CN108929408B - Preparation method and application of stevioside imprinted material - Google Patents
Preparation method and application of stevioside imprinted material Download PDFInfo
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Abstract
The invention discloses a preparation method and application of a stevioside imprinted material, wherein the preparation method comprises the following steps: firstly, ethanol is used as a solvent, methacrylic acid (MAA) is used as a monomer, ammonium persulfate is used as an initiator, and the temperature is 50 ℃, so that the grafted primary amine microsphere PMAA/PAS with the optimal condition is prepared. Then, a novel surface imprinting technology is adopted to prepare the stevioside surface molecularly imprinted material MIP-PMAA/PAS. And (3) adding a stevia sugar ethanol solution and glutaraldehyde serving as an initiator into the saturated adsorbed grafted primary amine microspheres to prepare the imprinted material MIP-PMAA/PAS. The resulting imprinted polymers were characterized by infrared spectroscopy (FTIR). Finally, factors influencing the resolution performance of the imprinting material are explored. The molecularly imprinted polymer prepared by the molecular imprinting technology can specifically identify template molecules, and has the characteristics of simple preparation, good selectivity and high stability.
Description
Technical Field
The invention relates to a preparation method and application of a stevioside imprinted material, and belongs to the field of molecular imprinted materials.
Background
Stevioside, (total steviol glycosides), commonly known as stevioside, is a natural sweetener with high sweetness and low calorie extracted from stevia leaves. Stevioside has the characteristics of low heat, high sweetness, good taste quality, high temperature resistance, good stability and the like. At present, the method for separating and purifying stevioside commonly used in factories is a resin adsorption-elution method. At present, adsorption resins for extracting and separating stevioside in China mainly comprise AB8, ADS-7 and the like, but stevia molecules have larger polarity and are difficult to separate from pigment molecules, so that a series of problems which are difficult to solve exist in an extraction process. For example, although a resin having a high stevia sugar adsorption capacity is used, the adsorption amount of the pigment is increased, and if a resin having a low adsorption amount of the pigment is used, the adsorption amount of the stevia sugar is decreased. Therefore, it is desirable to prepare a resin which can adsorb a large amount of stevia molecules, but has a small adsorption amount to other molecules.
Stevioside has the characteristics of high sweetness, low calorie, good taste quality, good stability and the like. Hardly digested and decomposed in the human body, and thus does not generate too much heat. Therefore, the stevioside is beneficial to regulating blood sugar and has auxiliary curative effect on patients with diabetes and obesity. Because it is not easily affected by temperature, pH, and microbial fermentation, it is not easy to deteriorate when added into food, and is easy to store for a long time. Therefore, stevia sugar is widely used in medicine, food, beverage and other fields in many countries and regions, is called as the "best natural sweetener", is the sugar with the most development value after sucrose and beet sugar, and is internationally called as the "third sugar source".
At present, the methods for extracting stevioside commonly used at home and abroad comprise an ultrasonic extraction method, a recrystallization method, a hot water extraction method, an enzyme extraction method and the like, but the methods have some defects such as high cost, low purification rate and the like.
(1) Leaching method
The method is divided into water extraction and organic solvent extraction, wherein the enzyme inactivation is serious after the extraction of the former, and the volatilization is large and the loss is serious after the extraction of the latter. And then, removing impurities from the extracting solution, concentrating, drying and crystallizing to obtain the pure stevioside. Fengshen investigated the optimum conditions for the extraction process, using ethanol as solvent for extraction, followed by concentration, desalting, deodorizing, and crystallization to give white amorphous forms of steviol glycosides. However, the method is complicated to operate and is not suitable for industrial production.
(2) Ultrasonic extraction method
The ultrasonic extraction technology is to use the cavitation of ultrasonic wave to accelerate the destruction of plant cell wall and the release of bioactive substance for convenient extraction. Liudongqiang et al uses an ultrasonic method, uses water as an extracting agent, the extraction rate of stevioside is 12.78%, and the purity reaches 92.15% after separation and purification. The stevioside is extracted by an ultrasonic method, so that the cost is low and the purity is high. But the process is complex, the extraction rate is not high, and the method is not suitable for industrial production.
(3) Recrystallization process
Recrystallization is a process in which the steviol glycosides are separated from each other by their solubility in a solvent. By the aid of plum culture and the like at university of south Jiangnan, the stevioside is recrystallized by using a methanol/isopropanol mixed solvent, and the high-purity RA stevioside is obtained. However, the recrystallization method generally has a large loss and a low purification rate.
