CN110927152A - Method for rapidly detecting amylose and amylopectin in highland barley - Google Patents
Method for rapidly detecting amylose and amylopectin in highland barley Download PDFInfo
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Abstract
The invention relates to a method for rapidly detecting amylose and amylopectin in highland barley, which comprises the following steps of ⑴ preparing an amylose standard solution and an amylopectin standard solution, ⑵ preparing an amylose determination solution and an amylopectin determination solution, ⑶ determining a determination wavelength and a reference wavelength of the amylose and the amylopectin by an isoabsorption dual-wavelength elimination method, ⑷ determining absorbance values of the amylose determination solution and the amylopectin determination solution at a determination wavelength 563 nm and a reference wavelength 511 nm of the amylose and at a determination wavelength 542nm and a reference wavelength 722 nm of the amylopectin respectively, ⑸ drawing a standard curve, ⑹ adding absolute ethyl alcohol into highland barley powder, swirling to wet a sample, ⑺ preparing a starch extraction solution, ⑻ preparing the starch determination solution, ⑼ determining the absorbance values of the starch determination solution, and then checking corresponding starch quality on the dual-wavelength amylose standard curve and the amylopectin standard curve respectively to calculate the amylose content and the amylopectin content of the amylose and the amylopectin in the highland barley respectively, wherein the sum of the amylose content and the amylopectin content is the total starch content.
Description
Technical Field
The invention relates to a method for rapidly detecting amylose and amylopectin, in particular to a method for rapidly detecting amylose and amylopectin in highland barley.
Background
The highland barley is an important plateau cereal crop growing in the northwest and southwest of China, particularly in the places of Tibet, Qinghai, Gansu and the like, also called naked barley, rice barley and highland barley, and has the academic name ofHordeum vulgareL. var.nudumHook, f., one of the barley varieties of the genus barley of the family triticum aestivum, family gramineae. The highland barley is used as the traditional main crop of Tibetan people, has been developed to a product generally popular with the masses from 'special use for agriculture and herdsmen' hundreds of years ago, and is developed into a special product. In recent years, along with the change of the concept of people, the vigorous advocation of green foods is followed and the people are again favored by the urbanized people. However, in the traditional concept, the highland barley of Tibetan nationality in China is required to be made into the tsamba, which has rough taste and is not easy to be absorbed by human bodies, and if the highland barley can be further processed into food with good taste and easy to be absorbed, the utilization space of the highland barley can be widened.
The starch is one of the main components of the highland barley, accounts for about 65 percent of the dry weight of grains, and comprises amylose starch and amylopectin starch. The starch has unique components, generally contains 74% -78% of amylopectin, and some amylopectin is close to 100%. The amylopectin content is in positive correlation with the dry matter water absorption rate, the correlation coefficient is 0.735, and the amylopectin content is in negative correlation with the dry matter loss rate and the protein loss rate, and the correlation coefficients are-0.731 and-0.854 respectively; the increased content of amylopectin optimizes the quality of starch as a whole, and can be used as a good thickening agent, an emulsifier, an adhesive and a suspending agent to be widely applied to the industries of food, paper, textile and adhesive. The content of amylose is an important factor influencing the sensory quality and the processing characteristics of the grains, and the content of amylose can be used as an important index for evaluating the quality of the grains. In addition, the properties of starch, such as gelatinization, viscosity, solubility, swelling capacity, digestibility and resistance, are also closely related to the amylose content. Therefore, the content determination of amylose and amylopectin has important significance for reasonable processing of grains, reasonable utilization of starch, agricultural seed selection, breeding and the like.
The method for measuring the starch content comprises indirect measurement (including national standard enzymolysis method and starch measurement kit method)Enzymatic hydrolysis-anthrone colorimetry, hydrochloric acid hydrolysis-DNS colorimetry, enzyme-electrode method, HPLC method, GC method); direct assay (including CaCl)2HOAC extraction method, concanavalin method, double-wavelength colorimetric method, near infrared spectroscopic analysis method, optical rotation method, differential thermal analysis method) and the like, wherein ⑴ national standard enzyme method, enzymolysis-anthrone colorimetric method and concanavalin method have complicated and time-consuming operation steps, various reagent types and strict operation requirements, a kit method has high cost, complicated and time-consuming determination process and is not suitable for determination of large-batch samples, a gas chromatography method and a liquid chromatography method have many advantages, but used test instruments and reagents are expensive and cannot be popularized, a hydrochloric acid hydrolysis-DNS colorimetric method often needs to consider various factors such as reducing sugar content in a sample to be determined, soluble substance type in the sample, heat sharing time, DNS amount, instrument performance and sensitivity, and the like, and has poor method reproducibility, and the methods can only determine the content of total starch ⑵ CaCl2The method has the advantages of simple sample preparation, high analysis speed, simultaneous determination of multiple components, online analysis, nondestructive analysis, no use of toxic organic reagents, no pollution and the like, the optical rotation method has higher precision and smaller variation degree, the differential thermal analysis method does not need to pre-treat the sample, the operation is quick and convenient, the method has better practicability, but can only determine the total starch content, the ⑶ dual-wavelength spectrophotometry is a common method for determining the starch content, the contents of amylose and amylopectin in the sample are determined through a standard curve, and the sum of the contents is the total starch content.
The research contents about the highland barley starch mainly comprise the chemical composition and the process property of the highland barley starch, the characteristics of starch granules, an amylase extraction process, the preparation of resistant starch, the polymorphism of highland barley starch granules and the relation between the polymorphism and the starch content, the influence of the genotype and the environmental effect on the content of the highland barley starch and the like, and the related literature reports about the content of the starch in the highland barley and the content determination of amylose and amylopectin forming the starch are few. At present, the amylose of rice, corn and millet seeds is measured (NY/T2639-2014, GB/T15683-2008 and GB 7648-1987), but the methods are complicated and time-consuming in operation steps, and only can measure the amylose group 1 value; in addition, the content of amylose and amylopectin in fresh corn is measured (DB 32/T2265-2012), but in the method, a sample needs to be degreased for 4 hours in advance and then a sample solution is prepared, the content of crude fat in the highland barley is less than 2%, the final detection result is not affected, the degreasing treatment is not needed in advance, and the operation step of degreasing is time-consuming and labor-consuming for detecting hundreds of thousands of samples.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method for simply, accurately and efficiently and quickly detecting amylose and amylopectin in highland barley.
