CN110632201A - Method for accurately detecting content of galactooligosaccharide in milk powder - Google Patents
Method for accurately detecting content of galactooligosaccharide in milk powder Download PDFInfo
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- CN110632201A CN110632201A CN201910929749.7A CN201910929749A CN110632201A CN 110632201 A CN110632201 A CN 110632201A CN 201910929749 A CN201910929749 A CN 201910929749A CN 110632201 A CN110632201 A CN 110632201A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/96—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation using ion-exchange
Abstract
The invention relates to a method for accurately detecting the content of galacto-oligosaccharide in milk powder, which comprises the steps of preparing a standard solution by adopting a 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance, weighing galacto-oligosaccharide raw materials, dissolving the galacto-oligosaccharide raw materials by using water, preparing standard curve solutions with different concentrations, carrying out ion chromatography detection on the solution, and using the chromatographic peak of the obtained 3-0-beta-D-galacto-pyranosyl-D-glucopyranose standard substance to determine the galacto-oligosaccharide disaccharide chromatographic peak in the galacto-oligosaccharide solution; weighing a milk powder sample to prepare a sample solution, carrying out ion chromatography detection, taking the qualitative galactooligosaccharide disaccharide in the galactooligosaccharide raw material solution as a quantitative peak, quantifying by an external standard method, and calculating the content of the galactooligosaccharide in the sample solution. The method solves the problem that the existing method can not accurately determine the galactooligosaccharide and the content of the galactooligosaccharide in the milk powder, and has the advantages of low detection limit and accurate quantification.
Description
Technical Field
The invention relates to the technical field of analysis and detection, in particular to a method for accurately detecting the content of galactooligosaccharide in milk powder.
Background
The galactooligosaccharide is an oligosaccharide mixture with different polymerization degrees or the same polymerization degree but containing a plurality of different glycosidic bond isomers, and the molecular structure is generally that 1-7 galactosyl groups are connected to galactose or glucose molecules, namely Gal- (Gal) n-Glc/Gal (n is 0-6). The galacto-oligosaccharide is a functional oligosaccharide with natural attribute, can be utilized by probiotics such as bifidobacterium, lactobacillus and the like in human intestinal tracts, has a strong proliferation effect on the intestinal probiotics, and can promote gastrointestinal peristalsis and improve gastrointestinal functions. In 2008, the national ministry of health issued "bulletin on approval of new resource food such as galactooligosaccharide", and the galactooligosaccharide was approved as a new resource food, and can be widely applied to infant food, dairy products, beverages, baked food and candies. The new resource food is used for food production and processing, and should meet the relevant laws, regulations and standard regulations.
In view of the complexity of the galactooligosaccharide component, the prior art cannot prepare a standard for each component thereof, so that quantitative detection of each component cannot be accomplished. At present, detection methods at home and abroad mainly comprise thin-layer chromatography, gas chromatography, liquid chromatography, ion chromatography and the like, wherein high performance liquid chromatography-differential detection methods are generally applied, but the differential detector has poor sensitivity, the milk powder matrix is complex, and the chromatographic column separation effect is not ideal. The ion chromatography for detecting the galactooligosaccharide is a relatively new detection technology, has the advantages of high sensitivity, good separation degree and high separation efficiency, and is increasingly applied to the detection of the galactooligosaccharide.
Foreign galactooligosaccharide ion chromatography has a detection method of AOAC 2001.02 trans-galactooligosaccharide ion chromatography in selected food, but because the principle of the method uses an enzymolysis method and the calculation is carried out by the difference value before and after the enzymolysis, the method is not suitable for the detection of the milk powder for the sample with high lactose content.
The patent publication No. CN105548436A uses a galactooligosaccharide raw material supplied by a customer as a standard and makes a standard curve. Weighing two samples with the same mass, adding a galacto-oligosaccharide raw material into one sample, then adding water for dissolving, directly adding water for dissolving the other sample, pretreating the two samples to obtain a sample injection solution, and injecting the standard curve solution and the sample solution into an ion chromatograph respectively for detection. And selecting specific peaks according to the spectrogram comparison, and quantifying by using the selected specific peaks.
The method does not use a galactooligosaccharide standard product, so that the selected quantitative peak has great subjectivity, and the specific peak which is selected by the method cannot be accurately judged to be the galactooligosaccharide peak. In previous experiments, the inventor finds that a galactooligosaccharide raw material is injected by adopting the prior method of the patent CN105548436A, and then spectrogram comparison is carried out by injecting standard products of kestose, nystose and kestose and the galactooligosaccharide raw material (see figure 1), wherein specific peaks with retention time of 11.400min, 26.534min and retention time of 35.344min selected by the prior patent method respectively comprise kestose, nystose and kestose. Therefore, the specific peaks selected by the method disclosed in the prior patent include not only galacto-oligosaccharide but also fructo-oligosaccharide (fructo-oligosaccharide includes kestose, nystose and hexa-fructose). Since fructo-oligosaccharide, a functional saccharide, is generally added into infant milk powder, the specific quantitative peak selected when the method is used for determining the content of galacto-oligosaccharide is inaccurate, so that the measured result is also inaccurate. In addition, when the method disclosed by the patent is used for measuring the content of the galactooligosaccharide, two samples need to be processed simultaneously, one is normally processed and the other is subjected to labeling processing, so that the experimental steps are complicated, and the working efficiency is low.