(4) Enzyme extraction method
The enzyme extraction technology is a biological engineering technology widely used in the traditional Chinese medicine industry in recent years, plant tissues are decomposed through mild enzyme reaction by selecting proper enzyme, the release of effective components is accelerated, and cellulase is mostly researched in the aspect of plant extraction at present. The extraction rate of stevioside in stevia rebaudiana is 12.94% by using cellulase. The enzyme extraction method shortens the extraction time, but the use conditions of the enzyme are harsh, and the repeated utilization rate is not high.
The purification method of stevioside comprises the following steps: the raw stevioside solution obtained by leaching method contains many impurities such as inorganic salts, pigments, organic acids, proteins and the like, and the common methods for removing the impurities include a physical impurity removal method, a chemical impurity removal method, a resin adsorption method and the like: (1) physical impurity removal method: the physical impurity removal method generally adopts methods such as centrifugation, filtration and the like to remove impurities such as pigments, organic acids and the like in the stevioside stock solution. The original stevioside liquid is separated and purified by an ultrafiltration membrane and a reverse osmosis membrane by Chenshaopang, Linguang and the like, and the impurity removal rate reaches over 90 percent. However, this method has a long production cycle and high running cost, and is not suitable for industrial use. (2) Chemical impurity removal method: the chemical flocculation method is one of the main methods for separating stevioside in China at present, and impurities are removed mainly by adding a flocculating agent into a stock solution. The principle is that most of flocculated substances are charged, and can adsorb groups with opposite charges, so that charge neutralization is carried out, and the flocculated substances lose stability. Finally, impurities are subjected to coagulation through the adhesion, bridging and crosslinking effects. The chemical flocculation method can be used for removing 80% of organic impurities in the stock solution. (3) Macroporous resin adsorption method: the macroporous adsorption resin separation technology is a novel separation technology and is started in the end of the 60 s of the 20 th century. The macroporous resin has many advantages which other substances do not have, such as stable physical and chemical properties, large specific surface area, good selectivity, mild adsorption conditions, high recycling rate and long service life. Therefore, macroporous resins are widely used industrially. The macroporous resin adsorption selectively adsorbs stevioside by using different polarities, and then the resin is eluted by using an organic solvent to obtain a stevioside solution with higher purity. Chentianhong et al performed adsorption elution performance studies on stevioside by using PYR resin. Although macroporous resins have some selectivity, they also adsorb some impurities.
Disclosure of Invention
The invention aims to provide a preparation method and application of a stevioside imprinted material.
The invention provides a preparation method of a stevioside imprinted material, which comprises the steps of taking methacrylic acid MAA as a monomer and ammonium persulfate as an initiator, carrying out graft polymerization on the surface of modified polystyrene primary amine microsphere PAS to obtain grafted particles PMAA/PAS, and then carrying out an isothermal adsorption test of the grafted particles PMAA/PAS on a stevioside solution; then, preparing the MIP-PMAA/PAS as a molecular surface imprinting material by using stevioside as a template molecule and glutaraldehyde as a cross-linking agent.
In the present invention, the primary amine resin is modified and grafted with methacrylic acid (MAA) to have a carboxylic acid functional group. In the process of cross-linking imprinting, the functional monomer is not only copolymerized with the added cross-linking agent, but also has a proper functional group in the molecule to interact with the template molecule. Since there is a hydrogen bonding interaction between the carboxylate and amino groups and the oxygen atoms of steviol glycosides, binding sites with specific binding properties can be formed, and the carboxyls on monomeric methacrylic acid can generate O-h. After the template molecules are washed away, three-dimensional holes with spatial structures matched with those of stevioside molecules are left in the polymer, and the holes can only adsorb stevioside, so that the stevioside can be separated and purified by using the holes on the surfaces of the polystyrene primary amine microspheres.
The preparation method specifically comprises the following steps:
(1) firstly, ethanol is used as a solvent, methacrylic acid is used as a monomer, ammonium persulfate is used as an initiator, and the temperature is 40-70 ℃ to prepare a grafted primary amine microsphere PMAA/PAS;
adding 0.1g of primary polystyrene amine resin into a four-neck flask, adding 7-11ml of DMF, soaking for 8-14h, adding 1-3ml (4-5% of the total mass) of MAA, stirring and heating by using a water bath kettle, raising the temperature, adding 0.02-0.03g of initiator ammonium persulfate (0.5-1.5% of the mass of a monomer) when the temperature reaches 30-40 ℃, and starting to react; refluxing and stirring at 40-70 deg.C, washing with distilled water for 7-9 hr, vacuum filtering, and drying in vacuum oven for 20-30 hr to obtain grafted particles PMAA/SiO2。
(2) Then preparing a stevioside surface molecular imprinting material MIP-PMAA/PAS by adopting a surface imprinting technology:
adding a solvent stevioside ethanol solution and glutaraldehyde as initiators into the saturated adsorbed grafted primary amine microspheres to prepare a imprinted material MIP-PMAA/PAS;
taking 0.05-0.15g of saturated stevia sugar adsorbed grafted microspheres PMAA/SiO2Vacuum drying; adding 40-60mL of stevioside ethanol solution as a solvent; then 0.05-0.15mL of glutaraldehyde is added as a cross-linking agent; refluxing and stirring for 5-7 hours at the temperature of 35-50 ℃; vacuum drying, adding into a four-neck flask, adding mixed solution of methanol and acetic acid, stirring at room temperature for 3-5 hr, and washing off stevioside template; after vacuum drying, obtaining a stevioside surface molecularly imprinted material MIP-PMAA/PAS;
the concentration of the ethanol solution of stevioside is 1g/L, and the volume ratio of methanol to acetic acid is 3:1-5: 1.
