Pseudo-ginseng herb residue polysaccharide enzymolysis optimization method and process

Abstract

The invention relates to the technical field of resource recovery, in particular to a pseudo-ginseng residue polysaccharide enzymolysis optimization method and process. The method comprises the following steps: respectively putting pseudo-ginseng dregs into different enzyme solutions for enzymolysis to obtain polysaccharide yield, and screening an enzyme preparation according to the polysaccharide yield; based on the screened enzyme preparation, carrying out a single factor test on the pseudo-ginseng dregs, and based on the result of the single factor test, carrying out response surface method optimization on the polysaccharide enzymatic hydrolysis test on the pseudo-ginseng dregs. The invention aims to optimize the enzymolysis process of the pseudo-ginseng dregs by combining a response surface method, so that the polysaccharide yield can be increased, and a scientific basis is provided for high-value utilization of the pseudo-ginseng dregs, thereby promoting the sustainable development of the traditional Chinese medicine industry and reducing the environmental pollution.

Description

Technical Field

[0001] This invention relates to the field of resource recycling technology, and in particular to an optimized method and process for enzymatic hydrolysis of polysaccharides from Panax notoginseng residue. Background Technology

[0002] Traditional Chinese medicine (TCM) resources are an important natural resource in my country and are of strategic significance for the sustainable development of the pharmaceutical industry. However, with the rapid development of the TCM industry, the amount of medicinal residue generated has been increasing year by year. Statistics show that my country produces approximately 60-70 million tons of TCM residue annually. Currently, this residue is mainly disposed of through open-air dumping, landfilling, or incineration, which not only causes a serious waste of land resources but also pollutes soil, water, and air due to its susceptibility to mold and decay and the emission of foul odors. These disposal methods not only threaten the environment but also lead to a significant waste of TCM resources. In recent years, with technological advancements and increased awareness of good manufacturing practices, TCM enterprises have begun to focus on the resource utilization of medicinal residue, striving to turn waste into treasure.

[0003] However, existing extraction processes still limit the extraction of effective components from medicinal residues, resulting in generally low extraction rates and resource waste. Only about 5%–10% of the effective components in plant-based medicinal materials are extracted. Medicinal residues are still rich in active substances such as polysaccharides, flavonoids, saponins, cellulose, lignin, and trace elements. Currently, the development and utilization of medicinal residues mainly include preparing microbial protein feed through microbial fermentation or processing them into organic fertilizer to achieve ecological recycling. Using mixed bacteria and synergistic enzyme fermentation to prepare protein feed increases the crude protein content by 9.17%, while simultaneously reducing the crude fiber content, degrading cellulose and hemicellulose in the medicinal residues, and improving the nutritional components and efficacy of the medicinal residues.

[0004] Panax notoginseng, as an important traditional Chinese medicine, possesses various biological activities, including immunomodulatory, anti-inflammatory, antioxidant, anti-tumor, and cardiovascular protective effects. Currently, Panax notoginseng processing mainly focuses on extracting saponins, resulting in the disposal of approximately 1000 tons of Panax notoginseng residue annually. This residue still retains many active ingredients, such as notoginsenosides, total polysaccharides, total saponins, and total flavonoids, which have significant development potential. Failure to utilize these resources would lead to substantial waste and loss. However, research on polysaccharide extraction processes from Panax notoginseng residue is currently limited; therefore, improving the polysaccharide extraction rate from Panax notoginseng residue is of great importance. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide an optimized method and process for the enzymatic hydrolysis of polysaccharides from Panax notoginseng residue. By combining response surface methodology to optimize the enzymatic hydrolysis process of Panax notoginseng residue, the polysaccharide yield can be improved, providing a scientific basis for the high-value utilization of Panax notoginseng residue, thereby promoting the sustainable development of the traditional Chinese medicine industry and reducing environmental pollution.

[0006] The present invention solves the above-mentioned technical problems through the following technical means:

[0007] An optimized method for enzymatic hydrolysis of polysaccharides from Panax notoginseng residue, the method comprising:

[0008] The residue of Panax notoginseng was placed in different enzyme solutions for enzymatic hydrolysis to obtain the polysaccharide yield. Based on the polysaccharide yield, enzyme preparations were screened.

[0009] Based on the screened enzyme preparation, single-factor experiments were conducted on Panax notoginseng residue. Based on the results of the single-factor experiments, the polysaccharide enzymatic hydrolysis experiment of Panax notoginseng residue was optimized using response surface methodology.

