CN111995697A - Extraction and purification method of Lithocarpus litseifolius polysaccharide - Google Patents
Extraction and purification method of Lithocarpus litseifolius polysaccharide Download PDFInfo
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention discloses an extraction and purification method of Lithocarpus litseifolius polysaccharide, which comprises the following steps: (1) pretreating Lithocarpus litseifolius leaves to obtain Lithocarpus litseifolius leaf powder; (2) removing fat-soluble compounds in Lithocarpus litseifolius leaf powder; (3) extracting Lithocarpus litseifolius (hook.) F.H.) leaves with liposoluble compounds removed under pressure with water as extraction solvent; (4) centrifuging the extract, and collecting supernatant to obtain Lithocarpus litseifolius leaf crude polysaccharide extract. The polysaccharide is extracted from the Lithocarpus litseifolius leaves by adopting a pressurized solvent auxiliary extraction method, the optimal extraction process parameters of the Lithocarpus litseifolius leaf polysaccharide extracted by the pressurized solvent are optimized by combining a single-factor multilevel experiment with a Box-Behnken center combined design-response surface method, and the purpose of efficiently extracting the polysaccharide is realized. The method has the advantages of high extraction efficiency, low extraction cost, short extraction time, low energy consumption, simple, stable and reliable extraction process and suitability for popularization and application.
Description
Technical Field
The invention relates to a method for extracting polysaccharide from plants, in particular to a method for extracting and purifying Lithocarpus litseifolius polysaccharide, belonging to the field of extracting and purifying Lithocarpus litseifolius polysaccharide.
Background
Lithocarpus litseifolius (Hance) Chun, a family Fagaceae, a tree of the genus Lithocarpus, is distributed in the southern provinces of the southern slope of Qinling mountain, China. Tender leaves are sweet and sticky when chewed, and most of the leaves in the southern areas of Yangtze river are used as tea substitutes, so the tea is called sweet tea. The Lithocarpus litseifolius sweet tea has been eaten for thousands of years in China, is a plant which can be used as both medicine and food and has three functions of tea, sugar and medicine, and is most abundant in Sichuan, Hunan and Yunnan resources. The Lithocarpus litseifolius has huge biological resource amount, and the annual yield of the Lithocarpus litseifolius can reach tens of thousands of tons only. Regarding the medicinal function of sweet tea, according to records of TCM journal and reports of related literature: can be used for preventing and treating hypertension, damp-heat dysentery, skin pruritus, superficial infection and malignant boil. And has effects in nourishing liver and kidney, regulating stomach function, lowering adverse qi, moistening lung, relieving cough, relieving sleepiness, and relieving hangover. The compendium of materia medica records that the smell is sweet and flat, the toxicity is low, the hemorrhoid is cured, the bleeding and bloody dysentery are stopped, the thirst is quenched, the blood is activated, and the urination is promoted. Lithocarpus litseifolius has been approved as a new food material in 2107 years.
Polysaccharides (Polysaccharides) are biopolymer compounds formed by polymerization of more than 10 monosaccharides via glycosidic bonds, and have the general formula [ C ]6(H2O)5]mTypically m is much greater than 20. At present, plant polysaccharides have been receiving the attention of more and more researchers, and the international scientific community even proposes the 21 st century as the century for polysaccharides; a large number of researches show that the plant polysaccharide has various biological activities, including antioxidant, blood lipid reducing, blood glucose reducing, anti-inflammatory, antitumor, anti-radiation, antibacterial, antiviral, immunoregulatory, liver protecting and other health care effects. Therefore, the plant polysaccharide has been widely applied to the food and drug field. The extraction and preparation of the polysaccharide are the key points of the fine and deep processing of the product.
So far, no extraction method of lithocarpus litseifolius leaf polysaccharide is reported. Therefore, the method for efficiently preparing the lithocarpus litseifolius leaf polysaccharide becomes the primary problem to be solved urgently for the industrial development of the lithocarpus litseifolius leaf polysaccharide.
