CN109142281B - Method for detecting content and structure of resistant starch of potatoes based on field flow separation technology - Google Patents
Method for detecting content and structure of resistant starch of potatoes based on field flow separation technology Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
- G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
Abstract
The invention provides a method for detecting the content and the structure of resistant starch of potatoes based on a field flow separation technology. The method comprises the following steps: a. hydrolyzing the non-resistant starch of potato; b. dissolving potato resistant starch; c. measuring the content and the structure of the resistant starch of the potato by field flow separation; d. and calculating the content of the resistant starch of the potatoes. According to the method, the content of the resistant starch of the potatoes is detected by adopting the asymmetric field flow separation combined multi-angle laser light scattering detector and the differential refraction detector, and the whole process of detecting the resistant starch of the potatoes is 4 hours, so that the time consumption is short; while providing a resistant starch turning radius and molecular weight distribution. Furthermore, the method of the present invention employs 50 mM NaNO3+3 mM NaN3The aqueous solution is used as a mobile phase, and the cost is low. Compared with the current general AOAC resistant starch detection method, the method has obvious advantages.
Description
Technical Field
The invention relates to a method for detecting resistant starch of potatoes, in particular to a method for detecting the content and the structure of the resistant starch of potatoes based on a field flow separation technology.
Background
With the improvement of living standard of people, people pay more and more attention to eating high-nutrition and low-calorie functional food. Starch is an important raw material in the food industry and is classified into three categories according to its rate and extent of digestion in the body: fast digestible starch, slow digestible starch and resistant starch. The resistant starch is a functional food component with low calorie and similar dietary fiber, has similar functions to the dietary fiber, is superior to the dietary fiber in taste and certain performance, and is helpful for preventing diabetes, controlling weight, improving immunity of organism, preventing colon cancer, etc.
The potatoes are the fourth world staple food crops of non-cereals, and have wide planting area and high yield in China. The potato starch has larger granules, smooth surface and higher content of resistant starch. The structure of starch (radius of rotation and molecular weight distribution) is an important factor affecting the viscosity characteristics, gelatinization characteristics, degree of swelling and retrogradation of starch. After the resistant starch is added into the starch product, the texture and the structure of the quick-frozen dough can be maintained, the expansion degree and the brittleness of the expanded food are improved, the death of probiotics in the yoghourt is prevented, and the taste, the color and the texture of the baked food are improved. Therefore, the method has important significance for the content of the resistant starch of the potato and the structural characterization thereof.
The main existing method for detecting the resistant starch of the potatoes is a Megazyme resistant starch kit method (AOAC method 2002.02): the samples were incubated for 16 h with alpha-amylopsin and Amyloglucosidase (AMG) in a 37 ℃ water bath with shaking, the non-resistant starch was hydrolyzed to D-glucose by the combined action of the two enzymes, and after the incubation was finished, the reaction was stopped by adding an equal volume of ethanol. The solution was centrifuged, and the resulting solid was dissolved in 2M (mol/L) KOH and vigorously stirred in an ice-water bath. The solution was made neutral with acetate buffer and the starch was hydrolyzed to D-glucose with AMG. D-glucose was measured with glucose oxidase/peroxidase reagent (GOPOD) to obtain the resistant starch content.
However, the AOAC kit method is costly, time-consuming and incapable of providing information on the rotation radius and molecular weight distribution of the resistant starch.
Disclosure of Invention
The invention aims to provide a method for detecting the content and the structure of resistant starch of potatoes based on a field flow separation technology, and aims to solve the problems that the cost is high, the time consumption is long, and the information of the rotating radius and the molecular weight distribution of the resistant starch cannot be provided simultaneously when the AOAC kit method is used for detecting the content of the resistant starch of potatoes.