[0010] In one embodiment, when the Panax notoginseng residue is placed in different enzyme solutions for enzymatic hydrolysis, the different enzyme solutions include cellulase solution, pectinase solution, papain solution, β-glucosidase solution and snail enzyme solution.

[0011] And / or, when performing the enzymatic hydrolysis, the solution includes pure water, the material-to-liquid ratio is 15 mL / g, the hydrolysis temperature is 50°C, the enzyme addition is 1%, and the hydrolysis time is 2 h.

[0012] Based on the above technical means, by enzymatically hydrolyzing Panax notoginseng residue under the same conditions with different enzymes, the enzyme that can obtain the highest polysaccharide yield can be screened out.

[0013] In one embodiment, when conducting the single-factor experiment, the factors in the single factor include the material-to-liquid ratio, the amount of enzyme added, the enzymatic hydrolysis temperature, and the enzymatic hydrolysis time.

[0014] Based on the above technical means, by examining the effects of factors such as the material-to-liquid ratio, enzyme addition amount, enzymatic hydrolysis temperature, and enzymatic hydrolysis time on the production of polysaccharides through enzymatic hydrolysis, it is convenient to optimize these factors.

[0015] In one embodiment, the response surface methodology optimization is further performed by bivariate correlation analysis, using the results of the single-factor experiment obtained from the bivariate correlation analysis, and then performing the response surface methodology optimization.

[0016] In one embodiment, the bivariate correlation analysis uses Pearson, Kendall, and Spearman correlation coefficients to analyze the correlation between the material-liquid ratio, enzyme dosage, hydrolysis temperature, and hydrolysis time and the polysaccharide.

[0017] Based on the above technical means, key influencing factors are identified through bivariate correlation analysis, which facilitates subsequent optimization using response surface methodology.

[0018] This application also discloses an enzymatic hydrolysis process for polysaccharides from Panax notoginseng residue, including the following steps:

[0019] Place the Panax notoginseng residue in a pure aqueous solution, add snail enzyme, and enzymatically hydrolyze for 30-90 minutes at an enzymatic hydrolysis temperature of 40-65℃.

[0020] Preferably, the Panax notoginseng residue is dried and passed through a 40-mesh sieve when placed in a pure aqueous solution.

[0021] Further preferably, when Panax notoginseng residue is added to pure aqueous solution, the material-to-liquid ratio is 17 mL / g.

[0022] Preferably, the snail enzyme is added at an amount of 1-3.5%.

[0023] Further preferably, the amount of snail enzyme added is 2.6%.

[0024] Preferably, the enzymatic hydrolysis temperature is 42°C.

[0025] Preferably, the enzymatic hydrolysis time is 60 min.

[0026] According to the above technical methods, the polysaccharide yield of Panax notoginseng residue is highest under the following conditions: material-to-liquid ratio of 17 mL / g, snail enzyme addition of 2.6%, enzymatic hydrolysis temperature of 42℃, and enzymatic hydrolysis time of 60 min.

[0027] The present application, employing the above-described scheme, has at least the following beneficial effects:

[0028] In this application, the process of extracting polysaccharides from Panax notoginseng residue by snail enzyme hydrolysis was optimized using response surface methodology (RSM). The optimized process not only improved the polysaccharide yield but also increased the resource utilization rate of Panax notoginseng residue, providing a feasible solution for the green development of the traditional Chinese medicine industry. Attached Figure Description

[0029] This application can be further illustrated by the non-limiting embodiments given in the accompanying drawings;

[0030] Figure 1 This is a flowchart illustrating the optimized enzymatic hydrolysis method for Panax notoginseng residue polysaccharides in this application;

[0031] Figure 2 This is the glucose standard curve in this application;

[0032] Figure 3 This is a graph showing the effect of enzyme addition on polysaccharide yield in Example 2 of this application;

[0033] Figure 4 This is a graph showing the effect of the material-to-liquid ratio on the polysaccharide yield in Example 2 of this application;

[0034] Figure 5 This is a graph showing the effect of temperature on polysaccharide yield in Example 2 of this application;

[0035] Figure 6 This is a graph showing the effect of enzymatic hydrolysis time on polysaccharide yield in Example 2 of this application;

[0036] Figure 7 This is a correlation analysis graph of the factors and polysaccharide yield in Example 3 of this application;

[0037] Figure 8 These are 3D diagrams and contour plots of the response surface optimization of the enzymatic hydrolysis process of Panax notoginseng residue in Example 3 of this application;

[0038] Figure 9 This is a comparison chart of the predicted and actual values ​​of the ANN model test set (A) and the RSM (B) model in Example 3 of this application. Detailed Implementation

[0039] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0040] Firstly, such as Figure 1 As shown, the optimized enzymatic hydrolysis method for Panax notoginseng residue polysaccharides of the present invention includes:

[0041] The residue of Panax notoginseng was placed in different enzyme solutions for enzymatic hydrolysis to obtain the polysaccharide yield. Based on the polysaccharide yield, enzyme preparations were screened.

