CN116223409A - Method for detecting total sugar content in wheat gluten - Google Patents
Method for detecting total sugar content in wheat gluten Download PDFInfo
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Abstract
The invention relates to a method for detecting total sugar content in wheat gluten, which comprises the following steps: s1, manufacturing a standard curve; s2, sample pretreatment: adding pepsin into the wheat gluten sample for pretreatment; s3, color reaction: adding a color reagent into the test solution pretreated in the step S2 to obtain a sample solution and performing color development treatment; s4, measuring absorbance of the sample: measuring and reading absorbance of the sample obtained after the color development treatment in the step S3 by adopting a spectrophotometer, and calculating the glucose content in the sample solution by using a standard curve; s5, blank test; s6, calculating a result: and (3) calculating the total sugar content in the sample according to the glucose content in the sample solution obtained in the step (S4) and the glucose content in the blank solution obtained in the step (S5). Compared with the prior art, the invention has the advantages of good stability, high accuracy and good repeatability of detection results, avoids the influence of the hydration viscoelasticity of the wheat gluten, identifies the quality of fish meal and improves the quality of subsequent products.
Description
Technical Field
The invention relates to the field of detection, in particular to a method for detecting total sugar content in wheat gluten.
Background
Wheat gluten is a wheat product obtained by taking wheat or wheat flour as a raw material and separating non-protein components such as starch or other carbohydrates in the wheat or the wheat flour, and has high viscoelasticity after hydration, and is also called active wheat gluten. Gluten meal is rich in high-quality vegetable proteins, and is widely paid attention to in place of fish meal in livestock and poultry feed. The quality index of GB/T21924-2008 gluten powder specifies crude protein (NX 6.25, dry basis)/% > 85% (first order), 80% (second order) without specifying the content of starch or other carbohydrates. In practical applications, different processes for the production of gluten may result in different nutritional qualities due to the different starch or other carbohydrates, i.e. the total sugar content (calculated as glucose).
Chinese patent CN201711375648.7 discloses a method for detecting sugar content in feed, comprising the steps of grinding feed sample into powder, eluting with diethyl ether, grinding into slurry in water bath of 40-45 ℃, adding water, grinding into slurry in water bath of 40-45 ℃, cooling to room temperature, filtering, collecting filtrate and constant volume to 100mL, diluting to obtain pretreated sample; glucose, galactose and pentose are mixed and then dissolved in water to prepare glucose-galactose-pentose standard solution, a standard curve is drawn, and finally, the determination and calculation of total sugar in a sample are carried out. The sample test recovery rate of the invention is 92-103.8%, and the error is smaller as the total sugar concentration in the sample is increased, the sensitivity can reach 5 mug/100 mL, the accuracy of the determination of the total sugar and the reducing sugar in the feed can be improved, the detection limit is reduced, an expensive chromatograph is not needed, the pretreatment work is simple, and the cost is low. The invention refers to bran, bean pulp and corn as main raw materials of feed, and the invention uses glucose-galactose-pentose standard solution to determine the total sugar content of feed samples according to the nutritional ingredients of the bran, the bean pulp and the corn. The invention is not directed to gluten, and the invention is not suitable for measuring the total sugar content of gluten, as is known from the high viscoelasticity of gluten after hydration.
Chinese patent CN201210397189.3 discloses a method for detecting polysaccharide content in wolfberry polysaccharide extract doped with saccharide interfering substance, comprising the following steps: (1) Dissolving fructus Lycii polysaccharide extract mixed with saccharide interfering substance in water, adding phosphate buffer solution and amylase, stirring, incubating, intermittently stirring, taking out, and heating to boil; transferring and supplementing water to constant volume after the boiling liquid is cooled, shaking up and filtering, discarding the primary filtrate, and collecting the rest filtrate; (2) Adding absolute ethyl alcohol into the filtrate, shaking uniformly, standing, and centrifuging to obtain a precipitate A; (3) Washing the precipitate with hot ethanol, repeating for several times, and mixing to obtain precipitate B; dissolving the precipitate B with water and fixing the volume to obtain a sample solution; (4) Glucose is used as a standard substance to manufacture a standard curve, then the absorbance value of the sample solution is measured, and the polysaccharide concentration in the sample solution is calculated; (5) And calculating according to a formula to obtain the polysaccharide content in the wolfberry polysaccharide extract doped with the saccharide interfering substance. The invention uses glucose as a standard substance to manufacture a standard curve, then the absorbance value of the test sample solution is measured, and the polysaccharide concentration in the test sample solution is calculated. However, the object to be tested is a wolfberry polysaccharide extract, and the wolfberry polysaccharide extract does not contain protein or has low protein content.
