CN113135998B

CN113135998B - Preparation method of selenium-rich starch

Info

Publication number: CN113135998B
Application number: CN202110365143.2A
Authority: CN
Inventors: 尹艳镇; 石成; 张瑞瑞; 刘子杰; 梁兴唐; 黄秋港; 钟书明; 马庆满; 徐磊廷
Original assignee: Beibu Gulf University
Current assignee: Shien Enshi Biomedical Development Co ltd; Wuhan Topaz Intellectual Property Operation Co.,Ltd.
Priority date: 2021-04-02
Filing date: 2021-04-02
Publication date: 2022-02-01
Anticipated expiration: 2041-04-02
Also published as: CN113135998A

Abstract

The invention relates to the technical field of starch modification, in particular to a preparation method of selenium-rich starch. The method comprises the following steps: (1) placing proper amount of selenium powder and sodium borohydride at round bottomAdding a proper amount of mixed solution of ethanol and water into a flask, and reacting at room temperature under the protection of nitrogen to obtain a NaSeH stock solution; (2) placing starch octenylsuccinate (OSA starch, 10g) in a round-bottom flask, adding a mixed solvent of ethanol and water, stirring for 30min, and uniformly mixing; (3) dropwise adding a NaSeH stock solution and a sodium hydroxide solution into a reaction system under an ice bath condition, and stopping the reaction after reacting for a certain time; (4) and (3) adjusting the pH of the reaction solution to 3-4, dropwise adding the solution into ethanol solvent to precipitate starch, stirring for 20 minutes, filtering and collecting a filter cake, sequentially washing the filter cake with 75% ethanol and absolute ethanol, and drying the filter cake in a vacuum drying oven to obtain the selenized starch sample. The series Se-starch prepared by the invention shows typical GPx enzymatic catalytic behavior, and the catalytic activity is 1.53 multiplied by 10 of classical small-molecule antioxidant selenase PhSePh⁵And (4) doubling.

Description

Preparation method of selenium-rich starch

Technical Field

The invention relates to the technical field of starch modification, in particular to a preparation method of selenium-rich starch.

Background

With the continuous enhancement of health and health care consciousness of people, the development of functional food which has positive effect on maintaining the health of human body is of great significance. The trace element selenium can participate in the synthesis of 25 selenium-containing proteins in human bodies, particularly can participate in the selenocysteine which forms a catalytic center of glutathione peroxidase (GPx), plays a role in removing excessive free radicals in human bodies, and plays a role in protecting human bodies from oxidative damage and resisting diseases such as inflammation, cancer, keshan disease and the like.

In view of the important antioxidation of the selenium element, a new technology for developing selenium-rich functional products is developed, and a new idea for scientific selenium supplement is provided, which is the research focus in the field of functional foods. The development of selenium-rich starch has attracted particular attention from researchers because starch occupies a large proportion of human dietary structure. At present, the development of the selenium-rich starch mainly comprises the following four measures: (1) screening selenium content in the culturable selenium-rich soil guarantee product; (2) activating the passivated selenium element in the soil to improve the absorption rate; (3) cultivating crops such as wheat, rice, potato and the like with strong selenium absorption capacity; (4) the exogenous selenium fertilizer is applied to improve the conversion and enrichment amount of the selenium of crops.

Although the above measures have achieved good results, there are two major problems in the development field of selenium-rich starch: firstly, the time period for the plant to grow, transform and absorb selenium is long, and the influence of environmental change is large, so that the selenium content in the selenium-rich starch is unstable; and secondly, the selenium-rich starch can play the role of oxidation resistance only by being converted into GPx by a human body and cannot directly play the role of oxidation resistance, thereby bringing great limitations to market acceptance and industrial popularization.

Therefore, inspired by GPx catalytic process, the invention proposes a novel method for directly modifying the catalytic center of natural GPx to the starch framework material, establishes a novel strategy for directly endowing the GPx with the catalytic activity of the selenium-rich starch without biotransformation, solves the limitation problem of developing the selenium-rich starch in the traditional planting industry, and provides a novel idea for developing the selenium-rich starch different from the traditional planting industry.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide a preparation method of selenium-rich starch.

The technical scheme provided by the invention is as follows:

a preparation method of selenium-rich starch comprises the following steps:

(1) placing a proper amount of selenium powder and sodium borohydride into a 100mL round-bottom flask, adding a proper amount of mixed solution of ethanol and water, and reacting at room temperature for 2 hours under the protection of nitrogen to obtain a NaSeH stock solution for later use;

(2) placing octenyl succinic acid starch ester (OSA starch, 10g) in a 250mL round-bottom flask, adding 100mL of mixed solvent of ethanol and water into the flask, and stirring for 30min to uniformly mix the system;

(3) slowly dropwise adding a NaSeH stock solution and a 0.1mol/L sodium hydroxide solution into a reaction system under an ice bath condition, and stopping the reaction after reacting for a certain time;

(4) and (3) adjusting the pH of the reaction solution to 3-4, dropwise adding the solution into a beaker filled with a large amount of ethanol solvent to precipitate starch, stirring for 20 minutes, filtering and collecting a filter cake, sequentially washing the filter cake with 75% ethanol and absolute ethanol, and drying the washed filter cake in a vacuum drying oven at 50 ℃ for 24 hours to obtain the selenized starch sample.

Preferably, the volume ratio of ethanol to water in the mixed solvent in step (2) is 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3: 7.

Preferably, the ratio of the NaSeH to the double bond in the NaSeH stock solution in step (2) is selected from 1:1, 1.2:1, 1.5:1, 1.8:1 and 2.2: 1.

Preferably, the pH of the reaction system in the step (2) is controlled to 8 to 10.

Preferably, the temperature of the reaction system in the step (2) is controlled to be 30 to 70 ℃.