The invention is realized by the following steps: a method for detecting the content and the structure of resistant starch of potatoes based on a field flow separation technology comprises the following steps:
a. hydrolyzing the non-resistant starch of potato;
a-1, weighing a proper amount of potato starch in a sample bottle, adding a sodium acetate buffer solution into the sample bottle, and then preheating in a water bath at 37 ℃;
a-2, adding a mixed enzyme of alpha-amylopsin and amyloglucosidase into the sample bottle;
a-3, continuously stirring at 160 rpm, and incubating in a water bath at 37 ℃ for 2 h;
a-4, transferring the sample bottle from a water bath at 37 ℃ to a water bath at 90 ℃, standing, and inactivating digestive enzymes;
a-5, taking out the sample bottle from the water bath at 90 ℃, and naturally cooling to room temperature;
b. dissolving potato resistant starch;
b-1, putting the sample obtained in the step a-5 into a sample bottle, adding a NaOH solution, carrying out water bath at 70 ℃, and magnetically stirring at 400 rpm to dissolve the resistant starch;
b-2, adding carrier liquid for asymmetric field flow separation, wherein the carrier liquid for asymmetric field flow separation is NaNO3And NaN3The aqueous solution is put in a water bath at 70 ℃ and magnetically stirred at 400 rpm;
b-3, adding HCl solution, carrying out water bath at 70 ℃, and continuing magnetic stirring at 400 rpm;
b-4, taking the sample bottle out of the water bath, and naturally cooling to room temperature;
c. measuring the resistant starch of the potatoes by adopting a field separation technology;
analyzing the sample obtained in the step b-4 by adopting an asymmetric field flow separation combined multi-angle laser light scattering detector and a refractive index detector to obtain a dRI peak area M1, a rotation radius and a molecular weight distribution of the resistant starch;
d. detecting the content of resistant starch of the potatoes;
d-1, weighing a proper amount of potato starch in a sample bottle, adding a NaOH solution, carrying out water bath at 70 ℃, and carrying out magnetic stirring at 400 rpm;
d-2, adding carrier liquid for asymmetric field flow separation, wherein the carrier liquid for asymmetric field flow separation is NaNO3And NaN3The aqueous solution is put in a water bath at 70 ℃ and magnetically stirred at 400 rpm;
d-3, adding an HCl solution, carrying out water bath at 70 ℃, and continuing magnetic stirring at 400 rpm;
d-4, taking the sample bottle out of the water bath, and naturally cooling to room temperature;
d-5, analyzing the sample obtained in the step d-4 by adopting an asymmetric field flow separation combined multi-angle laser light scattering detector and a differential refraction detector to obtain a dRI peak area M2 of the potato starch;
d-6, calculating the content of the resistant starch of the potatoes according to the following formula:
potato resistant starch content = M1/M2 × 100%.
When the asymmetric field flow separation combined multi-angle laser light scattering detector and the refractive index detector are used for analyzing the sample in the step c and the step d-5, the flow speed of the detector is 1 mL/min; the elution flow rate is exponential decay cross flow rate, and is reduced from 1.4 mL/min to 0.05 mL/min within 19 min, and the half life period is 4 min.
The carrier liquid used for the asymmetric field flow separation is NaNO3And NaN3And NaNO, and350 mM, NaN3At 3 mM, the pH of the carrier liquid is 7.00.
According to the method, the content of the resistant starch of the potatoes is detected by adopting the asymmetric field flow separation combined multi-angle laser light scattering detector and the differential refraction detector, and the whole process of detecting the resistant starch of the potatoes is 4 hours, so that the time consumption is short; while providing a resistant starch turning radius and molecular weight distribution. Furthermore, the method of the present invention employs 50 mM NaNO3 + 3 mM NaN3The aqueous solution is used as a mobile phase, and the cost is low. Compared with the current general AOAC resistant starch detection method, the method has obvious advantages.
Drawings
FIG. 1 is a graph of the radius of rotation profile of potato resistant starch.
FIG. 2 is a graph of the molecular weight distribution of potato resistant starch.
Detailed Description
The method for detecting the content and the structure of the resistant starch of the potatoes based on the field flow separation technology comprises the following steps:
1. hydrolyzed potato non-resistant starch
(1) Accurately weighing 100 mg of potato starch, placing in a 20 mL sample bottle, adding 6 mL of 0.1M sodium acetate buffer (pH 5.2, containing 1 mM CaCl)2). After the screw cap of the sample bottle is screwed down, the sample bottle is preheated for 5 min in a water bath at 37 ℃.