Chinese patent CN201410158074.8 discloses a method for determining polysaccharide content in wolfberry extract, comprising the following steps: (1) pretreatment of medlar extract; (2) preparation of reducing sugar and total sugar test solution; (3) preparation of a standard curve; (4) preparation of a reference solution of reducing sugar and total sugar; (5) Measuring absorbance of the sample solution of reducing sugar and total sugar, checking standard yeast, and calculating polysaccharide content. The invention adopts lead acetate to pretreat medlar extract, and the deproteinized solution is transparent and clear and is suitable for photometry measurement. The invention adopts lead acetate to precipitate protein.
Therefore, how to accurately and reliably detect the total sugar content (calculated by glucose) of wheat gluten has important significance for purchasing, storing and reasonably and efficiently using the wheat gluten.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a method for detecting the total sugar content in wheat gluten.
The aim of the invention can be achieved by the following technical scheme:
a detection method of total sugar content in wheat gluten comprises the following specific steps:
s1, standard curve preparation: preparing a plurality of bottles of glucose standard solution, and drawing a standard curve by taking the concentration of glucose as an ordinate and the absorbance value as an abscissa;
s2, sample pretreatment: adding pepsin into the wheat gluten sample for pretreatment;
s3, color reaction: adding a color reagent into the sample solution pretreated in the step S2 to obtain a sample solution, and performing color development treatment on the sample solution;
s4, measuring absorbance of the sample: measuring and reading absorbance of the sample obtained after the color development treatment in the step S3 by adopting a spectrophotometer, and calculating the glucose content in the sample solution by using a standard curve;
s5, blank test: the wheat gluten sample is not added, the rest operation steps are the same as the step S2 to the step S4, and the glucose content in the blank solution is calculated by using a standard curve;
s6, calculating a result: and (3) calculating the total sugar content in the sample according to the glucose content in the sample solution obtained in the step (S4) and the glucose content in the blank solution obtained in the step (S5), wherein the total sugar content is calculated according to glucose.
Further, in step S1, the standard curve includes the following steps:
s1.1, accurately transferring 0mL, 0.2mL, 0.6mL, 1.0mL, 1.2mL, 1.6mL and 2.0mL of glucose standard solution with the concentration of 1mg/mL into 7 test tubes, and adding water to make the volume of the glucose standard solution to be 2mL, wherein the glucose concentration is respectively 7 standard solutions of 0mg/mL, 0.008mg/mL, 0.024mg/mL, 0.040mg/mL, 0.048mg/mL, 0.064mg/mL and 0.080 mg/mL;
s1.2, adding 1.5mL of 3, 5-dinitrosalicylic acid reagent into 7 standard solutions, heating in boiling hot water bath for 5 minutes, taking out, immediately placing in cold water, cooling to room temperature, fixing the volume to 25mL, and shaking uniformly;
s1.3, respectively measuring the absorbance of 7 standard solutions heated in the step S1.2 at the wavelength of 540nm by using a spectrophotometer;
s1.4, drawing a standard curve by taking absorbance values as an abscissa and glucose concentration as an ordinate according to the measurement result of the step S1.3.
Further, in step S2, the sample pretreatment includes the steps of:
s2.1, degreasing: placing a proper amount of wheat gluten sample in a funnel containing filter paper, washing fat in the sample with a solvent, and obtaining a defatted wheat gluten sample after the solvent is completely volatilized;
s2.2, dissolving protease in the gluten powder sample: mixing the defatted gluten sample obtained in the step S2.1 with pepsin solution to obtain completely dissolved gluten solution;
s2.3, hydrolysis: adding hydrochloric acid solution into the wheat gluten solution obtained in the step S2.2, and hydrolyzing to obtain sample hydrolysate;
s2.4, adjusting pH: adding sodium hydroxide solution into the sample hydrolysate obtained in the step S2.3 to adjust the pH value, so as to obtain the sample hydrolysate with the pH value adjusted;
s2.5, precipitating protein: adding lead acetate solution into the sample hydrolysate after the pH adjustment obtained in the step S2.4, standing for 10-15min, and then adding sodium sulfate solution to obtain solution and residue;
s2.6, filtering: and (3) filtering the solution and residues obtained in the step S2.5, filtering, and discarding the primary filtrate, wherein the filtrate is used for testing.
In step S2.1, the wheat gluten sample is passed through a 60-80 mesh screen in advance.
In the above step S2.1, the solvent is petroleum ether or diethyl ether.
In the step S2.2, the mass-volume ratio of the wheat gluten sample to the pepsin solution is 1: : (5-20).
The above further, in step S2.2, the pepsin concentration in the pepsin solution is 0.5% -1.5%.