Preferably, the reaction time of the reaction system in the step (2) is controlled to be 1 to 8 hours.

Compared with the prior art, the invention has the following beneficial effects:

(1) according to the invention, waxy starch modified octenyl succinate starch ester is used as a raw material, and a new strategy for directly endowing selenium-rich waxy starch (Se-starch) GPx with catalytic activity without biotransformation is creatively established.

(2) The invention utilizes¹The method proves that the selenium element is successfully modified on the starch skeleton by characterization methods such as H NMR, EDS, XPS, SEM, XRD, FT-IR and the like, the prepared series of Se-starch shows typical GPx enzymatic catalytic behavior, and the catalytic activity is 1.53 multiplied by 10 of classical small-molecule antioxidant selenase PhSeSePh⁵And (4) doubling.

(3) The invention shows that the catalytic mechanism analysis of Se-starch80 with the highest catalytic activity: the selenization modification reaction enables the surface of the starch to present a weathered structure of folds and grooves, which is beneficial to forming a hydrophobic microenvironment, enhancing the substrate binding capacity and promoting the catalytic activity to be improved; meanwhile, effective matching of the catalytic center with the hydrophobic microenvironment is also important for maintaining high catalytic activity.

(4) The invention provides a new concept for developing selenium-rich starch different from the traditional planting industry, and can provide a theoretical basis for developing novel selenium-rich functional foods, selenium-rich additives and medicaments related to oxidative diseases.

Drawings

FIG. 1 is a schematic diagram of Se-starch prepared from OSA starch as raw material;

FIG. 2 is a schematic of the nucleophilic addition reaction of sodium hydroselenide with OSA starch to produce Se-starch 80;

FIG. 3 is of OSA starch and Se-starch80¹H NMR spectrum;

FIG. 4 shows XPS spectrum (A), EDS spectrum and Se mapping (B) of Se-starch 80; SEM photographs of OSA starch (C) and Se-starch80 (D);

FIG. 5 is FT-IR line (A) and XRD line (B) of OSA starch and Se-starch 80;

FIG. 6 is a graph showing the change in selenium content and oxidation resistant catalytic rate for Se-starch preparation by varying reaction time (A), reaction temperature (B), NaSeH to double bond molar ratio (C) and ethanol volume ratio (D); a dispersion state diagram (E) of the waxy starch OSA starch in reaction systems with different ethanol volumes;

FIG. 7 shows the initial temperature (Ti) of the thermogravimetric analysis determined samples: (a) OSA starch; (b) (c), (d), (e) Se-starch (ethanol volume of 80%, 30%, 60%, 90%); (f) se-starch (NaSeH to double bond molar ratio of 2.2); (g) se-starch (reaction temperature 70 ℃); (h) se-starch (reaction time is 1 hour) (except the reaction conditions noted in the figure, the other preparation conditions of the samples a-h are optimized optimal process conditions);

FIG. 8 shows the results of reaction between ArSH (TNB, NBT) and ROOH (CUOOH, H)₂O₂) Determining the GPx catalytic activity of Se-starch80 for a substrate;

FIG. 9 shows the change of H in the concentration of fixed TNB of 0.15mM₂O₂(A) And CUOOH (B) at concentrations of 0.05, 0.10, 0.25, 0.5, 1.0, 2.5 and 5.0mM, respectively, the catalytic rate of Se-starch 80; NBT was fixed at a concentration of 0.15mM, and H was changed₂O₂(C) And CUOOH (D) at concentrations of 0.05, 0.10, 0.25, 0.5, 1.0, 2.5 and 5.0mM, respectively, catalytic rate of Se-starch 80;

FIG. 10 shows the catalytic rates (v) of Se-starch80 at pH 7.0(50mM PBS) and 25 ℃ for the reduction of peroxide (ROOH, 250. mu.M) by 150. mu.M TNB (or NBT)₀) Wherein (A) CUOOH, TNB; (B) h₂O₂,TNB；(C)CUOOH,NBT；(D)H₂O₂,NBT；

Fig. 11 is (a): fluorescence spectrum lines of pyrene molecule (a) in OSA starch solution, pyrene molecule (b) in OSA starch solution, and pyrene molecule (c) in Se-starch80 solution; (B) oxidation-resistant catalytic rate for preparing Se-starch samples under different ethanol volume reaction systems and pyrene molecule I in the presence of Se-starch₁/I₃And (5) a variation graph.

Detailed Description

The present invention will be described in further detail with reference to specific examples.

1 experiment

1.1 preparation of selenium-enriched waxy starch (Se-starch)

Placing a proper amount of selenium powder and sodium borohydride into a 100mL round-bottom flask, adding a proper amount of mixed solution of ethanol and water, and reacting at room temperature for 2 hours under the protection of nitrogen to obtain a NaSeH stock solution for later use.

Starch octenylsuccinate (OSA starch, 10g) was placed in a 250mL round-bottomed flask, 100mL of a mixed solvent of ethanol and water (the volume ratio of ethanol to water was selected from 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, etc. at different ratios) was added to the flask, and the mixture was stirred for 30min to mix the system uniformly. Under the ice bath condition, NaSeH stock solution (different proportions of NaSeH and double bonds are selected to be 1:1, 1.2:1, 1.5:1, 1.8:1, 2.2:1 and the like) and 0.1mol/L sodium hydroxide are slowly dripped into a reaction system, the pH value of the reaction system is controlled to be between 8 and 10, and the reaction is stopped after reaction for certain time (different reaction times of 1h, 2h, 4h, 6h, 8h and the like are selected) at certain temperature (different reaction temperatures of 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and the like). And (3) adjusting the pH of the reaction solution to 3-4, dropwise adding the solution into a beaker filled with a large amount of ethanol solvent to precipitate starch, stirring for 20 minutes, filtering and collecting a filter cake, sequentially washing the filter cake with 75% ethanol and absolute ethanol, and drying in a vacuum drying oven at 50 ℃ for 24 hours to obtain a selenized starch sample. The selenium content of the starch is measured by an atomic fluorescence spectrophotometer (the sample with the highest catalytic activity is marked by Se-starch80, and the selenium content is 0.032 mg/kg). In addition, EDS determines the selenium content of 30g/kg on the surface of Se-starch80 starch.