In the step (2) of the method, 0.1g of saturated adsorbed grafted primary amine microspheres are selected, 50mL of 1g/L stevioside ethanol solution is added as a solvent, the temperature is 40 ℃, the reaction time is 6h, and the mass fraction of the cross-linking agent is 0.238% (taking template molecules as a reference substance); the selectivity coefficient of the obtained imprinted material was 7.88.
The invention provides application of the stevioside imprinted material prepared by the preparation method in molecular imprinting extraction of stevioside.
The application is characterized in that the separation effect of the stevia molecular imprinting material is the best when the mixed aqueous solution of the stevia sugar and the glucose which are 1g/L is adsorbed, the temperature is 20 ℃, the oscillation time is 1.5 h, and the pH value of the solution is 6. The maximum static adsorption capacity and the maximum dynamic adsorption capacity of the finally obtained stevioside molecular imprinting material are 203.48 mg/g and 217.69 mg/g respectively.
The invention has the beneficial effects that:
the method can efficiently and quickly identify and adsorb the template molecule stevioside from the stevioside crude extract, thereby improving the yield and purity of the stevioside, simultaneously reducing the material loss and energy consumption, and reducing or avoiding the emission of environmental pollutants.
Drawings
FIG. 1 is an infrared spectrum of an unmodified primary amine microsphere PAS, a grafted microsphere PMAA/PAS and a stevia molecular imprinting material MIP-PMAA/PAS obtained in example 1.
FIG. 2 is the effect of template molecule concentration on the resolution performance of the imprinted material.
FIG. 3 is a graph showing the effect of blotting temperature on the resolution performance of blotting materials.
FIG. 4 shows the effect of reaction time on the resolution performance of imprinted materials.
FIG. 5 is a graph showing the effect of the amount of cross-linking agent on the resolution performance of imprinted materials.
FIG. 6 is an isothermal adsorption curve of non-imprinted material NMIP-PMAA/PAS on stevia and glucose.
FIG. 7 is an isothermal adsorption curve of the imprinted material MIP-PMAA/PAS on stevioside and glucose.
FIG. 8 is a graph of the dynamic binding of non-imprinted material NMIP-PMAA/PAS to stevia and glucose.
FIG. 9 is a dynamic binding curve of imprinted material MIP-PMAA/PAS to stevioside and glucose.
FIG. 10 shows the effect of the concentration of aqueous solution of stevioside on the resolution performance of imprinted materials.
FIG. 11 is a graph showing the effect of the resolution temperature on the resolution performance of the imprinted material.
FIG. 12 is a graph showing the effect of solution pH on the amount of adsorption during resolution.
FIG. 13 shows the repeated utilization rate of the imprinted material MIP-PMAA/PAS.
Detailed Description
The present invention is further illustrated by, but is not limited to, the following examples.
Example 1: preparation method of stevioside imprinted material
The method comprises the following steps:
(1) a preparation method of a grafted microsphere PMAA/PAS,
0.1g of the washed primary amine resin beads were weighed in a four-necked flask, 10ml of DMF (N, N-dimethylformamide) was added thereto, and the mixture was sealed and immersed for ten hours. 1.4 mM AA, 0.0274g ammonium persulfate and 50mL ethanol were added as a solvent, and the mixture was stirred under reflux at 50 ℃ for 8 hours. After the reaction is finished, carrying out suction filtration and vacuum drying for later use.
(2) Preparation of stevioside surface imprinted material MIP-PMAA/PAS
0.1g of the graft microspheres saturated and adsorbed with stevioside was taken, vacuum dried, and placed in a four-neck flask. 50mL of 1g/L ethanol solution of stevioside was added as a solvent, and 0.1mL of glutaraldehyde was added as a crosslinking agent. The mixture was stirred under reflux at 40 ℃ for 6 hours. Vacuum drying, placing into a four-neck flask, adding methanol and acetic acid (V)1：V2=4: 1) the mixed solution is stirred for 3.5 hours at normal temperature, and the stevioside template is washed off. And (4) drying in vacuum to obtain the stevioside surface molecularly imprinted material MIP-PMAA/PAS.