In order to solve the problems, the method for rapidly detecting amylose and amylopectin in highland barley comprises the following steps:
⑴ preparation of standard solutions:
i preparation of amylose standard solution:
accurately weighing 50mg of potato amylose standard substance with the purity of more than 99.5%, placing the potato amylose standard substance into a 50 mL volumetric flask, adding 10 mL of 1mol/L sodium hydroxide solution, placing the potato amylose standard substance into a boiling water bath for dissolving and dispersing, taking out, cooling to room temperature, adding distilled water for diluting to a scale, and shaking up to obtain an amylose standard solution with the concentration of 1.0 mg/mL;
ii preparation of amylopectin standard solution:
accurately weighing 50mg of potato amylopectin standard substance with purity of more than 99.5%, adding 10 mL of 1mol/L sodium hydroxide solution into a 50 mL volumetric flask, dissolving and dispersing in boiling water bath, taking out, cooling to room temperature, adding distilled water to dilute to a scale, shaking up to obtain an amylopectin standard solution with concentration of 1.0 mg/mL;
⑵, accurately transferring 1.0 mL of the amylose standard solution and 1 part of the amylopectin standard solution respectively, placing the solutions in 25 mL volumetric flasks, adding 15 mL of water, adjusting the pH to 3.0-4.0 by using a glacial acetic acid solution with the concentration of 1mol/L, adding 0.20-0.30 mL of iodine reagent into the sample measuring solution, adding water into the sample measuring solution to a constant volume of 25 mL, shaking up, and preparing an amylose measuring solution and an amylopectin measuring solution respectively;
⑶ determination of the measured and reference wavelengths of amylose and amylopectin by isoabsorptive dual wavelength elimination:
① determination of amylose and reference wavelength by determining the same absorbance A in the amylopectin absorption curveSupport 1And ASupport 2Corresponding wavelengths are respectively lambda1And λ2；λ2The value of A on the corresponding amylose absorption curve is denoted AStraight 2So that λ2As the measurement wavelength of amylose; lambda [ alpha ]1The value of A on the corresponding amylose absorption curve is denoted AStraight 1So that λ1As reference wavelength for amylose;
② determination of amylopectin and reference wavelength by determining the maximum absorbance A in the absorption curve of amylopectinmaxCorresponding lambda4As the measurement wavelength of amylopectin; at λ4Find A on the corresponding amylose absorption curveStraight 4Continue to find the sum A on the curveStraight 4A with the same absorbanceStraight 3Finding the corresponding A on the amylopectin absorption curveStraight 3The same wavelength lambda3As reference wavelength for amylopectin;
⑷ standing the amylose measurement solution and the amylopectin measurement solution for 15 min, respectively measuring absorbance values at a measurement wavelength 563 nm and a reference wavelength 511 nm of amylose and at a measurement wavelength 542nm and a reference wavelength 722 nm of amylopectin with a 1 cm cuvette;
⑸ standard curves are plotted:
a dual wavelength amylose standard curve:
accurately transferring 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL and 0.6mL of amylose standard solution with the concentration of 1.0 mg/mL into 6 volumetric flasks with the volume of 25 mL respectively, preparing an amylose determination solution according to the step ⑵, determining absorbance values at the wavelengths of 563 nm and 511 nm by using a 1 cm cuvette according to the step ⑷, and determining the difference between the two absorbances, namely △ AStraight bar=Aλ2- Aλ1=A563 nm- A511 nmDrawing a dual-wavelength amylose standard curve by taking the mass of the amylose as the ordinate and the mass of the amylose as the abscissa;
b dual wavelength amylopectin standard curve:
accurately transferring 0.5 mL, 1.0 mL, 1.5mL, 2.0 mL and 3.0mL of amylopectin standard solution with concentration of 1.0 mg/mL into 6 volumetric flasks with volume of 25 mL respectively, preparing determination solution of amylopectin according to the step ⑵, determining absorbance values at wavelength of 542nm and 722 nm respectively by using a 1 cm cuvette according to the step ⑷, and determining difference between two absorbances, namely △ ABranch stand=Aλ4- Aλ3= A542 nm- A722 nmDrawing a dual-wavelength amylopectin standard curve by taking the ordinate as the mass of the amylopectin and the abscissa as the mass of the amylopectin;
⑹ placing 50-150 mg of crushed highland barley powder sieved by a 60-mesh sieve into a 25 mL volumetric flask, adding 0.5 mL of absolute ethyl alcohol, and vortexing to wet a sample;
⑺ adding 0.5-1.5 mL of 1mol/L sodium hydroxide solution into the wetted sample, uniformly mixing by vortex, heating in a boiling water bath for 2 min, taking out, vortex for 30 s, repeating the steps for 3-5 times, cooling to room temperature, metering to 25 mL, and shaking up to obtain starch extract;
⑻ accurately transferring 1.0 mL of the starch extracting solution into a 25 mL volumetric flask, and preparing a starch determination solution according to the step ⑵;
⑼, standing the starch determination solution for 15 min, determining absorbance values at wavelengths of 563 nm, 511 nm, 542nm and 722 nm by using a 1 cm cuvette respectively according to the step ⑷, and then checking the starch quality corresponding to the difference between the two corresponding absorbances of the starch determination solution on the dual-wavelength amylose and amylopectin standard curves respectively, thereby calculating the amylose and amylopectin contents in the highland barley respectively, wherein the sum of the amylose and the amylopectin contents is the total starch content.