Disclosure of Invention
The invention aims to solve the problems in the prior art and provides a method for accurately detecting the content of galactooligosaccharide in milk powder, wherein 3-0-beta-D-galactopyranosyl-D-glucopyranose is used as a standard substance to qualitatively identify galactooligosaccharide disaccharide in a galactooligosaccharide raw material and further determine the content of galactooligosaccharide in the milk powder, so that the problems that the prior method cannot accurately determine the galactooligosaccharide and cannot accurately determine the content of the galactooligosaccharide in the milk powder are solved. The method has the advantages of simple pretreatment, low detection limit and accurate quantification.
The technical scheme of the invention is as follows:
the invention provides a method for accurately detecting the content of galactooligosaccharide in milk powder, which comprises the following steps:
preparing a standard solution by using a 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance, weighing galactooligosaccharide raw materials, dissolving the galactooligosaccharide raw materials by using water to prepare standard curve solutions with different concentrations, carrying out ion chromatography detection on the standard solution and the standard curve solution, and using the obtained 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance peak to determine the galactooligosaccharide disaccharide peak in the galactooligosaccharide solution;
weighing and dissolving a milk powder sample, preparing a sample solution after extraction and purification, carrying out ion chromatography detection, taking the determined galacto-oligosaccharide disaccharide in the galacto-oligosaccharide raw material solution as a quantitative peak, quantifying by an external standard method, and calculating the content of the galacto-oligosaccharide in the sample solution.
Further, the chromatographic conditions were as follows: the mobile phase comprises a mobile phase A, a mobile phase B, a mobile phase C and a mobile phase D; gradient elution is adopted, the elution time is 55min, and the gradient elution program is as follows:
by optimizing the flow phase proportion and time in the gradient elution program, the positions of the chromatographic peaks in the chromatogram are adjusted, and the interference of other saccharides such as kestose and the like on 3-0-beta-D-galactopyranosyl-D-glucopyranose is ensured.
Further, the mobile phase A is water; the mobile phase B is 200mmol/L sodium hydroxide solution; the mobile phase C is a mixed solution of 150mmol/L sodium hydroxide solution and 500mmol/L sodium acetate solution; the mobile phase D is a 100mmol/L sodium acetate solution.
Furthermore, the chromatographic conditions also include that the chromatographic column adopts a Dionex CarboPac PA20 ion chromatographic separation column with the specification of 3x150mm to be used together with a Dionex CarboPac PA20 ion chromatographic protective column with the specification of 3x30 mm; the flow rate is 0.45 mL/min; the sample injection amount is 20 mu L; the column temperature is controlled at 35 ℃, and the column pressure is controlled within the range of 2500 psi-3500 psi.
And further. The preparation method of the standard solution comprises the following steps: accurately weighing a proper amount of 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance, dissolving with water, and preparing into a standard substance solution with the concentration of 100 mu g/mL.
Further, the preparation method of the standard curve solution comprises the following steps: accurately weighing galactooligosaccharide raw materials, dissolving with water and diluting to scale, preparing standard solution with concentration of 1000 μ g/mL according to purity conversion, diluting with primary water and preparing standard curve solution with concentration of 25 μ g/mL, 50 μ g/mL, 100 μ g/mL, 150 μ g/mL, 200 μ g/mL
Further, the preparation method of the sample solution comprises the following steps: accurately weighing an appropriate amount of milk powder, placing in a volumetric flask, adding water, shaking, dissolving in water bath and ultrasonic, placing to room temperature, adding acetonitrile to constant volume to scale, shaking, centrifuging the prepared sample solution, collecting supernatant, adding water for diluting by 10 times, shaking, purifying with a purifying column, and filtering with a filter membrane.
Further, the temperature of the water bath is 60 ℃; the ultrasonic time is 10 min; the rotating speed of the centrifugation is 8000r/min, and the time is 2 min; the purification column is a 1.0cc OnGuard RP column, and the filter membrane is a 0.22 μm nylon filter membrane.
The invention has the beneficial effects that:
1. the method adopts the 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance to determine the galactooligosaccharide disaccharide in the raw material, uses the galactooligosaccharide raw material to prepare a standard curve solution, and uses the 3-0-beta-D-galactopyranosyl-D-glucopyranose in the raw material as a quantitative peak to calculate the galactooligosaccharide content in the milk powder.