Disclosure of Invention
The invention mainly aims to provide a method for efficiently extracting polysaccharide from lithocarpus litseifolius;
the above object of the present invention is achieved by the following technical solutions:
a method for extracting Lithocarpus litseifolius polysaccharide comprises the following steps:
(1) pretreating Lithocarpus litseifolius leaves to obtain Lithocarpus litseifolius leaf powder; (2) removing fat-soluble compounds in Lithocarpus litseifolius leaf powder; (3) extracting Lithocarpus litseifolius (hook.) F.H.) leaves with liposoluble compounds removed under pressure with water as extraction solvent; (4) centrifuging the extract, and collecting supernatant to obtain Lithocarpus litseifolius leaf crude polysaccharide extract.
As a preferred embodiment of the present invention, the present invention further comprises: and (3) carrying out the following purification treatment on the obtained lithocarpus litseifolius leaf crude polysaccharide extract to obtain a lithocarpus litseifolius leaf polysaccharide extract pure product: (a) concentrating the extract of crude polysaccharide from Lithocarpus litseifolius leaf; (b) removing starch and protein from the concentrated product; (c) adding ethanol for precipitating, dissolving precipitate with water, dialyzing, and drying.
Wherein the method for removing starch and protein in the concentrated product comprises the following steps: adding alpha-amylase to carry out hydrolysis reaction to remove starch in the lithocarpus litseifolius leaf crude polysaccharide extract, inactivating the amylase, and centrifuging to remove denatured enzyme and other proteins; preferably, 95% ethanol is added into the lithocarpus litseifolius leaf crude polysaccharide extract for alcohol precipitation; centrifuging the precipitated product, taking the precipitate, redissolving the precipitate by water, and dialyzing the precipitate by a dialysis bag with the molecular weight cutoff of 3.5 kDa.
As a preferred embodiment of the present invention, the method for pretreating leaves of lithocarpus litseifolius in step (1) comprises: drying Lithocarpus litseifolius leaf, pulverizing, and sieving to obtain powder; wherein, the sieving is most preferably 60-mesh sieving.
As a preferred embodiment of the present invention, the method for removing the fat-soluble compounds from the lithocarpus litseifolius leaf powder in step (2) comprises: mixing Lithocarpus litseifolius leaf powder with ethanol, and performing ultrasonic treatment; centrifuging the product after ultrasonic treatment, and taking the precipitate to obtain the product. In order to achieve better effect of removing fat-soluble compounds, the obtained precipitate is preferably mixed with ethanol uniformly and then is subjected to ultrasonic treatment again, and the product after ultrasonic treatment is centrifuged to obtain the precipitate; the ethanol can be 20-95% ethanol; according to g: mL, the proportion of the lithocarpus litseifolius tea powder to the ethanol is 1: (5-30); the ultrasonic treatment conditions are as follows: the ultrasonic power is 500-800W, and the ultrasonic treatment time is 10-60 min; preferably, the ethanol is 80% ethanol; according to g: mL, the proportion of the lithocarpus litseifolius tea powder to the ethanol is 1: 10; the ultrasonic treatment conditions are as follows: the ultrasonic power is 640W, and the ultrasonic treatment time is 300 min.
As a preferred embodiment of the present invention, the amount of the extraction solvent added in step (3) is calculated according to the amount of lithocarpus litseifolius leaf powder, and the ratio of the lithocarpus litseifolius leaf powder to the extraction solvent is 1: (10-50); the ratio of Lithocarpus litseifolius leaf powder to extraction solvent can be 1: (20-30), preferably, the ratio of the lithocarpus litseifolius leaf powder to the extraction solvent is 1: 20-24; the time for the pressurized extraction can be 20-60min, preferably 30-50min, and most preferably 40-41 min; the pressure for pressure extraction can be 0.02-0.10 Mpa; preferably 0.08 to 0.09 MPa.