1.2 characterization of Se-starch80 Structure

The infrared spectrum (FT-IR) of Se-starch80 was recorded with a Frontier Fourier transform infrared spectrometer with a resolution of 4cm^-1Scanning times 32, scanning range 4000-^-1. After the Se-starch80 is sprayed with gold, the appearance of the Se-starch80 is observed in a scanning electron microscope (JEOL FESEM 6700F), a Se element distribution diagram (Mapping) is recorded, and the Se content of the Se-starch80 surface area is determined by EDS. The X-ray spectrometer measures the XRD spectral line of Se-starch80, the tube pressure is 40kv, the current is 40mA, the scanning range is 5 degrees to 40 degrees, and the step width is 0.05 degrees.

1.3 GPx enzyme Activity assay of Se-starch80

The catalytic activity of Se-starch80 is determined by a direct test method by using an Shimadzu 2600 UV-visible spectrophotometer. Testing of catalytic rates (v) with CUOOH and TNB as substrates₀) A typical procedure is as follows: buffer solution with a certain volume, TNB solution (100 mu L), bionic GPx solution to be detected or blank solution are sequentially added into a reaction pool with 1 mL. The solution is stirred and mixed evenly in a 1mL reaction tank, and the temperature is kept for 2 minutes. Then, the catalytic reaction was initiated by adding CUOOH solution (100. mu.L), and the change in absorbance of TNB was monitored at 410nm, and TNB (. epsilon.) at 410nm was assayed using the nonenzymatic reaction as a blank₄₁₀＝13600M^–1cm^–1pH 7.0) absorbance, from which the Se-starch80 catalyzed initial reaction rate v is calculated₀。

2 results and discussion

2.1 preparation and Structure characterization of selenium-enriched waxy starch

Aiming at the development of selenium enrichment in the traditional planting industryThe research is intended to creatively provide a new method for directly modifying the catalytic center of natural GPx to the molecular chain segment of the starch, and provide a new idea for developing the selenium-rich waxy starch different from the traditional planting industry. The assumed figure of Se-Starch prepared by using OSA Starch as a raw material is shown in figure 1, and the selenium-rich waxy Starch with the best antioxidant activity synthesized by the optimized preparation process is marked as Se-Starch 80. Wherein, the reaction formula of nucleophilic addition of NaSeH and double bond in OSA Starch is shown in figure 2 (taking the preparation of Se-Starch as an example), in order to prove the successful modification of selenium element in Starch skeleton, the method utilizes¹H NMR, EDS, XPS, SEM, XRD, FT-IR, etc.

As shown in figure 3, the signal peak of the double bond hydrogen No. 4 in the nuclear magnetic spectrum of the OSA starch before selenylation modification is obvious, and the signal peak of the double bond hydrogen No. 4 in the Se-starch80 after selenylation modification is basically disappeared, which proves that the selenylation modification reaction causes the structural change of the double bond group of the OSA starch. Since NaSeH is a nucleophilic reagent with strong nucleophilicity, a similar mercapto group and double bond 'click' reaction can occur, and the double bond in the reaction system is the only active group capable of reacting with NaSeH, the disappearance of the double bond signal is presumed to be caused by the nucleophilic addition reaction of NaSeH and the double bond.

In addition, the distribution and the form of the selenium element in the starch are directly characterized by XPS and EDS, and FIG. 4A is a full-range XPS line of Se-starch80, and an electronic signal appears at about 54eV, which shows that the selenium element in the Se-starch80 is mainly in a negative divalent form. Since the negative bivalent selenium in the reactant NaSeH is easily oxidized into zero-valent selenium in the air, and the negative bivalent form of the amino acid form organic selenium in the selenium-rich functional food is generally stable, the selenium in the Se-starch80 obtained by exposing the reactant NaSeH to the air is not yet oxidized into zero-valent selenium after separation and purification, and the NaSeH is proved to be successfully modified on a starch skeleton. EDS characterization showed that the mass fraction of selenium in Se-starch80 was 3% (FIG. 4B), and Se mapping photographs showed that selenium was uniformly distributed on the surface of starch granules. The reason why the EDS-determined selenium content (mass fraction 3%) is greatly different from the atomic fluorescence spectroscopy-determined selenium content (0.033mg/kg) may be because EDS determines the selenium content in a localized region of the surface and atomic fluorescence spectroscopy determines the average selenium content of the sample, which also indicates that selenium in Se-starch80 is mainly distributed on the surface of starch.

The NaSeH is a nucleophilic reagent and has strong basicity, and the sodium hydroxide is additionally added into a selenylation modification reaction system to maintain the basicity stability of the reaction system, so that the basicity reaction condition can generate gelatinization damage effect on the surface structure of Se-starch. And (5) representing the change of the Se-starch surface structure after selenylation modification by using SEM, XRD and FT-IR. Fig. 4C is an SEM photograph of OSA starch before modification by selenization reaction, and fig. 4D is an SEM photograph of Se-starch80 after modification by selenization reaction, comparing the efflorescent gelatinization phenomenon that gelatinization causes the surface of starch to exhibit wrinkles and grooves. In addition, 3000-3500cm in the FT-IR line of Se-starch80^-1The signal peak is weaker than that of OSA (fig. 5A), probably caused by that the surface structure of the starch is damaged after selenylation modification and water molecules associated through hydrogen bond action are reduced, and the experimental phenomenon that the surface structure of the starch is damaged to a certain extent is further verified. The study on the changes of the crystal domain structures of the starch before and after modification by the selenization reaction shows that the crystal domain structures of the OSA starch and the Se-starch80 are not obviously changed (FIG. 5B). The analysis of characterization results of SEM, XRD and FT-IR shows that the selenylation modification reaction can cause the surface structure of the starch to generate larger gelatinization destruction effect and has little influence on the structure of the starch crystal region, thus laying a foundation for the antioxidant mechanism explained in the following.

