Preparation method of lotus seed resistant starch

Abstract

The invention discloses a preparation method of lotus seed resistant starch, and belongs to the technical field of lotus seed processing. Comprising the following steps: S1, preparing lotus seed starch; s21, adding a proper amount of distilled water into lotus seed starch to prepare a starch milk solution with a certain concentration, carrying out hot pressing gelatinization, sequentially adding pepsin and pullulanase, carrying out sufficient enzymolysis, and carrying out high-temperature enzyme deactivation; s22, adding chitosan grafted with disulfide bonds into the milk solution subjected to enzyme deactivation, and centrifuging and filtering after full reaction; s23, adding a glutathione-magnetic silicon dioxide composite material into the milk solution filtered in the step S22, fully reacting in an inert atmosphere, centrifuging, and filtering; and S24, refrigerating the milk solution filtered in the step S23, and then drying, crushing, sieving and purifying to obtain the lotus seed resistant starch. By improving the prior art and optimizing process parameters, high-yield and high-purity resistant starch can be obtained, the yield is not lower than 62.72%, and the purity is not lower than 98.15%.

Description

Technical Field

[0001] The present invention relates to the technical field of lotus seed processing, and specifically to a preparation method of lotus seed resistant starch. Background Art

[0002] Lotus seeds are rich in physiological active substances such as vitamins, minerals, phospholipids, alkaloids, flavonoids, water-soluble polysaccharides, and superoxide dismutase (SOD), and have physiological functions such as maintaining blood sugar stability, antioxidant, anti-aging, regulating gastrointestinal function, enhancing immunity, skin whitening and anti-wrinkle, anti-inflammatory and antiviral, and protecting liver function. The starch content in lotus seeds is extremely high, which is the main flavor component of the delicate fragrance of lotus seeds. Among them, amylose in lotus seed starch accounts for about 40% of the total starch, and amylose is more likely to retrograde to form RS3 resistant starch than amylopectin.

[0003] Resistant starch (RS) is a type of starch that cannot be utilized and absorbed in the small intestine of healthy individuals, but it can be fermented or partially fermented by colonic flora in the colon and has physiological functions similar to soluble dietary fiber, including preventing gastrointestinal diseases and cardiovascular diseases, reducing the risk of ulcerative colitis and colon cancer, promoting bacterial growth and the absorption of mineral elements, and enhancing disease resistance.

[0004] According to different resistance principles, resistant starch can be divided into 5 categories: namely, RS1 physically embedded starch, where the embedding effect of the cell wall and protein is the main factor affecting resistance; RS2 resistant starch granules, including resistant natural starch granules and ungelatinized starch granules; RS3 retrograded or crystalline starch, which is formed by the retrogradation and crystallization of amylose in starch paste after low-temperature cooling to form aged starch that is difficult to be enzymatically hydrolyzed; RS4 chemically modified starch, which can resist the digestion of α-amylase, such as acetylated starch, hydroxypropyl starch, and crosslinked starch, etc.; RS5 amylose-lipid complex, also known as starch lipid.

[0005] Currently, autoclaving method, microwave method, ultrasonic-autoclaving method, enzymatic hydrolysis-autoclaving method, etc. are often used to prepare lotus seed RS3 resistant starch, but the yield is lower than 58%. For example, the application number is CN202311097534.6, and the name is a highly probiotic lotus seed resistant starch prepared by enzymatic hydrolysis and co-pressure treatment and its preparation method and application. The yield of the resistant starch prepared by this method is 58.64%.

[0006] Based on this, it is extremely important to provide a preparation method that can effectively improve the yield of lotus seed resistant starch. Summary of the Invention

[0007] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a preparation method of lotus seed resistant starch to at least achieve the purpose of improving the yield of lotus seed resistant starch.