The method takes tea powder prepared by grinding finished lithocarpus litseifolius tea as a raw material, the extraction rate of lithocarpus litseifolius leaf polysaccharide calculated by a weighing method is taken as a reference index, the optimal extraction process conditions for extracting the lithocarpus litseifolius leaf polysaccharide by a pressurized solvent are optimized by combining a single-factor multilevel experiment with a Box-Behnken center combined design-response surface method, the method mainly comprises the steps of material-liquid ratio of the lithocarpus litseifolius tea powder to ultrapure water, extraction time and extraction pressure, and the extraction material-liquid ratio is finally determined to be 1: 24. the extraction time is 41min, the extraction pressure is 0.09Mpa, and the extraction rate of Lithocarpus litseifolius leaf polysaccharide is 4.65 + -0.12% as determined by three verification tests under the condition.
Compared with the prior art, the invention mainly has the following beneficial effects:
(1) the polysaccharide is extracted from the lithocarpus litseifolius leaves by adopting a pressurized solvent auxiliary extraction method, and the method can effectively reduce the surface tension and viscosity of the solvent, so that the polysaccharide can better permeate into the lithocarpus litseifolius leaves, and the aim of efficiently extracting the polysaccharide is fulfilled; the method has the advantages of high extraction efficiency, low extraction cost, short extraction time, simple, stable and reliable extraction process and low extraction energy consumption, and the popularization and application of the method play an important role in promoting the industrial development of Lithocarpus litseifolius leaf polysaccharide.
(2) The response surface optimization method for the technological conditions of the Lithocarpus litseifolius leaf polysaccharide extracted by the aid of the pressurized solvent provides reliable reference for the application of the pressurized solvent in the practical production of the Lithocarpus litseifolius leaf polysaccharide, and provides technical support for the research and development of the Lithocarpus litseifolius leaf polysaccharide as a functional food.
Drawings
FIG. 1 is a flow chart of a pressurized solvent-assisted extraction method for efficiently preparing Lithocarpus litseifolius (sweet tea) polysaccharides.
FIG. 2 is a graph showing the effect of Lithocarpus litseifolius tea powder and ultrapure water feed liquid on the extraction rate of Lithocarpus litseifolius polysaccharide.
FIG. 3 is a graph showing the effect of extraction time on extraction rate of Lithocarpus litseifolius leaf polysaccharide.
FIG. 4 is a graph showing the effect of extraction pressure on the extraction rate of Lithocarpus litseifolius leaf polysaccharide.
FIG. 5 is a three-dimensional graph of the response surface of the extraction ratio of the extraction material to the extraction time to the extraction rate of Lithocarpus litseifolius polysaccharide.
FIG. 6 is a three-dimensional graph of the response surface of the extraction ratio of the extraction material to the extraction pressure on the extraction rate of Lithocarpus litseifolius polysaccharide.
FIG. 7 is a three-dimensional graph of the response surface of extraction time and extraction pressure to Lithocarpus litseifolius polysaccharide extraction rate.
FIG. 8 is a contour plot of the response surface of the extraction ratio of the extraction material to liquid and the extraction time versus the extraction rate of Lithocarpus litseifolius leaf polysaccharide.
FIG. 9 is a contour plot of the response surface of the extraction ratio of the extraction material to liquid and the extraction pressure on the extraction rate of Lithocarpus litseifolius polysaccharide.
FIG. 10 is a contour plot of the response surface of extraction time and pressure versus extraction rate of Lithocarpus litseifolius polysaccharide.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. It is to be understood that the described embodiments are exemplary only and are not limiting upon the scope of the invention. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.
Example 1 extraction of Lithocarpus litseifolius tea polysaccharides
(1) Drying the finished Lithocarpus litseifolius (wall.) Sk in a constant temperature hot air oven at 45 deg.C to constant weight, grinding with a Super Mill-1500 powder grinding machine, sieving with a 60 mesh sieve, packaging into a sealed bag, and storing at-20 deg.C.
