Immobilized compound enzyme for high-yield production of maltotetraose, hydrolyzed plant starch with soothing and itching relieving effects, and preparation method and application of immobilized compound enzyme

Abstract

The invention relates to the technical field of daily chemicals, and particularly discloses an immobilized compound enzyme for high-yield production of maltotetraose, hydrolyzed plant starch with soothing and itching relieving effects, and a preparation method and application of the immobilized compound enzyme. The compound enzyme is immobilized, the immobilized compound enzyme capable of producing maltotetraose at high yield is prepared, the problems that the compound enzyme is poor in stability and prone to inactivation are solved, the enzymolysis reaction condition is widened, industrial production of maltotetraose prepared through an enzymolysis method is facilitated, and the production cost of maltotetraose is reduced; the preparation method of the hydrolyzed plant starch and the like are optimized, a variable-temperature enzymolysis technology is innovatively adopted, the problems that alpha-starch and beta-amylase are not specific in enzyme catalysis and starch is randomly decomposed in the reaction period are solved, generation of by-products is reduced, and the finally prepared hydrolyzed plant starch is high in maltotetraose ratio, good in hydrolysis effect and good in hydrolysis effect. The skin-care composition is suitable for being used as a raw material of cosmetics.

Description

Technical Field

[0001] This application relates to the field of daily chemical products technology, and in particular to an immobilized complex enzyme with high maltotetrasaccharide production, hydrolyzed plant starch with soothing and antipruritic effects, its preparation method and application. Background Technology

[0002] Maltotetraose (M4) is a functional oligosaccharide composed of four glucose molecules linearly linked by α-1,4 glycosidic bonds, and it has broad application potential in sports nutrition foods, daily chemicals, and other fields. Compared to other short-chain sugars, such as maltose (M2) or maltotriose (M3), maltotetraose, as a novel functional oligosaccharide, can exhibit better functional properties under certain conditions, such as better moisturizing properties and various biological activities like oxidation, thus meeting consumers' dual needs for cosmetic efficacy and safety.

[0003] Common methods for preparing maltodextrose include acid hydrolysis and enzymatic hydrolysis. Acid hydrolysis requires strong acids such as hydrochloric acid and sulfuric acid to break the α-1,4 glycosidic bonds of starch at high temperatures (100-120℃), generating a mixture of oligosaccharides. Acid hydrolysis is highly random, yielding less than 20% maltodextrose, necessitating chromatographic separation or membrane filtration purification, resulting in extremely high costs. Therefore, current commercially available methods for preparing maltodextrose primarily focus on enzymatic hydrolysis. This method offers advantages such as a wide range of substrate selectivity (e.g., starch, maltodextrin, amylose) and relatively high conversion efficiency. However, enzymatic hydrolysis suffers from poor enzyme stability and easy inactivation, making the reaction conditions more demanding and unsuitable for large-scale production. Furthermore, the maltodextrose content in the enzymatically prepared product is typically around 40%, representing a relatively low proportion of the enzymatic hydrolysis product. Summary of the Invention

[0004] To address the issues of poor enzyme stability and easy inactivation during the enzymatic hydrolysis of maltotetrasaccharides, and the low proportion of maltotetrasaccharides in the product, this application aims to overcome the shortcomings of the prior art by providing an immobilized complex enzyme with high maltotetrasaccharide yield, hydrolyzed plant starch with soothing and antipruritic effects, its preparation method, and its applications. This application provides an immobilized complex enzyme that can hydrolyze corn starch, wheat starch, etc., to prepare hydrolyzed plant starch with a high maltotetrasaccharide content.

[0005] To achieve the above objectives, the technical solution adopted in this application is as follows:

[0006] This application provides a method for preparing an immobilized complex enzyme that produces high levels of maltotetraose, comprising the following steps:

[0007] S1. Add calcium salt and organic ligand to organic solvent, mix and heat, then wash and dry the obtained precipitate to obtain immobilized enzyme carrier.

[0008] S2. Add the immobilized enzyme carrier obtained in step S1 to water, then add the complex enzyme, stir the reaction and let it stand, then wash and dry the precipitate to obtain the immobilized complex enzyme with high maltotetrasaccharide production.

[0009] This application immobilizes a complex enzyme used for enzymatic hydrolysis of plant starch to prepare an immobilized complex enzyme capable of high-yield maltotetrasaccharide. This solves the problems of poor stability and easy inactivation of the complex enzyme, relaxes the conditions for enzymatic hydrolysis, facilitates the industrial production of maltotetrasaccharide by enzymatic hydrolysis, and reduces the production cost of maltotetrasaccharide.

[0010] In a preferred embodiment of the method for preparing the immobilized complex enzyme with high maltotetrasaccharide yield described in this application, in step S1, the calcium salt includes calcium chloride and / or calcium nitrate.

[0011] And / or, the organic ligand includes terephthalic acid;

[0012] And / or, the organic solvent includes at least one of ethanol, glycerol, propylene glycol, and butanediol;

[0013] In step S2, the complex enzyme includes α-amylase and β-amylase.

[0014] In the technical solution of this application, the Ca in the calcium salt 2+ It can maintain the activity and stability of α-amylase.

[0015] Complex enzymes include α-amylase and β-amylase. α-amylase is an endonuclease that randomly hydrolyzes the α-1,4-glycosidic bonds within starch molecules, but not the α-1,6-glycosidic bonds. It can break down starch into various oligosaccharides, and its advantage is high hydrolysis efficiency, capable of degrading some starch into maltodextrins in a relatively short time. β-amylase, on the other hand, is an exonuclease that continuously hydrolyzes the α-1,4-glycosidic bonds from the non-reducing end of the polysaccharide chain. It cannot break the α-1,6 bonds. When encountering amylopectin, it cleaves two glucose units at a time (generating maltose), and can continuously hydrolyze starch chains from the non-reducing end to produce maltose.

[0016] This application immobilizes α-amylase and β-amylase, which are used for enzymatic hydrolysis of plant starch, to prepare an immobilized composite enzyme capable of high maltotetrasaccharide production. This solves the problems of poor stability and easy inactivation of α-amylase and β-amylase, relaxes the conditions for enzymatic hydrolysis, and is beneficial for the industrial production of maltotetrasaccharide by enzymatic hydrolysis, thereby reducing the production cost of maltotetrasaccharide. In the enzymatic hydrolysis process of this application, α-amylase and β-amylase work synergistically, generating more maltotetrasaccharide during the hydrolysis process.

[0017] This application utilizes the aforementioned calcium salts and organic ligands to better improve the reusability, heat resistance, and acid resistance of the immobilized complex enzyme. Experiments in this application have shown that using α-amylase and β-amylase as the immobilized enzymes yields the best results; the interaction between these two enzymes enhances the reusability, heat resistance, and acid resistance of the final immobilized complex enzyme. Furthermore, the use of the aforementioned calcium salts, organic ligands, and complex enzymes can also better increase the maltotetrasaccharide content in hydrolyzed plant starch.

[0018] In a preferred embodiment of the method for preparing the immobilized complex enzyme with high maltotetrasaccharide yield described in this application, in step S1, the molar ratio of calcium element in the calcium salt to the organic ligand is (1-1.8):1;

[0019] And / or, the mass ratio of the calcium salt to the organic solvent is 1:(30-80);

[0020] In step S2, the mass ratio of the immobilized enzyme carrier to water is 1:(100-300);

[0021] And / or, the mass ratio of the immobilized enzyme carrier to the complex enzyme is 1:(0.05-0.3).

[0022] Preferably, the mass ratio of α-amylase to β-amylase is 1:(2-4).

[0023] The above-mentioned formulation can better improve the reusability, heat resistance and acid resistance of the immobilized complex enzyme, and can also increase the content of maltodextrose in hydrolyzed plant starch, ultimately improving the soothing and antipruritic effects of the final product.