(2) To the sample bottle was added 4.0 mL of the mixed enzyme (containing. alpha. -amylopsin (142U/mL) and AMG (15U/mL)).
(3) Stirring was continued at 160 rpm and incubation was carried out at 37 ℃ for 2 h.
(4) The sample bottle is transferred from the water bath at 37 ℃ to the water bath at 90 ℃, and is placed for 2 min to inactivate the digestive enzyme.
(5) The sample bottle was taken out of the 90 ℃ water bath and naturally cooled to room temperature.
2. Dissolving potato resistant starch
(1) 1 mL of the sample obtained in step 1 was added to 1 mL of 1M NaOH solution, and the mixture was magnetically stirred at 400 rpm for 2 min in a water bath at 70 ℃ to dissolve the resistant starch.
(2) The carrier liquid (50 mM NaNO) used for the asymmetric field flow separation was added in an amount of 8 mL3 + 3 mM NaN3pH 7.00), magnetically stirred at 400 rpm in a water bath at 70 ℃ for 1 h.
(3) 1 mL of 1M HCl solution was added and the magnetic stirring was continued at 400 rpm for 5 min at 70 ℃ in a water bath.
(4) And taking the sample bottle out of the water bath, naturally cooling to room temperature, and then fixing the volume to 10 mL.
3. Determination of resistant starch content and structure of potato by field flow separation
(1) The instrument comprises the following steps: asymmetric field-flow separation combines a multi-angle laser light scattering detector and a refractive index detector (AF 4-MALS-dRI).
(2) Liquid carrying: 50 mM NaNO3 + 3 mM NaN3，pH 7.00。
(3) Sample introduction amount: 50 μ L.
(4) Detector flow rate: 1 mL/min.
(5) Elution flow rate: the cross flow velocity is exponentially decayed, the flow velocity is reduced from 1.4 mL/min to 0.05 mL/min within 19 min, and the half life period is 4 min. Specific cross-flow rate measurement procedures are shown in table 1.
(6) After cooling the potato resistant starch sample (i.e. the sample obtained in step 2 above) to room temperature, the dRI peak area M1 of the digestion residue (resistant starch) and the radius of rotation (M1) were obtained by AF4-MALS-dRI analysisR g) And molecular weight of (M w) And (4) distribution.
4. Calculation of resistant starch content in Potato
(1) 10 mg of potato starch was added to 1 mL of 1M NaOH solution and stirred magnetically at 400 rpm for 2 min in a water bath at 70 ℃.
(2) The carrier liquid (50 mM NaNO) used for the asymmetric field flow separation was added in an amount of 8 mL3 + 3 mM NaN3pH 7.0), magnetic stirring was carried out at 400 rpm in a water bath at 70 ℃ for 2 h.
(3) 1 mL of 1M HCl solution was added and the magnetic stirring was continued at 400 rpm for 5 min at 70 ℃ in a water bath.
(4) And taking the sample bottle out of the water bath, naturally cooling to room temperature, and then fixing the volume to 10 mL.
(5) The potato starch (i.e., the sample obtained in step 4 (4)) was analyzed under the AF4-MALS-dRI analysis conditions in step 3, yielding a dRI peak area of the potato starch of M2.
Potato resistant starch content = M1/M2 × 100%.
The AF4-MALS-dRI spectrum and the radius of rotation and the molecular weight distribution of the potato starch can be measured according to the 4 steps. As shown in FIGS. 1 and 2, FIG. 1 is a view showing a rotational radius distribution of a resistant starch of potato, and FIG. 2 is a view showing a rotational radius distribution of a resistant starch of potatoMolecular weight distribution of potato resistant starch. As can be seen from FIGS. 1 and 2, the distribution range of the radius of rotation of the potato resistant starch is: 19.9 nm to 172 nm; the molecular weight distribution range of the potato resistant starch is as follows: 2X 105 g/mol ~2×108g/mol. The AF4-MALS-dRI analysis method of the potato resistant starch has better reproducibility.
