CN108409872B - Preparation method of octenyl succinic anhydride modified starch ester by double-enzyme enzymatic hydrolysis - Google Patents
Preparation method of octenyl succinic anhydride modified starch ester by double-enzyme enzymatic hydrolysis Download PDFInfo
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Abstract
The invention discloses a preparation method of octenyl succinic anhydride modified starch ester by double-enzyme enzymatic hydrolysis. The preparation method comprises the steps of preparing octenyl succinic anhydride starch ester; amylase enzymatic hydrolysis: adjusting the pH value to be acidic, dissolving amylase by using a buffer solution, adding the dissolved amylase into the octenyl succinic acid starch ester, and reacting under stirring. Compared with the traditional method, the surface tension, the viscosity and the molecular weight of the prepared octenyl succinic acid modified starch ester are changed, compared with the product prepared by the traditional process, the octenyl succinic acid starch ester produced by using the enzymatic hydrolysis of alpha-beta double amylase has better emulsification stability, the research has high theoretical research value and practical application significance, the starch is endowed with new performance and functionality, the starch application field is further widened, and a new way is provided for improving the starch application performance and the emulsifier production process of starch derivatives.
Description
Technical Field
The invention belongs to the technical field of macromolecular emulsifier preparation, and particularly relates to a preparation method of octenyl succinic anhydride modified starch ester by double-enzyme enzymatic hydrolysis.
Background
Some natural biological macromolecules, such as gum arabic, guar gum, casein, whey protein, soy protein, various water-soluble polymers, etc., are commonly used as emulsion stabilizers in food, pharmaceutical, cosmetic and other industrial systems. The oil is one of three nutrients for human body, is a good heat energy nutrient, and has important physiological function in human body. Generally speaking, gum arabic and guar gum are expensive, the emulsifying performance is not ideal, protein emulsifying agents have good emulsifying effect, but have isoelectric point effect and strict requirement on system pH, so that the industrial preparation of a natural polymer emulsifying agent which is low in price, wide in raw materials and free of requirement on system pH becomes a research hotspot.
Because starch has the characteristics of wide source, low price, reproducibility, safety and no toxicity, octenyl succinic anhydride has amphiphilic property because of having a hydrophobic 8-carbon long-chain alkyl group and a hydrophilic dicarboxylic acid structure, carboxyl of the anhydride can also generate esterification reaction with hydroxyl of starch in a weak alkaline environment, starch octenyl succinate (hereinafter referred to as OSA) is directly applied to an emulsion system as an emulsion stabilizer in the traditional process, and the emulsion stability is unsatisfactory. The stability of both oil-in-water and oil-in-water emulsions is affected by electrostatic repulsion, surface tension, fluid movement due to density differences, and forces of Brownian motion memory osmotic pressure due to its deflocculation, which in turn are affected by the degree of substitution and molecular weight of the starch.
Disclosure of Invention
This section is for the purpose of summarizing some aspects of embodiments of the invention and to briefly introduce some preferred embodiments. In this section, as well as in the abstract and the title of the invention of this application, simplifications or omissions may be made to avoid obscuring the purpose of the section, the abstract and the title, and such simplifications or omissions are not intended to limit the scope of the invention.
The present invention has been made in view of the above-mentioned technical drawbacks.
Therefore, in one aspect of the invention, the invention overcomes the defects in the prior art and provides a preparation method for performing double-enzyme enzymatic hydrolysis on octenyl succinic anhydride modified starch ester.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of octenyl succinic anhydride modified starch ester by double-enzyme enzymatic hydrolysis comprises the following steps,
preparation of starch octenyl succinate anhydride ester: adjusting the pH of natural starch milk to be alkaline, stirring the starch milk, dropwise adding octenyl succinic anhydride into the starch milk to perform octenyl succinic esterification reaction, and then adjusting the pH to be acidic to obtain octenyl succinic starch ester after the octenyl succinic esterification reaction;
amylase enzymatic hydrolysis: adjusting the pH value to be acidic, dissolving amylase by using a buffer solution, adding the dissolved amylase into the octenyl succinic acid starch ester, and reacting under stirring.
