Optimization method for extracting polysaccharide from lycium barbarum branches

Abstract

The invention relates to the technical field of natural product extraction, and discloses an optimization method for extracting polysaccharide from Chinese wolfberry branches, which comprises the following steps: firstly, pretreating the Chinese wolfberry branches, including cleaning, drying, crushing and cryopreservation; secondly, extracting polysaccharide by adopting a hot water extraction method, and optimizing the extraction efficiency through a single factor experiment, an orthogonal experiment and a response surface method; then, multiple extraction methods are combined to optimize the extraction steps, and the polysaccharide yield is increased; and finally determining the polysaccharide content by using a sulfuric acid-phenol method. The optimization method for extracting the polysaccharide from the lycium barbarum branches is easy and convenient to operate and low in cost, useful components are extracted from agricultural waste or agricultural non-important by-products so as to be reused, and the obtained lycium barbarum branch polysaccharide has the remarkable immunoregulatory activity and can be used for preparing the immunoregulatory polysaccharide of the lycium barbarum branches. The method can be widely applied to the fields of food, medicine, cosmetics, agricultural cultivation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of natural product extraction, and particularly relates to an optimized method for extracting polysaccharides from wolfberry branches. Background Art

[0002] Wolfberry is a traditional Chinese medicinal material and nourishing food, with a variety of bioactive components, among which polysaccharides are one of the main active components. Wolfberry polysaccharides have various biological activities such as antioxidant, immunomodulatory, and anti-tumor activities, and are applied in the fields of food, medicine, and cosmetics. However, current research on wolfberry polysaccharides mainly focuses on wolfberry fruits, and relatively little research has been done on polysaccharides in wolfberry branches.

[0003] Wolfberry branches, as by-products in the wolfberry planting process, are usually regarded as agricultural waste and discarded or used as fuel, without being fully utilized. In fact, wolfberry branches also contain rich polysaccharide components, and the price of wolfberry branches is much lower than that of wolfberry fruits, having potential development and utilization value. Therefore, extracting polysaccharides from wolfberry branches can not only improve the comprehensive utilization rate of wolfberry resources, but also provide raw materials for the development of new functional foods and drugs.

[0004] Currently, the main methods for extracting polysaccharides include hot water extraction method, ultrasonic-assisted extraction method, microwave-assisted extraction method, and enzymatic hydrolysis method, etc. Among them, the hot water extraction method is widely used in the extraction of polysaccharides due to its advantages such as simple operation, low cost, and little environmental damage. However, a single extraction method often has problems such as the destruction of polysaccharide activity and low extraction rate. Therefore, how to improve the biological activity and extraction rate of polysaccharides is an urgent problem to be solved currently. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides an optimized method for extracting polysaccharides from wolfberry branches, and optimizes the extraction conditions through single-factor experiments and orthogonal experiments to improve the extraction efficiency of polysaccharides. At the same time, it can promote the diversified utilization of agricultural waste - wolfberry branches, and provide a solution for the recycling and reuse of current agricultural waste resources.

[0006] The optimized method for extracting polysaccharides from wolfberry branches provided by the present invention includes the following steps:

[0007] S1. Pretreatment: Wash, dry, and crush fresh wolfberry branches, store them frozen, and grind them when in use to obtain wolfberry branch powder;

[0008] S2. Hot water extraction: Weigh a certain amount of wolfberry branch powder, add distilled water, and perform water bath extraction in hot water. Centrifuge the extraction solution and then collect the supernatant;

[0009] S3. Filtration: Filter the supernatant through a gauze and then perform suction filtration through filter paper in two steps;

[0010] S4, Soluble polysaccharide sedimentation: Add absolute ethanol to the filtered supernatant and sediment it in a refrigerated environment;

[0011] S5, Lipid and pigment removal: Centrifuge the sedimented filtrate, and sequentially add ethanol, isopropanol, and petroleum ether to remove pigments and lipids;

[0012] S6, Freeze-dry the extract in a freeze dryer to obtain wolfberry branch polysaccharide;

[0013] S7, Single-factor experiment: Examine the effects of solid-liquid ratio, extraction temperature, and extraction time on the polysaccharide extraction rate respectively;