[0042] Based on the screened enzyme preparation, single-factor experiments were conducted on Panax notoginseng residue. Based on the results of the single-factor experiments, the polysaccharide enzymatic hydrolysis experiment of Panax notoginseng residue was optimized using response surface methodology.

[0043] In this embodiment, by first placing the Panax notoginseng residue in different enzyme solutions for enzymatic hydrolysis, the enzyme preparation that produces the highest polysaccharide yield is screened out. Based on this enzyme preparation and the polysaccharide yield, single-factor experiments are conducted. According to the results of the single-factor experiments, the response surface methodology is used to optimize the different influencing factors of the Panax notoginseng residue polysaccharide enzymatic hydrolysis experiment, thereby obtaining the optimal Panax notoginseng residue polysaccharide enzymatic hydrolysis process.

[0044] In one specific embodiment, when the Panax notoginseng residue is placed in different enzyme solutions for enzymatic hydrolysis, the different enzyme solutions include cellulase solution, pectinase solution, papain solution, β-glucosidase solution and snail enzyme solution.

[0045] In this embodiment, the Panax notoginseng residue is the biomass residue remaining after ethanol extraction of saponins from Panax notoginseng rhizomes, containing structural polysaccharides, pectin, residual saponins, and a small amount of protein. Based on the main components of the residue—cellulose, pectin, and protein—enzymatic hydrolysis experiments were conducted using cellulase (which decomposes cellulose), pectinase (which decomposes pectin), papain (which decomposes protein), β-glucosidase (which decomposes glucan), and snail enzyme (which contains multiple enzymes and decomposes complex polysaccharides). This yielded an enzyme preparation that produced the highest polysaccharide content.

[0046] In one specific embodiment, during enzymatic hydrolysis, the solution includes pure water, the material-to-liquid ratio is 15 mL / g, the hydrolysis temperature is 50°C, the enzyme addition amount is 1%, and the hydrolysis time is 2 h.

[0047] By performing enzymatic hydrolysis in pure water and limiting the material-to-liquid ratio, hydrolysis temperature, enzyme dosage, and hydrolysis time, the enzyme itself becomes the only variable, thus enabling the screening of the optimal enzyme preparation.

[0048] In one specific implementation, when conducting the single-factor experiment, the factors in the single factor include the material-to-liquid ratio, the amount of enzyme added, the enzymatic hydrolysis temperature, and the enzymatic hydrolysis time.

[0049] By examining the effects of factors such as the material-to-liquid ratio, enzyme dosage, hydrolysis temperature, and hydrolysis time on polysaccharide production, it is possible to optimize these factors.

[0050] In one specific implementation, the response surface methodology optimization further includes bivariate correlation analysis. The results of the single-factor experiment are analyzed using the bivariate correlation analysis, and then the response surface methodology optimization is performed. The bivariate correlation analysis uses Pearson, Kendall, and Spearman correlation coefficients to analyze the correlation between the material-liquid ratio, enzyme addition amount, enzymatic hydrolysis temperature, and enzymatic hydrolysis time and the polysaccharide.

[0051] Key influencing factors were identified through bivariate correlation analysis, which facilitated subsequent optimization using response surface methodology.

[0052] Therefore, the optimized enzymatic hydrolysis method for Panax notoginseng residue polysaccharides in this application specifically includes the following methods:

[0053] Panax notoginseng residue was placed in cellulase solution, pectinase solution, papain solution, β-glucosidase solution and snail enzyme solution respectively. Enzymatic hydrolysis was carried out in pure water under the conditions of material-to-liquid ratio of 15 mL / g, enzymatic hydrolysis temperature of 50℃, enzyme addition of 1%, and enzymatic hydrolysis time of 2 h to obtain polysaccharide yield, total flavonoid content and saponin content. Based on the polysaccharide yield, snail enzyme was selected as the enzyme preparation for the enzymatic hydrolysis of Panax notoginseng residue.