2.2 preparation process optimization of selenium-rich waxy starch

The research selects the antioxidant activity of Se-starch as an evaluation index, optimizes the preparation process and establishes a new method for preparing the selenium-rich waxy starch. Based on earlier stage research, a simple and effective direct method is selected to determine the antioxidant activity, and a basis is provided for the optimization of the preparation process of Se-starch.

As shown in FIG. 6, factors such as the selenization reaction time (A), the reaction temperature (B), the molar ratio (C) of NaSeH to double bonds and the volume ratio (D) of ethanol were varied by a single-factor method,preparation of a series of Se-starchs and determination of the catalytic activity (i.e.catalysis of the initial reaction rate v)₀) And further carrying out correlation analysis on the reaction conditions, the selenium content and the catalytic activity to optimize the preparation process of the Se-starch.

As can be seen from fig. 6A, the selenium content and the catalytic activity were steadily increased from 1 hour to 6 hours; the selenium content and the catalytic activity cannot be improved by prolonging the reaction time after 6 hours, so that the optimized reaction time is selected to be 6 hours.

As can be seen from fig. 6B, when the reaction temperature is below 60 ℃, the selenium content does not change much and the catalytic activity decreases continuously, and both the selenium content and the catalytic activity decrease significantly at 70 ℃. The optimized reaction temperature was chosen to be 30 ℃ since increasing the reaction temperature leads to a continuous decrease in catalytic activity.

As can be seen from fig. 6C, the molar ratio of NaSeH to double bonds increased from 1.0 to 2.2, the selenium content increased significantly and the catalytic activity decreased insignificantly, and the optimized molar ratio of NaSeH to double bonds was selected to be 1.5, considering that the selenium content of Se-starch was high at a molar ratio of 1.5 and that unreacted NaSeH did not remain easily during the post-treatment.

As shown in fig. 6D, when the volume ratio of ethanol is increased from 30% to 90%, the selenium content and the catalytic activity generally show a wave-valley-shaped trend, and when the volume ratio of ethanol is 80%, the catalytic activity is the maximum and the selenium content of the selenium-rich waxy starch is higher, so that the volume ratio of ethanol selected and optimized is 80%. In conclusion, the optimized preparation process conditions are as follows: the selenization reaction time is 6 hours, the reaction temperature is 30 ℃, the molar ratio of NaSeH to double bonds is 1.5, the volume proportion of ethanol is 80 percent, and the Se-starch prepared under the condition has the highest catalytic activity of 3.64 mu M.min^-1The selenium-rich waxy starch is expressed by Se-starch80, and the influence mechanism of each reaction factor on the selenium content and the catalytic activity is analyzed later.

Slight structural changes of the natural enzyme GPx can cause larger changes of catalytic activity, and the catalytic activity of the bionic GPx is also greatly influenced by the structure of the framework material. Therefore, a thermogravimetric analyzer is adopted to determine the thermal decomposition initial temperature (Ti) of Se-starch prepared under different modification conditions, so that the change rule of the surface structure of the starch is analyzed, and a basis is provided for further correlation analysis of the relationship between the starch structure and the catalytic activity.

FIG. 7 is a bar graph of the thermal decomposition onset temperature Ti for 7 selenium-enriched waxy starch samples (a-h), where: a (OSA starch) 261.4 ℃; b. c, d, e (Se-starch prepared by 80%, 30%, 60%, 90% ethanol volume) are 268.9 deg.C, 278.4 deg.C, 264.0 deg.C, 266.7 deg.C; f (Se-starch prepared with a molar ratio of NaSeH to double bonds of 2.2) 277.4 ℃; g (Se-starch prepared at a reaction temperature of 70 ℃) is 263.4 ℃; h (Se-starch prepared with a reaction time of 1 hour) is 277.4 ℃.

It can be seen that the Ti of Se-starch samples (b-h) prepared by changing different conditions is higher than that of OSA starch (a), which shows that the influence rule of selenization modification on the series Se-starch structure is consistent. During the thermal decomposition, heat is transferred from outside to inside to the inside of the starch granule, and the initial temperature of the thermal decomposition is usually that water molecules associated with the surface of the starch granule through hydrogen bonding are firstly dissociated from the starch structure. Therefore, the Ti values for b-h are all higher, indicating that the corresponding water molecules associated with the starch surface structure have been partially dissociated during the reaction, which is consistent with the SEM characterization observing that the starch surface structure exhibits efflorescence-like phenomena with folds and grooves and the FT-IR characterization resulting in a reduction of water molecules associated with hydrogen bonding. Based on the analysis, it can be reasonably presumed that the gelatinization in the selenization modification process causes the surface structure of the Se-starch to be greatly damaged, and bound water outside the starch is dissociated in the reaction process, so that less crystal water can be dissociated on the surface of the starch at a lower temperature in the thermal decomposition process, and a higher temperature is required to transfer effective heat to the interior of the starch, so that associated crystal water in the Se-starch is dissociated. This speculation can be confirmed by the variation in Ti in fig. 7: when the reaction system is not well dispersed (d), the polarity of the reaction system is relatively smaller (e) or the reaction time is shorter (h), the Se-starch surface gelatinization effect is weaker, and the increase of Ti is relatively smaller (2-5 ℃); when the polarity of a reaction system is relatively larger (c), the molar ratio of NaSeH to double bonds is higher (f) or the reaction temperature is higher (g), the surface gelatinization effect of Se-starch is stronger, the increase of Ti is relatively larger (16-27 ℃), and the change rule of Ti can provide a basis for researching the catalytic mechanism of Se-starch later.

