Method for detecting content of modified starch in paper and paperboard

Abstract

The invention provides a method for detecting the content of modified starch in paper and paperboards, which comprises the following steps: A) soaking paper to be detected in an acidic buffer solution, and scattering to obtain a mixed system; b) adding an enzyme solution into the mixed system, and performing enzymolysis to obtain enzymatic hydrolysate; and C) adding the enzymatic hydrolysate into a 3, 5-dinitrosalicylic acid developing solution, heating for inactivation, measuring a light absorption value, substituting the measured light absorption value into a glucose standard curve equation, and calculating the content of glucose, thereby calculating the content of starch. According to the method for determining the starch content in the paper and the paperboard through the enzymolysis method, the starch content in the paper can be calculated by determining the content of the enzymolysis product glucose in the solution, the method is easy to operate, the test result is accurate and reliable, and the determination of the starch content in the paper can be met.

Description

Technical Field

[0001] The invention relates to the field of detection technology, in particular to a method for detecting the content of modified starch in paper and paperboard. Background Art

[0002] As a renewable green natural resource, starch has been widely used in the papermaking industry, and its usage is second only to pulp fiber and filler. Original starch is mainly used for interlayer spraying to increase the bonding area between fibers, make the paper thickness uniform, and improve the tensile strength, stiffness, bursting resistance, and interlayer bonding strength of the paper to varying degrees; modified starch is widely used in papermaking wet cloth as a reinforcing agent and retention and drainage aid to improve the strength of paper and improve the surface properties of paper sheets; starch and modified starch are also widely used as surface sizing agents to improve paper strength and improve the printing adaptability of paper sheets. For papermaking companies, by understanding the retention rate of starch in paper, they can adjust the production process and control product quality; for paper research, understanding the starch content in paper is conducive to studying the effect of starch on the performance of different papers and developing new products. Therefore, it is very important to study the determination method of starch content in different papers.

[0003] At present, there is no standard method for determining the starch content in paper in my country. The US TAPPI T 419om-02 standard provides a method for determining the starch content in paper. However, this method is only suitable for the quantitative determination of native starch in paper and starch modified only by conventional oxidation technology or enzyme conversion. It is not suitable for cationic starch, substituted starch, grafted starch or starch bound to resin. Domestic researchers have established a method for determining the starch content in paper products using a spectrophotometer based on the principle that starch will generate a blue complex when it encounters iodine. Whether it is the TAPPI T 419om-02 standard or the starch content determination method established by domestic researchers, hot water or boiling water is required to extract the starch from the paper. Since the starch used in papermaking is generally modified starch, such as cationic starch. Due to electrostatic adsorption, cationic starch has a strong adsorption force with paper fibers, and it is difficult to completely extract cationic starch from paper with hot or boiling water. In addition, since there are many factors that affect the maximum absorption wavelength of starch-iodine complex, such as temperature, iodine-potassium iodide concentration, starch polymerization degree, etc., in short, the shortcomings in starch separation and quantification on paper and paperboard will affect the reliability of the above standards and research methods.

[0004] Therefore, it is necessary to find a method that is simple to operate, has reliable test results, and can meet the requirements of determining the starch content in paper. Summary of the invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a method for detecting the content of modified starch in paper and paperboard. The method provided by the present invention is simple, and the measurement result is accurate and reliable.

[0006] The existing method for determining the starch content in paper and paperboard requires hot water extraction to separate the starch, which is complicated to operate, the starch extraction is not thorough, and the hemicellulose may be degraded during the extraction process, affecting the accuracy of the measurement result. The method for determining the starch content in paper and paperboard by enzymatic hydrolysis of the present invention can calculate the starch content in the paper by measuring the content of glucose, the enzymatic hydrolysis product in the solution. The method adopts enzymatic hydrolysis, which can enzymatically hydrolyze both native starch and modified starch, and can meet the measurement of various starch contents in paper. The method does not need to separate the starch in the paper and paperboard, the method is simple to operate, the test conditions are mild, and the test results are accurate and reliable.

[0007] The present invention provides a method for detecting the content of modified starch in paper and paperboard, comprising the following steps:

[0008] A) soaking the test paper in an acidic buffer and then breaking it up to obtain a mixed system;

[0009] B) adding an enzyme solution to the mixed system to perform enzymolysis to obtain an enzymolysis solution;

[0010] C) Add the enzymatic hydrolysate to 3,5-dinitrosalicylic acid colorimetric solution, inactivate it by heating, measure the light absorption value, and substitute the measured light absorption value into the glucose standard curve equation to calculate the glucose content, thereby calculating the starch content.