[0014] S8, Orthogonal experiment optimization: Select the relatively prominent data in the single-factor experiment and conduct a three-factor and three-level experimental design to obtain the best ratio combination;

[0015] S9, Response surface method optimization: On the basis of the single-factor experiment, conduct a response surface experiment using Box-Behnken design. Select the solid-liquid ratio, extraction temperature, and extraction time as independent variables, and the polysaccharide extraction rate as the response value to design a three-factor and three-level response surface experiment;

[0016] S10, High-temperature and high-pressure assisted hot water extraction method to improve the polysaccharide extraction efficiency;

[0017] S11, Use the phenol-sulfuric acid method to determine the polysaccharide content.

[0018] Preferably, in step S1, the wolfberry branches used are pulverized and ground twice and passed through a 60-mesh sieve when in use.

[0019] Preferably, in step 2, the ratio of wolfberry branch powder to distilled water is 1:35, the temperature of the hot water used in the water bath is 90 °C, and the extraction time in the water bath is 120 min.

[0020] Preferably, in step 3, filtration is carried out twice. A gauze is used to filter the wolfberry branch powder residue, and a filter paper is used to filter larger impurities.

[0021] Preferably, in step 4, the amount of absolute ethanol added to the supernatant is 3-4 times the volume of the supernatant, and it is sedimented in a 4 °C refrigerator for 10-12 h.

[0022] Preferably, in step 5, the specific operation of centrifugation is to place the filtrate in a high-speed refrigerated centrifuge and centrifuge at 4 °C for 10 min, with the rotation speed set at 5500 rpm to remove the grayish-white impurities, and then collect the filtrate.

[0023] Preferably, in the step 5, after centrifugation and removal of impurities, 4 volumes of absolute ethanol are added to the collected filtrate, and the mixture is precipitated in a refrigerator at 4 °C for 10 h, then centrifuged at 5000 rpm for 10 min. The waste liquid is discarded, and the precipitate is retained. Then, absolute ethanol is added again to the precipitate, and the mixture is centrifuged at 5000 rpm for 10 min. The waste liquid is discarded, and the precipitate is retained;

[0024] Then, isopropanol is added, and the mixture is precipitated and centrifuged twice, and the waste liquid is discarded, and the precipitate is retained;

[0025] Finally, petroleum ether is added, and the mixture is precipitated and centrifuged twice, and the waste liquid is discarded, and the precipitate is retained

[0026] Compared with the related technology, the optimized method for extracting polysaccharides from wolfberry branches provided by the present invention has the following beneficial effects:

[0027] 1. The extraction cost is low. The wolfberry branches used in the experiment are usually treated as agricultural waste, with rich resources and low prices. The instruments used in the experiment are relatively common, and the extraction difficulty is low, which can meet the extraction needs of researchers and relevant practitioners;

[0028] 2. It realizes the purpose of extracting utilizable resources from agricultural waste or by-products, promotes the innovation of extraction technology, can be used for large-scale production as an immunomodulator to a certain extent, and also promotes the sustainable development of the agricultural economy. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 It is a graph showing the effect of extraction temperature on the polysaccharide yield of wolfberry branches in the present invention;

[0030] Figure 2 It is a graph showing the effect of liquid-to-solid ratio on the polysaccharide yield of wolfberry branches in the present invention;

[0031] Figure 3 It is a graph showing the effect of extraction time on the polysaccharide yield of wolfberry branches in the present invention;

[0032] Figure 4 It is a standard curve graph of glucose in the present invention;

[0033] Figure 5 It is a residual normal probability graph and a graph showing the correspondence between predicted values and actual values in the present invention;

[0034] Figure 6 It is a graph showing the correspondence between predicted values and actual values in the present invention;

[0035] Figure 7 It is a contour graph and a response surface graph of each factor in the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0036] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0037] The optimized method for extracting polysaccharides from Lycium barbarum branches provided by the present invention comprises the following steps:

[0038] (1) Take fresh and pest-free Lycium barbarum branches and put them into a pulverizer for the first pulverization. After pulverization, store them in a low-temperature environment.

