阅读说明：本技术 酶法制备大米淀粉的工艺 (Process for preparing rice starch by enzyme method ) 是由 李梦露 于 2018-06-20 设计创作，主要内容包括：本发明提供了淀粉制备技术领域内的酶法制备大米淀粉的工艺,包括以下步骤,(1)大米粉的制备:将30g大米清洗后在150mL蒸馏水中浸泡18h,打浆后离心,干燥,过200目筛；(2)大米粉中直链淀粉含量的测定：采用直链淀粉分析试剂盒,根据Con A法进行测定；(3)大米淀粉的酶法制备；(4)蛋白酶水解大米蛋白水解度的测定；(5)反应条件的确定；(6)进一步纯化大米淀粉：纤维素酶的处理、超声波处理；(7)Alcalase和protease N共同水解分离大米淀粉和蛋白质；本发明反应时间快,成本低。(The invention provides a process for preparing rice starch by an enzyme method in the technical field of starch preparation, which comprises the following steps of (1) preparing rice flour, namely cleaning 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, centrifuging, drying, and sieving by a 200-mesh sieve; (2) determination of amylose content in rice flour: determining by using an amylose analysis kit according to a Con A method; (3) preparing rice starch by an enzyme method; (4) measuring the proteolysis degree of the protease hydrolyzed rice; (5) determining reaction conditions; (6) further purification of the rice starch: treating cellulase and performing ultrasonic treatment; (7) hydrolyzing Alcalase and protease N together to separate rice starch and protein; the invention has the advantages of fast reaction time and low cost.)

1. The process for preparing rice starch by an enzyme method is characterized by comprising the following steps:

(1) the rice flour is prepared by cleaning 30g rice, soaking in 150mL distilled water for 18h, pulping, centrifuging, drying, and sieving with 200 mesh sieve;

(2) determination of amylose content in rice flour: determining by using an amylose analysis kit according to a Con A method;

(3) the rice starch is prepared by an enzyme method: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, carrying out enzymatic hydrolysis reaction, carrying out hydrolysis at the optimum pH and the optimum temperature of each protease in a full-load manner by adopting a Ph-Stat method, adding 500U of protease into each milliliter of reaction liquid, reacting for 4h, carrying out centrifugal separation on the reaction liquid after the reaction is finished, removing supernatant, scraping yellow substances on the surface of precipitate, repeatedly washing the precipitate with distilled water for a plurality of times until the pH value of the washing liquid is neutral, drying the precipitate, and sieving a dried sample with a 200-mesh sieve to obtain rice starch;

(4) determination of degree of proteolysis of protease-hydrolyzed rice: the value of the Degree of Hydrolysis (DH) can be directly calculated according to the consumption of alkali in the process of hydrolyzing rice protein by protease:

；

wherein B is the amount of sodium hydroxide solution (mL) consumed in the hydrolysis process;

N_bis the equivalent concentration of the solution;

alpha is alpha-amino dissociation degree;

MP is the total amount of substrate protein;

h_totthe number of gram equivalents of peptide bonds per gram of protein (taken as 8.13 mmol);

(5) determination of reaction conditions: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, and performing enzymatic hydrolysis reaction with two selected proteases Alcalase and protease N respectively, wherein the temperature of the hydrolysis reaction is kept at the optimum action temperature of 50 ℃ for the two proteases;

(6) further purification of the rice starch: treating cellulase and performing ultrasonic treatment;

(7) alcalase and protease N cohydrolysis to separate rice starch and protein: cleaning 30g rice, soaking in 150mL distilled water for 18h, pulping, performing enzyme hydrolysis reaction at 50 deg.C, adjusting pH to 9.5, 10, and 1 respectively0.5, the adding amount of Alcalase is 70, 100 or 120U/mL (in terms of volume of reaction liquid), the protease N is added when the pH value of the hydrolysate is reduced to be close to the optimal pH value of the protease N, 70, 100 or 120U/mL (in terms of volume of reaction liquid) is added and measured, the reaction time is 4 hours, the content of residual protein in the prepared starch is designed by taking the initial pH value of the reaction, the using amounts of Alcalase and protease N as factors and the content of residual protein in the prepared starch as an index₉（3³) And (4) performing orthogonal experiments.