(3) Characterization of stevia sugar surface imprinted material MIP-PMAA/PAS
And (3) measuring the infrared spectrum of the MIP-PMAA/PAS by adopting a potassium bromide tabletting method, and determining whether the crosslinking is successful or not by observing each functional group contained in the material.
As can be seen from the infrared spectrum of FIG. 1, 3750 cm-1Is the stretching vibration peak of N-H, 1583 cm-1，1485 cm-1Bending vibration peak of N-H, 3445 cm-1And 1687 cm-1Is the vibrational shrinkage peak for-OH. 3445 cm in the PMAA/PAS spectrum-1the-OH expansion of (C) is obviously enhanced, and a new 700cm appears-1And a characteristic absorption peak of 3445 cm-1The characteristic peak at (a) is obviously strengthened, which indicates that MAA is successfully grafted on the primary amine microspheres. 700cm can be observed on MIP-PMAA/PAS-1，1740 cm-1There is a characteristic absorption peak of the fatty aldehyde indicating that the crosslinking reaction has occurred.
Example 2: preparation method of stevioside imprinted material
The method comprises the following steps:
(1) a preparation method of a grafted microsphere PMAA/PAS,
0.1g of the washed primary amine resin beads were weighed in a four-necked flask, and 8ml of DMF (N, N-dimethylformamide) was added thereto, followed by closed immersion for ten hours. 2.0 mM AA, 0.0274g ammonium persulfate and 50mL ethanol were added as solvent, and the mixture was stirred under reflux at 60 ℃ for 8 hours. After the reaction is finished, carrying out suction filtration and vacuum drying for later use.
(2) Preparation of stevioside surface imprinted material MIP-PMAA/PAS
0.1g of the graft microspheres saturated and adsorbed with stevioside was taken, vacuum dried, and placed in a four-neck flask. 50mL of 1g/L ethanol solution of stevioside was added as a solvent, and 0.1mL of glutaraldehyde was added as a crosslinking agent. The mixture was stirred under reflux at 50 ℃ for 6 hours. Vacuum drying, placing into a four-neck flask, adding methanol and acetic acid (V)1：V2= 3.5: 1) the mixed solution is stirred for 3.5 hours at normal temperature, and the stevioside template is washed off. And (4) drying in vacuum to obtain the stevioside surface molecularly imprinted material MIP-PMAA/PAS.
Example 3: preparation method of stevioside imprinted material
The method comprises the following steps:
(1) a preparation method of a grafted microsphere PMAA/PAS,
0.2g of the washed primary amine resin beads were weighed into a four-necked flask, and 14ml of DMF (N, N-dimethylformamide) was added thereto, followed by closed immersion for ten hours. 4.0 mM AA, 0.0274g ammonium persulfate and 50mL ethanol were added as a solvent, and the mixture was stirred under reflux at 50 ℃ for 8 hours. After the reaction is finished, carrying out suction filtration and vacuum drying for later use.
(2) Preparation of stevioside surface imprinted material MIP-PMAA/PAS
0.2g of the graft microspheres saturated and adsorbed with stevioside was taken, vacuum dried, and placed in a four-neck flask. 50mL of 1g/L ethanol solution of stevioside was added as a solvent, and 0.4mL of glutaraldehyde was added as a crosslinking agent. The mixture was stirred under reflux at 50 ℃ for 6 hours. Vacuum drying, placing into a four-neck flask, adding methanol and acetic acid (V)1：V2= 4.5: 1) the mixed solution is stirred for 3.5 hours at normal temperature, and the stevioside template is washed off. And (4) drying in vacuum to obtain the stevioside surface molecularly imprinted material MIP-PMAA/PAS.
Example 4: study on resolution performance of imprinted material MIP-PMAA/PAS
1. Effect of template molecule concentration on imprinted materials
0.1g of saturated adsorbed grafted microspheres PMAA/PAS and 0.1mL of glutaraldehyde are added into five four-mouth flasks, and 0.1g/L, 0.5g/L, 1.0g/L, 2.0g/L and 3.0g/L ethanol solutions of stevioside are added. The temperature is set at 50 ℃ and the mixture is stirred under reflux for 8 h. And after the reaction is finished, eluting the template, performing suction filtration and vacuum drying.
The difference in the resolving power of the blotting material when an aqueous solution of stevioside and an ethanol solution of stevioside were used as solvents can be seen in FIG. 2.
As is clear from FIG. 2, the effect of ethanol solution using stevioside is better than that of aqueous solution of stevioside. This is because the solubility of stevioside in ethanol is greater than that in water, and therefore more imprinted cavities can be formed in the ethanol solvent, so that the selectivity coefficient becomes large.