[0024] In a preferred embodiment of the method for preparing the immobilized complex enzyme with high maltotetrasaccharide yield described in this application, the mixing process in step S1 includes stirring and / or ultrasonic treatment.

[0025] The stirring conditions are: a rotation speed of 120–300 rpm and a time of 10–30 min;

[0026] And / or, the conditions for the ultrasonic treatment are: power of 300-600W and time of 8-20min;

[0027] And / or, the conditions for the heat treatment are: temperature of 90-140°C and time of 40-60h.

[0028] In a preferred embodiment of the method for preparing the high-yield maltotetrasaccharide immobilized complex enzyme described in this application, the washing step S1 includes rinsing with deionized water 2 to 5 times.

[0029] In a preferred embodiment of the method for preparing the high-yield maltotetrasaccharide immobilized complex enzyme described in this application, the drying conditions in step S1 are: a temperature of 50–70°C and a time of 5–10 h.

[0030] In a preferred embodiment of the method for preparing the immobilized complex enzyme with high maltotetrasaccharide yield described in this application, the operation of adding the complex enzyme in step S2 includes adding it in several installments, with the number of installments being 3 to 6.

[0031] As a preferred embodiment of the method for preparing the high-yield maltotetrasaccharide immobilized complex enzyme described in this application, in step S2, the stirring reaction conditions are: a rotation speed of 150-200 rpm and a time of 30-60 min.

[0032] And / or, the settling time is 10 to 30 minutes;

[0033] And / or, the cleaning includes rinsing with deionized water 2 to 5 times;

[0034] And / or, the drying conditions are: a temperature of 50–70°C and a time of 5–10 hours.

[0035] This application also provides an immobilized complex enzyme with high maltotetraose production prepared by the above-mentioned method for preparing immobilized complex enzymes.

[0036] The immobilized complex enzyme obtained using the preparation method of this application exhibits excellent stability and reusability, with minimal loss of enzyme activity even after five reuses. Furthermore, the immobilized complex enzyme of this application demonstrates excellent heat and acid resistance. Using the immobilized complex enzyme of this application can also better improve the soothing and antipruritic efficacy of the final product.

[0037] This application also provides the application of the above-mentioned immobilized complex enzyme with high maltotetrasaccharide content in the preparation of hydrolyzed plant starch with high maltotetrasaccharide content.

[0038] This application also provides a method for preparing hydrolyzed plant starch with soothing and antipruritic effects, comprising the following steps:

[0039] 1) Mix plant starch with water, add pH adjuster, heat and stir to obtain a suspension;

[0040] 2) Add the immobilized complex enzyme of high-yield maltotetrasaccharide to the suspension, perform stepwise enzymatic hydrolysis, and remove the immobilized complex enzyme of high-yield maltotetrasaccharide to obtain the enzymatic hydrolysate.

[0041] 3) The enzymatic hydrolysate is subjected to membrane concentration treatment to obtain hydrolyzed plant starch with soothing and antipruritic effects.

[0042] In the technical solution of this application, the hydrolyzed plant starch obtained by the preparation method of this application has a very high maltodextrose content, reaching over 50% in the final product. If unimmobilized α-amylase and β-amylase are directly used to enzymatically hydrolyze the plant starch, the enzymatic hydrolysis process involves multiple temperature variations. During this process, the unimmobilized α-amylase and β-amylase have very low heat resistance and cannot effectively perform enzymatic hydrolysis, resulting in a very low maltodextrose content in the final product. Furthermore, when using unimmobilized α-amylase and β-amylase to enzymatically hydrolyze plant starch, the instability of α-amylase and β-amylase during the hydrolysis process is very poor, leading to a poor enzymatic hydrolysis effect on the plant starch and a poor soothing and antipruritic effect in the final product.

[0043] Furthermore, hydrolyzed plant starch can effectively inhibit the influx of calcium ions. Experiments showed that, compared with the model control group, hydrolyzed plant starch could downregulate the relative expression level of IL-6 mRNA by 51.75%, indicating that the hydrolyzed plant starch of this application can effectively inhibit the expression of the pro-inflammatory cytokine IL-6 gene. Therefore, the hydrolyzed plant starch of this application exhibits excellent soothing and antipruritic effects.

[0044] In a preferred embodiment of the method for preparing hydrolyzed plant starch according to this application, in step 1), the plant starch includes corn starch and / or wheat starch;

[0045] And / or, the mass ratio of the plant starch to water is 1:(3-5);

[0046] And / or, the pH adjuster includes at least one of HCl solution, citric acid solution, and lactic acid solution; the concentration of the pH adjuster is 0.5–8 wt%.

[0047] In a preferred embodiment of the method for preparing hydrolyzed plant starch according to this application, in step 1), a pH adjuster is added to adjust the pH of the mixture of plant starch and water to 4.5-6.0.

[0048] In a preferred embodiment of the method for preparing hydrolyzed plant starch according to this application, in step 1), the heating and stirring temperature is 75-85°C, and the heating and stirring time is 60-100 min.

[0049] In a preferred embodiment of the method for preparing hydrolyzed plant starch as described in this application, in step 2), the amount of the immobilized complex enzyme for high-yield maltotetrasaccharide is 1-3% of the mass of the plant starch.

[0050] In a preferred embodiment of the method for preparing hydrolyzed plant starch according to this application, step 2) includes the step of stepwise enzymatic hydrolysis:

[0051] 21) Cool the suspension to 55-65°C, stir at 80-120 rpm, and keep warm for 25-45 min to obtain a first enzymatic hydrolysate;

[0052] 22) Cool the primary enzymatic hydrolysate to 35-45°C, stir at 80-120 rpm, and keep warm for 40-60 min to obtain the secondary enzymatic hydrolysate;

[0053] 23) Cool the secondary enzymatic hydrolysate to 15-25°C, stir at 80-120 rpm, keep warm for 15-30 min, and then remove the immobilized complex enzyme of the high-yield maltotetrasaccharide to obtain the enzymatic hydrolysate.

[0054] This application employs variable-temperature enzymatic hydrolysis technology to hydrolyze plant starch. During the enzymatic hydrolysis process, the temperature is gradually reduced from high to low, precisely controlling the activity and reaction rate of the immobilized complex enzyme. This reduces the problems of nonspecific catalysis by α-amylase and β-amylase, random decomposition during the reaction, and easy generation of by-products, and further increases the content of maltotetrasaccharide in the hydrolysate.

[0055] The enzymatic hydrolysis process has a significant impact on the maltotetraose content in hydrolyzed plant starch. This application adopts a stepped temperature variable enzymatic hydrolysis method, which can significantly improve the conversion rate of maltotetraose in plant starch and increase the maltotetraose content.

[0056] As a preferred embodiment of the method for preparing hydrolyzed plant starch according to this application, in step 3), the membrane concentration treatment conditions include: the filtration membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 400 Da-600 Da, the inlet pressure is 0.4 MPa to 0.8 MPa, and the concentration is to 40-60% of the original volume of the enzymatic hydrolysate.

[0057] In this application, membrane concentration treatment can remove some impurities from the enzymatic hydrolysate, increase the concentration of various active substances in the final product, and enhance the skin care effect of subsequent hydrolyzed plant starch.

[0058] This application also provides hydrolyzed plant starch with soothing and antipruritic effects prepared by the above-mentioned method of preparing hydrolyzed plant starch.

[0059] This application also provides the application of the above-mentioned hydrolyzed plant starch in the preparation of cosmetics, wherein the amount of the hydrolyzed plant starch with soothing and antipruritic effects added to the cosmetics is 0.1 to 30 wt%.

