CN114277071B - Preparation method of gel-enhanced heat reversible starch - Google Patents
Preparation method of gel-enhanced heat reversible starch Download PDFInfo
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Abstract
The invention discloses a preparation method of gel-enhanced heat reversible starch, and belongs to the technical field of starch modification. The invention takes corn starch as raw material, adds proper amount of 4-alpha-glycosyltransferase, selects proper addition amount and action time and amylosucrase, and selects proper addition amount and action time to prepare the thermo-reversible starch-based gel with enhanced gel strength. The preparation method has the advantages of high efficiency, simple equipment and good product effect. The method of the invention improves the problem of low gel strength of gelatinized corn starch, can obtain gel with enhanced strength in a short time, is suitable for preparing jam, sticky soup, sauce and cake, and has wider application range.
Description
Technical Field
The invention relates to a preparation method of gel-enhanced heat reversible starch, and belongs to the technical field of starch modification.
Background
Starch is an important source of carbohydrates in the human diet and has a wide range of applications in the food industry. Starch gels are polysaccharide gels formed during cooling after gelatinization of starch, and are popular because they are non-toxic and do not readily produce flatulence. However, starch gels have limited application in repeated heat processing products because they cannot recover the solution after reheating.
Since 4-alpha-glycosyltransferases can undergo disproportionation, i.e., extend the amylose moiety to the outer side chain of amylopectin, they are widely used in enzymatic modification of starch to produce thermoreversible starch gels. The Korean Park team and the Netherlands van der Maarel team successfully modified rice starch and potato starch to form a thermoreversible phenomenon. Oh et al succeeded in modifying cornstarch particles to form a thermoreversible phenomenon.
In the present laboratory preliminary study Wang Yu attempted modification with potato starch as substrate using 4- α -glycosyltransferase from source Thermoproteus uzoniensis, resulting in thermoreversible properties but reduced gel strength; the cassava starch is optimized to obtain the thermal reversibility, the gel is in a state of a bias flow body, and the strength is extremely low; and corn starch is not always thermally reversible.
However, no one has used 4-alpha-glycosyltransferase to modify gelatinized corn starch at home and abroad so far, probably because the length of the amylopectin external side chain of the corn starch is shorter than that of potato starch, the effect of disproportionation reaction of the 4-alpha-glycosyltransferase on the external side chain of the corn starch is poor, corn is a main raw material for starch production in China, and in recent years, the corn starch accounts for more than 90% of the total starch yield, so that the modification of the gel property of the corn starch in the heat reversible starch has important research value in the food industry.
Therefore, how to carry out thermal reversible modification on corn starch, and enhance gel strength while changing the molecular structure of the starch is a technical problem to be solved urgently.
Disclosure of Invention
The first object of the present invention is a process for the preparation of a gel-enhanced thermoreversible starch comprising the steps of:
(1) Dissolving corn starch and sucrose in a buffer solution for pulping, preparing a corn starch sucrose solution, and heating for gelatinization to obtain gelatinized corn starch;
(2) Cooling the gelatinized corn starch obtained in the step (1), regulating the pH to 5.0-12.0, adding 4-alpha-glycosyltransferase and amylosucrase for enzymolysis, inactivating enzyme, drying and grinding to obtain gel-enhanced heat reversible starch;
in one embodiment of the invention, the mass concentration of the corn starch in the corn starch sucrose solution in the step (1) is 0.5% -10%; the preferred corn starch concentration is 2%.
In one embodiment of the invention, the concentration of sucrose in the corn starch sucrose solution in step (1) is 0.1 to 0.3mol/L; the preferred concentration of sucrose is 0.2mol/L.
In one embodiment of the present invention, the temperature of the thermal gelatinization in step (1) is 80 to 120 ℃.
In one embodiment of the present invention, the buffer in step (1) is Tris-HCl buffer, the concentration is 0.02-0.05 mol/L, and the pH is 7-8.
In one embodiment of the present invention, the amount of 4-alpha-glycosyltransferase added in step (2) is from 1 to 20U/g of 4-alpha-glycosyltransferase per starch, preferably from 10 to 12U/g of 4-alpha-glycosyltransferase, and most preferably from 10U/g of 4-alpha-glycosyltransferase.
In one embodiment of the present invention, the source of 4- α -glycosyltransferase (Tu αGT) in step (2) is Thermoproteus uzoniensis, which is disclosed in publication CN 110656139A.
