阅读说明：本技术 一种酶法改性淀粉-甘油酯复合物及其制备方法 (Enzyme-modified starch-glyceride compound and preparation method thereof ) 是由 刘鹏飞 崔波 邹飞雪 吴正宗 郭丽 方奕珊 袁超 于滨 陶海腾 于 2021-02-05 设计创作，主要内容包括：本发明公开了一种酶法改性淀粉-甘油酯复合物及其制备方法,属于农产品加工技术领域。本发明以淀粉和甘油酯为原料,采用麦芽糖淀粉酶转糖苷辅助普鲁兰酶脱支技术,改变玉米淀粉的链长分布,增加糊化体系中可参与络合反应的线性淀粉链的释放量,促进玉米淀粉-甘油酯复合物的形成。本发明克服了传统物理法络合指数低和化学法不安全的缺陷,具有高效、安全的特点,制备的酶法改性淀粉-甘油酯复合物抗消化淀粉含量高,具有良好的耐酶解性能,且热稳定性较高,在食品加工领域具有良好的应用前景。(The invention discloses an enzyme-method modified starch-glyceride compound and a preparation method thereof, and belongs to the technical field of agricultural product processing. The invention takes starch and glyceride as raw materials, adopts a maltogenic amylase transglycosylation auxiliary pullulanase debranching technology, changes the chain length distribution of corn starch, increases the release amount of linear starch chains which can participate in a complex reaction in a pasting system, and promotes the formation of a corn starch-glyceride compound. The method overcomes the defects of low complexation index and unsafe chemical method in the traditional physical method, has the characteristics of high efficiency and safety, and the prepared enzyme-method modified starch-glyceride compound has high content of resistant digestible starch, good enzymolysis resistance, higher thermal stability and good application prospect in the field of food processing.)

1. A preparation method of an enzyme-modified starch-glyceride compound is characterized by comprising the following steps:

(1) and (3) starch enzymolysis: adding starch into buffer solution to obtain 5-10wt% starch milk solution, gelatinizing at 90-95 deg.C for 20-40min, cooling to 50-55 deg.C, adding maltogenic amylase for enzymolysis, and inactivating enzyme after enzymolysis;

(2) debranching treatment: cooling the enzymatic hydrolysate deactivated in the step (1) to 55-60 ℃, adjusting the pH value, adding pullulanase for debranching treatment, and deactivating enzyme after debranching treatment;

(3) preparation of starch-glyceride complexes: and (3) adding the dissolved glyceride into the enzymatic hydrolysate subjected to debranching treatment in the step (2), stirring and reacting at 90-95 ℃ for 50-100min, cooling to room temperature after reaction, extracting with alcohol, performing vacuum freeze drying, and crushing to obtain the modified starch-glyceride compound.

2. The method according to claim 1, wherein the starch of step (1) is corn starch; the glyceride in the step (3) is one of glycerol monolaurate, glycerol monopalmitate, glycerol monomyristate and glycerol monostearate.

3. The method according to claim 1, wherein the maltogenic amylase in step (1) is added in an amount of 3-18U/g dry starch, and the enzymolysis time is 20-40 min; the dosage of the pullulanase in the step (2) is 30-70U per gram of dry starch, and the debranching treatment time is 30-70 min.

4. The method according to claim 1, wherein the glyceride is used in an amount of 3 to 8% based on the starch in the step (3).

5. The preparation method according to claim 1, wherein the alcohol extraction method in step (3) comprises: adding 2-3 times volume of 50vt% ethanol solution into the enzymolysis solution, centrifuging, washing for 2-3 times, and collecting the lower layer precipitate.

6. The process according to claim 1, wherein the buffer solution in the step (1) is a sodium acetate buffer solution having a pH value of 6.8 to 7.0; in the step (2), the pH value is adjusted to 4.5-5.0.

7. The method according to claim 1, wherein the enzyme deactivation treatment in the steps (1) and (2) is heating the enzymatic hydrolysate at 90-95 ℃ for 10-15 min.

8. The method according to claim 5, wherein the centrifugation in the step (3) is at 4500rpm for 10 min; the vacuum freeze drying time is 24-48 h.

9. A modified starch-glyceride complex prepared by the preparation method according to any one of claims 1 to 8.

Technical Field

The invention belongs to the technical field of agricultural product processing, and particularly relates to an enzyme-method modified starch-glyceride compound and a preparation method thereof.

