Method for optimizing preparation, separation and purification process of lambda-carrageenan oligosaccharide by response surface method

Abstract

The invention discloses a method for optimizing lambda-carrageenan oligosaccharide preparation, separation and purification processes through a response surface method. The method comprises the following steps: preparing a mixed solution of lambda-carrageenan, H2O2 and vitamin C; performing water bath on the mixed solution, performing ultrafiltration to obtain a filtrate, performing dialysis, concentration, alcohol precipitation and freeze-drying to obtain lambda-carrageenan oligosaccharide, and performing chromatographic separation and purification by using a gel column. The lambda-carrageenan oligosaccharide is prepared by adopting an H2O2-vitamin C oxidation method, the preparation process of the lambda-carrageenan oligosaccharide is optimized by combining a single factor with a response surface experiment, and the optimal preparation conditions are as follows: the concentration of an H2O2 solution is 3.3% (equimolar vitamin C is added), the water bath temperature is 43 DEG C, and the water bath time is 4.6 hours. Compared with lambda-carrageenan, after the lambda-carrageenan oligosaccharide obtained by the process is subjected to gel chromatography, the molecular weight is reduced, the purity is improved, and the main chemical structure is not changed. The method has the advantages of being simple in process, low in cost, environmentally friendly, high in product purity, complete in structure and the like, and a theoretical basis is provided for research and production of the lambda-carrageenan oligosaccharide.

Description

Technical Field

[0001] This invention relates to the field of polysaccharide derivative preparation technology, specifically to a method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology. Background Technology

[0002] Carrageenan is a sulfated polysaccharide with a linear structure extracted from red algae. Its basic structure consists of repeating disaccharide units composed of galactose and 3,6-anhydrogalactose linked by alternating α-1,3 glycosidic bonds and β-1,4 bonds. Based on the substitution position and number of sulfate groups on the sugar units, it can be classified into κ-, ι-, and λ-types. λ-carrageenan has the highest degree of sulfated formation, with each disaccharide unit containing an average of 3 sulfate ester groups, accounting for approximately 41% (w / w) of the disaccharide unit structure, thus exhibiting the highest negative charge density and unique hydration properties. λ-carrageenan oligosaccharides are obtained by degrading λ-carrageenan. Compared to λ-carrageenan, they have a lower molecular weight, better water solubility, and higher and greater bioavailability. Current research has found that λ-carrageenan oligosaccharides possess anticoagulant, antioxidant, anti-inflammatory, antitumor, hypoglycemic, and lipid-lowering activities. They have broad application potential in functional foods and pharmaceuticals. Therefore, the efficient preparation of λ-carrageenan oligosaccharides has become an important research issue.

[0003] The main methods for preparing carrageenan oligosaccharides currently include physical methods, enzymatic methods, and chemical methods. Physical methods primarily utilize ultraviolet light, ultrasound, and microwaves to degrade carrageenan; however, due to their low degradation efficiency, they are generally used as auxiliary methods. Enzymatic hydrolysis has the advantages of mild reaction and single product, but it suffers from drawbacks such as high enzyme cost, easy inactivation, and difficulty in large-scale application. Chemical methods mainly include acid hydrolysis and oxidation. Acid hydrolysis is a vigorous reaction that easily causes the loss of active groups such as sulfate ester groups, while oxidation has high degradation efficiency, a relatively mild degradation process, and produces highly active products.

[0004] Current optimization methods for the oxidative degradation of carrageenan often rely on single-factor controlled variable methods to find optimal parameters. This approach fails to effectively assess and optimize the interactions between multiple parameters, resulting in insufficient accuracy and reliability of the obtained "optimal conditions," making it difficult to guide stable and efficient large-scale production. Response surface methodology (RSM), by establishing mathematical models, can systematically study the impact of various factors and their interactions on the response value, efficiently and accurately pinpointing the optimal conditions. Box-Behnken design within RSM requires fewer experiments and is highly efficient, suitable for process optimization involving 3 to 5 factors. Therefore, utilizing Box-Behnken design within RSM to efficiently consider interactions between multiple variables, improve accuracy, and optimize the oxidative preparation of λ-carrageenan oligosaccharides is of great significance. Summary of the Invention

[0005] The purpose of this invention is to address the shortcomings and defects of existing technologies by providing a method for optimizing the preparation, separation, and purification process of λ-carrageenan oligosaccharides using response surface methodology.