[0039] (2) Put the frozen and pulverized Lycium barbarum branch powder into a pulverizer for secondary pulverization, pass through a 60-mesh sieve, and dry to obtain the final Lycium barbarum branch powder.

[0040] (3) Add the pulverized Lycium barbarum branch powder to a conical flask, add distilled water according to a liquid-to-material ratio of 35:1, put it into a constant-temperature water bath, adjust to 90 °C, extract for 120 min, and collect the filtrate.

[0041] (4) Filter the collected filtrate through four layers of gauze to remove macromolecular impurities, and then perform suction filtration with filter paper to make the obtained filtrate clear and transparent.

[0042] (5) Put the filtrate into a high-speed refrigerated centrifuge and centrifuge at 4 °C for 10 min, set the rotation speed at 5500 rpm, remove the grayish-white impurities, and collect the filtrate.

[0043] (6) Add four times the volume of absolute ethanol, precipitate in a 4 °C refrigerator for 10 h, then centrifuge at 5000 rpm for 10 min, discard the waste liquid, and retain the precipitate.

[0044] (7) Add ethanol to the precipitate, centrifuge at 5000 rpm for 10 min, discard the waste liquid, and then add isopropanol and petroleum ether in sequence, with the steps as before.

[0045] (8) Put the extract into a freeze dryer for freeze-drying to obtain the polysaccharides from Lycium barbarum branches.

[0046] The determination of the polysaccharide extraction rate adopts the phenol-sulfuric acid method.

[0047] Phenol-sulfuric acid method:

[0048] (1) Preparation of glucose standard solution: Accurately weigh 0.1 g of glucose standard, dissolve it in a beaker, and make up the volume to 1000 ml in a volumetric flask, and shake well.

[0049] (2) Establishment of glucose standard curve: Precisely pipette 0, 0.1, 0.2, 0.4, 0.8, 1 ml from the prepared standard solution, and make up the volume to 1 ml with distilled water. Add 1.0 ml of 5% phenol solution to each stoppered test tube, shake well, and then quickly add 5.0 ml of concentrated sulfuric acid, and shake well. Another 0.1 g of polysaccharide sample is made up to 100 ml in water, and 1.0 ml is precisely pipetted from it, and phenol and concentrated sulfuric acid are added in sequence.

[0050] (3) Let it stand for 5 min, then heat it in a boiling water bath for 15 min. After taking it out, quickly cool it to room temperature, and detect the absorbance at 490 nm on an enzyme-labeling instrument. Repeat three times and take the average value.

[0051] Next, the method for extracting polysaccharides from wolfberry branches will be analyzed from three aspects: single-factor experiment, orthogonal experiment, and response surface experiment.

[0052] 1. Single-factor experiment analysis

[0053] 1.1 Effect of extraction temperature on the polysaccharide yield of wolfberry branches

[0054] The effect of extraction temperature on the polysaccharide yield of wolfberry branches is as Figure 1 shown. The significant temperature factors are 80 °C, 90 °C, and 100 °C.

[0055] It can be seen from the figure that the polysaccharide yield of wolfberry branches increases with the increase of extraction temperature. At lower extraction temperatures, the activity of polysaccharide molecules is low and the solubility is not high, resulting in low extraction efficiency. As the temperature increases, the activity of polysaccharide molecules increases and reaches a peak at 90 °C, and the solubility further increases. However, continuous increase in temperature will cause the degradation of polysaccharide molecules, resulting in a decrease in yield.

[0056] 1.2 Effect of liquid-to-solid ratio on the polysaccharide yield of wolfberry branches

[0057] The effect of liquid-to-solid ratio on the polysaccharide yield of wolfberry branches is as Figure 2 shown. The significant liquid-to-solid ratio factors are 1:25 (g / mL), 1:30 (g / mL), and 1:35 (g / mL).

[0058] It can be seen from the figure that the polysaccharide yield of wolfberry branches increases with the increase of liquid-to-solid ratio. After the ratio continues to increase and exceeds 30:1 (ml:g), it begins to decrease, and the polysaccharide yield of wolfberry branches reaches the highest at 4.47% when the liquid-to-solid ratio is 30:1 (ml:g). The initial increase in yield in the experiment is because when the liquid volume increases, the water-soluble polysaccharides in wolfberry branches are fully contacted with the solvent and hydrolyzed and dissolved. When the solvent volume is too large, it may lead to an increase in the dissolution of impurities in the extraction process, which will instead reduce the purity and yield of polysaccharides.

