A method for preparing highly digestion-resistant recrystallized nano starch

Abstract

The present invention discloses a method for preparing highly digestible recrystallized nano starch, which comprises the following steps: (1) bio-enzyme treatment: treating a starch suspension with a bio-enzyme to obtain an enzymatic hydrolyzate, and inactivating the enzyme in the enzymatic hydrolyzate; (2) alcohol precipitation treatment: subjecting the inactivated enzymatic hydrolyzate to alcohol precipitation at a super-gelatinization temperature, centrifuging to obtain a precipitate, regenerating, and freeze-drying to obtain nano starch crystals; and (3) high-temperature and high-humidity treatment: compounding the nano starch crystals with pure water, heat-insulating the mixture in a high-temperature environment, centrifuging, obtaining a precipitate, and freeze-drying to obtain the highly digestible recrystallized nano starch. By using the enzymatic starch suspension, introducing ethanol at a super-gelatinization temperature, constant temperature stirring, and high-humidity and high-heat treatment, the processes of regulating starch chain length distribution, promoting starch chain aggregation, increasing starch chain freedom and migration ability, and accelerating starch chain migration, rearrangement, and crystallization are connected in series, thereby forming a method for preparing highly digestible recrystallized nano starch.

Description

Technical Field

[0001] The invention relates to recrystallized nano starch, in particular to a preparation method of highly digestion-resistant recrystallized nano starch. Background Art

[0002] Nanostarch crystals are modified starches that have been modified physically, chemically, or enzymatically to nanoscale dimensions (typically less than 1000 nm). These starches possess a higher surface area, greater dispersibility, and greater modifiability than natural starches. They are primarily used in food packaging, as emulsifiers / stabilizers, and as drug delivery materials. However, due to their disrupted crystalline structure, nanostarch is easily digested, which conflicts with its "resistance" property. Therefore, developing nanostarch that combines nanoscale dimensions with a high resistant starch content has become a technical challenge and a research focus. Summary of the Invention

[0003] In order to solve the above problems, the present invention provides a method for preparing highly digestion-resistant recrystallized nano starch.

[0004] The present invention provides the following technical solution: a method for preparing highly digestion-resistant recrystallized nano starch, comprising the following steps:

[0005] (1) Bio-enzyme treatment: using bio-enzyme to treat starch suspension to obtain enzymatic solution, and then inactivate the enzyme in the enzymatic solution;

[0006] (2) Alcohol precipitation treatment: The enzymatic hydrolysate after enzyme inactivation is subjected to alcohol precipitation treatment at super gelatinization temperature, and the precipitate is centrifuged and then regenerated and freeze-dried to obtain nano-starch crystals;

[0007] (3) High temperature and high humidity treatment: The nano starch crystals are compounded with pure water, kept warm in a high temperature environment, centrifuged, and the precipitate is freeze-dried to obtain the highly digestible recrystallized nano starch.

[0008] In the present invention, digestion-resistant starch refers to the sum of the digestion-resistant starch content and the slowly digestible starch content.

[0009] Furthermore, the biological enzyme used to hydrolyze α-1,6-glycosidic bonds is one or both of debranching enzyme and pullulanase, with a concentration of 10-250 npun per gram of starch and a treatment time of 1-5 h.

[0010] Furthermore, the source of the starch is any one of corn, potato, wheat, cassava, sweet potato, and rice, and the liquid of the starch suspension is a phosphate buffer solution with a pH of 5-6.

[0011] Furthermore, the enzyme is inactivated at 80-100°C.

[0012] Furthermore, the alcohol precipitation treatment conditions are as follows: pouring anhydrous ethanol into the enzyme-inactivated hydrolysate at a super gelatinization temperature until the volume ratio of ethanol to the enzyme-inactivated hydrolysate is 1-3:2, while maintaining stirring during the process for 10-90 minutes, and the super gelatinization temperature range is 70-100°C.

[0013] Furthermore, the regeneration conditions are: regeneration temperature is 0-5°C, and regeneration time is 24-72 h.

[0014] Furthermore, the mass ratio of nano starch crystals to pure water is 2:3~2:8.

[0015] Furthermore, the conditions for the heat preservation treatment in a high temperature environment are: the temperature setting range is 70-100° C., and the heat preservation time is 5-30 min.

[0016] Furthermore, the centrifugation conditions are: relative centrifugal force of 1000-5000×g, and centrifugation time of 10 min.

[0017] Furthermore, the freeze-drying conditions are: the freezing temperature is below -50°C, and the drying time is 24 to 48 hours.

[0018] The beneficial effects of the present invention are as follows:

[0019] (1) The process of enzymatic directional debranching, water activity regulation, alcohol precipitation-induced nucleation, and high-temperature wet heat recrystallization is integrated in series to achieve the full process control of starch chain uniform dispersion-directional aggregation-structural rearrangement-dense crystallization. Through the specific hydrolysis of α-1,6-glycosidic bonds and precise induced aggregation in solution, the formation of nano starch crystals is effectively promoted. Combined with high humidity and heat conditions, the migration of starch chains and serialized crystallization are further promoted, significantly improving the starch's resistance to enzymatic digestion.

[0020] (2) Compared with the traditional nano starch preparation method, the present invention emphasizes the dual optimization of structural reconstruction and crystal order, which not only obtains nanoparticles with uniform particle size and dense structure, but also maintains or increases the content of digestion-resistant starch. It is particularly suitable for high value-added application fields such as sugar-controlled foods, intestinal sustained-release carriers and functional dietary fibers.