In the step S2.3, the hydrochloric acid solution is preheated to 42-45 ℃ in advance, the concentration of the hydrochloric acid solution is 20-95%, the concentration of the hydrochloric acid solution is preferably 20-25%,
in the above step S2.3, the hydrolysis time is 0.5-2 hours, the hydrolysis temperature is 40-100deg.C, the hydrolysis time is preferably 1 hour, and the hydrolysis temperature is preferably 90deg.C.
In the step S2.4, the sample hydrolysate obtained in the step S2.3 is cooled first, the phenolphthalein indicator solution is dripped into the sample hydrolysate, the sodium hydroxide solution is added to adjust the pH, and finally the sample hydrolysate is tested by using a precision pH test paper.
The above further, in step S2.4, the pH is 7.0.
In the step S2.5, the concentration of the lead acetate solution is 200g/L, and the addition amount is 10-25mL.
In the step S2.5, the standing time is 10-15min.
In the step S2.5, the concentration of the sodium sulfate solution is 100g/L, and the addition amount is 10-25mL.
In step S2.6, the primary filtrate is 10-30mL.
Further, in step S3, the color reaction includes the steps of:
s3.1, sucking the test solution pretreated in the step S2, adding water to make the volume of the test solution be 2mL, adding a 3, 5-dinitrosalicylic acid reagent, and shaking uniformly;
and S3.2, placing the test solution processed in the step S3.1 into a boiling water bath, heating for 5 minutes, taking out, cooling to room temperature, fixing the volume by water, and fully shaking to obtain the sample solution.
In the step S3.1, the amount of the sample removed is adjusted according to the total sugar content of the sample, wherein the total sugar content is calculated as glucose.
In the step S3.1, the amount of the removed sample solution after the pretreatment is 0.2 to 0.5mL.
Further, in step S4, absorbance of the sample obtained after the color development treatment in step S3 was measured at a wavelength of 540nm with a spectrophotometer.
Further, in step S6, the calculation formula 1) in the measurement calculation step is
Wherein X is the total sugar content in the sample, calculated as glucose, and the numerical value is expressed as percent;
m-mass of gluten sample, g;
g, calculating the glucose content in the sample solution by using a standard curve, and mg;
g0-calculating the glucose content in the blank solution by using a standard curve, and mg;
V total (S) -total volume of sample solution obtained during color development, mL;
V taking out The volume of the pretreated test solution is removed during color development, and the volume is mL.
Compared with the prior art, the invention has the following advantages:
1. the method is characterized in that protease is added to avoid the influence of too high viscosity of a wheat gluten sample on the subsequent measurement of total sugar, the interference of the wheat gluten hydration viscoelasticity on the measurement of the total sugar (calculated by glucose) is removed through pepsin, the wheat gluten has high viscoelasticity after hydration, the crude protein content (dry basis) of the wheat gluten is up to 80% or more, if a sample is directly dissolved in water for measurement, the wheat gluten hydration viscoelasticity can influence the hydrolysis of the total sugar (calculated by glucose), and a larger deviation exists in a detection result, so that the method adopts specific enzyme for dissolving to destroy the protein structure, thereby reducing the viscoelasticity generated by the wheat gluten hydration, ensuring that the sample to be measured is more fully hydrolyzed, and the detection result is more stable and reliable;
2. the method has the advantages that relevant standards for measuring the total sugar content (calculated by glucose) of the wheat gluten are not formulated, namely, the starch or other carbohydrate content of the wheat gluten is not specified in GB/T21924-2008 wheat gluten, the hydrolysis degree of the total sugar (calculated by glucose) of a sample and the accuracy of a detection result are verified by optimizing the hydrochloric acid concentration, the reaction temperature and the reaction time of an acid hydrolysis reaction system and using starch (GR or reference level), and the detection result is good in stability, high in accuracy and good in repeatability.
Drawings
FIG. 1 is a graph of glucose calibration in an example of the present invention.
Detailed Description
The invention will now be described in detail with reference to the drawings and specific examples.
A detection method of total sugar content in wheat gluten comprises the following specific steps:
s1, standard curve preparation: preparing a plurality of bottles of glucose standard solution, and drawing a standard curve by taking the concentration of glucose as an ordinate and the absorbance value as an abscissa;
s2, sample pretreatment: adding pepsin into the wheat gluten sample for pretreatment;
s3, color reaction: adding a color reagent into the sample solution pretreated in the step S2 to obtain a sample solution, and performing color development treatment on the sample solution;
s4, measuring absorbance of the sample: measuring and reading absorbance of the sample obtained after the color development treatment in the step S3 by adopting a spectrophotometer, and calculating the glucose content in the sample solution by using a standard curve;
s5, blank test: the wheat gluten sample is not added, the rest operation steps are the same as the step S2 to the step S4, and the glucose content in the blank solution is calculated by using a standard curve;
s6, calculating a result: and (3) calculating the total sugar content in the sample according to the glucose content in the sample solution obtained in the step (S4) and the glucose content in the blank solution obtained in the step (S5), wherein the total sugar content is calculated according to glucose.