(2) Accurately weighing 2g of Lithocarpus litseifolius tea powder, adding into a 50mL centrifuge tube, vortex uniformly mixing, adding 20mL of 80% ethanol (1: 10, W/v), and performing ultrasonic treatment for 30min at 640W by using a PL-S80T ultrasonic cleaner. After sample balancing, the sample is centrifuged for 10min at 4000g by an X3R type high-speed refrigerated centrifuge, 20mL of 80% ethanol is added into the sediment after supernatant fluid is discarded, and ultrasonic treatment is carried out for 30min at 80% power by using a PL-S80T ultrasonic cleaner. After the sample was equilibrated, the sample was centrifuged at 4000g for 10min by a high-speed refrigerated X3R centrifuge, and the supernatant was discarded to retain the precipitate.
(3) Accurately measuring 60mL (1: 30, w/v) of ultrapure water, washing the precipitate obtained in the step (2) from which the fat-soluble compound is removed from a centrifuge tube into a 250mL conical flask, and shaking up; subsequently, using a GI54DS autoclave to perform auxiliary extraction on the Lithocarpus litseifolius leaf polysaccharide, setting the extraction time to be 40min and the extraction pressure to be 0.06 MPa; after extraction, the sample in the conical flask is respectively filled into two 50mL centrifuge tubes, balanced, centrifuged for 15min at 4000g, and after centrifugation, the supernatant (Lithocarpus litseifolius crude polysaccharide aqueous extract) is collected, and the precipitate is extracted again according to the steps. And adding 20mL of ultrapure water into the precipitate obtained after the second extraction, shaking uniformly, centrifuging for 15min at 4000g, and collecting the supernatant. Finally, the two extracts were mixed and the supernatant washed, rotary evaporated at 80 ℃ using a rotary evaporator, and concentrated to 1/3 in the original volume.
(4) Adding 17.5mg of peaking alpha-amylase (which is high temperature resistant alpha-amylase, 40000U/g, purchased from Beijing Solibao science and technology Co., Ltd.) (5U/mL) into the crude polysaccharide water extract of Lithocarpus litseifolius to react for 4h in a water bath kettle at 80 ℃ to remove starch in the crude polysaccharide water extract of Lithocarpus litseifolius; after the reaction is finished, the temperature of the water bath kettle is raised to 95 ℃, the peak alpha-amylase is inactivated by water bath for 30min, then the sample is taken out and cooled to the room temperature, and the denatured enzyme and a small amount of protein in the sample are removed by centrifugation for 15min at 4000 g.
(5) Precipitating the extracting solution obtained in the step (4) with 95% ethanol with three times of volume, completing the alcohol precipitation step on a constant-temperature heating type magnetic stirrer, dropwise adding the 95% ethanol, and after the step is completed, placing the sample in a refrigerator at 4 ℃ for precipitation overnight; centrifuging at 4000g for 15min the next day to obtain Lithocarpus litseifolius leaf polysaccharide precipitate; then, adding 30mL of 80% ethanol into the precipitate, shaking up, and washing the precipitate by centrifuging for 15min at 4000 g; finally, the precipitate was reconstituted with 60 ℃ ultra pure water (if insoluble precipitate appeared, insoluble material was removed by centrifugation at 4000g for 15 min).
(6) Further and rapidly purifying the redissolved Lithocarpus litseifolius leaf polysaccharide extract by using a dialysis bag (molecular weight cut-off of 3.5kDa), wherein the specific method comprises the following steps: subpackaging Lithocarpus litseifolius leaf polysaccharide extract in dialysis bag, dialyzing the dialysis bag in dialysate (ultrapure water), replacing ultrapure water every 2 hr, and detecting conductivity of dialysate before water replacement until the conductivity is not changed before and after dialysis. According to the actual operation condition, the starch hydrolysate and other small molecular substances in the sample can be completely dialyzed for about 3 days; and finally, collecting the sample in the dialysis bag, and freeze-drying to obtain purified Lithocarpus litseifolius leaf polysaccharide, wherein the freeze-drying time is 48h, and immediately collecting the sample after drying and weighing the sample by using an analytical balance. The extraction rate calculation formula is as follows:
the extraction rate is A/B multiplied by 100%
Wherein A is the weight (2g) of Lithocarpus litseifolius tea powder; b is the weight of Lithocarpus litseifolius leaf polysaccharide.