2. The method provided by the invention is simple to operate, the hot water is used for directly extracting the galacto-oligosaccharide in the milk powder, the galacto-oligosaccharide is directly measured, and only sample treatment is needed when the content measurement is carried out, and the treatment of a labeled sample is not needed, so that the workload is reduced, and the working efficiency is improved.
Drawings
FIG. 1 is a chromatogram obtained by stacking galacto-oligosaccharide raw material and fructo-oligosaccharide standard substance by the detection method of CN 105548436A.
Detailed Description
The technical solutions of the present invention will be described in detail and fully with reference to the following specific embodiments, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
For a further understanding of the present invention, reference will now be made in detail to the following examples.
Examples
The invention provides a method for accurately detecting galactooligosaccharides in milk powder, which comprises the following specific steps:
(1) preparation of standard solutions, standard curve solutions and sample solutions:
the preparation method of the standard solution comprises the following steps: accurately weighing a proper amount of 1.0mg of 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance in a 10mL volumetric flask, dissolving the standard substance in water and diluting the solution to a scale to prepare a standard substance solution with the concentration of 100 mu g/mL;
the preparation method of the standard curve solution comprises the following steps: accurately weighing 10.0mg (converted according to the purity of the raw material) of the galacto-oligosaccharide raw material in a 10mL volumetric flask, dissolving the galacto-oligosaccharide raw material in water and diluting the galacto-oligosaccharide raw material to a scale, and preparing standard curve solutions with the concentrations of 25 mu g/mL, 50 mu g/mL, 100 mu g/mL, 150 mu g/mL and 200 mu g/mL;
the preparation method of the sample solution comprises the following steps: accurately weighing 0.5g (accurate to 0.001g) of appropriate amount of milk powder, placing into a 10mL volumetric flask, adding 4mL of water, shaking to dissolve, placing into a water bath at 60 ℃ for ultrasonic treatment for 10min, placing to room temperature, diluting to a certain volume with acetonitrile, shaking uniformly, and preparing into a sample solution with a concentration of 50 mug/mL; placing the prepared sample solution into a centrifuge, setting the centrifuge at 8000r/min, centrifuging for 2min, taking 1mL of supernatant, adding water to dilute to 10mL, shaking uniformly, and filtering by a 1.0cc OnGuard RP column and a 0.22 μm filter membrane for later use.
(2) Chromatographic conditions are as follows:
the instrument comprises the following steps: ion chromatograph (Thermo ICS 5000)
A detector: ampere detector
A chromatographic column: ion chromatography separation column Dionex CarboPac PA 203 x150mm
Ion chromatography protective column Dionex CarboPac PA 203 x30mm
Flow rate: 0.45mL/min
Column temperature: 35 deg.C
Column pressure: 2500 psi-3500 psi
Mobile phase: the mobile phase A is water; the mobile phase B is 200mmol/L sodium hydroxide solution; the mobile phase C is a mixed solution of 150mmol/L sodium hydroxide solution and 500mmol/L sodium acetate solution; the mobile phase D is a 100mmol/L sodium acetate solution.
Gradient elution procedure:
elution time: 55min
(3) The determination method comprises the following steps:
sequentially injecting the standard solution and the standard curve solution according to the set chromatographic conditions, recording a chromatogram, and preparing a linear correlation working curve according to the peak area and concentration data of the galacto-oligosaccharide disaccharide of the standard solution, wherein the linear relation of the curve is good, and R is2And (3) more than or equal to 0.999, substituting the peak area of the galactooligosaccharide disaccharide of the sample solution for calculation to obtain the concentration of the sample solution, and calculating the content of the galactooligosaccharide in the sample according to the dilution times of the sample solution and the weighed sample mass.
The above description is only for the preferred embodiment of the present invention and should not be taken as limiting the invention, and any modifications, equivalents, improvements and the like made within the scope of the present invention should be included in the patent protection scope of the present invention.
Claims (8)
1. A method for accurately detecting the content of galactooligosaccharide in milk powder is characterized by comprising the following steps:
preparing a standard solution by using a 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance, weighing galactooligosaccharide raw materials, dissolving the galactooligosaccharide raw materials by using water to prepare standard curve solutions with different concentrations, carrying out ion chromatography detection on the standard solution and the standard curve solution, and using the chromatographic peak of the obtained 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance to determine the chromatographic peak of galactooligosaccharide disaccharide in the galactooligosaccharide solution;
weighing and dissolving a milk powder sample, preparing a sample solution after extraction and purification, carrying out ion chromatography detection, taking the determined galacto-oligosaccharide disaccharide in the galacto-oligosaccharide raw material solution as a quantitative peak, quantifying by an external standard method, and calculating the content of the galacto-oligosaccharide in the sample solution.