[0059] 1.3 Effect of extraction time on the polysaccharide yield of wolfberry branches

[0060] The effect of extraction time on the polysaccharide yield of wolfberry branches is as Figure 3 shown. The significant time factors are 90 min, 120 min, and 150 min.

[0061] It can be seen from the figure that when the extraction time is short (60 °C), the solvent and polysaccharide molecules are not fully fused, the solubility is low, the extraction is incomplete, resulting in a low yield. As the extraction time prolongs, the polysaccharide molecules have enough time to dissolve in the solvent (120 min), and the extraction efficiency reaches the peak. However, too long contact time may cause the degradation of polysaccharide molecules or reaction with other components, resulting in a reverse increase in the yield.

[0062] 1.4 Establishment of glucose standard curve

[0063] The glucose standard curve is as shown in Figure 4 The calculation formula for the polysaccharide content is as follows:

[0064] Polysaccharide content (%) = (A sample - b) / a × V constant volume / m sample × 1000 × 100%

[0065] Among them, A is the absorbance of the sample solution, a is the slope of the standard curve, b is the intercept of the standard curve, V is the constant volume of the sample (ml), m is the mass of the polysaccharide sample weighed (g), and 1000 is the unit conversion coefficient.

[0066] The absorbance of the polysaccharide in wolfberry branches was measured at 490 nm, and the polysaccharide content was found to be 28.43%.

[0067] 2. Orthogonal test analysis

[0068] 2.1 Orthogonal and Box-Behnken response surface test design

[0069] Table 1 Orthogonal and Box-Behnken design test factor level table

[0070]

[0071]

[0072] Based on the results of single-factor experiments, extraction temperature, extraction time, and liquid-to-material ratio were selected. According to the principles of orthogonal experiments and Box-Behnken experiments, three-factor three-level orthogonal and response surface analyses were performed to optimize the extraction process of wolfberry branch polysaccharides.

[0073] 2.2 Analysis of orthogonal experiment results

[0074] Table 2 Orthogonal experiment design and result analysis

[0075]

[0076]

[0077] On the basis of the results of single-factor experiments, taking the yield of wolfberry branch polysaccharides as the investigation index, a 4-factor 3-level L9(34 ) orthogonal experiment. As can be seen from the table, the optimal extraction process for the polysaccharide yield of wolfberry branches is A2B2C3D3. Since item D is the blank group, that is, the extraction temperature is 90°C, the extraction time is 120 min, the liquid-to-solid ratio is 35:1, and the polysaccharide yield of wolfberry branches is 4.80%. The factors affecting the polysaccharide yield of wolfberry branches are extraction temperature > extraction time > liquid-to-solid ratio. At the same time, three repeated experiments were conducted to verify that the yield is 4.74 ± 0.39%

[0078] 2.3 Variance analysis of orthogonal experiment

[0079] Table 3 Variance analysis table

[0080]

[0081] Note: * Indicates significant difference (P < 0.05); ** Indicates extremely significant difference (P < 0.01).

[0082] 3. Box-Behnken response surface experiment analysis

[0083] Table 4 Box-Behnken response surface experiment analysis

[0084]

[0085]

[0086] Binary regression fitting equation:

[0087] Coded equation: Y = 4.63 + 0.4500 A + 0.1625 B + 0.1975 C - 0.1425 AB - 0.1225 AC + 0.0975 BC - 0.6778 A 2 -0.3678 B 2 -0.1027C 2

[0088] Actual equation: Y = -75.1275 + 0.1395 A + 0.1267 B + 0.4286 C - 0.0005 AB - 0.0025 AC + 0.0007 BC - 0.0068 A 2 -0.0004 B 2 -0.0041C 2

[0089] The Design-Expert 13 software was used for regression fitting, and the data in the table was subjected to quadratic regression analysis. The regression model for the effects of three factors, extraction temperature (A), extraction time (B), and liquid-to-solid ratio (C), on the polysaccharide yield (Y) of wolfberry branches was obtained as Y = -75.1275 + 0.1395A + 0.1267B + 0.4286C - 0.0005AB - 0.0025AC + 0.0007BC - 0.0068A 2 - 0.0004B 2 - 0.0041C 2

[0090] The analysis of variance of each term in the regression equation shows that the observed residual normal probability is shown in Figure 5 , and most of the scatter points are located on the straight line, indicating that the model has good adaptability.