[0054] Based on snail enzyme, single-factor enzymatic hydrolysis experiments were conducted on Panax notoginseng residue. The single-factor factors included the material-to-liquid ratio, enzyme dosage, hydrolysis temperature, and hydrolysis time. Based on the results of the single-factor experiments, Pearson, Kendall, and Spearman correlation coefficients (range -1 to 1) were used to analyze the correlation between the material-to-liquid ratio, enzyme dosage, temperature, and time and the polysaccharide yield to identify key influencing factors. SPSS software was used for calculations when analyzing the single-factor experimental data. The results showed that the material-to-liquid ratio, enzyme dosage, and temperature had strong correlations with the polysaccharide yield, while the correlation with time was weak. Therefore, the response surface methodology (RSM) optimization experiment fixed the hydrolysis time at 60 min. The RSM experiment was then designed using Design-Expert 12 software, selecting the material-to-liquid ratio, enzyme dosage, and hydrolysis temperature as the three factors, with the polysaccharide yield as the response value, to design experiments and optimize the enzymatic hydrolysis process parameters.

[0055] Then, an artificial neural network (ANN) model was constructed using a multilayer perceptron (MLP) in SPSS software, including an input layer (material-liquid ratio, enzyme dosage, temperature), a hidden layer, and an output layer (polysaccharide yield) (based on SERGEIO, VITALIIC, VITALIIM. Application of Machine Learning Methods for Estimating the Fuel Consumption of Locomotives for Switching Service[J]. Transportation Research Procedia, 2021, 54: 802-807.). The hidden layer had one node, the activation function was hyperbolic tangent, and the training algorithm was gradient descent. The training, validation, and test sets were divided in a 7:2:1 ratio (based on response surface methodology data). This model was used to perform simulation experiments to fit the response surface results and virtual samples to obtain the optimal polysaccharide target, using R... 2 The model performance was evaluated and compared with response surface methodology to screen and validate the optimal experimental parameters for the enzymatic hydrolysis of polysaccharides from Panax notoginseng residue. The formula for the hyperbolic tangent function is as follows:

[0056] Where e is the base of the natural logarithm.

[0057] The gradient descent method is the batch gradient descent method in the existing technology.

[0058] The enzymatic hydrolysis process of Panax notoginseng residue polysaccharide of the present invention will be described in detail below.

[0059] Medicinal materials and reagents:

[0060] The residue from Panax notoginseng was provided by Yunnan Baiyao Group Co., Ltd. Anhydrous glucose was from Sinopharm Chemical Reagent Co., Ltd.; anthrone and sodium nitrite were from Tianjin Fuchen Chemical Reagent Factory; sodium hydroxide was from Beijing Chemical Plant; methanol was from Beijing Bailingwei Technology Co., Ltd.; rutin (reference standard) was from Beijing Century Aoke Biotechnology Co., Ltd.; Panax notoginseng saponins R1, Re, Rg1, Rb1, Rd, and F2 (reference standards) were all purchased from Beijing Solarbio Technology Co., Ltd. Cellulase was from Dalian Meilun Biotechnology Co., Ltd.; pectinase and papain were from Solarbio; snail enzyme was from Shanghai Yuanye Biotechnology Co., Ltd.

[0061] Instruments and equipment:

[0062] ZN-20L mini pulverizer, Beijing Xingshilihe Technology Development Co., Ltd.; analytical balance, Sartorius Scientific Instruments (Beijing) Co., Ltd.; SL-100 shaker, Yongkang Songqing Hardware Factory; SLXFATS microplate reader, Porton Instruments, Inc.; low-speed centrifuge, Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd.; SCIENTZ-50F freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.

[0063] Methods for determining the content of enzymatic hydrolysis products:

[0064] A precise 1.00 mmol / g anhydrous glucose standard stock solution was prepared, and glucose standard solutions of different concentration gradients were prepared by dilution. 0.12 mL of glucose standard solution and 0.12 mL of purified water were added to anthrone-sulfuric acid solution at a ratio of 1:5. After thorough shaking and mixing, the solution was placed in a boiling water bath for 10 minutes. After cooling to room temperature, the absorbance at 620 nm was measured, and a standard curve was plotted. The standard regression equation was y = 0.2743x - 0.0002, and the correlation coefficient R0 was [value missing]. 2 =0.9995, showing a good linear relationship. The sample solution was processed in the same way, and the absorbance at 620 nm was measured after diluting the sample solution by a certain factor.

[0065] When using the anthrone-sulfuric acid method, a standard curve was established using Abs(x) - concentration (y). Glucose standard solutions with concentrations ranging from 0.4 to 0.00625 mg / mL were prepared, and the results are as follows: Figure 2 .

[0066] The saponin components (ginsenoside R1, ginsenoside Re, ginsenoside Rg1, ginsenoside Rb1, ginsenoside Rd, and ginsenoside F2) were detected by HPLC. Acetonitrile (C) and water (D) were used as the mobile phase, the flow rate was 1.0 mL / min, the detection wavelength was 203 nm, the column temperature was 35 ℃, and the injection volume was 20 μL.

