Method for measuring content of starch in stem paste

Abstract

The invention relates to the technical field of chemical analysis, in particular to a method for analyzing the content of starch in stem paste, which comprises the following steps: precipitating the starch dissolved in the stem paste by using ethanol, separating the starch from a complex stem paste matrix, separating the starch from other interfering substances such as saccharides, determining the content of amylopectin in the stem paste, and determining the content of the starch in the stem paste. According to the method, amylose and amylopectin are finally converted into glucose according to the proportion of the amylose and hydrolysis by using an enzyme with relatively high specificity, then the content of the glucose in the solution is measured by an ion chromatography method, so that the content of the starch is represented, and the method has the advantages of sensitivity in measurement and high accuracy in measurement of the content of the starch in the stem paste.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for determining starch content in stem paste. Background Art

[0002] There is currently no standard method for determining the starch content in tobacco stems. The starch content in tobacco is usually analyzed by spectrophotometry and continuous flow color development, drawing on the method for determining the starch content in tobacco; however, there are three reasons why the results of this analysis are not accurate: first, it is difficult to eliminate the interference caused by the complex matrix and darker color of the stems; second, the pre-treatment object of the starch in tobacco is solid, while the analysis target of the starch content in the stems is tobacco stem extract, which is a solution. The solution uses 80% ethanol-saturated sodium chloride solution to wash sugar. During the process of passing through the C3 sand core sintered glass funnel, the starch in the stems cannot be fully precipitated, and the particles easily enter the sand core part, blocking the micropores in the sand core, and the starch in the stems cannot be completely extracted, resulting in a large measurement error; third, because the retained part of the starch cannot be controlled when passing through the sand core, the test result will be lower than the detection limit of the flow analysis, and the result is unstable. Therefore, the stability and accuracy of the currently used method for determining the starch content in stems are relatively poor.

[0003] Therefore, it is necessary to provide a novel starch content determination and analysis method in the stem paste. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a method for measuring and analyzing the starch content in stem paste.

[0005] The technical solution of the present invention is as follows.

[0006] The present invention provides a method for determining the starch content in a stem paste, the method comprising the following steps:

[0007] (1) Add 25 mL of 80% ethanol-saturated sodium chloride solution to 1.500 g of stem extract, ultrasonicate at 300 W for 25 min, centrifuge and discard the supernatant, add 75% ethanol solution to the obtained precipitate, shake repeatedly to wash the precipitate, and centrifuge to obtain a precipitate;

[0008] (2) adding α-amylase and 25 ml of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 5.5 to the precipitate obtained in step (1), and performing enzymatic hydrolysis at 55° C. for 3.0 h; then, adding saccharifying enzyme, pullulanase and 25 ml of citric acid-sodium citrate buffer at pH 4.5, and performing enzymatic hydrolysis at 55° C. for 2.5 h; after cooling, filtering under reduced pressure, and diluting to 100 mL with water;

[0009] (3) Take 1 mL of the solution obtained in step (2), dilute to 250 mL with water, and then pass through C18 The impurities are removed by a small column and filtered with a 0.22 μm water system membrane, and then the filtrate is subjected to ion chromatography-pulsed amperometry analysis, the analysis includes:

[0010] A PA-10 sugar column was used, with a column temperature of 30°C, a flow rate of 1.0 mL / min, and an injection volume of 25 μL;

[0011] Mobile phase A was ultrapure water, and mobile phase B was 250 mmol / L NaOH solution;

[0012] Isocratic elution was performed with 80% volume of mobile phase A and 20% volume of mobile phase B for 13 min;

[0013] Calculate the chromatographic peak area (Y);

[0014] (4) Calculate the glucose concentration X according to the following formula:

[0015] Y = 1.6432X – 0.0431, R 2 =0.9992, where X represents the glucose concentration and Y represents the chromatographic peak area; then the glucose content W is calculated. 葡萄糖 ;

[0016] Further, the starch content was calculated according to the following formula:

[0017] W 淀粉 =W 葡萄糖 ×0.9

[0018] Among them, W 淀粉 is the starch content, W 葡萄糖 is the glucose content after enzymatic hydrolysis, and 0.9 is the conversion coefficient of glucose content to starch content.