2.3 catalytic Activity and catalytic mechanism of Se-starch80

The direct measurement method of the catalytic activity is based on an improved measurement method reported by professor Hilvert, and can simply and effectively evaluate the anti-oxidation catalytic activity of Se-starch. Se-starch80 with the highest catalytic activity is selected as an example in the research to carry out the research on catalytic behavior and catalytic mechanism. First, the catalytic activity of Se-starch80 was measured using thiophenol substrates and peroxide substrates as the double substrates (see FIG. 8 for the catalytic reaction). The method adopts thiophenol substrate as 3-carboxyl-4-nitrothiophenol (TNB) or 4-Nitrothiophenol (NBT); the peroxide substrate is cumene hydroperoxide (CUOOH) or hydrogen peroxide (H)₂O₂). Wherein TNB has one more carboxyl group than NBT; CUOOH ratio H₂O₂One p-cumyl is added, the different substrates have different structures, so that the GPx catalytic activity presents obvious difference, and the specific catalytic activity is shown in table 1. It is noteworthy that Se-starch80 is 1.53X 10 of classical small molecule biomimetic GPx (PhSeSePh) under the same assay conditions⁵Doubling; compared with the reported micelle bionic GPx (micelle catalyst) with high substrate recognition capability and catalytic activity, the highest catalytic activity of Se-starch80 (3.64 mM. min.)^-1) The highest catalytic activity (2.25 mM. min.) compared with that of Micellar Catalyst^-1) The improvement of 61.8 percent and better catalytic activity make the industrialized application of Se-starch80 more advantageous.

TABLE 1 catalysis Rate (v) of Se-starch80 catalysis of reduction of peroxides (ROOH, 250 μ M) by thiophenol (ArSH, 150 μ M)₀)

a measurement of. + -. SD in 50mM PBS at pH 7.0, 3 replicates per sample, calculated to evaluate catalytic ability.

In addition, as shown in FIG. 9, the concentration of thiophenol substrate was fixed at 0.15mM, and the concentrations of peroxide were changed at 0.05, 0.10, 0.25, 0.5, 1.0, 2.5 and 5.0mM, respectively, and activity of Se-starch80 was measured and kinetic parameters of the catalytic reaction were calculated (see Table 2). When two thiophenol substrates and two peroxide substrates are combined for catalytic activity determination, Se-starch80 can show typical saturated kinetic catalytic behavior similar to natural GPx, which shows that the idea of establishing a new strategy for directly endowing GPx catalytic activity to selenium-rich starch without biotransformation in the research is successfully realized.

Molecular structure of bound substrate (see scheme 3), catalytic activity (Table 1) and maximum reaction Rate (K)_cat) Mie constant (K)_m) Isoenzymology parameters (Table 2) analysis, when using CUOOH + NBT two hydrophobic more substrate combination for catalytic activity determination, Se-starch80 catalytic activity and K_catThe results are the maximum, which shows that hydrophobic microenvironment can be formed in the Se-starch80 starch skeleton surface structure after selenylation modification, and the specific binding can be performed on hydrophobic substrates, so that the catalytic rate is improved. While changing the Michaelis constant K of Se-starch80 catalytic reaction when the catalytic activity of thiophenol substrates is determined by using CUOOH + TNB combination_mThe minimum, however, indicates that the substrate combination of CUOOH + TNB is the best substrate for Se-starch 80. When Se-starch80 surface structure is damaged and more associated water molecules are released after selenylation modification, and the freedom degree of starch molecule chain segments is increased, the capability of forming hydrogen bond combination on the starch surface is stronger, because TNB has one more carboxyl group compared with NBT, Se-starch80 may form too strong combination effect on TNB substrate through stronger hydrogen bond effect, so that the TNB substrate is difficult to dissociate after the catalytic reaction is completed, and other substrates complete effective catalytic process, which may lead to strong substrate combination capability (K) of Se-starch80 on CUOOH + TNB (K-B)_mLess) and less catalytic activity. Therefore, in the Se-starch80 catalytic process, a hydrophobic microenvironment contributes to improving the catalytic activity, and another catalytic factor substrate recognition site can play a role in inhibiting the catalytic activity due to the influence of substrate dissociation through hydrogen bond action. In order to further clarify the influence mechanism of the hydrophobic microenvironment and the substrate recognition site on the Se-starch80 catalytic behavior, a multivariate enzyme activity test method and a pyrene fluorescent probe method are utilized for research.

TABLE 2 saturation kinetic constants of Se-starch80 catalytic reaction

The multielement enzyme activity test method mainly combines substrates with different structures and measures the catalytic activity, and indirectly researches the influence mechanism of hydrophobic microenvironment and substrate recognition sites on the catalytic activity through the activity difference. To more intuitively present the change in catalytic activity, CUOOH + tnb (a) is plotted in fig. 10; h₂O₂+TNB(B)；CUOOH+NBT(C)；H₂O₂+ nbt (d) histogram of catalytic activity for four combinations.