Compared with the prior art, the invention has the following advantages:
1. the invention respectively carries out single-factor cycle test on 5 factors of sample amount, sodium hydroxide concentration, alkali extraction dissolution vortex frequency, chromogenic pH value adjustment and addition of a color reagent iodine reagent, and designs five-factor three-level orthogonal test to determine complete extraction and color development of two kinds of starch.
2. In the invention, in consideration of principle, in a two-component mixture of a and b according to the principle of an equal absorption dual-wavelength elimination method, namely the principle of overlapping absorption spectra, if the interference of b is eliminated to measure a, two wavelengths lambda with equal absorbance can be selected from the absorption spectrum of b1And λ2Separately preparing amylose and amylopectin dual-wavelength standard curves respectively, reflecting linear relations of amylose and amylopectin with different concentrations, generating pure blue and purple red properties respectively according to the action of the amylose and the amylopectin with iodine reagents, scanning reaction liquid of the two starches and the iodine reagents at full wavelength (450 nm-900 nm) by using a visible spectrophotometer, determining respective reference wavelength and determination wavelength 4 data in the same coordinate system by using a graph method, determining the contents of the amylose and the amylopectin in a sample respectively through the dual-wavelength standard curves, wherein the sum of the two is the total starch content, and the mutual interference effect of the two starches is not considered, so that the sensitivity and the selectivity of the determination can be improved.
3. The method is considered from the preparation of the standard solution, has no degreasing step, adopts the potato high-purity amylose and amylopectin standard substances (the purity is more than 99.5 percent) provided by Shanghai Huichi biological technology company Limited, has a qualified certificate, and has determined uncertainty, and in addition, the 2 standard substances are easy to purchase and have reasonable price.
4. In consideration of sample preparation, even if the highland barley contains less than 2% of crude fat, the highland barley and NaOH solution react and hydrolyze under heating condition in the sample solution preparation process to obtain higher fatty acid sodium, white higher fatty acid is separated out after glacial acetic acid is added for adjusting acidity, and the white higher fatty acid can be removed together with sample residues after centrifugation, so that the subsequent color development step is not influenced, the operation steps are reduced, and the detection efficiency is improved.
5. The method has the characteristics of low measurement cost, high precision, accurate result and good repeatability and stability, can obtain 3 groups of numerical values of amylose, amylopectin and total starch, greatly improves the working efficiency, and is suitable for measuring large-batch samples; meanwhile, the content of the starch in the highland barley and the content of amylose and amylopectin forming the starch are also determined, and the method has important significance for reasonable processing of the highland barley, reasonable utilization of the starch, agricultural seed selection, breeding and the like.
6. The invention establishes a method for rapidly detecting amylose and amylopectin in highland barley, and the detection method has the characteristics of better linear relation, precision, stability, repeatability, reproducibility, recovery rate, quantitative limit, detection limit and the like through a methodology verification experiment and a comparison verification experiment between laboratories.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a scanned graph (450 nm-900 nm) of the absorption curves of amylose and amylopectin in the present invention.
FIG. 2 is a dual wavelength amylose calibration curve of the present invention.
FIG. 3 is a standard curve for dual wavelength amylopectin according to the present invention.
In the figure: 1. amylose absorption curve; 2. amylopectin absorption curve.
Detailed Description
A method for rapidly detecting amylose and amylopectin in highland barley comprises the following steps:
⑴ preparation of standard solutions:
i preparation of amylose standard solution:
accurately weighing 50mg of potato amylose standard substance with the purity of more than 99.5%, placing the potato amylose standard substance into a 50 mL volumetric flask, adding 10 mL of 1mol/L sodium hydroxide solution, placing the potato amylose standard substance into a boiling water bath for dissolving and dispersing, taking out, cooling to room temperature, adding distilled water for diluting to a scale, and shaking up to obtain an amylose standard solution with the concentration of 1.0 mg/mL;
ii preparation of amylopectin standard solution:
accurately weighing 50mg of potato amylopectin standard substance with purity of more than 99.5%, adding 10 mL of 1mol/L sodium hydroxide solution into a 50 mL volumetric flask, dissolving and dispersing in boiling water bath, taking out, cooling to room temperature, adding distilled water to dilute to a scale, shaking up to obtain an amylopectin standard solution with concentration of 1.0 mg/mL;
⑵, accurately transferring 1.0 mL of the amylose standard solution and 1 part of the amylopectin standard solution respectively, placing the solutions in 25 mL volumetric flasks, adding 15 mL of water, adjusting the pH to 3.0-4.0 by using a glacial acetic acid solution with the concentration of 1mol/L, adding 0.20-0.30 mL of iodine reagent into the sample measuring solution, adding water into the sample measuring solution to a constant volume of 25 mL, shaking up, and preparing an amylose measuring solution and an amylopectin measuring solution respectively;
⑶ determination of the measured and reference wavelengths of amylose and amylopectin by isoabsorptive dual wavelength elimination:
① determination of amylose and reference wavelength by determining the same absorbance A in the amylopectin absorption curveSupport 1And ASupport 2Corresponding wavelengths are respectively lambda1And λ2；λ2The value of A on the corresponding amylose absorption curve is denoted AStraight 2So that λ2As the measurement wavelength of amylose; lambda [ alpha ]1The value of A on the corresponding amylose absorption curve is denoted AStraight 1So that λ1As reference wavelength for amylose;