[0011] Preferably, the formula for calculating starch content is as follows:

[0012]

[0013] X: starch content in paper sample, %;

[0014] V: total volume of enzymatic reaction solution;

[0015] V1: The volume of the test solution taken for DNS colorimetric determination;

[0016] m: The mass of glucose calculated by substituting the average absorbance value of the sample into the standard curve equation, mg;

[0017] 0.9: Conversion factor between glucose and starch;

[0018] M: Paper sample mass, g.

[0019] Preferably, the glucose standard curve equation is m=0.4106x+0.002, R 2 =0.999, where m is the glucose content and x is the absorbance;

[0020] The concentration of the glucose standard solution is 0.1 g·L -1 ~1g·L-1 .

[0021] Preferably, the acidic buffer solution in step A) is an acetic acid-sodium acetate buffer solution, and the pH of the buffer solution is 3-5.

[0022] Preferably, the enzyme solution is saccharifying enzyme and / or α-amylase; the enzyme addition amount is 2.64 mg·mL -1 ~5 mg mL -1 .

[0023] Preferably, when the starch content in paper and paperboard is less than 4%, the enzyme solution is saccharifying enzyme; when the starch content is greater than 4%, the enzyme is α-amylase and saccharifying enzyme;

[0024] Preferably, the enzymolysis temperature is 40-55°C; the enzymolysis time is 35-75 min.

[0025] Preferably, the operation of judging whether the enzymatic hydrolysis is complete is: taking 1-2 drops at regular intervals and placing them on the surface of the white substrate, and adding I2-KI solution to check whether the hydrolysis is complete.

[0026] Preferably, the light absorption value is measured at a wavelength of 540 nm.

[0027] Preferably, the detection limit of glucose is 1.22 mg / L -1 The detection limit of starch is 1.1 mg / L -1 The standard curve of starch is c=0.4106A+0.002, and the starch content range that can be measured is 0.16% to 35.7%.

[0028] Compared with the prior art, the present invention provides a method for detecting the content of modified starch in paper and paperboard, comprising the following steps: A) soaking the paper to be tested in an acidic buffer solution and then breaking it up to obtain a mixed system; B) adding an enzyme solution to the mixed system for enzymolysis to obtain an enzymolysis solution; C) adding the enzymolysis solution to a 3,5-dinitrosalicylic acid colorimetric solution, heating and inactivating it, measuring the light absorption value, and substituting the measured light absorption value into the glucose standard curve equation to calculate the glucose content, thereby calculating the starch content. The enzymolysis method of the present invention can calculate the starch content in paper by measuring the content of glucose, a hydrolysis product in the solution. The method is simple to operate and the test results are accurate and reliable, which can meet the requirements for determining the starch content in paper. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The response surface and contour plots of the effects of time and enzyme dosage on glucose content in Example 2;

[0030] Figure 2The response surface and contour plot of temperature and enzyme dosage to glucose content in Example 2;

[0031] Figure 3 The response surface and contour diagram of temperature and time to glucose content in Example 2;

[0032] Figure 4 The response surface and contour plots of the effects of time and enzyme dosage on glucose content in Example 3;

[0033] Figure 5 The response surface and contour plot of temperature and enzyme dosage to glucose content in Example 3;

[0034] Figure 6 Response surface and contour plot of temperature and time to glucose content in Example 3. DETAILED DESCRIPTION

[0035] The present invention provides a method for detecting the content of modified starch in paper and paperboard. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and they all fall within the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to achieve and apply the technology of the present invention.

[0036] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0037] The present invention uses saccharifying enzyme or α-amylase and saccharifying enzyme dual enzyme system to enzymatically hydrolyze starch in paper and paperboard to produce glucose, and uses DNS method to quantitatively determine the glucose content, and then calculates the starch content from the glucose content. The method is simple to operate, and the test results are accurate and reliable, which can meet the requirements of the determination of various starch contents in paper.