Through detection, the extraction rate of the polysaccharide in the embodiment is 4.54%, and the extraction yield of the polysaccharide is 90.8 mg.
Example 2 extraction of Lithocarpus litseifolius tea polysaccharides
The difference from example 1 is that: in the step (3), the material-liquid ratio is 1:24 when high-pressure extraction is adopted, the extraction time is 41min, and the extraction pressure is 0.09 Mpa; the rest is the same as in example 1.
The extraction yield of the polysaccharide in this example was 4.65%, and the extraction yield of the polysaccharide was 93.0 mg.
Example 3 extraction of Lithocarpus litseifolius tea polysaccharides
The difference from example 1 is that: in the step (3), the material-liquid ratio is 1:20 when high-pressure extraction is adopted, the extraction time is 40min, and the extraction pressure is 0.02 Mpa; the rest is the same as in example 1.
Through detection, the extraction rate of the embodiment is 3.29%, and the extraction yield of the polysaccharide is 65.8 mg.
Example 4 extraction of Lithocarpus litseifolius tea polysaccharides
The difference from example 1 is that: in the step (3), the material-liquid ratio is 1:30 when high-pressure extraction is adopted, the extraction time is 40min, and the extraction pressure is 0.06 MPa; the rest is the same as in example 1.
Through detection, the extraction rate of the polysaccharide in the embodiment is 3.96%, and the extraction yield of the polysaccharide is 79.2 mg.
Example 5 extraction of Lithocarpus litseifolius tea polysaccharides
The difference from example 1 is that: in the step (3), the material-liquid ratio is 1:40 when high-pressure extraction is adopted, the extraction time is 40min, and the extraction pressure is 0.06 MPa; the rest is the same as in example 1.
Through detection, the extraction rate of the polysaccharide in the embodiment is 3.82%, and the extraction yield of the polysaccharide is 76.4 mg.
Example 6 extraction of Lithocarpus litseifolius tea polysaccharides
The difference from example 1 is that: in the step (3), the material-liquid ratio is 1:20 when high-pressure extraction is adopted, the extraction time is 50min, and the extraction pressure is 0.06 Mpa; the rest is the same as in example 1.
Through detection, the extraction rate of the polysaccharide in the embodiment is 3.83%, and the extraction yield of the polysaccharide is 76.6 mg.
Example 7 extraction of Lithocarpus litseifolius tea polysaccharides
The difference from example 1 is that: in the step (3), the material-liquid ratio is 1:20 when high-pressure extraction is adopted, the extraction time is 20min, and the extraction pressure is 0.06 MPa; the rest is the same as in example 1.
Through detection, the extraction rate of the polysaccharide in the embodiment is 2.38%, and the extraction yield of the polysaccharide is 56.6 mg.
Experimental example 1 optimization experiment of pressure solvent-assisted extraction process conditions of lithocarpus litseifolius (sweet tea) polysaccharides
1. Single factor multiple level experiment
According to the pressurized solvent-assisted extraction method for efficiently preparing lithocarpus litseifolius (sweet tea) polysaccharide in example 1, 3 factors influencing the extraction rate of lithocarpus litseifolius polysaccharide are screened, namely: the proportion, the extraction time and the extraction pressure of the lithocarpus litseifolius leaf powder and the ultrapure water as raw materials; taking the extraction rate of Lithocarpus litseifolius leaf polysaccharide as a reference index, and performing single-factor multi-level experiment.