② determination of amylopectin and reference wavelength by determining the maximum absorbance A in the absorption curve of amylopectinmaxCorresponding lambda4As the measurement wavelength of amylopectin; at λ4Find the corresponding amylose absorption kojiOn-line AStraight 4Continue to find the sum A on the curveStraight 4A with the same absorbanceStraight 3Finding the corresponding A on the amylopectin absorption curveStraight 3The same wavelength lambda3As reference wavelength for amylopectin;
⑷ standing the amylose measurement solution and the amylopectin measurement solution for 15 min, respectively measuring absorbance values at a measurement wavelength 563 nm and a reference wavelength 511 nm of amylose and at a measurement wavelength 542nm and a reference wavelength 722 nm of amylopectin with a 1 cm cuvette;
⑸ standard curves are plotted:
a dual wavelength amylose standard curve:
accurately transferring 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL and 0.6mL of amylose standard solution with the concentration of 1.0 mg/mL into 6 volumetric flasks with the volume of 25 mL respectively, preparing an amylose determination solution according to the step ⑵, determining absorbance values at the wavelengths of 563 nm and 511 nm by using a 1 cm cuvette according to the step ⑷, and determining the difference between the two absorbances, namely △ AStraight bar=Aλ2- Aλ1=A563 nm- A511 nmDrawing a dual-wavelength amylose standard curve by taking the mass of the amylose as the ordinate and the mass of the amylose as the abscissa;
b dual wavelength amylopectin standard curve:
accurately transferring 0.5 mL, 1.0 mL, 1.5mL, 2.0 mL and 3.0mL of amylopectin standard solution with concentration of 1.0 mg/mL into 6 volumetric flasks with volume of 25 mL respectively, preparing determination solution of amylopectin according to the step ⑵, determining absorbance values at wavelength of 542nm and 722 nm respectively by using a 1 cm cuvette according to the step ⑷, and determining difference between two absorbances, namely △ ABranch stand=Aλ4- Aλ3= A542 nm- A722 nmDrawing a dual-wavelength amylopectin standard curve by taking the ordinate as the mass of the amylopectin and the abscissa as the mass of the amylopectin;
⑹ placing 50-150 mg of crushed highland barley powder sieved by a 60-mesh sieve into a 25 mL volumetric flask, adding 0.5 mL of absolute ethyl alcohol, and vortexing to wet a sample;
⑺ adding 0.5-1.5 mL of 1mol/L sodium hydroxide solution into the wetted sample, uniformly mixing by vortex, heating in a boiling water bath for 2 min, taking out, vortex for 30 s, repeating the steps for 3-5 times, cooling to room temperature, metering to 25 mL, and shaking up to obtain starch extract;
⑻ accurately transferring 1.0 mL of the starch extracting solution into a 25 mL volumetric flask, and preparing a starch determination solution according to the step ⑵;
⑼, standing the starch determination solution for 15 min, determining absorbance values at wavelengths of 563 nm, 511 nm, 542nm and 722 nm by using a 1 cm cuvette respectively according to the step ⑷, and then checking the starch quality corresponding to the difference between the two corresponding absorbances of the starch determination solution on the dual-wavelength amylose and amylopectin standard curves respectively, thereby calculating the amylose and amylopectin contents in the highland barley respectively, wherein the sum of the amylose and the amylopectin contents is the total starch content.
Examples
In the concrete implementation of said invention, according to the principle of equal-absorption double-wavelength elimination method-two-component mixture of a and b whose absorption spectra are overlapped, if the interference of b is eliminated to measure a, two wavelengths lambda whose absorbances are equal can be selected from absorption spectrum of b1And λ2And simultaneously, according to the properties of pure blue and purple red generated by the action of amylose and amylopectin with an iodine reagent, respectively, carrying out full-wavelength scanning (450 nm-900 nm) on reaction liquid of the two starches and the iodine reagent by using a visible spectrophotometer, and determining respective measuring wavelength and reference wavelength in the same coordinate system by using a graph method.
1. Experimental methods
Preparation of a reagent:
preparing 1mol/L sodium hydroxide solution: weighing 40 g of sodium hydroxide solid (accurate to +/-0.001 g), adding water to dissolve, transferring to a 1000 mL volumetric flask, mixing uniformly, and fixing the volume.
Preparation of 1mol/L glacial acetic acid solution: transferring 57.3 mL of glacial acetic acid into a 1000 mL volumetric flask, adding water for dilution, uniformly mixing, and fixing the volume.
Preparing an iodine reagent: weighing 2 g potassium iodide (accurate to +/-0.001 g), adding water to form a saturated solution, adding 0.2 g iodine (accurate to +/-0.001 g), transferring to an l00 mL volumetric flask after the iodine is completely dissolved, mixing uniformly, and fixing the volume. It is used when prepared at present and stored in dark.
Preparation of a standard solution:
1.0 mg/mL amylose standard solution preparation: accurately weighing 50.0 mg of potato amylose standard (accurate to +/-0.1 mg) with the purity of more than 99.5 percent, putting the potato amylose standard into a 50 mL volumetric flask, adding 10 mL of 1mol/L sodium hydroxide solution, dissolving and dispersing in a boiling water bath, taking out, cooling to room temperature, adding water to dilute to a scale, and shaking up.
1.0 mg/mL amylopectin standard solution preparation: accurately weighing 50.0 mg of potato amylopectin standard product (accurate to +/-0.1 mg) with purity of more than 99.5%, placing the potato amylopectin standard product in a 50 mL volumetric flask, adding 10 mL of 1mol/L sodium hydroxide solution, placing the product in a boiling water bath for dissolving and dispersing, taking out the product, cooling the product to room temperature, adding water for diluting the product to a scale, and shaking the product uniformly.
