Method for detecting amyloid ratio of starch

Abstract

The invention discloses a method for detecting the amylopectin ratio of starch. The method comprises the following steps: preparing an Au NPs (at) pullulanase hybrid enzyme biosensor, and detecting the ratio (amylopectin ratio) of amylose to amylopectin in a starch sample by utilizing the Au NPs (at) pullulanase hybrid enzyme biosensor through cyclic voltammetry. By preparing the Au NPs (at) pullulanase hybrid enzyme biosensor and combining a CV method, an accurate and regular result is obtained in the process of detecting amylopectin and amylopectin oxidation signals, so that the amylopectin amylopectin direct-branch ratio can be rapidly determined, the method is low in detection limit, high in sensitivity and simple and rapid in detection process, rapid and quantitative analysis of the amylopectin amylopectin direct-branch ratio in rice starch can be realized, and the method is suitable for large-scale popularization and application. Compared with a traditional detection technology, the modified working electrode has the advantages that the pretreatment process is simple, operation is easy, time consumption is low, cost is low, detection equipment is high in portability and wider in application range, and the modified working electrode has reusability, can be used for batch detection of samples and is particularly suitable for detection of the straight count ratio in rice starch.

Description

Technical Field

[0001] The invention belongs to the technical field of grain detection, and relates to a method for detecting the straight-branched ratio of starch, and specifically to a method for detecting the straight-branched ratio in rice starch. Background Art

[0002] Rice is rich in natural starch. Starch is one of the main polysaccharides used by plants for energy storage and one of the main sources of energy required by the human body. Starch and its derivatives are widely used as a plant extract in the food field. Starch is mainly composed of amylose and amylopectin. The ratio of the two is closely related to the physical and chemical properties of starch, such as gelatinization, aging, and gelation. The straight-chain to branch-chain ratio of starch (i.e., the ratio of amylose to amylopectin) not only determines the taste quality and gel consistency of rice, but also has important significance for the evaluation of its food processing adaptability. It can be said that it determines the economic value of starch to a certain extent. Therefore, the detection of the straight-chain to branch-chain ratio of starch is widely used in the fields of breeding of starch-containing cereal crops, high-value utilization of starch, and food quality assurance. At the same time, the detection of the straight-chain to branch-chain ratio of starch also plays an important role in the development of food formulas and the selection of equipment and parameter settings in the food processing process. In recent years, with the rapid and high-quality development of the food industry, the control standards for food raw materials, food formulas, food processing technology, and product quality have become increasingly higher. Therefore, the detection technology of the straight-chain to branch-chain ratio of starch has gradually attracted attention.

[0003] At present, there are many methods for detecting the straight-branch ratio of starch, such as iodine colorimetry, iodine potentiometric titration, size exclusion chromatography (also known as gel chromatography), concanavalin method, near-infrared spectroscopy, differential scanning calorimetry, etc. Among them, the iodine colorimetry method has a simple principle and low testing cost; the iodine potentiometric titration method has high sensitivity, good result reproducibility, and is less affected by temperature; the size exclusion chromatography test results are accurate and reliable, and can be tested automatically; the concanavalin method is suitable for the detection of a variety of starches with high accuracy; the near-infrared spectroscopy method is simple and convenient; and the differential scanning calorimetry method is easy to automate. However, the above conventional detection methods have cumbersome operating steps, high technical requirements for detection personnel, high detection costs, and non-portable detection equipment, and their applications are relatively limited.

[0004] In view of this, it is necessary to further improve the detection method of starch straight-branch ratio in the prior art. Summary of the invention

[0005] To this end, the technical problem to be solved by the present invention is that the traditional method for detecting the straight-to-branch ratio of starch is cumbersome to operate, costly, and the detection equipment is not portable, thus proposing a method for detecting the straight-to-branch ratio of starch which is simple to operate, low in cost, has a portable detection device, and has high detection efficiency.

[0006] In order to solve the above technical problems, the technical solution of the present invention is:

[0007] The present invention provides a method for detecting the straight-branched ratio of starch, which comprises the following steps:

[0008] preparing an Au NPs@pullulanase hybrid enzyme biosensor, mixing a pullulanase enzyme solution and a nanogold solution at a volume ratio of 1:3-3:1 at 55-60° C. to obtain a hybrid enzyme solution, and electro-depositing the hybrid enzyme solution on a pretreated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor;

[0009] The ratio of amylose to amylopectin in a starch sample is detected by cyclic voltammetry: the starch sample to be tested is prepared as a starch solution, and mixed with a buffer solution to obtain a mixed solution to be tested; the Au NPs@pullulanase hybrid enzyme biosensor, a reference electrode, and a blank electrode are placed in the mixed solution to be tested, and the ratio of amylose to amylopectin in the starch sample is determined.

[0010] Preferably, the nano gold solution is prepared by the following steps:

[0011] The mass concentration is 0.5×10 -4 g / L-1.5×10 -4 g / L HAuCl4 was heated to boiling;

[0012] The HAuCl4 and the sodium citrate solution with a mass concentration of 5-15 g / L are mixed evenly according to a volume ratio of 10:0.6-1.5, and heated until the solution turns wine red, and the heating is stopped;

[0013] The solution is cooled to room temperature to obtain a nano-gold solution, in which the average particle size of the gold nanoparticles is 13-15 nm.

[0014] Preferably, the hybrid enzyme solution is prepared by culturing pullulanase solution and nanogold solution in a water bath at 55-60° C. for 2 hours in a certain proportion; the enzyme activity of the pullulanase solution is 40-160U.