[0006] To achieve the above objectives, the present invention employs the following technical solution: a method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology, comprising the following steps: preparing a mixture of λ-carrageenan solution and H2O2 solution containing an equimolar amount of vitamin C; bathing the mixture in a constant temperature water bath to obtain a degradation solution; passing the degradation solution sequentially through 10 kDa and 5 kDa ultrafiltration membranes, collecting the filtrate with a molecular weight less than 5 kDa; dialyzing the filtrate and concentrating it, adding anhydrous ethanol for precipitation, collecting the precipitate, resolving it in pure water, and freeze-drying it to obtain the initial product of λ-carrageenan oligosaccharides; separating and purifying the initial product of λ-carrageenan oligosaccharides using gel column chromatography, concentrating and freeze-drying it to obtain purified λ-carrageenan oligosaccharides; wherein, the concentrations of H2O2 and vitamin C and the water bath parameters are determined by optimization using response surface methodology, the optimization including: based on the results of single-factor experiments, using the concentration of H2O2, water bath temperature, and water bath time as independent variables, and the yield of λ-carrageenan oligosaccharides as the response value, and using Design Expert... The optimal process conditions were obtained through experimental design and analysis using software 13.0 and Box-Behnken response surface methodology.

[0007] Furthermore, the concentration of the λ-carrageenan solution is 1 wt%.

[0008] Furthermore, the concentration of the H2O2 solution is 2-6% (w / v).

[0009] Furthermore, the water bath time is 1-5 hours.

[0010] Furthermore, the temperature of the water bath is 30-70℃.

[0011] Furthermore, the optimal process conditions are: H2O2 concentration of 3.31%, water bath temperature of 42.66℃, and water bath time of 4.57h.

[0012] Furthermore, the gel column chromatography uses Chromdex 30PG gel medium.

[0013] Furthermore, the ultrafiltration membrane is a regenerated cellulose membrane, and the ultrafiltration process is carried out at room temperature and an operating pressure not exceeding 2.5 Bar.

[0014] Furthermore, the alcohol precipitation involves adding 4-6 times the volume of anhydrous ethanol to the concentrated liquid and allowing it to stand at 4°C for 8-12 hours; the drying is freeze drying under the following conditions: cold trap temperature -60°C, vacuum degree less than 10 Pa, and drying time 48 hours.

[0015] A λ-carrageenan oligosaccharide prepared by the method described above.

[0016] After adopting the above technical solution, the beneficial effects of the present invention are as follows: 1. Simple and efficient process, environmentally friendly: This invention uses the oxidation method of H2O2 and vitamin C solution to prepare λ-carrageenan oligosaccharides, which has the characteristics of simple operation, high yield and low cost. The preparation process does not generate environmental pollution, thus meeting the requirements of economy and environmental protection.

[0017] 2. Optimized Methodology, Significantly Improved Accuracy: This invention determines the optimal range of key factors based on single-factor experiments, and then uses the Box-Behnken response surface methodology to study the interaction between H2O2 solution concentration, water bath time, and water bath temperature. Multiple linear and quadratic regression models are established to efficiently analyze the relationships between variables and improve the accuracy of the results. This method overcomes the limitations of traditional single-factor methods, achieving high efficiency and significantly improving process accuracy.

[0018] 3. Effective purification methods and improved product purity: After obtaining the initial product, the present invention uses gel column chromatography for separation and purification, which can remove components and impurities with uneven molecular weight distribution in the initial product, and further improve the component uniformity and purity of the product.

[0019] 4. The product structure is intact, and the active groups are preserved: Analysis of the product revealed that, under optimal preparation conditions, the molecular weight of the λ-carrageenan oligosaccharide is lower than that of λ-carrageenan, falling within the range of oligosaccharides, while the main skeletal structure and functional groups remain unchanged. This demonstrates that while achieving efficient degradation, the active structural basis is perfectly preserved. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a schematic diagram illustrating the effect of H2O2 concentration on the yield of λ-carrageenan oligosaccharides in this invention.

[0022] Figure 2 This is a schematic diagram illustrating the effect of water bath time on the yield of λ-carrageenan oligosaccharides in this invention.

[0023] Figure 3 This is a schematic diagram illustrating the effect of water bath temperature on the yield of λ-carrageenan oligosaccharides in this invention.

[0024] Figure 4This is a response surface plot showing the effect of H2O2 concentration and water bath time on the yield of λ-carrageenan oligosaccharides in this invention.

[0025] Figure 5 This is a response surface plot showing the effect of H2O2 concentration and water bath temperature on the yield of λ-carrageenan oligosaccharides in this invention.

[0026] Figure 6 This is a response surface plot showing the effect of water bath time and water bath temperature on the yield of λ-carrageenan oligosaccharides in this invention.

[0027] Figure 7 This is the elution curve of λ-carrageenan oligosaccharide gel filtration column chromatography in this invention.