Determination of amylose and amylopectin measurement wavelengths and of reference wavelengths:
the absorption curves of two starch standard solutions are superposed in the same coordinate system (450 nm-900 nm), and according to the principle that a certain solute in the solution absorbs under two wavelengths, the absorption difference value of the two wavelengths is in direct proportion to the concentration of the solute, the respective measuring wavelength and the reference wavelength of the two starches are determined by an equal absorption dual-wavelength elimination method: when determining the amylose content, the amylose absorption peak wavelength lambda is selected2As the measurement wavelength, a perpendicular line to the x-axis is drawn at the position of this wavelength, and this line intersects with the absorption spectrum of amylopectin, which is an interfering component (absorbance A)Support 2) From this point, a straight line parallel to the x-axis is drawn, which in turn intersects the absorption spectrum of amylopectin at the isoabsorption point (absorbance A)Support 1，ASupport 1=ASupport 2) Then select the wavelength λ corresponding to the intersection point1As a reference wavelength for amylose, thereby eliminating lambda at the measurement wavelength2Interference of amylopectin with determination of amylose content. Similarly, when determining the amylopectin content, the amylopectin is selectedPowder absorption peak wavelength lambda4As the measurement wavelength, a perpendicular line to the x-axis is drawn at the position of this wavelength, and this line intersects with the absorption spectrum of amylose, an interfering component, at a certain point (absorbance A)Straight 4) From this point, a line is drawn parallel to the x-axis, which in turn intersects the absorption spectrum of amylose at the isoabsorbance point (absorbance A)Straight 3，AStraight 3=AStraight 4) Then select the wavelength λ corresponding to the intersection point3As a reference wavelength for amylopectin starch, thereby eliminating the wavelength λ at the measurement wavelength4Interference of amylose on determination of amylopectin content.
Drawing a dual-wavelength amylose standard curve:
accurately transferring amylose standard solution with concentration of 1.0 mg/mL, respectively placing 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL and 0.6mL into 6 25 mL volumetric flasks according to the method described above, and performing quantitative analysis according to the difference between the two absorbances, namely △ AStraight bar= Aλ2- Aλ1=A563 nm- A511 nmAnd (3) drawing a dual-wavelength amylose standard curve by taking the ordinate as the mass of the amylose and the abscissa as the mass of the amylose.
Drawing a dual-wavelength amylopectin standard curve:
accurately transferring 0.5 mL, 1.0 mL, 1.5mL, 2.0 mL and 3.0mL of amylopectin standard solution with concentration of 1.0 mg/mL into 6 volumetric flasks with 25 mL, respectively, and performing the above-mentioned method according to the difference between the two absorbances of △ ABranch stand= Aλ4- Aλ3=A542 nm- A722 nmAnd drawing a dual-wavelength amylopectin standard curve by taking the ordinate as the mass of the amylopectin and the abscissa as the mass of the amylopectin.
Examination of the optimal conditions for the preparation of the test solution:
50mg, 100 mg and 150mg of the highland barley powder (highland barley of short mango and short mango four long collected from Yushu of Qinghai) which is crushed and sieved by a 60-mesh sieve are placed in a 25 mL volumetric flask, 0.5 mL of absolute ethyl alcohol is added, and the sample is vortexed to be wetted. Adding 0.5 mL, 1.0 mL and 1.5mL of 1mol/L sodium hydroxide solution, uniformly mixing by vortex, heating in a boiling water bath for 2 min, and taking out and vortex for 30 s; repeating the above steps for 3, 4 and 5 times, cooling to room temperature, metering to 25 mL, shaking, and filtering to obtain starch extractive solution. Accurately transferring 1.0 mL of starch extracting solution, placing the starch extracting solution into a 25 mL volumetric flask, adding 15 mL of water, adjusting the pH to 3.0, 3.5 and 4.0 by using a glacial acetic acid solution with the concentration of 1mol/L, then adding 0.20 mL, 0.25 mL and 0.30 mL of iodine reagent into the sample measuring solution, adding water into the sample measuring solution to a constant volume, and shaking up to obtain the starch measuring solution.
And (3) determining the content of amylose and amylopectin in the highland barley:
the measurement and reference wavelengths of amylose and amylopectin were determined by isoabsorptive dual wavelength elimination. Standing the starch determination solution for 15 min, determining absorbance values at wavelengths of 563 nm, 511 nm, 542nm and 722 nm respectively by using a 1 cm cuvette, and then respectively checking the starch quality corresponding to two absorbance differences corresponding to the starch determination solution on a dual-wavelength amylose and amylopectin standard curve, thus respectively calculating the amylose and amylopectin contents in the highland barley, wherein the sum of the amylose and the amylopectin contents is the total starch content of the highland barley.
The mass fractions of amylose, amylopectin and total starch content (moisture in GB/T20264, GB/T21305 on a dry basis) were calculated according to formula ⑴, formula ⑵ and formula ⑶, respectively:
in the formula:
Two replicates of each sample were taken and the arithmetic mean was taken as the measurement result, leaving the 1 st decimal place. The absolute difference between two independent determinations under repeated conditions must not exceed 5% of the arithmetic mean.
Methodology validation experiment:
and (3) precision experiment: accurately transferring a prepared starch extract (highland barley, four-long Erighur and two-short-awn, respectively, from Yushu, Qinghai), and repeatedly measuring absorbance A at 4 wavelengths of 511 nm, 542nm, 563 nm, and 722 nm511 nm、A542 nm、A563 nm、A722 nmAnd (4) calculating the RSD value of the amylose content and the amylopectin content for 6 times, and detecting the precision of the method.
Stability test: accurately transferring a prepared starch extract (highland barley, four-long two-short awn, and white highland barley from Yushu, Qinghai) according to the above method, standing at room temperature, and measuring absorbance A at 4 wavelengths of 511 nm, 542nm, 563 nm, and 722 nm every 1 h511 nm、A542 nm、A563 nmAnd A722 nmAnd calculating RSD values of amylose and amylopectin contents, and detecting the stability of the starch determination solution.
And (3) repeatability experiment: accurately transferring five prepared starch extractive solutions (highland barley, four long two short awn, and white highland barley from Yushu, Qinghai), and measuring absorbance A at 4 wavelengths of 511 nm, 542nm, 563 nm, and 722 nm511 nm、A542 nm、A563 nmAnd A722 nmAnd calculating RSD values of amylose and amylopectin contents, and detecting the reproducibility of the method.