[0019] Preferably, in step (1), the centrifugation after ultrasound is performed at a speed of 5000 r / min for 5 min.

[0020] Preferably, in step (1), the centrifugation after adding the ethanol solution to wash the precipitate is performed at a speed of 5000 r / min for 5 min.

[0021] Preferably, in step (1), 8-12 mL of 75% ethanol solution is added, the precipitate is repeatedly shaken and centrifuged for 3-5 times, wherein the centrifugation is performed at a speed of 5000 r / min for 5 min.

[0022] Preferably, in step (2), the total amount of α-amylase, saccharifying enzyme and pullulanase added is 200 U per gram of stem extract.

[0023] Preferably, in step (2), the added amounts of α-amylase, saccharifying enzyme and pullulanase are calculated in an enzyme activity unit ratio of 1:1:1.

[0024] Preferably, in step (2), the enzymatic hydrolysis is carried out in a constant temperature water bath.

[0025] The following is a detailed description of the present invention.

[0026] To achieve the above object, the technical solution adopted by the present invention is: a method for analyzing the starch content in stem paste, comprising the steps of:

[0027] A. Preparation of standard working solution:

[0028] A1. Accurately weigh 0.1000g of glucose standard, dissolve it in ultrapure water and dilute it to 100mL volumetric flask, shake well to obtain 1.00mg / mL mixed standard stock solution;

[0029] A2. Accurately pipette 1 mL of the mixed standard stock solution and dilute it to a 25 mL volumetric flask to obtain a 0.040 mg / mL mixed standard solution;

[0030] A3. Accurately pipette 0.05, 0.125, 0.25, 0.5, and 1 mL of the standard solution into 5 mL volumetric flasks to obtain a series of mixed standard solutions with concentrations of 0.4, 1, 2, 4, and 8 mg / L, respectively.

[0031] A4. Establishment of standard curve

[0032] The glucose concentration in the standard solution was determined by ion chromatography, and the standard curve equation was obtained as follows:

[0033] Y = 1.6432X – 0.0431, R 2 =0.9992

[0034] Where X represents the glucose concentration and Y represents the chromatographic peak area;

[0035] The detection limit LOD of glucose was calculated to be 0.79 mg / g stem paste according to the signal-to-noise ratio (S / N=3), and the quantification limit LOQ was calculated to be 2.56 mg / g stem paste according to the signal-to-noise ratio (S / N=10). The detection limit LOD and quantification limit LOQ of starch were 0.72 mg / g stem paste and 2.30 mg / g stem paste, respectively.

[0036] B. Pretreatment of stem paste samples

[0037] B1. Accurately weigh 1.500g of stem paste, add 25mL of 80% ethanol-saturated sodium chloride solution, place in a 500ml conical flask, ultrasonicate for 25min, centrifuge at 5000r / min for 5min, discard the supernatant, wash the lower precipitate with 75% ethanol solution 3-5 times, shake and wash the precipitate repeatedly, and centrifuge;

[0038] B2. Determination of amylose: Weigh 10 mg of amylose as the standard, add 0.1 mL of anhydrous ethanol and 0.9 mL of water, mix well, seal with a sealing film, and place in a boiling water bath until dissolved, which is a 10 mg / mL amylose standard solution. Dilution of the standard solution: Take 40 μL of the 10 mg / mL standard solution, add 960 μL of a mixed solution of amylase (liquid) and DNS color developer, mix well, and prepare a 0.4 mg / mL standard solution.

[0039] Accurately weigh 1.500 g of stem paste, add 25 mL of 80% ethanol-saturated sodium chloride solution, place in a 500 ml conical flask, ultrasonicate at 300 W for 25 min, centrifuge at 5000 r / min for 5 min, discard the supernatant, wash the lower precipitate with about 10 mL of 75% ethanol solution for 3-5 times, shake and wash the precipitate repeatedly, and centrifuge to obtain a precipitate.