[0060] This application optimizes the preparation methods of immobilized complex enzymes and hydrolyzed plant starch, and innovatively adopts variable-temperature enzymatic hydrolysis technology to reduce the problem of nonspecific catalysis by α-starch and β-amylase and random decomposition of starch during the reaction, thereby reducing the generation of by-products. The hydrolyzed plant starch with soothing and antipruritic effects has a high proportion of maltodextrose and good skin care effects, making it suitable as a raw material for cosmetics.

[0061] This application also uses an immobilized complex enzyme to hydrolyze plant starch, and the resulting maltotetrasaccharide has a good soothing effect, can inhibit abnormal influx of calcium ions into cells and IL-6 gene expression, and is suitable as an active ingredient in cosmetics.

[0062] Compared with the prior art, this application has the following beneficial effects:

[0063] This application provides an immobilized complex enzyme with high maltodextrose production, hydrolyzed plant starch with soothing and antipruritic effects, its preparation method, and its applications. This application immobilizes the complex enzyme to prepare an immobilized complex enzyme capable of high maltodextrose production, solving the problems of poor enzyme stability and easy inactivation. It also relaxes the conditions for enzymatic hydrolysis, facilitating the industrial production of maltodextrose through enzymatic hydrolysis and reducing its production cost. Furthermore, this application optimizes the preparation method of hydrolyzed plant starch, innovatively employing variable-temperature enzymatic hydrolysis technology. This reduces the problem of nonspecific catalysis by α-amylase and β-amylase, which randomly decomposes starch during the reaction, thus reducing the generation of byproducts. The resulting hydrolyzed plant starch not only has a high maltodextrose content but also good skincare effects, making it suitable as a raw material for cosmetics. This application also uses an immobilized complex enzyme to hydrolyze plant starch, and the resulting maltodextrose has excellent soothing effects, inhibiting abnormal calcium ion influx and IL-6 gene expression, making it suitable as a functional ingredient in cosmetics. Attached Figure Description

[0064] Figure 1 This is a fluorescence staining image of the negative control group in Experiment 5;

[0065] Figure 2 This is a fluorescence staining image of the model control group in Experimental Example 5;

[0066] Figure 3 This is a fluorescent staining image from Example 12 of Experimental Example 5. Detailed Implementation

[0067] To better illustrate the purpose, technical solution, and advantages of this application, the following description will be provided in conjunction with the accompanying drawings and specific embodiments.

[0068] In the following examples and comparative examples, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. Furthermore, the raw materials used in each parallel experiment are the same.

[0069] The CAS numbers for α-amylase, β-amylase, pullulanase, and pullulanase used in this application are 9000-90-2, 9000-91-3, and 9075-68-7, respectively. Commercially available enzymes with CAS numbers 9000-90-2, 9000-91-3, and 9075-68-7 can also be used in this application.

[0070] In the proposed method for preparing hydrolyzed plant starch, the immobilized complex enzyme for high-yield maltotetrasaccharide is a solid with relatively large particles, which can be removed by conventional solid-liquid separation methods such as tweezers, gauze filtration, or centrifugation.

[0071] The corn starch and hydrolyzed starch used in this application are commercially available products.

[0072] Example 1, a high-maltotetraose-producing immobilized complex enzyme and a method for preparing the same

[0073] This embodiment provides an immobilized complex enzyme that produces high levels of maltotetraose, and its preparation method includes:

[0074] S1. 3.675 g of calcium chloride dihydrate (CaCl2·2H2O) and 3.322 g of terephthalic acid (H2BDC) (molar ratio of 1.25:1) were added to 280 mL of ethanol, stirred at 150 rpm for 15 min, sonicated at 500 W for 10 min, mixed evenly, and heated at 120 °C for 48 h. The obtained precipitate was washed three times with deionized water and dried at 60 °C for 8 h to obtain the immobilized enzyme carrier CaMT.

[0075] S2. Add 0.3g of immobilized enzyme carrier CaMT to 60g of water, and add 0.03g of a complex enzyme containing α-amylase and β-amylase (mass ratio of α-amylase to β-amylase is 1:3) in four portions. Stir at 180rpm for 45min, let stand for 15min, remove the precipitate, wash the obtained precipitate three times with deionized water, and dry at 60℃ for 8h to obtain the immobilized complex enzyme with high maltotetrasaccharide production.

[0076] Examples 2-9, a high-maltotetraose-producing immobilized complex enzyme and a method for preparing the same

[0077] Examples 2-9 provide an immobilized complex enzyme with high maltotetrasaccharide production and its preparation method, respectively. The difference between them and Example 1 is that some parameters in step S1 or step S2 are different from those in Example 1. The differences between Examples 2-9 and Example 1 are shown in Table 1 below.

[0078] Table 1

[0079]

[0080]

[0081] Example 10, a high-maltotetraose-producing immobilized complex enzyme and a method for preparing the same

[0082] This embodiment provides an immobilized complex enzyme that produces high levels of maltotetraose, and its preparation method includes:

[0083] S1. 4.410 g of calcium chloride dihydrate (CaCl2·2H2O) and 3.322 g of terephthalic acid (H2BDC) (molar ratio of 1.5:1) were added to 280 mL of glycerol and stirred at 120 rpm for 30 min. The mixture was then sonicated at 300 W for 20 min. After mixing thoroughly, the mixture was heated at 100 °C for 60 h. The precipitate was washed 5 times with deionized water and dried at 50 °C for 10 h to obtain the immobilized enzyme carrier CaMT.

[0084] S2. Add 0.3g of immobilized enzyme carrier CaMT to 30g of water, and add 0.03g of a complex enzyme containing α-amylase and β-amylase (mass ratio of α-amylase to β-amylase is 1:2.5) in four portions. Stir at 200rpm for 30min, let stand for 10min, remove the precipitate, wash the obtained precipitate five times with deionized water, and dry at 50℃ for 10h to obtain the immobilized complex enzyme with high maltotetrasaccharide production.

[0085] Example 11, a high-maltotetraose-producing immobilized complex enzyme and a method for preparing the same

[0086] This embodiment provides an immobilized complex enzyme that produces high levels of maltotetraose, and its preparation method includes:

[0087] S1. 4.116 g of calcium chloride dihydrate (CaCl2·2H2O) and 3.322 g of terephthalic acid (H2BDC) (molar ratio of 1.4:1) were added to 280 mL of 1,2-butanediol and stirred at 300 rpm for 10 min. The mixture was then sonicated at 600 W for 8 min. After mixing thoroughly, the mixture was heated at 130 °C for 40 h. The precipitate was washed twice with deionized water and dried at 70 °C for 5 h to obtain the immobilized enzyme carrier CaMT.

[0088] S2. Add 0.3g of immobilized enzyme carrier CaMT to 90g of water, and add 0.24g of a complex enzyme containing α-amylase and β-amylase (mass ratio of α-amylase to β-amylase is 1:3.5) in four portions. Stir at 150rpm for 60min, let stand for 30min, remove the precipitate, wash the obtained precipitate twice with deionized water, and dry at 70℃ for 5h to obtain the immobilized complex enzyme with high maltotetrasaccharide production.

[0089] Comparative Example 1, an immobilized complex enzyme and a method for preparing the same

[0090] This comparative example provides an immobilized complex enzyme, the preparation method of which includes:

[0091] S1. 3.784 g of aluminum chloride hexahydrate (AlCl3·6H2O) and 3.322 g of terephthalic acid (H2BDC) (molar ratio of 1.25:1) were added to 280 mL of ethanol, stirred at 150 rpm for 15 min, sonicated at 500 W for 10 min, mixed evenly, and heated at 120 °C for 48 h. The obtained precipitate was washed three times with deionized water and dried at 60 °C for 8 h to obtain the immobilized enzyme carrier AlMT.