2. The process for preparing rice starch by an enzymatic method according to claim 1, wherein the step (2) comprises the following steps:

(201) accurately weighing 20mg of rice flour in a test tube with a plug, adding 1mL of DMSO, uniformly mixing in a vortex mixer, adding 2mL of 95% ethanol, uniformly oscillating, adding 4mL of ethanol, uniformly mixing, standing for 15min, 2000g, and centrifuging for 5 min;

(202) adding 0.2mL of DMSO into the precipitate, heating in a boiling water bath for 15min, adding 2mL of Con A, mixing uniformly, and diluting to 5mL (solution A) with Con A;

(203) taking 1.0mL of the solution A, putting the solution A into a 2.0mL centrifuge tube, adding 0.50mL of Con A solution, uniformly mixing for multiple times, and standing for 1 h;

(204) taking 0.5mL of the solution A, adding 4mL of 100mmol/L sodium acetate buffer solution with pH4.5, 0.1mL of amyloglucosidase and alpha-amylase solution, preserving the temperature for 10min at 40 ℃, taking 1.0mL, adding 4mL of GOPOD, preserving the temperature for 20min at 40 ℃, and then measuring the absorbance at 510 nm.

3. The process for enzymatically preparing rice starch according to claim 1, wherein in said step (5), the determination of the reaction conditions further comprises the determination of the reaction initiation pH, the determination of the amount of the enzyme to be added, the determination of the reaction time;

wherein the determination of the initial pH value of the reaction comprises the following steps: adjusting the pH value of the rice slurry to 10.0, 10.5 and 11.0 by using 1.0mol/L NaOH solution, adding Alcalase 100U/mL, reacting for 4h, then carrying out linchenxin, determining the optimal initial pH value of the reaction by using the protein content in the rice starch as an index in the same step (3), adjusting the initial pH value to 8.5 for protease N, and keeping the rest reaction conditions the same as Alcalase;

the determination of the reaction time comprises the following steps: adjusting the pH value of the rice pulp to 10.5 by using 1.0mol/L NaOH solution, adding 100U/mL Alcalase protease, respectively preserving the temperature for 2/4 h and 6h, centrifuging, determining the optimal reaction time by using the protein content in the rice starch as an index after the reaction is finished in the same way as the step (3), adjusting the initial pH value to 8.5 for protease N, and keeping the rest reaction conditions the same as Alcalase.

4. The process for enzymatically preparing rice starch according to claim 1, wherein in the step (5), the treatment with cellulase comprises the steps of: washing 30g of rice, soaking in 150mL of distilled water for 18h, pulping, adjusting the pH value of rice slurry to 5.0, adding 60U/mL of cellulase to react for 2h, adjusting the pH value of a reaction system to 10.5 or 8.5, adding Alcalase or protease N100U/mL to continue reacting for 4h, and then treating the rice for 4h, wherein the treatment is the same as the step (3).

5. The process for preparing rice starch by enzyme method according to claim 1, wherein in the step (5), the ultrasonic treatment is specifically carried out by ultrasonic treatment before adding Alcalase or protease N into the rice slurry, adopting 50% amplitude and 5s pulse, the total action time is 15min, then adding Alcalase or protease N100U/mL, reacting for 4h, and centrifuging, and the treatment is the same as that in the step (3).

Technical Field

The invention belongs to the technical field of starch preparation, and particularly relates to a process for preparing rice starch by an enzyme method.

Background

The starch separation method is different according to different raw materials, the key step in the processing of the corn and wheat starch is the separation of gluten protein, and the processing of the cassava starch mainly adopts a screening method. The separation of rice starch is difficult compared to corn and wheat starch mainly because the starch in the rice endosperm is tightly bound to protein components and the tiny particles make the rice starch difficult to precipitate in water, increasing the difficulty of separation and purification. However, in order to fully utilize the excellent characteristics of small particle size and white color of rice starch granules, the starch must be efficiently separated from other components, among which separation from protein is the first to be achieved.

At present, the industrial production of rice starch mainly adopts the process of alkaline hydrolysis protein combined with centrifugal separation, but a large amount of salt and alkaline waste liquid can be introduced in the alkaline treatment process, so that the cost of wastewater treatment and discharge is increased.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to overcome the defects in the prior art and provide the process for preparing the rice starch by the enzyme method, and the process does not introduce a large amount of salt and increase the burden of wastewater treatment.