[0015] Preferably, the working electrode is pretreated by the following steps:

[0016] Soak the gold electrode in the piranha solution for 2-3 minutes, then wash it with anhydrous ethanol and distilled water in turn;

[0017] The cleaned gold electrode is polished and then activated with an electrolyte, wherein the electrolyte is prepared from potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3;

[0018] The activated gold electrode was cleaned and dried with nitrogen.

[0019] Preferably, the piranha solution is prepared by mixing a 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:6-9.

[0020] Preferably, during the electrodeposition process, the deposition potential is -0.2-1.2 V, the scan rate is 0.02-0.1 v / s, and the deposition is performed by cyclic voltammetry for 30-120 cycles.

[0021] Preferably, in the process of detecting the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry, the scanning potential is 0.2-1.2 V, the scanning rate is 0.02-0.2 v / s, and the number of scanning circles is 30 circles.

[0022] Preferably, the reference electrode is an Ag / Agcl electrode, and the blank electrode is a Pt electrode.

[0023] Preferably, the buffer solution is a sodium acetate buffer solution with a molar concentration of 0.1-0.3 mol / L and a pH of 4.5.

[0024] Preferably, the gold electrode is polished by a suede cloth covered with aluminum powder; and the linear detection range of the cyclic voltammetry method for detecting the ratio of amylose to amylopectin in the starch sample is 0.5-10 mg / ml.

[0025] The above technical solution of the present invention has the following advantages compared with the prior art:

[0026] The detection method of the straight-chain-branch ratio of starch provided by the present invention comprises: preparing an Au NPs@pullulanase hybrid enzyme biosensor, and using the Au NPs@pullulanase hybrid enzyme biosensor to detect the ratio of straight-chain starch to branched starch (straight-chain-branch ratio) in a starch sample by cyclic voltammetry. By preparing the Au NPs@pullulanase hybrid enzyme biosensor, and combining the CV method, accurate and regular results are obtained in the process of detecting branched starch and branched starch oxidation signals, so that the straight-chain-branch ratio of starch can be quickly determined, the method has low detection limit, high sensitivity, and simple and fast detection process, and can realize rapid quantitative analysis of the straight-chain-branch ratio in rice starch, and compared with traditional detection technology, the pre-treatment process is simple, easy to operate, less time-consuming, low cost, high portability of detection equipment, wider application range, and the modified working electrode has reusability, can be used for batch detection of samples, and is particularly suitable for the detection of the straight-chain-branch ratio in rice starch. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0028] Figure 1 is the current response curve of the hybrid enzyme biosensor detecting starch solution under different enzyme activities;

[0029] Figure 2 is the current response curve at different volume ratios of pullulanase solution to nano-gold solution;

[0030] Figure 3 It is the current response curve under different scanning numbers;

[0031] Figure 4 It is the CV curve diagram of different starch solution concentrations. DETAILED DESCRIPTION

[0032] The following embodiments will clearly and completely describe the technical solutions in the embodiments of the present application.

[0033] The materials and equipment used in this application are as follows: distilled water (Shanghai Huxi Analytical Instrument Factory); pullulanase (Guangdong Wengjiang Chemical Reagent Co., Ltd.); chloroauric acid (Tianjin Tianli Chemical Reagent Co., Ltd.); sodium citrate (Tianjin Fuyu Fine Chemical Co., Ltd.); hydrogen peroxide (Tianjin Tianli Chemical Reagent Co., Ltd.); concentrated sulfuric acid (Xilong Chemical Co., Ltd.); sodium acetate buffer (Guangdong Wengjiang Chemical Reagent Co., Ltd.); potassium ferrocyanide Shandong Hengxin Chemical Co., Ltd.; potassium chloride Tianjin Guangfu Fine Chemical Research Institute; potassium ferrocyanide Tianjin Guangfu Fine Chemical Research Institute. Electrochemical workstation (Shanghai Chenhua Instrument Co., Ltd.); analytical balance (Ji'er International Trade Co., Ltd.); precision micropipette (Beijing Zhongxi Yuanda Technology Co., Ltd.); ultrasonic cleaning machine (Guangzhou Hu Ruiming Instrument Co., Ltd.); pH meter (Shanghai Chenhua Instrument Co., Ltd.); gold electrode (Shanghai Chenhua Instrument Co., Ltd.); reference electrode (Shanghai Chenhua Instrument Co., Ltd.); blank electrode (Shanghai Chenhua Instrument Co., Ltd.). If the specific conditions are not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased from the market.

[0034] Example 1

[0035] This embodiment provides a method for detecting the linear-branched ratio of starch, which is used for detecting the linear-branched ratio of starch in rice.

[0036] In this embodiment, the sources of raw materials are as follows:

[0037] The method for detecting the starch straight-branch ratio provided in this embodiment comprises the following steps:

[0038] S1. Preparation of Au NPs@pullulanase hybrid enzyme biosensor.

[0039] S11, preparation of gold nanoparticle solution:

[0040] First, 100 ml of the mass concentration of 0.5×10 -4 g / L HAuCl4 was heated to boiling for 2 minutes, then 6 ml of 5 g / L sodium citrate solution was added, and the solution was shaken to mix evenly, and heated to 94°C for 10 minutes to change the color of the solution from light yellow to wine red, and the heating was stopped. The obtained solution was cooled to room temperature to obtain a nanogold solution, in which the average particle size of gold nanoparticles (Au NPs) was 13 nm.