As a preferable scheme of the preparation method for the double-enzyme enzymatic hydrolysis of the octenyl succinic anhydride modified starch ester, the method comprises the following steps: in the natural starch milk, the mass concentration of the starch is 10-40%; adjusting the pH value to be alkaline, wherein the pH value is 8-10; stirring at the speed of 2000 r/min; dropwise adding octenyl succinic anhydride into the starch milk, wherein the concentration of the octenyl succinic anhydride is 5-11%; then, the pH is adjusted to be acidic, and the pH is adjusted to be 6.5; and performing octenyl succinate esterification reaction for 1-24 h at 30-60 ℃.
As a preferable scheme of the preparation method for the double-enzyme enzymatic hydrolysis of the octenyl succinic anhydride modified starch ester, the method comprises the following steps: in the natural starch milk, the mass concentration of the starch is 30%, the pH is adjusted to be alkaline, and the pH value is 9; stirring at the speed of 2000 r/min; dropwise adding octenyl succinic anhydride into the starch milk, wherein the mass concentration of the octenyl succinic anhydride is 7%, and diluting the octenyl succinic anhydride into a 95% ethanol solution to be dropwise added into the starch milk; then, the pH is adjusted to be acidic, and the pH is adjusted to be 6.5; the octenyl succinate esterification reaction is carried out, the reaction time is 4h, and the reaction temperature is 40 ℃.
As a preferable scheme of the preparation method for the double-enzyme enzymatic hydrolysis of the octenyl succinic anhydride modified starch ester, the method comprises the following steps: the amylase is used for enzymatic hydrolysis, and comprises the following steps: alpha amylase and beta amylase double enzyme enzymatic hydrolysis.
As a preferable scheme of the preparation method for the double-enzyme enzymatic hydrolysis of the octenyl succinic anhydride modified starch ester, the method comprises the following steps: the amylase is used for enzymatic hydrolysis, wherein the pH is adjusted to be acidic, the pH is adjusted to be 5.0, and the buffer solution is acetic acid-sodium acetate buffer solution.
As a preferable scheme of the preparation method for the double-enzyme enzymatic hydrolysis of the octenyl succinic anhydride modified starch ester, the method comprises the following steps: the alpha amylase and beta amylase double-enzyme enzymatic hydrolysis is carried out by firstly hydrolyzing with alpha amylase and then hydrolyzing with beta amylase, wherein the enzyme activity of the alpha amylase is 0.7-20U/g, and the reaction time of the alpha amylase hydrolysis is 0.5-1 h; the enzyme activity of the beta amylase is 2-13U/g, the reaction time of hydrolysis of the beta amylase is 0.5-3 h, enzymatic hydrolysis of the alpha amylase and the beta amylase is carried out, and the pH value of a hydrolysis system is controlled to be 5.0.
As a preferable scheme of the preparation method for the double-enzyme enzymatic hydrolysis of the octenyl succinic anhydride modified starch ester, the method comprises the following steps: the enzyme activity of the alpha amylase is 6.6U/g, the reaction time of alpha amylase hydrolysis is 0.5h, the enzyme activity of the beta amylase is 5U/g, and the reaction time of beta amylase hydrolysis is 1 h.
As a preferable scheme of the preparation method for the double-enzyme enzymatic hydrolysis of the octenyl succinic anhydride modified starch ester, the method comprises the following steps: the natural starch comprises one or more of corn starch, wheat starch, long-shaped rice starch, barley starch, waxy corn starch, potato starch, cassava starch, glutinous rice starch, bean starch, lotus root starch, water chestnut starch, lotus seed starch, banana starch, sweet potato starch or water chestnut starch.
As another aspect of the invention, the invention provides the use of octenyl succinic anhydride modified starch ester as an emulsifier.
The invention has the beneficial effects that: the starch raw material of the invention is natural and renewable, has a plurality of hydrophilic hydroxyls on the molecule, can be esterified with fatty acid, and can be used as a raw material for preparing the surfactant. The alpha amylase enzymatic hydrolysis octenyl succinic anhydride modified starch ester can obviously reduce the molecular weight of the product, thereby reducing the viscosity of the system, providing possibility for increasing the addition of an emulsifier on the premise of keeping the viscosity of the system not to be obviously changed, and increasing the emulsification stability. The beta amylase of the invention can hydrolyze the octenyl succinic anhydride enzymatically, can hydrolyze alpha-14 glycosidic bonds from the non-reducing end of starch so as to cut off disaccharides one by one, has resistance to octenyl succinic anhydride groups, stops reaction once the glucose units grafted with the octenyl succinic anhydride groups are hydrolyzed, ensures that the octenyl succinic anhydride groups are exposed at the tail end of a starch chain as much as possible, ensures that the octenyl succinic anhydride groups are fully contacted with an oil-water interface, and enhances the emulsifying capacity.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without inventive exercise. Wherein:
FIG. 1 is a graph showing the relationship between the concentration of starch and the degree and efficiency of substitution in example 1.