[0038] The present invention provides a method for detecting the content of modified starch in paper and paperboard, comprising the following steps:

[0039] A) soaking the test paper in an acidic buffer and then breaking it up to obtain a mixed system;

[0040] B) adding an enzyme solution to the mixed system to perform enzymolysis to obtain an enzymolysis solution;

[0041] C) Add the enzymatic hydrolysate to 3,5-dinitrosalicylic acid colorimetric solution, inactivate it by heating, measure the light absorption value, and substitute the measured light absorption value into the glucose standard curve equation to calculate the glucose content, thereby calculating the starch content.

[0042] The invention provides a method for detecting the content of modified starch in paper and paperboard. The paper and paperboard to be detected include but are not limited to cigarette tipping paper and white cardboard.

[0043] First, the test paper is soaked in an acidic buffer solution and then broken up to obtain a mixed system. The acidic buffer solution is a 0.05M acetic acid-sodium acetate buffer solution, and the pH of the buffer solution is 3-5; the pH can be 3, 4 or 5.

[0044] The present invention can make the enzyme react at a suitable pH by selecting the buffer system; at the same time, the reaction conditions are mild and will not degrade the cellulose and hemicellulose in the paper, thereby making the test result accurate.

[0045] The soaking in the present invention is preferably soaking overnight; the breaking is preferably breaking on a disperser, and then washing with a buffer solution for multiple times to obtain a mixed system.

[0046] Add an enzyme solution to the mixed system for enzymolysis to obtain an enzymolysis solution. The enzyme solution is saccharifying enzyme and / or α-amylase; the amount of the enzyme added is 2.64 mg·mL -1 ~5 mg mL -1 .

[0047] When the raw material is cigarette tipping paper, the enzyme solution is saccharifying enzyme;

[0048] In one embodiment of the present invention, the optimal reaction conditions are: the amount of saccharifying enzyme is 2.64 mg·mL -1 , the reaction time was 35 min and the reaction temperature was 53 °C.

[0049] When the raw material is white cardboard, the enzymes are α-amylase and saccharifying enzyme.

[0050] In one embodiment of the present invention, the optimal reaction conditions are:

[0051] The enzyme dosage is 5 mg mL -1 , the reaction time is 75min and the reaction temperature is 40℃.

[0052] The enzymolysis temperature of the present invention is 40-55° C. and the enzymolysis time is 35-75 min.

[0053] The operation to determine whether the enzymatic hydrolysis is complete is as follows: take 1 to 2 drops at regular intervals and place them on the surface of the white substrate, and add I2-KI solution to check whether the hydrolysis is complete.

[0054] The present invention creatively hydrolyzes starch in paper to obtain glucose without separating the starch in the paper by using the above-mentioned specific enzyme. The enzyme used is specific and only hydrolyzes starch, and has no effect on cellulose and hemicellulose in the paper. In addition, the enzymatic hydrolysis conditions are mild, the determination method is simple, and the result is accurate.

[0055] The enzymatic solution is added to the 3,5-dinitrosalicylic acid colorimetric solution and heated to inactivate the enzyme, preferably in a boiling water bath for 3 to 10 minutes.

[0056] After taking out, the mixture was rapidly cooled with running water and then filtered, and the light absorption value was measured. The light absorption value was measured at a wavelength of 540 nm.

[0057] Substitute the measured light absorption value into the glucose standard curve equation to calculate the glucose content.

[0058] The concentration of the glucose standard solution of the present invention is 0.1 g·L -1 ~1g·L -1 .

[0059] The glucose standard curve equation is m=0.4106x+0.002, R 2 =0.999, where m is the glucose content and x is the absorbance.

[0060] In one embodiment of the present invention, the glucose standard solution can be prepared as follows:

[0061] Accurately measure 0.0, 0.1, 0.2, 0.3, 0.4, and 0.5 mL of 0.5 g·L-1 glucose standard solution into 6 10 mL colorimetric tubes, and add deionized water to 0.5 mL.

[0062] Substitute the measured glucose content into the formula to calculate the starch content.

[0063] The formula for calculating starch content of the present invention is as follows:

[0064]

[0065] X: starch content in paper sample, %;

[0066] V: total volume of enzymatic reaction solution;

[0067] V1: The volume of the test solution taken for DNS colorimetric determination;

[0068] m: The mass of glucose calculated by substituting the average absorbance value of the sample into the standard curve equation, mg;

[0069] 0.9: Conversion factor between glucose and starch;

[0070] M: Paper sample mass, g.