[0041] S12. Prepare hybrid enzyme solution: incubate pullulanase solution and nanogold solution in a water bath at 60° C. for 2 h in a volume ratio of 1:1 to obtain a hybrid enzyme solution, wherein the enzyme activity of the pullulanase solution is 40 U. Refrigerate the hybrid enzyme solution at 4° C. for later use.

[0042] S13, electro-depositing the hybrid enzyme solution on the pre-treated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor:

[0043] The working electrode is pretreated by the following steps: the gold electrode is soaked in the piranha solution for 2 minutes, then taken out, and then ultrasonically cleaned in 99% anhydrous ethanol for 2 minutes, and then ultrasonically cleaned in distilled water for 2 minutes. The cleaned gold electrode is polished into a mirror surface with suede covered with a layer of aluminum powder (Al2O3), and after rinsing, it is placed in an electrolyte for activation treatment. In this embodiment, the piranha solution is prepared by a volume concentration of 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:6. The electrolyte is a mixed solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3, and the activation treatment time is 0.9 minutes. After the activation treatment, the working electrode is cleaned again and blown dry with nitrogen.

[0044] The obtained hybrid enzyme solution was mixed with a buffer solution in a volume ratio of 1:3 to obtain a premixed solution. The pretreated working electrode, reference electrode (Ag / AgCl electrode) and blank electrode (Pt electrode) were placed in the premixed solution. Cyclic voltammetry was used to scan 30 circles at a deposition potential of -0.2-1.2 V and a scan rate of 0.02 v / s, and 4 layers were electrodeposited to obtain an Au NPs@pullulanase hybrid enzyme biosensor, wherein the buffer solution was a sodium acetate buffer solution with a molar concentration of 0.1 mol / L and a pH of 4.5.

[0045] S2. Cyclic voltammetry (CV) was used to detect the ratio of amylopectin to branched starch in starch samples.

[0046] S21. Prepare the starch sample to be tested into a 1 mg / ml starch solution, and prepare a mixed solution to be tested by mixing the starch solution and a sodium acetate buffer solution with a molar concentration of 0.1 mol / L and a pH of 4.5 in a volume ratio of 1:2.

[0047] S22, the obtained Au NPs@pullulanase hybrid enzyme biosensor, reference electrode (Ag / AgCl electrode), and blank electrode (Pt electrode) were placed in the mixed solution to be tested, and the ratio of amylopectin to amylopectin in the starch sample was analyzed by CV method at a scanning potential of 0.2-1.2V and a scanning rate of 0.02v / s for 27 cycles. The linear detection range of the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry was 0.5-10mg / ml.

[0048] The method for detecting the straight-branch ratio of starch provided in this embodiment is to prepare an Au NPs@pullulanase hybrid enzyme biosensor and combine it with the CV method to obtain accurate and regular results in the process of detecting amylopectin and amylopectin oxidation signals, so that the straight-branch ratio of starch can be quickly determined. The method has a low detection limit, high sensitivity, and a simple and fast detection process, and can realize a rapid quantitative analysis of the straight-branch ratio in rice starch. The biosensor is prepared by a hydrothermal method. Compared with traditional detection technology, the pretreatment process is simple, easy to operate, less time-consuming, low cost, high portability of the detection equipment, and a wider range of applications. The modified working electrode is reusable and can be used for batch detection of samples, and is particularly suitable for the detection of the straight-branch ratio in rice starch.

[0049] The detection method of the starch straight-chain branch ratio provided in this embodiment is based on the principle that there is both electrostatic interaction and strong adsorption characteristics of chemical bond binding between Au NPs and the bare gold electrode. Based on these two characteristics and combined with enzyme biosensing technology, AuNPs are adsorbed on the Au electrode. At the same time, the -NH2 of glutamic acid in Pullulanase can be bonded to Au on the surface of AuNPs through a gold amine bond (Au-NH2), thereby producing a unique catalytic effect on the electrochemical behavior of amylose. Based on this principle, a hybrid enzyme modified electrode was prepared. Through the specific debranching effect of Pullulanase on starch, the hydroxyl radical (-OH) in amylose is exposed, and a dehydrogenation oxidation reaction occurs under the drive of the electrode, thereby generating a measurable electrical signal, thereby achieving a rapid detection effect of the ratio of amylose to amylopectin in rice starch.

[0050] Example 2

[0051] This embodiment provides a method for detecting the straight-branched ratio of starch, which is particularly used for detecting the straight-branched ratio of starch in rice. The method comprises the following steps:

[0052] S1. Preparation of Au NPs@pullulanase hybrid enzyme biosensor.

[0053] S11, preparation of gold nanoparticle solution:

[0054] First, 100 ml of the mass concentration of 1×10 -4 g / L HAuCl4 was heated to boiling for 2 minutes, then 6 ml of 10 g / L sodium citrate solution was added, the solution was shaken to mix evenly, heated to 95 ° C, 10 minutes, the solution color changed from light yellow to wine red, and the heating was stopped. The obtained solution was cooled to room temperature to obtain a nanogold solution, in which the average particle size of gold nanoparticles (Au NPs) was 13 nm.

[0055] S12. Prepare hybrid enzyme solution: incubate pullulanase solution and nanogold solution in a water bath at 60° C. for 2 h in a volume ratio of 1:2 to obtain a hybrid enzyme solution, wherein the enzyme activity of the pullulanase solution is 80 U. Refrigerate the hybrid enzyme solution at 4° C. for later use.