In one embodiment of the invention, the amylosucrase (NpAS) in step (2) is of source Neisseria polysaccharea, which is disclosed in NCBI as Gen-Bank: AJ011781.1.
In one embodiment of the invention, the amylosucrase in step (2) is added in an amount of 0.5 to 15U/g of amylosucrase/starch, preferably in an amount of 0.5U/g of amylosucrase.
In one embodiment of the present invention, the enzyme deactivation method in the step (2) is one of high temperature enzyme deactivation, acid-base enzyme deactivation and ethanol enzyme deactivation.
In one embodiment of the present invention, the temperature reduction in step (2) is to 35-90 ℃.
In one embodiment of the present invention, the drying method in step (2) is one of freeze drying, atmospheric drying, spray drying, drum drying, and microwave drying.
The second object of the invention is to provide the gel-enhanced heat-reversible starch prepared by the method.
The third object of the invention is to provide an application of the gel-enhanced heat reversible starch prepared by the method in the fields of food and medicine preparation.
The fourth object of the invention is to obtain a method for preparing a thermoreversible cornstarch gel, comprising the following steps: and dissolving the gel-enhanced heat-reversible starch in water, heating and stirring, and solidifying to obtain the gel-enhanced heat-reversible starch gel.
In one embodiment of the invention, the modified corn starch is dissolved in water to prepare the gel-enhanced heat-reversible starch slurry by dissolving the gel-enhanced heat-reversible starch in deionized water.
In one embodiment of the present invention, the gel-enhanced thermoreversible starch slurry has a mass concentration of 5-15%, preferably a gel-enhanced thermoreversible starch slurry mass concentration of 6%.
The fifth object of the present invention is to provide a gel-reinforced thermoreversible starch gel prepared by the above method.
The sixth object of the invention is to provide an application of the gel-enhanced thermoreversible starch gel prepared by the method in the fields of food and medicine preparation.
[ advantageous effects ]
(1) The method of the invention improves the problem of low gel strength of gelatinized corn starch, and can obtain gel with enhanced strength in a short time.
(2) The gel strength is improved by adding the amylosucrase, the invention is suitable for preparing jam, sticky soup, sauce and cake, and the application range is wider.
Drawings
FIG. 1 is a graph showing the effect of Tu. Alpha. GT modification of 0.5-4% corn starch concentration on thermoreversibility
FIG. 2 shows the effect of TuαGT on thermoreversibility
FIG. 3 shows the effect of time of action of sequential double enzyme reaction NpAS on thermoreversibility
FIG. 4 shows the effect of the time of action of the reverse-sequence double-enzyme reaction NpAS on the thermoreversibility
FIG. 5 shows the effect of sucrose concentration on thermoreversibility by one-pot double enzyme reaction
FIG. 6 shows the effect of the combined action time of TuαGT and NpAS on the thermoreversibility by the one-pot double-enzyme reaction
FIG. 7 shows the effect of the amount of TuαGT added in one-pot double enzyme reaction on the thermoreversibility
FIG. 8 shows the effect of one-pot double enzyme reaction NpAS addition on thermoreversibility
Detailed Description
The following is a specific description of the present invention:
preparation of the enzyme used in the present invention:
fermenting by shaking, centrifuging, precipitating, breaking the wall by ultrasonic to obtain crude enzyme, and purifying by a nickel column to finally obtain Tu alpha GT and NpAS pure enzymes.
(1) Tu alpha GT enzyme activity determination method
100. Mu.L of an appropriately diluted enzyme solution was added to a mixture of a 0.2% amylose solution of potato and a 1.0% maltose solution (0.02 mol/L Tris-HCl buffer, pH 7.5) and reacted at 75℃for 10 minutes. The reaction was stopped in a boiling water bath for 10min. The amylose content in the reaction solution was measured by an amylose-iodine solution method. Total enzyme activity is defined as the amount of enzyme required to degrade 0.5mg/mL amylose per minute.
The enzyme activity unit is defined as: the amount of enzyme required to degrade 0.5mg/mL amylose per minute.