1.1 Single-factor experiment of feed-liquid ratio of Lithocarpus litseifolius tea powder to ultrapure water
Selecting 5 levels to perform single-factor experiment of the feed-liquid ratio of lithocarpus litseifolius tea powder to ultrapure water: the fixed extraction time is 40min, the extraction pressure is 0.06MPa, and the extraction rate of Lithocarpus litseifolius polysaccharide is measured when the ratio (g/ml) of Lithocarpus litseifolius tea powder to ultrapure water is 1:10, 1:20, 1:30, 1:40 and 1:50 respectively.
The effect of the Lithocarpus litseifolius tea powder and the ultrapure water feed liquid ratio on the Lithocarpus litseifolius leaf polysaccharide extraction rate is shown in figure 2, and the test results show that when the extraction material-liquid ratio is 1:10, the Lithocarpus litseifolius leaf polysaccharide extraction rate is only about 3.20%, when the extraction material-liquid ratio is 1:20, the Lithocarpus litseifolius leaf polysaccharide extraction rate reaches about 4.0%, and with the increase of the extraction material-liquid ratio, the Lithocarpus litseifolius leaf polysaccharide extraction rate shows a remarkable reduction trend, so the optimal extraction material-liquid ratio is 1: 20.
1.2 Single factor experiment of extraction time
5 levels were selected for single factor experiments of extraction time: the ratio of the Lithocarpus litseifolius tea powder to the ultrapure water is fixed at 1:20(w/v), the extraction pressure is 0.06MPa, and the extraction rate of Lithocarpus litseifolius polysaccharide is measured at 20min, 30min, 40min, 50min and 60min respectively.
The effect of the extraction time on the extraction rate of Lithocarpus litseifolius leaf polysaccharide is shown in FIG. 3, and it can be seen from the results that the extraction rate of Lithocarpus litseifolius leaf polysaccharide is in a rising trend with the increase of the extraction time, and reaches the maximum value when the extraction time is 40 min. Subsequently, the extraction rate of Lithocarpus litseifolius leaf polysaccharide begins to decrease, so the optimal extraction time determined by single factor test is 40 min.
1.3 Single factor experiment of extraction pressure
5 levels were selected for single factor experiments of extraction pressure: the extraction rate of Lithocarpus litseifolius polysaccharide was measured at extraction pressures of 0.02, 0.04, 0.06, 0.08 and 0.10MPa, respectively, by setting the material-to-liquid ratio of Lithocarpus litseifolius tea powder to ultrapure water at 1:20(w/v) and the extraction time at 40 min.
The test results of the influence of the extraction pressure on the extraction rate of the Lithocarpus litseifolius polysaccharide are shown in fig. 4, and it can be seen from fig. 4 that the extraction rate of the Lithocarpus litseifolius polysaccharide increases and then decreases with the increase of the extraction pressure, and the extraction rate of the Lithocarpus litseifolius polysaccharide is the highest when the extraction pressure is 0.08 MPa. Therefore, the optimum extraction pressure determined by the single-factor test is 0.08 MPa.
2. Analytical test of response surface
According to the single-factor multilevel experiment result, the three-factor three-level response surface analysis test is designed by using the lithocarpus litseifolius tea powder and ultrapure water material-liquid ratio (A), the extraction time (B) and the extraction pressure (C) as independent variables, using the lithocarpus litseifolius leaf polysaccharide extraction rate as a response value and adopting a Box-Behnken center combined design method, wherein the design factors and the levels of the response surface test are shown in Table 1.
TABLE 1 response surface test design factors and levels
According to the Box-Behnke test design principle, the reaction conditions of Lithocarpus litseifolius leaf polysaccharide extraction are further optimized by utilizing three-factor three-level response surface design, 17 test points are implemented, wherein 12 analysis factors are adopted, and 5 central tests are adopted to estimate errors. Table 2 lists the response surface experimental matrices and experimental data.
Table 2 response surface experimental design and results
And (3) performing regression fitting analysis on the test result by adopting Design Expert 8.0.6 to generate a second-order polynomial equation, and obtaining a final equation with the coding factors as follows:
Y＝4.55+0.17A+0.28B+0.29C+0.11AB+0.097AC+0.043BC-0.47A2-0.52B2-0.27C2
wherein Y is the extraction rate of Lithocarpus litseifolius leaf polysaccharide; A. b and C are respectively the feed-liquid ratio (mL/g), extraction time (min) and extraction pressure (MPa) of the extraction.