[0008] The object of the present invention is achieved by the following technical solutions: A preparation method of lotus seed resistant starch, comprising the following steps: S1. Preparation of lotus seed starch; S2. Preparation of lotus seed resistant starch: S21. Add the lotus seed starch to an appropriate amount of distilled water to prepare a starch milk solution with a certain concentration. After pressure heat gelatinization, add pepsin and pullulanase in sequence. After sufficient enzymatic hydrolysis, inactivate the enzyme at high temperature; S22. Add chitosan grafted with disulfide bonds to the enzyme-inactivated milk solution. After sufficient reaction, centrifuge and filter; S23. Add glutathione-magnetic silica composite material to the milk solution filtered in S22. After sufficient reaction under an inert atmosphere, centrifuge and filter; S24. Refrigerate the milk solution filtered in S23 at 4°C under normal pressure for 12-20 h; then obtain crude lotus seed resistant starch after drying, pulverizing, and sieving; S25. Enzymatically hydrolyze the crude lotus seed resistant starch with α-amylase and glucoamylase in sequence. After enzymatic hydrolysis, inactivate the enzyme at high temperature, and then filter, wash, and dry to obtain lotus seed resistant starch.

[0009] As some feasible embodiments of the present application, in step S21, the concentration of the prepared starch milk solution is 20-30%.

[0010] As some feasible embodiments of the present application, in step S21, the gelatinization treatment conditions are: the pressure heat gelatinization treatment conditions are: the temperature is 105-20°C, the pressure is 0.1-0.3 MPa, and the time is 8-15 min.

[0011] As some feasible embodiments of the present application, the conditions for enzymatic hydrolysis of the protease are: pH is 2-2.5, temperature is 30-45°C, time is 1-2 h, and the enzyme dosage is 5-15 U / g (dry basis starch); the conditions for enzymatic hydrolysis of the pullulanase are: pH is 5-6, temperature is 75-85°C, time is 8-10 h, and the enzyme dosage is 15-25 U / g (dry basis starch).

[0012] As some feasible embodiments of the present application, the pH regulator used for pH adjustment is a mixed solution of citric acid and sodium citrate with a concentration of 0.2-0.3 mol / L.

[0013] As some feasible embodiments of the present application, in step S22, the addition amount of chitosan grafted with disulfide bonds is 10-15% of the total mass of the enzyme-inactivated milk solution in step S22; the reaction conditions after adding chitosan grafted with disulfide bonds are: maintaining at 75-85°C for 4-5 h.

[0014] As some feasible embodiments of the present application, in step S23, the mass of the glutathione-magnetic silica composite added is 20-30% of the total weight of the milk solution filtered in S22; after adding the glutathione-magnetic silica composite, ultrasonic treatment is performed at 30-45 °C for 15 min, and the ultrasonic power is 200-300 w.

[0015] As some feasible embodiments of the present application, in step S23, the preparation method of the glutathione-magnetic silica composite is as follows: S2301: Add an organic solvent and glutaraldehyde to magnetic silica with a particle size of 100-400 nm, fully react and then centrifuge, collect the solid and wash it; S2302: Add glutathione, an organic solvent and deionized water to the washed solid, fully react and then centrifuge, take out the solid, and after washing and drying, obtain the glutathione-magnetic silica composite.

[0016] As some feasible embodiments of the present application, in step S25, the mass ratio of crude lotus seed resistant starch, α-amylase and glucoamylase is 10: (1-1.5): (0.5-1.5).

[0017] The beneficial effects of the present invention are: 1. By adding chitosan grafted with disulfide bonds to the starch milk solution after debranching with pullulanase, it can be compounded with some debranched starch and amylopectin, and precipitate some amylopectin, thereby effectively removing amylopectin in the starch milk solution and increasing the yield of resistant starch; by grafting disulfide bonds on the chitosan molecule, steric hindrance can be formed on the surface of the chitosan molecule, thereby effectively reducing its binding degree with amylose, and further effectively reducing the loss of amylose to avoid adverse effects on the preparation of resistant starch.

[0018] 2. By adding glutathione-magnetic silica composite material to the starch emulsion solution with chitosan grafted with disulfide bonds, the magnetic silica can adsorb part of the branched starch (because it has more branches, it is easier to be adsorbed), and the thiol, amino, carboxyl and other groups in glutathione can interact non-covalently with the amino groups of chitosan grafted with disulfide bonds, thereby achieving the purpose of specific adsorption of chitosan grafted with disulfide bonds. In addition, the thiol in glutathione can reduce the disulfide bonds in chitosan, thereby opening the disulfide bonds to reduce the chitosan molecules. The steric hindrance of the magnetic silica particles promotes the non-covalent interaction between the thiol, amino, carboxyl and other groups and chitosan, thereby maximally combining the chitosan grafted with disulfide bonds with the glutathione-magnetic silica composite material, thereby achieving the separation of the chitosan grafted with disulfide bonds from the starch solution, so as to maximize the purpose of increasing the yield and purity of the resistant starch; in addition, the glutathione-magnetic silica composite material can also adsorb the amino acids after protease hydrolysis, thereby reducing the content of amino acids in the milk solution and improving the purity of the crude resistant starch.