The results of the AF4-MALS-dRI assay for resistant potato starch are shown in Table 2. As can be seen from Table 2, the content of resistant starch in potato, measured by AF4-MALS-dRI, was 72.3%. Average weight average molecular weight of 2.93X 107g/mol, mean radius of rotation 87.7 nm. Compared with the existing method, the whole process of detecting the resistant starch of the potato by AF4-MALS-dRI is 4 hours, and the time consumption is short; the carrier liquid used for separation is 50 mM NaNO3 + 3 mM NaN3The cost of the aqueous solution is low; meanwhile, the method can provide the information of the rotating radius and the molecular weight distribution of the resistant starch and has better reproducibility.
The method for detecting the content of the resistant starch in the potatoes by adopting the asymmetric field flow separation combined multi-angle laser light scattering detector and the differential refraction detector has short analysis time, and can provide the rotating radius and the molecular weight distribution of the resistant starch. The method uses 50 mM NaNO3 + 3 mM NaN3The aqueous solution is used as a mobile phase, and the cost is low. The AF4 method of the present invention has certain advantages over the current AOAC resistant starch assay, as shown in Table 3.
Claims (6)
1. A method for detecting the content and the structure of resistant starch of potatoes based on a field flow separation technology is characterized by comprising the following steps:
a. hydrolyzing the non-resistant starch of potato;
a-1, weighing a proper amount of potato starch in a sample bottle, adding a sodium acetate buffer solution into the sample bottle, and then preheating in a water bath at 37 ℃;
a-2, adding a mixed enzyme of alpha-amylopsin and amyloglucosidase into the sample bottle;
a-3, continuously stirring and incubating in a water bath at 37 ℃;
a-4, transferring the sample bottle from a water bath at 37 ℃ to a water bath at 90 ℃, standing, and inactivating digestive enzymes;
a-5, taking out the sample bottle from the water bath at 90 ℃, and naturally cooling to room temperature;
b. dissolving potato resistant starch;
b-1, putting the sample obtained in the step a-5 into a sample bottle, adding a NaOH solution, carrying out water bath at 70 ℃, and carrying out magnetic stirring to dissolve the resistant starch;
b-2, adding carrier liquid for asymmetric field flow separation, wherein the carrier liquid for asymmetric field flow separation is NaNO3And NaN3The aqueous solution is put in water bath at 70 ℃ and stirred by magnetic force;
b-3, adding HCl solution, carrying out water bath at 70 ℃, and continuing magnetic stirring;
b-4, taking the sample bottle out of the water bath, and naturally cooling to room temperature;
c. measuring the resistant starch of the potatoes by adopting a field separation technology;
analyzing the sample obtained in the step b-4 by adopting an asymmetric field flow separation combined multi-angle laser light scattering detector and a refractive index detector to obtain a dRI peak area M1, a rotation radius and a molecular weight distribution of the resistant starch;
d. detecting the content of resistant starch of the potatoes;
d-1, weighing a proper amount of potato starch in a sample bottle, adding a NaOH solution, carrying out water bath at 70 ℃, and carrying out magnetic stirring;
d-2, adding carrier liquid for asymmetric field flow separation, wherein the carrier liquid for asymmetric field flow separation is NaNO3And NaN3The aqueous solution is put in water bath at 70 ℃ and stirred by magnetic force;
d-3, adding HCl solution, carrying out water bath at 70 ℃, and continuing magnetic stirring;
d-4, taking the sample bottle out of the water bath, and naturally cooling to room temperature;
d-5, analyzing the sample obtained in the step d-4 by adopting an asymmetric field flow separation combined multi-angle laser light scattering detector and a differential refraction detector to obtain a dRI peak area M2 of the potato starch;
d-6, calculating the content of the resistant starch of the potatoes according to the following formula:
potato resistant starch content = M1/M2 × 100%.