[0092] S2. Add 0.3g of immobilized enzyme carrier AlMT to 60g of water, and add 0.03g of a complex enzyme containing α-amylase and β-amylase (mass ratio of α-amylase to β-amylase is 1:3) in four portions. Stir at 180rpm for 45min, let stand for 15min, remove the precipitate, wash the obtained precipitate three times with deionized water, and dry at 60℃ for 8h to obtain the immobilized complex enzyme.

[0093] Comparative Example 2, an immobilized complex enzyme and a method for preparing the same

[0094] This comparative example provides an immobilized complex enzyme, the preparation method of which includes:

[0095] S1. Add 3.408 g of zinc chloride (ZnCl2) and 3.322 g of terephthalic acid (H2BDC) (molar ratio of 1.25:1) to 280 mL of ethanol, stir at 150 rpm for 15 min, sonicate at 500 W for 10 min, mix evenly, heat at 120 °C for 48 h, wash the obtained precipitate three times with deionized water, and dry at 60 °C for 8 h to obtain the immobilized enzyme carrier ZnMT;

[0096] S2. Add 0.3g of immobilized enzyme carrier ZnMT to 60g of water, and add 0.03g of a complex enzyme containing α-amylase and β-amylase (mass ratio of α-amylase to β-amylase is 1:3) in four portions. Stir at 180rpm for 45min, let stand for 15min, remove the precipitate, wash the obtained precipitate three times with deionized water, and dry at 60℃ for 8h to obtain the immobilized complex enzyme.

[0097] Comparative Example 3, an immobilized complex enzyme and a method for preparing the same

[0098] This comparative example provides an immobilized complex enzyme, the preparation method of which includes:

[0099] S1. 3.675 g of calcium chloride dihydrate (CaCl2·2H2O) and 4.203 g of trimesic acid (H3BTC) (molar ratio of 1.25:1) were added to 280 mL of ethanol, stirred at 150 rpm for 15 min, sonicated at 500 W for 10 min, mixed evenly, and heated at 120 °C for 48 h. The obtained precipitate was washed three times with deionized water and dried at 60 °C for 8 h to obtain the immobilized enzyme carrier CaNS.

[0100] S2. Add 0.3g of immobilized enzyme carrier CaNS to 60g of water, and add 0.03g of a complex enzyme containing α-amylase and β-amylase (mass ratio of α-amylase to β-amylase is 1:3) in four portions. Stir at 180rpm for 45min, let stand for 15min, remove the precipitate, wash the obtained precipitate three times with deionized water, and dry at 60℃ for 8h to obtain the immobilized complex enzyme.

[0101] Comparative Example 4, an immobilized complex enzyme and a method for preparing the same

[0102] This comparative example provides an immobilized complex enzyme, the preparation method of which includes:

[0103] S1. 3.675 g of calcium chloride dihydrate (CaCl2·2H2O) and 3.322 g of terephthalic acid (H2BDC) (molar ratio of 1.25:1) were added to 280 mL of ethanol, stirred at 150 rpm for 15 min, sonicated at 500 W for 10 min, mixed evenly, and heated at 120 °C for 48 h. The obtained precipitate was washed three times with deionized water and dried at 60 °C for 8 h to obtain the immobilized enzyme carrier CaMT.

[0104] S2. Add 0.3g of immobilized enzyme carrier CaMT to 60g of water, and add 0.03g of a complex enzyme containing α-amylase and pullulanase (mass ratio of α-amylase to pullulanase is 1:3) in four portions. Stir at 180rpm for 45min, let stand for 15min, remove the precipitate, wash the obtained precipitate three times with deionized water, and dry at 60℃ for 8h to obtain the immobilized complex enzyme.

[0105] Comparative Example 5, an immobilized complex enzyme and a method for preparing the same

[0106] This comparative example provides an immobilized complex enzyme, the preparation method of which includes:

[0107] S1. 3.675 g of calcium chloride dihydrate (CaCl2·2H2O) and 3.322 g of terephthalic acid (H2BDC) (molar ratio of 1.25:1) were added to 280 mL of ethanol, stirred at 150 rpm for 15 min, sonicated at 500 W for 10 min, mixed evenly, and heated at 120 °C for 48 h. The obtained precipitate was washed three times with deionized water and dried at 60 °C for 8 h to obtain the immobilized enzyme carrier CaMT.

[0108] S2. Add 0.3g of immobilized enzyme carrier CaMT to 60g of water, and add 0.03g of a complex enzyme containing maltose amylase and β-amylase (mass ratio of maltose amylase to β-amylase is 1:3) in four portions. Stir at 180rpm for 45min, let stand for 15min, remove the precipitate, wash the obtained precipitate three times with deionized water, and dry at 60℃ for 8h to obtain the immobilized complex enzyme.

[0109] Comparative Example 6, an immobilized complex enzyme and a method for preparing the same

[0110] This comparative example provides an immobilized complex enzyme. The difference between the preparation method of this example and that of Example 1 is that the complex enzyme in step S2 of Comparative Example 5 is replaced with an equal mass of α-amylase, while the rest is the same as that of Example 1.

[0111] Comparative Example 7, an immobilized complex enzyme and a method for preparing the same

[0112] This comparative example provides an immobilized complex enzyme. The difference between the preparation method of this example and that of Example 1 is that the complex enzyme in step S2 of Comparative Example 6 is replaced with an equal mass of β-amylase, while the rest is the same as that of Example 1.

[0113] Comparative Example 8, an immobilized complex enzyme and a method for preparing the same

[0114] This comparative example provides an immobilized complex enzyme. The difference between the preparation method of this example and that of Example 1 is that the heating temperature of Comparative Example 8 is changed to 180°C, while the rest are the same as those of Example 1.

[0115] Comparative Example 9, an immobilized complex enzyme and a method for preparing the same

[0116] This comparative example provides an immobilized complex enzyme. The difference between this method and Example 1 is that the amount of immobilized enzyme carrier and complex enzyme added in Comparative Example 9 is changed from 0.3g:0.03g to 0.3g:0.15g. All other aspects are the same as in Example 1.

[0117] Example 12, a hydrolyzed plant starch having soothing and anti-itching effects and a method for preparing the same

[0118] This embodiment provides a method for preparing hydrolyzed plant starch with soothing and antipruritic effects, including the following steps:

[0119] 1) Mix corn starch with 4 times its weight of water, add 5 wt% hydrochloric acid solution to adjust the pH to 5.5, stir at 80℃ for 80 min to obtain a suspension;

[0120] 2) Add 2% (by weight of corn starch) of the immobilized complex enzyme of high-yield maltotetrasaccharide from Example 1 to the suspension for stepwise enzymatic hydrolysis. Cool to 60°C and incubate at 100 rpm for 35 min to obtain the first hydrolysate; cool to 40°C and incubate at 100 rpm for 50 min; cool to 20°C and incubate at 100 rpm for 20 min. Remove the immobilized complex enzyme of high-yield maltotetrasaccharide to obtain the hydrolysate.

[0121] 3) The enzymatic hydrolysate is concentrated by membrane treatment (conditions: the filter membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 500 Da, and the inlet pressure is 0.6 MPa) to concentrate it to 50% of the volume of the enzymatic hydrolysate, thus obtaining hydrolyzed plant starch with soothing and antipruritic effects.

[0122] Example 13, a hydrolyzed plant starch having soothing and anti-itching effects and a method for preparing the same

[0123] This embodiment provides a method for preparing hydrolyzed plant starch with soothing and antipruritic effects, including the following steps:

[0124] 1) Mix corn starch with 4 times its weight of water, add 5 wt% hydrochloric acid solution to adjust the pH to 5.5, stir at 80℃ for 80 min to obtain a suspension;

[0125] 2) Add 2% (by weight of corn starch) of the immobilized complex enzyme of high-yield maltotetrasaccharide from Example 1 to the suspension for stepwise enzymatic hydrolysis. Cool to 65°C and incubate at 100 rpm for 35 min to obtain the first hydrolysate; cool to 45°C and incubate at 100 rpm for 50 min; cool to 25°C and incubate at 100 rpm for 20 min. Remove the immobilized complex enzyme of high-yield maltotetrasaccharide to obtain the hydrolysate.