[0028] Figure 8 This is a high-performance liquid chromatography gel permeation chromatogram of λ-carrageenan and λ-carrageenan oligosaccharides in this invention.

[0029] Figure 9 These are scanning electron microscope images of λ-carrageenan (A) and λ-carrageenan oligosaccharide (B) in this invention.

[0030] Figure 10 This is the Fourier transform infrared spectrum of λ-carrageenan and λ-carrageenan oligosaccharide in this invention. Detailed Implementation Example 1: Single-factor experiment

[0031] To investigate the individual effects of each key factor on the yield of λ-carrageenan oligosaccharides, single-factor experiments were conducted.

[0032] Basic experimental conditions: Accurately weigh λ-carrageenan, dissolve it in pure water at 80℃ with stirring, and prepare a 1wt% solution. Add hydrogen peroxide solution containing an equimolar amount of vitamin C and mix thoroughly.

[0033] Factors and levels to be considered: 1) H2O2 concentration: With a fixed water bath time of 4h and a temperature of 40℃, the effect of H2O2 concentrations of 2%, 3%, 4%, 5%, and 6% (w / v) on the yield was investigated.

[0034] 2) Water bath time: With a fixed H2O2 concentration of 3% and a temperature of 40℃, the effect of water bath time on yield was investigated for 1h, 2h, 3h, 4h and 5h.

[0035] 3) Water bath temperature: With a fixed H2O2 concentration of 3% and a time of 4 hours, the effect of water bath temperatures of 30℃, 40℃, 50℃, 60℃ and 70℃ on the yield was investigated.

[0036] Yield determination and results: After the reaction was completed, the oligosaccharide yield was calculated according to the methods for determining reducing sugars and total sugars. Results are as follows: Figure 1-3As shown in the figure. The results indicate that the oligosaccharide yield reached its highest value under each single-factor condition when the hydrogen peroxide concentration was 3%, the water bath time was 4 h, and the water bath temperature was 40 °C. Therefore, this condition was selected as the central point for the response surface methodology.

[0037] Example 2: Response Surface Methodology Optimization Experiment Based on the results of single-factor experiments, the degradation process was optimized using a Box-Behnken design (BBD).

[0038] Experimental Design: Using Design-Expert 13.0 software, a three-factor, three-level response surface methodology experiment was designed with hydrogen peroxide concentration (A), water bath time (B), and water bath temperature (C) as independent variables and λ-carrageenan oligosaccharide yield (Y) as the response value. Factor codes and levels are shown in Table 1.

[0039] Table 1: Factors and Levels in Box-Behnken Experimental Design Experiments and Results: A total of 17 experiments were conducted. The design scheme and measured yield results are shown in Table 2.

[0040] Table 2: Box-Behnken Experimental Design and Results Model establishment and analysis: Multiple regression fitting was performed on the experimental data to obtain the quadratic polynomial regression equation of the yield (Y) on the encoded independent variables A, B, and C: Y = -164.42 + 32.08A + 20.73B + 4.64C - 0.09AB - 0.08AC - 0.21BC - 4.25A² - 1.25B² - 0.04C².

[0041] Analysis of variance (ANOVA) was performed on the model, and the results are shown in Table 3. The model has a large F-value (89.53), a highly significant P-value (<0.0001), and no significant lack-of-fit terms (P=0.3630>0.05). Furthermore, the coefficient of determination R²=0.9914, and the adjusted coefficient of determination R²adj=0.9803, indicating that the model is highly significant, has a good fit, and accurately reflects the relationship between factors and response values. The order of influence of each factor is: temperature (C) > concentration (A) > time (B). The interaction terms AC and BC have significant effects (P<0.05). Response surface plots can visually reflect the influence of each factor on the response value, facilitating the analysis and study of the interactions between variables and identifying extreme values. The steeper the slope of the response surface plot, the more significant the interaction between the two factors and the greater their impact on the response value. Figure 4-6 As shown, Figure 5 and Figure 6 The slope of the three-dimensional curved surface is higher than Figure 4This indicates that the interaction between concentration and temperature, and between time and temperature, is more significant than that between concentration and time, which is consistent with the model analysis results.

[0042] Table 3: Analysis of Variance Table for Regression Model Note: P < 0.05 is significant, P < 0.01 is highly significant. Model Validation: The optimal preparation process, calculated by software, was determined to be: concentration 3.31%, temperature 42.66℃, and extraction time 4.57h. For ease of practical operation, this was modified to: concentration 3.3%, extraction temperature 43℃, and extraction time 4.6h. Three repeated experiments were conducted to verify the yields, which were 33.98%, 34.06%, and 33.87%, respectively. The average yield of λ-carrageenan oligosaccharides was 33.97%, with a deviation of less than 1% from the predicted value of 34.92%, demonstrating the reliability of the response surface methodology optimization scheme.