[0021] (3) The process conditions of the present invention are mild, safe and controllable. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a process flow chart of a method for preparing highly digestion-resistant recrystallized nano starch of the present invention;

[0023] Figure 2Scanning electron microscope images (SEM) of the highly digestion-resistant recrystallized nano-starch prepared in the present invention, wherein (A) is a SEM image of the product prepared in Example 1, (B) is a SEM image of the product prepared in Example 2, (C) is a SEM image of the product prepared in Example 3, (D) is a SEM image of the product prepared in Example 4, and (E) is a SEM image of the product prepared in Example 5;

[0024] Figure 3 These are scanning electron microscope images (SEM) of the nano starch crystals prepared in Comparative Examples 1 to 3 of the present invention, wherein (A) is a SEM image of the product prepared in Comparative Example 1, (B) is a SEM image of the product prepared in Comparative Example 2, and (C) is a SEM image of the product prepared in Comparative Example 3;

[0025] Figure 4 The distribution of starch components in the examples of the present invention and the comparative examples is shown: resistant starch (RS), slowly digestible starch (SDS) and rapidly digestible starch (RDS). DETAILED DESCRIPTION

[0026] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined purpose of the invention, the specific implementation methods, structures, features and effects of the present invention are described in detail below in conjunction with the accompanying drawings and preferred embodiments.

[0027] This method utilizes enzymatic hydrolysis of a starch suspension, the introduction of ethanol at supergelatinization temperature, constant temperature stirring, and high-humidity and high-heat treatment to cascade processes such as regulating starch chain length distribution, promoting starch chain aggregation, increasing starch chain freedom and migration capacity, and accelerating starch chain migration, rearrangement, and crystallization. This method forms a set of methods for preparing highly digestible recrystallized nanostarch. This method produces nanostarch crystals with a digestible starch content of >70% and a relative crystallinity of >50%, promising a new method for producing highly digestible recrystallized nanostarch.

[0028] The embodiments of the present invention are further described below with reference to a number of embodiments.

[0029] Example 1

[0030] A method for preparing highly digestion-resistant recrystallized nano starch comprises the following steps: treating 100 g of corn starch suspension with a debranching enzyme at a concentration of 10 npun per gram of starch for 1 hour to obtain an enzymatic hydrolysate, wherein the corn starch suspension is prepared from 5 g of corn starch and 95 g of phosphate buffer (pH = 6); inactivating the enzyme at 80°C; then pouring anhydrous ethanol into the inactivated enzymatic hydrolysate at 75°C until the volume ratio of ethanol to the inactivated enzymatic hydrolysate is 1:2; stirring is maintained during the process for 10 minutes; centrifuging to obtain a precipitate for retrogradation and freeze-drying; and retrogradation conditions include a retrogradation temperature of 0°C and a retrogradation time of 24 hours to obtain nano starch crystals; recombining the nano starch crystals with pure water at a mass ratio of 2:3, and heat-insulating the mixture in a high temperature environment with a temperature setting range of 70°C and a heat-insulating time of 30 minutes. min; centrifuge, obtain the precipitate and freeze-dry. The freeze-drying conditions are as follows: freezing temperature is -50°C, drying time is 48h, and highly digestion-resistant recrystallized nano-starch is obtained.

[0031] In this embodiment, the relative centrifugal force is 1000×g and the centrifugation time is 10 min.

[0032] (1) Nanoscale morphology observation: The starch particles were sieved through a 200-mesh sieve, and a small amount was evenly applied to the conductive adhesive using a toothpick. The particles were then sprayed with gold in a vacuum environment. Field emission scanning electron microscopy was used for observation, and the accelerating voltage was set to 3 kV. In this example, the starch morphology was observed to be nanoscale, with a relatively smooth surface and a densely packed granular structure, such as Figure 2 (A) in the.

[0033] (2) Particle size determination: Weigh 20 mg of starch granules into a 50 ml centrifuge tube and add 20 ml of deionized water to make a 1 mg / ml (w / v) suspension. Homogenize at 15,000 rpm for 2 min to evenly disperse the starch granules in the aqueous phase. Pipette 1 ml of the liquid into a sample cell and measure the particle size distribution at 25°C using a nanoparticle and zeta potential analyzer. The particle size of the recrystallized nanostarch in this example was 859 nm.

[0034] (3) Determination of digestibility starch content: 790 ml of 0.2 mol / L sodium acetate solution was mixed with 210 ml of 0.2 mol / L acetic acid solution, and 0.04 mol of calcium chloride was added. The mixture was stirred until completely mixed and dissolved to obtain a buffer solution. 2 g of pancreatic enzyme was weighed and added to 20 ml of the above buffer solution. After stirring for 20 min, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was collected and mixed with saccharifying enzyme at a volume ratio of 170:7 to prepare a mixed enzyme solution. 40 mg of starch granules passed through a 60-mesh sieve were weighed into each group of labeled centrifuge tubes. 4 ml of acetate buffer, 0.8 ml of mixed enzyme solution, and 3 glass beads were added. The tubes were placed in a constant temperature water bath and shaken at 270 rpm at 37°C. Samples were taken at 0 min, 20 min, and 120 min, and 24 ml of anhydrous ethanol was added to inactivate the enzyme. The supernatant digestion solution was obtained after centrifugation at 4000 rpm for 10 min. The glucose content in the supernatant digestion solution was determined using a glucose oxidase detection kit. The glucose content measured in the supernatant digestion solution at 0, 20, and 120 min was marked as G0, G20, and G120, respectively. The RDS, SDS, and RS contents were calculated as follows:

[0035] RDS (%) = (G20 − G0) × 0.9 × 100 / 0.04;

[0036] SDS (%) = (G120 – G20) × 0.9 × 100 / 0.04;

[0037] RS(%) = 1 − RDS(%) − SDS(%);

[0038] Among them, 0.04 represents the total amount of starch is 0.04 g, and 0.9 represents the glucose conversion coefficient.

