Method for optimizing eisenia foetida earthworm polysaccharide extraction conditions by using response surface method and application of eisenia foetida earthworm polysaccharide extraction conditions

Abstract

The invention belongs to the technical field of polysaccharide extraction, and discloses a method for optimizing eisenia foetida earthworm polysaccharide extraction conditions by utilizing a response surface method.The method comprises the steps that a Box-Behnken response surface design method is utilized, three factors including reaction temperature, reaction time and a material-liquid ratio are selected as variables, the optimal condition in a single-factor experiment is selected as the center, and the reaction temperature, the reaction time and the material-liquid ratio are optimized; taking equal ranges above and below as variable ranges of the response surface; performing polynomial fitting regression on the variables by taking the extraction rate of the earthworm polysaccharide as a response value to obtain a regression equation; and calculating the variables through a regression equation to obtain the extraction condition when the eisenia fetida polysaccharide extraction rate is maximum. According to the method disclosed by the invention, suitable conditions for extracting the eisenia fetida earthworm polysaccharide are determined, a reference basis is provided for development and utilization of the eisenia fetida polysaccharide, and a more-developed and more-basis thought is provided for full development and utilization of eisenia fetida resources.

Description

Technical Field

[0001] The invention belongs to the technical field of polysaccharide extraction, and in particular to a method and application of optimizing the extraction conditions of Eisenia fetida and Pheretima polysaccharide by utilizing response surface methodology. Background Art

[0002] Earthworms, also known as Eisenia foetida, are a traditional Chinese medicine with a long history of application. As early as in the "Shennong's Classic of Materia Medica", it was recorded that it has the effects of clearing away heat and calming the nerves, unblocking the meridians, relieving asthma, and diuresis; Li Shizhen said in the "Compendium of Materia Medica" that it has the effects of unblocking the meridians and activating the meridians, and promoting blood circulation and removing blood stasis. Eisenia foetida, scientifically known as Eisenia foetida, is an annelid of the genus Eisenia in the family Lumbricidae, class Oligochaeta. It belongs to the same order as the source species of the traditional Chinese medicine "earthworms" in my country, and has similar pharmacological activities. Eisenia foetida has the advantages of small system, short growth cycle, fast reproduction, and strong adaptability. It has become one of the most common cultivated earthworm varieties in the world, and is also a key species for scientific research at home and abroad. Animal polysaccharides have a variety of biological activities and are widely used in the development of functional foods and clinical drugs. Therefore, the development and utilization of animal polysaccharides and related research work have become a research hotspot in the fields of natural medicine, life sciences, and biomaterials. Studies have shown that the polysaccharides from Eisenia fetida show rich diversity in pharmacological activity, especially in anti-coagulation and thrombolysis.

[0003] As an animal polysaccharide, Eisenia fetida polysaccharide has a complex structure. Its polysaccharide is usually combined with proteins, lipids, etc., which makes its extraction rate low. Currently, there is no complete and effective method for extracting Eisenia fetida polysaccharide.

[0004] At present, there is no special extraction technology for earth dragon polysaccharides, and no one has ever specially extracted earth dragon polysaccharides. The common technology for extracting polysaccharides is water extraction and alcohol precipitation, but the conditions of the water extraction and alcohol precipitation method are not completely suitable for extracting earth dragon polysaccharides. The extraction rate of earth dragon polysaccharides extracted by the unoptimized water extraction and alcohol precipitation method is low. At the same time, the high temperature of the water extraction and alcohol precipitation method may cause the polysaccharides to be inactivated, and the sugar content of the earth dragon polysaccharides extracted by the unoptimized water extraction and alcohol precipitation method is low. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies in the prior art and provide a method and application for optimizing the extraction conditions of polysaccharides from Eisenia fetida using response surface methodology.