And when the ethanol solution concentration of the stevioside is 1g/L, the selectivity coefficient of the molecular imprinting material MIP-PMAA/PAS on the surface of the stevioside is the largest. The reason is that with the increase of the concentration of the stevioside aqueous solution, hydrogen bonds are formed between the stevioside and the imprinting material with a higher probability, more template molecule-functional monomer complexes are formed, the number of imprinting holes in the imprinting material is increased, and the selectivity coefficient is gradually increased. However, when the concentration of the solution is higher than 1g/L, the adsorption amount of the grafted particles to the stevioside molecules reaches the balance, the number of formed cavities is not increased any more, and therefore, the selectivity coefficient is not changed almost any more.
Influence of reaction temperature on blotting Material
0.1g of saturated and adsorbed grafted microspheres PMAA/PAS and 0.1mL of glutaraldehyde were added to five four-necked flasks, respectively, and 50mL of a 1g/L ethanol solution of stevioside was added. The temperature is set to 20 ℃, 30 ℃, 40 ℃, 50 ℃ and 60 ℃ respectively, and the reflux stirring is carried out for 8 hours. And after the reaction is finished, eluting the template, performing suction filtration and vacuum drying.
As can be seen from fig. 3, the selectivity coefficient of the stevia molecular imprinting material is the greatest when the temperature is 40 ℃. As can be seen from the figure, as the blotting temperature increases, the selectivity coefficient becomes larger; the temperature reaches the maximum when reaching 40 ℃; as temperature continues to rise, the selectivity coefficient decreases instead. This is because the crosslinking reaction is endothermic and cannot provide sufficient energy for the reaction at a low temperature, so that the degree of completion of the crosslinking reaction is low at a low temperature, and the number of imprinted holes formed is small, resulting in a small selectivity coefficient; however, when the temperature is too high, the interaction force between the stevia sugar molecules and MIP-PMAA/PAS is electrostatic interaction and hydrogen bond interaction, and the interaction belongs to physical adsorption. With the increase of the temperature, the stevioside template molecules are easy to fall off from the imprinted material MIP-PMAA/PAS, so that the formation of imprinted pores is not facilitated, and after the temperature is higher than 40 ℃, the negative influence is dominant, and the imprinted pores are reduced, so that the imprinting effect is obviously reduced, and the selectivity coefficient is also reduced.
Influence of reaction time on blotting Material
0.1g of saturated and adsorbed grafted microspheres PMAA/PAS and 0.1mL of glutaraldehyde were added to five four-necked flasks, respectively, and 50mL of 1g/L ethanol solution of stevioside was added. The temperature is set to be 40 ℃, and the reflux stirring time is 2 h, 4h, 6h and 8 h respectively. And after the reaction is finished, eluting the template, performing suction filtration and vacuum drying.
As can be seen from FIG. 4, the selectivity coefficient is maximized at 6 h. When the imprinting time is less than 6h, the imprinting reaction can not be completely carried out due to too short reaction time; when the imprinting time is too long, the stevioside template molecules adsorbed on the grafted microspheres PMAA/PAS may fall off, so that imprinting cavities are reduced, the adsorption quantity is reduced, and the selectivity coefficient is reduced.
Influence of the amount of crosslinker on the imprinted Material
0.1g of saturated and adsorbed grafted microspheres PMAA/PAS and 50mL of 1g/L ethanol solution of stevioside were added into five four-necked flasks, respectively. Adding cross-linking agent glutaraldehyde in the mass fractions of 0.038% (0.015 mL), 0.138% (0.06 mL), 0.238% (0.10 mL), 0.338% (0.14 mL) and 0.438% (0.18 mL), and refluxing and stirring for 6 hours. And after the reaction is finished, eluting the template, performing suction filtration and vacuum drying.
0.02 g of the dried blotting material is weighed respectively and placed in a 25 mL conical flask, 10mL of 1g/L aqueous solution of stevioside is added, the temperature is 20 ℃, and the constant temperature oscillation is carried out for 1.5 h. After completion of shaking, 1mL of the supernatant was diluted 25-fold, and the absorbance at wavelength of 195 nm was measured. The adsorption amount was calculated by the following formula:
in the formula C0（mol/L）、Ce(mol/L) is the concentration of stevioside in the solution before and after adsorption respectively; v (L) is the volume of the stevioside solution; m (g) is the mass of the functional grafting particles PMAA/PAS.