2. The method for detecting the content and the structure of resistant starch in potatoes based on the field flow separation technology as claimed in claim 1, wherein the flow rate of the detector is 1 mL/min when the asymmetric field flow separation combined with the multi-angle laser light scattering detector and the differential refraction detector is used for analyzing the sample in the steps c and d-5; the elution flow rate is exponential decay cross flow rate, and is reduced from 1.4 mL/min to 0.05 mL/min within 19 min, and the half life period is 4 min.
3. The method for detecting the content and the structure of the resistant starch of potatoes based on the field flow separation technology as claimed in claim 1, wherein the carrier liquid used for the asymmetric field flow separation is NaNO3And NaN3And NaNO, and350 mM, NaN3At 3 mM, the pH of the carrier liquid is 7.00.
4. The method for detecting the content and the structure of the resistant starch of potatoes based on the field flow separation technology as claimed in claim 1, wherein the stirring speed in the step a-3 is 160 rpm, and the potato is incubated in a water bath at 37 ℃ for 2 h.
5. The method for detecting the content and the structure of the resistant starch in the potatoes based on the field flow separation technology as claimed in claim 1, wherein the magnetic stirring speed in the steps b-1, b-2, b-3, d-1, d-2 and d-3 is 400 rpm.
6. The method for detecting the content and the structure of the resistant starch of potatoes based on the field flow separation technology as claimed in claim 1, whereinThe pH of the sodium acetate buffer solution in the step a-1 is 5.2, and the sodium acetate buffer solution contains 1 mM CaCl2。
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| CN109827931A (en) * | 2019-03-08 | 2019-05-31 | 河北大学 | A kind of method dissolving cornstarch and the method for detecting the cornstarch radius of gyration and molecular weight distribution |
| CN109991067A (en) * | 2019-04-30 | 2019-07-09 | 河北大学 | A kind of accurate method for detecting the gastrodia elata polysaccharide radius of gyration and molecular weight distribution |
| CN114136960B (en) * | 2021-12-01 | 2023-09-15 | 华南农业大学 | Method for batch measurement of rice resistant starch content |
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| Amylose and amylopectin in starch by asymmetric flow field-flow fractionation with multi-angle light scattering and refractive index detection (AF4eMALSeRI);Eija Chiaramonte et al.;《Journal of Cereal Science》;20120508;第56卷;第457-463页 * |
| Molecular characterization of starches by AF4-MALS-RI: An alternative procedure;Luis A.Bello-Perez et al.;《Journal of Cereal Science》;20170329;第75卷;第132-134页 * |
| Study on antidiabetic activity of wheat and barley starch using asymmetrical flow field-flow fractionation coupled with multiangle light scattering;Dou Haiyang et al.;《Journal of Chromatography A》;20140312;第1340卷;第116页第2.1-2.4节 * |
| 抗性淀粉测定方法探讨及预处理对宜糖米抗性淀粉质量分数的影响;周颖等;《食品与生物技术学报》;20170430;第36卷(第4期);第416-419页 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2796408C2 (en) * | 2021-09-16 | 2023-05-23 | Федеральное государственное бюджетное научное учреждение «Федеральный научный центр пищевых систем им. В.М. Горбатова» РАН | Method for determining the degree of resistance of starches and starch products |
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| Publication number | Publication date |
|---|---|
| CN109142281A (en) | 2019-01-04 |
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Application publication date: 20190104 Assignee: BEIJING YONGKANGNIAN HEALTH TECHNOLOGY CO.,LTD. Assignor: HEBEI University Contract record no.: X2024990000043 Denomination of invention: A method for detecting the content and structure of potato resistant starch based on field flow separation technology Granted publication date: 20201229 License type: Common License Record date: 20240117 Application publication date: 20190104 Assignee: Huishi Huanyu (Beijing) Pharmaceutical Research Institute Co.,Ltd. Assignor: HEBEI University Contract record no.: X2024990000040 Denomination of invention: A method for detecting the content and structure of potato resistant starch based on field flow separation technology Granted publication date: 20201229 License type: Common License Record date: 20240117 |