[0040] Weigh 1 g of precipitate, add 25 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer with a pH value of 5.5 and pullulanase, hydrolyze at 80°C for 20 minutes, extract in a water bath for 30 minutes, then centrifuge at 5000 r / min and 25°C for 5 minutes, discard the supernatant, add 1 mL of amylase and phosphate buffer with a pH value of 5.5 to the precipitate, oscillate for 5 minutes, water bath at 90°C for 10 minutes, cool and centrifuge for 5 minutes, take the supernatant, which is the enzymatic hydrolyzate of the stem paste precipitate, and use the DNS method for colorimetric determination.

[0041] The absorbance at 550 nm and 485 nm was measured with a spectrophotometer. The absorbance at 550 nm was recorded as A assay, A standard and A blank, respectively, and the absorbance at 485 nm was recorded as A1 assay, A1 standard and A1 blank, respectively, and ΔA assay = (A assay - A blank) - (A1 assay - A1 blank), ΔA standard = (A standard - A blank) - (A1 standard - A1 blank) was calculated.

[0042] Calculation of amylose content: amylose content (mg / g stem paste mass) = 2 × ΔA determination / (ΔA standard × W), "W" represents the total mass of the stem paste. It was determined that amylose / amylopectin in the stem paste = 1:5.

[0043] B3. Enzymes are specific. Pullulanase is a specific starch debranching enzyme that can specifically cut the α-1,6-glycosidic bonds in the amylopectin branches. It can cut the entire amylopectin branch, including the smallest unit branch, to form straight-chain starch. Glycosylase can hydrolyze the α-1,4-glucosidic bonds from the non-reducing sugar end of starch to release single β-D-glucose one by one. In addition, it can also hydrolyze the α-1,6-glucosidic bonds and α-1,3-glycosidic bonds, completely hydrolyzing the amylopectin in the stem paste into glucose. According to the measured ratio of straight and amylopectin in the stem paste (amylose / amylopectin = 1:5), combined with the characteristics of various enzymes hydrolyzing glycosidic bonds and the comprehensive results, 1-2 mL of 2000 U / g α-amylase was added to the precipitate, and 25 ml of NaH2PO4-Na2HPO4 buffer solution was added to the stem paste precipitate sample, and enzymolysis was performed in a constant temperature water bath at 55°C for 3.0 h; then 25 ml of citric acid-sodium citrate buffer solution with a pH of 4.5, 5000 U / g saccharifying enzyme and 5000 U / g pullulanase were added, and enzymolysis was performed in a constant temperature water bath at 55°C for 2.5 h. After cooling, the sample liquid was filtered and the volume was fixed to a 100 mL volumetric flask for standby use;

[0044] Pipette 1 mL into a 250 mL volumetric flask and pass the enzymatic solution through C 18 The impurities were removed from the small column, and after the 0.22μm water system membrane, the glucose concentration X was obtained by ion chromatography, and the glucose content W was further calculated. 葡萄糖 ;

[0045] C. Result determination

[0046] The starch content is converted by the glucose content. The conversion formula is as follows:

[0047] W 淀粉 =W 葡萄糖 *0.9

[0048] Among them, W 淀粉 is the starch content, W 葡萄糖 is the glucose content after enzymatic hydrolysis, and 0.9 is the conversion coefficient of glucose content to starch content.

[0049] The present invention provides a method for accurately analyzing the content of starch in stem paste, the method comprising:

[0050] Firstly, the starch in the stem paste was precipitated with 80% ethanol-saturated sodium chloride solution, and the precipitate was repeatedly washed with 75% ethanol solution to eliminate the interference and negative effects of soluble sugar and other ions on the detection, thereby separating the starch from the complex stem paste matrix;

[0051] Then, by analyzing the content ratio of straight and branched starch in the precipitate and comprehensively considering the differences in the positions of glycosidic bonds, three enzymes (amylase, saccharifying enzyme, and pullulanase) were selected to hydrolyze starch, and the starch was fully converted into glucose through a highly specific enzymatic hydrolysis method.

[0052] After enzymatic hydrolysis, other interfering substances are separated through further treatments such as impurity removal and filtration, and then the glucose content in the solution is determined by ion chromatography to characterize the starch content.