[0019] 3. The present invention improves the existing technology and optimizes the process parameters to obtain high-yield and high-purity resistant starch. Specifically, pepsin, pullulanase, chitosan grafted with disulfide bonds and glutathione-magnetic silica composite material are first added to lotus seed starch in sequence, and then refrigerated and purified. The yield of the obtained resistant starch is not less than 62.72%, and the purity is not less than 98.15%. DETAILED DESCRIPTION

[0020] A method for preparing lotus seed resistant starch comprises the following steps: S1. Preparation of lotus seed starch; S2. Preparation of lotus seed resistant starch: S21, adding an appropriate amount of distilled water to the lotus seed starch to prepare a starch milk solution of a certain concentration, adding pepsin and pullulanase in sequence after autoclaving and gelatinization, and after sufficient enzymolysis, inactivating the enzymes at high temperature; S22, adding chitosan grafted with disulfide bonds to the enzyme-inactivated milk solution, centrifuging and filtering after sufficient reaction; S23, adding glutathione-magnetic silica composite material to the milk solution filtered in S22, and after sufficient reaction under inert atmosphere, centrifuging and filtering; S24, cooling the milk solution filtered in S23 to room temperature, and refrigerating at 4° C. and normal pressure for 12 to 20 hours; then drying, crushing, and sieving to obtain lotus seed crude resistant starch; S25, enzymolyzing the lotus seed crude resistant starch with α-amylase and glucoamylase successively, inactivating the enzymes at high temperature after enzymolysis, and then filtering, washing and drying to obtain lotus seed resistant starch.

[0021] In the above solution, in step S21, lotus seed starch is first formulated into a starch milk solution and gelatinized by autoclaving treatment, so that the starch granules expand, the amylose overflows and undergoes rearrangement through retrogradation to form resistant starch. Then pepsin is added, which can hydrolyze the proteins in lotus seed starch, avoiding the protection of starch by proteins and preventing starch aging, thereby reducing the content of RS3 resistant starch. By adding pullulanase, the α-1,6 glycosidic bonds in amylopectin can be hydrolyzed, so that the branches of amylopectin are cut off, increasing the number of linear starches, reducing the hindrance of amylopectin to the movement of amylose segments, promoting the retrogradation of amylose, and increasing the content of RS3 resistant starch.

[0022] After the amylopectin is debranched by pullulanase, the steric hindrance of amylopectin is reduced, and at the same time, the debranched starch obtained exposes more -OH and reactive sites compared with amylopectin.

[0023] In step S22, by adding chitosan to the milk solution, it can complex with the -OH and reactive sites of the debranched starch, thereby removing part of the debranched starch to improve the purity of RS3 resistant starch. At the same time, the added chitosan has electronegativity, which can precipitate amylopectin, thereby further reducing the content of amylopectin and further increasing the yield of resistant starch.

[0024] Furthermore, since chitosan will also complex with part of the amylose in addition to complexing with the debranched starch, the content of amylose is reduced and the yield of resistant starch is decreased. Based on this, a disulfide bond is grafted onto chitosan in the present invention. The disulfide bond can increase the steric hindrance of chitosan molecules, thereby reducing the contact opportunity between the active sites of amylose and chitosan and minimizing the degree of their complexation to the greatest extent.

[0025] In actual implementation, since the complex of chitosan and debranched starch is colloidal and stably exists in the starch milk solution, if the chitosan grafted with disulfide bonds is not removed, it will form an obstacle between the amylose molecules, reducing the contact opportunity of amylose, thereby reducing the yield of resistant starch, and at the same time, it will seriously reduce the purity of resistant starch.