[0039] The measured results of this example showed that the value of G120 was 0.644, the value of G20 was 0.63, and the value of G0 was 0.62. The content of resistant starch (RS) in the recrystallized nano starch was 44%, the content of slowly digestible starch (SDS) was 31%, and the total content of resistant starch was 75%.

[0040] Example 2

[0041] A method for preparing highly digestion-resistant recrystallized nano-starch comprises treating 100 g of potato starch suspension with pullulanase at a concentration of 60 npun per gram of starch for 2 h to obtain an enzymatic hydrolyzate, wherein the potato starch suspension is prepared from 5 g of potato starch and 95 g of phosphate buffer (pH = 5). The enzymatic hydrolyzate is inactivated at 85°C, and then anhydrous ethanol is poured into the inactivated enzymatic hydrolyzate at 80°C until the volume ratio of ethanol to the inactivated enzymatic hydrolyzate reaches 1.5:2. The process is stirred for 30 min, and the precipitate is obtained by centrifugation for retrogradation and freeze-drying. The retrogradation conditions are as follows: retrogradation temperature of 1°C, retrogradation time of 36 h, to obtain nano starch crystals; compound the nano starch crystals with pure water, with a mass ratio of nano starch crystals to pure water of 2:4, and keep them warm in a high temperature environment, with the temperature setting range being 76°C and the warming time being 12 min; centrifuge, obtain the precipitate and freeze-dry it, with freeze drying conditions as follows: freezing temperature at -55°C and drying time at 42 h, to obtain highly digestion-resistant recrystallized nano starch.

[0042] In this embodiment, the relative centrifugal force is 2000×g and the centrifugation time is 10 min.

[0043] (1) Nanoscale morphology observation: The starch particles were sieved through a 200-mesh sieve, a small amount was picked up with a toothpick and evenly spread on the conductive glue, and gold was sprayed under a vacuum environment. The observation was performed using a field emission scanning electron microscope with an accelerating voltage set to 3 kV. In this example, the starch particles were observed to have a nanoscale morphology, a dense ridge-like structure and a nanoscale network structure, such as Figure 2 (B) in the.

[0044] (2) Particle size determination: Weigh 20 mg of starch granules into a 50 ml centrifuge tube and add 20 ml of deionized water to a 1 mg / ml (w / v) suspension. Homogenize at 15,000 rpm for 2 min to evenly disperse the starch granules in the aqueous phase. Pipette 1 ml of the solution into a sample cell and measure the particle size distribution using a nanoparticle and zeta potential analyzer at 25°C. The particle size of the recrystallized nanostarch in this example was 624 nm.

[0045] (3) Determination of digestibility starch content: 790 ml of 0.2 mol / l sodium acetate solution was mixed with 210 ml of 0.2 mol / l acetic acid solution, and 0.04 mol of calcium chloride was added. The mixture was stirred until completely mixed and dissolved to obtain a buffer solution. 2 g of pancreatic enzyme was weighed and added to 20 ml of the above buffer solution. After stirring for 20 min, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was collected and mixed with saccharifying enzyme at a volume ratio of 170:7 to prepare a mixed enzyme solution. 40 mg of starch granules passed through a 60-mesh sieve were weighed into each group of labeled centrifuge tubes. 4 ml of acetate buffer, 0.8 ml of mixed enzyme solution and 3 glass beads were added. The tubes were placed in a constant temperature water bath and shaken at 270 rpm at 37 °C. Samples were taken at 0 min, 20 min, and 120 min, and 24 ml of anhydrous ethanol was added to inactivate the enzyme. The supernatant digestion solution was centrifuged at 4000 rpm for 10 min, and the glucose content in the supernatant digestion solution was determined using a glucose oxidase detection kit. The glucose content measured in the supernatant digestion solution at 0, 20, and 120 min was marked as G0, G20, and G120, respectively. The RDS, SDS, and RS content were calculated as follows:

[0046] RDS (%) = (G20 − G0) × 0.9 × 100 / 0.04;

[0047] SDS (%) = (G120 – G20) × 0.9 × 100 / 0.04;

[0048] RS(%) = 1 − RDS(%) − SDS(%);

[0049] Among them, 0.04 represents the total amount of starch is 0.04 g, and 0.9 represents the glucose conversion coefficient.

[0050] The measured results for this example show G120 values ​​of 0.89, G20 values ​​of 0.889, and G0 values ​​of 0.879. Therefore, the content of RS in the recrystallized nanostarch is 61%, the content of SDS is 15%, and the total content of resistant starch is 76%.

[0051] Example 3

[0052] A method for preparing highly digestion-resistant recrystallized nano starch comprises the following steps: treating 100 g of wheat starch suspension with a debranching enzyme at a concentration of 110 npun per gram of starch for 3 h to obtain an enzymatic hydrolysate, wherein the wheat starch suspension is prepared from 5 g of wheat starch and 95 g of phosphate buffer (pH = 6); inactivating the enzyme at 90°C, and then pouring anhydrous ethanol into the inactivated enzymatic hydrolysate at 90°C until the volume ratio of ethanol to the inactivated enzymatic hydrolysate is 2:2; stirring is maintained during the process for 50 min; centrifuging to obtain a precipitate, performing retrogradation and freeze-drying treatment, wherein the retrogradation temperature is 2°C and the retrogradation time is 48 h, to obtain nano starch crystals; recombining the nano starch crystals with pure water, wherein the mass ratio of the nano starch crystals to the pure water is 2:5, and the mixture is kept warm in a high temperature environment, wherein the temperature setting range is 82°C and the holding time is 19 min; centrifuging to obtain a precipitate, and freeze-drying is performed, wherein the freeze-drying conditions are as follows: the freezing temperature is -60°C and the drying time is 36 h, the obtained highly digestion-resistant recrystallized nano starch.