[0056] S13, electro-depositing the hybrid enzyme solution on the pre-treated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor:

[0057] The working electrode is pretreated by the following steps: the gold electrode is soaked in the piranha solution for 2 minutes, then taken out, and then ultrasonically cleaned in 99% anhydrous ethanol for 2 minutes, and then ultrasonically cleaned in distilled water for 2 minutes. The cleaned gold electrode is polished to a mirror surface with suede covered with a layer of aluminum powder (Al2O3), and after rinsing, it is placed in an electrolyte for activation treatment. In this embodiment, the piranha solution is prepared by a volume concentration of 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:7. The electrolyte is a mixed solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3, and the activation treatment time is 1.8 minutes. After the activation treatment, the working electrode is cleaned again and blown dry with nitrogen.

[0058] The obtained hybrid enzyme solution was mixed with a buffer solution in a volume ratio of 1:3 to obtain a premixed solution. The pretreated working electrode, reference electrode (Ag / AgCl electrode) and blank electrode (Pt electrode) were placed in the premixed solution. At a deposition potential of -0.2-1.2 V and a scan rate of 0.05 v / s, cyclic voltammetry was used to scan 40 circles and electrodeposit 4 layers to obtain an Au NPs@pullulanase hybrid enzyme biosensor, wherein the buffer solution was a sodium acetate buffer solution with a molar concentration of 0.3 mol / L and a pH of 4.5.

[0059] S2. Cyclic voltammetry (CV) was used to detect the ratio of amylopectin to branched starch in starch samples.

[0060] S21. Prepare the starch sample to be tested into a 1 mg / ml starch solution, and prepare a mixed solution to be tested by mixing the starch solution and a sodium acetate buffer solution with a molar concentration of 0.3 mol / L and a pH of 4.5 in a volume ratio of 1:2.

[0061] S22, the obtained Au NPs@pullulanase hybrid enzyme biosensor, reference electrode (Ag / AgCl electrode), and blank electrode (Pt electrode) were placed in the mixed solution to be tested, and the ratio of amylopectin to amylopectin in the starch sample was analyzed by CV method at a scanning potential of 0.2-1.2V and a scanning rate of 0.04v / s for 30 cycles. The linear detection range of the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry was 0.5-10mg / ml.

[0062] Example 3

[0063] This embodiment provides a method for detecting the straight-branched ratio of starch, which is particularly used for detecting the straight-branched ratio of starch in rice. The method comprises the following steps:

[0064] S1. Preparation of Au NPs@pullulanase hybrid enzyme biosensor.

[0065] S11, preparation of gold nanoparticle solution:

[0066] First, 100 ml of the mass concentration of 1.5×10 -4 g / L HAuCl4 was heated to boiling for 2 minutes, then 15 ml of 15 g / L sodium citrate solution was added, the solution was shaken to mix evenly, heated to 96 ° C, 10 minutes, the solution color changed from light yellow to wine red, and the heating was stopped. The obtained solution was cooled to room temperature to obtain a nanogold solution, in which the average particle size of gold nanoparticles (Au NPs) was 15 nm.

[0067] S12. Prepare hybrid enzyme solution: incubate pullulanase solution and nanogold solution in a water bath at 60° C. for 2 h at a volume ratio of 2:1 to obtain a hybrid enzyme solution, wherein the enzyme activity of the pullulanase solution is 120 U. Refrigerate the hybrid enzyme solution at 4° C. for later use.

[0068] S13, electro-depositing the hybrid enzyme solution on the pre-treated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor:

[0069] The working electrode is pretreated by the following steps: the gold electrode is soaked in the piranha solution for 2 minutes, then taken out, and then ultrasonically cleaned in 99% anhydrous ethanol for 2 minutes, and then ultrasonically cleaned in distilled water for 2 minutes. The cleaned gold electrode is polished into a mirror surface with suede covered with a layer of aluminum powder (Al2O3), and after rinsing, it is placed in an electrolyte for activation treatment. In this embodiment, the piranha solution is prepared by a volume concentration of 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:9. The electrolyte is a mixed solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3, and the activation treatment time is 2.7 minutes. After the activation treatment, the working electrode is cleaned again and blown dry with nitrogen.

[0070] The obtained hybrid enzyme solution was mixed with a buffer solution in a volume ratio of 1:3 to obtain a premixed solution. The pretreated working electrode, reference electrode (Ag / AgCl electrode) and blank electrode (Pt electrode) were placed in the premixed solution. At a deposition potential of -0.2-1.2 V and a scan rate of 0.1 v / s, cyclic voltammetry was used to scan 110 circles and electrodeposit 4 layers to obtain an Au NPs@pullulanase hybrid enzyme biosensor, wherein the buffer solution was a sodium acetate buffer solution with a molar concentration of 0.2 mol / L and a pH of 4.5.

[0071] S2. Cyclic voltammetry (CV) was used to detect the ratio of amylopectin to branched starch in starch samples.

[0072] S21. Prepare the starch sample to be tested into a 1 mg / ml starch solution, and prepare a mixed solution to be tested by mixing the starch solution and a sodium acetate buffer solution with a molar concentration of 0.2 mol / L and a pH of 4.5 in a volume ratio of 1:2.

[0073] S22, the obtained Au NPs@pullulanase hybrid enzyme biosensor, reference electrode (Ag / AgCl electrode), and blank electrode (Pt electrode) were placed in the mixed solution to be tested, and the ratio of amylopectin to amylopectin in the starch sample was analyzed by CV method at a scanning potential of 0.2-1.2V and a scanning rate of 0.2v / s for 30 cycles. The linear detection range of the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry was 0.5-10mg / ml.