[0026] Based on this, in step S23, glutathione-magnetic silica composite is added. Among them, magnetic silica can adsorb part of amylopectin (because it has more branches and is more easily adsorbed), and groups such as sulfhydryl, amino, and carboxyl in glutathione can have non-covalent interactions with the amino groups of chitosan grafted with disulfide bonds, thereby realizing the specific adsorption of chitosan grafted with disulfide bonds. In addition, the sulfhydryl group in glutathione can reduce the disulfide bond in chitosan, thereby opening the disulfide bond to reduce the steric hindrance of chitosan molecules and promote the non-covalent interactions between groups such as sulfhydryl, amino, and carboxyl and the amino groups of chitosan, so as to maximize the combination between chitosan grafted with disulfide bonds and glutathione-magnetic silica composite, and then achieve the purpose of separating chitosan grafted with disulfide bonds from the starch solution; in addition, glutathione-magnetic silica composite can also adsorb amino acids after protease hydrolysis, thereby reducing the content of amino acids in the milk solution. In summary, after adding glutathione-magnetic silica composite, the yield and purity of resistant starch can be effectively improved.

[0027] In step S23, an inert atmosphere is limited to avoid the oxidation of sulfhydryl groups, which makes it difficult for them to effectively act on amino groups and effectively reduce disulfide bonds, resulting in incomplete removal of chitosan and thus reducing the yield and purity of resistant starch.

[0028] The milk solution after the action of chitosan grafted with disulfide bonds and glutathione-magnetic silica composite is a milk solution almost free of amylopectin. Based on this, in step S24, the milk solution is directly refrigerated to make a large number of linear starch molecules overflow from the starch granules, realizing retrogradation rearrangement and preparing crude resistant starch.

[0029] Finally, through the purification in step S25, impurities in the resistant starch can be effectively removed and the purity of the resistant starch can be improved.

[0030] In summary, through the optimization and improvement of the existing preparation process, the present invention can effectively improve the yield and purity of resistant starch. Specifically, the yield of the prepared resistant starch is not less than 62.72%, and the purity is not less than 98.15%.

[0031] In order to further improve the yield of resistant starch, in some feasible embodiments of the present application, the concentration of the starch milk solution is further defined, that is, in step S21, the concentration of the prepared starch milk solution is 20-30%. In actual implementation, the concentration of the starch milk is extremely important, which directly determines the yield of resistant starch. Specifically, when its concentration is higher than 30%, it is difficult for starch granules to fully expand and gelatinize. Due to the excessive viscosity of the system and the influence of α-1,6-glycosidic bonds, it is not easy for amylose to approach each other to form resistant starch; when the starch milk is lower than 20%, the probability of free amylose molecules approaching each other decreases, which is not conducive to the formation of resistant starch.

[0032] In order to further improve the yield of resistant starch, in some feasible embodiments of the present application, the gelatinization conditions are further defined, that is, in step S21, the gelatinization treatment conditions are as follows: the pressure-heating gelatinization treatment conditions are: the temperature is 105-120 °C, the pressure is 0.-0.3 MPa, and the time is 8-15 min. When the temperature is too low, the heat of the starch milk solution is insufficient, resulting in incomplete gelatinization and making it difficult to form resistant starch in the later stage; when the temperature is too high, the gel formed by the starch milk degrades, the system becomes thinner, and the molecular movement accelerates, increasing the difficulty of amylose molecules contacting each other to form hydrogen bonds, which is not conducive to the formation of resistant starch.

[0033] In order to further improve the yield and purity of resistant starch, in some feasible embodiments of the present application, the conditions of protease and pullulanase are further defined, that is, the conditions for protease hydrolysis are: pH is 2-2.5, temperature is 30-45 °C, time is 1-2 h, and the enzyme dosage is 5-15 U / g (dry basis starch); the hydrolysis conditions of pullulanase are: pH is 5-6, temperature is 75-85 °C, time is 8-10 h, and the enzyme dosage is 15-25 U / g (dry basis starch).

[0034] In actual implementation, the hydrolysis conditions are extremely important, which directly affect the effect of the system, as well as the final yield and purity of resistant starch. If the addition amount of pullulanase is too much or the action time is too long, it may cause excessive hydrolysis of starch, resulting in a decrease in the amylose / branched chain ratio, and then leading to a decrease in the yield of resistant starch.