[0053] In this embodiment, the relative centrifugal force is 3000×g and the centrifugation time is 10 min.

[0054] (1) Nanoscale morphology observation: The starch particles were sieved through a 200-mesh sieve, and a small amount was picked up with a toothpick and evenly spread on the conductive glue. The particles were then sprayed with gold in a vacuum environment. Field emission scanning electron microscopy was used for observation, and the accelerating voltage was set to 3 kV. In this example, the starch particles were observed to have a nanoscale morphology, and also showed a dense ridge-like structure and a nanoscale network structure, such as Figure 2 (C) in.

[0055] (2) Particle size determination: Weigh 20 mg of starch granules into a 50 ml centrifuge tube and add 20 ml of deionized water to make a 1 mg / ml (w / v) suspension. Homogenize at 15,000 rpm for 2 min to evenly disperse the starch granules in the aqueous phase. Pipette 1 ml of the liquid into a sample cell and measure the particle size distribution at 25°C using a nanoparticle and zeta potential analyzer. The particle size of the recrystallized nanostarch in this example was 748 nm.

[0056] (3) Determination of digestibility starch content: 790 ml of 0.2 mol / l sodium acetate solution was mixed with 210 ml of 0.2 mol / l acetic acid solution, and 0.04 mol of calcium chloride was added. The mixture was stirred until completely mixed and dissolved to obtain a buffer solution. 2 g of pancreatic enzyme was weighed and added to 20 ml of the above buffer solution. After stirring for 20 min, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was collected and mixed with saccharifying enzyme at a volume ratio of 170:7 to prepare a mixed enzyme solution. 40 mg of starch granules passed through a 60-mesh sieve were weighed into each group of labeled centrifuge tubes. 4 ml of acetate buffer, 0.8 ml of mixed enzyme solution and 3 glass beads were added. The tubes were placed in a constant temperature water bath and shaken at 270 rpm at 37 °C. Samples were taken at 0 min, 20 min, and 120 min, and 24 ml of anhydrous ethanol was added to inactivate the enzyme. The supernatant digestion solution was centrifuged at 4000 rpm for 10 min, and the glucose content in the supernatant digestion solution was determined using a glucose oxidase detection kit. The glucose content measured in the supernatant digestion solution at 0, 20, and 120 min was marked as G0, G20, and G120, respectively. The RDS, SDS, and RS content were calculated as follows:

[0057] RDS (%) = (G20 − G0) × 0.9 × 100 / 0.04;

[0058] SDS (%) = (G120 – G20) × 0.9 × 100 / 0.04;

[0059] RS(%) = 1 − RDS(%) − SDS(%);

[0060] Among them, 0.04 represents the total amount of starch is 0.04 g, and 0.9 represents the glucose conversion coefficient.

[0061] In this example, the measured G120 values ​​were 0.936, G20 values ​​were 0.933, and G0 values ​​were 0.925. Therefore, the content of RS in the recrystallized nanostarch was 74%, the content of SDS was 6%, and the total content of resistant starch was 80%.

[0062] Example 4

[0063] A method for preparing highly digestible recrystallized nano starch comprises the following steps: treating 100 g of cassava starch suspension with pullulanase at a concentration of 160 npun per gram of starch for 4 h to obtain an enzymatic hydrolyzate, wherein the cassava starch suspension is prepared from 5 g of cassava starch and 95 g of phosphate buffer (pH = 5); inactivating the enzymatic hydrolyzate at 95°C, and then pouring anhydrous ethanol into the enzymatic hydrolyzate at 90°C until the volume ratio of ethanol to the enzymatic hydrolyzate reaches 2.5:2; stirring is maintained during the process for 70 min; centrifuging to obtain a precipitate, performing retrogradation and freeze-drying treatment, wherein the retrogradation temperature is 3°C and the retrogradation time is 60 h, and nano starch crystals are obtained; the nano starch crystals are compounded with pure water, wherein the mass ratio of the nano starch crystals to the pure water is 2:6, and the mixture is kept warm in a high temperature environment, wherein the temperature setting range is 90°C and the holding time is 25 min; centrifuging to obtain a precipitate and freeze-drying, wherein the freeze-drying conditions are: the freezing temperature is -65°C and the drying time is 30 min. h, the obtained highly digestion-resistant recrystallized nano starch.

[0064] In this embodiment, the relative centrifugal force is 4000×g and the centrifugation time is 10 min.

[0065] (1) Nanoscale morphology observation: The starch particles were sieved through a 200-mesh sieve, a small amount was picked up with a toothpick and evenly spread on the conductive glue, and gold was sprayed under a vacuum environment. Field emission scanning electron microscopy was used for observation, and the accelerating voltage was set to 3 kV. In this example, the starch particles were observed to have a nanoscale morphology, showing a dense ridge-like structure and a nanoscale network structure, such as Figure 2 (D) in the.

[0066] (2) Particle size determination: Weigh 20 mg of starch granules into a 50 ml centrifuge tube and add 20 ml of deionized water to make a 1 mg / ml (w / v) suspension. Homogenize at 15,000 rpm for 2 min to evenly disperse the starch granules in the aqueous phase. Pipette 1 ml of the liquid into a sample cell and measure the particle size distribution at 25°C using a nanoparticle and zeta potential analyzer. The particle size of the recrystallized nanostarch in this example was 636 nm.

