CN115718150A - Method for detecting content of oligoisomaltose in formula milk powder - Google Patents
Method for detecting content of oligoisomaltose in formula milk powder Download PDFInfo
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Abstract
The invention discloses a method for detecting the content of oligomeric isomaltose in formula milk powder, which comprises the following steps: s1: preparing hydrochloric acid, a sodium hydroxide solution, a PBS buffer solution and a beta-galactosidase-glucose oxidase solution; s2: respectively weighing the standard substance of each sugar, and preparing into standard working solutions with different concentrations; s3: adopting a high performance liquid chromatography to obtain peak area values of standard working solutions with different concentrations, and establishing a standard curve by taking the peak area values as vertical coordinates and the concentration values of the corresponding standard working solutions as horizontal coordinates; s4: weighing a sample to be detected, and adding a beta-galactosidase-glucose oxidase solution to prepare a liquid to be detected; s5: and obtaining the peak area value of the liquid to be detected by using a high performance liquid chromatography, searching the concentration of each sugar in the liquid to be detected in a corresponding standard curve according to the peak area value, and obtaining the content of each sugar in the sample to be detected by using a conversion formula. The invention achieves the aim of accurately detecting the content of the oligoisomaltose in the formula milk powder.
Description
Technical Field
The invention relates to the technical field of chemical analysis, in particular to a method for detecting the content of isomaltose hypgather in formula milk powder.
Background
Isomaltooligosaccharide (IMO) is a starch sugar product prepared from starch or starchy material by enzymatic conversion, refining, and concentrating. The main component is alpha-1,6-glucoside bond-bound Isomaltose (IG) 2 ) Panose (P), isomaltotriose (IG) 3 ) Etc. oligosaccharides (IG) n ). Isomaltooligosaccharides are non-digestible oligosaccharides with a number of physiological functions including: promoting the growth of probiotics, regulating intestinal flora, improving constipation, synthesizing essential vitamins for human body, promoting mineral absorption, improving immunity of organism, preventing rectal cancer and the like, so that the health-care food is widely applied to the industries of food, health-care products, medicines and the like.
At present, in China, no standard document exists in the aspect of detection of isomaltose hypgather in food, only GB/T20881-2017 isomaltose hypgather exists, the standard is only suitable for detection of pure isomaltose hypgather products (isomaltose hypgather pulp or powdered sugar), and for food with complex ingredients, no relevant national standard detection method exists. Because the main component of isomaltooligosaccharide is disaccharide which is formed by connecting two glucose molecules by alpha-1,6 glycosidic bonds, if the sample to be detected contains disaccharide except isomaltooligosaccharide, such as lactose, the peak time of the disaccharide is overlapped with that of the isomaltooligosaccharide, so that the qualitative and quantitative determination of the isomaltooligosaccharide cannot be carried out by using the high performance liquid chromatography, and the corresponding high performance liquid chromatogram of the isomaltooligosaccharide and the lactose is shown in figures 1 and 2, and the peak time of the isomaltooligosaccharide and the lactose is consistent, so that the accurate qualitative and quantitative determination cannot be carried out.