[0074] Example 4

[0075] This embodiment provides a method for detecting the straight-branched ratio of starch, which is particularly used for detecting the straight-branched ratio of starch in rice. The method comprises the following steps:

[0076] S1. Preparation of Au NPs@pullulanase hybrid enzyme biosensor.

[0077] S11, preparation of gold nanoparticle solution:

[0078] First, 100 ml of the mass concentration of 0.9×10 -4 g / L HAuCl4 was heated to boiling for 2 minutes, then 8 ml of 11 g / L sodium citrate solution was added, and the solution was shaken to mix evenly, and heated to 97 ° C for 10 minutes to change the color of the solution from light yellow to wine red, and the heating was stopped. The obtained solution was cooled to room temperature to obtain a nanogold solution, in which the average particle size of gold nanoparticles (Au NPs) was 14 nm.

[0079] S12. Prepare hybrid enzyme solution: incubate pullulanase solution in a water bath at 60° C. for 2 h at a volume ratio of 1:3 to obtain a hybrid enzyme solution, wherein the enzyme activity of the pullulanase solution is 80 U. Refrigerate the hybrid enzyme solution at 4° C. for later use.

[0080] S13, electro-depositing the hybrid enzyme solution on the pre-treated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor:

[0081] The working electrode is pretreated by the following steps: the gold electrode is soaked in the piranha solution for 2 minutes, then taken out, and then ultrasonically cleaned in 99% anhydrous ethanol for 2 minutes, and then ultrasonically cleaned in distilled water for 2 minutes. The cleaned gold electrode is polished into a mirror surface with suede covered with a layer of aluminum powder (Al2O3), and after rinsing, it is placed in an electrolyte for activation treatment. In this embodiment, the piranha solution is prepared by a volume concentration of 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:7. The electrolyte is a mixed solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3, and the activation treatment time is 3.6 minutes. After the activation treatment, the working electrode is cleaned again and blown dry with nitrogen.

[0082] The obtained hybrid enzyme solution was mixed with a buffer solution in a volume ratio of 1:3 to obtain a premixed solution. The pretreated working electrode, reference electrode (Ag / AgCl electrode) and blank electrode (Pt electrode) were placed in the premixed solution. At a deposition potential of -0.2-1.2 V and a scan rate of 0.06 v / s, cyclic voltammetry was used to scan 80 circles and electrodeposit 4 layers to obtain an Au NPs@pullulanase hybrid enzyme biosensor, wherein the buffer solution was a sodium acetate buffer solution with a molar concentration of 0.2 mol / L and a pH of 4.5.

[0083] S2. Cyclic voltammetry (CV) was used to detect the ratio of amylopectin to branched starch in starch samples.

[0084] S21. Prepare the starch sample to be tested into a 1 mg / ml starch solution, and prepare a mixed solution to be tested by mixing the starch solution and a sodium acetate buffer solution with a molar concentration of 0.2 mol / L and a pH of 4.5 in a volume ratio of 1:2.

[0085] S22, the obtained Au NPs@pullulanase hybrid enzyme biosensor, reference electrode (Ag / AgCl electrode), and blank electrode (Pt electrode) were placed in the mixed solution to be tested, and the ratio of amylopectin to amylopectin in the starch sample was analyzed by CV method at a scanning potential of 0.2-1.2V and a scanning rate of 0.12v / s for 28 cycles. The linear detection range of the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry was 0.5-10mg / ml.

[0086] Example 5

[0087] This embodiment provides a method for detecting the straight-branched ratio of starch, which is particularly used for detecting the straight-branched ratio of starch in rice. The method comprises the following steps:

[0088] S1. Preparation of Au NPs@pullulanase hybrid enzyme biosensor.

[0089] S11, preparation of gold nanoparticle solution:

[0090] First, 100 ml of the mass concentration of 1.3×10 -4 g / L HAuCl4 was heated to boiling for 2 minutes, then 12 ml of 12 g / L sodium citrate solution was added, the solution was shaken to mix evenly, heated to 98 ° C, 10 minutes, the solution color changed from light yellow to wine red, and the heating was stopped. The obtained solution was cooled to room temperature to obtain a nanogold solution, in which the average particle size of gold nanoparticles (Au NPs) was 15 nm.

[0091] S12. Prepare hybrid enzyme solution: incubate pullulanase solution and nanogold solution in a water bath at 56° C. for 2 h in a volume ratio of 1:3 to obtain a hybrid enzyme solution, wherein the enzyme activity of the pullulanase solution is 120 U. Refrigerate the hybrid enzyme solution at 4° C. for later use.

[0092] S13, electro-depositing the hybrid enzyme solution on the pre-treated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor:

[0093] The working electrode is pretreated by the following steps: the gold electrode is soaked in the piranha solution for 2 minutes, then taken out, and then ultrasonically cleaned in 99% anhydrous ethanol for 2 minutes, and then ultrasonically cleaned in distilled water for 2 minutes. The cleaned gold electrode is polished into a mirror surface with suede covered with a layer of aluminum powder (Al2O3), and after rinsing, it is placed in an electrolyte for activation treatment. In this embodiment, the piranha solution is prepared by a volume concentration of 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:8. The electrolyte is a mixed solution prepared by potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3, and the activation treatment time is 4.5 minutes. After the activation treatment, the working electrode is cleaned again and blown dry with nitrogen.