[0067] (3) Determination of digestibility starch content: 790 ml of 0.2 mol / l sodium acetate solution was mixed with 210 ml of 0.2 mol / l acetic acid solution, and 0.04 mol of calcium chloride was added. The mixture was stirred until completely mixed and dissolved to obtain a buffer solution. 2 g of pancreatic enzyme was weighed and added to 20 ml of the above buffer solution. After stirring for 20 min, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was collected and mixed with saccharifying enzyme at a volume ratio of 170:7 to prepare a mixed enzyme solution. 40 mg of starch granules passed through a 60-mesh sieve were weighed into each group of labeled centrifuge tubes. 4 ml of acetate buffer, 0.8 ml of mixed enzyme solution and 3 glass beads were added. The tubes were placed in a constant temperature water bath and shaken at 270 rpm at 37 °C. Samples were taken at 0 min, 20 min, and 120 min, and 24 ml of anhydrous ethanol was added to inactivate the enzyme. The supernatant digestion solution was centrifuged at 4000 rpm for 10 min, and the glucose content in the supernatant digestion solution was determined using a glucose oxidase detection kit. The glucose content measured in the supernatant digestion solution at 0, 20, and 120 min was marked as G0, G20, and G120, respectively. The RDS, SDS, and RS content were calculated as follows:

[0068] RDS (%) = (G20 − G0) × 0.9 × 100 / 0.04;

[0069] SDS (%) = (G120 – G20) × 0.9 × 100 / 0.04;

[0070] RS(%) = 1 − RDS(%) − SDS(%);

[0071] Among them, 0.04 represents the total amount of starch is 0.04 g, and 0.9 represents the glucose conversion coefficient.

[0072] In this example, the measured values ​​of G120 were 0.787, G20 was 0.785, and G0 was 0.777. Therefore, the content of RS in the recrystallized nanostarch was 76%, the content of SDS was 5%, and the total content of resistant starch was 81%.

[0073] Example 5

[0074] A method for preparing highly digestion-resistant recrystallized nano starch comprises the following steps: treating 100 g of a sweet potato starch suspension with a debranching enzyme and a pullulanase at a mass ratio of 1:1 at a concentration of 250 npun per gram of starch for 5 h to obtain an enzymatic hydrolyzate, wherein the sweet potato starch suspension is prepared from 5 g of sweet potato starch and 95 g of phosphate buffer (pH = 5.5); inactivating the enzyme at 100°C; then pouring anhydrous ethanol into the enzymatic hydrolyzate at 95°C until the volume ratio of ethanol to the enzymatic hydrolyzate is 3:2; stirring is maintained during the process for 90 min; centrifuging to obtain a precipitate, performing retrogradation and freeze-drying treatment, and performing retrogradation treatment under the following retrogradation conditions: a retrogradation temperature of 5°C and a retrogradation time of 24 h to obtain nano starch crystals; recombining the nano starch crystals with pure water at a mass ratio of nano starch crystals to pure water of 2:8; and heat-insulating the mixture in a high temperature environment with a temperature setting range of 100°C and a heat preservation time of 30 min. min; centrifuge, obtain the precipitate and freeze-dry. The freeze-drying conditions are as follows: freezing temperature is -70℃, drying time is 24 h, and highly digestion-resistant recrystallized nano-starch is obtained.

[0075] In this embodiment, the relative centrifugal force is 5000×g and the centrifugation time is 10 min.

[0076] (1) Nanoscale morphology observation: The starch particles were sieved through a 200-mesh sieve, and a small amount was picked up with a toothpick and evenly spread on the conductive glue. The particles were then sprayed with gold in a vacuum environment. Field emission scanning electron microscopy was used for observation, and the accelerating voltage was set to 3 kV. In this example, the starch particles were observed to have a nanoscale morphology, and also showed a dense ridge-like structure and a nanoscale network structure, such as Figure 2 (E) in.

[0077] (2) Particle size determination: Weigh 20 mg of starch granules into a 50 ml centrifuge tube and add 20 ml of deionized water to a 1 mg / ml (w / v) suspension. Homogenize at 15,000 rpm for 2 min to evenly disperse the starch granules in the aqueous phase. Pipette 1 ml of the liquid into a sample cell and measure the particle size distribution using a nanoparticle and zeta potential analyzer at 25°C. The particle size of the nano starch crystals in this example was 758 nm.

[0078] (3) Determination of digestibility starch content: 790 ml of 0.2 mol / l sodium acetate solution was mixed with 210 ml of 0.2 mol / l acetic acid solution, and 0.04 mol of calcium chloride was added. The mixture was stirred until completely mixed and dissolved to obtain a buffer solution. 2 g of pancreatic enzyme was weighed and added to 20 ml of the above buffer solution. After stirring for 20 min, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was collected and mixed with saccharifying enzyme at a volume ratio of 170:7 to prepare a mixed enzyme solution. 40 mg of starch granules passed through a 60-mesh sieve were weighed into each group of labeled centrifuge tubes. 4 ml of acetate buffer, 0.8 ml of mixed enzyme solution and 3 glass beads were added. The tubes were placed in a constant temperature water bath and shaken at 270 rpm at 37 °C. Samples were taken at 0 min, 20 min, and 120 min, and 24 ml of anhydrous ethanol was added to inactivate the enzyme. The supernatant digestion solution was centrifuged at 4000 rpm for 10 min, and the glucose content in the supernatant digestion solution was determined using a glucose oxidase detection kit. The glucose content measured in the supernatant digestion solution at 0, 20, and 120 min was marked as G0, G20, and G120, respectively. The RDS, SDS, and RS contents were calculated as follows:

[0079] RDS (%) = (G20 − G0) × 0.9 × 100 / 0.04;

[0080] SDS (%) = (G120 – G20) × 0.9 × 100 / 0.04;

[0081] RS(%) = 1 − RDS(%) − SDS(%);

[0082] Among them, 0.04 represents the total amount of starch is 0.04 g, and 0.9 represents the glucose conversion coefficient.