[0126] 3) The enzymatic hydrolysate is concentrated by membrane treatment (conditions: the filter membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 500 Da, and the inlet pressure is 0.6 MPa) to concentrate it to 50% of the volume of the enzymatic hydrolysate, thus obtaining hydrolyzed plant starch with soothing and antipruritic effects.

[0127] Example 14, a hydrolyzed plant starch having soothing and anti-itching effects and a method for preparing the same

[0128] This embodiment provides a method for preparing hydrolyzed plant starch with soothing and antipruritic effects, including the following steps:

[0129] 1) Mix corn starch with 4 times its weight of water, add 5 wt% hydrochloric acid solution to adjust the pH to 5.5, stir at 80℃ for 80 min to obtain a suspension;

[0130] 2) Add 2% (by weight of corn starch) of the immobilized complex enzyme of high-yield maltotetrasaccharide from Example 1 to the suspension, and perform stepwise enzymatic hydrolysis. Cool to 55°C and incubate at 100 rpm for 35 min to obtain the first hydrolysate; cool to 35°C and incubate at 100 rpm for 50 min; cool to 15°C and incubate at 100 rpm for 20 min, and remove the immobilized complex enzyme of high-yield maltotetrasaccharide to obtain the hydrolysate.

[0131] 3) The enzymatic hydrolysate is concentrated by membrane treatment (conditions: the filter membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 500 Da, and the inlet pressure is 0.6 MPa) to concentrate it to 50% of the volume of the enzymatic hydrolysate, thus obtaining hydrolyzed plant starch with soothing and antipruritic effects.

[0132] Example 15, a hydrolyzed plant starch having soothing and anti-itching effects and a method for preparing the same

[0133] The difference between Example 15 and Example 12 is that in Example 15, the pH in step 1) is changed from 5.5 to 4.5, while the rest of the steps and parameters are the same as in Example 12.

[0134] Example 16, a hydrolyzed plant starch having soothing and anti-itching effects and a method for preparing the same

[0135] The difference between Example 16 and Example 12 is that in Example 16, the pH in step 1) is changed from 5.5 to 6.0, while the rest of the steps and parameters are the same as in Example 12.

[0136] Example 17, a hydrolyzed plant starch having soothing and anti-itching effects and a method for preparing the same

[0137] This embodiment provides a method for preparing hydrolyzed plant starch with soothing and antipruritic effects, including the following steps:

[0138] 1) Mix wheat starch with 3 times its weight of water, add 0.5 wt% citric acid solution to adjust the pH to 5.8, stir at 75℃ for 100 min to obtain a suspension;

[0139] 2) Add 3% by weight of wheat starch of the immobilized complex enzyme of high-yield maltotetrasaccharide from Example 10 to the suspension for stepwise enzymatic hydrolysis. Cool to 62°C and incubate at 80 rpm for 25 min to obtain the first hydrolysate; cool to 42°C and incubate at 80 rpm for 60 min; cool to 22°C and incubate at 80 rpm for 15 min. Remove the immobilized complex enzyme of high-yield maltotetrasaccharide to obtain the hydrolysate.

[0140] 3) The enzymatic hydrolysate is concentrated by membrane treatment (conditions: the filter membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 600 Da, and the inlet pressure is 0.4 MPa) to concentrate it to 60% of the volume of the enzymatic hydrolysate, thus obtaining hydrolyzed plant starch with soothing and antipruritic effects.

[0141] Example 18, a hydrolyzed plant starch having soothing and anti-itching effects and a method for preparing the same

[0142] This embodiment provides a method for preparing hydrolyzed plant starch with soothing and antipruritic effects, including the following steps:

[0143] 1) Mix wheat starch with 5 times its weight of water, add 8 wt% lactic acid solution to adjust the pH to 4.3, and stir at 85℃ for 60 min to obtain a suspension;

[0144] 2) Add 1% by weight of wheat starch of the immobilized complex enzyme of high-yield maltotetrasaccharide from Example 11 to the suspension for stepwise enzymatic hydrolysis. Cool to 58°C and incubate at 120 rpm for 45 min to obtain the first hydrolysate; cool to 38°C and incubate at 120 rpm for 40 min; cool to 18°C ​​and incubate at 120 rpm for 30 min. Remove the immobilized complex enzyme of high-yield maltotetrasaccharide to obtain the hydrolysate.

[0145] 3) The enzymatic hydrolysate is concentrated by membrane treatment (conditions: the filter membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 400 Da, and the inlet pressure is 0.8 MPa) to concentrate it to 40% of the volume of the enzymatic hydrolysate, thus obtaining hydrolyzed plant starch with soothing and antipruritic effects.

[0146] Comparative Example 10, a hydrolyzed plant starch and a method for preparing the same

[0147] The difference between Comparative Example 10 and Example 12 is that in Comparative Example 12, the immobilized complex enzyme of high-yield maltotetrasaccharide in Example 1 in step 2) is replaced with an equal mass of α-amylase and β-amylase (mass ratio of 1:3), while the remaining steps and parameters are the same as in Example 12.

[0148] Comparative Example 11, a hydrolyzed plant starch and a method for preparing the same

[0149] The difference between Comparative Example 11 and Example 12 is that in Comparative Example 11, the immobilized complex enzyme of high-yield maltotetrasaccharide in Example 1 in step 2) is replaced with the immobilized complex enzyme of Comparative Example 1 in the same mass ratio. The remaining steps and parameters are the same as in Example 12.

[0150] Comparative Example 12, a hydrolyzed plant starch and a method for preparing the same

[0151] The difference between Comparative Example 12 and Example 12 is that in Comparative Example 12, the immobilized complex enzyme of high-yield maltotetrasaccharide in Example 1 in step 2) is replaced with the immobilized complex enzyme of Comparative Example 2 in the same mass ratio. The remaining steps and parameters are the same as in Example 12.

[0152] Comparative Example 13, a hydrolyzed plant starch and a method for preparing the same

[0153] The difference between Comparative Example 13 and Example 12 is that in Comparative Example 13, the immobilized complex enzyme of high-yield maltotetrasaccharide in Example 1 in step 2) is replaced with the immobilized complex enzyme of Comparative Example 3 in the same mass ratio. The remaining steps and parameters are the same as in Example 12.

[0154] Comparative Example 14, a hydrolyzed plant starch and a method for preparing the same

[0155] The difference between Comparative Example 14 and Example 12 is that in Comparative Example 14, the immobilized complex enzyme of high-yield maltotetrasaccharide in Example 1 in step 2) is replaced with the immobilized complex enzyme of Comparative Example 6 in the same mass ratio. The remaining steps and parameters are the same as in Example 12.

[0156] Comparative Example 15, a hydrolyzed plant starch and a method for preparing the same

[0157] The difference between Comparative Example 15 and Example 12 is that in Comparative Example 15, the immobilized complex enzyme of high-yield maltotetrasaccharide in Example 1 in step 2) is replaced with the immobilized complex enzyme of Comparative Example 7 in the same mass ratio. The remaining steps and parameters are the same as in Example 12.

[0158] Comparative Example 16, a hydrolyzed plant starch and a method for preparing the same

[0159] This comparative example provides a method for preparing hydrolyzed plant starch, including the following steps:

[0160] 1) Mix corn starch with 4 times its weight of water, add 5 wt% hydrochloric acid solution to adjust the pH to 5.5, stir at 80℃ for 80 min to obtain a suspension;

[0161] 2) Add 2% by weight of corn starch of the immobilized complex enzyme of high maltotetrasaccharide production in Example 1 to the suspension, cool to 60°C, keep warm at 100 rpm for 105 min, and take out the immobilized complex enzyme of high maltotetrasaccharide production to obtain the enzymatic hydrolysate.