Further, in step S1, the standard curve includes the following steps:
s1.1, accurately transferring 0mL, 0.2mL, 0.6mL, 1.0mL, 1.2mL, 1.6mL and 2.0mL of glucose standard solution with the concentration of 1mg/mL into 7 test tubes, and adding water to make the volume of the glucose standard solution to be 2mL, wherein the glucose concentration is respectively 7 standard solutions of 0mg/mL, 0.008mg/mL, 0.024mg/mL, 0.040mg/mL, 0.048mg/mL, 0.064mg/mL and 0.080 mg/mL;
s1.2, adding 1.5mL of 3, 5-dinitrosalicylic acid reagent into 7 standard solutions, heating in boiling hot water bath for 5 minutes, taking out, immediately placing in cold water, cooling to room temperature, fixing the volume to 25mL, and shaking uniformly;
s1.3, respectively measuring the absorbance of 7 standard solutions heated in the step S1.2 at the wavelength of 540nm by using a spectrophotometer;
s1.4, drawing a standard curve by taking absorbance values as an abscissa and glucose concentration as an ordinate according to the measurement result of the step S1.3.
Further, in step S2, the sample pretreatment includes the steps of:
s2.1, degreasing: placing a proper amount of wheat gluten sample in a funnel containing filter paper, washing fat in the sample with a solvent, and obtaining a defatted wheat gluten sample after the solvent is completely volatilized;
s2.2, dissolving protease in the gluten powder sample: mixing the defatted gluten sample obtained in the step S2.1 with pepsin solution to obtain completely dissolved gluten solution;
s2.3, hydrolysis: adding hydrochloric acid solution into the wheat gluten solution obtained in the step S2.2, and hydrolyzing to obtain sample hydrolysate;
s2.4, adjusting pH: adding sodium hydroxide solution into the sample hydrolysate obtained in the step S2.3 to adjust the pH value, so as to obtain the sample hydrolysate with the pH value adjusted;
s2.5, precipitating protein: adding lead acetate solution into the sample hydrolysate after the pH adjustment obtained in the step S2.4, standing for 10-15min, and then adding sodium sulfate solution to obtain solution and residue;
s2.6, filtering: and (3) filtering the solution and residues obtained in the step S2.5, filtering, and discarding the primary filtrate, wherein the filtrate is used for testing.
In step S2.1, the wheat gluten sample is passed through a 60-80 mesh screen in advance.
In the above step S2.1, the solvent is petroleum ether or diethyl ether.
In the step S2.2, the mass-volume ratio of the wheat gluten sample to the pepsin solution is 1: : (5-20).
The above further, in step S2.2, the pepsin concentration in the pepsin solution is 0.5% -1.5%.
In the step S2.3, the hydrochloric acid solution is preheated to 42-45 ℃ in advance, the concentration of the hydrochloric acid solution is 20-95%, the concentration of the hydrochloric acid solution is preferably 20-25%,
in the above step S2.3, the hydrolysis time is 0.5-2 hours, the hydrolysis temperature is 40-100deg.C, the hydrolysis time is preferably 1 hour, and the hydrolysis temperature is preferably 90deg.C.
In the step S2.4, the sample hydrolysate obtained in the step S2.3 is cooled first, the phenolphthalein indicator solution is dripped into the sample hydrolysate, the sodium hydroxide solution is added to adjust the pH, and finally the sample hydrolysate is tested by using a precision pH test paper.
The above further, in step S2.4, the pH is 7.0.
In the step S2.5, the concentration of the lead acetate solution is 200g/L, and the addition amount is 10-25mL.
In the step S2.5, the standing time is 10-15min.
In the step S2.5, the concentration of the sodium sulfate solution is 100g/L, and the addition amount is 10-25mL.
In step S2.6, the primary filtrate is 10-30mL.
Further, in step S3, the color reaction includes the steps of:
s3.1, sucking the test solution pretreated in the step S2, adding water to make the volume of the test solution be 2mL, adding a 3, 5-dinitrosalicylic acid reagent, and shaking uniformly;
and S3.2, placing the test solution processed in the step S3.1 into a boiling water bath, heating for 5 minutes, taking out, cooling to room temperature, fixing the volume by water, and fully shaking to obtain the sample solution.
In the step S3.1, the amount of the sample removed is adjusted according to the total sugar content of the sample, wherein the total sugar content is calculated as glucose.
In the step S3.1, the amount of the removed sample solution after the pretreatment is 0.2 to 0.5mL.