[0071] The detection limit of glucose in the present invention is 1.22 mg / L -1 The detection limit of starch is 1.1 mg / L -1 The standard curve of starch is c=0.4106A+0.002.

[0072] The present invention provides a method for detecting the content of modified starch in paper and paperboard, comprising the following steps: A) soaking the paper to be tested in an acidic buffer solution and then breaking it up to obtain a mixed system; B) adding an enzyme solution to the mixed system for enzymolysis to obtain an enzymolysis solution; C) adding the enzymolysis solution to a 3,5-dinitrosalicylic acid colorimetric solution, heating and inactivating it, measuring the light absorption value, and substituting the measured light absorption value into a glucose standard curve equation to calculate the glucose content, thereby calculating the starch content. The method for measuring the starch content in paper and paperboard by enzymolysis of the present invention can calculate the starch content in the paper by measuring the content of glucose, a product of enzymolysis, in the solution. The method is simple to operate, and the test results are accurate and reliable, and can meet the requirements for measuring the starch content in the paper.

[0073] In the actual testing process, according to the amount of starch in the paper sample, a single enzyme or a compound enzyme can be selected, and the sample mass, the volume of the reaction solution and the amount of enzyme used can be appropriately adjusted to make the absorbance value of the test solution fall within the range of the standard curve. To ensure the accuracy of the test results, a standard curve should be drawn at the same time for each measurement.

[0074] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0075] The numerical ranges and parameters involved in the present invention have presented the relevant numerical values ​​in the specific embodiments as accurately as possible. However, any numerical value essentially inevitably contains standard deviations due to individual testing methods. Therefore, unless otherwise explicitly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have certain reasonable deviations within a certain range, for example: within 1% or 0.5%.

[0076] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0077] In order to further illustrate the present invention, a method for detecting the content of modified starch in paper and paperboard provided by the present invention is described in detail below in conjunction with examples.

[0078] Experimental materials and instruments

[0079] Experimental Materials

[0080] Paper sample provided by Hubei China Tobacco Technology Center.

[0081] 3,5-Dinitrosalicylic acid (DNS) colorimetric solution, Feijing Biotechnology Co., Ltd.; starch and cationic starch, Jiangxi Liuhe Chemical Industry Co., Ltd.; saccharifying enzyme (enzyme activity of 130000U / g), Hunan Hongyingxiang Bioengineering Co., Ltd.; α-amylase (enzyme activity of 4000U / g), Beijing Shuangxuan Microbial Culture Medium Products Factory; glucose, glacial acetic acid, sodium acetate, iodine, potassium iodide, etc. were all analytical grade.

[0082] 1.1.2 Instruments

[0083] V-1100D visible spectrophotometer, Shanghai Meipuda Instrument Co., Ltd.; SHZ-82B water bath constant temperature oscillator, Changzhou Guowang Instrument Manufacturing Co., Ltd.

[0084] Experimental methods

[0085] Determination of moisture content in paper samples

[0086] The moisture content in the paper sample is determined in accordance with GB / T462-2008 "Determination of moisture content in paper, paperboard and pulp analysis samples" to calculate the absolute dry weight of the sample.

[0087] Preparation of glucose standard solution: Accurately weigh 0.5 g of glucose dried to constant weight at 105°C, add a small amount of distilled water to dissolve it, and then make up to 1000 mL with distilled water.

[0088] Preparation of 10g / L starch solution: Accurately weigh 5.0000g of starch or cationic starch into a 500mL beaker, add a small amount of water and stir evenly, add to boiling water, continue heating and boiling for 5mins, cool to room temperature, and then make up to 500mL.

[0089] Preparation of glucose standard curve Accurately measure 0.0, 0.1, 0.2, 0.3, 0.4, 0.5mL to a concentration of 0.5g·L -1 Put the glucose standard solution in 6 10mL colorimetric tubes, add deionized water to 0.5mL, add 0.5mL DNS reagent, mix well and place in a boiling water bath for 5mins to develop color, take out and quickly cool with running water, add 4.0mL of deionized water to each, shake well, adjust to zero with tube 0 at a wavelength of 540nm, and read the absorbance value A540 of each tube respectively. The measured glucose standard curve equation is: m=0.4106x+0.002, R 2=0.999, where m is the glucose content and x is the absorbance.