FIG. 2 is a graph showing the relationship between the reaction time and the degree and efficiency of substitution in example 1.
FIG. 3 is a graph showing the relationship between the reaction temperature and the degree and efficiency of substitution in example 1.
FIG. 4 is a graph showing the relationship between the reaction pH and the degree and efficiency of substitution in example 1.
FIG. 5 shows the emulsifying rate of the system after the alpha-amylase with different enzyme activities hydrolyzes octenyl succinic anhydride starch ester and is used as an emulsifier to emulsify medium-chain fatty acid-water.
FIG. 6 shows the emulsifying rate of the system after the beta-amylase with different enzyme activities hydrolyzes octenyl succinic anhydride starch ester and is used as an emulsifier to emulsify medium-chain fatty acid water.
FIG. 7 shows the storage stability of medium-chain fatty acid-water emulsified system using hydrolyzed starch ester as emulsifier after the starch ester of octenyl succinic anhydride is hydrolyzed by alpha, beta amylase with different enzyme activities.
FIG. 8 shows the effect of the synergistic effect of the enzymatic hydrolysis time of alpha-and beta-amylase on emulsion stability.
FIG. 9 is a graph comparing the rheological storage modulus of starch octenyl succinate prepared by different methods.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with examples are described in detail below.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Furthermore, reference herein to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one implementation of the invention. The appearances of the phrase "in one embodiment" in various places in the specification are not necessarily all referring to the same embodiment, nor are separate or alternative embodiments mutually exclusive of other embodiments.
Example 1: preparation of starch octenylsuccinate
Preparing starch milk with a certain concentration (w/v), putting the starch milk into a beaker, adjusting the pH value of the starch milk to a certain range, uniformly mixing, transferring the starch into a constant-temperature water bath at a certain temperature, and slowly and uniformly adding octenyl succinic anhydride diluted in a 95% ethanol solution into the starch milk within 0.5 hour at a stirring speed of 2000 r/min. The pH of the system is controlled to be in a certain range by using 2 percent NaOH solution. After continuing the reaction for a certain time, the pH of the system was adjusted to 6.5 with 2% HCl. And then transferring the starch milk to a Buchner funnel, performing suction filtration and washing twice by using 75% ethanol, performing centrifugal washing twice by using distilled water, dehydrating, drying overnight in a drying oven at 40 ℃, and finally grinding and sieving the dried esterified starch to obtain the finished product of the octenyl succinic acid starch ester.
Single factor test:
(1) effect of starch milk concentration
The experimental conditions for this influencing factor were: the addition amount of the OSA is 7% of the dry weight of the starch, the reaction time is 4h, the reaction temperature is 35 ℃, the pH value of a reaction system is 8.0-9.0, starch milk with the concentration of 10%, 15%, 20%, 25%, 30% and 40% is prepared respectively, and the substitution degree and the substitution efficiency of the obtained product are related to the concentration of the starch milk.
FIG. 1 is a single factor experiment on starch concentration during octenyl succinic acid modification, and it can be seen that esterification has the highest degree of substitution and reaction efficiency at a starch concentration of 30%.
(2) Influence of reaction time
The experimental conditions for this influencing factor were: the addition amount of the OSA is 7% of the dry weight of the starch, the concentration of the prepared starch milk is 35%, the reaction temperature is 35 ℃, the pH value of the reaction system is 8.0-9.0, and the reaction time is 1-24 hours respectively, so that the relationship between the substitution degree of the product and the reaction time is obtained.
FIG. 2 is a single-factor experiment regarding the reaction time during the modification of octenyl succinic acid, and it can be seen that the esterification has the highest degree of substitution and reaction efficiency when the reaction time is 4 hours.