[0091] The corresponding relationship between the predicted value and the actual value is as shown in Figure 6 . The diagonal line represents the special case where the experimental value and the expected value are exactly the same. The closer the true value is to the predicted value, the better the fitting degree of the expected model and the experiment. It can be seen from the figure that most of the points are distributed on or near the straight line, and the discreteness is small.

[0092] Table 5 Results of the analysis of variance of the response surface regression model for wolfberry branch polysaccharides

[0093]

[0094] Note: * Indicates significant difference (P < 0.05); ** Indicates extremely significant difference (P < 0.01).

[0095] The results of the analysis of variance are shown in the table. The P value of the model is less than 0.001, and the p value of the lack-of-fit term is 0.7865 > 0.05, indicating that the model is significant, has a good fit with the experiment, and R2 is infinitely close to 1, indicating good linear relationship. The coefficient of variation C.V. < 4%, so this model is applicable to the theoretical prediction of the polysaccharide yield extraction experiment in wolfberry branches. The order of the influence of the three factors on the polysaccharide yield is extraction temperature (A) > liquid-to-solid ratio (C) > extraction time (B). This result is different from that of the orthogonal experiment, but the difference in the K values of extraction time (B) and liquid-to-solid ratio (C) in the orthogonal experiment is small, so this result is in line with the prediction. Among the quadratic terms, A2 and B2 have extremely significant effects on the polysaccharide yield, and C2 has a significant effect. Among the interaction effects of the three influencing factors, AB has an extremely significant effect, and AC and BC have significant effects.

[0096] As shown in Figure 7As shown in the figure, the steepness of the response surface indicates the influence of the research factors on the results. The steeper the surface, the greater the influence of the factors on the results, and vice versa. The shape of the contour map reflects the degree of interaction between the factors. An ellipse indicates that the interaction between the factors is significant, and a circle indicates that the interaction between the factors is not significant. As can be seen from the figure, as the level of the research factors gradually increases, the polysaccharide production of wolfberry branches first increases and then decreases. The response surface is convex and the contour lines are elliptical, indicating that the interaction between the factors is significant and has a maximum value.

[0097] Optimal process verification:

[0098] The theoretical optimal extraction process provided by Design Expert 13 software is: extraction temperature of 93.0975℃, extraction time of 129.408min, liquid-to-solid ratio of 34.2566mL / g. Under this extraction process, the theoretical predicted value of the yield of Lycium barbarum branch polysaccharide is 4.79%. Taking into account the actual operation, the process was adjusted, and the experiment was repeated three times under the conditions of extraction temperature of 93℃, extraction time of 130min, and liquid-to-solid ratio of 35ml / g. The yield at this time was 4.85±0.23%.

[0099] 4. High temperature and high pressure assisted hot water extraction

[0100] 4.1 Orthogonal test assisted verification

[0101] Distilled water was added to the wolfberry branch raw material at a liquid-to-solid ratio of 35:1, and high-pressure steaming was carried out for 30 minutes at 112 degrees Celsius and an absolute pressure of 154 kPa (pressure data is quoted from NIST water vapor table). Subsequently, the raw material was placed in a water bath and hot water extraction was carried out at 90°C for 120 minutes. The experiment was repeated three times and the result was 4.79±0.47%, which was a small improvement compared with the ordinary hot water alcohol extraction method, and fluctuated with the expected yield value. This may be because while the integrity of the polysaccharide structure is maintained by high pressure, the excessively high temperature also destroys the structure of the polysaccharide, resulting in a decrease in yield.