[0090] Glucoamylase hydrolyzed starch solution

[0091] At a reaction temperature of 50°C, the amount of 10 g / L starch solution was 2 mL, and the amount of saccharifying enzyme was 2 mg mL -1 , 1.7mg·mL -1 , 1.3mg·mL -1 , 1mg·mL -1 , 0.7mg·mL -1 , 0.35mg·mL -1 When the pH value of the reaction solution was 4.8 and the total volume was 150 mL, the starch solution was enzymatically hydrolyzed. A sample was taken every 10 minutes for DNS determination. At the same time, 1 to 2 drops were placed on a white porcelain plate and 1 drop of I2-KI solution was added to check whether the hydrolysis was complete.

[0092] The starch recovery test was conducted at a reaction temperature of 50°C and a saccharifying enzyme dosage of 1.3 mg mL -1 The dosage of 10g / L starch solution was 2mL, 4mL, 6mL, 8mL, and 10mL. When the pH value of the reaction solution was 4.8 and the total volume was 150mL, the starch solution was enzymatically hydrolyzed. After 1h of reaction, samples were taken for DNS determination. At the same time, 1 to 2 drops were placed on a white porcelain plate, and 1 drop of I2-KI solution was added to check whether the hydrolysis was complete.

[0093] Enzymatic hydrolysis of starch in paper samples and determination of glucose in enzymatic hydrolysate

[0094] Weigh 1.0g or 0.5g of the sample according to the starch content in the sample, add acetic acid-sodium acetate buffer solution with a pH value of 4.8 to soak overnight, break it up on a disperser, wash it with buffer solution for several times, pour it into a 250mL or 500mL stoppered conical flask, place it in a water bath constant temperature oscillator and keep it warm for 30 minutes, then add saccharifying enzyme or a mixture of saccharifying enzyme and α-amylase to make the total volume of the solution 150mL or 300mL. Start timing, take out 1-2 drops every 10 minutes and place them on a white porcelain plate, add 1 drop of I2-KI solution to check whether the hydrolysis is complete; at the same time, use a pipette to draw 0.5 mL of enzymatic solution, add 0.5 mL of DNS reagent, heat in a boiling water bath for 5 minutes to react, take it out and quickly cool it with running water, add 4.0 mL of distilled water, shake it well, filter it with a filter head, and measure the light absorption value at a wavelength of 540 nm. Substitute the measured absorbance value into the equation in 1.2.1 to calculate the glucose content in the enzymatic solution.

[0095] Calculation of starch content

[0096]

[0097] X: starch content in paper, %;

[0098] V: total volume of enzymatic reaction solution;

[0099] 0.5: The volume of the test solution taken for determination by DNS colorimetry;

[0100] m: The mass of glucose calculated by substituting the average absorbance value of the sample into the standard curve equation, mg;

[0101] 0.9: Conversion factor between glucose and starch;

[0102] M: paper sample mass, g.

[0103] Example 1

[0104] Starch solution hydrolysis test

[0105] In order to determine the experimental conditions for hydrolyzing starch with saccharifying enzyme, starch recovery rate tests were carried out at different saccharifying enzyme dosages. The absorbance values ​​at different times are shown in Table 1, and the starch recovery rate results are shown in Table 2.

[0106] As can be seen from Table 1, under different enzyme dosages, the absorbance remained basically unchanged when the solution reacted for 40 minutes, which is consistent with the result that starch turns blue when exposed to iodine. Therefore, the starch recovery rate can be calculated based on the absorbance of the reaction solution at 40 minutes. As can be seen from Table 3, when the enzyme dosage is greater than 0.13%, the starch recovery rate meets the experimental conditions.

[0107] Table 1 Effect of saccharifying enzyme dosage on starch hydrolysis

[0108]

[0109] Table 2 Starch recovery rate at different saccharifying enzyme dosages

[0110]

[0111] Starch recovery rate at different starch dosages

[0112] In order to test the accuracy of starch hydrolysis by saccharifying enzyme, the starch recovery rate test was carried out at different starch dosages, and the recovery rate values ​​are shown in Table 3. It can also be seen from Table 3 that the recovery rate of pure starch meets the determination requirements.