As can be seen from FIG. 10, Se-starch80 exhibited higher catalytic activity when activity assay was performed using more hydrophobic substrate, e.g., catalytic activity A when peroxide substrate was immobilized as CUOOH<C (fixed as H)₂O₂When B is<D) In that respect In the A and C two groups of catalytic activity measuring systems, only substrate molecules NBT and TNB have different structures, and NBT has one less carboxyl group compared with TNB, so that the difference of hydrophilic and hydrophobic effects and hydrogen bonding effects of Se-starch80 and the substrate can be caused, and the difference of catalytic activity is further caused. The strong capability of Se-starch80 to form hydrogen bonds with TNB with one more carboxyl group leads the contribution of a substrate recognition site to the substrate binding capability in the catalysis process to be larger than that of a hydrophobic microenvironment, but when the action of the substrate recognition site is too strong, TNB is taken as a substrate and shows lower catalytic activity (A)<C or B<D) This may be due to the poor dissociation capability of TNB after catalysis is completed due to the strong hydrogen bonding. Furthermore, when the thiophenol substrate is TNB, the peroxidation substrate CUOOH is more than H₂O₂One more p-cumyl group, stronger hydrophobicity and higher catalytic activity (A)>B) In that respect When a more hydrophobic substrate combination (CUOOH + NBT) is used, the higher catalytic activity is shown, which indicates that the hydrophobic microenvironment is more important than the substrate recognition site for maintaining the high catalytic activity of Se-starch80, and this is consistent with the conclusion that the substrate recognition site affects the substrate dissociation through hydrogen bonding and thus inhibits the catalytic activity. To more intuitively demonstrate the formation of a hydrophobic microenvironment in the Se-starch80 starch backbone,and (5) performing characterization by using a pyrene molecular fluorescence probe method.

The research shows that: when combined with different materials, the pyrene molecule has a peak intensity I of about 370nm₁And peak intensity I around 384nm₃Variation of the ratio, when pyrene molecules are combined with more hydrophobic materials or dispersions I₁/I₃And smaller, the characteristic has been successfully used for researching the formation mechanism of a hydrophobic microenvironment in the bionic GPx skeleton. By referring to the earlier stage research method, the concentration of the pyrene probe molecule is determined to be 10^-6Fluorescence spectra of OSA Starch + pyrene molecule (FIG. 11A a), pyrene molecule (FIG. 11A b) and Se-Starch80 + pyrene molecule (FIG. 11A c) at mol/L, and I of a three-group pyrene molecule system of a, b and c was calculated₁/I₃1.75, 1.69 and 1.66 respectively. Comparison of the I of pyrene molecule in the presence of Se-starch80₁/I₃At the minimum, the Se-starch80 can provide a hydrophobic microenvironment beneficial to the combination of pyrene molecules, and has a positive effect on improving the substrate combination capacity and the catalytic activity of the Se-starch 80. The formation of the hydrophobic micro-environment in the Se-starch80 is probably formed by reaggregation of octenyl succinate groups and starch molecule chain segments in a certain micro-area due to the fact that the free mobility of the starch molecule chain segments is increased after the surface structure of the starch is damaged.

In conclusion, in the Se-starch80 catalytic process, a hydrophobic microenvironment and a substrate recognition site can be formed, which plays an important role in improving the substrate binding capacity, but the hydrophobic microenvironment is more important than the substrate recognition site in maintaining the high catalytic activity of the Se-starch80, and the conclusion also provides theoretical support for the following explanation of the influence mechanism of the preparation process on the catalytic activity.

2.4 mechanism of influence of preparation Process on catalytic Activity

As can be seen from FIG. 6, for increasing the selenium content of Se-starch, the molar ratio (C) of NaSeH to double bonds and the volume ratio (D) of ethanol have significant effects; the two factors of the reaction temperature (B) and the volume ratio (D) of ethanol have obvious influence on improving the catalytic activity of Se-starch. The influence mechanism of each factor of the preparation process on the Se-starch80 catalytic activity is analyzed by combining the influence mechanism of the hydrophobic microenvironment and the substrate recognition site on the Se-starch80 catalytic activity.

Firstly, when the reaction time and process conditions are optimized, the reaction temperature is 40 ℃, the molar ratio of NaSeH to double bonds is 1:1, and the volume ratio of ethanol is 30%, and the catalytic activity and selenium content change of the series Se-starch prepared by changing the reaction time are shown in figure 6A. From 1 hour to 6 hours, Se-starch has a steady increase in both selenium content and catalytic activity, with a greater proportion of catalytic activity, about 36.69%, compared to selenium content.

Further, it is understood from FIG. 7 that the Ti contents of OSA starch and Se-starch prepared by 1 hour and 6 hours (other conditions are optimum process conditions) are 261.4 ℃, 263.4 ℃ and 268.9 ℃, respectively, and that the influence of gelatinization on the surface structure of Se-starch increases with time. According to the research of a catalytic mechanism of Se-starch80, a hydrophobic microenvironment formed in a starch skeleton has a large influence on catalytic activity. In conclusion, the Se-starch prepared in the reaction time of 6 hours has longer gelatinization, can increase the degree of freedom of the movement of molecular chain segments on the surface of starch, is favorable for forming a hydrophobic microenvironment, and further improves the catalytic rate. Therefore, the optimal reaction time of the selenylation modification is determined to be 6 hours through the process optimization of the reaction time, and the moderate gelatinization of the starch surface under the condition is favorable for improving the catalytic activity of Se-starch.