Most of the formula milk powder and related products contain lactose, so the prior detection method cannot carry out accurate qualitative and quantitative determination on the formula milk powder.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for detecting the content of isomaltose hypgather in a formula milk powder, which is used for solving the technical problem that the existing detection method cannot accurately determine qualitatively and quantitatively aiming at the formula milk powder, so that the aim of accurately detecting the content of isomaltose hypgather in the formula milk powder is fulfilled.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a method for detecting the content of oligoisomaltose in formula milk powder comprises the following steps:
step S1: preparing a hydrochloric acid solution, a sodium hydroxide solution, a PBS buffer solution and a beta-galactosidase-glucose oxidase solution;
step S2: respectively weighing an isomaltose standard substance, a panose standard substance and an isomaltotriose standard substance to prepare an isomaltose standard working solution, a panose standard working solution and an isomaltotriose standard working solution with different concentrations;
and step S3: adopting a high performance liquid chromatography to obtain peak area values of isomaltose, panose and isomaltotriose in standard working solutions with different concentrations in a chromatogram according to certain chromatographic conditions, and establishing an isomaltose standard curve, a panose standard curve and an isomaltotriose standard curve by taking the peak area values as ordinate and the concentration values of the corresponding standard working solutions as abscissa;
and step S4: weighing a sample to be detected, and adding the beta-galactosidase-glucose oxidase solution to prepare a liquid to be detected;
step S5: and obtaining peak area values of isomaltose, panose and isomaltotriose in the liquid to be detected in a chromatogram by using a high performance liquid chromatography, searching concentrations of the isomaltose, panose and isomaltotriose in the liquid to be detected in a corresponding standard curve according to the peak area values, and obtaining the contents of the isomaltose, panose and isomaltotriose in the sample to be detected by using a conversion formula.
As a preferred embodiment of the present invention, in step S4, the process of preparing the solution to be measured is:
precisely weighing 2-5 g of a sample to be measured;
adding 20ml of water with the temperature of 45-50 ℃ into the sample to be detected, uniformly mixing, standing for 10min, and cooling to room temperature;
adjusting the pH value to 1.7 +/-0.1 by using the hydrochloric acid solution, and standing for 1min;
adjusting the pH value to 4.5 +/-0.1 by using the sodium hydroxide solution;
adding 5mL of the beta-galactosidase-glucose oxidase solution, shaking uniformly, placing in a constant-temperature water bath kettle at 60 ℃ for shaking for 60min, rapidly cooling, adding water to a constant volume of 50mL, shaking uniformly, and filtering with a membrane to a sample bottle to serve as a liquid to be detected.
As a preferred embodiment of the present invention, the chromatographic conditions comprise: amino chromatographic column, mobile phase, detector temperature, column temperature, flow rate, and sample size;
wherein the amino chromatographic column has a column length of 250mm, an inner diameter of 4.6mm and a membrane thickness of 5 μm; the mobile phase consists of 73% acetonitrile and 27% water by weight percentage; the temperature of the detector is 35 ℃, the column temperature is 35 ℃, the flow rate is 1.0mL/min, and the sample injection amount is 10 mu L.
As a preferred embodiment of the present invention, the concentration of the hydrochloric acid solution is 5.0mol/L or 0.1mol/L.
In a preferred embodiment of the invention, the concentration of the sodium hydroxide solution is 5.0mol/L and/or 0.1mol/L.
In a preferred embodiment of the present invention, the PBS buffer has a pH of 6.0, and is prepared by the following steps: 1.1g of potassium dihydrogenphosphate and 0.3g of dipotassium hydrogenphosphate were weighed and made up to 50mL with water.
As a preferred embodiment of the present invention, the preparation process of the β -galactosidase-glucose oxidase solution is: weighing 0.8g of beta-galactosidase and 0.5g of glucose oxidase in the same colorimetric tube, dissolving the beta-galactosidase and the glucose oxidase in 10mL of PBS buffer solution, and uniformly mixing to prepare the product for use.
In a preferred embodiment of the invention, the activity of the beta-galactosidase is more than or equal to 240U/mg, and the activity of the glucose oxidase is more than or equal to 15U/mg.
In a preferred embodiment of the present invention, the isomaltose standard, panose standard and isomaltotriose standard all have a purity of greater than 95%.
In a preferred embodiment of the present invention, the water in the hydrochloric acid solution and the water in the sodium hydroxide solution are all primary water as defined in GB/T6682.
Compared with the prior art, the invention has the beneficial effects that:
(1) According to the detection method provided by the invention, beta-galactosidase is used for carrying out enzymolysis on lactose in the formula milk powder, so that the interference of lactose is removed, and the high performance liquid chromatography is used for realizing accurate qualitative and quantitative detection on isomaltooligosaccharide in the formula milk powder;
(2) The invention establishes a unified sample pretreatment method for the formula milk powder raw materials and the products thereof, provides technical support for the detection of the content of isomaltooligosaccharide in the formula milk powder, fills the blank of the existing analysis technology, and assists in the development and supervision of the industry.