[0083] In this example, the measured values ​​of G120 were 0.68, G20 was 0.678, and G0 was 0.67. Therefore, the content of RS in the recrystallized nanostarch was 77%, the content of SDS was 5%, and the total content of resistant starch was 82%.

[0084] Comparative Example 1

[0085] The difference between this comparative example and Example 1 is that there is no high temperature and high humidity treatment step.

[0086] 100 g of corn starch suspension was treated with a debranching enzyme at a concentration of 10 npun per gram of starch for 1 hour to obtain an enzymatic hydrolysate. The corn starch suspension was prepared by mixing 5 g of corn starch with 95 g of phosphate buffer (pH = 6). The enzyme was inactivated at 90°C, and then anhydrous ethanol was poured into the inactivated enzymatic hydrolysate at 75°C until the volume ratio of ethanol to the inactivated enzymatic hydrolysate reached 1:2. The process was stirred for 10 min, and the precipitate was obtained by centrifugation for retrogradation and freeze-drying. The retrogradation conditions were as follows: retrogradation temperature at 0°C and retrogradation time for 24 h to obtain nano-starch crystals.

[0087] In this embodiment, the relative centrifugal force is 1000×g and the centrifugation time is 10 min.

[0088] (1) Nanoscale morphology observation: The starch particles were passed through a 200-mesh sieve, a small amount was picked up with a toothpick and evenly spread on the conductive glue, and gold was sprayed under a vacuum environment. Field emission scanning electron microscopy was used for observation, and the acceleration voltage was set to 3 kV. In this example, the starch particles were observed to have a relatively loose nano starch particle structure, such as Figure 3 (A) in the.

[0089] (2) Particle size determination: Weigh 20 mg of starch granules into a 50 ml centrifuge tube and add 20 ml of deionized water to make a 1 mg / ml (w / v) suspension. Homogenize at 15,000 rpm for 2 min to evenly disperse the starch granules in the aqueous phase. Pipette 1 ml of the liquid into a sample cell and measure the particle size distribution using a nanoparticle and zeta potential analyzer at 25°C. The particle size of the nano starch crystals in this example was 914 nm.

[0090] (3) Determination of digestibility starch content: 790 ml of 0.2 mol / l sodium acetate solution was mixed with 210 ml of 0.2 mol / l acetic acid solution, and 0.04 mol of calcium chloride was added. The mixture was stirred until completely mixed and dissolved to obtain a buffer solution. 2 g of pancreatic enzyme was weighed and added to 20 ml of the above buffer solution. After stirring for 20 min, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was collected and mixed with saccharifying enzyme at a volume ratio of 170:7 to prepare a mixed enzyme solution. 40 mg of starch granules passed through a 60-mesh sieve were weighed into each group of labeled centrifuge tubes. 4 ml of acetate buffer, 0.8 ml of mixed enzyme solution and 3 glass beads were added. The tubes were placed in a constant temperature water bath and shaken at 270 rpm at 37 °C. Samples were taken at 0 min, 20 min, and 120 min, and 24 ml of anhydrous ethanol was added to inactivate the enzyme. The supernatant digestion solution was centrifuged at 4000 rpm for 10 min, and the glucose content in the supernatant digestion solution was determined using a glucose oxidase detection kit. The glucose content measured in the supernatant digestion solution at 0, 20, and 120 min was marked as G0, G20, and G120, respectively. The RDS, SDS, and RS content were calculated as follows:

[0091] RDS (%) = (G20 − G0) × 0.9 × 100 / 0.04;

[0092] SDS (%) = (G120 – G20) × 0.9 × 100 / 0.04;

[0093] RS(%) = 1 − RDS(%) − SDS(%);

[0094] Among them, 0.04 represents the total amount of starch is 0.04 g, and 0.9 represents the glucose conversion coefficient.

[0095] In this comparative example, the measured values ​​for G120 were 0.7864, G20 0.786, and G0 0.758. Therefore, the content of resistant starch (RS) in the nano-starch crystals was 36%, the content of slowly digestible starch (SDS) was 1%, and the total digestible starch content was 37%. This demonstrates that high temperature and high humidity are important process measures for increasing the content of digestible starch.

[0096] Comparative Example 2

[0097] The difference between this comparative example and Example 1 is that there is no biological enzyme treatment and enzyme inactivation treatment.

[0098] Take 100g of corn starch suspension, which is prepared by 5g of corn starch and 95g of phosphate buffer (pH=6). Pour anhydrous ethanol into the corn starch suspension at 75℃ until the volume ratio of ethanol to corn starch suspension is 1:2. Keep stirring during the process for 10 minutes. Centrifuge to obtain a precipitate for retrogradation and freeze-drying. The retrogradation conditions are as follows: the retrogradation temperature is 0℃ and the retrogradation time is 24 hours to obtain nano-starch crystals. The nano-starch crystals are compounded with pure water at a mass ratio of nano-starch crystals to pure water of 2:3. The mixture is kept warm in a high temperature environment with a temperature setting range of 70℃ and a holding time of 30 minutes. Centrifuge and freeze-dry the precipitate. The freeze-drying conditions are as follows: the freezing temperature is -50℃ and the drying time is 48 hours to obtain recrystallized starch.