Secondly, when the reaction temperature and process conditions are optimized, the reaction time is 6 hours, the molar ratio of NaSeH to double bonds is 1:1, the volume ratio of ethanol is 30%, and the catalytic activity and selenium content change of the series Se-starch prepared by changing the reaction temperature are shown in figure 6B. As shown in fig. 6B, when the reaction temperature is below 60 ℃, the selenium content is not greatly changed and the catalytic activity is continuously reduced; both the selenium content and the catalytic activity are significantly reduced at 70 ℃ and are mainly related to the reverse reaction (elimination) of the addition reaction at 70 ℃. For the catalytic activity, increasing the reaction temperature leads to the continuous decrease of the catalytic activity, and from fig. 7, it can be seen that the Ti of the OSA-starch prepared by OSA-starch and the reaction temperature of 30 ℃ and 70 ℃ (other conditions are the optimal process conditions) are 261.4 ℃, 268.9 ℃ and 288.9 ℃. Unlike the previous samples prepared at 6 hours of reaction where the Ti was increased by 7.49 ℃ over the 1 hour samples, the samples prepared at 70 ℃ had a Ti increase of 27.48 ℃ over the 30 ℃ samples, indicating that the gelatinization effect due to the reaction temperature was more influential than the reaction time. The higher temperature condition of 70 ℃ can generate stronger gelatinization, so that more associated crystal water is released on the surface of the starch, the freedom degree of molecular chain segments is increased, and the change can have two effects on the Se-starch prepared: on one hand, a substrate recognition site with stronger hydrogen bonding capacity is formed, and the improvement of catalytic activity is possibly inhibited; on the other hand, the degree of freedom of movement of a starch molecule chain segment is higher, the molecule chain segment is probably wound, the steric effect is generated on the combination of the substrate, the embedding phenomenon of the chain segment on the catalytic center is brought, the combination of the substrate and the catalytic center is prevented, the improvement of the catalytic activity is inhibited, and similar phenomena are also reported in the early reported temperature response bionic GPx work. Therefore, the optimal reaction temperature is determined to be 30 ℃ based on the process optimization of the reaction temperature, and the comprehensive analysis of the catalytic activity shows that the degree of freedom of the starch molecule chain segment in the starch skeleton is increased at a higher reaction temperature, so that the hydrogen bond function of the substrate recognition site is stronger, the steric hindrance function is increased, the embedding of the catalytic center is realized, and the inhibition effect on the improvement of the catalytic activity is realized.

Thirdly, when the process conditions of the molar ratio of the NaSeH to the double bonds are optimized, the reaction time is 6 hours, the reaction temperature is 30 ℃, the volume ratio of ethanol is 30%, and the catalytic activity and the selenium content of the series Se-starch prepared by changing the molar ratio of the NaSeH to the double bonds are shown in figure 6C. Since the catalytic activity of Se-starch under different process conditions does not change greatly, the optimization of the molar ratio process from the perspective of the catalytic activity is not considered. When the molar ratio is higher than 1.5:1, the selenium content of the selenium-enriched waxy starch is higher, but the phenomenon that excessive unreacted NaSeH is difficult to effectively remove can occur when the molar ratio is continuously increased. Considering that the potential application of Se-starch is selenium-enriched functional food or food additive, the higher selenium content and the less NaSeH residue are more beneficial to industrial development and application, the molar ratio of the NaSeH and the double bonds in the selenylation modification reaction is selected to be 1.5: 1.

Finally, when the technological conditions of the ethanol volume ratio are optimized, the reaction time is 6 hours, the reaction temperature is 30 ℃, the molar ratio of NaSeH to double bonds is 1.5:1, and the catalytic activity and selenium content change of the series Se-starch prepared by changing the ethanol volume ratio are shown in figure 6D. As shown in FIG. 6D, the volume ratio of ethanol influences the selenium-rich starchThe most significant factor of the selenium content of the flour sample, the increase of the ethanol volume proportion from 30% to 90%, the selenium content and the catalytic activity showed a wave-valley-like trend overall. Fig. 6E shows a dispersion state of the OSA starch raw material in reaction systems with different ethanol ratios, when the ethanol is less than 30%, the amphiphilicity of the OSA starch makes the OSA starch to exhibit the characteristics of an emulsifier, a solid product is difficult to obtain by ethanol precipitation separation after selenylation modification reaction, gelatinization damage of the starch surface structure is very serious under the condition, catalytic factors such as hydrophobic microenvironment and the like are difficult to form, and an effective antioxidant catalysis process cannot be completed, so that the selenium-enriched waxy starch is prepared without selecting ethanol with a volume less than 30%. When the volume of the ethanol is 50-70%, the phenomenon that a starch sample adheres to a reactor and the starch sample is aggregated into lumps and cannot be effectively dispersed occurs in the OSA starch during reaction, particularly the starch adhesion phenomenon is the most serious at 60% and 70%, the reaction efficiency of the OSA starch and NaSeH is very low under the condition, and the selenium content of the prepared Se-starch is greatly reduced. When the volume proportion of ethanol is higher than 70%, OSA starch is easy to disperse in a reaction system although not dissolved, the efficiency of heterogeneous reaction is higher, and the Se-starch selenium content prepared under the condition is gradually increased. The general trend of the Se-starch is consistent with the change of the selenium content, and the ethanol volume proportion is analyzed in two stages of 30-60% and 70-90%. First, when the ethanol volume ratio is analyzed to be 30% to 60%, it is understood from fig. 7 that Ti for preparing Se-starch is 261.4 ℃, 278.4 ℃ and 264.0 ℃ respectively when the OSA starch and the reaction system ethanol volume ratio is 30% and 60%. When the ethanol volume proportion is increased from 30 percent to 60 percent, the selenium content and the catalytic activity are rapidly reduced. It is noteworthy that the Ti of the prepared samples was lowest in the Se-starch series at 60% ethanol volume, and the selenium content and catalytic activity of the prepared samples were lowest at this point, probably due to the starch structure undergoing the weakest gelatinization in this condition, which is caused by the non-uniform dispersion of OSA starch in the system, sticking to the reactor, and low reaction efficiency. As the hydrophobic microenvironment is more important to maintain the high-efficiency antioxidant activity of Se-starch80 than the substrate recognition site, the hydrophobic microenvironment of Se-starch (30-50 percent of ethanol volume ratio) is directly shown by the pyrene fluorescent probe moleculesAnd (5) carrying out characterization. As shown in FIG. 11B, I₁/I₃The ratio of (A) to (B) increases with the proportion of ethanol, indicating that the hydrophobic microenvironment gradually weakens, which is also the key reason for the tendency of the catalytic activity to decrease. And when the volume proportion of ethanol is 60 percent, I₁/I₃The decrease compared to 30-50% may be related to uneven starch dispersion and low reaction efficiency under the reaction conditions, where the structural properties of Se-starch are more similar to the unreacted OSA starch and the catalytic activity is minimal. Secondly, the stage of re-analyzing that the volume proportion of ethanol is 70-90%, because Se-starch80 prepared when the volume proportion of ethanol is 80% has the best antioxidant activity, the hydrophobic microenvironment and the substrate recognition site in the starch skeleton have the best substrate binding capacity at the moment. Preparation of I of starch when the ethanol volume fraction varies from 70% to 90%₁/I₃The sustained decrease (fig. 11B), illustrating a sustained increase in the hydrophobic microenvironment in the starch backbone. It is noteworthy that the hydrophobic microenvironment is strongest when the ethanol volume fraction is 90% (I)₁/I₃The lowest ratio) but the catalytic activity was low by 80%, which is probably due to the catalytic center being embedded to some extent under this condition. As can be seen from fig. 6E, when the ethanol system contains 90% and 100% of suspended fine particles in the solution, it may be that when the ethanol content in the system is more, the polarity of the solvent system is smaller, the dissolving capacity for starch is deteriorated, the starch molecule chain segment is more easily folded, and is easily re-aggregated to reduce the surface polarity, and the acting force between the fine starch particles is weak, thereby causing the suspension phenomenon of more particles. At this time, the folding of the starch molecule chain segment may cause the embedding phenomenon of partial catalytic center, so that the catalytic center is difficult to effectively combine with the substrate to complete the catalytic process, and the catalytic activity is reduced. Therefore, the strong hydrophobic microenvironment is not the only factor for maintaining the high catalytic activity of Se-starch, and if the catalytic center can not be effectively combined with the substrate to complete the catalytic process, the enhancement of the catalytic activity of Se-starch is not facilitated. Therefore, the optimal ethanol volume proportion is optimally determined to be 80%, and comprehensive analysis on the catalytic activity shows that a hydrophobic microenvironment is an important factor for maintaining the high catalytic activity of Se-starch, and the effective matching of a catalytic center and the hydrophobic microenvironment is also a key factor.