[0094] The obtained hybrid enzyme solution was mixed with a buffer solution in a volume ratio of 1:3 to obtain a premixed solution. The pretreated working electrode, reference electrode (Ag / AgCl electrode) and blank electrode (Pt electrode) were placed in the premixed solution. Cyclic voltammetry was used to scan 100 circles at a deposition potential of -0.2-1.2 V and a scan rate of 0.04 v / s, and 4 layers were electrodeposited to obtain an Au NPs@pullulanase hybrid enzyme biosensor, wherein the buffer solution was a sodium acetate buffer solution with a molar concentration of 0.25 mol / L and a pH of 4.5.

[0095] S2. Cyclic voltammetry (CV) was used to detect the ratio of amylopectin to branched starch in starch samples.

[0096] S21. Prepare the starch sample to be tested into a 1 mg / ml starch solution, and prepare a mixed solution to be tested by mixing the starch solution and a sodium acetate buffer solution with a molar concentration of 0.25 mol / L and a pH of 4.5 in a volume ratio of 1:2.

[0097] S22, the obtained Au NPs@pullulanase hybrid enzyme biosensor, reference electrode (Ag / AgCl electrode), and blank electrode (Pt electrode) were placed in the mixed solution to be tested, and the ratio of amylopectin to amylopectin in the starch sample was analyzed by CV method at a scanning potential of 0.2-1.2V and a scanning rate of 0.16v / s for 27 cycles. The linear detection range of the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry was 0.5-10mg / ml.

[0098] Example 6

[0099] This embodiment provides a method for detecting the straight-branched ratio of starch, which is particularly used for detecting the straight-branched ratio of starch in rice. The method comprises the following steps:

[0100] S1. Preparation of Au NPs@pullulanase hybrid enzyme biosensor.

[0101] S11, preparation of gold nanoparticle solution:

[0102] First, 100 ml of the mass concentration of 1.3×10 -4 g / L HAuCl4 was heated to boiling for 2 minutes, then 8 ml of 6 g / L sodium citrate solution was added, and the solution was shaken to mix evenly, and heated to 99°C for 10 minutes until the color of the solution changed from light yellow to wine red, and the heating was stopped. The obtained solution was cooled to room temperature to obtain a nanogold solution, in which the average particle size of gold nanoparticles (Au NPs) was 13 nm.

[0103] S12. Prepare hybrid enzyme solution: incubate pullulanase solution and nanogold solution in a water bath at 55° C. for 2 h at a volume ratio of 2:1 to obtain a hybrid enzyme solution, wherein the enzyme activity of the pullulanase solution is 160 U. Refrigerate the hybrid enzyme solution at 4° C. for later use.

[0104] S13, electro-depositing the hybrid enzyme solution on the pre-treated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor:

[0105] The working electrode is pretreated by the following steps: the gold electrode is soaked in the piranha solution for 2 minutes, then taken out, and then ultrasonically cleaned in 99% anhydrous ethanol for 2 minutes, and then ultrasonically cleaned in distilled water for 2 minutes. The cleaned gold electrode is polished to a mirror surface with suede covered with a layer of aluminum powder (Al2O3), and after rinsing, it is placed in an electrolyte for activation treatment. In this embodiment, the piranha solution is prepared by a volume concentration of 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:7. The electrolyte is a mixed solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3, and the activation treatment time is 5.4 minutes. After the activation treatment, the working electrode is cleaned again and blown dry with nitrogen.

[0106] The obtained hybrid enzyme solution was mixed with a buffer solution in a volume ratio of 1:3 to obtain a premixed solution. The pretreated working electrode, reference electrode (Ag / AgCl electrode) and blank electrode (Pt electrode) were placed in the premixed solution. Cyclic voltammetry was used to scan 50 circles at a deposition potential of -0.2-1.2 V and a scan rate of 0.08 v / s, and 4 layers were electrodeposited to obtain an Au NPs@pullulanase hybrid enzyme biosensor, wherein the buffer solution was a sodium acetate buffer solution with a molar concentration of 0.15 mol / L and a pH of 4.5.

[0107] S2. Cyclic voltammetry (CV) was used to detect the ratio of amylopectin to branched starch in starch samples.

[0108] S21. Prepare the starch sample to be tested into a 1 mg / ml starch solution, and prepare a mixed solution to be tested by mixing the starch solution and a sodium acetate buffer solution with a molar concentration of 0.15 mol / L and a pH of 4.5 in a volume ratio of 1:2.

[0109] S22, the obtained Au NPs@pullulanase hybrid enzyme biosensor, reference electrode (Ag / AgCl electrode), and blank electrode (Pt electrode) were placed in the mixed solution to be tested, and the ratio of amylopectin to amylopectin in the starch sample was analyzed by CV method at a scanning potential of 0.2-1.2V and a scanning rate of 0.09v / s for 30 cycles. The linear detection range of the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry was 0.5-10mg / ml.

[0110] Example 7

[0111] This embodiment provides a method for detecting the straight-branched ratio of starch, which is particularly used for detecting the straight-branched ratio of starch in rice. The method comprises the following steps:

[0112] S1. Preparation of Au NPs@pullulanase hybrid enzyme biosensor.

[0113] S11, preparation of gold nanoparticle solution:

[0114] First, 100 ml of the mass concentration of 1.4×10 -4 g / L HAuCl4 was heated to boiling for 2 minutes, then 14 ml of 9 g / L sodium citrate solution was added, the solution was shaken to mix evenly, heated to 100 ° C, 10 minutes, the solution color changed from light yellow to wine red, and the heating was stopped. The obtained solution was cooled to room temperature to obtain a nanogold solution, in which the average particle size of gold nanoparticles (Au NPs) was 14 nm.