[0099] In this embodiment, the relative centrifugal force is 1000×g and the centrifugation time is 10 min.

[0100] (1) Nanoscale morphology observation: The starch particles were sieved through a 200-mesh sieve, a small amount was picked up with a toothpick and evenly spread on the conductive glue, and gold was sprayed under a vacuum environment. The observation was performed using a field emission scanning electron microscope with an accelerating voltage set to 3 kV. In this example, the starch morphology was observed to be loose starch particles with a scale of more than 1 un. Figure 3 (B) in the.

[0101] (2) Particle size determination: Weigh 20 mg of starch granules into a 50 ml centrifuge tube and add 20 ml of deionized water to a 1 mg / ml (w / v) suspension. Homogenize at 15,000 rpm for 2 min to evenly disperse the starch granules in the aqueous phase. Pipette 1 ml of the liquid into a sample cell and measure the particle size distribution at 25°C using a nanoparticle and zeta potential analyzer. The particle size of the recrystallized starch in this example was 1127 nm.

[0102] (3) Determination of digestibility starch content: 790 ml of 0.2 mol / l sodium acetate solution was mixed with 210 ml of 0.2 mol / l acetic acid solution, and 0.04 mol of calcium chloride was added. The mixture was stirred until completely mixed and dissolved to obtain a buffer solution. 2 g of pancreatic enzyme was weighed and added to 20 ml of the above buffer solution. After stirring for 20 min, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was collected and mixed with saccharifying enzyme at a volume ratio of 170:7 to prepare a mixed enzyme solution. 40 mg of starch granules passed through a 60-mesh sieve were weighed into each group of labeled centrifuge tubes. 4 ml of acetate buffer, 0.8 ml of mixed enzyme solution and 3 glass beads were added. The tubes were placed in a constant temperature water bath and shaken at 270 rpm at 37 °C. Samples were taken at 0 min, 20 min, and 120 min, and 24 ml of anhydrous ethanol was added to inactivate the enzyme. The supernatant digestion solution was centrifuged at 4000 rpm for 10 min, and the glucose content in the supernatant digestion solution was determined using a glucose oxidase detection kit. The glucose content measured in the supernatant digestion solution at 0, 20, and 120 min was marked as G0, G20, and G120, respectively. The RDS, SDS, and RS contents were calculated as follows:

[0103] RDS (%) = (G20 − G0) × 0.9 × 100 / 0.04;

[0104] SDS (%) = (G120 – G20) × 0.9 × 100 / 0.04;

[0105] RS(%) = 1 − RDS(%) − SDS(%);

[0106] Among them, 0.04 represents the total amount of starch is 0.04 g, and 0.9 represents the glucose conversion coefficient.

[0107] In this comparative example, the measured G120 values ​​were 0.902, G20 values ​​were 0.896, and G0 values ​​were 0.866. Therefore, the content of resistant starch (RS) in the recrystallized starch was 20%, the content of slowly digestible starch (SDS) was 13%, and the total content of resistant starch was 33%. This indicates that hydrolysis at a certain enzyme concentration is necessary to achieve nanoscale production.

[0108] Comparative Example 3

[0109] The difference between this comparative example and Example 1 is that no alcohol precipitation treatment is performed.

[0110] 100 g of corn starch suspension was treated with a debranching enzyme at a concentration of 10 npun per gram of starch for 1 h to obtain an enzymatic hydrolysate. The corn starch suspension was prepared by mixing 5 g of corn starch with 95 g of phosphate buffer (pH = 6). The enzymatic hydrolysate was inactivated at 90°C and the precipitate was obtained by centrifugation for retrogradation and freeze-drying. The retrogradation conditions were as follows: retrogradation temperature at 0°C and retrogradation time for 24 h to obtain nano-starch crystals. The nano-starch crystals were then compounded with pure water at a mass ratio of 2:3, and the mixture was kept warm in a high temperature environment at 70°C for 30 min. The precipitate was then centrifuged and freeze-dried. The freeze-drying conditions were as follows: freezing temperature at -50°C and drying time for 48 h to obtain recrystallized starch.

[0111] In this embodiment, the relative centrifugal force is 1000×g and the centrifugation time is 10 min.

[0112] (1) Nanoscale morphology observation: The starch particles were sieved through a 200-mesh sieve, a small amount was picked up with a toothpick and evenly spread on the conductive glue, and gold was sprayed under a vacuum environment. The observation was performed using a field emission scanning electron microscope with an accelerating voltage set to 3 kV. In this example, the starch morphology was observed to be dense starch particles with a size of more than 1 μm, without a network structure, such as Figure 3 (C) in.

[0113] (2) Particle size determination: Weigh 20 mg of starch granules into a 50 ml centrifuge tube and add 20 ml of deionized water to make a 1 mg / ml (w / v) suspension. Homogenize at 15,000 rpm for 2 min to evenly disperse the starch granules in the aqueous phase. Pipette 1 ml of the liquid into a sample cell and measure the particle size distribution using a nanoparticle and zeta potential analyzer at 25°C. The particle size of the nano starch crystals in this example was 1374 nm.