In conclusion, the selenylation modification reaction process can enable the surface of the starch to be properly gelatinized, a hydrophobic microenvironment and a substrate recognition site which are beneficial to substrate combination are formed, and the combination capability of Se-starch to the substrate is improved; the hydrophobic microenvironment is more important for improving the catalytic activity of Se-starch than the substrate recognition site, mainly has the inhibition effect on the catalytic activity due to the over-strong hydrogen bond effect and steric hindrance increase of the substrate recognition site and the embedding effect on the catalytic center, and the effective matching of the catalytic center and the hydrophobic microenvironment is a key factor for maintaining the high catalytic activity of Se-starch.

3 conclusion

The invention creatively constructs a natural GPx catalytic center in a waxy starch skeleton by utilizing the nucleophilic addition reaction of double bond groups in NaSeH and OSA starch, and establishes a new strategy for directly endowing GPx catalytic activity to selenium-enriched starch without biotransformation.

The research shows that: selenium in the selenium-rich waxy starch exists mainly in a negative divalent form and shows a typical saturation kinetic catalytic behavior, and the catalytic activity of Se-starch80 is 1.53 multiplied by 10 of PhSeSePh⁵The Catalyst is 61.78% higher than the highest catalytic activity of Micellar Catalyst. The research shows that: the strong basicity of NaSeH and the alkaline condition of a reaction system cause the surface of starch to present more efflorescent gelatinization action structures with folds and grooves, which is favorable for forming a hydrophobic microenvironment and a substrate recognition site; the hydrophobic microenvironment is more important for improving the catalytic activity of Se-starch than the recognition site, and the over-strong hydrogen bond effect and steric hindrance effect of the substrate recognition site are increased, and the embedding effect on the catalytic center has an inhibiting effect on the catalytic activity. The novel method for directly endowing the selenium-rich waxy starch GPx with catalytic activity without biotransformation, which is established by the work, can provide a theoretical basis for the development of novel selenium-rich additives and antioxidant drugs.

The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.

Claims

1. The preparation method of the selenium-rich starch is characterized by comprising the following steps:

(2) placing OSA starch10 g of starch octenyl succinate in a 250mL round-bottom flask, adding 100mL of mixed solvent of ethanol and water into the flask, and stirring for 30min to uniformly mix the system;

(3) slowly dropwise adding the NaSeH stock solution and 0.1mol/L sodium hydroxide solution into a reaction system under the ice bath condition, and stopping the reaction after reacting for 1-8 hours;

(4) adjusting the pH of the reaction solution to 3-4, dropwise adding the solution into a beaker filled with a large amount of ethanol solvent to precipitate starch, stirring for 20 minutes, filtering and collecting a filter cake, sequentially washing the filter cake with 75% ethanol and absolute ethanol, and drying the washed filter cake in a vacuum drying oven at 50 ℃ for 24 hours to obtain a selenylation starch sample;

in the step (2), the volume ratio of ethanol to water in the mixed solvent is 9:1, 8:2, 7:3, 6:4, 5:5, 4:6 or 3: 7;

the ratio of the NaSeH to the double bond in the NaSeH stock solution in the step (2) is 1:1, 1.2:1, 1.5:1, 1.8:1 or 2.2: 1.

2. The method for preparing the selenium-enriched starch according to claim 1, wherein the pH value of the reaction system is controlled to be 8-10 in the step (2).

3. The method for preparing the selenium-enriched starch according to claim 1, wherein the temperature of the reaction system in the step (2) is controlled to be 30-70 ℃.