[0006] The technical solution adopted by the present invention to solve its technical problem is:

[0007] A method for optimizing the extraction conditions of Eisenia fetida polysaccharide from earthworms using a response surface methodology, wherein the method utilizes the Box-Behnken response surface design method, selects three factors, namely, reaction temperature, reaction time and solid-liquid ratio, as variables, selects the optimal condition in a single factor experiment as the center, and takes equal ranges above and below the optimal condition as the variable range of the response surface; takes the extraction rate of earthworm polysaccharide as a response value, performs polynomial fitting regression on the above variables, and obtains a regression equation; calculates the variables using the regression equation, and obtains the extraction conditions when the extraction rate of earthworm polysaccharide is the maximum.

[0008] Furthermore, the regression equation is:

[0009] Y=51.98-0.6412A+0.6500B+2.45C-6.53AB-0.9475AC-0.5600BC-8.64A 2 -1.85B 2 -3.07C 2

[0010] , where Y is the extraction rate of Eisenia fetida polysaccharide, A is the temperature variable, B is the time variable, C is the solid-liquid ratio variable, the temperature variable unit is ℃, and the time variable unit is h.

[0011] Further, the following steps are included:

[0012] Step 1, sample removal: dissolving the Eisenia fetida sample in anhydrous ethanol, centrifuging, collecting the precipitate, and drying the precipitate;

[0013] Step 2, extraction: the dried sample is first subjected to water extraction, suction filtration, concentration, and then alcohol extraction, centrifugation, and freeze-drying to obtain crude polysaccharides of Eisenia fetida;

[0014] Step 3, dialyzing the obtained crude polysaccharide of Eisenia fetida and earthworm to remove the pigment, and finally obtaining polysaccharide of Eisenia fetida and earthworm.

[0015] Furthermore, in step 1, the centrifugation condition is: 5000 rpm for 5 min.

[0016] Furthermore, in the step 2, the conditions for water extraction are: adding ultrapure water to the precipitate obtained in step 1 according to the material-liquid ratio, and bathing at different temperatures for different periods of time.

[0017] Furthermore, in the step 2, the conditions for alcohol extraction are: adding four times the volume of anhydrous ethanol to the water extract until the ethanol concentration is not less than 75%, shaking well, and refrigerating and standing overnight.

[0018] Furthermore, in step 2, the centrifugation condition is: 5000 rpm for 10 min.

[0019] Furthermore, in step three, the dialysis time is 24 hours, wherein the water is changed every six hours.

[0020] Furthermore, in step 3, the pigment removal uses activated carbon to adsorb the pigment. Activated carbon is a safe substance that does not release harmful substances, and the use and treatment process of using activated carbon to adsorb pigments is relatively simple and easy to operate.

[0021] The above method is used in the extraction of polysaccharides from Eisenia fetida.

[0022] The advantages and positive effects achieved by the present invention are:

[0023] 1. Based on the single factor experiment, the present invention uses the response surface method to optimize the extraction process conditions of polysaccharides from Eisenia fetida. The three factors of reaction temperature, reaction time and solid-liquid ratio were selected to have a significant effect on the extraction rate of polysaccharides from Eisenia fetida. The regression curve was fitted, and the appropriate conditions for extracting polysaccharides from Eisenia fetida were finally determined. The appropriate conditions were reaction temperature of 67°C, reaction time of 3.6h, solid-liquid ratio of 1:22, and the polysaccharide extraction rate was 35.73% before optimization. After optimization, the polysaccharide extraction rate can reach 56.31%, which provides a reference basis for the development and utilization of polysaccharides from Eisenia fetida, and provides a more pioneering and more reliable idea for the full development and utilization of Eisenia fetida resources.

[0024] 2. The present invention uses a water extraction and alcohol precipitation method to extract polysaccharides from Eisenia fetida. In order to improve the extraction rate of polysaccharides from Eisenia fetida, the present invention uses a response surface method to regress polynomial fitting, establish a regression equation, calculate the regression equation, and obtain the optimal conditions for extracting polysaccharides from earthworms. This method can effectively optimize the multi-factor experiment to obtain the best combination conditions.