(2) NpAS enzyme activity determination method
100. Mu.L of an appropriately diluted enzyme solution was added to a mixture of waxy corn starch (0.1%) and sucrose (0.05 mol/L Tris-HCl buffer, pH 7.0) and reacted at 35℃for 10 minutes. The reaction was stopped in a boiling water bath for 10min. The reducing sugar content in the reaction solution was measured by DNS method. Total enzyme activity is defined as the amount of enzyme required to produce 1 μmol fructose per minute.
The enzyme activity unit is defined as: the amount of enzyme required to produce 1. Mu. Mol of fructose per minute.
(3) The starch conversion was calculated as:
wherein: x is starch conversion; c0 is the amylose content of the raw starch; c1 is the amylose content of the enzymatically modified starch.
(4) Gel strength determination method:
the gel strength of the samples was measured using a rotarheometer. The gel samples were placed uniformly on the rheometer test platform. The gel strength of the starch gel was determined using an advanced rotarheometer using TA company, usa, at 25 ℃. A stainless steel flat plate clamp with the diameter of 20mm is selected, the measuring distance is 1000 mu m, the strain value is 1%, and the oscillation frequency is 1Hz.
(5) Measurement method of thermoreversibility:
the thermoreversible properties of the samples were measured using a rotarheometer. The gel samples were placed uniformly on the rheometer test platform. The rheological properties of the starch gel were measured at 5-90℃using an advanced rotary rheometer using the company TA, USA.
A stainless steel flat plate clamp with the diameter of 20mm is selected, the measuring distance is 1000 mu m, the strain value is 1 percent, the oscillation frequency is 1Hz,
the temperature rise rate is 10 ℃/min, and the change of the storage modulus G 'and the loss modulus G' of the starch gel along with the temperature rise is measured.
The buffer used in the examples of the present invention was formulated as 0.05mol/L Tris-HCl buffer, pH7.0.
Example 1
(1) Corn starch and sucrose are dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of corn starch of 2 percent and the concentration of sucrose of 0.2mol/L, and the solution is completely gelatinized at 120 ℃.
(2) The temperature is reduced to 35 ℃, the pH is adjusted to 7.0, 10U/g Tu alpha GT and 0.5U/g NpAS are added simultaneously for reaction for 3 hours, then enzyme is deactivated, dried and ground to obtain the modified corn starch.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel.
The prepared starch gel has thermal reversibility and gel strength of 11.4Pa.
Example 2
(1) Optimum reaction pH of double enzyme
The enzyme activities were measured at the respective optimum temperatures using Tu. Alpha. GT and NpAS buffers at the respective optimum pH values, and the measured enzyme activities are shown in Table 1, and the optimum pH values of the double enzymes were selected. The optimal reaction pH was 7.0.
TABLE 1 determination of the optimal reaction pH for the double enzymes
(2) Temperature stability of Tu alpha GT
And (3) preserving the heat at different temperatures (35-45 ℃), taking out part of enzyme liquid every 1h, rapidly cooling, and measuring the residual total enzyme activity, wherein the measured residual total enzyme activity is shown in Table 2. The results show that: at 35-45 deg.c, tu alpha GT has relative enzyme activity maintained over 80%.
TABLE 2 measurement of temperature stability of Tu alpha GT
(3) Temperature stability of NpAS
At different temperatures (35-45 ℃) and after heat preservation, part of enzyme liquid is taken out every 1h, and is rapidly cooled, the residual total enzyme activity is measured, and the measured residual total enzyme activity is shown in Table 3. The results show that: at 35 ℃, the relative enzyme activities of the NpAS are all kept above 80%.
TABLE 3 determination of temperature stability of NpAS
Example 3
(1) Corn starch is dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of 0.5%, 1%, 2%, 3%, 4% and the sucrose concentration of 0.2mol/L, and the solution is completely gelatinized at 120 ℃.
(2) The temperature is reduced to 75 ℃, the pH is adjusted to 7.0, 10U/g Tu alpha GT is added for reaction for 4 hours, then enzyme is deactivated, dried and ground to obtain the modified corn starch.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel. The gel properties obtained are shown in Table 4.
TABLE 4 influence of corn starch concentration on starch gel Properties
| Starch concentration (%) | 0.5％ | 1％ | 2％ | 3％ | 4％ |
| Gel strength (Pa) | 2.0 | 1.3 | 2.3 | 1.1 | 4.5 |
| Thermal reversible property | Has the following components | Without any means for | Has the following components | Without any means for | Without any means for |
Example 4
(1) The corn starch is dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of 2% of the corn starch, and the solution is completely gelatinized at 120 ℃.