[0067] The aluminum nitrate-sodium nitrite colorimetric method was used. The extract was diluted to 2 mL with 70% ethanol, and then 0.3 mL of 5% NaNO2 solution and 10% Al(NO3)3 solution were added sequentially. The mixture was shaken and allowed to stand. Then, 10% NaOH solution was added and allowed to stand for 15 min. The absorbance (Abs) was measured at 510 nm and the value was recorded to calculate the total flavonoid content.

[0068] Example 1: Effects of different enzymes on the enzymatic hydrolysis effect and composition of Panax notoginseng residue

[0069] Multiple reaction vessels were prepared, and equal amounts of Panax notoginseng residue were placed in pure aqueous solutions in each vessel at a material-to-liquid ratio of 15 mL / g. Cellulase, pectinase, papain, β-glucosidase, snail enzyme, and a compound enzyme (a mixture of cellulase, pectinase, and papain in a mass ratio of 2:2:1) were then added to each vessel. A control vessel without added enzymes was used. The amount of each enzyme added was 1%. The temperature was set at 50℃ and the enzymatic hydrolysis time was 2 hours. The enzymatic hydrolysis reaction was carried out, and each experiment was repeated three times. The average value was taken. The results are shown in Table 1.

[0070] Table 1. Effects of different enzyme treatments on the yield of polysaccharides and content of active components in Panax notoginseng residue.

[0071]

[0072]

[0073] Table 1 shows that snail enzyme polysaccharide yield was the highest, reaching 4.687%, significantly higher than the control group (1.711%) and other single enzymes. Pectinase (4.178%) and cellulase (4.022%) performed well in decomposing pectin and cellulose, while the compound enzyme showed significantly higher levels of total flavonoids (1.47 mg / g) and saponins (e.g., R1 0.836 mg / g, Rg1 0.685 mg / g) than other single enzyme groups, demonstrating its advantage in multi-component retention. Papain and β-glucosidase yields were low, reflecting limited substrate specificity and matching degree with the polysaccharide structure of the drug residue. Some saponins (e.g., Rd, F2) were not detected under single enzyme treatment, possibly due to unfavorable enzymatic hydrolysis conditions (e.g., pH or temperature) or insufficient sensitivity of the detection method.

[0074] The high efficiency of snail enzymes stems from their complex enzyme system (containing cellulase, pectinase, papain, etc.), which synergistically decomposes the complex cell walls and pectin layer of Panax notoginseng residue, releasing polysaccharides. After ethanol extraction, the residual polysaccharides in Panax notoginseng residue mainly exist in the form of cellulose-pectin complexes. The synergistic effect of snail enzymes significantly improves the solubility of polysaccharides. The advantage of the complex enzymes in saponin retention may be due to the optimized ratio of its enzyme components, reducing the hydrolysis or conversion of saponins. The control group had a higher flavonoid content of 1.45 mg / g, indicating that flavonoids are easily soluble under water extraction conditions, while polysaccharides and some saponins require enzymatic hydrolysis for release. The significant difference between snail enzymes and complex enzymes in polysaccharide yield and saponin content suggests their potential in different application scenarios.

[0075] Example 2: Effect of single-factor experiments on enzymatic hydrolysis process

[0076] Dried Panax notoginseng residue powder that has passed through a 40-mesh sieve was used as the experimental material, and purified water was used as the solvent. The polysaccharide yield was used as the indicator, and each experiment was conducted in triplicate. With other conditions kept constant, the effects of the following factors on the polysaccharide yield were investigated: material-to-liquid ratio (1:10 mL / g, 1:15 mL / g, 1:20 mL / g, 1:25 mL / g, 1:30 mL / g, 1:35 mL / g), snail enzyme dosage (1%, 1.5%, 2%, 2.5%, 3%, 3.5%), enzymatic hydrolysis temperature (40℃, 45℃, 50℃, 55℃, 60℃, 65℃), and enzymatic hydrolysis time (30 min, 45 min, 60 min, 75 min, 90 min).

[0077] The test results are as follows Figure 3 , Figure 4 , Figure 5 , Figure 6 As shown.