[0115] S12. Prepare hybrid enzyme solution: incubate pullulanase solution and nanogold solution in a water bath at 60° C. for 2 h in a volume ratio of 3:1 to obtain a hybrid enzyme solution, wherein the enzyme activity of the pullulanase solution is 200 U. Refrigerate the hybrid enzyme solution at 4° C. for later use.

[0116] S13, electro-depositing the hybrid enzyme solution on the pre-treated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor:

[0117] The working electrode is pretreated by the following steps: the gold electrode is soaked in the piranha solution for 2 minutes, then taken out, and then ultrasonically cleaned in 99% anhydrous ethanol for 2 minutes, and then ultrasonically cleaned in distilled water for 2 minutes. The cleaned gold electrode is polished into a mirror surface with suede covered with a layer of aluminum powder (Al2O3), and after rinsing, it is placed in an electrolyte for activation treatment. In this embodiment, the piranha solution is prepared by a volume concentration of 30% hydrogen peroxide solution and a 98% concentrated sulfuric acid solution in a volume ratio of 3:8. The electrolyte is a mixed solution of potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3, and the activation treatment time is 6.3 minutes. After the activation treatment, the working electrode is cleaned again and blown dry with nitrogen.

[0118] The obtained hybrid enzyme solution was mixed with a buffer solution in a volume ratio of 1:3 to obtain a premixed solution. The pretreated working electrode, reference electrode (Ag / AgCl electrode) and blank electrode (Pt electrode) were placed in the premixed solution. Cyclic voltammetry was used to scan 120 circles at a deposition potential of -0.2-1.2 V and a scan rate of 0.03 v / s, and 4 layers were electrodeposited to obtain an Au NPs@pullulanase hybrid enzyme biosensor, wherein the buffer solution was a sodium acetate buffer solution with a molar concentration of 0.3 mol / L and a pH of 4.5.

[0119] S2. Cyclic voltammetry (CV) was used to detect the ratio of amylopectin to branched starch in starch samples.

[0120] S21. Prepare the starch sample to be tested into a 1 mg / ml starch solution, and prepare a mixed solution to be tested by mixing the starch solution and a sodium acetate buffer solution with a molar concentration of 0.3 mol / L and a pH of 4.5 in a volume ratio of 1:2.

[0121] S22, the obtained Au NPs@pullulanase hybrid enzyme biosensor, reference electrode (Ag / AgCl electrode), and blank electrode (Pt electrode) were placed in the mixed solution to be tested, and the ratio of amylopectin to amylopectin in the starch sample was analyzed by CV method at a scanning potential of 0.2-1.2V and a scanning rate of 0.17v / s for 29 cycles. The linear detection range of the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry was 0.5-10mg / ml.

[0122] Experimental example

[0123] 1. Determination of the optimal enzyme activity of Au NPs@pullulanase hybrid enzyme biosensor:

[0124] The current response curve of the Au NPs@pullulanase hybrid enzyme biosensor obtained by the method provided in the embodiment of the present application to detect starch solution (1 mg / ml) was tested. The current response curve test results when other reaction conditions were the same and only the enzyme activity of the pullulanase enzyme was different were as follows: Figure 1 shown.

[0125] As can be seen from the figure, under different enzyme activities of 40U, 80U, 120U, 160U, and 200U, as the enzyme activity increases, the current response signal first increases and then decreases. When the enzyme activity is 80U, the current response signal is the strongest. Therefore, the optimal enzyme activity is 80U.

[0126] 2. When preparing Au NPs@pullulanase hybrid enzyme biosensor, the optimal volume ratio of pullulanase solution to nanogold solution was determined:

[0127] The current response curve of the Au NPs@pullulanase hybrid enzyme biosensor obtained by the method provided in the embodiment of the present application in the electrolyte was tested. The current response curve when the volume ratio of the pullulanase enzyme solution to the nanogold solution was different was the same under other reaction conditions. The test results are as follows: Figure 2 shown.

[0128] It can be seen from the figure that when the volume ratio of pullulanase solution to nanogold solution is 1:2, the peak current increases significantly, while when the volume ratio of pullulanase solution to nanogold solution is 1:1, 1:3, 2:1, and 3:1, the peak current shows a downward trend. Therefore, it can be determined that when preparing Au NPs@pullulanase hybrid enzyme biosensor, the volume ratio of pullulanase solution to nanogold solution is 1:2, which is the optimal ratio.

[0129] 3. Determination of the optimal scanning number when detecting the straight-branch ratio in starch by cyclic voltammetry:

[0130] The Au NPs@pullulanase hybrid enzyme biosensor prepared by the method provided in Example 2 was tested, and the corresponding signal test results of the sensor are as follows: Figure 3 As shown in the figure, it can be seen that the corresponding signal of the sensor gradually increases with the increase of the number of scanning circles, and the rising trend of the response signal gradually decreases. When scanning to the 27th circle, the response signal of the sensor reaches the maximum value. Therefore, the number of scanning circles can be set to 27-30 circles.

[0131] 4. Test the detection characteristics of Au NPs@pullulanase hybrid enzyme biosensor at different concentrations of the tested starch solution:

[0132] The Au NPs@pullulanase hybrid enzyme biosensor prepared by the method provided in Test Example 2 was used to test the linear-branch ratio of starch solutions of different concentrations. The prepared enzyme biosensor was placed in a mixed solution of 0.5 mg / L, 1 mg / L, and 1.5 mg / L starch solution and sodium acetate buffer solution (0.3 mol / L) to be tested, and the oxidation current signal was tested. The test results are as follows: Figure 4 shown.