Background

The starch is composed of branched amylopectin and linear amylose, wherein the amylose is a linear polysaccharide formed by connecting anhydroglucose residues through alpha-1 → 4 glycosidic bonds and can form a complex with different hydrophobic guest molecules through hydrophobic interaction. Under proper conditions, when guest molecules such as fatty acid, glyceride, emulsifier and the like exist in a starch solution, the starch chain is curled under the action of hydrogen bonds in amylose molecules to generate an internal hydrophobic structure, and the amylose and hydrophobic groups of the guest molecules are subjected to a complexing action through a hydrophobic interaction to perform an embedding action on the guest molecules, so that an amylose-lipid complex is formed.

As a traditional staple food, the digestion, absorption and metabolism characteristics of starch are important factors influencing the realization of the nutritional function of starch-based food. With the change of life style and dietary structure of people and the aging degree of society

The incidence of non-infectious chronic diseases such as diabetes, cardiovascular diseases, obesity and the like is increasing, and low-sugar and low-energy food is receiving wide attention. Thus, the digestibility of starch has been the focus of attention. Complexing starch with lipids can increase the resistant starch content to some extent while reducing the fast-digestible starch content. The obtained compound has health promoting effects similar to dietary fiber, such as controlling blood sugar, improving intestinal microbial flora, preventing colon cancer, etc., and can increase nutritive value of food.

The formation and functional properties of starch-lipid complexes can be influenced by many factors, such as starch characteristics (amylose content, molecular chain length, source, etc.), lipid structure (type, chain length and degree of unsaturation), processing methods and processing conditions, among which amylose content is one of the most important influencing factors. Currently, the preparation methods of amylose-lipid complexes include physical preparation methods and chemical preparation methods. The physical preparation method has the defects of low complexing index, low production efficiency and the like of the compound; the chemical preparation method has the problems of low safety, complex process, environmental pollution and the like. There is a strong need for a safe and efficient method for the preparation of amylose-lipid complexes. The enzyme method is adopted to replace a chemical method and an inefficient physical method which have certain safety problems, and is one of the research hotspots at home and abroad at present. In the conventional enzyme method, debranching is performed on starch by using debranching enzymes (pullulanase and isoamylase) and then the debranching is complexed with guest molecules, so that the formation of a complex can be promoted to a certain extent, for example, Chinese patent CN111675830A, but the branching density of natural starch is low, the number of linear starch chains which are hydrolyzed by the pullulanase or the isoamylase in the debranching process and are suitable for complex formation is limited, and the treatment effect is poor by using a single debranching enzyme. In addition, the starch-lipid complex prepared in the prior art has the problem of low thermal stability, which affects the application of the starch-lipid complex at high temperature.

In conclusion, the research and development of the preparation method of the starch-glyceride compound with high complexation index, high production efficiency, high content of the digestion-resistant starch and good thermal stability has important significance.

Disclosure of Invention

Aiming at the problems of low complexation index, low production efficiency, low content of anti-digestion starch and poor thermal stability of a starch-lipid compound in the prior art, the invention provides an enzyme method modified starch-glyceride compound and a preparation method thereof.

The invention is realized by the following technical scheme:

a preparation method of an enzyme-modified starch-glyceride compound comprises the following steps:

(1) and (3) starch enzymolysis: adding starch into buffer solution to obtain 5-10wt% starch milk solution, gelatinizing at 90-95 deg.C for 20-40min, cooling to 50-55 deg.C, adding maltogenic amylase for enzymolysis, and inactivating enzyme after enzymolysis;

(2) debranching treatment: cooling the enzymatic hydrolysate deactivated in the step (1) to 55-60 ℃, adjusting the pH value, adding pullulanase for debranching treatment, and deactivating enzyme after debranching treatment;

(3) preparation of starch-glyceride complexes: and (3) adding the dissolved glyceride into the enzymatic hydrolysate subjected to debranching treatment in the step (2), stirring and reacting at 90-95 ℃ for 50-100min, cooling to room temperature after reaction, extracting with alcohol, performing vacuum freeze drying, and crushing to obtain the modified starch-glyceride compound.

Further, the starch in the step (1) is corn starch; the glyceride in the step (3) is one of glycerol monolaurate, glycerol monopalmitate, glycerol monomyristate and glycerol monostearate.

Further, the dosage of the maltogenic amylase in the step (1) is 3-18U per gram of dry starch, and the enzymolysis time is 20-40 min; the dosage of the pullulanase in the step (2) is 30-70U per gram of dry starch, and the debranching treatment time is 30-70 min.

Further, the amount of the glyceride used in the step (3) is 3-8% of the amount of the starch.