[0113] Table 3 Recovery rate of different starch dosage

[0114]

[0115] Example 2

[0116] Determination of starch content in cigarette tipping paper

[0117] The previous exploratory tests found that the starch content in ordinary tipping paper is low, and saccharifying enzyme can completely hydrolyze the starch contained therein. And with the increase of enzyme dosage, the glucose content also shows an increasing trend. After the enzyme dosage reaches a certain level, the increase of glucose content tends to be flat; the glucose content shows an increasing trend with the extension of reaction time, but after reaching a certain time, the glucose content tends to be stable; temperature has little effect on the reaction.

[0118] Response surface experiment

[0119] According to the results of the single-factor exploratory test, a response surface experiment with three factors and three levels and 17 test points was designed, with the three parameters of saccharifying enzyme addition (X1), reaction time (X2), and temperature (X3) as independent variables and glucose content as the response value. The horizontal design is shown in Table 4, and the analysis results are shown in Table 5.

[0120] Table 4 Response surface analysis factors and levels

[0121]

[0122] Table 5 Box-Behnken test design and results

[0123]

[0124] Establishment of regression equation and variance analysis

[0125] Regression analysis was performed on the data in Table 5 to obtain a quadratic multinomial regression model of glucose content on enzyme dosage, time and temperature: Y = -0.002 + 0.0014X1 + 0.0061X2 + 0.0002X3 + 0.0003X1X2 + 0.0000X1X3 + 0.0001X2X3 + 0.0008X1 2 +0.0019X2 2 +0.0007X3 2 .

[0126] The model and coefficients were tested for significance, and the results are shown in Table 6. 2 The value is 0.9828, indicating that only about 1.72% cannot be explained by the equation, and the lack-of-fit error is not significant (P<0.0001), indicating that the model is significant and has a good fit. As can be seen from Table 6, the effects of the first-order terms X1 and X2 on the response value are extremely significant, while the effect of X3 on the response value is not significant, that is, the effects of enzyme dosage and reaction time on starch hydrolysis are significant, while the effect of temperature is small, which is consistent with the results of the single-factor test. The second-order term X1 2 , X2 2 、X3 2The P values ​​of the three experimental factors on starch hydrolysis are also less than 0.01, indicating that the changes in the response values ​​are relatively complex, and the effects of the three experimental factors on starch hydrolysis are not simple linear relationships, and the surface effect is significant; the P value of the interaction term X1X2 is 0.021, and the P values ​​of X1X3 and X2X3 are greater than 0.05, indicating that the interactions of reaction time, enzymatic hydrolysis temperature, and enzyme dosage are not obvious, and the effects are not significant.

[0127] Table 6 Analysis of variance for starch hydrolysis

[0128]

[0129] Note: ** indicates highly significant (P < 0.01); * indicates significant difference (P < 0.05)

[0130] Response surface analysis and optimization

[0131] Figure 1-Figure 3 The response surface diagram and contour diagram are made based on the regression analysis results. The slope change of the response surface and the shape of the contour lines can reflect the degree of influence of the factors on the glucose content and their interaction. When the slope of the response surface is steep, it indicates that the response value is very sensitive to changes in the treatment conditions; if the response surface is relatively flat, it means that the response value is not sensitive to changes in the treatment conditions. The shape of the contour line can reflect the strength of the interaction. The ellipse indicates that the interaction between the two factors is significant, and the circle indicates that the interaction between the two factors is negligible. Figure 1-Figure 3 It can be seen that the enzyme dosage and reaction time have a significant effect on the glucose content of starch hydrolysis, and there are significant interactions between X1 and X2, X2 and X3, that is, enzyme dosage and reaction time, and reaction time and temperature.

[0132] Optimal reaction conditions and verification experiments

[0133] The optimal reaction conditions were analyzed by Design-Expert V 13.0.5 software: the dosage of saccharifying enzyme was 2.64 mg·mL -1 , the reaction time was 35.12 min, the reaction temperature was 52.82 °C, and the theoretical concentration of glucose in starch hydrolysis under these conditions was 0.070 mg·mL -1 Considering the actual operation, the condition was corrected to 2.64 mg·mL of saccharifying enzyme -1 , reaction time 35mins, reaction temperature 53℃. Under these conditions, through 6 parallel experiments, the concentration of glucose hydrolyzed by starch in ordinary tipping paper under the best conditions was 0.072mg·mL -1 , the comparison error is 2.8%, the starch content in ordinary tipping paper is 1.45%, and the RSD value is 1.02%.