[0035] In order to further improve the yield of resistant starch, in some feasible embodiments of the present application, the regulators used for pH adjustment of protease and pullulanase are further defined, that is, the pH regulator used for pH adjustment is a mixed solution of citric acid and sodium citrate with a concentration of 0.2-0.3 mol / L.

[0036] In order to further improve the yield and purity of resistant starch, as some feasible embodiments of the present application, the dosage of chitosan and reaction conditions are further defined. That is, in step S22, the addition amount of chitosan grafted with disulfide bonds is 10-15% of the total mass of the milk solution after enzyme inactivation in step S22; the reaction conditions after adding chitosan grafted with disulfide bonds are: maintaining at 75-85 °C for 4-5 h.

[0037] In order to further improve the yield and purity of resistant starch, as some feasible embodiments of the present application, the dosage of glutathione-magnetic silica composite and process parameters are further defined. That is, in step S23, the added mass of the glutathione-magnetic silica composite is 20-30% of the total weight of the milk solution filtered in S22; after adding the glutathione-magnetic silica composite, ultrasonic treatment is carried out at 30-45 °C for 15 min, and the ultrasonic power is 200-300 w.

[0038] In order to further improve the yield and purity of resistant starch, as some feasible embodiments of the present application, the preparation method of the glutathione-magnetic silica composite is further defined. That is, in step S23, the preparation method of the glutathione-magnetic silica composite is as follows: S2301: Add an organic solvent and glutaraldehyde to magnetic silica with a particle size of 100-400 nm, fully react and then centrifuge, collect the solid and wash it; S2302: Add glutathione, an organic solvent and deionized water to the washed solid, fully react and then centrifuge, take out the solid, and after washing and drying, obtain the glutathione-magnetic silica composite.

[0039] In order to further improve the resistant starch and purity, as some feasible embodiments of the present application, the mass ratio of crude lotus seed resistant starch, α-amylase and glucoamylase is further defined. That is, as some feasible embodiments of the present application, in step S25, the mass ratio of crude lotus seed resistant starch, α-amylase and glucoamylase is 10: (1-1.5): (0.5-1.5).

[0040] The following further elaborates in detail on the preparation method of the lotus seed resistant starch described in the present application in combination with specific embodiments.

[0041] Example 1 A preparation method of lotus seed resistant starch includes the following steps: S1. Preparation of lotus seed starch: After thawing fresh frozen lotus seeds in an airtight environment, add distilled water with a mass twice that of the lotus seeds and blend into lotus seed pulp. Then, pass the pulp through a 100-mesh gauze, add distilled water with a mass twice that of the original pulp for dilution, and let it stand and precipitate at 25°C for 10 h. After the starch has completely settled, discard the supernatant, wash the precipitate with distilled water, redissolve the precipitate, let it stand at 25°C until the starch and water are completely separated, and then discard the supernatant. Finally, wash the precipitate with distilled water and dry it in an oven at 52°C for 4 h to obtain lotus seed starch.

[0042] S2. Preparation of lotus seed resistant starch: S21. Take a certain mass of the lotus seed starch prepared above, add 10 mL of distilled water to prepare a starch milk solution with a mass fraction of 25%. Gelatinize it thoroughly at a temperature of 115°C, a pressure of 0.3 MPa, and a time of 12 min. Then, add pepsin [enzymolysis conditions: pH 2 - 2.5, temperature 45°C, time 1.5 h, enzyme dosage 10 U / g (dry basis starch), enzyme activity 8000 U / g] and pullulanase [enzymolysis conditions: pH 5.5 - 6, temperature 80°C, time 9 h, enzyme dosage 20 U / g (dry basis starch), enzyme activity 1000 U / mL] in sequence. After thorough enzymolysis, inactivate the enzyme in a boiling water bath for 15 min. Among them, the pH regulator used for pepsin and pullulanase is a mixed solution of citric acid and sodium citrate with a concentration of 0.2 mol / L.

[0043] S22. Add chitosan grafted with disulfide bonds with a mass of 15% of the total mass of the enzyme-inactivated milk solution to the enzyme-inactivated milk solution, and keep it at 75°C for 4 h. Then, centrifuge and filter.