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
Drawings
FIG. 1 is a chromatogram of a working isomaltose solution according to the present invention;
FIG. 2-is a chromatogram of a lactose working solution according to the invention;
FIG. 3-is a chromatogram of standard working solution concentration 5 of an embodiment of the present invention;
FIG. 4-is a chromatogram of a first test solution of example 1 of the present invention;
FIG. 5-is a chromatogram of a second test solution according to example 2 of the present invention;
FIG. 6-is a chromatogram of a third test solution according to example 3 of the present invention;
FIG. 7-is a chromatogram of a test solution of a formula milk powder without isomaltooligosaccharides according to a comparative example of the present invention;
FIG. 8-is a chromatogram of a test solution of a formula containing isomaltooligosaccharide and added with a β -galactosidase-glucose oxidase solution according to a comparative example of the present invention.
Detailed Description
The method for detecting the content of the oligoisomaltose in the formula milk powder provided by the invention comprises the following steps:
step S1: preparing hydrochloric acid solution, sodium hydroxide solution, PBS buffer solution and beta-galactosidase-glucose oxidase solution.
Further, the concentration of the hydrochloric acid solution comprises 5.0mol/L and 0.1mol/L, and the preparation process specifically comprises the following steps:
measuring 45mL of hydrochloric acid (analytically pure), diluting with primary water specified in GB/T6682, and diluting to 100mL to obtain a hydrochloric acid solution with the concentration of 5.0 mol/L;
9mL of hydrochloric acid (analytically pure) is measured, diluted by primary water specified in GB/T6682 and metered to 1000mL to obtain a hydrochloric acid solution with the concentration of 0.1mol/L.
One high-concentration hydrochloric acid solution (5.0 mol/L), one low-concentration hydrochloric acid solution (0.1 mol/L) and 2 hydrochloric acid solutions are used for adjusting the pH value of the solution.
Further, the concentration of the sodium hydroxide solution comprises 5.0mol/L and 0.1mol/L, and the preparation process is as follows:
weighing 20g of sodium hydroxide (analytically pure), adding 50mL of primary water specified by GB/T6682, cooling, and then using the primary water specified by GB/T6682 to fix the volume to 100mL to obtain a sodium hydroxide solution with the concentration of 5.0 mol/L;
0.4g of sodium hydroxide (analytically pure) is weighed, 50mL of primary water specified in GB/T6682 is added, after cooling, the primary water specified in GB/T6682 is used for fixing the volume to 100mL, and the sodium hydroxide solution with the concentration of 0.1mol/L is obtained.
A high concentration sodium hydroxide solution (5.0 mol/L), a low concentration sodium hydroxide solution (0.1 mol/L), 2 concentrations of sodium hydroxide solution can be used to adjust the pH of the solution.
Further, the pH value of the PBS buffer solution is 6.0, and the preparation process comprises the following steps: 1.1g of potassium dihydrogen phosphate (analytically pure) and 0.3g of dipotassium hydrogen phosphate (analytically pure) were weighed out and made up to 50mL with primary water as specified in GB/T6682.
Further, the preparation process of the beta-galactosidase-glucose oxidase solution is as follows: weighing 0.8g of beta-galactosidase and 0.5g of glucose oxidase in the same colorimetric tube, dissolving the beta-galactosidase and the glucose oxidase in 10mL of PBS buffer solution, and mixing the beta-galactosidase and the glucose oxidase uniformly to prepare the product for use.
Furthermore, the activity of the beta-galactosidase is more than or equal to 240U/mg, and the activity of the glucose oxidase is more than or equal to 15U/mg.
Step S2: respectively weighing an isomaltose standard substance, a panose standard substance and an isomaltotriose standard substance, and preparing into isomaltose standard working solution, panose standard working solution and isomaltotriose standard working solution with different concentrations.