[0025] 3. The present invention adopts water extraction and alcohol precipitation method to extract polysaccharides from Eisenia fetida and earthworm, which can not only protect the biological activity of polysaccharides but also improve the extraction rate of polysaccharides from Eisenia fetida and earthworm. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a graph showing the effect of reaction temperature on the extraction rate of polysaccharides from Eisenia fetida in the examples of the present invention;

[0027] Figure 2 This is a graph showing the effect of reaction time on the extraction rate of polysaccharides from Eisenia fetida in an embodiment of the present invention;

[0028] Figure 3 This is a graph showing the effect of the solid-liquid ratio on the extraction rate of polysaccharides from Eisenia fetida in the examples of the present invention;

[0029] Figure 4This is a graph showing the effect of reaction temperature and reaction time on the extraction rate of polysaccharides from Eisenia fetida in an embodiment of the present invention;

[0030] Figure 5 This is a graph showing the effects of reaction temperature and material-liquid ratio on the extraction rate of polysaccharides from Eisenia fetida in an embodiment of the present invention;

[0031] Figure 6 This is a graph showing the effects of reaction time and material-liquid ratio on the extraction rate of polysaccharides from Eisenia fetida in an embodiment of the present invention;

[0032] Figure 7 The Fourier infrared spectrum of the polysaccharide of Eisenia fetida from the present invention;

[0033] Figure 8 These are actual photographs of the inactivated Eisenia fetida earthworm protein powder (left) and Eisenia fetida earthworm polysaccharide (right) in the present invention. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the embodiments. The following embodiments are descriptive rather than restrictive, and the protection scope of the present invention cannot be limited by the following embodiments.

[0035] The various experimental operations involved in the specific embodiments are all routine techniques in the art. For parts not specially annotated in this document, ordinary technicians in the art can implement them by referring to various commonly used reference books, scientific and technological literature or related instructions, manuals, etc. before the filing date of this invention.

[0036] The structures, proportions, sizes, etc. illustrated in the present invention are only used to match the contents disclosed in the specification so as to facilitate understanding and reading by persons familiar with the technology. They are not used to limit the conditions under which the present invention can be implemented, and therefore have no substantial technical significance. Any structural modification, change in proportion or adjustment of size should still fall within the scope of the technical contents disclosed in the present invention without affecting the effects and purposes that can be achieved by the present invention.

[0037] A method for optimizing the extraction conditions of Eisenia fetida polysaccharide from earthworms using a response surface methodology, wherein the method utilizes the Box-Behnken response surface design method, selects three factors, namely, reaction temperature, reaction time and solid-liquid ratio, as variables, selects the optimal condition in a single factor experiment as the center, and takes equal ranges above and below the optimal condition as the variable range of the response surface; takes the extraction rate of earthworm polysaccharide as a response value, performs polynomial fitting regression on the above variables, and obtains a regression equation; calculates the variables using the regression equation, and obtains the extraction conditions when the extraction rate of earthworm polysaccharide is the maximum.

[0038] Preferably, the regression equation is:

[0039] Y=51.98-0.6412A+0.6500B+2.45C-6.53AB-0.9475AC-0.5600BC-8.64A 2 -1.85B 2 -3.07C 2

[0040] , where Y is the extraction rate of Eisenia fetida polysaccharide, A is the temperature variable, B is the time variable, C is the solid-liquid ratio variable, the temperature variable unit is ℃, and the time variable unit is h.

[0041] Preferably, the method comprises the following steps:

[0042] Step 1, sample removal: dissolving the Eisenia fetida sample in anhydrous ethanol, centrifuging, collecting the precipitate, and drying the precipitate;

[0043] Step 2, extraction: the dried sample is first subjected to water extraction, suction filtration, concentration, and then alcohol extraction, centrifugation, and freeze-drying to obtain crude polysaccharides of Eisenia fetida;

[0044] Step three, dialyzing the obtained crude polysaccharide of Eisenia fetida to remove the pigment, and finally obtaining polysaccharide of Eisenia fetida and earthworm.

[0045] Preferably, in step 1, the centrifugation condition is: 5000 rpm for 5 min.

[0046] Preferably, in step 2, the conditions for water extraction are: adding ultrapure water to the precipitate obtained in step 1 according to the material-liquid ratio, and bathing at different temperatures for different periods of time.