[0044] Among them, the preparation method of chitosan grafted with disulfide bonds is as follows: Add 1.8 g of chitosan to 70 mL of deionized water, then add 1 mol / L acetic acid to adjust the pH to 3, and keep stirring until the chitosan is completely dissolved. Then, add deionized water to make the volume up to 90 mL to obtain a 20 g / L chitosan solution. Then, based on 90 mL of the 20 g / L chitosan solution, calculate and weigh α-lipoic acid according to the molar ratio of a single chitosan repeating unit to α-lipoic acid of 3:1. Add α-lipoic acid and the same mass of anhydrous Na 2 SO 3 to 30 mL of deionized water respectively to obtain an α-lipoic acid solution. Add 5.6 mmol / L EDC, stir at room temperature for 1 h, then add the above 90 mL of chitosan solution, adjust the pH of the solution to 6 with 0.1 mol / L NaOH, and then make the volume up to 150 mL with deionized water. Finally, stir at room temperature for 4 h to obtain chitosan grafted with disulfide bonds.

[0045] S23. Add glutathione-magnetic silica composite material accounting for 25% of the total weight of the milk solution filtered in S22 to the milk solution filtered in S22, ultrasonicate at 45 °C for 15 min in an inert atmosphere (such as nitrogen), with an ultrasonic power of 300 W, and then centrifuge and filter.

[0046] Among them, the preparation method of the glutathione-magnetic silica composite material is as follows: S2301. Add 1.5 g of nano-Fe 3 O 4 to 600 mL of ethanol, then add 15 mL of water and 15 mL of ammonia water, ultrasonicate at a temperature of 40 °C and a power of 300 W for 30 min; then add 4 mL of TEOS, and stir and react at 70 °C for 6 h, then perform magnetic separation, wash the precipitate with water, and dry it in vacuum to obtain magnetic silica.

[0047] S2302. Take 0.9 g of magnetic silica and add it to 45 mL of ethanol, ultrasonicate at room temperature for 30 min (power: 300 W), then add 0.9 mL of 50% glutaraldehyde, stir at 55 °C for 4 h, cool, perform magnetic separation, discard the supernatant, and collect the solid product.

[0048] S2303. Wash the solid product 3 times with deionized water and ethanol respectively, then soak the washed product in an appropriate amount of ethanol solution for 6 h, and finally perform magnetic separation and discard the supernatant.

[0049] S2304. Add 2 g of glutathione, 50 mL of absolute ethanol, and 80 mL of deionized water to the solid product in step S2303, then stir at 55 °C for 2 h, then perform magnetic separation and wash 3 times with ethanol and deionized water respectively, and finally dry at 60 °C for 4 h to obtain the glutathione-magnetic silica composite material.

[0050] S24. Cool the milk solution filtered in S23 to room temperature, refrigerate it at 4 °C and normal pressure for 20 h; then dry it at 53 °C for 4 h, and then pulverize it and pass it through a 100-mesh sieve to obtain crude lotus seed resistant starch; S25. Add distilled water with a mass three times that of the crude lotus seed resistant starch, then adjust the pH to 5.5, add α-amylase (activity 2000 U / g), and treat at 60 °C for 2 h. Then cool to room temperature and add glucoamylase (enzyme activity 5000 U / g), and treat at 55 °C for 1 h. Then heat the enzymolysis solution to 100 °C and maintain for 10 min. Then cool to room temperature, centrifuge to obtain the precipitate, mix the precipitate with distilled water with a volume four times that of the precipitate, and stir at 20 - 30 °C for 1 h. Then centrifuge to obtain the precipitate, wash the precipitate three times with 95% ethanol, and dry at 53 °C for 4 h to obtain lotus seed resistant starch. Among them, the mass ratio of the crude lotus seed resistant starch, α-amylase, and glucoamylase is 10:1:0.9.