The purpose of the invention is realized as follows: the process for preparing rice starch by an enzyme method comprises the following steps,

(1) the rice flour is prepared by cleaning 30g rice, soaking in 150mL distilled water for 18h, pulping, centrifuging, drying, and sieving with 200 mesh sieve;

(2) determination of amylose content in rice flour: determining by using an amylose analysis kit according to a Con A method;

(3) the alkaline preparation method of the rice starch comprises the following steps: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, carrying out enzymatic hydrolysis reaction, carrying out hydrolysis at the optimum pH and the optimum temperature of each protease in a full-load manner by adopting a Ph-Stat method, adding 500U of protease into each milliliter of reaction liquid, reacting for 4h, carrying out centrifugal separation on the reaction liquid after the reaction is finished, removing supernatant, scraping yellow substances on the surface of precipitate, repeatedly washing the precipitate with distilled water for a plurality of times until the pH value of the washing liquid is neutral, drying the precipitate, and sieving a dried sample with a 200-mesh sieve to obtain rice starch;

(4) determination of degree of proteolysis of protease-hydrolyzed rice: the value of the Degree of Hydrolysis (DH) can be directly calculated according to the consumption of alkali in the process of hydrolyzing rice protein by protease:

；

wherein B is the amount of sodium hydroxide solution (mL) consumed in the hydrolysis process;

N_bis the equivalent concentration of the solution;

alpha is alpha-amino dissociation degree;

MP is the total amount of substrate protein;

h_totthe number of gram equivalents of peptide bonds per gram of protein (taken as 8.13 mmol);

(5) determination of reaction conditions: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, and performing enzymatic hydrolysis reaction with two selected proteases Alcalase and protease N respectively, wherein the temperature of the hydrolysis reaction is kept at the optimum action temperature of 50 ℃ for the two proteases;

(6) further purification of the rice starch: treating cellulase and performing ultrasonic treatment;

(7) alcalase and protease N cohydrolysis to separate rice starch and protein: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, carrying out enzymatic hydrolysis reaction, keeping the temperature of the hydrolysis reaction at 50 ℃, adjusting the pH value of a reaction system to be 9.5, 10 and 10.5 respectively, adding 70, 100 or 120U/mL (in terms of the volume of reaction liquid) of Alcalase, adding 70, 100 or 120U/mL (in terms of the volume of reaction liquid) of Procalase N when the pH value of hydrolysis liquid is reduced to be close to the optimal pH value of the Procalase N, reacting for 4h, designing L by taking the content of residual protein in the prepared starch as an index by taking the initial pH value of the reaction, the dosage of Alcalase and the Procalase N as factors₉（3³) And (4) performing orthogonal experiments.

In order to realize the determination of the amylose content in the rice flour, the step (2) specifically comprises the following steps:

(201) accurately weighing 20mg of rice flour in a test tube with a plug, adding 1mL of DMSO, uniformly mixing in a vortex mixer, adding 2mL of 95% ethanol, uniformly oscillating, adding 4mL of ethanol, uniformly mixing, standing for 15min, 2000g, and centrifuging for 5 min;

(202) adding 0.2mL of DMSO into the precipitate, heating in a boiling water bath for 15min, adding 2mL of Con A, mixing uniformly, and diluting to 5mL (solution A) with Con A;

(203) taking 1.0mL of the solution A, putting the solution A into a 2.0mL centrifuge tube, adding 0.50mL of Con A solution, uniformly mixing for multiple times, and standing for 1 h;

(204) taking 0.5mL of the solution A, adding 4mL of 100mmol/L sodium acetate buffer solution with pH4.5, 0.1mL of amyloglucosidase and alpha-amylase solution, preserving the temperature for 10min at 40 ℃, taking 1.0mL, adding 4mL of GOPOD, preserving the temperature for 20min at 40 ℃, and then measuring the absorbance at 510 nm.

In order to realize the determination of the breakage rate of the rice starch granules, in the step (5), the determination of the reaction conditions further comprises the determination of the initial pH value of the reaction, the determination of the enzyme adding amount and the determination of the reaction time;

wherein the determination of the initial pH value of the reaction comprises the following steps: adjusting the pH value of the rice slurry to 10.0, 10.5 and 11.0 by using 1.0mol/L NaOH solution, adding Alcalase 100U/mL, reacting for 4h, then carrying out linchenxin, determining the optimal initial pH value of the reaction by using the protein content in the rice starch as an index in the same step (3), adjusting the initial pH value to 8.5 for protease N, and keeping the rest reaction conditions the same as Alcalase;

the determination of the reaction time comprises the following steps: adjusting the pH value of the rice pulp to 10.5 by using 1.0mol/L NaOH solution, adding 100U/mL Alcalase protease, respectively preserving the temperature for 2/4 h and 6h, centrifuging, determining the optimal reaction time by using the protein content in the rice starch as an index after the reaction is finished in the same way as the step (3), adjusting the initial pH value to 8.5 for protease N, and keeping the rest reaction conditions the same as Alcalase.