[0162] 3) The enzymatic hydrolysate is concentrated by membrane treatment (conditions: the filter membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 500 Da, and the inlet pressure is 0.6 MPa) to concentrate it to 50% of the volume of the enzymatic hydrolysate to obtain hydrolyzed plant starch.

[0163] Comparative Example 17, a hydrolyzed plant starch and a method for preparing the same

[0164] This comparative example provides a method for preparing hydrolyzed plant starch, including the following steps:

[0165] 1) Mix corn starch with 4 times its weight of water, adjust the pH to 5.5 with 5wt% hydrochloric acid solution, and stir at 80℃ for 80 min to obtain a suspension;

[0166] 2) Add 2% by weight of corn starch of the immobilized complex enzyme of high maltotetrasaccharide production in Example 1 to the suspension, cool to 40°C, keep warm at 100 rpm for 105 min, and take out the immobilized complex enzyme of high maltotetrasaccharide production to obtain the enzymatic hydrolysate.

[0167] 3) The enzymatic hydrolysate is concentrated by membrane treatment (conditions: the filter membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 500 Da, and the inlet pressure is 0.6 MPa) to concentrate it to 50% of the volume of the enzymatic hydrolysate to obtain hydrolyzed plant starch.

[0168] Comparative Example 18, a hydrolyzed plant starch and a method for preparing the same

[0169] This comparative example provides a method for preparing hydrolyzed plant starch, including the following steps:

[0170] 1) Mix corn starch with 4 times its weight of water, adjust the pH to 5.5 with 5wt% hydrochloric acid solution, and stir at 80℃ for 80 min to obtain a suspension;

[0171] 2) Add 2% by weight of corn starch of the immobilized complex enzyme of high maltotetrasaccharide production in Example 1 to the suspension, cool to 20°C, keep warm at 100 rpm for 105 min, and take out the immobilized complex enzyme of high maltotetrasaccharide production to obtain the enzymatic hydrolysate.

[0172] 3) The enzymatic hydrolysate is concentrated by membrane treatment (conditions: the filter membrane is an ultrafiltration membrane with a nominal molecular weight cutoff of 500 Da, and the inlet pressure is 0.6 MPa) to concentrate it to 50% of the volume of the enzymatic hydrolysate to obtain hydrolyzed plant starch.

[0173] Test Example 1, enzyme hydrolysis effect test

[0174] Test samples: Immobilized complex enzymes of Examples 1-11 and Comparative Examples 1-9.

[0175] Test Procedure: A 2 wt% sample of corn starch was subjected to enzymatic hydrolysis in a mixture of corn starch and water (corn starch to water ratio of 1:4, pH adjusted to 6.0 with 5 wt% hydrochloric acid solution). The hydrolysis temperature was 65℃, and the hydrolysis time was 2 h. After hydrolysis, the sample was washed twice with PBS solution. The sample was then added to a new hydrolysis experiment, and the hydrolysis experiment was repeated 4 times. The enzyme activity of unimmobilized β-amylase after soaking at 50℃ for 10 min was taken as 100% (for Comparative Example 4, pullulanase activity after soaking at 50℃ for 10 min was taken as 100%; for Comparative Example 6, α-amylase activity after soaking at 50℃ for 10 min was taken as 100%). The relative enzyme activity of the sample before hydrolysis, after one hydrolysis, and after five hydrolysis cycles was calculated.

[0176] The test results are shown in Table 2 below.

[0177] Table 2

[0178]

[0179]

[0180] Test Example 2, heat stability test

[0181] Test samples: Immobilized complex enzymes of Examples 1-11 and Comparative Examples 1-9.

[0182] Control group: Unimmobilized β-amylase.

[0183] Test procedure: The test samples were immersed in deionized water at 50℃, 60℃ and 70℃ for 2 hours respectively. The enzyme activity of hyaluronidase before and after immersion was tested. The enzyme activity of unimmobilized β-amylase after immersion at 50℃ for 10 min was taken as 100% (for Comparative Example 4, the enzyme activity of pullulanase after immersion at 50℃ for 10 min was taken as 100%; for Comparative Example 6, the enzyme activity of α-amylase after immersion at 50℃ for 10 min was taken as 100%). The relative enzyme activity of the test samples at different temperatures was calculated.

[0184] The test results are shown in Table 3 below.

[0185] Table 3

[0186]

[0187] Test Example 3, acid resistance effect test

[0188] Test samples: Immobilized complex enzymes of Examples 1-11 and Comparative Examples 1-9.

[0189] The immobilized complex enzyme was immersed in deionized water and acidic solution respectively to investigate its acid resistance.

[0190] Control group: Unimmobilized β-amylase.

[0191] Test procedure: The test samples were immersed in deionized water at 50℃ and a solution at 50℃ and pH 4.5 (the pH was adjusted to 4.0 with 5wt% hydrochloric acid solution) for 2 hours. The activity of β-amylase in the test samples was measured. The enzyme activity of unimmobilized β-amylase after immersion in deionized water at 50℃ for 10 minutes was taken as 100%. The relative activities of the immobilized complex enzyme in deionized water and acidic solution were calculated to evaluate the acid resistance effect of the immobilized complex enzyme.

[0192] The test results are shown in Table 4 below.

[0193] Table 4

[0194]

[0195]

[0196] As shown in Tables 2-4, the immobilized complex enzyme of this application exhibits excellent stability and reusability, with minimal loss of enzyme activity even after five repeated uses. Furthermore, the immobilized complex enzyme of this application demonstrates excellent heat and acid resistance.

[0197] The reusability, heat resistance, and acid resistance of the immobilized complex enzymes in Examples 1-10 are superior to those in Comparative Examples 1-3 and Comparative Example 8. This indicates that in this application, the type of metal and ligand in the immobilized enzyme carrier material, as well as the preparation temperature of the immobilized carrier, affect the degree of immobilization of the immobilized enzyme carrier, thereby affecting the immobilization rate and related properties of the final immobilized complex enzyme. In the immobilized complex enzymes of this application, the metal of the immobilized enzyme carrier is preferably a calcium salt, the ligand is preferably terephthalic acid, and the heat treatment temperature is preferably 90-140℃. The reusability, heat resistance, and acid resistance of the immobilized complex enzymes in Examples 1-10 are superior to those in Comparative Examples 4-7. This indicates that in the immobilized complex enzymes of this application, the type of enzyme and the ratio between different enzymes affect the reusability, heat resistance, and acid resistance of the final product. In this application, α-amylase and β-amylase are the most effective enzymes for immobilization. The interaction between the two enzymes can improve the reusability, heat resistance, and acid resistance of the final immobilized complex enzyme. The reusability, heat resistance, and acid resistance of the immobilized complex enzymes in Examples 1-10 are better than those in Comparative Example 9, indicating that the ratio of carrier to enzyme has a significant impact on the reusability, heat resistance, and acid resistance of the final immobilized complex enzyme. Preferably, the mass ratio of the immobilized enzyme carrier to the complex enzyme in this application is 1:(0.05-0.3).

[0198] Test Example 4, maltotetraose content test

[0199] Test samples: hydrolyzed plant starch from Examples 12-18 and Comparative Examples 10-18.