[0051] Example 2 A method for preparing lotus seed resistant starch, comprising the following steps: S1. Preparation of lotus seed starch (same as Example 1): S2. Preparation of lotus seed resistant starch: S21. Take a certain mass of the lotus seed starch and add 10 mL of distilled water to prepare a starch milk solution with a mass fraction of 25%. Fully gelatinize at a temperature of 117 °C, a pressure of 0.2 MPa, and a time of 13 min. Then sequentially add pepsin [enzymolysis conditions: pH 2 - 2.5, temperature 45 °C, time 1.7 h, enzyme dosage 10 U / g (dry basis starch), enzyme activity 8000 U / g] and pullulanase [enzymolysis conditions: pH 5.5 - 6, temperature 80 °C, time 9.2 h, enzyme dosage 20 U / g (dry basis starch), enzyme activity 1000 U / mL]. After sufficient enzymolysis, inactivate the enzyme in a boiling water bath for 15 min. Among them, the pH regulator used for pepsin and pullulanase is a mixed solution of citric acid and sodium citrate with a concentration of 0.2 mol / L.

[0052] S22. Add chitosan grafted with disulfide bonds (preparation method same as Example 1) accounting for 13% of the total mass of the enzyme-inactivated milk solution to the enzyme-inactivated milk solution, and maintain at 80 °C for 4.2 h. Then centrifuge and filter.

[0053] S23. Add glutathione-magnetic silica composite material (preparation method same as Example 1) accounting for 25% of the total weight of the milk solution filtered in S22 to the milk solution filtered in S22, ultrasonicate in an inert atmosphere (such as nitrogen) at 40 °C for 15 min, with an ultrasonic power of 300 w. Then centrifuge and filter.

[0054] S24. Cool the milk solution filtered in S23 to room temperature, and refrigerate at 4 °C and normal pressure for 22 h. Then dry at 50 °C for 4.5 h, and then obtain crude lotus seed resistant starch after pulverization and passing through a 100-mesh sieve; S25. Add distilled water with a mass three times that of the crude lotus seed resistant starch, then adjust the pH to 5.5, add α-amylase (activity 2000 U / g), and treat at 60 °C for 2.5 h. Then cool to room temperature and add glucoamylase (enzyme activity 5000 U / g), and treat at 55 °C for 1.5 h. Then heat the enzymatic hydrolysate to 100 °C and maintain for 10 min. Then cool to room temperature, centrifuge to obtain the precipitate, mix the precipitate with distilled water with a volume four times that of the precipitate, and stir at 20 - 30 °C for 1 h. Then centrifuge to obtain the precipitate, and wash the precipitate three times with 95% ethanol, and dry at 55 °C for 4 h to obtain lotus seed resistant starch. Among them, the mass ratio of the crude lotus seed resistant starch, α-amylase, and glucoamylase is 10:1.2:1.2.

[0055] Comparative Example 1 Compared with Example 1, chitosan grafted with disulfide bonds and glutathione-magnetic silica composite are not added, that is, steps S22 and S23 are removed.

[0056] Comparative Example 2 Compared with Example 1, the chitosan grafted with disulfide bonds is changed to chitosan.

[0057] Comparative Example 3 Compared with Example 1, the glutathione-magnetic silica composite is not added.

[0058] The yields and purities of the lotus seed resistant starch obtained in Examples 1 - 2 and Comparative Examples 1 - 3 are shown in Table 1.

[0059] Among them, the calculation method of the resistant starch yield is the prior art, and specifically refer to the following technology: Add the prepared lotus seed resistant starch to a citric acid-disodium hydrogen phosphate buffer solution with a pH of 6.0, then add an excessive amount of thermostable α-amylase (500 U / g), and enzymatically hydrolyze at 90 °C for 2 h. Adjust the pH to 4.0 - 4.5 with 4 mol / L citric acid, add an excessive amount of glucoamylase (5000 U / L), and enzymatically hydrolyze at 60 °C for 1 h. Then centrifuge the sample at 4000 r / min for 10 min, discard the supernatant, wash the precipitate with distilled water, and centrifuge, repeating 2 times. Add 5 mL of 2 mol / L KOH to the precipitate, shake vigorously for 30 min to fully dissolve the resistant starch. Adjust the pH to 4.0 - 4.5 with 1 mol / L acetic acid solution, add an excessive amount of glucoamylase, and enzymatically hydrolyze at 60 °C for 1 h. Centrifuge the sample at 4000 r / min for 10 min, collect the supernatant into a 100 mL volumetric flask, wash the precipitate with distilled water, centrifuge, repeat 2 times, combine the supernatant, and finally make up the volume. Determine its reducing sugar content by the DNS method.