Furthermore, the purity of the isomaltose standard substance, panose standard substance and isomaltotriose standard substance is more than 95 percent, or the standard substance is certified by the state and awarded with a standard substance certificate.
And step S3: adopting a high performance liquid chromatography, obtaining peak area values of isomaltose, panose and isomaltotriose in standard working solutions with different concentrations in a chromatogram according to certain chromatographic conditions, and establishing an isomaltose standard curve, a panose standard curve and an isomaltotriose standard curve by taking the peak area values as ordinate and the concentration values of the corresponding standard working solutions as abscissa.
Further, the high performance liquid chromatography employs an apparatus including: high performance liquid chromatograph, mobile phase vacuum filtration degasser, microporous filter membrane and analytical balance.
Further, the high performance liquid chromatograph was equipped with a differential refractive detector and a column thermostat system, and the accuracy of the analytical balance was 0.1mg.
Further, the chromatographic conditions include: amino chromatography column, mobile phase, detector temperature, column temperature, flow rate, and sample size.
Further, the amino column had a column length of 250mm, an inner diameter of 4.6mm and a film thickness of 5 μm, or a column having equivalent performance.
Further, the mobile phase consisted of 73% acetonitrile and 27% water, the acetonitrile being chromatographically pure.
Further, the temperature of the detector was 35 ℃, the column temperature was 35 ℃, the flow rate was 1.0mL/min, and the amount of sample was 10. Mu.L.
And step S4: weighing a sample to be detected, and adding the beta-galactosidase-glucose oxidase solution to prepare a liquid to be detected.
Further, the preparation process of the liquid to be detected is as follows:
accurately weighing 2-5 g (accurate to 0.0001g, the content of various sugars is in the linear range of a standard curve, otherwise, the sample weighing amount can be properly increased or decreased or dilution treatment is carried out) of a sample, adding 20mL of water with the temperature of 45-50 ℃ (primary water specified by GB/T6682) into a 100mL beaker, uniformly mixing, standing for 10min, and cooling to room temperature;
adjusting the pH value of the solution to 1.7 +/-0.1 by using a hydrochloric acid solution, standing for 1min, and adjusting the pH value to 4.5 +/-0.1 by using a sodium hydroxide solution;
putting the beaker with the sample into an ultrasonic oscillator for ultrasonic treatment for 10min, transferring the solution in the beaker into a 50mL centrifuge tube, washing the beaker with water (primary water specified by GB/T6682), combining the washing solutions into the 50mL centrifuge tube, adding 5mL beta-galactosidase-glucose oxidase, shaking up, placing the beaker in a constant temperature water bath kettle at 60 ℃ for shaking up for 60min, rapidly cooling, transferring into a 50mL volumetric flask, adding water (primary water specified by GB/T6682) to reach 50mL volume, shaking up, and filtering the solution into a sample bottle to serve as a solution to be measured.
Step S5: and obtaining peak area values of isomaltose, panose and isomaltotriose in the liquid to be detected in a chromatogram by using a high performance liquid chromatography, searching concentrations of the isomaltose, panose and isomaltotriose in the liquid to be detected in a corresponding standard curve according to the peak area values, and obtaining the contents of the isomaltose, panose and isomaltotriose in the sample to be detected by using a conversion formula.
Furthermore, the content of each sugar in the sample to be tested is calculated according to the following conversion formula:
wherein X is the content of each sugar (isomaltose, panose and isomaltotriose) in the sample to be tested, and the unit is gram per hundred grams (g/100 g); c is the concentration of each sugar in the solution to be measured in terms of mL per mL (mg/mL); v is the constant volume in milliliters (mL); d is the dilution factor, m is the mass of the sample, and the unit is gram (g); 100 is a conversion factor, and 1000 is a conversion factor.