2. The method for accurately detecting the content of galactooligosaccharide in milk powder according to claim 1, wherein the chromatographic conditions are as follows: the mobile phase comprises a mobile phase A, a mobile phase B, a mobile phase C and a mobile phase D; gradient elution is adopted, the elution time is 55min, and the gradient elution program is as follows:
0-15.0 min, wherein the volume percent of the mobile phase A is 50%, the volume percent of the mobile phase B is 48%, and the volume percent of the mobile phase C is 0; the volume percentage of the mobile phase D was 2%;
15.0-30.0 min, the content of the mobile phase A is 50-22%, the content of the mobile phase B is 48%, and the content of the mobile phase C is 0; the mobile phase D is 2-30%;
30.0-35.0 min, wherein the mobile phase A is 22%, the mobile phase B is 48% and the mobile phase C is 0; mobile phase D is 30%;
35.0-35.1 min, the mobile phase A is 22-0 percent, the mobile phase B is 48-0 percent, and the mobile phase C is 0-100 percent; the mobile phase D is from 30 to 0;
35.1-45.0 min, wherein the mobile phase A is 0, the mobile phase B is 0, and the mobile phase C is 100%; mobile phase D is 0;
45.0-45.1 min, the mobile phase A is 0, the mobile phase B is 0-100%, and the mobile phase C is 100-0%; mobile phase D is 0;
45.1-50.0 min, the mobile phase A is 0, the mobile phase B is 100%, and the mobile phase C is 0; mobile phase D is 0;
50.0-50.1 min, the mobile phase A is 0-50%, the mobile phase B is 100-48%, and the mobile phase C is 0; the mobile phase D is 0-2%;
50.1-55.0 min, 50% of mobile phase A, 48% of mobile phase B and 0% of mobile phase C; the mobile phase D was 2%.
3. The method for accurately detecting the content of galactooligosaccharide in milk powder according to claim 2, wherein the mobile phase A is water; the mobile phase B is 200mmol/L sodium hydroxide solution; the mobile phase C is a mixed solution of 150mmol/L sodium hydroxide solution and 500mmol/L sodium acetate solution; the mobile phase D is a 100mmol/L sodium acetate solution.
4. The method for accurately detecting the content of galactooligosaccharides in the milk powder according to claim 2, wherein the chromatographic conditions further comprise that the chromatographic column is measured by using a 3x150mm ion chromatographic separation column Dionex CarboPac PA20 matched with a 3x30mm ion chromatographic protection column Dionex CarboPac PA 20; the flow rate is 0.45 mL/min; the sample injection amount is 20 mu L; the column temperature is controlled at 35 ℃, and the column pressure is controlled within the range of 2500 psi-3500 psi.
5. The method for accurately detecting the content of galactooligosaccharide in milk powder according to claim 1, wherein the standard solution is prepared by the following steps: accurately weighing a proper amount of 3-0-beta-D-galactopyranosyl-D-glucopyranose standard substance, dissolving with water, and preparing into a standard substance solution with the concentration of 100 mu g/mL.
6. The method for accurately detecting the content of galactooligosaccharide in milk powder according to claim 1, wherein the standard curve solution is prepared by the following steps: accurately weighing galactooligosaccharide raw materials, dissolving the galactooligosaccharide raw materials with water and diluting the galactooligosaccharide raw materials to a scale, preparing a standard solution with the concentration of 1000 mu g/mL according to the conversion of the purity, and preparing standard curve solutions with the concentrations of 25 mu g/mL, 50 mu g/mL, 100 mu g/mL, 150 mu g/mL and 200 mu g/mL by diluting the galactooligosaccharide raw materials with primary water.
7. The method for accurately detecting the content of galactooligosaccharide in milk powder according to claim 1, wherein the sample solution is prepared by the following steps: accurately weighing an appropriate amount of milk powder in a volumetric flask, adding water to dissolve the milk powder, diluting to a certain volume with acetonitrile to obtain a sample solution, centrifuging the prepared sample solution, taking supernatant, adding water to dilute by 10 times, shaking uniformly, purifying by a purification column, and filtering by a filter membrane.
8. The method for accurately detecting the content of galactooligosaccharide in milk powder of claim 7, wherein the purification column is a 1.0cc OnGuard RP column and the filter membrane is a 0.22 μm nylon filter membrane.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111537662A (en) * | 2020-06-01 | 2020-08-14 | 江苏中烟工业有限责任公司 | Detection method for simultaneously determining cellulose, hemicellulose and lignin in tobacco and tobacco products |
| CN112526021A (en) * | 2020-03-13 | 2021-03-19 | 内蒙古伊利实业集团股份有限公司 | Method for detecting 2' -fucosyllactose in milk |
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Application publication date: 20191231 |
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| RJ01 | Rejection of invention patent application after publication |