[0053] The method provided by the present invention has the advantages of being sensitive and highly accurate in determining the starch content in the stem paste. Experiments have shown that the starch content in the stem paste sample is determined by the method of the present invention, and the average starch content in the stem paste is 6.42%, and the relative standard deviation (RSD, n=5) is 1.24%; when low and medium amounts of soluble corn starch are added to the stem paste sample, the recovery rate is 98.9% to 101.3%.

[0054] The invention solves the serious influence of low test data and unstable test results caused by the sand core sintered glass funnel in the continuous flow method for determining starch in tobacco and tobacco products in the tobacco industry standard YCT216-2013, and has the advantages of strong ability to remove matrix interference, high sensitivity and high accuracy, and is very suitable for the determination of starch content in a complex system such as stem paste. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0056] Figure 1 : The standard curve of glucose content determination in Example 1.

[0057] Figure 2 : Ion chromatogram of glucose standard in Example 1.

[0058] Figure 3 : Starch degradation rate curve of different enzyme combinations in Example 3. Among them, 1 represents "α-amylase + pullulanase", 2 represents "α-amylase", 3 represents "α-amylase + pullulanase + saccharifying enzyme", and 4 represents "α-amylase + saccharifying enzyme".

[0059] Figure 4 : Standard curve of starch content determination in Comparative Example 1.

[0060] Figure 5 : The standard curve diagram of starch content determination in Comparative Example 1.

[0061] Figure 6 : The content determination spectrum of the starch sample in Comparative Example 1. DETAILED DESCRIPTION

[0062] The present invention is described below with reference to specific examples. It will be appreciated by those skilled in the art that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0063] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0064] Example 1 Establishment of standard curve for determination of glucose content

[0065] Accurately weigh 0.1000 g of glucose standard, dissolve it in ultrapure water, dilute it to 100 mL volumetric flask, and shake well to obtain 1.00 mg / mL mixed standard stock solution.

[0066] Accurately pipette 1 mL of the mixed standard stock solution and dilute it to a 25 mL volumetric flask to obtain a 0.040 mg / mL mixed standard solution.

[0067] Accurately pipette 0.05, 0.125, 0.25, 0.5, and 1 mL of the mixed standard solution and dilute them into 5 mL volumetric flasks to obtain a series of diluted mixed standard solutions with concentrations of 0.4, 1, 2, 4, and 8 mg / L, respectively.

[0068] Ion chromatography-pulsed amperometry was used using the ion chromatography conditions shown in Tables 1 and 2:

[0069] Table 1. Ion chromatography-pulsed amperometry detection conditions

[0070]

[0071] Table 2. Isocratic elution conditions of mobile phase

[0072]

[0073] The glucose concentration in the series of mixed standard solutions was determined, and the standard curve equation was obtained as follows:

[0074] Y=1.6432X+0.0431,R 2 =0.9992

[0075] Where X represents the glucose concentration and Y represents the chromatographic peak area. The standard curve is shown in Figure 1 ; Chromatogram is shown in Figure 2 .

[0076] Example 2 Determination of the ratio of straight and amylopectin in stem paste

[0077] Weigh 10 mg of amylose as a standard, add 0.1 mL of anhydrous ethanol and 0.9 mL of water, mix well, seal with a sealing film, and place in a boiling water bath until dissolved, which is a 10 mg / mL amylose standard solution. Take 40 μL of the 10 mg / mL amylose standard solution, add 960 μL of a mixed solution of amylase (liquid) and DNS color developer, mix well, and prepare a 0.4 mg / mL standard solution for use.

[0078] Accurately weigh 1.500 g of stem paste, add 25 mL of 80% ethanol-saturated sodium chloride solution, place in a 500 ml conical flask, ultrasonicate at 300 W for 25 min, centrifuge at 5000 r / min for 5 min, discard the supernatant, wash the lower precipitate with 10 mL of 75% ethanol solution for 3-5 times, shake and wash the precipitate repeatedly, and centrifuge to obtain a precipitate.

[0079] Weigh 1 g of precipitate, add 25 mL of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer with a pH value of 5.5 and pullulanase, hydrolyze at 80°C for 20 minutes, extract in a water bath for 30 minutes, then centrifuge at 5000 r / min and 25°C for 5 minutes, discard the supernatant, add 1 mL of amylase and phosphate buffer with a pH value of 5.5 to the precipitate, oscillate for 5 minutes, water bath at 90°C for 10 minutes, cool and centrifuge for 5 minutes, take the supernatant, which is the enzymatic hydrolyzate of the stem paste precipitate, and use the DNS method for colorimetric determination.