Further, the alcohol extraction method in the step (3) comprises the following steps: adding 2-3 times volume of 50vt% ethanol solution into the enzymolysis solution, centrifuging, washing for 2-3 times, and collecting the lower layer precipitate.

Further, the buffer solution in the step (1) is sodium acetate buffer solution with the pH value of 6.8-7.0; in the step (2), the pH value is adjusted to 4.5-5.0.

Further, the enzyme deactivation treatment in the step (1) and the step (2) means that the enzymolysis liquid is heated for 10-15min at 90-95 ℃.

Further, the centrifugation in the step (3) is 4500rpm for 10 min; the vacuum freeze drying time is 24-48 h.

In the invention, the modified starch-glyceride compound is prepared by the preparation method.

According to the method, the transglycosylation effect of the maltogenic amylase is utilized, the number of alpha-1, 6 glycosidic bonds is increased, the chain length distribution of the starch is changed, the branch density of the starch is regulated and controlled by controlling the addition amount of the enzyme, more linear starch chains suitable for inclusion complexation are generated in the subsequent debranching process, and the formation of a starch-glyceride compound is effectively promoted.

Advantageous effects

(1) The invention adopts the maltogenic amylase transglycosylation auxiliary pullulanase debranching technology, effectively changes the chain structure of corn starch, increases the release amount of linear starch chains which can participate in complex reaction in a pasting system, thereby increasing the composite probability of glyceride and amylose and promoting the formation of a corn starch-glyceride compound;

(2) the invention adopts an enzyme method to prepare the compound, can effectively improve the content of the digestion-resistant starch in the corn starch, improve the enzymolysis resistance of the corn starch, and enable the compound to have health functions beneficial to human bodies, such as controlling the reaction of blood sugar and insulin, controlling obesity, preventing colon cancer and the like;

(3) the preparation process is safe, efficient, green and environment-friendly, provides a new way for synthesizing the amylose-lipid complex, and has obvious advantages compared with a chemical preparation method and a physical preparation method.

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention in conjunction with the following examples, but it will be understood that the description is intended to illustrate the features and advantages of the invention further, and not to limit the invention.

The corn native starch used in the examples of the present invention had an amylose content of 25.3% and an amylopectin content of 74.7%.

Example 1

(1) And (3) starch enzymolysis: adding corn starch into sodium acetate buffer solution with pH value of 7.0 to prepare 6wt% starch milk solution, stirring and gelatinizing the starch milk solution at 95 ℃ for 30min, cooling to 55 ℃, adding maltogenic amylase according to the amount of 9U/g of starch dry powder, performing enzymolysis for 30min, and heating enzymolysis liquid at 90 ℃ for 15min for enzyme deactivation after enzymolysis;

(2) debranching treatment: cooling the maltogenic amylase enzymolysis liquid obtained in the step (1) to 58 ℃, adjusting the pH value of the enzymolysis liquid to 5.0, adding pullulanase according to the amount of 60U/g of starch dry powder, carrying out debranching treatment for 30min, and heating the enzymolysis liquid at 90 ℃ for 15min after the debranching treatment for enzyme deactivation;

(3) formation of enzymatically modified starch-glycerol monolaurate complex: dissolving 5% of monolaurin in anhydrous ethanol, slowly adding into the enzymatic hydrolysate of maltogenic amylase/pullulanase obtained in step (2), stirring at 90 deg.C for 70min, and cooling the mixed solution to room temperature;

(4) purifying the enzyme modified starch-glycerol monolaurate compound: and (3) adding the mixed solution obtained in the step (3) into a 3-fold volume of 50% ethanol solution, removing unreacted monolaurin in the mixed solution, then centrifuging at 4500rpm for 10min, washing for 2 times, removing supernatant, collecting lower-layer precipitate, carrying out vacuum freeze drying on the precipitate for 36h, crushing, and sieving with a 80-mesh sieve to obtain the enzyme-method modified starch-monolaurin compound.