TABLE 3 regression equation analysis of variance
Note: r2＝0.9982，R2 adj＝0.9959，C.V.＝0.84％，adeq.precision＝55.135；
aA, material-liquid ratio (mL/g); b, extracting time (min); c, extraction pressure (MPa);bsignificant difference (p)<0.05) and a significant difference (p)<0.01)。
Table 3 is an ANOVA of regression model with a quadratic regression model having a higher F value (430.96) and a lower P value (P)<0.0001) The applicability of the model is very remarkable. Coefficient of determination R of model20.9982, correction decision coefficient R2 adj0.9959, the model proved to have a high degree of fit to the actual experimental process. Therefore, the regression model has feasibility, and can be used for predicting the relation between the extraction rate of the lithocarpus litseifolius polysaccharides and the single-factor level change and the data fluctuation.
In this experiment, when p <0.05, this factor was shown to be significant, and when p <0.01, this factor was shown to be extremely significant. The mismatching terms P (0.0581, P >0.05) and F (6.00) indicate that the residuals are mainly composed of random errors and have negligible effect on the optimization results. In addition, C.V (coefficient of variation of Y in the equation) represents the confidence of the experiment, the larger the c.v. value is, the reliability of the experiment is low, and the c.v. value in the experiment is 0.84%, which indicates that the experiment operation is reliable and has sufficient accuracy and general applicability. In addition, the parameter optimization coefficients show that the linear coefficients (a, B, C), quadratic terms (AA, BB, CC) and interaction coefficients (AB, AC, BC) have a great influence on the extraction rate of the corymbose litse leaves of litsea pungens (p < 0.05).
Fig. 5, 6, and 7 show three-dimensional response surface maps (3D) of the response surface prediction results, and fig. 8, 9, and 10 show contour maps of the response surface prediction results. In general, the response surface with the elliptical profile indicates that the interaction between the corresponding variables is significant, while the circular profile indicates that the interaction between the corresponding variables is not significant. As can be seen from fig. 5 to 10, the interaction between the feed-liquid ratio (a) of lithocarpus litseifolius leaf powder and ultrapure water, the extraction pressure (C), the extraction time (B), and the extraction pressure (C) is significant; although the two-dimensional contour diagrams of the feed-to-liquid ratio (a) and the extraction time (B) of the lithocarpus litseifolius tea powder and the ultrapure water are not perfectly elliptical, it is clear from table 3 that the interaction between the feed-to-liquid ratio (a) and the extraction time (B) of the lithocarpus litseifolius tea powder and the ultrapure water is also significant.
According to the regression model constructed in the steps, statistical software Design-Expert 8.0.6 is applied to further optimize the pressurized solvent assisted extraction process of lithocarpus litseifolius (sweet tea) polysaccharides, and the result shows that the lithocarpus litseifolius polysaccharides with the maximum extraction rate can be obtained under the following optimal reaction conditions: the extraction ratio of material to liquid is 24.08mL/g, the extraction time is 41.31min, and the extraction pressure is 0.09 MPa. The predicted value of the extraction rate of the Lithocarpus litseifolius leaf polysaccharide is 4.67%. Considering the actual operation, the above conditions are adjusted to 24mL/g of extraction feed-liquid ratio, 41min of extraction time and 0.09MPa of extraction pressure, and the extraction feed-liquid ratio is 1: and (3) performing three verification tests under the conditions of 24 (g: mL), 41min of extraction time and 0.09Mpa of extraction pressure, wherein the extraction rate of the lithocarpus litseifolius leaf polysaccharide is 4.65 +/-0.12 percent, which is not much different from the theoretical prediction value, and the process for extracting the lithocarpus litseifolius leaf polysaccharide under the assistance of the pressurized solvent after the Box-Behnken central combination design-response surface method optimization is feasible and can be popularized in industrial application.