[0133] It can be seen from the figure that the oxidation response signal of the hybrid enzyme biosensor gradually increases with the increase of starch solution concentration, and the change of the response signal has a certain regularity. min ) is the amylose content (C 直 ), maximum peak current value (A max ) is the total starch content, therefore, the amylopectin content (C 支 )=A max —A min , thus through C 直 / C 支 The linear-branch ratio of starch can be calculated. The calculation results of the linear-branch ratio of the above three concentrations of starch solutions are shown in the electrochemical method column in Table 1.

[0134] The amylose content of the three concentrations of starch solution was measured by traditional iodine colorimetry, and the amylopectin content of the three concentrations of starch solution was measured by fluorescence method. The amylose / amylopectin ratio was calculated by dividing the amylose content by the amylopectin content. The calculation results of the amylose / amylopectin ratio are shown in the iodine colorimetry / fluorescence method column in Table 1.

[0135] Table 1

[0136]

[0137] The results in Table 1 show that the starch straight-branch ratio measured by the Au NPs@pullulanase hybrid enzyme biosensor prepared in Example 2 is not much different from the detection results of the traditional iodine colorimetry and fluorescence methods, indicating that the constructed Au NPs@pullulanase hybrid enzyme biosensor can be applied to the detection of the straight-branch ratio in rice starch, and the detection method provided by the present invention has simple detection steps and high detection efficiency, and the straight-branch ratio result of starch can be obtained without combining the two detections.

[0138] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the present invention.

Claims

1. A method for detecting the straight-branched ratio of starch, characterized in that: The steps include: preparing an Au NPs@pullulanase hybrid enzyme biosensor, mixing a pullulanase enzyme solution and a nanogold solution at a volume ratio of 1:3-3:1 at 55-60° C. to obtain a hybrid enzyme solution, and electro-depositing the hybrid enzyme solution on a pretreated working electrode surface to obtain the Au NPs@pullulanase hybrid enzyme biosensor; The ratio of amylose to amylopectin in a starch sample is detected by cyclic voltammetry: the starch sample to be tested is prepared as a starch solution, and mixed with a buffer solution to obtain a mixed solution to be tested; the Au NPs@pullulanase hybrid enzyme biosensor, a reference electrode, and a blank electrode are placed in the mixed solution to be tested, and the ratio of amylose to amylopectin in the starch sample is determined.

2. The method for detecting the starch straight-branch ratio according to claim 1, characterized in that: The nano gold solution is prepared by the following steps: The mass concentration is 0.5×10 -4 g / L-1.5×10 -4 g / L HAuCl4 was heated to boiling; The HAuCl4 and the sodium citrate solution with a mass concentration of 5-15 g / L are mixed evenly according to a volume ratio of 10:0.6-1.5, and heated until the solution turns wine red, and the heating is stopped; The solution is cooled to room temperature to obtain a nano-gold solution, in which the average particle size of the gold nanoparticles is 13-15 nm.

3. The method for detecting the starch straight-branch ratio according to claim 2, characterized in that: The hybrid enzyme solution is prepared by culturing the pullulanase solution and the nano-gold solution in a water bath at 55-60° C. for 2 hours in a certain proportion; the enzyme activity of the pullulanase solution is 40-160U.

4. The method for detecting the straight-branched ratio of starch according to any one of claims 1 to 3, characterized in that: The working electrode is pretreated by the following steps: Soak the gold electrode in the piranha solution for 2-3 minutes, then wash it with anhydrous ethanol and distilled water in turn; The cleaned gold electrode is polished and then activated with an electrolyte, wherein the electrolyte is prepared from potassium chloride, potassium ferrocyanide, and potassium ferrocyanide in a molar ratio of 50:3:3; The activated gold electrode was cleaned and dried with nitrogen.

5. The method for detecting the starch straight-branch ratio according to claim 4, characterized in that: The piranha solution is prepared by mixing a hydrogen peroxide solution with a volume concentration of 30% and a concentrated sulfuric acid solution with a volume ratio of 98% in the range of 3:6-9.

6. The method for detecting the starch straight-branch ratio according to claim 5, characterized in that: During the electrodeposition process, the deposition potential is -0.2-1.2 V, the scan rate is 0.02-0.1 V / s, and the deposition is performed by cyclic voltammetry for 30-120 cycles.

7. The method for detecting the starch straight-branch ratio according to claim 6, characterized in that: In the process of detecting the ratio of amylose to amylopectin in the starch sample by cyclic voltammetry, the scanning potential is 0.2-1.2V, the scanning rate is 0.02-0.2v / s, and the number of scanning circles is 27-30 circles.

8. The method for detecting the starch straight-branch ratio according to claim 7, characterized in that: The reference electrode is an Ag / Agcl electrode, and the blank electrode is a Pt electrode.

9. The method for detecting the starch straight-branch ratio according to claim 8, characterized in that: The buffer solution is a sodium acetate buffer solution with a molar concentration of 0.1-0.3 mol / L and a pH of 4.

5.

10. The method for detecting the starch straight-branch ratio according to claim 4, characterized in that: The gold electrode is polished by a suede cloth covered with aluminum powder; the linear detection range of the ratio of amylose to amylopectin in the starch sample detected by the cyclic voltammetry method is 0.5-10 mg / ml.

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