Further, in step S4, absorbance of the sample obtained after the color development treatment in step S3 was measured at a wavelength of 540nm with a spectrophotometer.
Further, in step S6, the calculation formula 1) in the measurement calculation step is
Wherein X is the total sugar content in the sample, calculated as glucose, and the numerical value is expressed as percent;
m-mass of gluten sample, g;
g, calculating the glucose content in the sample solution by using a standard curve, and mg;
g0-calculating the glucose content in the blank solution by using a standard curve, and mg;
V total (S) -total volume of sample solution obtained during color development, mL;
V taking out The volume of the pretreated test solution is removed during color development, and the volume is mL.
In the examples below, the reagents used were as follows:
1. pepsin: biochemical grade pepsin with activity of 1:3000;
2. pepsin solution (prepared immediately prior to use): diluting 6.1mL of concentrated hydrochloric acid into 1000mL of water (pH is approximately equal to 1-2), heating to 42-45 ℃, adding 10g of biochemical pepsin with activity of 1:3000 (non-biochemical pepsin can not be used), slowly stirring until dissolving, and not heating pepsin solution on a heating plate or overheating during preparation;
3. hydrochloric acid solution (1+3): 100mL of hydrochloric acid is measured and mixed with 300mL of water;
4. phenolphthalein indicator (1%): 1g of the mixture is weighed and dissolved in 100mL of 95% ethanol, and the mixture is uniformly mixed;
5. sodium hydroxide solution (400 g/L): weighing 40g of sodium hydroxide, adding water for dissolution, cooling to room temperature, and diluting to 100mL;
6. lead acetate solution (200 g/L): weighing 200g of lead acetate, adding water for dissolution, cooling to room temperature, and diluting to 1000mL;
7. sodium sulfate solution (100 g/L): weighing 100g of sodium sulfate, adding water for dissolution, cooling to room temperature, and diluting to 1000mL;
8. sodium hydroxide solution (2 mol/L): weighing 80.0g of sodium hydroxide in a 500mL beaker, and dissolving the sodium hydroxide in water to a constant volume into a 1000mL volumetric flask;
9.3, 5-dinitrosalicylic acid (DNS) reagent: 6.3g of DNS and 262mL of 2mol/L sodium hydroxide solution were added to a solution containing 185g of sodium potassium tartrate (C 4 H 4 O 6 KNa·4H 2 O) in 500mL of hot water, 5g of phenol and 5g of sodium sulfite (Na 2 SO 3 ) Stirring for dissolving, cooling, fixing volume to 1000mL with water, and storing in a brown bottle for later use.
10. Glucose standard solution (1 mg/mL): accurately weighing 0.1000g of glucose (C) which is dried for 2 hours at 80 DEG C 6 H 12 O 6 ) Standard substance, dissolved to volume 100mL. Is prepared in the prior art.
The invention is further illustrated by the following figures and examples.
Examples
The invention provides a method for detecting total sugar content in wheat gluten, which comprises the following specific steps:
s1, taking a proper amount of sample (passing through a 60-80 mesh sieve) and placing the sample in a funnel with slow filter paper, washing fat in the sample by using 50mL of petroleum ether or diethyl ether for five times, discarding the petroleum ether or the diethyl ether, and placing the sample in a sample bottle (bag) for standby after the petroleum ether or the diethyl ether volatilizes completely;
s2, dissolving protease in the gluten powder sample: weighing a proper amount of 1-2 g of the defatted wheat gluten sample obtained in the step S1, marking the sample as m in a 250mL grinding conical flask, and adding 10-20 mL of newly prepared pepsin solution preheated to 42-45 ℃ to obtain completely dissolved wheat gluten solution;
s3, hydrolysis: adding 30-50 mL of hydrochloric acid (1+3) into the wheat gluten solution obtained in the step S2, shaking uniformly, connecting a conical flask with a condenser tube, refluxing for 1h at 90 ℃ (or placing in a water bath at 90 ℃ for 1h, uniformly mixing every 15 min) to obtain sample hydrolysate;
s4, adjusting pH: after the reflux is finished, immediately cooling, adding 3 drops of phenolphthalein indicator liquid after the sample hydrolysate obtained in the step S3 is cooled, preparing reddish color by using sodium hydroxide solution (400 g/L), and simultaneously testing by using precise pH test paper to ensure that the pH of the sample hydrolysate is about 7.0, thereby obtaining the sample hydrolysate with the pH adjusted;
s5, precipitating protein: adding 20mL of lead acetate solution (200 g/L) into the sample hydrolysate after the pH adjustment obtained in the step S4 to precipitate protein, shaking uniformly, standing for 10min, adding 20mL of sodium sulfate solution (100 g/L), shaking uniformly, and removing excessive lead to obtain a mixed solution;
s6, filtering: after shaking up, transferring the solution and the residues obtained in the step S5 into a 250mL volumetric flask, washing the conical flask with water, combining the washing with the volumetric flask, and diluting with water to a scale. Filtering, discarding 20mL of primary filtrate, and obtaining filtrate for to-be-detected use;