(2) The temperature is reduced to 75 ℃, the pH is adjusted to 7.0, 10U/g Tu alpha GT is added for reaction for 4 hours, then enzyme is deactivated, dried and ground to obtain the modified corn starch.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel. The tuαgt reaction times were 1h, 2h, 3h, and 4h, respectively, and the gel properties obtained are shown in table 5.
TABLE 5 influence of the time of action of Tu alpha GT on the gel properties of starch
| Time of action of Tu alpha GT (h) | 1 | 2 | 3 | 4 |
| Conversion (%) | 60.30 | 71.90 | 72.67 | 78.08 |
| Gel strength (Pa) | 6.1 | 3.1 | 5.5 | 2.3 |
| Thermal reversible property | Without any means for | Without any means for | Without any means for | Has the following components |
Example 5
(1) Corn starch and sucrose are dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of corn starch of 2%, and the concentration of sucrose of 0.1mol/L, 0.2mol/L and 0.3mol/L, and the solution is completely gelatinized at 120 ℃.
(2) The temperature is reduced to 35 ℃, the pH is adjusted to 7.0, 10U/g Tu alpha GT and 0.5U/g NpAS are added simultaneously for reaction for 3 hours, then enzyme is deactivated, dried and ground to obtain the modified corn starch.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel. Sucrose concentrations were 0.1M to 0.3M, respectively, and the results are shown in Table 6.
TABLE 6 influence of sucrose concentration on starch gel properties
| Sucrose concentration (mol/L) | 0.1 | 0.2 | 0.3 |
| Gel strength (Pa) | 4.6 | 11.4 | 2.3 |
| Thermal reversible property | Without any means for | Has the following components | Without any means for |
Example 6
(1) Corn starch and sucrose are dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of corn starch of 2 percent and the concentration of sucrose of 0.2mol/L, and the solution is completely gelatinized at 120 ℃.
(2) The temperature is reduced to 35 ℃, the pH is adjusted to 7.0, 10U/g Tu alpha GT and 0.5U/g NpAS are added simultaneously for reaction for 3 hours, then enzyme is deactivated, dried and ground to obtain the modified corn starch.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel. The combined action time of the double enzymes is respectively 1h, 2h, 3h and 4h, and the results are shown in Table 7.
TABLE 7 Effect of double enzyme coaction time on starch gel Properties
| Double enzyme coaction time (h) | 1 | 2 | 3 | 4 |
| Conversion (%) | 61.73 | 66.25 | 71.67 | 73.06 |
| Gel strength (Pa) | 3.1 | 88.6 | 11.4 | 20.6 |
| Thermal reversible property | Without any means for | Without any means for | Has the following components | Without any means for |
Example 7
(1) Corn starch and sucrose are dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of corn starch of 2 percent and the concentration of sucrose of 0.2mol/L, and the solution is completely gelatinized at 120 ℃.
(2) The temperature is reduced to 35 ℃, the pH is adjusted to 7.0, 1-20U/g Tu alpha GT and 0.5U/g NpAS are added at the same time, the reaction is carried out for 3 hours, then enzyme is deactivated, and the modified corn starch is obtained by drying and grinding.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel. Tu. Alpha. GT is added in the amount of 1-20U/g, and the results are shown in Table 8.
TABLE 8 influence of Tu alpha GT addition on starch gel Properties
| Tu alpha GT additive amount (U/g) | 1 | 5 | 10 | 12 | 15 | 20 |
| Gel strength (Pa) | 69.1 | 31.0 | 11.4 | 2.0 | 0.6 | 2.4 |
| Thermal reversible property | Without any means for | Without any means for | Has the following components | Has the following components | Without any means for | Without any means for |
Example 8
(1) Corn starch and sucrose are dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of corn starch of 2 percent and the concentration of sucrose of 0.2mol/L, and the solution is completely gelatinized at 120 ℃.
(2) The temperature is reduced to 35 ℃, the pH is adjusted to 7.0, 10U/g Tu alpha GT and 0.5-15U/g NpAS are added at the same time, the reaction is carried out for 3 hours, then enzyme is deactivated, and the modified corn starch is obtained by drying and grinding.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel. The amount of NpAS added was 0.5-15U/g, respectively, and the results are shown in Table 9.