(3) Influence of reaction temperature
The experimental conditions for this influencing factor were: the addition amount of the OSA is 7% of the dry weight of the starch, the concentration of the prepared starch milk is 10%, the reaction time is 3.5h, the pH of a reaction system is 8.0-9.0, and the relationship between the substitution degree of the product and the reaction temperature is obtained when the reaction temperature is respectively 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃ and 90 ℃.
FIG. 3 is a single-factor experiment regarding the reaction temperature during modification of octenyl succinic acid, and it can be seen that esterification has the highest degree of substitution and reaction efficiency when the reaction temperature is 40 ℃.
(4) Influence of the pH of the reaction System
The experimental conditions for this influencing factor were: the addition amount of the OSA is 7% of the dry weight of the starch, the concentration of the prepared starch milk is 30%, the reaction time is 4h, the reaction temperature is 30 ℃, the pH of the reaction system is respectively controlled at 8.0, 9.0, 10.0, 11.0 and 12.0, and the relationship between the substitution degree of the product and the pH of the reaction system is obtained.
Fig. 4 is a one-factor experiment on the pH of the system during modification of octenyl succinic acid, and it can be seen that esterification has the highest degree of substitution and reaction efficiency when the pH of the system is 9.
(5) Effect of Octenylsuccinic anhydride concentration
The concentration of the octenyl succinic anhydride prepared respectively is 5 wt%, 7 wt%, 9 wt% and 11 wt%, and experimental results show that the effect is optimal when the concentration of the octenyl succinic anhydride is 7 wt%, and the starch emulsification stability is optimal. The addition amount of octenyl succinic acid is most preferable to 7% because of the tendency that the addition amount of octenyl succinic acid reagent has a positive correlation with the degree of substitution of starch, and the reaction efficiency is lower as the addition amount of octenyl succinic acid is higher.
Example 2: determination of the degree of substitution of starch octenylsuccinate
The specific method comprises the following steps:
accurately weighing 1.5g-2g of octenyl succinic acid canna edulis ker starch ester by an electronic balance, dissolving in 50mL of 95% isopropanol solution, placing on a magnetic stirrer, stirring for 10min, adding 15mL of 2.0mol/L hydrochloric acid-isopropanol solution, and continuing to magnetically stir for 30 min. The solution was then transferred to a buchner funnel for dehydration and washed several times with 90% isopropanol solution until chloride free checked with 0.1mol/L silver nitrate. Then the solid sample is transferred into a 250mL conical flask, 100mL distilled water is added, 2 drops of phenolphthalein are added immediately after heating in a boiling water bath for 20min and shaking up, and the pink color is titrated with 0.1mol/L NaOH while the solution is hot. The formula for calculating the degree of substitution of the product is as follows:
in the formula: 0.1624: molar mass of glucose residues, g/mmol; 0.21: (ii) the molar mass of octenyl succinic anhydride, g/mmol; c: the molar concentration of NaOH standard solution, mol/L; v: titrating the volume of NaOH consumed by the sample, mL; w: mass of sample weighed, g.
Example 3: enzymatic hydrolysis of starch octenylsuccinate Using alpha Amylase alone
Firstly, the octenyl succinic anhydride starch ester prepared in the example 1 is prepared into 10 percent suspension by buffer solution, and the suspension is continuously stirred in water bath at 40 ℃.
② dissolving the alpha-amylase by acetic acid-sodium acetate buffer solution, 4000r/min, freezing and centrifuging for 30 min. Taking the supernatant for later use.
Thirdly, adding alpha-amylase with enzyme activity of 0.7, 3.5, 6.6 and 19.8U/g into the suspension of the octenyl succinate starch ester, and carrying out water bath constant temperature oscillation reaction for a certain time under the conditions of a certain reaction temperature and a pH value of 4.5.
Fourthly, adjusting the pH value of the reaction system to 2 and inactivating the enzyme.
FIG. 5 is a graph showing the emulsion rate after accelerated storage for 30 days after the preparation of an emulsion by hydrolyzing OSA starch with only alpha amylase, and it can be seen that the starch has the best emulsification properties when the alpha amylase concentration is 6.6U/g.
Example 4: enzymatic hydrolysis of starch octenylsuccinate Using beta Amylase alone
Firstly, the octenyl succinic anhydride starch ester prepared in the example 1 is prepared into 10 percent suspension by buffer solution, and the suspension is continuously stirred in water bath at 40 ℃.