[0043] Example 3: Preparation of λ-carrageenan oligosaccharides Degradation: Accurately weigh λ-carrageenan and dissolve it in pure water at 80℃ to prepare a 1wt% solution. Add hydrogen peroxide solution containing an equimolar amount of vitamin C to the solution to make the final concentration of H2O2 in the mixture 3.3% (w / v). Place the mixture in a constant temperature water bath at 43℃ for 4.6 h to obtain the degradation solution.

[0044] Ultrafiltration: The degradation solution was first ultrafiltered at room temperature using a regenerated cellulose ultrafiltration membrane with a molecular weight cutoff of 10 kDa, under an operating pressure not exceeding 2.5 Bar, and the permeate was collected. This permeate was then ultrafiltered a second time using a regenerated cellulose ultrafiltration membrane with a molecular weight cutoff of 5 kDa, under the same conditions, and finally the filtrate with a molecular weight less than 5 kDa was collected.

[0045] Dialysis: The filtrate was placed in a 500 Da dialysis bag and placed in pure water. The water was changed every 4 hours for a total of 72 hours. The solution was concentrated to 1 / 5 of its original volume under reduced pressure at 50°C. Five times the volume of the concentrated solution of anhydrous ethanol was added, and the solution was placed in a refrigerator at 4°C for 12 hours for alcohol precipitation.

[0046] Freeze-drying: After alcohol precipitation, the sample was centrifuged at 10,000 rpm for 20 min. The supernatant was discarded, the precipitate was collected, reconstituted with pure water, and placed in a freeze dryer. Under the conditions of cold trap temperature -60℃ and vacuum degree less than 10 Pa, the sample was freeze-dried for 48 h to obtain the initial product of λ-carrageenan oligosaccharide.

[0047] Example 4: Isolation and purification of λ-carrageenan oligosaccharides Approximately 500 mg of the initial product obtained in Example 3 was dissolved in 5 ml of ultrapure water and centrifuged at 8000 rpm for 10 min. The supernatant was collected and filtered through a 0.45 μm microporous membrane. The filtrate was loaded onto a pre-equilibrated gel chromatography column (Chromdex 30PG, 2.6 cm × 100 cm), with a loading volume of 1% of the column volume. After all the sample solution had entered the chromatography column, ultrapure water was used for elution at a flow rate of 2.0 ml / min. The eluent was collected using an automatic collector, with 5 ml collected per tube, eluting at 1 column volume. The collection was monitored online using a differential detector. The elution curve is shown below. Figure 7 As shown, the eluent corresponding to the main peak (symmetrical and concentrated peak shape) was collected. The eluents were combined, concentrated under reduced pressure, and freeze-dried to obtain the purified λ-carrageenan oligosaccharide. Three parallel experiments were performed, and the average yield of the purified product (mass of purified product / mass of unpurified product) was 75.00%.

[0048] Example 5: Product structure characterization and performance analysis The purified λ-carrageenan oligosaccharide obtained in Example 4 was compared with the total sugar content, sulfate group content, molecular weight and characterization structure of λ-carrageenan.

[0049] Total sugar content: determined by the phenol-sulfuric acid method. The total sugar content of λ-carrageenan was 67.82% ± 0.87%, while the total sugar content of λ-carrageenan oligosaccharides was 75.65% ± 0.39%, indicating a significant improvement in purity.

[0050] Sulfate content: determined using the gelatin-barium chloride method. The sulfate content of the raw material λ-carrageenan was 32.17% ± 0.45%, and the sulfate content of λ-carrageenan oligosaccharides was 39.61% ± 0.93%. The results show that the degradation and purification process of this invention did not cause sulfate loss, but the relative sulfate content in the product increased due to the removal of impurities.

[0051] Molecular weight: Absolute molecular weight was determined by gel permeation chromatography. λ-carrageenan oligosaccharides and λ-carrageenan were dissolved separately in 0.1M NaNO3 solution (containing 0.02% NaN3, w / w) to a final concentration of 1 mg / mL, and then filtered through a 0.45 μm filter before analysis. Figure 8The HPLC gel permeation chromatogram of λ-carrageenan oligosaccharides is shown. The chromatographic system used was a gel chromatography-differential chromatography-multi-angle laser light scattering system. The column and elution conditions were: Shodex SB 805-803 tandem gel column, column temperature 45℃, injection volume 100 μL, mobile phase 0.1 M NaNO3 solution, flow rate 0.6 mL / min, and isocratic elution gradient for 75 min. The chromatographic data were processed using ASTRA 6.1 software, and the average absolute molecular weight was calculated. λ-carrageenan oligosaccharides: 1742 Da; λ-carrageenan: 732.3 kDa. The results demonstrate that the method of this invention can effectively reduce the molecular weight of λ-carrageenan, achieving the molecular weight standard for oligosaccharides.