[0102] 4.2 Box-Behnken response surface test auxiliary verification

[0103] Distilled water was added to the wolfberry branch raw material at a liquid-to-solid ratio of 35:1, and high-pressure steaming was carried out for 30 minutes at 112 degrees Celsius and an absolute pressure of 154 kPa (pressure data is quoted from NIST water vapor table). Then, hot water extraction was carried out in a water bath at 93°C, 130 minutes, and a liquid-to-solid ratio of 35:1. The experiment was repeated three times and the result was 4.86±0.19%. Compared with the ordinary hot water extraction method, the extraction efficiency was slightly increased and the extraction was more stable.

Claims

1. An optimized method for extracting polysaccharides from wolfberry branches, characterized in that, It includes the following steps: S1. Pretreatment: Wash, dry and crush fresh wolfberry branches, store them frozen, grind them when in use, and pass through a 60-mesh sieve to obtain wolfberry branch powder; S2. Hot water extraction: Weigh a certain amount of wolfberry branch powder, add distilled water, carry out water bath extraction in hot water, centrifuge the extraction solution, and then collect the supernatant; S3. Filtration: The supernatant is filtered through a gauze and then by suction filtration with filter paper in two steps; S4. Sedimentation of soluble polysaccharides: Add anhydrous ethanol to the filtered supernatant and sediment it in a refrigerated environment; S5. Removal of lipids and pigments: Centrifuge the sedimented filtrate, and successively add ethanol, isopropanol, and petroleum ether to remove pigments and lipids; S6. Put the extract into a freeze dryer for freeze drying to obtain wolfberry branch polysaccharides; S7. Single factor experiment: Respectively investigate the effects of solid-liquid ratio, extraction temperature, and extraction time on the polysaccharide extraction rate; S8. Orthogonal experiment optimization: Select the more prominent data in the single factor experiment, conduct a three-factor and three-level experimental design to obtain the best ratio combination; S9. Response surface method optimization: On the basis of the single factor experiment, conduct a response surface experiment using Box-Behnken design. Select the solid-liquid ratio, extraction temperature, and extraction time as independent variables, and the polysaccharide extraction rate as the response value to design a three-factor and three-level response surface experiment; S10. High temperature and high pressure assisted hot water extraction method to improve the polysaccharide extraction rate; S11. Use the phenol-sulfuric acid method to determine the polysaccharide content.

2. The optimized method for extracting polysaccharides from wolfberry branches according to claim 1, characterized in that, In the step S1, the used wolfberry branches are ground twice and passed through a 60-mesh sieve when in powder form.

3. The optimized method for extracting polysaccharides from Lycium barbarum branches according to claim 1, characterized in that, In the step 2, the ratio of wolfberry branch powder to distilled water is 1:35, the temperature of the hot water used in the water bath is 90 °C, and the extraction time in the water bath is 120 min.

4. The optimized method for extracting polysaccharides from wolfberry branches as described in claim 1, wherein In the step 3, filtration is carried out twice. The gauze is used to filter the wolfberry branch powder residue, and the filter paper is used to filter larger impurities.

5. The optimized method for extracting polysaccharides from wolfberry branches according to claim 1, characterized in that, In the step 4, the amount of anhydrous ethanol added to the supernatant is 3-4 times the volume of the supernatant, and it is sedimented in a 4 °C refrigerator for 10-12 h.

6. The optimized method for extracting polysaccharides from wolfberry branches according to claim 1, characterized in that, In the step 5, the specific operation of centrifugation is to put the filtrate into a high-speed refrigerated centrifuge and centrifuge at 4 °C for 10 min, with the rotation speed set at 5500 rpm, remove the grayish-white impurities, and then collect the filtrate.

7. The optimized method for extracting polysaccharides from wolfberry branches as described in claim 5, characterized in that, In the filtrate collected after centrifugation and removal of impurities in the step 5, add 4 times the volume of anhydrous ethanol, precipitate in a 4 °C refrigerator for 10 h, then centrifuge at 5000 rpm for 10 min, discard the waste liquid, retain the precipitate, then add anhydrous ethanol to the precipitate again, centrifuge at 5000 rpm for 10 min, discard the waste liquid, retain the precipitate; Then, add isopropanol, carry out precipitation and centrifugation twice, discard the waste liquid, and retain the precipitate; Finally, add petroleum ether, carry out precipitation and centrifugation twice, discard the waste liquid, and retain the precipitate.

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