s7, manufacturing a standard curve: accurately transferring 0mL, 0.2mL, 0.6mL, 1.0mL, 1.2mL, 1.6mL and 2.0mL glucose standard solution into 7 25mL test tubes with stopper scales, adding water to make the volume to 2mL, adding 1.5mL 3, 5-dinitrosalicylic acid reagent, heating in boiling water bath for 5min, taking out, immediately placing in cold water, cooling to room temperature, fixing the volume, and shaking uniformly. The concentration of the obtained glucose standard solution is respectively 0mg/mL, 0.008mg/mL, 0.024mg/mL, 0.040mg/mL, 0.048mg/mL, 0.064mg/mL and 0.080mg/mL. Colorimetric determination is carried out by taking 0mg/mL glucose standard solution as a blank and a 1cm cuvette at a wavelength of 540nm, and a standard curve is drawn by taking the glucose concentration (mg/mL) as an ordinate (y) and the absorbance value as an abscissa (x), wherein the glucose standard curve is shown in figure 1;
s8, color reaction: accurately transferring a proper amount of 0.2-0.5mL of the filtrate pretreated in the step S6 (the transfer amount can be adjusted according to the total sugar content of the sample, and the total sugar content is calculated by glucose) into a 25mL test tube with a stopper, adding water to make the volume be 2mL, adding 1.5mL of 3, 5-dinitrosalicylic acid reagent, and heating in a boiling water bath for 5min. Taking out, immediately placing in cold water, cooling to room temperature, fixing the volume to 25mL, obtaining sample liquid, and shaking uniformly. (note: the standard curve and the test sample should be subjected to color development reaction simultaneously);
s9, sample measurement: the absorbance at 540nm was measured using a spectrophotometer. Measuring the absorbance of the sample solution, and calculating the total sugar content in the sample solution by using a standard curve, wherein the total sugar content is calculated by glucose;
s10, blank test: the wheat gluten sample is not added, the rest operation steps are the same as the step S2 to the step S9, and the glucose content in the blank solution is calculated by using a standard curve;
s11, calculating a result: comparing the absorbance obtained in the step S9 with the standard curve in the step S7, calculating the total sugar content in the sample according to the formula 1),
the calculation formula 1) in the measurement calculation step is
Wherein X is the total sugar content in the sample, calculated as glucose, and the numerical value is expressed as percent;
m-mass of gluten sample, g;
g, calculating the glucose content in the sample solution by using a standard curve, and mg;
g0-calculating the glucose content in the blank solution by using a standard curve, and mg;
V total (S) -total volume of sample solution obtained during color development, mL;
V taking out The volume of the pretreated test solution is removed during color development, and the volume is mL.
The experimental results are as follows:
on the premise of not adding pepsin solution, the same wheat gluten is directly adopted to optimize the hydrolysis condition:
(1) Optimization of hydrochloric acid concentration: respectively hydrolyzing with 20%, 40%, 50%, 60%, 80%, 95% (V: V) hydrochloric acid, keeping the hydrolysis temperature (100deg.C) and hydrolysis time (2 h) unchanged, and counting total sugar (calculated as glucose) content of wheat gluten under different hydrochloric acid concentrations.
TABLE 1 Total sugar content of wheat gluten with different hydrochloric acid concentrations
| Hydrochloric acid concentration | 20％ | 40％ | 50％ | 60％ | 80％ | 95％ |
| Gluten total sugar content (in% based on glucose) | 15.21 | 13.07 | 10.10 | 5.29 | 1.61 | 1.47 |
Note that: the addition amount of hydrochloric acid was 50ml.
The experimental results show that the total sugar content (calculated by glucose) of the gluten tends to decrease with increasing hydrochloric acid concentration, and only a small part of the total sugar in the gluten is hydrolyzed when the hydrochloric acid concentration exceeds 80%. Therefore, the concentration of hydrolyzed hydrochloric acid in gluten is preferably 20% hydrochloric acid.
(2) Optimization of hydrolysis time: respectively carrying out hydrolysis in the reaction time of 0.5h, 1.0h, 1.5h and 2.0h, keeping the hydrolysis temperature (100 ℃) and the hydrochloric acid concentration (20%) unchanged, and counting the total sugar (calculated by glucose) content of the wheat gluten under different hydrolysis time.
TABLE 2 Total sugar content of wheat gluten at various hydrolysis times
The experimental result shows that the total sugar content (calculated by glucose) of the wheat gluten reaches the maximum hydrolysis degree at 1.0h, and the detection result is not obviously changed along with the extension of the hydrolysis time. In order to maximize the hydrolysis of gluten and to shorten the experimental time, the hydrolysis time of gluten is preferably 1.0h.