Example 2

(1) And (3) starch enzymolysis: adding corn starch into sodium acetate buffer solution with pH value of 6.8 to prepare 8wt% starch milk solution, stirring and gelatinizing the starch milk solution at 95 ℃ for 25min, cooling to 52 ℃, adding maltogenic amylase according to the amount of 12U/g starch dry powder, performing enzymolysis for 25min, heating enzymolysis liquid at 95 ℃ for 10min after enzymolysis, and inactivating enzyme;

(2) debranching treatment: cooling the maltogenic amylase enzymolysis liquid obtained in the step (1) to 60 ℃, adjusting the pH value of the enzymolysis liquid to 4.7, adding pullulanase according to the amount of 40U/g of starch dry powder, carrying out debranching treatment for 45min, and heating the enzymolysis liquid at 95 ℃ for 10min after the debranching treatment for enzyme deactivation treatment;

(3) formation of enzymatically modified starch-monopalmitin complex: dissolving monopalmitin accounting for 4% of the starch amount with absolute ethanol, slowly adding into the maltogenic amylase/pullulanase enzymatic hydrolysate obtained in the step (2), keeping the temperature and stirring at 95 ℃ for 60min, and cooling the mixed solution to room temperature after reaction;

(4) purifying the modified starch-monopalmitin compound by an enzyme method: and (3) adding the mixed solution obtained in the step (3) into 2-fold volume of 50% ethanol solution, removing unreacted monopalmitin in the mixed solution, then centrifuging at 4500rpm for 10min, washing for 3 times, removing supernatant, collecting lower-layer precipitate, carrying out vacuum freeze drying on the precipitate for 36h, crushing, and sieving with a 80-mesh sieve to obtain the enzyme-method modified starch-monopalmitin compound.

Example 3

(1) And (3) starch enzymolysis: adding corn starch into sodium acetate buffer solution with pH value of 7.0 to prepare 7wt% starch milk solution, stirring and gelatinizing the starch milk solution at 90 deg.C for 40min, cooling to 50 deg.C, adding maltogenic amylase according to the amount of 6U/g starch dry powder, performing enzymolysis for 40min, heating hydrolysate at 90 deg.C for 15min after enzymolysis, and inactivating enzyme;

(2) debranching treatment: cooling the maltogenic amylase enzymolysis liquid obtained in the step (1) to 55 ℃, adjusting the pH value of the enzymolysis liquid to 4.5, adding pullulanase according to the amount of 55U/g of starch dry powder, carrying out debranching treatment for 40min, and heating the enzymolysis liquid at 90 ℃ for 15min after the debranching treatment for enzyme deactivation;

(3) formation of enzymatically modified starch-monomyristin complex: dissolving monomyristoyl glyceride accounting for 6% of the starch amount by using absolute ethyl alcohol, slowly adding the solution into the maltogenic amylase/pullulanase enzymatic hydrolysate obtained in the step (2), keeping the temperature and stirring the solution at 90 ℃ for 75min, and cooling the mixed solution to room temperature after reaction;

(4) purification of the enzyme-modified starch-glyceryl monomyristate complex: and (3) adding the mixed solution obtained in the step (3) into a 3-fold volume of 50% ethanol solution, removing unreacted monomyristoyl glyceride in the mixed solution, then centrifuging at 4500rpm for 10min, washing for 2 times, removing supernatant, collecting lower-layer precipitate, carrying out vacuum freeze drying on the precipitate for 36h, and crushing and sieving with a 80-mesh sieve to obtain the enzyme modified starch-monomyristoyl glyceride compound.

Example 4

(1) And (3) starch enzymolysis: adding corn starch into sodium acetate buffer solution with pH value of 7.0 to prepare 5wt% starch milk solution, stirring and gelatinizing starch milk at 95 deg.C for 30min, cooling to 52 deg.C, adding maltogenic amylase according to 15U/g starch dry powder amount, performing enzymolysis for 20min, heating enzymolysis solution at 95 deg.C for 10min, and inactivating enzyme;

(2) debranching treatment: cooling the maltogenic amylase enzymolysis liquid obtained in the step (1) to 58 ℃, adjusting the pH value of the enzymolysis liquid to 5.0, adding pullulanase according to the amount of 60U/g of starch dry powder, carrying out debranching treatment for 35min, and heating the enzymolysis liquid at 95 ℃ for 10min after the debranching treatment for enzyme deactivation treatment;

(3) formation of enzymatically modified starch-glyceryl monostearate complex: dissolving glyceryl monostearate accounting for 3% of the starch amount in absolute ethanol, slowly adding into the maltogenic amylase/pullulanase hydrolysate obtained in the step (2), keeping the temperature and stirring at 95 ℃ for 65min, and cooling the mixed solution to room temperature after reaction;

(4) purifying the modified starch-glyceryl monostearate compound by an enzyme method: and (3) adding the mixed solution obtained in the step (3) into 2-fold volume of 50% ethanol solution, removing unreacted glyceryl monostearate in the mixed solution, then centrifuging at 4500rpm for 10min, washing for 3 times, removing supernatant, collecting lower-layer precipitate, carrying out vacuum freeze drying on the precipitate for 36h, crushing, and sieving with a 80-mesh sieve to obtain the enzyme-modified starch-glyceryl monostearate compound.