In order to treat cellulase, in the step (5), the treatment of cellulase comprises the steps of: washing 30g of rice, soaking in 150mL of distilled water for 18h, pulping, adjusting the pH value of rice slurry to 5.0, adding 60U/mL of cellulase to react for 2h, adjusting the pH value of a reaction system to 10.5 or 8.5, adding Alcalase or protease N100U/mL to continue reacting for 4h, and then treating the rice for 4h, wherein the treatment is the same as the step (3).

As a further improvement of the invention, in the step (5), the ultrasonic treatment is specifically that before adding Alcalase or protease N into the rice slurry, the rice slurry is treated by ultrasonic treatment, 50% of amplitude is adopted, 5s pulse is adopted, the total action time is 15min, then Alcalase or protease N100U/mL is added, the rice slurry is reacted for 4h, and then the rice slurry is centrifuged, and the treatment is the same as that in the step (3).

Detailed Description

A process for preparing rice starch by enzyme method comprises (1) preparing rice flour by cleaning rice 30g, soaking in 150mL distilled water for 18h, pulping, centrifuging, drying, and sieving with 200 mesh sieve;

(2) determination of amylose content in rice flour: determining by using an amylose analysis kit according to a Con A method;

(3) the alkaline preparation method of the rice starch comprises the following steps: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, carrying out enzymatic hydrolysis reaction, carrying out hydrolysis at the optimum pH and the optimum temperature of each protease in a full-load manner by adopting a Ph-Stat method, adding 500U of protease into each milliliter of reaction liquid, reacting for 4h, carrying out centrifugal separation on the reaction liquid after the reaction is finished, removing supernatant, scraping yellow substances on the surface of precipitate, repeatedly washing the precipitate with distilled water for a plurality of times until the pH value of the washing liquid is neutral, drying the precipitate, and sieving a dried sample with a 200-mesh sieve to obtain rice starch;

(4) determination of degree of proteolysis of protease-hydrolyzed rice: the value of the Degree of Hydrolysis (DH) can be directly calculated according to the consumption of alkali in the process of hydrolyzing rice protein by protease:

；

wherein B is the amount of sodium hydroxide solution (mL) consumed in the hydrolysis process;

N_bis the equivalent concentration of the solution;

alpha is alpha-amino dissociation degree;

MP is the total amount of substrate protein;

h_totthe number of gram equivalents of peptide bonds per gram of protein (taken as 8.13 mmol);

(5) determination of reaction conditions: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, and performing enzymatic hydrolysis reaction with two selected proteases Alcalase and protease N respectively, wherein the temperature of the hydrolysis reaction is kept at the optimum action temperature of 50 ℃ for the two proteases;

(6) further purification of the rice starch: the method comprises the following steps of cellulase treatment and ultrasonic treatment, wherein the cellulase treatment comprises the following steps: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, adjusting the pH value of rice pulp to 5.0, adding 60U/mL of cellulase to react for 2h, adjusting the pH value of a reaction system to 10.5 or 8.5, adding Alcalase or protease N100U/mL to continue reacting for 4h, and carrying out the same treatment as the step (3); the ultrasonic treatment is specifically that before adding Alcalase or protease N into the rice pulp, the rice pulp is treated by ultrasonic wave, 50% of amplitude is adopted, 5s of pulse is adopted, the total action time is 15min, then Alcalase or protease N100U/mL is added, the rice pulp is reacted for 4h and then centrifuged, and the treatment is the same as that in the step (3);

(7) alcalase and protease N cohydrolysis to separate rice starch and protein: washing 30g of rice, soaking the rice in 150mL of distilled water for 18h, pulping, carrying out enzymatic hydrolysis reaction, keeping the temperature of the hydrolysis reaction at 50 ℃, adjusting the pH value of a reaction system to be 9.5, 10 and 10.5 respectively, adding 70, 100 or 120U/mL (in terms of the volume of reaction liquid) of Alcalase, adding 70, 100 or 120U/mL (in terms of the volume of reaction liquid) of Procalase N when the pH value of hydrolysis liquid is reduced to be close to the optimal pH value of the Procalase N, reacting for 4h, designing L by taking the content of residual protein in the prepared starch as an index by taking the initial pH value of the reaction, the dosage of Alcalase and the Procalase N as factors₉（3³) And (4) performing orthogonal experiments.