Claims (10)
1. The extraction method of Lithocarpus litseifolius polysaccharide is characterized by comprising the following steps:
(1) pretreating Lithocarpus litseifolius leaves to obtain Lithocarpus litseifolius leaf powder; (2) removing fat-soluble compounds in Lithocarpus litseifolius leaf powder; (3) extracting Lithocarpus litseifolius (hook.) F.H.) leaves with liposoluble compounds removed under pressure with water as extraction solvent; (4) centrifuging the extract, and collecting supernatant to obtain Lithocarpus litseifolius leaf crude polysaccharide extract.
2. The extraction method according to claim 1, wherein the amount of the extraction solvent added in the step (3) is calculated based on the amount of Lithocarpus litseifolius leaf powder, and the ratio of Lithocarpus litseifolius leaf powder to the extraction solvent is 1: (10-50); preferably, the ratio of lithocarpus litseifolius leaf powder to the extraction solvent is 1: (20-30); most preferably, the ratio of Lithocarpus litseifolius leaf powder to extraction solvent is 1: 20-24.
3. The extraction process according to claim 1, wherein the pressure extraction time in the step (3) is 20 to 60 min; preferably 30-50 min; most preferably 40-41 min.
4. The extraction process according to claim 1, wherein the pressure of said pressure extraction in the step (3) is 0.02 to 0.10 Mpa; preferably 0.08 to 0.09 MPa.
5. The extraction method according to claim 1, wherein the pretreatment method of Lithocarpus litseifolius leaves in step (1) comprises: drying Lithocarpus litseifolius leaf, pulverizing, and sieving.
6. The extraction process according to claim 1, wherein the method for removing the fat-soluble compounds from the Lithocarpus litseifolius leaf powder in step (2) comprises: mixing Lithocarpus litseifolius leaf powder with ethanol, and performing ultrasonic treatment; centrifuging the product after ultrasonic treatment, and taking the precipitate to obtain the product.
7. The extraction method according to claim 6, wherein the precipitate obtained in step (2) is subjected to ultrasonic treatment again after being mixed with ethanol uniformly, and the product after ultrasonic treatment is centrifuged to obtain the precipitate;
preferably, the ethanol is 20-95% ethanol; according to g: mL, the proportion of the lithocarpus litseifolius tea powder to the ethanol is 1: (5-30); the ultrasonic treatment conditions are as follows: the ultrasonic power is 500-800W, and the ultrasonic treatment time is 10-60 min;
more preferably, the ethanol is 80% ethanol; according to g: mL, the proportion of the lithocarpus litseifolius tea powder to the ethanol is 1: 10; the ultrasonic treatment conditions are as follows: the ultrasonic power is 640W, and the ultrasonic treatment time is 300 min.
8. The extraction method according to claim 1, further comprising: the obtained Lithocarpus litseifolius leaf crude polysaccharide extract is subjected to purification treatment as follows: (1) concentrating the extract of crude polysaccharide from Lithocarpus litseifolius leaf; (2) removing starch and protein from the concentrated product; (3) adding ethanol for alcohol precipitation, re-dissolving the precipitate with water, dialyzing, and drying to obtain pure Lithocarpus litseifolius leaf polysaccharide extract.
9. The extraction process of claim 8, wherein the step of removing starch and protein from the concentrated product comprises: adding alpha-amylase to carry out hydrolysis reaction to remove starch in the Lithocarpus litseifolius leaf crude polysaccharide extract, inactivating amylase, and centrifuging to remove denatured enzyme and other proteins.
10. The extraction method according to claim 8, wherein in the step (3), 95% ethanol is added to the lithocarpus litseifolius crude polysaccharide extract for alcohol precipitation; centrifuging the precipitated product, taking the precipitate, redissolving the precipitate by water, and dialyzing the precipitate by a dialysis bag with the molecular weight cutoff of 3.5 kDa.
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