Recovery rate experiment: weighing 9 parts of crushed 60-mesh highland barley sample (four-long two-short awn white highland barley collected from Qinghai jade tree) powder with known amylose and amylopectin contents by adopting a sample adding recovery method, wherein each part is 50mg (accurate to +/-1 mg), adding 2.5 mL, 5.0 mL and 7.5 mL of amylose (0.9901 mg/mL) and amylopectin standard solution (5.005 mg/mL) with low, medium and high concentrations respectively and 3 parts of each concentration respectively according to the method, and measuring at 511 nm, 542nm, 563 nm and 722 nm4 wavelengthsAbsorbance A511 nm、A542 nm、A563 nmAnd A722 nmAnd calculating the average recovery rate and the RSD value of the amylose content and the amylopectin content, and detecting the recovery rate of the method.
Quantitative limit and detection limit experiments: according to the validated methodology content of Q2 (R1) analytical methods in ICH guidelines, the limit of quantitation (QL =10 × σ/S) and the limit of detection (DL = 3.3 × σ/S) are calculated from the standard deviation (σ) of a certain amount of blank response and the slope (S) of the standard curve of the analyte. Following the above procedure, a dual wavelength standard curve was drawn to determine the slope S, and 10 portions of 1.0 mL of the sample blank were accurately pipetted and the absorbance A was measured at 4 wavelengths 511 nm, 542nm, 563 nm, and 722 nm511 nm、A542 nm、A563 nmAnd A722 nmThe standard deviation σ of the response values of the 10 blank samples was determined.
Laboratory comparison verification experiment:
2 highland barley samples (highland barley of origin coming from the Carpenty department of agriculture and animal science of Qinghai university and highland barley of Changbao from the Yushu tree) which are submitted by the institute of science and technology of Qinghai university are spot-inspected by a laboratory of the northwest China plateau biological research center of the Chinese academy of sciences and samples are prepared uniformly, the content of amylose and amylopectin in the 2 highland barley samples is determined, and simultaneously the samples are sent to the ecological center of the institute of sciences of the northwest China and other two laboratories of the geological environment geological survey institute of hydrogeological engineering of Qinghai province for comparison and verification experiments between laboratories, each laboratory measures 6 times, and the reproducibility of the detection method is realized.
2. Results and analysis of the experiments
Measurement results of amylose and amylopectin measurement wavelengths and reference wavelengths:
the absorption curves of the two starch standard solutions are superposed in the same coordinate system (450 nm-900 nm), and the result shown in figure 1 can be obtained. From the results, it can be seen that: the measurement wavelength and the reference wavelength of amylose are respectively lambda2= 563 nm and λ1=511 nm; the measurement wavelength and the reference wavelength of amylopectin are respectively lambda4= 542nm and λ3= 722 nm。
Drawing a standard curve of the dual-wavelength amylose and amylopectin:
a dual-wavelength amylose standard curve, namely measuring the absorbance values of amylose standard solutions with different concentrations at 511 nm and 563 nm respectively, and using the difference value of the two absorbances to △A straight= A563 nm- A511 nmThe ordinate is the amylose mass (mg) and the abscissa is the abscissa, and a dual wavelength amylose standard curve is plotted, see FIG. 2. As can be seen from FIG. 2, the amylose content is in a good linear relationship within the range of 0.198mg to 0.594 mg, and the regression equation isy= 0.3099x- 0.0165，R 2=0.9991。
A dual-wavelength amylopectin standard curve, which is to measure the absorbance values of the amylopectin standard solution with different concentrations at 542nm and 722 nm respectively and to obtain △ difference value of the two absorbancesA branch= A542 nm– A722 nmAs an ordinate, the amylopectin mass (mg) was plotted as an abscissa, and a standard curve of the amylopectin with a double wavelength was plotted, as shown in FIG. 3. As can be seen from FIG. 3, the amylopectin content is in a good linear relationship within the range of 0.50 mg-3.00 mg, and the regression equation isy= 0.1366x- 0.0199，R 2=0.9995。
Examination of the optimal conditions for the preparation of the test solution:
in order to comprehensively investigate all factors of complete extraction and color development of the two starches, an orthogonal experiment is designed on the basis of a single-factor experiment. A sample weighing amount (A), a sodium hydroxide concentration (B), an alkali extraction dissolution vortex frequency (C), a color development pH value adjustment (D) and a color development reagent addition amount (E) are used as investigation objects, and a five-factor three-level orthogonal experiment is designed to determine the optimal conditions for complete extraction and color development of two kinds of starch. The factors and specific horizontal points are shown in Table 1, and selectedL 27（35) Orthogonal table, orthogonal experimental results and analysis, variance analysis results are shown in table 2 and table 3 respectively.
Table 1 various factors and levels of orthogonal experimental design
TABLE 2 orthogonal experimental results and analysis
As can be seen from Table 2, the primary and secondary sequence of the factors affecting the complete color development and extraction of two starches is as follows: sample weighing (A)>Concentration of sodium hydroxide (B)>Number of vortexes of alkali extraction solution (C)>Amount of color reagent iodine added (E)>And (D) adjusting the color development pH value. FromkValue analysis, the optimal extraction and color development condition for making the total starch content be highest is A2B2C3D2E2And the result of the orthogonal experiment is A2B2C3D1E3. The orthogonal design has no repeated test, and the analysis of variance by using SPSS 19.0 software shows that ss (color development pH value adjustment D) = 15.975 is minimum, the influence on the whole test result is minimum, and therefore the ss is used as error estimation to test the significance of the action of other factors, and the result is shown in Table 3.
TABLE 3 analysis of variance results
As can be seen from Table 3, when the coloration pH was adjusted to an error (D), the total starch content was significantly affected by the amount of the sample (A), the concentration of sodium hydroxide (B), and the number of alkali dissolution vortexes (C) (C)PLess than 0.05), the addition of the color reagent iodine reagent (E) has no significant influence on the total starch content (P> 0.05). From this, the visual analysis and the analysis of variance result are consistent. The optimal extraction and color development complete condition is selected as A by combining various factors2B2C3D2E2Namely, the sample weighing amount is 100 mg, the concentration of sodium hydroxide is 1.0 mol/L, the number of alkali extraction dissolution vortexes is 5, the color development pH value is adjusted to pH3.5, and the addition amount of a color development reagent iodine reagent is 0.25 mL.