[0134] Example 3

[0135] Determination of starch content in white cardboard

[0136] It was found from the preliminary exploratory tests that the starch content in white cardboard is relatively high. Using saccharifying enzyme alone cannot completely hydrolyze the starch it contains. It requires the synergistic effect of α-amylase to completely hydrolyze the starch contained therein.

[0137] Experimental design and results

[0138] According to the results of the single-factor exploratory test, a response surface experiment with three factors and three levels and 17 test points was designed, with the three parameters of α-amylase and saccharifying enzyme addition (X1), reaction time (X2), and temperature (X3) as independent variables and glucose content as the response value. The horizontal design is shown in Table 7, and the analysis results are shown in Table 8.

[0139] Table 7 Response surface analysis factors and levels

[0140]

[0141]

[0142] Table 8 Box-Behnken test design and results for determination of starch content in white cardboard

[0143]

[0144] Establishment of regression equation and variance analysis

[0145] Regression analysis was performed on the data in Table 8 to obtain the quadratic polynomial regression equation of glucose content on the amount of α-amylase and saccharifying enzyme, reaction time and reaction temperature: Y = -0.0132 + 0.0067X1 + 0.0564X2 + 0.0001X3 + 0.0015X1X2 + 0.0009X1X3 + 0.0004X2X3 + 0.0006X1 2 +0.0188X2 2 +0.0007X3 2

[0146] The regression equation and coefficients were tested for significance, and the results are shown in Table 9. 2 The value is 0.9886, indicating that only about 1.14% cannot be explained by the equation, and the lack-of-fit error is not significant (P<0.0001), indicating that the model is significant and has a good fit. As can be seen from Table 9, the first-order terms X1 and X2 have a very significant effect on the response value, while X3 has no significant effect on the response value, that is, the amount of α-amylase and saccharifying enzyme and the reaction time have a significant effect on starch hydrolysis, while the temperature has a small effect. The second-order term X2 2The P value of the interaction terms X1X2 and X1X3 is <0.05, indicating that the enzyme dosage has a significant interaction with the reaction time and reaction temperature, and the P value of X2X3 is >0.05, indicating that the interaction between reaction time and temperature is not obvious.

[0147] Table 9 Analysis of variance of starch hydrolysis in white cardboard

[0148]

[0149] Note: ** indicates highly significant (P < 0.01); * indicates significant difference (P < 0.05)

[0150] Response surface analysis

[0151] Figure 4-Figure 6 The response surface diagram and contour diagram are made based on the regression analysis results. Figure 4-Figure 6 It can be seen that the enzyme dosage and reaction time have a significant effect on the glucose content of starch hydrolysis in white cardboard, while the temperature has no obvious effect; X1 and X2, that is, the interaction between enzyme dosage and reaction time is significant.

[0152] Optimal reaction conditions and verification experiments

[0153] The optimal conditions were analyzed by Design-Expert V 13.0.5 software: the enzyme dosage was 5 mg·mL -1 , the reaction time is 115min, the reaction temperature is 60℃, and the theoretical concentration of glucose under these conditions is 0.220mg·mL -1 Under this condition, through 6 parallel experiments, the concentration of glucose in starch hydrolysis in white cardboard under the best condition was 0.218 mg·mL -1 The comparison error was 0.9%, the starch content in the white cardboard was measured to be 13.12%, and the RSD value was 0.8%.

[0154] Linearity and detection limit of the method

[0155] After deducting the absorbance of the blank sample, the concentration value corresponding to the absorbance of 0.01 is the detection limit. At this time, the corresponding glucose concentration of the solution is about 1.22 mg / L, and the starch concentration is about 1.1 mg / L.

[0156] Table 10 Linear relationship and detection limit of the method

[0157]

[0158] Spike recovery

[0159] Weigh the test paper sample, add starch solution with a starch content close to or double that in the paper sample to the reaction solution, react, and measure the spike recovery rate. The results are shown in Table 12. As can be seen from Table 12, the recovery rate is 96.2% to 102%.

[0160] Table 11 Spiked recovery

[0161]

[0162]

[0163] Glucose amylase or a compound enzyme of amylase and glucose amylase can hydrolyze the starch in the dispersed paper sample into glucose. The starch content in the paper sample can be calculated by measuring the glucose content by spectrophotometry. This method uses ordinary instruments, is simple to operate, has good precision, high recovery rate, and accurate and reliable test results. It can be used to determine the starch content in paper and paperboard.