[0078] Depend on Figure 3 It was found that the polysaccharide yield reached its highest level of 11.730% when the enzyme addition was 2.5%, significantly higher than that when the enzyme addition was 1.0% (8.323%). However, the polysaccharide yield decreased slightly after exceeding 2.5%, for example, it decreased by 12.9% when the enzyme addition was 3.5%. Appropriate enzyme addition enhanced the binding efficiency between the enzyme and the substrate, promoting the release of polysaccharides from the cell walls of the drug residue. Excessive enzyme dosage may trigger competition between enzyme molecules or non-specific reactions, leading to a decrease in efficiency. This highlights the high efficiency of snail enzymes and the synergistic effect of their complex enzyme system; therefore, an enzyme addition of 2.5% was chosen as the central point for subsequent optimization.

[0079] Depend on Figure 4It was found that the highest polysaccharide yield (11.167%) was achieved at a solid-liquid ratio of 15 mL / g, which was 34.17% higher than that at 10 mL / g. The yield decreased significantly beyond 15 mL / g, reaching only 7.028% at 30 mL / g. An appropriate solid-liquid ratio facilitates sufficient contact between the residue and the solvent, promoting polysaccharide dissolution and maintaining enzyme activity. An excessively high solid-liquid ratio may dilute the enzyme concentration or increase the dissolution of impurities, reducing extraction efficiency. This experiment showed a higher yield at 15 mL / g, indicating that the polysaccharide structure of the Panax notoginseng residue is more suitable for these conditions; therefore, the optimal solid-liquid ratio was determined to be 15 mL / g.

[0080] Depend on Figure 5 The results showed that the highest polysaccharide yield (9.552%) was achieved at 45℃, significantly higher than the 8.289% at 40℃. The polysaccharide yield decreased above 45℃. 45℃ may be the optimal temperature for snail enzymes, where enzyme activity reaches its peak, promoting the decomposition of cellulose and pectin. High temperatures may lead to enzyme denaturation or polysaccharide degradation, reducing the yield. The higher polysaccharide yield at 45℃ suggests the temperature sensitivity of snail enzymes and their superiority under mild conditions; therefore, 45℃ can be selected as the optimal extraction temperature for future studies.

[0081] Depend on Figure 6 It was found that the highest polysaccharide yield (8.323%) was achieved at a hydrolysis time of 60 min, significantly higher than the 7.881% at 30 min. The yield decreased after 60 min, reaching only 7.464% at 90 min. Enzymatic hydrolysis within 60 min fully releases the polysaccharides; extending the reaction time may lead to polysaccharide hydrolysis or enzyme inactivation. The time factor has a significant impact, but its effect is weaker than that of the material-to-liquid ratio and the amount of enzyme added. Therefore, 60 min was fixed as the optimal time condition for subsequent optimization, effectively reflecting the high efficiency of the snail enzyme.

[0082] Example 3: Optimization of enzymatic hydrolysis process parameters for Panax notoginseng residue

[0083] S1. Bivariate Correlation Analysis:

[0084] The correlation coefficients (Pearson, Kendall, and Spearman correlation coefficients, ranging from -1 to 1) were used to analyze the correlation between the solid-liquid ratio, enzyme dosage, hydrolysis temperature, and hydrolysis time and the polysaccharide yield to identify key influencing factors. The analysis was based on single-factor experimental data and calculated using SPSS software. The results are as follows: Figure 7 As shown, from Figure 7It was found that the solid-liquid ratio, enzyme dosage, and temperature were strongly correlated with the polysaccharide yield, while the time correlation was weak. The high correlation of enzyme dosage reflects its dominant role in enzyme-substrate binding efficiency, the solid-liquid ratio affects solute diffusion and enzyme activity, and temperature regulates enzyme catalytic efficiency. The weak correlation of time indicates that 60 min is close to enzymatic hydrolysis equilibrium, and extending the reaction time has limited contribution. Based on this, the solid-liquid ratio, enzyme dosage, and temperature can be selected as key factors for response surface methodology (RSM). Therefore, the RSM experiment fixed the hydrolysis time at 60 min and examined the three factors of solid-liquid ratio, enzyme dosage, and hydrolysis temperature.

[0085] S2. Response surface methodology:

[0086] Based on the results of single-factor experiments, a response surface methodology (RSM) experiment was designed using Design-Expert 12 software. The material-to-liquid ratio (A), enzyme dosage (B), and temperature (C) were selected as the three factors, with polysaccharide yield as the response value. Seventeen sets of experiments were designed to optimize the enzymatic hydrolysis process parameters. The experimental results are shown in Tables 3-4 and 4. Figure 8 As shown in the table, Table 3 presents the response surface methodology and results of the enzymatic hydrolysis process of Panax notoginseng residue; Table 4 presents the analysis of variance of the regression model for the response surface methodology of the enzymatic hydrolysis process of Panax notoginseng residue. Snail enzyme was used in all experiments, with a fixed hydrolysis time of 60 min. Process optimization and screening were conducted according to the principles of response surface methodology. The experimental factors and level table design are shown in Table 2.