(3) Optimization of reaction temperature: respectively carrying out hydrolysis at the reaction temperature of 40 ℃, 50 ℃, 60 ℃, 70 ℃,80 ℃, 90 ℃ and 100 ℃, keeping the hydrolysis time (1 h) and the hydrochloric acid concentration (20%) unchanged, and counting the total sugar (calculated by glucose) content of the gluten powder at different reaction temperatures.
TABLE 3 Total sugar content of wheat gluten at different hydrolysis temperatures
The experimental result shows that when the hydrolysis temperature is less than or equal to 50 ℃, only a small part of total sugar in the wheat gluten is hydrolyzed. As the hydrolysis temperature increases, the total sugar content (in% by glucose) of the gluten tends to increase, reaching a maximum degree of hydrolysis at 90 ℃, and then tending to stabilize. Therefore, the hydrolysis temperature of gluten is preferably 90℃or higher.
The drying weight loss of the soluble starch (AR) is less than or equal to 13.0 percent, the burning residue (calculated by sulfate) is less than or equal to 0.5 percent, and the hydrolysis degree of the total sugar (calculated by glucose) of the sample is verified, so that the accuracy of the detection result can be known: the starch content is more than or equal to 86.5%, the theoretical total sugar content (calculated by glucose) is more than or equal to 96.11%, the measured value is 96.22%, and the requirements are met.
(4) The invention removes the interference of the hydration viscoelasticity of the wheat gluten on the measurement of total sugar (calculated by glucose) by adding pepsin solution on the basis of the optimal hydrolysis condition (hydrochloric acid concentration of 20%, water temperature of more than 90 ℃ and hydrolysis time of 1.0 h) of the wheat gluten, and verifies the influence of the hydrolysis temperature on the total sugar content (calculated by glucose) of the wheat gluten again in an enzymatic reaction system, thereby further optimizing the reaction temperature.
TABLE 4 Total sugar content of wheat gluten at different hydrolysis temperatures
The experimental result shows that with the increase of the hydrolysis temperature, the total sugar content (calculated by glucose) of the wheat gluten tends to be increased firstly and then decreased, and the maximum hydrolysis degree is achieved at 90 ℃. At the same temperature, the detection result of the invention is mostly higher than the detection value of direct hydrolysis of wheat gluten. Therefore, by adding the pepsin solution, a rapid enzymatic reaction system is formed, so that the total sugar (calculated by glucose) in the wheat gluten can be fully hydrolyzed.
The total gluten sugar content of the different gluten samples was measured by the method of this example and the specific results are shown in table 5.
TABLE 5 Total sugar content of gluten from different gluten samples
| Sample name | Total sugar content (in% based on glucose) |
| |
15.06 |
| Gluten flour 2 | 13.50 |
| Gluten powder 3 | 14.78 |
The foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The method for detecting the total sugar content in the wheat gluten is characterized by comprising the following specific steps of:
s1, standard curve preparation: preparing a plurality of bottles of glucose standard solution, and drawing a standard curve by taking the concentration of glucose as an ordinate and the absorbance value as an abscissa;
s2, sample pretreatment: adding pepsin into the wheat gluten sample for pretreatment;
s3, color reaction: adding a color reagent into the sample solution pretreated in the step S2 to obtain a sample solution, and performing color development treatment on the sample solution;
s4, measuring absorbance of the sample: measuring and reading absorbance of the sample obtained after the color development treatment in the step S3 by adopting a spectrophotometer, and calculating the glucose content in the sample solution by using a standard curve;
s5, blank test: the wheat gluten sample is not added, the rest operation steps are the same as the step S2 to the step S4, and the glucose content in the blank solution is calculated by using a standard curve;
s6, calculating a result: and (3) calculating the total sugar content in the sample according to the glucose content in the sample solution obtained in the step (S4) and the glucose content in the blank solution obtained in the step (S5), wherein the total sugar content is calculated according to glucose.
2. The method for detecting total sugar content in wheat gluten according to claim 1, wherein in step S1, the standard curve comprises the steps of:
s1.1, accurately transferring 0mL, 0.2mL, 0.6mL, 1.0mL, 1.2mL, 1.6mL and 2.0mL of glucose standard solution with the concentration of 1mg/mL into 7 test tubes, and adding water to make the volume of the glucose standard solution to be 2mL, wherein the glucose concentration is respectively 7 standard solutions of 0mg/mL, 0.008mg/mL, 0.024mg/mL, 0.040mg/mL, 0.048mg/mL, 0.064mg/mL and 0.080 mg/mL;
s1.2, adding 1.5mL of 3, 5-dinitrosalicylic acid reagent into 7 standard solutions, heating in boiling hot water bath for 5 minutes, taking out, immediately placing in cold water, cooling to room temperature, fixing the volume to 25mL, and shaking uniformly;
s1.3, respectively measuring the absorbance of 7 standard solutions heated in the step S1.2 at the wavelength of 540nm by using a spectrophotometer;
s1.4, drawing a standard curve by taking absorbance values as an abscissa and glucose concentration as an ordinate according to the measurement result of the step S1.3.