In order to realize the determination of the amylose content in the rice flour, the step (2) specifically comprises the following steps:

(201) accurately weighing 20mg of rice flour in a test tube with a plug, adding 1mL of DMSO, uniformly mixing in a vortex mixer, adding 2mL of 95% ethanol, uniformly oscillating, adding 4mL of ethanol, uniformly mixing, standing for 15min, 2000g, and centrifuging for 5 min;

(202) adding 0.2mL of DMSO into the precipitate, heating in a boiling water bath for 15min, adding 2mL of Con A, mixing uniformly, and diluting to 5mL (solution A) with Con A;

(203) taking 1.0mL of the solution A, putting the solution A into a 2.0mL centrifuge tube, adding 0.50mL of Con A solution, uniformly mixing for multiple times, and standing for 1 h;

(204) taking 0.5mL of the solution A, adding 4mL of 100mmol/L sodium acetate buffer solution with pH4.5, 0.1mL of amyloglucosidase and alpha-amylase solution, preserving the temperature for 10min at 40 ℃, taking 1.0mL, adding 4mL of GOPOD, preserving the temperature for 20min at 40 ℃, and then measuring the absorbance at 510 nm.

Wherein the determination of the initial pH value of the reaction comprises the following steps: adjusting the pH value of the rice slurry to 10.0, 10.5 and 11.0 by using 1.0mol/L NaOH solution, adding Alcalase 100U/mL, and carrying out linchenxin after 4h of reaction, wherein the treatment method after the reaction is the same as the step (3), the optimal initial pH value of the reaction is determined by using the protein content in the rice starch as an index, for proteaseN, the initial pH value is adjusted to 8.5, and the rest reaction conditions are the same as Alcalase;

the determination of the reaction time comprises the following steps: adjusting the pH value of the rice pulp to 10.5 by using 1.0mol/L NaOH solution, adding 100U/mL Alcalase protease, respectively preserving the temperature for 2/4 h and 6h, centrifuging, determining the optimal reaction time by using the protein content in the rice starch as an index after the reaction is finished in the same way as the step (3), adjusting the initial pH value to 8.5 for protease N, and keeping the rest reaction conditions the same as Alcalase.

TABLE 1 Effect of different initial reaction pH values on the residual protein content of rice starch

；

As can be seen from Table 1, at an initial pH of 10.5, the protein content of the rice starch obtained in the 4-hour reaction was reduced to 0.69%, which was lower than 0.93% in the pH-Stst method, and the protease amount was reduced from 500U/mL to 100U/mL, since gluten, which is a main protein component of rice protein, is an alkali-soluble protein, it was more favorable for the dissolution and hydrolysis of protein at a high pH, and the initial pH of the reaction was further increased to 11.0 while keeping the enzyme addition amount and the reaction time constant, and as a result, the protein content was increased to 0.76%, which was too high, so that the protease was always operated at a pH higher than its optimum pH, and the effect was weak, and therefore, the initial pH of Alcalase natural hydrolysis was selected to be 10.5.

The enzyme dosage directly influences the degree of combination with the substrate, the fixed reaction time of the experiment is 4h, the initial pH value of the reaction is 10.5, the addition amount of Alcalase is changed, and 60, 100, 200 or 300U of enzyme activity is added into each milliliter of reaction liquid respectively, which is shown in Table 2; it can be seen that when the enzyme dosage is increased from 60U/mL to 100U/mL, the residual protein content is significantly reduced, and when the enzyme dosage is increased continuously, the influence on the residual protein content is not great, and from the production cost, the best hydrolysis effect is realized by experiments with the least possible enzyme dosage, so the enzyme dosage of 100U/mL is most suitable.

TABLE 2 Effect of different Alcalases on the residual protein content of rice starch

。

The reaction time also has an influence on the hydrolysis degree of the protease, generally, the longer the reaction time is, the lower the protein content in the obtained rice starch is, but the longer the reaction time is, the cost is increased, so that the moderate reaction time is also determined, 2h, 4h and 6h are respectively selected as the time of the enzymolysis reaction in the experiment, and the result is shown in table 3, and it can be seen that the reaction time is most suitable, and the reduction of the protein content is not obvious when the reaction time is further prolonged.

TABLE 3 Effect of different reaction times on the residual protein content of rice starch

。

The present invention is not limited to the above embodiments, and based on the technical solutions disclosed in the present invention, those skilled in the art can make some substitutions and modifications to some technical features without creative efforts based on the disclosed technical solutions, and these substitutions and modifications are all within the protection scope of the present invention.