② dissolving the beta-amylase by acetic acid-sodium acetate buffer solution, 4000r/min, freezing and centrifuging for 30 min. Taking the supernatant for later use.
Thirdly, adding enzyme activities with the enzyme activities of 2, 5, 10 and 13U/g into the suspension of the octenyl succinate starch ester, and carrying out water bath constant temperature oscillation reaction for a certain time under the conditions of a certain reaction temperature and a pH value of 5.0.
Adjusting the pH value of the reaction system to 2, inactivating enzyme, washing with water, centrifuging, and drying in an oven at 45 ℃ for later use.
FIG. 6 is a graph showing the emulsion rate after accelerated storage for 30 days after the preparation of an emulsion by hydrolyzing OSA starch with beta amylase alone, and it can be seen that the starch has the best emulsification properties when the beta amylase concentration is 5U/g.
Example 5: example 3 modified Octenylsuccinate starch ester emulsified Medium chain fatty acid (MCT) nanoemulsion experiment prepared solely by enzymatic hydrolysis with alpha-Amylase
The starch ester of octenyl succinic anhydride hydrolyzed by the enzyme activity of alpha amylase in the enzymolysis degree of 0.7, 3.5, 6.6 and 19.8U/g in the embodiment 3 is dissolved in a beaker at 40 ℃, 10 percent MCT grease is added, the mixture is emulsified and dispersed by a high-speed shearing dispersion machine at 10000r/min, and then the mixture is immediately homogenized by a high-pressure homogenizer with the homogenization pressure of 40MPa and the homogenization times of 2 times.
Example 6: experiment for measuring emulsification rate of modified starch octenyl succinate-MCT nanoemulsion prepared by enzymatic hydrolysis method of alpha-amylase only in example 3
And (3) accelerating and storing the MCT emulsion in an oven at 45 ℃, and after the emulsion is obviously layered, determining the emulsification rate of the sample according to the following formula:
wherein the total height of the sample is HE, the height of the clarifying layer at the bottom of the sample is HC, H is the emulsification rate, and the larger the emulsification rate is, the more stable the emulsion is.
The results of the experiment are shown in FIG. 5.
Example 7: example 4 emulsification of MCT nanoemulsion experiment Using modified starch octenyl succinate prepared by enzymatic hydrolysis with beta-Amylase alone
Dissolving octenyl succinic anhydride starch ester which is hydrolyzed by beta amylase with the enzyme activity of 2, 5, 10 and 13U/g respectively in the embodiment 4 at 40 ℃, adding 10 percent MCT grease, emulsifying and dispersing by a high-speed shearing dispersion machine at 10000r/min, immediately homogenizing by a high-pressure homogenizer with the homogenizing pressure of 40MPa and the homogenizing times of 2 times.
Example 8: example 4 determination of emulsification Rate of modified Octenylsuccinate starch ester-MCT nanoemulsion Using enzymatic hydrolysis with beta-Amylase alone
And (3) accelerating and storing the MCT emulsion in an oven at 45 ℃, and after the emulsion is obviously layered, determining the emulsification rate of the sample according to the following formula:
wherein the total height of the sample is HE, the height of the clarifying layer at the bottom of the sample is HC, H is the emulsification rate, and the larger the emulsification rate is, the more stable the emulsion is.
The results of the experiment are shown in FIG. 6.
Example 9: preparation of alpha-beta-diastase enzymatic hydrolysis octenyl succinic acid starch ester
According to the specific enzymolysis processes in the embodiment 6 and the embodiment 8, after starch ester is hydrolyzed by respectively selecting different alpha amylase enzyme activities, the enzymolysis is carried out by beta amylase with the enzyme activities of 2, 5, 10 and 13U/g respectively.