[0047] Preferably, in step 2, the conditions for alcohol extraction are: adding four times the volume of anhydrous ethanol to the water extract until the ethanol concentration is not less than 75%, shaking well, and refrigerating and standing overnight.

[0048] Preferably, in step 2, the centrifugation condition is: 5000 rpm for 10 min.

[0049] Preferably, in step 3, the dialysis time is 24 hours, wherein the water is changed every six hours.

[0050] Preferably, in step 3, the pigment removal uses activated carbon to adsorb the pigment. Activated carbon is a safe substance that does not release harmful substances, and the use and treatment process of using activated carbon to adsorb pigments is relatively simple and easy to operate.

[0051] The above method is used in the extraction of polysaccharides from Eisenia fetida.

[0052] Specifically, the relevant preparation and detection are as follows:

[0053] A method for optimizing the extraction conditions of polysaccharides from Eisenia fetida using response surface methodology, the steps are as follows:

[0054] 1. Dissolve 1g of Eisenia fetida sample in 10ml of anhydrous ethanol, centrifuge at 5000rpm for 5min to remove impurities and precipitate, and dry at 60℃. Add ultrapure water to the dried sample at a material-liquid ratio of 1:10-1:30, heat it in a water bath at 40℃-80℃ for 2h-6h, and extract the Eisenia fetida earthworm polysaccharide from the sample. Filter the sample after the water bath, concentrate the volume to 3-5ml, and add 4 times the volume of anhydrous ethanol. Let it stand at 4℃ for 8h, centrifuge the sample after alcohol precipitation at 5000rpm for 10min, and freeze-dry to obtain the Eisenia fetida earthworm polysaccharide. Redissolve the obtained Eisenia fetida earthworm polysaccharide in water, dialyze it with a 10000Da dialysis bag for 8h, change the water every 2h, add activated carbon and shake it repeatedly to remove the pigment, and further purify the Eisenia fetida earthworm polysaccharide.

[0055] 2. The response surface method was used to determine the variables for extracting polysaccharides from earthworms and lumbricoides. A three-factor response surface analysis was performed with the extraction rate of polysaccharides from earthworms and lumbricoides as the response value. Then, a regression analysis was performed on the data to obtain the multivariate quadratic regression equation:

[0056] Y=51.98-0.6412A+0.6500B+2.45C-6.53AB-0.9475AC-0.5600BC-8.64A 2 -1.85B 2 -3.07C 2

[0057] Wherein, Y is the extraction rate of polysaccharides from Eisenia fetida, A is the reaction temperature, B is the reaction time, and C is the solid-liquid ratio. The optimal extraction conditions were finally determined to be: reaction temperature of 67°C, reaction time of 3.6h, and solid-liquid ratio of 1:22. The estimated extraction rate of polysaccharides from Eisenia fetida is 52.77%.

[0058] 3. The calculation formula for the extraction rate of Eisenia fetida polysaccharide is: Y (%) = the mass of Eisenia fetida polysaccharide / the content of sugar in each milligram of Eisenia fetida sample × 100%.

[0059] 4. The content of Eisenia fetida polysaccharide was determined by phenol-sulfuric acid method. The calculation formula of Eisenia fetida polysaccharide content is: Eisenia fetida polysaccharide content (%) = concentration of Eisenia fetida polysaccharide in solution / solution concentration

[0060] ×100%.

[0061] 5. The phenol-sulfuric acid method in step 4 is as follows: Draw a standard curve: take 0 μL, 40 μL, 80 μL, 120 μL, 160 μL, and 200 μL of a 100 μg / mL glucose standard solution, and add ultrapure water to make up to 200 μL in turn, add 100 μL of a phenol solution with a mass concentration of 6% in turn, quickly add 500 μL of concentrated sulfuric acid in turn, shake well and bathe in a 90°C constant temperature water bath for 5 minutes, then quickly cool to room temperature in a cold water bath, and then measure the absorption value of the solution at 488 nm. Finally, draw a standard curve with concentration (x) as the horizontal coordinate and absorbance value (Y) as the vertical coordinate. Dilute the sample to be tested according to a certain multiple, dilute the sample to 200 μg / mL, take 200 μL of the solution to be tested, and measure it according to the above-mentioned determination method.