As can be seen from FIG. 5, the selectivity coefficient of the imprinted material MIP-PMAA/PAS reaches the maximum when the mass fraction of glutaraldehyde is 2.38%. Initially, the selectivity coefficient increases with increasing amounts of glutaraldehyde, and reaches a maximum of 7.88 at 0.238%. Then, as the amount of glutaraldehyde increases, the selectivity coefficient gradually decreases again. The reason is that the crosslinking degree is insufficient due to too small dosage of the crosslinking agent, and the crosslinking agent is not enough to form enough imprinting holes, so that the adsorption performance of the imprinting material is reduced, and the selectivity coefficient is reduced; when the amount of glutaraldehyde is too high, the excessive cross-linking agent can cause the degree of cross-linking to be too large, so that the imprinting cavity is reduced, the template molecule stevioside is squeezed out, and the selectivity coefficient of the imprinting material MIP-PMAA/PAS on the stevioside is reduced.
Example 5: imprinted material recognition selectivity
1. Isothermal binding curve
0.02 g of imprinted material MIP-PMAA/PAS prepared under the optimal conditions is weighed in seven conical flasks, 10mL of stevia aqueous solution is added, and the concentration is 0.2 g/L, 0.4 g/L, 0.6 g/L, 0.8 g/L, 1.0g/L, 1.2g/L and 1.4 g/L respectively. Shaking at constant temperature of 20 deg.C for 1.5 h. After completion of shaking, 1mL of the supernatant was diluted 25-fold, and the absorbance at wavelength of 195 nm was measured. The amount of adsorption was calculated using equation 2.1.
FIGS. 6 and 7 are the isothermal adsorption curves of imprinted material MIP-PMAA/PAS and non-imprinted material NMIP-PMAA/PAS on stevioside and glucose.
As can be seen from FIG. 6, the non-imprinted material NMIP-PMAA/PAS has an adsorption effect on both stevioside and glucose, wherein the adsorption amount on stevioside reaches 203.48 mg/g, and the adsorption amount on glucose reaches 160.13 mg/g, which indicates that the non-imprinted material has no selective recognition on stevioside and glucose. FIG. 7 shows that the adsorption amount of the imprinted material MIP-PMAA/PAS on stevioside is still 203.48 mg/g, but the adsorption amount on glucose is reduced to 45.23 mg/g. The change of the adsorption quantity shows that the imprinted material MIP-PMAA/PAS has better selective recognition capability and binding capability on the template molecule stevioside. The difference is mainly caused by that the imprinting material contains a cavity which is matched with the stevioside template molecule, but the cavity is not matched with the glucose molecule. Therefore, the imprinted material MIP-PMAA/PAS has low absorption amount on glucose but has high absorption amount on stevioside.
Dynamic binding curve
The selective performance of the imprinted material MIP-PMAA/PAS on stevioside is determined by using a dynamic adsorption method. At 20 ℃, a glass tube with the inner diameter of 6 mm is filled with imprinting material MIP-PMAA/PAS with the mass of 0.6685 g, the Volume of a packed Bed (Bed Volume, BV) is 2 mL, 1.0g/L of stevioside aqueous solution is led to PASs through the glass tube in a counter-current mode, the flow rate is controlled to be 5 BV/h, the collection interval of effluent is 3 BV, and the stevioside concentration of the effluent is determined by measuring the absorbance of the effluent. The glucose concentration of the effluent was measured by the same dynamic adsorption experiment using MIP-PMAA/PAS while keeping the other conditions constant. The corresponding dynamic binding curves are also plotted. The adsorption amounts of the above two molecules were calculated.
And drawing a dynamic adsorption curve of NMIP-PMAA/PAS on stevioside and glucose according to the same adsorption condition.
As can be seen from FIG. 8, the non-imprinted material has a small ability to resolve stevia sugar and glucose. However, as can be seen from fig. 9, the resolving power of the imprinted material for stevioside and glucose is obviously enhanced. The dynamic adsorption curve shows that the imprinted material MIP-PMAA/PAS has strong binding capacity and recognition capacity on the template molecule stevioside.
Example 6: effect of resolution conditions on the resolving power of imprinted materials
1. Effect of resolution concentration on resolution Properties of imprinted Material
0.02 g of imprinted material MIP-PMAA/PAS prepared under the optimal conditions is weighed in seven conical flasks, 10mL of stevia aqueous solution is added, and the concentration is 0.2 g/L, 0.4 g/L, 0.6 g/L, 0.8 g/L, 1.0g/L, 1.2g/L and 1.4 g/L respectively. Shaking at constant temperature of 20 deg.C for 1.5 h. After completion of shaking, 1mL of the supernatant was diluted 25-fold, and the absorbance at wavelength of 195 nm was measured. The adsorption amount was calculated by the following formula:
as can be seen from FIG. 10, when the concentration of the aqueous solution of stevioside was less than 1g/L, the adsorption reached equilibrium due to the low concentration of the solution, and therefore the selectivity coefficient was low. As the solution concentration increases, the selectivity coefficient increases. When the concentration of the solution is higher than 1g/L, the adsorption quantity is not changed any more, and the selectivity coefficient is balanced.