[0087] Table 2. Response Surface Experimental Factors and Levels

[0088] level Material-to-liquid ratio (mL / g) Enzyme addition amount (%) Temperature (°C) -1 10 2 40 0 15 2.5 45 1 20 3 50

[0089] Table 3. Response surface design and results of enzymatic hydrolysis process of Panax notoginseng residue

[0090]

[0091] Table 4. Analysis of Variance for Regression Models

[0092]

[0093]

[0094] Note: *P<0.05, significant; **P<0.01, highly significant.

[0095] The regression equation is Y = -146.87 + 0.89145A + 29.926B + 4.9986C + 0.1478AB - 0.01238AC - 0.2972BC - 0.02217A 2 -3.419B 2 -0.04523C 2 R 2=0.9978, indicating a very high model fit and reliable prediction accuracy. Enzyme dosage (F=897.25, P<0.0001) and material-to-liquid ratio (F=48.08, P=0.0002) were the dominant factors, with interaction terms AB (F=87.43), AC (F=61.34), and BC (F=353.52) all being highly significant (P<0.0001). Temperature had no significant effect as a single factor (P=0.8902), but its quadratic term (P<0.0001) and interaction terms with other factors (such as BC, P<0.0001) were highly significant. Temperature remains an indispensable key factor in the model and optimization. Based on the F-values, the order of influence of the three factors on the enzymatic hydrolysis yield of Panax notoginseng residue is B>A>C, i.e., enzyme dosage > material-to-liquid ratio > temperature.

[0096] Figure 8 The strong interaction between the material-to-liquid ratio and the enzyme dosage was visually demonstrated. The optimization results predicted the optimal process as follows: material-to-liquid ratio 17.362 mL / g, enzyme dosage 2.609%, temperature 42.232℃, and polysaccharide yield 12.031%. For ease of operation, the ratio was adjusted to 17 mL / g, enzyme dosage 2.6%, and temperature 42℃. The validation experiment yielded 12.119%, with a relative error of 0.73% compared to the predicted value, validating the high accuracy of the model. Compared to the highest yield of 11.730% in the single-factor experiment, the optimized yield increased by 3.3%, and by 158% compared to the single enzyme treatment. The material-to-liquid ratio of 17 mL / g is superior to the single-factor optimal of 15 mL / g, possibly due to its enhanced interaction with the enzyme dosage, which improves enzyme-substrate contact efficiency (AB interaction, P<0.0001). The temperature of 42℃ is lower than the single-factor optimal (45℃), reflecting that the model balances the requirements of enzyme activity and polysaccharide stability, highlighting the advantages of snail enzyme and RSM optimization.

[0097] S3. Artificial Neural Network Model:

[0098] An artificial neural network (ANN) model was constructed using a multilayer perceptron (MLP) in SPSS software. The model included an input layer (material-to-liquid ratio, enzyme dosage, and temperature), hidden layers, and an output layer (polysaccharide yield). The hidden layer had one node, with hyperbolic tangent activation. Gradient descent was used for training. The training, validation, and test sets were partitioned in a 7:2:1 ratio (based on 17 sets of data from response surface methodology experiments). This model was used to fit the response surface results and virtual samples in simulation experiments to obtain the optimal polysaccharide target. The R-squared value was calculated. 2 The model performance was evaluated and compared with that of a response surface model. Results are as follows: Figure 9 As shown:

[0099] Depend on Figure 9 It can be seen that the R-value of the ANN model on the training set is... 2 =0.849, the goodness of fit is lower than RSM(R 2=0.9978), which may be due to the limited amount of data restricting the ANN's ability to capture complex nonlinear relationships. The hidden layer structure and parameter settings also need further optimization. RSM, based on BBD design, is suitable for small sample scenarios and shows higher prediction accuracy, but its generalization ability may be limited by the experimental range. ANN may be more suitable for capturing complex patterns of multi-factor interactions after the amount of data increases. The RSM model in this application (R 2 =0.9978) is more accurate, verifying its superiority in the enzymatic hydrolysis of Panax notoginseng residue.

[0100] Model performance comparison

[0101] Table 5 Comparison of performance metrics between RSM and ANN models

[0102]

[0103] The performance comparison results of the models (Table 5) show that the response surface model (R) 2 =0.9978) shows a significant advantage in fitting modeling data, with its RMSE (0.051) and MAE (0.042) being much lower than the corresponding values ​​(R²) of artificial neural network models on the test set. 2 =0.849, RMSE=0.177, MAE=0.26). This highlights the superior performance of the RSM method in predicting the enzymatic hydrolysis process parameters of Panax notoginseng residue under the specific dataset and small sample conditions of this application.