[0164] Comparative Example 1

[0165] References for the existing research methods of the present invention: Xu Rong, Xie Tangtang, Chen Guiqiang, et al. The starch content in paper products was determined by spectrophotometry, and the results are shown in the following table.

[0166] Table 12 Comparison between the method of the present invention and the existing method

[0167]

[0168] As can be seen from the table, the starch content measured by the existing research method is lower than that of this method. The main reason is that the existing method uses hot water extraction, and it is difficult to extract modified starch such as cationic starch in paper and paperboard, so the test result is low. Similarly, the method of TAPPI T 419om-02 further confirms that hot water extraction has the disadvantage of not being able to completely extract starch from paper and paperboard. In addition, the spiked recovery rate and relative deviation of the method of the present invention are better than those of the existing research method and the method of TAPPI T419om-02. This is mainly because the existing method and the method of TAPPI T 419om-02 use the colored complex generated by starch and iodine reagent and use spectrophotometry to directly determine. However, the complexes generated by amylose and amylopectin with iodine have different colors. Amylose is a linear polymer formed by glucose residues connected by α-1,4 glycosidic bonds. The complex generated with iodine is blue with an absorption wavelength of 500 to 800nm. In addition to the main chain of glucose residues connected by α-1,4 glycosidic bonds, the branched chains in the amylopectin molecule are also connected by α-1,6 glycosidic bonds. The complex with iodine is purple-red and has an absorption wavelength of 500-600nm. Since starch is a mixture of amylose and amylopectin, when using the single wavelength method, the absorption peaks of amylose and amylopectin overlap, which will interfere with each other and affect the accuracy of the results. Based on the above analysis, it can be seen that the method of the present invention is simple to operate and the test results are accurate and reliable, which can meet the determination of various starch contents in paper.

[0169] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for detecting the content of modified starch in paper and paperboard, characterized in that: The steps include: A) soaking the test paper in an acidic buffer and then breaking it up to obtain a mixed system; B) adding an enzyme solution to the mixed system to perform enzymolysis to obtain an enzymolysis solution; C) Add the enzymatic hydrolysate to 3,5-dinitrosalicylic acid colorimetric solution, inactivate it by heating, measure the light absorption value, and substitute the measured light absorption value into the glucose standard curve equation to calculate the glucose content, thereby calculating the starch content.

2. The detection method according to claim 1, characterized in that: The formula for calculating starch content is as follows: X: starch content in paper sample, %; V: total volume of enzymatic reaction solution; V1: The volume of the test solution taken for DNS colorimetric determination; m: The mass of glucose calculated by substituting the average absorbance value of the sample into the standard curve equation, mg; 0.9: Conversion factor between glucose and starch; M: Paper sample mass, g.

3. The detection method according to claim 1, characterized in that: The glucose standard curve equation is m=0.4106x+0.002, R 2 =0.999, where m is the glucose content and x is the absorbance; The concentration of the glucose standard solution is 0.1 g·L -1 ~1g·L -1 .

4. The detection method according to claim 1, characterized in that: Step A) The acidic buffer solution is an acetic acid-sodium acetate buffer solution, and the pH of the buffer solution is 3-5.

5. The detection method according to claim 1, characterized in that: The enzyme solution is saccharifying enzyme and / or α-amylase; the amount of enzyme added is 2.64 mg·mL -1 ~5 mg mL -1 .

6. The detection method according to claim 1, characterized in that: When the starch content in paper and paperboard is less than 4%, the enzyme solution is saccharifying enzyme; when the starch content is greater than 4%, the enzymes are α-amylase and saccharifying enzyme.

7. The detection method according to claim 1, characterized in that: The enzymolysis temperature is 45-55° C. and the enzymolysis time is 35-75 min.

8. The detection method according to claim 1, characterized in that: The operation to determine whether the enzymatic hydrolysis is complete is as follows: take 1 to 2 drops at regular intervals and place them on the surface of the white substrate, and add I2-KI solution to check whether the hydrolysis is complete.

9. The detection method according to claim 1, characterized in that: The light absorption value is measured at a wavelength of 540 nm.

10. The detection method according to claim 1, characterized in that: The detection limit of glucose is 1.22 mg / L -1 , the detection limit of starch is 1.1mg / L -1 The standard curve of starch is c=0.4106A+0.002, and the starch content range that can be measured is 0.16% to 35.7%.

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