And (3) determining the content of amylose and amylopectin in the highland barley:
the contents of amylose and amylopectin in highland barleys of different origins and varieties in the Qinghai environment are measured by an isoabsorptive dual-wavelength elimination method, and the sum of the contents is the total starch content, and the result is shown in table 4.
Table 4 results of amylose and amylopectin contents in highland barley of different origins and varieties in Qinghai (g/100 g dw,n= 3）
as can be seen from Table 4: the amylopectin content in highland barley in Qinghai is higher than the amylose content, and the amylopectin content is about 5.4 times higher than the amylose content. The highest content of total starch of the highland barley of Source blue from Qinghai, which is called much, is 75.4 g/100g DW, and the lowest content of total starch of the highland barley of Changmang white from Qinghai Yu Tree is 64.0 g/100g DW.
Methodology validation experiment:
and (3) precision experiment: accurately transferring a prepared starch extract (highland barley, four-long Erighur and two-short-awn, respectively, from Yushu, Qinghai), and repeatedly measuring absorbance A at 4 wavelengths of 511 nm, 542nm, 563 nm, and 722 nm511 nm、A542 nm、A563 nm、A722 nmThe results show that the RSD values of the amylose content and the amylopectin content in the starch determination solution are 0.62 percent and 0.45 percent respectively.
Stability test: accurately transferring a prepared starch extract (highland barley, four-long two-short awn, and white highland barley from Yushu, Qinghai) according to the above method, standing at room temperature, and measuring absorbance A at 4 wavelengths of 511 nm, 542nm, 563 nm, and 722 nm every 1 h511 nm、A542 nm、A563 nm、A722 nmThe result shows that the starch determination solution has good stability within 12 hours, and the calculated RSD values of the amylose content and the amylopectin content are 1.5 percent and 1.8 percent respectively.
And (3) repeatability experiment: accurately transferring five prepared starch extractive solutions (semen Avenae Nudae, four-long-two-short-awn, and white semen Avenae Nudae, collected from Yushuang, Qinghai, and Yushui) at 511 nm, 542nm, 563 nm, and 722 nmDetermination of the absorbance A at nm511 nm、A542 nm、A563 nm、A722 nmThe results show that the reproducibility of the detection method is good, and the calculated RSD values of the amylose content and the amylopectin content are 1.1% and 1.3% respectively.
Recovery rate experiment: weighing 9 parts of crushed 60-mesh highland barley sample (four-long two-short-awn white highland barley collected from Qinghai jade tree) powder with known amylose and amylopectin contents by adopting a sample adding recovery method, wherein each part is 50mg (accurate to +/-1 mg), accurately adding 2.5 mL, 5.0 mL and 7.5 mL of amylose (0.9901 mg/mL) and amylopectin standard solution (5.005 mg/mL) with low, medium and high concentrations respectively and 3 parts of each concentration according to the method, and measuring absorbance A at 511 nm, 542nm, 563 nm and 722 nm511 nm、A542 nm、A563 nm、A722 nmThe results are shown in tables 5 and 6. The calculated average recovery rates of the amylose content and the amylopectin content are 91.14% and 91.73% respectively, and the RSD content is 1.70% and 2.25% respectively, and the result shows that the detection method has good accuracy.
TABLE 5 amylose recovery test results: (n= 9）
TABLE 6 amylopectin recovery test results: (n= 9）
Quantitative limit and detection limit experiments:
following the above procedure, a dual wavelength standard curve was drawn to determine the slope S, and 10 portions of 1.0 mL of the sample blank were accurately pipetted and the absorbance A was measured at 4 wavelengths 511 nm, 542nm, 563 nm, and 722 nm511 nm、A542 nm、A563 nmAnd A722 nmThe standard deviation σ of the response values of the 10 blank samples was determined. The quantitative limit (QL =10 × σ/S) and the detection limit (DL = 3.3 × σ/S) are determined from the calculation formula, and the result is obtainedThe results are shown in tables 7 and 8.
TABLE 7 amylose quantitation and detection limits Experimental results: (n= 10）
TABLE 8 amylopectin quantification and detection limits experimental results: (n= 10）
Laboratory comparison verification experiment:
2 highland barley samples (highland barley of origin coming from the Carpenty department of agriculture and animal science of Qinghai university and highland barley of Changbao from the Yushu tree) which are submitted by the institute of science and technology of Qinghai university are spot-inspected by the laboratory of the northwest China plateau biological research center of the Chinese academy of sciences and samples are prepared uniformly, the content of amylose and amylopectin in the 2 highland barley samples is determined, and simultaneously the samples are sent to the ecological center of the institute of North West plateau biological research of the Chinese academy of sciences and other two laboratories of the geological environment geological survey institute of hydrogeological engineering of Qinghai province for comparison and verification experiments between laboratories, each laboratory measures 6 times, and the results are shown in tables 9-11. The calculation shows that the indoor RSD values of the amylose and amylopectin contents in the Qinghai source flower highland barley and the Changmai white highland barley of the Qinghai jade tree, which are measured in different laboratories, are all less than 2.5%, and the indoor RSD values are all less than 4.2%, so that the result shows that the detection method has good reproducibility.
Table 9 analysis of amylose and amylopectin content in 2 highland barleys measured in the laboratory of the testing center (g/100 gDW,n= 6）
table 101 # the content of amylose and amylopectin in 2 highland barleys tested in the laboratory (g/100 g DW,n= 6）
table 112 # lab measurements of amylose and amylopectin content in 2 highland barleys (g/100 g DW,n= 6）
3. conclusion of the experiment
The invention respectively carries out single-factor cycle test by taking 5 factors of sample weight, sodium hydroxide concentration, alkali extraction dissolution vortex frequency, chromogenic pH value adjustment and addition of a chromogenic reagent iodine reagent, and designs five-factor three-level orthogonal test, so that when the sample weight is 100 mg, the sodium hydroxide concentration is 1.0 mol/L, the alkali extraction dissolution vortex frequency is 5 times, the chromogenic pH value is adjusted to pH3.5, and the addition of the chromogenic reagent iodine reagent is 0.25 mL, two kinds of starch are completely extracted and developed, and early-stage preparation is prepared for subsequent experiments.