[0080] The absorbance values ​​of the amylose standard solution and the supernatant at 550nm and 485nm were measured by a spectrophotometer. The measuring tube (adding the stem extract precipitation enzymatic hydrolyzate), standard tube (adding the amylose standard solution), and blank tube (phosphate buffer) at 550nm were recorded as A measurement, A standard, and A blank, respectively. Similarly, the measuring tube, standard tube, and blank tube measured at 485nm were recorded as A1 measurement, A1 standard, and A1 blank, respectively. The absorbance measured by each tube was used to calculate: ΔA measurement = (A measurement - A blank) - (A1 measurement - A1 blank), ΔA standard = (A standard - A blank) - (A1 standard - A1 blank).

[0081] Calculate the amylose content: amylose content (mg / g stem extract mass) = 2×ΔA assay / (ΔA standard×W), where “W” represents the total mass of the precipitate.

[0082] The amylopectin content was calculated based on the total mass of 1 g of the precipitate; the straight chain starch / amylopectin content in the stem paste was determined to be 1:5.

[0083] Example 3 Determination of starch content in stalk paste

[0084] Pullulanase is a specific starch debranching enzyme that can specifically cut the α-1,6-glycosidic bonds in the amylopectin branches. It can cut the entire amylopectin branch, including the smallest unit branch, to form straight-chain starch; saccharifying enzyme can hydrolyze the α-1,4-glucosidic bonds of starch from the non-reducing sugar end to release single β-D-glucose one by one. In addition, it can also hydrolyze the α-1,6-glucosidic bonds and α-1,3-glycosidic bonds, and completely hydrolyze the amylopectin in the stem paste into glucose. According to the measured ratio of straight amylopectin in the stem paste (amylose / amylopectin = 1:5), combined with the characteristics of various enzymes in hydrolyzing glycosidic bonds and the comprehensive results, α-amylase, saccharifying enzyme and pullulanase were preliminarily selected, and the enzymatic hydrolysis effects of these enzymes were compared.

[0085] Accurately weigh 1.500g of stem paste, add 25mL of 80% ethanol-saturated sodium chloride solution, place in a 500ml conical flask, ultrasonicate at 300W for 25min, centrifuge at 5000r / min for 5min, discard the supernatant, wash the lower precipitate with 10mL of 75% ethanol solution for 3-5 times, shake the precipitate repeatedly, centrifuge at 5000r / min for 5min to obtain a precipitate.

[0086] (1) Add a certain amount of α-amylase to the precipitate, and at the same time add 25 ml of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer with a pH of 5.5, and perform enzymolysis in a constant temperature water bath at 55°C for 3.0 h; then add 25 ml of citric acid-sodium citrate buffer with a pH of 4.5, a certain amount of saccharifying enzyme (5000 U / g) and a certain amount of pullulanase (5000 U / g), and perform enzymolysis in a constant temperature water bath at 55°C for 2.5 h. After cooling, use a Buchner funnel to decompress and filter the sample liquid, and dilute to a 100 mL volumetric flask with water to obtain an enzymolysis solution.

[0087] According to the total amount of the three enzymes (1:1:1) of 30U / g stem paste, 50U / g stem paste, 100U / g stem paste, and 200U / g stem paste, the above operation was performed to obtain multiple enzymatic hydrolysates.

[0088] (2) Add a certain amount of α-amylase to the precipitate, and at the same time add 25 ml of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer with a pH of 5.5, and perform enzymolysis in a constant temperature water bath at 55°C for 3.0 h; then add 25 ml of citric acid-sodium citrate buffer with a pH of 4.5 and a certain amount of pullulanase (5000 U / g), and perform enzymolysis in a constant temperature water bath at 55°C for 2.5 h. After cooling, use a Buchner funnel to decompress and filter the sample liquid, and dilute to a 100 mL volumetric flask with water to obtain an enzymolysis solution.