The principle on which the invention is based is as follows:
lactose is a disaccharide with D-glucose and D-galactose bonded by a beta-1,4 bond, also known as 1,4-galactoside glucose. Lactase is a digestive enzyme in human body, is the most important digestive enzyme in infants and young children taking breast milk/milk powder as a main nutrient source, is widely present in various animals, plants and microorganisms, and comprises beta-galactosidase, acid beta-galactosidase and iso-beta-galactosidase, and the latter two enzymes rarely play a role in lactose. The main function of lactase is to digest lactose, hydrolyse lactose into galactose and glucose, which can be absorbed by the human body. Therefore, the invention utilizes the beta-galactosidase to directionally enzymolyze the lactose in the formula milk powder to remove the interference of the lactose, and then is matched with other technical means such as high performance liquid chromatography and the like, thereby realizing the accurate determination of the content of the oligoisomaltose in the formula milk powder.
The following examples are further illustrative of the present invention, but the scope of the present invention is not limited thereto.
Preparing standard solution
Respectively weighing appropriate amounts of isomaltose standard substance, panose standard substance and isomaltotriose standard substance by using an analytical balance (with the precision of 0.1 mg) to prepare six standard working solutions with different concentrations, which are specifically shown in table 1:
TABLE 1 Standard working fluid concentration Table
Detecting the six standard working solutions with different concentrations by using a high performance liquid chromatograph, wherein the chromatographic conditions are as follows:
a) Amino chromatographic column: the length of the column is 250mm, the inner diameter is 4.6mm, and the thickness of the membrane is 5 μm;
b) Mobile phase: acetonitrile: water =73:27 (in weight ratios);
c) Detector temperature: 35 ℃;
d) Column temperature: 35 ℃;
e) Flow rate: 1.0mL/min;
f) Sample introduction amount: 10 μ L.
The test results are specifically shown in table 2:
TABLE 2 Standard working solution Peak area Table
| Peak area value of isomaltose | Peak area value of panose | Peak area value of isomaltotriose | |
| Concentration 1 | 25103.402768 | 12535.318406 | 5623.319866 |
| |
153733.182378 | 71193.400036 | 30598.721591 |
| Concentration 3 | 230094.241682 | 103106.789602 | 49228.507721 |
| Concentration 4 | 316814.023777 | 145410.629680 | 69096.378884 |
| Concentration 5 | 401031.891126 | 186901.691051 | 84285.739686 |
| Concentration 6 | 602893.581290 | 280095.089783 | 127974.460501 |
The chromatogram of the standard working solution having a concentration of 5 is shown in FIG. 3.
Taking the concentration values of the standard working solution with the six concentrations as a horizontal coordinate and the peak area value as a vertical coordinate, establishing a linear equation and obtaining corresponding correlation coefficients, as shown in table 3:
TABLE 3 Linear equation, correlation coefficient of isomaltose, panose and isomaltotriose
| Compound (I) | Linear equation of equations | Correlation coefficient |
| Isomaltose | Y＝58945X-14098 | 0.999900 |
| Pan candy | Y＝64240X-7585.2 | 0.999700 |
| Isomaltotriose | Y＝39002X-3251.5 | 0.999650 |
As can be seen from the correlation coefficients in table 3, isomaltose, isomaltotriose and panose are in good linear relationship.
Example 1
Weighing 2g of formula milk powder in a 100ml beaker, adding 20ml of 45 ℃ water, uniformly mixing, standing for 10min, and cooling to room temperature; adjusting pH to 1.6 with hydrochloric acid solution, and standing for 1min; adjusting the pH value to 4.4 by using a sodium hydroxide solution; putting the beaker into an ultrasonic oscillator for 10min, transferring the solution in the beaker into a 50mL centrifuge tube, washing the beaker with water (primary water specified by GB/T6682), combining the washing solutions into the 50mL centrifuge tube, adding 5mL beta-galactosidase-glucose oxidase, shaking up, placing the centrifuge tube in a constant temperature water bath kettle at 60 ℃ for 60min, quickly cooling, transferring into a 50mL volumetric flask, adding water (primary water specified by GB/T6682) to a constant volume of 50mL, shaking up, and filtering the solution into a sample bottle by using a filter membrane to serve as a first solution to be detected. And (3) carrying out chromatographic analysis on the first solution to be detected by using a high performance liquid chromatograph to obtain a chromatogram of the first solution to be detected, as shown in fig. 4.