TABLE 9 influence of the amount of NpAS added on the gel properties of starch
| NpAS addition amount (U/g) | 0.5 | 1 | 2 | 5 | 10 | 15 |
| Gel strength (Pa) | 11.4 | 15.6 | 104.1 | 11.3 | 6.2 | 85.2 |
| Thermal reversible property | Has the following components | Without any means for | Without any means for | Without any means for | Without any means for | Without any means for |
Comparative example 1
(1) Corn starch and sucrose are dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of corn starch of 2 percent and the concentration of sucrose of 0.2mol/L, and the solution is completely gelatinized at 120 ℃.
(2) And (3) reducing the temperature to 35 ℃, regulating the pH to 7.0, adding 0.5U/g NpAS for reaction for 1-4h, adding 10U/g Tu alpha GT for reaction for 4h after enzyme deactivation, then inactivating enzyme, drying and grinding to obtain the modified corn starch.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel. The NpAS duration of action was 1-4h, respectively, and the results are shown in Table 10.
TABLE 10 influence of NpAS action time on starch gel properties
| NpAS time of action (h) | 1 | 2 | 3 | 4 |
| Conversion (%) | 44.99 | 71.12 | 81.56 | 87.63 |
| Gel strength (Pa) | 4.2 | 20.5 | 93.2 | 93.6 |
| Thermal reversible property | Without any means for | Without any means for | Without any means for | Without any means for |
Comparative example 2
(1) Corn starch and sucrose are dissolved in buffer solution for size mixing to prepare a solution with the mass concentration of corn starch of 2 percent and the concentration of sucrose of 0.2mol/L, and the solution is completely gelatinized at 120 ℃.
(2) And (3) reducing the temperature to 35 ℃, regulating the pH to 7.0, adding 10U/g Tu alpha GT for reaction for 4 hours, adding 0.5U/g NpAS for reaction for 1-4 hours after enzyme deactivation, then inactivating enzyme, drying and grinding to obtain the modified corn starch.
(3) And dissolving the obtained modified corn starch in deionized water to prepare a 6% modified corn starch solution, heating and stirring at 100 ℃, and solidifying to obtain the modified starch gel. The NpAS duration of action was 1-4h, respectively, and the results are shown in Table 11.
TABLE 11 influence of NpAS action time on starch gel Properties
| NpAS time of action (h) | 1 | 2 | 3 | 4 |
| Conversion (%) | 54.78 | 72.35 | 79.86 | 83.48 |
| Gel strength (Pa) | 21.1 | 20.0 | 76.0 | 26.8 |
| Thermal reversible property | Without any means for | Without any means for | Without any means for | Without any means for |
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (7)
1. The preparation method of the gel-enhanced heat reversible starch is characterized by comprising the following steps of:
(1) Dissolving corn starch and sucrose in a buffer solution for pulping to prepare a solution with the mass concentration of the corn starch being 2% and the sucrose concentration being 0.2mol/L, and heating for gelatinization to obtain gelatinized corn starch;
(2) Cooling the gelatinized corn starch obtained in the step (1) to 35 ℃, regulating the pH to 7.0, simultaneously adding 4-alpha-glycosyltransferase and amylosucrase for enzymolysis for 3 hours, inactivating enzyme, drying and grinding to obtain gel-enhanced heat reversible corn starch;
the addition amount of the 4-alpha-glycosyltransferase in the step (2) is 10-12U/g of 4-alpha-glycosyltransferase/starch;
the amylosucrase in step (2) is added in an amount of 0.5U/g amylosucrase/starch.
2. The method of claim 1, wherein the temperature of the thermal gelatinization in step (1) is 80-120 ℃.
3. The gel-reinforced heat-reversible starch prepared by the method of any one of claims 1-2.
4. Use of the gel-enhanced thermally reversible starch according to claim 3 in the field of food and pharmaceutical preparation.
5. The preparation method of the thermoreversible corn starch gel is characterized by comprising the following steps of: the gel-reinforced heat-reversible starch gel is prepared by dissolving the gel-reinforced heat-reversible starch in water, heating and stirring, and solidifying.
6. The gel-enhanced thermoreversible starch gel prepared according to the method of claim 5.
7. Use of the gel-enhanced thermoreversible starch gel according to claim 6 in the field of food and pharmaceutical preparation.
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