[0052] Appearance morphology: The appearance morphology of the polysaccharide samples was observed using scanning electron microscopy. For example... Figure 9 As shown in the image, magnified at 2000×, λ-carrageenan (A) exhibits a dense lamellar structure, while λ-carrageenan oligosaccharide (B) transforms into a smooth, small, and dispersed fragment structure, visually reflecting the degradation effect.

[0053] Infrared absorption spectroscopy: Fourier transform infrared spectroscopy was used to study the changes in functional groups before and after the degradation of λ-carrageenan. Comparative spectra are shown below. Figure 10 As shown, λ-carrageenan oligosaccharides and λ-carrageenan are basically consistent in the positions of their main characteristic absorption peaks, such as the S=O stretching vibration near 1250 cm⁻¹ and the characteristic peak of 3,6-lactone galactose near 920 cm⁻¹, indicating that the degradation process did not destroy the main chain backbone and characteristic functional groups of λ-carrageenan.

[0054] Comparative example: The preparation process of κ-carrageenan oligosaccharides reported in the published patent CN117050209A was applied to λ-carrageenan.

[0055] The method is as follows: A 2wt% λ-carrageenan solution was prepared and dissolved in pure water by stirring. H₂O₂-vitamin C solution was added to bring the final concentration to 10 mM. The mixed solution was placed in a reaction environment at 50°C and 40 MPa for 30 min to obtain the λ-carrageenan degradation solution. Subsequent ultrafiltration, dialysis, alcohol precipitation, freeze-drying, and gel chromatography purification steps were the same as in Examples 3 and 4 of this invention.

[0056] The comparative product was subjected to three parallel experiments, and its oligosaccharide yield, total sugar content, and sulfate group content were 3.59%, 68.25%, and 38.69%, respectively. Compared with the embodiment of the present invention (yield 33.97%, total sugar 75.65%, sulfate group 39.61%), the optimized method of the present invention has a much higher product yield and higher product purity, while also retaining active sulfate groups, demonstrating significant overall advantages.

[0057] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention, as long as they do not depart from the spirit and scope of the technical solution of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology, characterized in that, Includes the following steps: S1, prepare a mixture of λ-carrageenan solution and H2O2 solution containing equimolar amounts of vitamin C; S2, the mixture is placed in a constant temperature water bath to obtain the degradation solution; S3, the degradation solution is passed through 10 kDa and 5 kDa ultrafiltration membranes in sequence, and the filtrate with a molecular weight of less than 5 kDa is collected; S4. After dialysis and concentration of the filtrate, anhydrous ethanol was added for alcohol precipitation. The precipitate was collected, reconstituted with pure water, and freeze-dried to obtain the initial product of λ-carrageenan oligosaccharide. S5 separated and purified the primary product of λ-carrageenan oligosaccharide using gel column chromatography, and obtained purified λ-carrageenan oligosaccharide after concentration and freeze-drying. The concentrations of H2O2 and Vitamin C in S1 and the water bath parameters in S2 were determined by response surface methodology. The optimization included: based on the results of single-factor experiments, using H2O2 concentration, water bath temperature, and water bath time as independent variables, and the yield of λ-carrageenan oligosaccharides as the response value, and using Box-Behnken response surface experimental design and analysis to obtain the optimal process conditions.

2. The method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology according to claim 1, characterized in that: The concentration of the λ-carrageenan solution in S1 is 1 wt%.

3. The method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology according to claim 1, characterized in that: The concentration of H2O2 solution in S1 is 2-6% (w / v).

4. The method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology according to claim 1, characterized in that: The water bath time in S2 is 1-5 hours.

5. The method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology according to claim 1, characterized in that: The temperature of the water bath in S2 is 30-70℃.

6. The method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology according to claim 1, characterized in that: The optimal process conditions in S1 and S2 are: H2O2 concentration of 3.31%, water bath temperature of 42.66℃, and water bath time of 4.57h.

7. The method for optimizing the preparation and purification process of λ-carrageenan oligosaccharides using response surface methodology according to claim 1, characterized in that: In S5, the gel medium used for gel column chromatography is Chromdex 30PG.

8. A λ-carrageenan oligosaccharide prepared by the method of any one of claims 1-7.

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