[0062] 6. Experimental statistics and statistical analysis

[0063] (1) The extraction conditions were determined by single factor experiments as follows: reaction temperature of 70 °C, reaction time of 3 h, and solid-liquid ratio of 1:20.

[0064] The specific steps are as follows:

[0065] The temperature variables used 40℃, 50℃, 60℃, 70℃ and 80℃ as different conditions. The water extraction and alcohol precipitation method was used to measure the extraction rate of Eisenia fetida polysaccharide under the conditions of reaction time 4h and solid-liquid ratio 1:20.

[0066] The time variables used were 2h, 3h, 4h, 5h, and 6h. The reaction temperature was 70℃ and the solid-liquid ratio was 1:20. The water extraction and alcohol precipitation method was used to measure the extraction rate of Eisenia fetida polysaccharide.

[0067] The solid-liquid ratio variables used were 1:10, 1:15, 1:20, 1:25, and 1:30, and the water extraction and alcohol precipitation method was used with a reaction time of 4h and a temperature of 70°C to measure the extraction rate of Eisenia fetida polysaccharide.

[0068] The water extraction and alcohol precipitation step is step 1 of the above-mentioned method for optimizing the extraction conditions of Eisenia fetida and earthworm polysaccharide using response surface methodology. The result is that the extraction rate of Eisenia fetida and earthworm polysaccharide is the highest when the temperature is 70°C, the time is 3 hours, and the solid-liquid ratio is 1:20.

[0069] The effect of reaction temperature on the extraction rate of polysaccharides from Eisenia fetida Figure 1 The effect of reaction time on the extraction rate of polysaccharides from Eisenia fetida is shown in Figure 2 The effect of the solid-liquid ratio on the extraction rate of polysaccharides from Eisenia fetida is shown in Figure 3 shown.

[0070] Depend on Figure 1It can be seen that as the reaction temperature increases, the polysaccharide extraction rate also increases. When the temperature reaches 70°C, the polysaccharide extraction rate of Eisenia fetida is the highest. Then, as the temperature increases, the extraction rate begins to decrease.

[0071] Depend on Figure 2 It can be seen that with the extension of reaction time, the polysaccharide extraction rate increases accordingly. When the reaction time is 3h, the polysaccharide extraction rate of Eisenia fetida is the highest, and then the extraction rate gradually decreases with the extension of reaction time.

[0072] Depend on Figure 3 It can be seen that with the increase in the volume of ultrapure water added, the polysaccharide extraction rate increases accordingly. When the solid-liquid ratio is 1:20, the polysaccharide extraction rate of Eisenia fetida and earthworm is the highest. After that, the volume of ultrapure water is increased, and the polysaccharide extraction rate gradually decreases to a flat level.

[0073] (2) Based on the single factor experiment, the optimal value of the extraction conditions was determined. The reaction temperature, reaction time and solid-liquid ratio were used as variables, and the extraction rate of Eisenia fetida polysaccharide was used as the response value. The Box-Behnken method was used to perform a three-factor three-level analysis to optimize the extraction conditions of Eisenia fetida polysaccharide. The experimental factor level design is shown in Table 1.

[0074] Table 1 Factors and levels of response surface analysis of Eisenia fetida polysaccharide

[0075]

[0076] According to the response surface design in Design-Expert 13 software, there were 17 groups of optimization experiments on the extraction conditions of polysaccharides from Eisenia fetida, including 5 groups of center point replicate experiments. The experimental design and response value results are shown in Table 2.

[0077] Table 2 Response surface experimental design and results

[0078]

[0079]

[0080] From the results of the response surface experimental design in Table 2, it can be seen that the extraction rate is highest when the temperature is around 70°C, the time is around 3h, and the solid-liquid ratio is around 1:20.

[0081] The data in Table 2 were subjected to multiple regression fitting using Design-Expert 13 software, with the extraction rate of polysaccharides from Eisenia fetida as the response value Y. The results are shown in Table 3.