[0200] Test Procedure: The determination of maltodextrose was performed according to the test method for maltodextrose in "Simultaneous Determination of Six Sugar-Related Substances in Glucose Raw Material by HPLC-ELSD". The specific test method is as follows: Maltodextrose standard was prepared into a 1 mg / mL reference solution using 75% (v / v) acetonitrile aqueous solution. The test sample was prepared into a 1 mg / mL sample solution using 75% (v / v) acetonitrile aqueous solution. The reference solution and sample solution were tested by high-performance liquid chromatography (HPLC). Chromatographic conditions: Column: Amide column; Mobile phase: Acetonitrile:Water = 75:25; Elution mode: Isocratic elution; Flow rate: 0.5 mL / min; Column temperature: 30℃; Injection volume: 10 μL; Detector: ELSD; Carrier gas: Nitrogen; Gas pressure: 40 psi; Evaporation temperature: 80℃; Drift tube temperature: 80℃; Gain: 100.

[0201] The maltodextrose content in the test samples was calculated using the following company:

[0202]

[0203] The test results are shown in Table 5 below.

[0204] Table 5

[0205] Group Maltotetraose content (%) Group Maltotetraose content (%) Example 12 59.4 Comparative Example 10 16.4 Example 13 58.2 Comparative Example 11 21.8 Example 14 56.9 Comparative Example 12 25.9 Example 15 57.8 Comparative Example 13 18.7 Example 16 58.3 Comparative Example 14 20.7 Example 17 53.8 Comparative Example 15 32.2 Example 18 55.2 Comparative Example 16 42.4 / / Comparative Example 17 31.6 / / Comparative Example 18 18.5

[0206] Test results show that the hydrolyzed plant starch prepared by the methods in Examples 12-18 of this application has a high maltotetrasaccharide content, with the final product containing over 50% maltotetrasaccharide. Comparing Examples 12-18 and Comparative Example 10, it is evident that directly using unimmobilized α-amylase and β-amylase to enzymatically hydrolyze plant starch involves multiple temperature variations. During this process, the unimmobilized α-amylase and β-amylase exhibit low heat resistance and cannot effectively perform enzymatic hydrolysis, resulting in a low maltotetrasaccharide content in the final product. Comparing Examples 12-18 and Comparative Examples 11-13, it is clear that while the enzyme types are the same, different immobilization carriers lead to significant differences in enzyme activity in the final products, resulting in substantial variations in the maltotetrasaccharide content of the hydrolyzed plant starch prepared from these examples. This application preferably uses an immobilized composite enzyme with CaMT (calcium salt selected from at least one of calcium chloride and calcium nitrate, and organic ligand selected from terephthalic acid) as the immobilized enzyme carrier for enzymatic hydrolysis. Comparative Examples 12-18 and Comparative Examples 14-15 show that the type of enzyme in the immobilized complex enzyme has a significant impact on the maltotetraose content of the final hydrolyzed plant starch. In the enzymatic hydrolysis process of this application, α-amylase and β-amylase work synergistically, generating more maltotetraose during hydrolysis. Comparative Examples 12-18 and Comparative Examples 16-18 show that the enzymatic hydrolysis process has a significant impact on the maltotetraose content of the hydrolyzed plant starch. Examples 12-18 of this application employ a stepped temperature variable-temperature enzymatic hydrolysis technology, which can significantly improve the conversion rate of maltotetraose in plant starch and increase the maltotetraose content.

[0207] Test Example 5, soothing and anti-itching effect test - calcium ion channel test

[0208] Test principle:

[0209] The TRPV1 receptor is a key sensor for skin stimulation. HaCaT cells express transient receptor potential vanillic acid isoform 1 (TRPV1), a capsaicin receptor. When the skin is stimulated by external factors (such as heat, chemicals, or mechanical damage), capsaicin (CAP) activates TRPV1, leading to the release of calcium ions (Ca). 2+ The opening of the channel triggered a large number of Ca 2+ internal flow. Ca 2+ Influx can trigger the release of neuropeptides (such as substance P), leading to neurogenic inflammation such as redness and fever, as well as neuronal depolarization and itch signal transmission. If the test sample can inhibit Ca... 2+ Abnormal influx can block the overactivation of TRPV1 and block the NF-κB pathway, thereby reducing skin inflammation, itching, and other symptoms, achieving a soothing and antipruritic effect.

[0210] Calcium indicators are bound to Ca 2+The molecule exhibiting enhanced fluorescence is shown later. Fluo-4AM is an acetylated methyl ester derivative of Fluo-4, in which two chlorine substituents are replaced by fluorine, resulting in enhanced fluorescence excitation at a wavelength of 488 nm, thus leading to a higher fluorescence signal level. The dissolved indicator can be directly added to a culture dish containing cultured cells to load these calcium ion indicators in the form of AM esters into the cells. The calcium ion fluorescent probe Fluo-4AM itself is almost non-fluorescent. Once inside the cell, after hydrolysis by intracellular esterases, the resulting Fluo-4 binds to calcium ions and emits green fluorescence. The influx of extracellular calcium ions or the release of intracellularly stored calcium ions increases the concentration of calcium ions in the cytoplasm, allowing Fluo-4 to bind more calcium ions, ultimately increasing the intensity of intracellular green fluorescence. Conversely, the outflow or storage of intracellular calcium ions decreases the concentration of calcium ions in the cytoplasm, reducing the amount of calcium ions that Fluo-4 can bind, ultimately decreasing the intensity of intracellular green fluorescence.

[0211] Test samples: Hydrolyzed plant starch from Examples 12-18 and Comparative Examples 10-18 were prepared into a 2% (w / w) solution using H-DMEM medium containing 10% fetal bovine serum as the solvent.

[0212] Test procedure: HaCaT cells in the logarithmic growth phase were seeded into 6-well culture plates (2 × 10⁻⁶ cells / well). 5 Cells were cultured in H-DMEM medium containing 10% fetal bovine serum (FBS) at 5% CO2 and 37°C for 24 h. The culture medium was then aspirated. 5 μg / mL LPS solution was added to the model control group and test sample group, while the negative control group received an equal volume of H-DMEM medium containing 10% FBS. The cells were cultured at 5% CO2 and 37°C for 18 h. The culture medium was then aspirated. The prepared test sample solution was added to the test sample group, while the model control group and negative control group received H-DMEM medium containing 10% FBS. The cells were cultured at 5% CO2 and 37°C for 24 h. Cells were treated according to a cell calcium channel detection kit. Fluorescence of Fluo-4 was detected using a laser confocal microscope, fluorescence microplate reader, fluorescence spectrophotometer, or flow cytometer to determine changes in intracellular calcium ion concentration. The fluorescence grayscale values ​​were converted using ImageJ to evaluate the relative fluorescence intensity between the test sample group and the model control group. The fluorescence staining image of the negative control group is shown below. Figure 1 As shown; fluorescence staining images of the model control group are shown below. Figure 2 As shown; a fluorescent staining photograph of Example 12 is shown. Figure 3 As shown.

[0213] The test results are shown in Table 6 below.

[0214] Table 6

[0215] Group Fluorescence intensity (10 3 )]]> Group Fluorescence intensity (10 3 )]]> Negative control group 0.29 Comparative Example 10 3.45 Model control group 3.84 Comparative Example 11 2.94 Example 12 1.45 Comparative Example 12 3.07 Example 13 1.58 Comparative Example 13 3.27 Example 14 1.67 Comparative Example 14 2.85 Example 15 1.62 Comparative Example 15 2.48 Example 16 1.70 Comparative Example 16 2.12 Example 17 1.81 Comparative Example 17 2.56 Example 18 1.93 Comparative Example 18 3.22

[0216] Test Example 6, soothing effect test (IL-6 gene expression test)

[0217] Test Principle: IL-6 (interleukin-6) is a key mediator in skin inflammation, mainly secreted by keratinocytes, macrophages, and T cells. Its levels rise rapidly when the skin is stimulated (e.g., by chemicals, ultraviolet radiation, pathogens), inducing the release of pro-inflammatory factors such as TNF-α and IL-1β, forming an inflammatory amplification circuit; it also activates the Ras-MAPK pathway, promoting cell proliferation and the release of inflammatory mediators. Inhibiting IL-6 overexpression can reduce inflammatory cell infiltration and increased vascular permeability; it can alleviate subjective discomfort such as redness, stinging, and itching, thus achieving a soothing effect.