Influence of resolution temperature on resolution performance of imprinting material
Taking five conical flasks, respectively weighing 0.02 g of imprinted material MIP-PMAA/PAS prepared under the optimal conditions,
10ml of 1.0g/L aqueous stevioside solution is added. Oscillating for 1.5 h at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C and 60 deg.C, respectively. After completion of shaking, 1mL of each supernatant was diluted 25-fold, and the absorbance at a wavelength of 195 nm was measured.
The adsorption amount was calculated by the following formula:
as shown in FIG. 11, the selectivity coefficient of the imprinted material MIP-PMAA/PAS for stevia molecules decreases with increasing resolution temperature. This is because the force of the imprinted material on the adsorption of stevioside comes from the interaction force of hydrogen bonds between the host and the guest. Since temperature has a great influence on the hydrogen bonding force, the hydrogen bonding between the components is weakened or even destroyed with the increase of temperature, thereby resulting in the decrease of the selectivity coefficient. The result shows that the imprinted material MIP-PMAA/PAS has the best adsorption performance on stevia molecules at 20 ℃, the interaction force of hydrogen bonds between the MIP-PMAA/PAS is strongest, and the selectivity coefficient of the imprinted material is the best.
Influence of solution pH on imprinted material resolution performance during resolution
Taking five conical flasks, respectively weighing 0.02 g of imprinted material MIP-PMAA/PAS prepared under the optimal conditions, and adding 10ml of 1.0g/L stevioside aqueous solution. The mixture was shaken for 1.5 h at a temperature of 20 ℃. The pH of the solution was adjusted to 2, 4, 6, 8, 10, respectively. After completion of shaking, 1mL of each supernatant was diluted 25-fold, and the absorbance at a wavelength of 195 nm was measured. The adsorption amount was calculated by the following formula:
as can be seen from fig. 12, the selectivity coefficient of MIP-PMAA/PAS for mandelic acid increases first with increasing pH, and then gradually decreases again after reaching a maximum, and reaches a maximum at pH = 6. This is because when the pH is less than 6, the protonation degree of amino groups is very high, but the ionization degree of carboxyl groups in stevia sugar molecules is very low, so the electrostatic action is very weak, and the hydrogen bond action is the main action of the grafted particles on the adsorption action of stevia sugar; when the pH value is gradually increased, the ionization degree of the carboxyl is also increased, the electrostatic interaction between the particle MIP-PMAA/PAS and the stevia sugar molecules is continuously enhanced, and the interaction of hydrogen bond interaction force still exists. Under the combined action of the two acting forces, the selectivity coefficient is increased. When the pH value is more than 6, the protonation degree of amino groups in MIP-PMAA/PAS molecules is weaker and weaker, carboxyl groups are changed into carboxylic acid compounds, so that hydrogen bonds are broken, the adsorption quantity is reduced, and the selectivity coefficient is gradually reduced. From the above, the optimum pH value of MIP-PMAA/PAS microspheres for stevioside adsorption is 6.
Example 7: investigating the specific adsorption of the imprinting material to the stevia molecule
Taking two erlenmeyer flasks, weighing 0.02 g of imprinted material MIP-PMAA/PAS respectively, and adding 10ml of 1.0g/L stevioside aqueous solution and 1g/L glucose solution respectively. The mixture was shaken for 1.5 h at a temperature of 20 ℃. The absorbance of the stevia solution was measured, and the concentration of the glucose solution was titrated using a titration method. Calculating the adsorption capacity, K, to two moleculesd(mL/g) and selectivity factor k:
in the formula, Kd(mL/g) is the partition coefficient of stevioside or glucose; qe(mg/g) and Ce(mg/mL) is the equilibrium adsorption amount and equilibrium concentration of the blotting material for stevioside, respectively.
Wherein k is the selectivity coefficient of the imprinted material MIP-PMAA/PAS to stevioside relative to glucose.
The adsorption capacity and selectivity coefficient of the non-imprinted material NMIP-PMAA/PAS to the non-imprinted material NMIP-PMAA/PAS are determined under the same conditions.
TABLE 1 Selectivity coefficients for imprinted and non-imprinted materials
As can be seen from Table 1, the non-imprinted material NMIP-PMAA/PAS adsorbs both stevioside and glucose, has a small selectivity coefficient, and has poor resolution capability on stevioside. However, the stevia molecular imprinting material MIP-PMAA/PAS adsorbs the stevia, but the adsorption quantity to glucose is reduced, the selectivity coefficient is increased, and the capability of splitting the stevia is enhanced. The prepared stevia sugar molecular imprinting material has certain resolution capability on the stevia sugar.