[0104] Example 4: Enzymatic hydrolysis process of polysaccharides from Panax notoginseng residue

[0105] The residue of Panax notoginseng was placed in a pure aqueous solution with a material-to-liquid ratio of 17 mL / g. 2.6% snail enzyme was added, and the mixture was enzymatically hydrolyzed at 42℃ for 60 min, with a polysaccharide yield of 12.119%.

[0106] Example 5: Utilization of polysaccharide enzymatic hydrolysis residue from Panax notoginseng residue

[0107] The polysaccharide residue of Panax notoginseng still contains residual polysaccharides, proteins and trace elements after enzymatic hydrolysis, and still has the potential for biotransformation.

[0108] The enzymatic hydrolysis residue was dried and pulverized. After high-temperature sterilization and cooling, *Lactobacillus acidophilus* was inoculated onto the residue for secondary fermentation at a liquid-to-solid ratio of 1:15 (mL / g). The fermentation endpoint was set at 48 hours on a shaker. Testing according to the method described in this application revealed that the polysaccharide content still accounted for 6.51% of the residue's dry basis.

[0109] This indicates that the residue after enzymatic hydrolysis can be added to animal diets after secondary fermentation, which helps improve animal growth performance and immunity. Studies on fermented medicinal residue feed have shown that the crude protein content in the residue can be increased from 12.51% to 21.65% through microbial fermentation. In studies on the fermentation of Panax notoginseng residue, probiotics mainly caused deglycosylation metabolism of saponins, and the contents of sugars and flavonoids were significantly increased, by 105.64% compared to the residue before fermentation.

[0110] In summary, this application successfully optimized the snail enzyme enzymatic extraction process for polysaccharides from Panax notoginseng residue using response surface methodology (RSM). Combining RSM with the optimization of polysaccharide extraction from Panax notoginseng residue overcomes the limitations of traditional methods. Results showed that snail enzyme outperformed other enzymes in polysaccharide yield (4.687%), and the composite enzyme exhibited significant advantages in the retention of flavonoids and saponins. The optimized process not only improved the resource utilization rate of Panax notoginseng residue but also provided a feasible solution for the green development of the traditional Chinese medicine industry. Through single-factor experiments and bivariate analysis, the material-to-liquid ratio, enzyme dosage, and temperature were identified as key factors. Response surface optimization determined the optimal process conditions to be a material-to-liquid ratio of 17 g / mg, an enzyme dosage of 2.6%, and a temperature of 42℃, verifying a polysaccharide yield of 12.119%, an improvement of 158% compared to single-enzyme treatment.

[0111] The above provides a detailed description of the optimized enzymatic hydrolysis method and process for polysaccharides from Panax notoginseng residue provided by this invention. The specific embodiments are provided only to aid in understanding the method and core concepts of this invention. It should be noted that those skilled in the art can make various improvements and modifications to this invention without departing from its principles, and these improvements and modifications also fall within the scope of protection of the claims of this invention.

[0112] It should be noted that: for experimental steps or conditions not specified in the examples, the procedures and conditions described in conventional experimental procedures in the literature of this art can be followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0113] The above examples are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by anyone under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

Claims

1. A method for enzymatic hydrolysis optimization of polysaccharides from Panax notoginseng residue, characterized in that, The method comprises: The method comprises: The method comprises:

2. The optimized enzymatic hydrolysis method for Panax notoginseng residue polysaccharides according to claim 1, characterized in that, The method comprises: The method comprises:

3. The method of claim 1 or 2, wherein the enzyme is selected from the group consisting of cellulase, hemicellulase, pectinase, xylanase, amylase, protease, lipase, and combinations thereof. The method comprises:

4. The method of claim 3, wherein the enzyme is a protease. The method comprises:

5. The method of claim 4, wherein the enzyme is a protease. The method comprises:

6. A polysaccharide enzymatic hydrolysis process for Panax notoginseng residue as described in any one of claims 1-5, characterized in that, The method comprises: The method comprises:

7. The enzymatic process of ginseng residue polysaccharide according to claim 6, characterized in that, The method comprises: The method comprises:

8. The enzymatic process of panax notoginseng residue polysaccharide according to claim 6 or 7, characterized in that, The method comprises:

9. The enzymatic process of ginseng residue polysaccharide according to claim 6 or 7, characterized in that, The method comprises:

10. 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