Example 10: effect of enzymolysis time synergy of alpha-beta double amylase on emulsion stability
The effect of the reaction time of the enzymatic hydrolysis of starch octenyl succinate by α - β -amylase on stability was investigated, and it can be seen from fig. 8 that the synergistic effect of the two enzymes has an accelerating effect on emulsion stability, wherein α -amylase achieves this acceleration by changing the molecular size of starch and β -amylase achieves this acceleration by changing the position of the hydrophobic group. Meanwhile, when the enzymolysis time of the alpha amylase exceeds 0.5 hour, the molecular size of the starch is reduced, which brings adverse effects on the stability of the emulsion, and when the enzymolysis time of the beta amylase exceeds 1 hour, although more hydrophobic groups may be exposed on the molecular surface, the particle size of the emulsion is further reduced, and the stability of the system is improved, because the enzymolysis time of the beta amylase is prolonged, the molecular weight of the starch is further reduced, and the molecular size of the starch is too small in cooperation with the reduction effect of the alpha amylase on the molecular weight of the starch, which is not easy to provide effective steric hindrance, so that the emulsification stability of the starch is reduced, thereby showing that when the enzymolysis time of the alpha amylase is 0.5 hour, and the enzymolysis time of the beta amylase is 1 hour, the prepared modified starch has the best emulsification property. The pH value of the system is adjusted to 5.0 in the reaction process.
Example 11: determination of storage stability of alpha-beta-double amylase enzymatic hydrolysis octenyl succinate starch ester-MCT nanoemulsion
Octenyl succinic anhydride starch esters obtained by enzymatic hydrolysis of different alpha amylases were used to emulsify MCT emulsions under the conditions described in examples 3 and 6, and the resulting emulsions were subjected to DLS techniques to determine the average particle size distribution of the emulsions over different storage times. The scattered light angle was fixed at 90 ℃ and the measurement temperature was 25. + -. 1 ℃. The light source used was a fixed light source, operating wavelength 658nm, power 30 mW.
The results of the experiment are shown in FIG. 7.
As can be seen from FIG. 7, when the alpha-amylase activity was 6.6U/g and the beta-amylase activity was 5U/g, the emulsification properties had a significant gain compared to the other examples. Both alpha-amylase and beta-amylase can cut alpha-1, 4 glycosidic bonds in starch, but alpha-amylase is endonuclease and can randomly cut alpha-1, 4 glycosidic bonds of starch in the starch, while beta-amylase is exonuclease and can only cut alpha-1, 4 glycosidic bonds one by one from a non-reducing end, while OSA group has a barrier effect on the cutting of the two enzymes, namely, when the two enzymes meet the group on the starch, the OSA group stops acting, and the starch has certain resistance. Meanwhile, due to the introduction of the hydrophobic OSA group, the amphiphilic property of the hydrophilic starch is endowed, so that an oil-water system can be well emulsified, and in addition, the mechanism for stabilizing the emulsion also comprises larger steric hindrance provided by a starch molecule and a part of electronegativity provided by the OSA group. On one hand, the optimal molecular size for providing steric hindrance can be obtained through the action of alpha amylase, and on the other hand, the intervention of the exo-beta amylase can enable more hydrophobic OSA groups to be exposed on the surface of the starch to a greater extent, so that the groups can be adsorbed to an oil-water interface more quickly, and a proper condition is provided for better emulsification.
FIG. 9 is a graph comparing the rheological storage modulus of starch octenyl succinate prepared by different methods.
The storage modulus of the starch can represent the elastic property of an emulsion interfacial film, and the strength of the emulsifying capacity is illustrated on the side face, and as can be seen in fig. 9, the emulsifying property of the starch octenyl succinate prepared by the synergistic modification of the alpha-beta double enzymes is obviously higher than that of a sample which is not treated by the enzymes, and meanwhile, the emulsifying property of the starch octenyl succinate modified by the synergistic modification of the double enzymes is also higher than that of two commercial products on the market at present.
When the double-enzyme synergistic method is adopted in the patent, the used enzymolysis sequence is firstly hydrolyzed by alpha amylase and then hydrolyzed by beta amylase, and the sequence cannot be changed, because if the beta amylase is firstly hydrolyzed, the hydrophobic end is exposed on the surface and then hydrolyzed by the alpha amylase, more new non-reducing ends can be hydrolyzed again, the function of increasing the proportion of the hydrophobic group at the non-reducing end cannot be realized, and the improvement of the emulsifying property is not facilitated, so the alpha amylase is firstly hydrolyzed, and then the beta amylase is hydrolyzed.
