved by more than 32.09% and 39.60%, respectively, compared to traditional single acid hydrolysis.

[0314] Accuracy analysis of enzymatic-acid hydrolysis method and single hydrolysis method for determining uronic acid content in acidic polysaccharides

[0315] The results of uronic acid content determination in acidic polysaccharides PGP, PEP, and PFP using three single hydrolysis methods—sulfuric acid hydrolysis (Comparative Examples 25-27), trifluoroacetic acid hydrolysis (Comparative Examples 28-30), degradase hydrolysis (Comparative Examples 31-33), and enzyme-acid hydrolysis (Examples 33, 35, and 47)—are shown in Table 11, compared with the results of colorimetric determination. Using the colorimetric determination of uronic acid content as a reference, the differences between the uronic acid content determination values ​​of PGP, PEP, and PFP obtained by the enzyme-acid hydrolysis method and the reference values ​​were all smaller than those obtained by the three single hydrolysis methods, and were highly consistent with the reference values, demonstrating the highest accuracy. The traditional one-step acid hydrolysis method resulted in uronic acid content values ​​in acidic polysaccharide samples that were all lower than the reference values. Specifically, the results obtained from hydrolyzing PGP were 58.40% lower than the reference value, and the results obtained from hydrolyzing PEP were 59.66% lower than the reference value. While enzyme-mediated hydrolysis improved the hydrolysis of uronic acid in PEP and PFP, resulting in a higher measured value compared to acid hydrolysis alone and close to the reference value determined by colorimetric method, it failed to effectively hydrolyze uronic acid in PGP. Therefore, enzyme-acid hydrolysis is more accurate than acid hydrolysis alone and enzyme-mediated hydrolysis in determining the monosaccharide composition of polysaccharides with low, medium, and high uronic acid content.

[0316] Table 11. Determination of uronic acid content of PGP, PEP, and PFP by different hydrolysis methods and colorimetric methods

[0317]

[0318] Note: "-" indicates that the component is not present.

[0319] VI. Intraday verification of the effectiveness of enzyme-acid hydrolysis in hydrolyzing acidic polysaccharides

[0320] Examples 62-64 represent the intraday validation of the enzymatic-acid hydrolysis method for low uronic acid content polysaccharide PGP (2M, 90 min), medium uronic acid content polysaccharide PEP (0.5M, 60 min), and high uronic acid content polysaccharide PEP (1M, 60 min). The experimental results are shown in Table 12. The contents of Rha and Xyl in PFP were 0.42% and 0.13%, respectively, which were extremely low. This may be the reason why the RSD values ​​of Rha and Xyl in PFP were greater than 3% but less than 10% in the intraday validation experiment. Apart from this, the RSD values ​​of the monosaccharide composition results of each polysaccharide were all less than 3%, which indicates that the enzymatic-acid hydrolysis method has high stability and accuracy.

[0321] Table 12 Intra-day validation RSD values ​​of PGP, PEP, and PFP hydrolyzed by enzyme-acid hydrolysis

[0322]

[0323] Note: "-" indicates that the component is not present; "*" indicates that the component is present in trace amounts and cannot be calculated.

[0324] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for determining the monosaccharide composition of an acidic polysaccharide, characterized in that, Includes the following steps: S1. Enzymatic hydrolysis: The acidic polysaccharide sample is mixed with the decomposing enzyme in a buffer system for enzymatic hydrolysis. After the reaction, the enzyme is inactivated to obtain the enzymatic hydrolysate. The temperature of the enzymatic hydrolysis reaction is 32~38℃ and the reaction time is 24~48 h. S2, Acid hydrolysis: Add acid to the enzymatic hydrolysate and carry out acid hydrolysis under heating conditions to obtain a hydrolysate containing monosaccharides. The temperature of the acid hydrolysis reaction is 95~105℃, the reaction time is 30~90 minutes, and the hydrogen ion concentration in the acid is 1~4M. S3. Separate and analyze the obtained hydrolysate to determine the monosaccharide composition of the acidic polysaccharide sample.

2. The method for determining the monosaccharide composition of an acidic polysaccharide according to claim 1, characterized in that, In step S1, the buffer solution is one of acetate-sodium acetate buffer, citrate-sodium citrate buffer, or phosphate buffer.

3. The method for determining the monosaccharide composition of an acidic polysaccharide according to claim 1, characterized in that, In step S1, the concentration of the breakdown enzyme in the reaction system is 0.008~0.05 g / mL; The concentration of the acidic polysaccharide sample in the reaction system is 0.005~0.015 g / mL.

4. The method for determining the monosaccharide composition of an acidic polysaccharide according to claim 1, characterized in that, In step S1, the mass ratio of the acidic polysaccharide sample to the breakdown enzyme is 0.1~0.625:

1.

5. The method for determining the monosaccharide composition of an acidic polysaccharide according to claim 1, characterized in that, In step S1, the acidic polysaccharide sample includes one or more of the following: polysaccharides with low uronic acid content, polysaccharides with medium uronic acid content, or polysaccharides with high uronic acid content. The low-uronic acid content polysaccharides include peach gum polysaccharide, the medium-uronic acid content polysaccharides include Yunnan olive polysaccharide, and the high-uronic acid content polysaccharides include passion fruit pectin.

6. The method for determining the monosaccharide composition of an acidic polysaccharide according to claim 1, characterized in that, In step S2, the acid solution is one or more of sulfuric acid solution, hydrochloric acid solution, or trifluoroacetic acid solution; The volume ratio of the enzymatic hydrolysate to the acid solution is 0.1~0.2:

1.

7. The method for determining the monosaccharide composition of an acidic polysaccharide according to claim 1, characterized in that, In step S2, the hydrolysate includes one or more of the following: fucose, rhamnose, arabinose, galactose, glucose, mannose, xylose, fructose, glucuronic acid, or galacturonic acid. In step S3, the monosaccharide composition that can be determined includes one or more of trehalose, rhamnose, arabinose, galactose, glucose, mannose, xylose, fructose, glucuronic acid, or galacturonic acid.

8. The method for determining the monosaccharide composition of an acidic polysaccharide according to claim 1, characterized in that, In step S3, the hydrolysate is separated and detected using high-performance anion exchange chromatography-pulse amperometric detection.

9. The method for determining the monosaccharide composition of an acidic polysaccharide according to claim 8, characterized in that, The high-performance anion exchange chromatography-pulse amperometric detection method is equipped with an anion exchange guard column and an analytical column, and uses an aqueous solution of sodium hydroxide and / or sodium acetate as the mobile phase for gradient elution. The flow rate for chromatographic analysis was 0.4–0.5 mL / min, the column temperature was 28–35 °C, and the data acquisition time was 50–70 min.

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