Claims (1)
1. A method for rapidly detecting amylose and amylopectin in highland barley comprises the following steps:
⑴ preparation of standard solutions:
i preparation of amylose standard solution:
accurately weighing 50mg of potato amylose standard substance with the purity of more than 99.5%, placing the potato amylose standard substance into a 50 mL volumetric flask, adding 10 mL of 1mol/L sodium hydroxide solution, placing the potato amylose standard substance into a boiling water bath for dissolving and dispersing, taking out, cooling to room temperature, adding distilled water for diluting to a scale, and shaking up to obtain an amylose standard solution with the concentration of 1.0 mg/mL;
ii preparation of amylopectin standard solution:
accurately weighing 50mg of potato amylopectin standard substance with purity of more than 99.5%, adding 10 mL of 1mol/L sodium hydroxide solution into a 50 mL volumetric flask, dissolving and dispersing in boiling water bath, taking out, cooling to room temperature, adding distilled water to dilute to a scale, shaking up to obtain an amylopectin standard solution with concentration of 1.0 mg/mL;
⑵, accurately transferring 1.0 mL of the amylose standard solution and 1 part of the amylopectin standard solution respectively, placing the solutions in 25 mL volumetric flasks, adding 15 mL of water, adjusting the pH to 3.0-4.0 by using a glacial acetic acid solution with the concentration of 1mol/L, adding 0.20-0.30 mL of iodine reagent into the sample measuring solution, adding water into the sample measuring solution to a constant volume of 25 mL, shaking up, and preparing an amylose measuring solution and an amylopectin measuring solution respectively;
⑶ determination of the measured and reference wavelengths of amylose and amylopectin by isoabsorptive dual wavelength elimination:
① determination of amylose and reference wavelength by determining the same absorbance A in the amylopectin absorption curveSupport 1And ASupport 2Corresponding wavelengths are respectively lambda1And λ2；λ2The value of A on the corresponding amylose absorption curve is denoted AStraight 2So that λ2As the measurement wavelength of amylose; lambda [ alpha ]1The value of A on the corresponding amylose absorption curve is denoted AStraight 1So that λ1As reference wavelength for amylose;
② determination of amylopectin and reference wavelength by determining the maximum absorbance A in the absorption curve of amylopectinmaxCorresponding lambda4As the measurement wavelength of amylopectin; at λ4Find A on the corresponding amylose absorption curveStraight 4Continue to find the sum A on the curveStraight 4A with the same absorbanceStraight 3Finding the corresponding A on the amylopectin absorption curveStraight 3The same wavelength lambda3As reference wavelength for amylopectin;
⑷ standing the amylose measurement solution and the amylopectin measurement solution for 15 min, respectively measuring absorbance values at a measurement wavelength 563 nm and a reference wavelength 511 nm of amylose and at a measurement wavelength 542nm and a reference wavelength 722 nm of amylopectin with a 1 cm cuvette;
⑸ standard curves are plotted:
a dual wavelength amylose standard curve:
accurately transferring 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL and 0.6mL of amylose standard solution with the concentration of 1.0 mg/mL into 6 volumetric flasks with the volume of 25 mL respectivelyPreparing an amylose determination solution in the step ⑵, determining absorbance values at 563 nm and 511 nm of wavelengths respectively by using a 1 cm cuvette according to the step ⑷, and determining the absorbance difference between the absorbance values, namely △ AStraight bar=Aλ2- Aλ1=A563 nm- A511 nmDrawing a dual-wavelength amylose standard curve by taking the mass of the amylose as the ordinate and the mass of the amylose as the abscissa;
b dual wavelength amylopectin standard curve:
accurately transferring 0.5 mL, 1.0 mL, 1.5mL, 2.0 mL and 3.0mL of amylopectin standard solution with concentration of 1.0 mg/mL into 6 volumetric flasks with volume of 25 mL respectively, preparing determination solution of amylopectin according to the step ⑵, determining absorbance values at wavelength of 542nm and 722 nm respectively by using a 1 cm cuvette according to the step ⑷, and determining difference between two absorbances, namely △ ABranch stand=Aλ4- Aλ3= A542 nm- A722 nmDrawing a dual-wavelength amylopectin standard curve by taking the ordinate as the mass of the amylopectin and the abscissa as the mass of the amylopectin;
⑹ placing 50-150 mg of crushed highland barley powder sieved by a 60-mesh sieve into a 25 mL volumetric flask, adding 0.5 mL of absolute ethyl alcohol, and vortexing to wet a sample;
⑺ adding 0.5-1.5 mL of 1mol/L sodium hydroxide solution into the wetted sample, uniformly mixing by vortex, heating in a boiling water bath for 2 min, taking out, vortex for 30 s, repeating the steps for 3-5 times, cooling to room temperature, metering to 25 mL, and shaking up to obtain starch extract;
⑻ accurately transferring 1.0 mL of the starch extracting solution into a 25 mL volumetric flask, and preparing a starch determination solution according to the step ⑵;
⑼, standing the starch determination solution for 15 min, determining absorbance values at wavelengths of 563 nm, 511 nm, 542nm and 722 nm by using a 1 cm cuvette respectively according to the step ⑷, and then checking the starch quality corresponding to the difference between the two corresponding absorbances of the starch determination solution on the dual-wavelength amylose and amylopectin standard curves respectively, thereby calculating the amylose and amylopectin contents in the highland barley respectively, wherein the sum of the amylose and the amylopectin contents is the total starch content.
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