The method comprises the steps of treating octenyl succinic acid starch ester by using alpha amylase with an endonuclease effect, randomly shearing starch chains, particularly modified starch chains in an amorphous region, to obtain octenyl succinic acid modified starch ester with a proper molecular weight, further, cutting off a disaccharide unit one by one from a non-reducing end of the starch chain by the beta amylase until encountering a grafted octenyl succinic anhydride group and stopping reaction because the beta amylase is an exoenzyme and the octenyl succinic anhydride is resistant to the enzyme, wherein the grafted octenyl succinic anhydride group is exposed at the tail end of the starch chain as much as possible in the process so as to be fully contacted with an oil-water interface.
Compared with the traditional method, the surface tension, the viscosity and the molecular weight of the prepared octenyl succinic acid modified starch ester are changed, compared with the product prepared by the traditional process, the octenyl succinic acid starch ester produced by using the enzymatic hydrolysis of alpha-beta double amylase has better emulsification stability, the research has high theoretical research value and practical application significance, the starch is endowed with new performance and functionality, the starch application field is further widened, and a new way is provided for improving the starch application performance and the emulsifier production process of starch derivatives.
The starch raw material of the invention is natural and renewable, has a plurality of hydrophilic hydroxyls on the molecule, can be esterified with fatty acid, and can be used as a raw material for preparing the surfactant. The alpha amylase enzymatic hydrolysis octenyl succinic anhydride modified starch ester can obviously reduce the molecular weight of the product, thereby reducing the viscosity of the system, providing possibility for increasing the addition of an emulsifier on the premise of keeping the viscosity of the system not to be obviously changed, and increasing the emulsification stability. The beta amylase of the invention can hydrolyze the octenyl succinic anhydride enzymatically, can hydrolyze alpha-14 glycosidic bonds from the non-reducing end of starch so as to cut off disaccharides one by one, has resistance to octenyl succinic anhydride groups, stops reaction once the glucose units grafted with the octenyl succinic anhydride groups are hydrolyzed, ensures that the octenyl succinic anhydride groups are exposed at the tail end of a starch chain as much as possible, ensures that the octenyl succinic anhydride groups are fully contacted with an oil-water interface, and enhances the emulsifying capacity.
It should be noted that the above-mentioned embodiments are only for illustrating the technical solutions of the present invention and not for limiting, and although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, which should be covered by the claims of the present invention.
Claims (3)
1. A preparation method of octenyl succinic anhydride modified starch ester by double-enzyme enzymatic hydrolysis is characterized in that: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
preparation of starch octenyl succinate anhydride ester: adjusting the pH value of natural starch milk with the mass concentration of 30% to 9, stirring the starch milk at the stirring speed of 2000r/min, dropwise adding octenyl succinic anhydride with the concentration of 7% into the starch milk, carrying out octenyl succinic acid esterification reaction for 4h at the temperature of 40 ℃, and then adjusting the pH value to 6.5 to obtain octenyl succinic acid starch ester after octenyl succinic acid esterification reaction;
amylase enzymatic hydrolysis: enzymatic hydrolysis is carried out by using alpha amylase and beta amylase, pH is adjusted to be acidic, amylase is dissolved by buffer solution and then added into octenyl succinic acid starch ester, and reaction is carried out under stirring; wherein, the pH is adjusted to be acidic, the pH is adjusted to be 5.0, and the buffer solution is acetic acid-sodium acetate buffer solution; the alpha amylase and beta amylase double-enzyme enzymatic hydrolysis is carried out by firstly hydrolyzing with alpha amylase and then hydrolyzing with beta amylase, the enzyme activity of the alpha amylase is 6.6U/g, and the reaction time of the alpha amylase hydrolysis is 0.5 h; the enzyme activity of the beta-amylase is 5U/g, and the hydrolysis reaction time of the beta-amylase is 1 h.
2. The process for preparing a starch ester modified with octenyl succinic anhydride by the enzymatic hydrolysis with two enzymes according to claim 1, wherein: the natural starch comprises one or more of corn starch, wheat starch, long-shaped rice starch, barley starch, waxy corn starch, potato starch, cassava starch, glutinous rice starch, bean starch, lotus root starch, water caltrop starch, lotus seed starch, banana starch, sweet potato starch or water chestnut starch.
3. Use of octenyl succinic anhydride modified starch ester produced by the production process according to claim 1 or 2 as an emulsifier.
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