[0089] According to the total amount of the two enzymes (1:1 enzyme activity unit) of 30U / g stem paste, 50U / g stem paste, 100U / g stem paste, and 200U / g stem paste, the above operation was performed to obtain multiple enzymatic hydrolysates.

[0090] (3) Add a certain amount of α-amylase to the precipitate, and at the same time add sodium dihydrogen phosphate-disodium hydrogen phosphate buffer solution with a pH of 5.5, and hydrolyze in a constant temperature water bath at 55°C for 3.0 hours; then add 25 ml of citric acid-sodium citrate buffer solution with a pH of 4.5, and hydrolyze in a constant temperature water bath at 55°C for 2.5 hours. After cooling, use a Buchner funnel to decompress and filter the sample liquid, and dilute to a 100 mL volumetric flask with water to obtain an enzymatic solution.

[0091] A plurality of enzymatic hydrolysates were obtained by processing according to the total amount of enzyme being 30U / g stem paste, 50U / g stem paste, 100U / g stem paste and 200U / g stem paste respectively.

[0092] (4) Add a certain amount of α-amylase to the precipitate, and add sodium dihydrogen phosphate-disodium hydrogen phosphate buffer with a pH of 5.5, and perform enzymolysis in a constant temperature water bath at 55°C for 3.0 hours; then add 25 ml of citric acid-sodium citrate buffer and a certain amount of saccharifying enzyme (5000 U / g), and perform enzymolysis in a constant temperature water bath at 55°C for 2.5 hours. After cooling, use a Buchner funnel to decompress and filter the sample liquid, and dilute to a 100 mL volumetric flask with water to obtain an enzymolysis solution.

[0093] According to the total amount of the two enzymes (1:1 enzyme activity unit) of 30U / g stem paste, 50U / g stem paste, 100U / g stem paste, and 200U / g stem paste, the above operation was performed to obtain multiple enzymatic hydrolysates.

[0094] Pipette 1 mL of each of the enzymatic hydrolysates from (1) to (4) above, dilute to 250 mL with water in a volumetric flask, load the diluted enzymatic hydrolysate into a syringe, and pressurize through C 18 The impurities were removed by a small column, and after filtration with a 0.22 μm water system membrane, ion chromatography-pulsed amperometry analysis was performed to obtain the chromatographic peak area. The ion chromatography detection conditions are shown in Example 1.

[0095] The glucose concentration was obtained according to the standard curve equation of Example 1 and converted into the glucose content W according to the volume. 葡萄糖 , further the glucose content W 葡萄糖 Converted into starch content, the conversion formula is as follows:

[0096] W 淀粉 =W 葡萄糖 ×0.9.

[0097] Calculate the starch degradation rate (the starch content obtained is divided by 1.500g of stem paste), and the results are shown in Figure 3 .Depend on Figure 3 It can be seen that when α-amylase+pullulanase+saccharifying enzyme (curve 3) is used, the highest starch degradation is achieved overall.

[0098] The starch content in the stem paste samples was measured 5 times in parallel using the combined enzymatic hydrolysis of the three enzymes as above, and the results are shown in Table 3. The average starch content in the stem paste samples measured 5 times was 6.42%, and the relative standard deviation (RSD, n=5) was 1.24%.

[0099] Table 3. Starch content in stem paste

[0100]

[0101] Low and medium amounts of soluble corn starch were added to the stem paste sample and the above process was used for re-measurement. The recovery rates were 98.9% to 101.3%, and the results are shown in Table 4. The recovery rate was calculated as follows: ("total amount of starch measured" - "the starch content detected without the addition of corn starch") / amount of corn starch added.

[0102] Table 4. Recovery of starch

[0103]

[0104] Comparative Example 1

[0105] The starch content in tobacco stems was determined using the industry standard YCT216-2013 Tobacco and Tobacco Products Starch Determination Method (continuous flow method). The results of the tobacco flow analyzer for determining the starch content in stem paste are as follows:

[0106] 1. Standard curve and detection limit and quantification limit

[0107] The standard curve of starch is a=-7.4258E-002; b=2.0284E-005, R 2 =1.0000. The detection limit LOD of starch is 0.46 mg / L, and the quantification limit LOQ is 1.53 mg / L. The standard curve is shown in Figure 4 ; The standard curve is shown in Figure 5 .