Example 2
Weighing 3g of formula milk powder in a 100ml beaker, adding 20ml of 47 ℃ water, uniformly mixing, standing for 10min, and cooling to room temperature; adjusting pH to 1.7 with hydrochloric acid solution, and standing for 1min; adjusting the pH value to 4.5 by using a sodium hydroxide solution; putting the beaker into an ultrasonic oscillator for 10min, transferring the solution in the beaker into a 50mL centrifuge tube, washing the beaker with water (primary water specified by GB/T6682), combining the washing solutions into the 50mL centrifuge tube, adding 5mL beta-galactosidase-glucose oxidase, shaking up, placing the centrifuge tube in a constant temperature water bath kettle at 60 ℃ for 60min, quickly cooling, transferring into a 50mL volumetric flask, adding water (primary water specified by GB/T6682) to a constant volume of 50mL, shaking up, and filtering the solution by a filter membrane into a sample bottle to serve as a second solution to be detected. And (3) performing chromatographic analysis on the second liquid to be detected by using a high performance liquid chromatograph to obtain a chromatogram of the second liquid to be detected, as shown in fig. 5.
Example 3
Weighing 5g of formula milk powder in a 100ml beaker, adding 20ml of 50 ℃ water, uniformly mixing, standing for 10min, and cooling to room temperature; adjusting pH to 1.8 with hydrochloric acid solution, and standing for 1min; adjusting the pH value to 4.6 by using a sodium hydroxide solution; putting the beaker into an ultrasonic oscillator for 10min, transferring the solution in the beaker into a 50mL centrifuge tube, washing the beaker with water (primary water specified by GB/T6682), combining the washing solutions into the 50mL centrifuge tube, adding 5mL beta-galactosidase-glucose oxidase, shaking up, placing the centrifuge tube in a constant temperature water bath kettle at 60 ℃ for 60min, quickly cooling, transferring into a 50mL volumetric flask, adding water (primary water specified by GB/T6682) to a constant volume of 50mL, shaking up, and filtering the solution by a filter membrane into a sample bottle to serve as a third solution to be detected. And (3) performing chromatographic analysis on the third liquid to be detected by using a high performance liquid chromatograph to obtain a chromatogram of the third liquid to be detected, as shown in fig. 6.
Comparative example
To verify that the beta-galactosidase-glucose oxidase solution in the claims can be added in an amount to completely remove interference from lactose, a milk formula without isomalto-oligosaccharide was taken and added with the beta-galactosidase-glucose oxidase solution, otherwise in accordance with example 1, and the chromatogram obtained is shown in fig. 7. As can be seen by comparing fig. 2 and fig. 7, at the time of lactose peak, fig. 7 has no chromatographic peak, indicating that lactose in the test solution has been completely removed.
The chromatographic chart of the prepared milk powder containing isomaltooligosaccharide, which is not treated by the beta-galactosidase-glucose oxidase solution, is shown in fig. 8, and is otherwise identical to that of example 1. As can be seen from the graphs in FIGS. 4 and 8, the peak appearance sequence is isomaltose, panose and isomaltotriose, and the peak area value of isomaltose in FIG. 8 is obviously smaller than that of isomaltose in FIG. 4 by comparing the contents, which shows that the beta-galactosidase-glucose oxidase can effectively remove lactose in the formula milk powder.