3. The method for detecting total sugar content in wheat gluten according to claim 1, wherein in step S2, the sample pretreatment comprises the steps of:
s2.1, degreasing: placing a proper amount of wheat gluten sample in a funnel containing filter paper, washing fat in the sample with a solvent, and obtaining a defatted wheat gluten sample after the solvent is completely volatilized;
s2.2, dissolving protease in the gluten powder sample: mixing the defatted gluten sample obtained in the step S2.1 with pepsin solution to obtain completely dissolved gluten solution;
s2.3, hydrolysis: adding hydrochloric acid solution into the wheat gluten solution obtained in the step S2.2, and hydrolyzing to obtain sample hydrolysate;
s2.4, adjusting pH: adding sodium hydroxide solution into the sample hydrolysate obtained in the step S2.3 to adjust the pH value, so as to obtain the sample hydrolysate with the pH value adjusted;
s2.5, precipitating protein: adding lead acetate solution into the sample hydrolysate after the pH adjustment obtained in the step S2.4, standing for 10-15min, and then adding sodium sulfate solution to obtain solution and residue;
s2.6, filtering: and (3) filtering the solution and residues obtained in the step S2.5, filtering, and discarding the primary filtrate, wherein the filtrate is used for testing.
4. A method for detecting total sugar content in wheat gluten as set forth in claim 3, wherein,
in the step S2.1, the wheat gluten sample is passed through 60-80 mesh holes in advance,
the solvent is petroleum ether or diethyl ether;
in the step S2.2, the mass-volume ratio of the wheat gluten sample to the pepsin solution is 1: (5-20),
the pepsin concentration in the pepsin solution is 0.5% -1.5%;
in the step S2.3, the hydrochloric acid solution is preheated to 42-45 ℃ in advance, the concentration of the hydrochloric acid solution is 20-95%,
the hydrolysis time is 0.5-2h, and the hydrolysis temperature is 40-100 ℃;
in the step S2.4, the sample hydrolysate obtained in the step S2.3 is firstly cooled, the phenolphthalein indicator liquid is dripped into the sample hydrolysate, the sodium hydroxide solution is added to adjust the pH value, and finally the pH value of the sample hydrolysate is tested by using a precise pH test paper,
the pH was 7.0.
5. The method for detecting total sugar content in wheat gluten according to claim 4, wherein in step S2.3, the concentration of the hydrochloric acid solution is 20% -25%, the hydrolysis time is 1h, and the hydrolysis temperature is 90 ℃.
6. A method for detecting total sugar content in wheat gluten according to claim 3, wherein in step S2.5, the concentration of the lead acetate solution is 200g/L, and the concentration of the sodium sulfate solution is 100g/L.
7. The method for detecting total sugar content in wheat gluten according to claim 1, wherein in step S3, the color reaction comprises the steps of:
s3.1, sucking the test solution pretreated in the step S2, adding water to make the volume of the test solution be 2mL, adding a 3, 5-dinitrosalicylic acid reagent, and shaking uniformly;
and S3.2, placing the test solution processed in the step S3.1 into a boiling water bath, heating for 5 minutes, taking out, cooling to room temperature, fixing the volume by water, and fully shaking to obtain the sample solution.
8. The method for detecting total sugar content in wheat gluten according to claim 7, wherein in step S3.1, the amount of the removed sample is adjusted according to the total sugar content of the sample, and the total sugar content is calculated as glucose.
9. The method for detecting total sugar content in wheat gluten according to claim 1, wherein in step S4, absorbance of the sample obtained after the color development treatment in step S3 is measured at a wavelength of 540nm with a spectrophotometer.
10. The method for detecting total sugar content in wheat gluten according to claim 1, wherein in step S6, the calculation formula 1) in the measurement calculation step is
Wherein X is the total sugar content in the sample, calculated as glucose, and the numerical value is expressed as percent;
m-mass of gluten sample, g;
g, calculating the glucose content in the sample solution by using a standard curve, and mg;
g0-calculating the glucose content in the blank solution by using a standard curve, and mg;
V total (S) -total volume of sample solution obtained during color development, mL;
V taking out The volume of the pretreated test solution is removed during color development, and the volume is mL.
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