[0108] 2. Precision and recovery

[0109] The starch content in the stem paste sample was measured, and the results are shown in Table 5. After the sample was measured 5 times in parallel, the average starch content in the stem paste sample was 0.076%, and the relative standard deviation (RSD, n = 5) was 1.083%. The measured value of the starch content in the sample was obviously low, which proved that this method could not effectively precipitate the starch in the stem paste, resulting in a low starch content measurement value, and was not suitable for the analysis of the starch content in the stem paste. The starch sample spectrum is shown in Figure 6 .

[0110] Table 5. Starch content in stem paste

[0111]

[0112] Similarly, low and medium amounts of soluble corn starch were added to the stem paste samples and the industry standard method was used to measure again for recovery verification. The measured recovery rate was 40% to 58%, and the results are shown in Table 6. This further shows that the industry standard method has a low determination result and is not suitable for the determination of starch in stem paste.

[0113] Table 6. Recovery of starch

[0114]

[0115]

[0116] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of the claims attached to the present invention.

Claims

1. A method for determining starch content in stem paste, characterized in that: The determination method comprises the following steps: (1) Add 25 mL of 80% ethanol-saturated sodium chloride solution to 1.500 g of stem extract, ultrasonicate at 300 W for 25 min, centrifuge and discard the supernatant, add 75% ethanol solution to the obtained precipitate, shake repeatedly to wash the precipitate, and centrifuge to obtain a precipitate; (2) adding α-amylase and 25 ml of sodium dihydrogen phosphate-disodium hydrogen phosphate buffer at pH 5.5 to the precipitate obtained in step (1), and performing enzymatic hydrolysis at 55° C. for 3.0 h; then, adding saccharifying enzyme, pullulanase and 25 ml of citric acid-sodium citrate buffer at pH 4.5, and performing enzymatic hydrolysis at 55° C. for 2.5 h; after cooling, filtering under reduced pressure, and diluting to 100 mL with water; (3) Take 1 mL of the solution obtained in step (2), dilute to 250 mL with water, and then pass through C 18 The impurities are removed by a small column and filtered with a 0.22 μm water system membrane, and then the filtrate is subjected to ion chromatography-pulsed amperometry analysis, the analysis includes: A PA-10 sugar column was used, with a column temperature of 30°C, a flow rate of 1.0 mL / min, and an injection volume of 25 μL; Mobile phase A was ultrapure water, and mobile phase B was 250 mmol / L NaOH solution; Isocratic elution was performed with 80% volume of mobile phase A and 20% volume of mobile phase B for 13 min; Calculate the chromatographic peak area (Y); (4) Calculate the glucose concentration X according to the following formula: Y = 1.6432X – 0.0431, R 2 =0.9992, where X represents the glucose concentration and Y represents the chromatographic peak area; then the glucose content W is calculated. 葡萄糖 ; Further, the starch content was calculated according to the following formula: 淀粉 =W 葡萄糖 ×0.

9.

2. The measuring method according to claim 1, characterized in that In step (1), the centrifugation after ultrasound is performed at a speed of 5000 r / min for 5 minutes.

3. The measuring method according to claim 1 or 2, characterized in that In step (1), the centrifugation after adding the ethanol solution to wash the precipitate is performed at a speed of 5000 r / min for 5 minutes.

4. The measuring method according to any one of claims 1 to 3, characterized in that In step (1), add 8-12 mL of 75% ethanol solution, shake repeatedly to wash the precipitate, and centrifuge 3-5 times.

5. The measuring method according to any one of claims 1 to 4, characterized in that In step (2), the total amount of α-amylase, saccharifying enzyme and pullulanase added is 200U per gram of stem paste.

6. The measuring method according to any one of claims 1 to 5, characterized in that In step (2), the added amounts of α-amylase, saccharifying enzyme and pullulanase are calculated in an enzyme activity unit ratio of 1:1:

1.

7. The measuring method according to any one of claims 1 to 6, characterized in that In step (2), the enzymatic hydrolysis is carried out in a constant temperature water bath.

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