Example 8: investigating recyclability of blotting Material
0.1g of blotting material MIP-PMAA/PAS which is saturated and adsorbs stevioside is taken and placed in a four-mouth flask. 40mL of a mixture of methanol and acetic acid (methanol: acetic acid =4: 1) was added thereto, and the mixture was stirred at room temperature for 3.5 hours, filtered under suction, and dried in vacuo. And (4) taking the dried imprinting material to repeat isothermal adsorption and elution experiments. The absorbance at 195 nm was measured, and the amount adsorbed was calculated according to equation 2.1. To investigate the reusability of the blotting material.
Fig. 13 shows the recycling rate of the imprinted material MIP-PMAA/PAS, which is determined to be 92.63% of the prepared stevia molecular imprinted material.
Claims (7)
1. A preparation method of a stevioside imprinted material is characterized by comprising the following steps: carrying out graft polymerization on the surface of the modified primary polystyrene amine microsphere PAS by using methacrylic acid MAA as a monomer and ammonium persulfate as an initiator to obtain graft particles PMAA/PAS, and then carrying out an isothermal adsorption test of the graft particles PMAA/PAS on a stevioside solution; then preparing a molecular surface imprinting material MIP-PMAA/PAS by using stevioside as a template molecule and glutaraldehyde as a cross-linking agent; after the template molecules are washed away, three-dimensional holes with spatial structures matched with those of stevioside molecules are left in the polymer, and the holes can only adsorb stevioside, so that the holes on the surfaces of the polystyrene primary amine microspheres are utilized to separate and purify the stevioside;
the preparation method specifically comprises the following steps:
(1) firstly, ethanol is used as a solvent, methacrylic acid is used as a monomer, ammonium persulfate is used as an initiator, and the temperature is 40-70 ℃ to prepare a grafted primary amine microsphere PMAA/PAS;
taking 0.1g of primary polystyrene amine resin, adding 7-11ml of DMF, soaking for 8-14h, adding 1-3ml of MAA, stirring and heating by using a water bath kettle, and adding an initiator to start reaction when the temperature reaches 30-40 ℃; refluxing and stirring at 40-70 ℃, repeatedly washing with distilled water for 7-9h, filtering, and drying in a vacuum oven for 20-30h to obtain grafted particles PMAA/PAS;
(2) then preparing a stevioside surface molecular imprinting material MIP-PMAA/PAS by adopting a surface imprinting technology:
adding a solvent stevioside ethanol solution and glutaraldehyde as initiators into a saturated adsorbed grafted primary amine microsphere PMAA/PAS to prepare a imprinted material MIP-PMAA/PAS;
taking 0.05-0.15g of saturated stevia sugar adsorbed grafted microspheres PMAA/PAS, and drying in vacuum; adding 40-60mL of stevioside ethanol solution as a solvent; then 0.05-0.15mL of glutaraldehyde is added as a cross-linking agent; refluxing and stirring for 5-7 hours at the temperature of 35-50 ℃; vacuum drying, adding into a four-neck flask, adding mixed solution of methanol and acetic acid, stirring at room temperature for 3-5 hr, and washing off stevioside template; and (4) drying in vacuum to obtain the stevioside surface molecularly imprinted material MIP-PMAA/PAS.
2. The method for preparing a stevioside imprinted material according to claim 1, characterized in that: in the step (1), the adding amount of MAA accounts for 4-5% of the total mass of the raw materials; the initiator is ammonium persulfate, and the using amount of the initiator is 0.5-1.5% of the mass of the monomer.
3. The method for preparing a stevioside imprinted material according to claim 1, characterized in that: in the step (2), 0.1g of grafted microsphere PMAA/PAS which is saturated and adsorbs stevioside is selected, 50mL of stevioside ethanol solution is added as a solvent, the temperature is 40 ℃, the reaction time is 6h, and the addition amount of the cross-linking agent accounts for 0.238 percent of the mass fraction of the stevioside template molecules; the selectivity coefficient of the obtained imprinted material was 7.88.
4. The method for preparing a stevioside imprinted material according to claim 1, characterized in that: the concentration of the ethanol solution of stevioside is 1g/L, and the volume ratio of methanol to acetic acid is 3:1-5: 1.
5. A stevioside imprinted material prepared by the preparation method of any one of claims 1 to 4.
6. The application of the stevioside imprinted material of claim 5 in extraction of stevioside by a molecular imprinting method.
7. Use according to claim 6, characterized in that: adsorbing a mixed aqueous solution of 1g/L of stevioside and glucose, wherein the temperature is 20 ℃, the oscillation time is 1.5 hours, and when the pH of the solution is 6, the splitting effect of the stevioside molecular imprinting material is the best; the maximum static adsorption capacity and the maximum dynamic adsorption capacity of the finally obtained stevioside molecular imprinting material are 203.48 mg/g and 217.69 mg/g respectively.
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