[0114] (3) Determination of digestibility starch content: 790 ml of 0.2 mol / l sodium acetate solution was mixed with 210 ml of 0.2 mol / l acetic acid solution, and 0.04 mol of calcium chloride was added. The mixture was stirred until completely mixed and dissolved to obtain a buffer solution. 2 g of pancreatic enzyme was weighed and added to 20 ml of the above buffer solution. After stirring for 20 min, the mixture was centrifuged at 4000 rpm for 10 min. The supernatant was collected and mixed with saccharifying enzyme at a volume ratio of 170:7 to prepare a mixed enzyme solution. 40 mg of starch granules passed through a 60-mesh sieve were weighed into each group of labeled centrifuge tubes. 4 ml of acetate buffer, 0.8 ml of mixed enzyme solution and 3 glass beads were added. The tubes were placed in a constant temperature water bath and shaken at 270 rpm at 37 °C. Samples were taken at 0 min, 20 min, and 120 min, and 24 ml of anhydrous ethanol was added to inactivate the enzyme. The supernatant digestion solution was centrifuged at 4000 rpm for 10 min, and the glucose content in the supernatant digestion solution was determined using a glucose oxidase detection kit. The glucose content measured in the supernatant digestion solution at 0, 20, and 120 min was marked as G0, G20, and G120, respectively. The RDS, SDS, and RS content were calculated as follows:

[0115] RDS (%) = (G20 − G0) × 0.9 × 100 / 0.04;

[0116] SDS (%) = (G120 – G20) × 0.9 × 100 / 0.04;

[0117] RS(%) = 1 − RDS(%) − SDS(%);

[0118] Among them, 0.04 represents the total amount of starch is 0.04 g, and 0.9 represents the glucose conversion coefficient.

[0119] In this comparative example, the measured values ​​for G120 were 0.993, G20 0.986, and G0 0.954. Therefore, the content of resistant starch (RS) in the recrystallized starch was 14%, the content of slowly digestible starch (SDS) 15%, and the total content of resistant starch was 29%. This demonstrates that alcohol precipitation is an important process for forming a nanoscale network structure.

[0120] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as above in terms of a preferred embodiment, it is not intended to limit the present invention. Any person skilled in the art can, without departing from the scope of the technical solution of the present invention, make some changes or modifications to equivalent embodiments using the technical contents disclosed above. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing highly digestion-resistant recrystallized nano starch, characterized in that: The steps include: (1) Bioenzyme treatment: using bioenzyme to treat starch suspension to obtain enzymatic hydrolysate, and then inactivating the enzyme in the enzymatic hydrolysate; the bioenzyme is a debranching enzyme, the concentration is 10-250 npun per gram of starch, and the treatment time is 1-5 h; (2) Alcohol precipitation treatment: The enzyme-inactivated hydrolysate is subjected to alcohol precipitation treatment at a super-gelatinization temperature, and the precipitate is obtained by centrifugation and then regenerated and freeze-dried to obtain nano-starch crystals; the alcohol precipitation treatment conditions are as follows: anhydrous ethanol is poured into the enzyme-inactivated hydrolysate at a super-gelatinization temperature until the volume ratio of ethanol to the enzyme-inactivated hydrolysate is 1-3:2, and the process is kept stirring for 10-90 min, and the super-gelatinization temperature is 70-100°C; (3) High temperature and high humidity treatment: The nano starch crystals are compounded with pure water, kept warm in a high temperature environment, centrifuged, and the precipitate is freeze-dried to obtain the highly digestible recrystallized nano starch; the mass ratio of nano starch crystals to pure water is 2:3~2:8; the conditions for the heat preservation treatment in a high temperature environment are: the temperature setting range is 70~100℃, and the heat preservation time is 5~30 min.

2. A method for preparing highly digestion-resistant recrystallized nano starch, characterized in that: The steps include: (1) Bioenzyme treatment: using bioenzyme to treat starch suspension to obtain enzymatic hydrolysate, and then inactivating the enzyme in the enzymatic hydrolysate; the bioenzyme is pullulanase, the concentration is 10-250 npun per gram of starch, and the treatment time is 1-5 h; (2) Alcohol precipitation treatment: The enzyme-inactivated hydrolysate is subjected to alcohol precipitation treatment at a super-gelatinization temperature, and the precipitate is obtained by centrifugation and then regenerated and freeze-dried to obtain nano-starch crystals; the alcohol precipitation treatment conditions are as follows: anhydrous ethanol is poured into the enzyme-inactivated hydrolysate at a super-gelatinization temperature until the volume ratio of ethanol to the enzyme-inactivated hydrolysate is 1-3:2, and the process is kept stirring for 10-90 min, and the super-gelatinization temperature is 70-100°C; (3) High temperature and high humidity treatment: The nano starch crystals are compounded with pure water, kept warm in a high temperature environment, centrifuged, and the precipitate is freeze-dried to obtain the highly digestible recrystallized nano starch; the mass ratio of nano starch crystals to pure water is 2:3~2:8; the conditions for the heat preservation treatment in a high temperature environment are: the temperature setting range is 70~100℃, and the heat preservation time is 5~30 min.

3. The preparation method according to claim 1 or 2, characterized in that The starch is sourced from any one of corn, potato, wheat, cassava, sweet potato and rice, and the liquid of the starch suspension is a phosphate buffer solution with a pH of 5-6.

4. The preparation method according to claim 1 or 2, characterized in that The enzyme is inactivated at 80-100°C.

5. The preparation method according to claim 1 or 2, characterized in that The regeneration conditions are as follows: the regeneration temperature is 0-5°C, and the regeneration time is 24-72 h.

6. The preparation method according to claim 1 or 2, characterized in that The centrifugal conditions are as follows: relative centrifugal force of 1000-5000×g, and centrifugal time of 10 min.

7. The preparation method according to claim 1 or 2, characterized in that The freeze-drying conditions are as follows: the freezing temperature is lower than -50°C, and the drying time is 24 to 48 hours.

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