[0060] The calculation formula for the yield of resistant starch is: Yield (%) = [(content of reducing sugar (g) × 0.9) / dry basis mass of lotus seed starch (g)] × 100%.

[0061] Table 1: It can be seen from Table 1 that: the yield of resistant starch prepared by the method in the present invention (only some process parameters are different between Example 1 and Example 2) is not less than 62.72%, and the purity is not less than 98.15%. Compared with Comparative Examples 1 to 3, both the yield and the purity are significantly improved.

Claims

1. A method for preparing lotus seed resistant starch, characterized in that: The steps include: S1. Preparation of lotus seed starch; S2. Preparation of lotus seed resistant starch: S21, adding an appropriate amount of distilled water to the lotus seed starch to prepare a starch milk solution of a certain concentration, adding pepsin and pullulanase in sequence after autoclaving and gelatinization, and after sufficient enzymolysis, inactivating the enzymes at high temperature; S22, adding chitosan grafted with disulfide bonds to the enzyme-inactivated milk solution, centrifuging and filtering after sufficient reaction; S23, adding glutathione-magnetic silica composite material to the milk solution filtered in S22, and after sufficient reaction under inert atmosphere, centrifuging and filtering; S24, refrigerating the milk solution filtered in S23 at 4° C. and normal pressure for 12-20 hours; then drying, crushing and sieving to obtain lotus seed crude resistant starch; S25, enzymolyzing the lotus seed crude resistant starch with α-amylase and glucoamylase successively, inactivating the enzymes at high temperature after enzymolysis, and then filtering, washing and drying to obtain lotus seed resistant starch.

2. The method for preparing lotus seed resistant starch according to claim 1, characterized in that: In step S21, the concentration of the prepared starch milk solution is 20-30%.

3. The method for preparing lotus seed resistant starch according to claim 1, characterized in that: In step S21, the autoclave gelatinization treatment conditions are: temperature of 105-120°C, pressure of 0.1-0.3 MPa, and time of 8-15 min.

4. The method for preparing lotus seed resistant starch according to claim 1, characterized in that: In step S21, the conditions for the protease hydrolysis are: pH 2-2.5, temperature 30-45°C, time 1-2h, and enzyme dosage 5-15U / g (dry basis starch); the conditions for the pullulanase hydrolysis are: pH 5-6, temperature 75-85°C, time 8-10h, and enzyme dosage 15-25U / g (dry basis starch).

5. The method for preparing lotus seed resistant starch according to claim 4, characterized in that: The pH adjuster used for pH adjustment is a mixed solution of citric acid and sodium citrate at 0.2 to 0.3 mol / L.

6. The method for preparing lotus seed resistant starch according to claim 1, characterized in that: In step S22, the amount of chitosan grafted with disulfide bonds added is 10-15% of the total mass of the latex solution after the enzyme is inactivated in step S22; the reaction conditions after adding chitosan grafted with disulfide bonds are: maintaining at 75-85° C. for 4-5 hours.

7. The method for preparing lotus seed resistant starch according to claim 1, characterized in that: In step S23, the mass of the glutathione-magnetic silica composite material added is 20-30% of the total weight of the latex solution filtered in S22; after adding the glutathione-magnetic silica composite material, ultrasonic treatment is performed at 30-45° C. for 15 minutes, and the ultrasonic power is 200-300w.

8. The method for preparing lotus seed resistant starch according to claim 7, characterized in that: In step S23, the preparation method of the glutathione-magnetic silica composite material is as follows: S2201: adding an organic solvent and glutaraldehyde to magnetic silica with a particle size of 100 to 400 nm, centrifuging after sufficient reaction, collecting the solid and washing; S2202: adding glutathione, an organic solvent and deionized water to the washed solid, centrifuging after sufficient reaction, taking out the solid, and washing and drying it to obtain a glutathione-magnetic silica composite material.

9. The method for preparing lotus seed resistant starch according to claim 1, characterized in that: In step S25, the mass ratio of lotus seed crude resistant starch, α-amylase and glucoamylase is 10:(1-1.5):(0.5-1.5).

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