[0082]

[0083]

[0084] Note: * indicates significant effect (p<0.05), ** indicates extremely significant effect (p<0.01).

[0085] Effects of reaction temperature and reaction time on the extraction rate of polysaccharides from Eisenia fetida Figure 4 The effects of reaction temperature and liquid-to-solid ratio on the extraction rate of polysaccharides from Eisenia fetida Figure 5 The effects of reaction time and solid-liquid ratio on the extraction rate of polysaccharides from Eisenia fetida Figure 6 As shown. Figure 4 The response surface shape shows that the extraction rate of polysaccharides from Eisenia fetida is significantly affected by reaction temperature and reaction time. Figure 5 The response surface shape shows that the extraction rate of polysaccharides from Eisenia fetida is significantly affected by reaction temperature and solid-liquid ratio. Figure 6 The shape of the response surface showed that the extraction rate of Eisenia fetida polysaccharide was not significantly affected by reaction time and solid-liquid ratio.

[0086] From the results of the multivariate regression fitting analysis in Table 3, we can see that the linear terms A and B of the regression model are not significant, C is extremely significant, and the quadratic term A is 2 , C 2 Very significant, B 2 The results are significant, the interaction terms AB are extremely significant, and AC and BC are not significant, indicating that there is no simple linear relationship between different extraction conditions and the extraction rate of polysaccharides from Eisenia fetida. After regression analysis of the experimental data, the quadratic multiple regression equation was obtained:

[0087] Y=51.98-0.6412A+0.6500B+2.45C-6.53AB-0.9475AC-0.5600BC-8.64A 2 -1.85B 2 -3.07C 2

[0088] The regression equation model is extremely significant (p<0.01); the lack of fit term p=0.2908>0.05, indicating that the lack of fit term is not significant, that is, the model fits this experiment well. The correlation coefficient of the model is R 2 =0.9811, adjustment coefficient R Adj 2 =0.9567, indicating that the model can explain 95.67% of the response value changes, that is, the model has a good fit with the actual experiment, the experimental error is small, and it is feasible to use this model to analyze the extraction conditions of Eisenia fetida polysaccharides.

[0089] (3) Verification experiment

[0090] According to the results obtained from the response surface experiment and the quadratic multinomial regression equation, the optimal extraction conditions of each factor when the extraction rate of Eisenia fetida polysaccharide was the highest were reaction temperature of 67°C, reaction time of 3.6h, solid-liquid ratio of 1:22, and the estimated extraction rate of Eisenia fetida polysaccharide was 52.77%. The extraction conditions were verified and repeated 3 times, and the test results are shown in Table 4.

[0091]

[0092] When three groups of experiments were conducted on the optimized extraction conditions, the average extraction rate of Eisenia fetida polysaccharide was 56.31%, which had a relative error of 2.5% from the 52.77% predicted by the regression equation, and was within the allowable error range (20%), indicating that the regression model used in the optimization experiment of the extraction conditions of Eisenia fetida polysaccharide using the response surface methodology is reliable.

[0093] 7. Qualitative identification of polysaccharides from Eisenia fetida

[0094] (1) Properties and solubility test of polysaccharide from Eisenia fetida: The purified extract is a yellow-brown flocculent powder, soluble in water, but insoluble in organic solvents such as ethanol and chloroform. Figure 8 shown.

[0095] (2) Iodine-potassium iodide test: The test results showed that the starch solution turned blue, while the sample solution and distilled water showed no obvious changes, indicating that the sample did not contain starch.

[0096] (3) Molish test: The results show that a purple-red interface appears in the sample, and the reaction is positive, indicating that the sample contains polysaccharides. However, the polysaccharide composition of Eisenia fetida is relatively complex, and further research is needed to identify its structure.

[0097] (4) Fourier transform infrared spectroscopy: Figure 7 This is the Fourier infrared spectrum of the polysaccharide of Eisenia fetida, 3399.88cm -1 The absorption peak at 1650.67 cm is the stretching vibration absorption peak of -OH. -1 The absorption peak at 1385.38 cm is the stretching vibration of carboxyl COO-. -1 The CH bending vibration absorption peak is at 1047.26cm -1 It is the angle-variation vibration absorption peak of alcohol hydroxyl group -OH.