[0218] This test uses LPS-induced mouse macrophages Raw264.7 as an in vitro inflammatory cell model and evaluates whether the test substance has in vitro soothing effects by measuring the relative expression level of inflammatory factor (IL-6) mRNA.

[0219] Test samples: Hydrolyzed plant starch from Examples 12-18 and Comparative Examples 10-18 were prepared into a 2% (w / w) solution using DMEM culture medium containing 10% FBS and 1% double antibiotics as the solvent.

[0220] Test procedure: Macrophages in the logarithmic growth phase were seeded into 6-well culture plates (2 × 10⁻⁶ cells / well). 5 Cells / well were added to DMEM culture medium containing 10% FBS and 1% penicillin-dextrin (hereinafter referred to as culture medium) and cultured at 5% CO2 and 37℃ for 24 h. The culture medium was then discarded. 5 μg / mL LPS solution was added to the model control group and the test sample group, and the same volume of culture medium was added to the negative control group. The cells were cultured at 5% CO2 and 37℃ for 6 h. The culture medium was then discarded. The prepared test sample solution was added to the test sample group, and the culture medium was added to the model control group and the negative control group. The cells were cultured at 5% CO2 and 37℃ for 24 h. After culture, total RNA was extracted from macrophages in each well, and reverse transcription was performed for quantitative real-time PCR. The results of the quantitative real-time PCR were analyzed using 2... － The △△CT method is used for calculation.

[0221] The test results are shown in Table 7 below.

[0222] Table 7

[0223] Group IL-6 gene relative expression amount Group IL-6 gene relative expression amount Negative control group 1.00 Comparative Example 10 2.91 Model control group 3.15 Comparative Example 11 2.84 Example 12 1.52 Comparative Example 12 2.71 Example 13 1.67 Comparative Example 13 2.78 Example 14 1.58 Comparative Example 14 2.46 Example 15 1.73 Comparative Example 15 2.15 Example 16 1.61 Comparative Example 16 2.23 Example 17 1.87 Comparative Example 17 2.64 Example 18 1.92 Comparative Example 18 2.90

[0224] The test results of Examples 5 and 6 show that, compared with the model control group, the fluorescence intensity of Example 12 was significantly reduced by 62.24%, and the fluorescence intensity of the other examples was also significantly reduced, indicating that the hydrolyzed plant starch of this application can effectively inhibit the influx of calcium ions. Compared with the model control group, Example 12 can downregulate the relative expression level of IL-6 mRNA by 51.75%, indicating that the hydrolyzed plant starch of this application can effectively inhibit the expression of the pro-inflammatory factor IL-6 gene.

[0225] The results above show that the hydrolyzed plant starch in this application exhibits excellent soothing and antipruritic effects. The soothing effects of Examples 12-18 are superior to Comparative Example 10, indicating that the use of unimmobilized α-amylase and β-amylase for enzymatic hydrolysis of plant starch results in poor stability of α-amylase and β-amylase during the hydrolysis process, leading to poor hydrolysis of plant starch and consequently, poor soothing and antipruritic effects in the final product. The soothing effects of Examples 12-18 are superior to Comparative Examples 11-13, indicating that the type of immobilized complex enzyme has a significant impact on the hydrolysis process. Different carriers used for the immobilized complex enzyme in this application result in significant differences in the enzyme activity of the final immobilized complex enzyme, and also significant differences in the active substances with soothing and antipruritic effects in the hydrolysis product. This application preferably uses an immobilized complex enzyme with CaMT (calcium salt selected from at least one of calcium chloride and calcium nitrate, and organic ligand selected from terephthalic acid) as the immobilized enzyme carrier for enzymatic hydrolysis. The soothing and antipruritic effects of Examples 12-18 are superior to those of Comparative Examples 14-15. The types of enzymes in the immobilized complex enzyme have a significant impact on the final enzymatic hydrolysis process of plant starch. In the enzymatic hydrolysis process of this application, α-amylase and β-amylase work synergistically, generating more soothing active substances during the hydrolysis process, resulting in better soothing and antipruritic effects of the final product. The soothing effects of Examples 12-18 are superior to those of Comparative Examples 16-18, indicating that temperature control during the enzymatic hydrolysis process has a significant impact on the soothing and antipruritic effects of the final product. This application uses a stepped variable-temperature enzymatic hydrolysis technology, which can significantly improve the soothing and antipruritic effects of the final product.

[0226] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit the scope of protection of this application. Although this application has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of this application without departing from the substance and scope of the technical solutions of this application.

Claims

1. A method for producing a high-maltotetraose-producing immobilized complex enzyme, characterized by, The method comprises the following steps: S1, adding calcium salt and organic ligand into organic solvent, mixing and treating uniformly, then heating, washing and drying the obtained precipitate to obtain an immobilized enzyme carrier; S2, adding the immobilized enzyme carrier obtained in step S1 into water, then adding complex enzyme, stirring and reacting, then standing, washing and drying the precipitate to obtain a high-maltotetraose-producing immobilized complex enzyme.

2. The method for preparing an immobilized complex enzyme according to claim 1, wherein In step S1, the calcium salt comprises calcium chloride or / and calcium nitrate; And / or, the organic ligand comprises terephthalic acid; And / or, the organic solvent comprises at least one of ethanol, glycerol, propylene glycol and butanediol; In step S2, the complex enzyme comprises α-amylase and β-amylase.

3. The method for preparing an immobilized complex enzyme according to claim 1, wherein the enzyme is a lipase. In step S1, the molar ratio of calcium element in the calcium salt to the organic ligand is (1-1.8):1; And / or, the mass ratio of the calcium salt to the organic solvent is 1:(30-80).

4. The method for preparing an immobilized complex enzyme according to claim 1, wherein In step S2, the mass ratio of the immobilized enzyme carrier to water is 1:(100-300); And / or, the mass ratio of the immobilized enzyme carrier to complex enzyme is 1:(0.05-0.3).

5. The method for preparing an immobilized complex enzyme according to claim 1, wherein In step S1, the mixing and treating comprises stirring and / or ultrasonic treating; The stirring treating is performed at a speed of 120-300 rpm for 10-30 min; And / or, the ultrasonic treating is performed at a power of 300-600 W for 8-20 min; And / or, the heating treating is performed at a temperature of 90-140℃ for 40-60 h.

6. The high-maltotetraose-producing immobilized complex enzyme prepared by the method of any one of claims 1-5.

7. The use of the high-maltotetraose-producing immobilized complex enzyme of claim 6 in preparing a high-maltotetraose-containing hydrolyzed plant starch.

8. A process for the preparation of hydrolyzed plant starch having soothing and anti-itching effects, characterized by, The method comprises the following steps: 1) mixing plant starch with water, adding pH regulator, heating and stirring to obtain a suspension; 2) adding the high-maltotetraose-producing immobilized complex enzyme of claim 6 into the suspension, performing gradient enzymatic hydrolysis, then removing the high-maltotetraose-producing immobilized complex enzyme to obtain an enzymatic hydrolysate; 3) performing membrane concentration on the enzymatic hydrolysate to obtain a hydrolyzed plant starch having soothing and anti-itch effects.

9. The hydrolyzed plant starch having soothing and anti-itch effects prepared by the method of claim 8.

10. Use of the hydrolyzed plant starch having soothing and anti-itching effects according to claim 9 in the preparation of a cosmetic product, characterized in that, The addition amount of the hydrolyzed plant starch having soothing and anti-itch effects in cosmetics is 0.1-30 wt%.

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