Comparative example 1

(1) Starch gelatinization: adding corn starch into sodium acetate buffer solution with pH value of 7.0 to prepare 6wt% starch milk solution, stirring and gelatinizing the starch milk at 95 deg.C for 30min, and cooling to 55 deg.C;

(2) debranching treatment: adjusting the pH value of the gelatinized starch obtained in the step (1) to 5.0, adding pullulanase according to the amount of 60U/g of starch dry powder, debranching for 30min, and heating the enzymolysis liquid at 90 ℃ for 15min for enzyme deactivation after debranching;

(3) formation of starch-glycerol monolaurate complex: dissolving 5% of monolaurin in anhydrous ethanol, slowly adding into the pullulanase enzymatic hydrolysate obtained in the step (2), keeping the temperature at 90 ℃, stirring for 70min, and cooling the mixed solution to room temperature after reaction;

(4) purification of starch-glycerol monolaurate complex: and (3) adding the mixed solution obtained in the step (3) into a 3-fold volume of 50% ethanol solution, removing unreacted monolaurin in the mixed solution, then centrifuging at 4500rpm for 10min, washing for 2 times, removing supernatant, collecting lower-layer precipitate, carrying out vacuum freeze drying on the precipitate for 36h, crushing, and sieving with a 80-mesh sieve to obtain the starch-monolaurin compound by the enzyme method.

Starch-lipid complex performance testing

(1) Determination of in vitro digestion performance of enzyme-method modified starch-glyceride compound

The glucose oxidase method is adopted to determine the contents of fast-digestion starch (RDS), slow-digestion starch (SDS) and Resistant Starch (RS) in the starch-lipid complex and the corn native starch, and the specific test method comprises the following steps: mixing 200 mg of a sample to be detected with water, gelatinizing the mixture at 95 ℃ for 20min, cooling the mixture to room temperature, placing the mixture in a constant-temperature water bath kettle at 37 ℃, adding a sodium acetate buffer solution with pH 5.2 after the temperature is balanced to constant volume of 15 mL, then adding 10 mL of mixed enzyme solution (290U/mL pancreatic alpha-amylase and 20U/mL amyloglucosidase), and fully reacting the mixture for 120min after the samples are uniformly mixed. Hydrolyzing for 20min and 120min, respectively taking out 0.5 ml of enzymolysis solution, rapidly adding appropriate amount of anhydrous ethanol to inactivate enzyme, standing, centrifuging, collecting appropriate amount of centrifuged supernatant, and measuring glucose content with glucose oxidase method.

The calculation formula is as follows:

RDS =（m₂₀ – m₀）× 0.9

SDS =（m₁₂₀ – m₂₀）× 0.9

RS =100 – (RDS+SDS)

in the formula: m is₀Is the free glucose content of the starch before the enzyme hydrolysis treatment; m is₂₀The glucose content is determined after 20min of amylase hydrolysis；m₁₂₀Is the glucose content measured after 120min of amylase hydrolysis.

(2) Determination of complex index of enzyme-method modified starch-glyceride compound

Weighing 5 g of sample to be tested, adding 25 ml of distilled water, heating and stirring in boiling water bath for 30min until the sample is completely gelatinized, cooling the sample to room temperature, performing vortex oscillation for 2min, centrifuging the sample at 3500 rpm for 20min, accurately weighing 500 μ l of supernatant in a test tube, adding 15 ml of distilled water and 2 ml of iodine solution (2.0% KI and 1.3% of I)₂) Then, the absorbance (A) at 690 nm was measured_s) Wherein the control group is absorbance of corn starch without glyceride (A)₀). The calculation formula is as follows:

CI （%） = 100 × （A₀ – A_s）/ A₀

the in vitro digestibility and complexation index of the enzymatically modified starch-glyceride complexes prepared in examples 1 to 4 and comparative example 1 were tested, and the test results are shown in table 1 below:

TABLE 1 starch-lipid Complex Performance test results

。

As can be seen from Table 1, the enzyme-method modified starch-glyceride composite prepared by using corn starch as a raw material has the total content of slowly digestible starch and resistant starch up to 47.9% (example 1), and compared with the corn native starch, the content of the enzymolysis-resistant starch is obviously improved. Compared with the traditional enzyme method, the complex index of the enzyme method modified starch-glyceride compound prepared by the invention is obviously increased.