[0098] 8. Detection of polysaccharide content in Eisenia fetida and earthworm: The polysaccharide content in Eisenia fetida detected by phenol-sulfuric acid method under the optimal conditions was 58%.

[0099] The present invention optimizes the extraction process conditions of polysaccharides from Eisenia fetida using response surface methodology on the basis of single factor experiments. It is determined that the three conditions of reaction temperature, reaction time and material-liquid ratio have a significant effect on the extraction rate of polysaccharides from Eisenia fetida. On the basis of the single factor experiment, a 3-factor 3-level experiment is designed, and the optimal extraction conditions for extracting polysaccharides from Eisenia fetida are finally determined, providing a reference for the development and utilization of polysaccharides from Eisenia fetida. The polysaccharide extraction rate before optimization is 35.73%, and the polysaccharide extraction rate after optimization can reach 56.31%.

[0100] Although the embodiments of the present invention are disclosed for illustrative purposes, those skilled in the art will appreciate that various substitutions, changes and modifications are possible without departing from the spirit and scope of the present invention and the appended claims. Therefore, the scope of the present invention is not limited to the contents disclosed in the embodiments.

Claims

1. A method for optimizing the extraction conditions of polysaccharides from Eisenia fetida using response surface methodology, characterized in that: The method uses the Box-Behnken response surface design method, selects three factors of reaction temperature, reaction time and solid-liquid ratio as variables, selects the optimal condition in the single factor experiment as the center, and takes equal ranges above and below it as the variable range of the response surface; takes the extraction rate of earthworm polysaccharide as the response value, performs polynomial fitting regression on the above variables, and obtains the regression equation; calculates the variables through the regression equation, and obtains the extraction conditions when the extraction rate of earthworm polysaccharide is the maximum.

2. The method according to claim 1, characterized in that: The regression equation is: Y=51.98-0.6412A+0.6500B+2.45C-6.53AB-0.9475AC-0.5600BC-8.64A 2 -1.85B 2 -3.07C 2 Wherein, Y is the extraction rate of polysaccharide from Eisenia fetida, A is the temperature variable, B is the time variable, C is the solid-liquid ratio variable, the unit of temperature variable is ℃, and the unit of time variable is h.

3. The method according to claim 1 or 2, characterized in that: The steps include: Step 1, sample removal: dissolving the Eisenia fetida sample in anhydrous ethanol, centrifuging, collecting the precipitate, and drying the precipitate; Step 2, extraction: the dried sample is first subjected to water extraction, suction filtration, concentration, and then alcohol extraction, centrifugation, and freeze-drying to obtain crude polysaccharides of Eisenia fetida; Step 3, dialyzing the obtained crude polysaccharide of Eisenia fetida and earthworm to remove the pigment, and finally obtaining polysaccharide of Eisenia fetida and earthworm.

4. The method according to claim 3, characterized in that: In the step 1, the centrifugal condition is: centrifugation at 5000 rpm for 5 min.

5. The method according to claim 3, characterized in that: In the step 2, the conditions for water extraction are: adding ultrapure water to the precipitate obtained in the step 1 according to the material-liquid ratio, and bathing at different temperatures for different periods of time.

6. The method according to claim 3, characterized in that: In the step 2, the conditions for alcohol extraction are: adding four times the volume of anhydrous ethanol to the water extract until the ethanol concentration is not less than 75%, shaking well, and refrigerating and standing overnight.

7. The method according to claim 3, characterized in that: In the step 2, the centrifugal condition is: 5000 rpm for 10 min.

8. The method according to claim 3, characterized in that: In step 3, the dialysis time is 24 hours, wherein the water is changed every six hours.

9. The method according to claim 3, characterized in that: In the step 3, the pigment is removed by using an activated carbon method to adsorb the pigment.

10. Use of the method according to any one of claims 1 to 9 in extracting polysaccharides from Eisenia fetida and Lumbricus foetida.

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