The peak area values of examples 1 to 3 can be obtained from fig. 4 to 6, and are specifically shown in table 4:
table 4 peak area values of examples 1 to 3
| Peak area of isomaltose | Peak area value of panose | Peak area value of isomaltotriose | |
| Example 1 | 46615.35 | 23378.48 | 10984.23 |
| Example 2 | 77856.21 | 38731.84 | 17848.58 |
| Example 3 | 145380 | 70161 | 35419 |
The concentrations of isomaltose peak, panose and isomaltotriose in the solutions to be tested of examples 1 to 3 were obtained according to the linear equation of isomaltose, panose and isomaltotriose, and are specifically shown in table 5:
TABLE 5 concentration of isomaltose, panose, isomaltotriose in examples 1 to 3
The contents of isomaltose, panose and isomaltotriose in the milk powder formulations of examples 1 to 3 were obtained by converting the concentrations of isomaltose, panose and isomaltotriose according to the above conversion formula, as shown in table 6:
TABLE 6 contents of isomaltose, panose and isomaltotriose in the milk formula of examples 1 to 3
| Isomaltose (g/100 g) | Panose (g/100 g) | Isomaltotriose (g/100 g) | |
| Example 1 (formula milk powder) | 2.7 | 1.2 | 0.9 |
| Example 2 (formula milk powder) | 2.6 | 1.2 | 0.9 |
| Example 3 (formula milk powder) | 2.6 | 1.2 | 0.9 |
In order to verify the feasibility and the accuracy of the invention, 3 concentration gradients of standard solutions were added based on example 3, 6 replicates were performed for each concentration, and the standard recovery rate was performed according to the above method, with the results shown in table 7 and the detection limit in table 8:
TABLE 7 recovery with addition of standard
TABLE 8 detection limits of the methods
| Isomaltose | Pan candy | Isomaltotriose | |
| Detection limit (g/100 g) | 0.6 | 0.5 | 0.5 |
Note: the method detection limit is the minimum concentration of the analyte that can be detected by a given analytical method under specific conditions with a reasonable confidence level; the invention compares the measurement signals of the known low-concentration analyte sample and the blank sample to determine the minimum concentration which is reliably detected, and the selected signal-to-noise ratio is 3:1.
As can be seen from tables 7 and 8, the recovery rate of the target compound in the invention reaches 98.3-100.2% by adding the standard, and the detection limit of the method reaches 0.5-0.6g/100g, which shows that the detection method provided by the invention has good recovery rate and higher accuracy.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.
Claims (10)
1. The method for detecting the content of oligoisomaltose in the formula milk powder is characterized by comprising the following steps:
step S1: preparing a hydrochloric acid solution, a sodium hydroxide solution, a PBS buffer solution and a beta-galactosidase-glucose oxidase solution;
step S2: respectively weighing an isomaltose standard substance, a panose standard substance and an isomaltotriose standard substance, and preparing into an isomaltose standard working solution, a panose standard working solution and an isomaltotriose standard working solution with different concentrations;
and step S3: adopting a high performance liquid chromatography to obtain peak area values of isomaltose, panose and isomaltotriose in standard working solutions with different concentrations in a chromatogram according to certain chromatographic conditions, and establishing an isomaltose standard curve, a panose standard curve and an isomaltotriose standard curve by taking the peak area values as ordinate and the concentration values of the corresponding standard working solutions as abscissa;
and step S4: weighing a sample to be detected, and adding the beta-galactosidase-glucose oxidase solution to prepare a liquid to be detected;
step S5: and obtaining peak area values of isomaltose, panose and isomaltotriose in the liquid to be detected in a chromatogram by using a high performance liquid chromatography, searching concentrations of the isomaltose, panose and isomaltotriose in the liquid to be detected in a corresponding standard curve according to the peak area values, and obtaining the contents of the isomaltose, panose and isomaltotriose in the sample to be detected by using a conversion formula.
2. The method for detecting the content of isomaltose hypgather in the formula milk powder according to claim 1, wherein in the step S4, the preparation process of the solution to be detected is as follows:
precisely weighing 2-5 g of a sample to be measured;
adding 20ml of water with the temperature of 45-50 ℃ into the sample to be detected, uniformly mixing, standing for 10min, and cooling to room temperature;
adjusting the pH value to 1.7 +/-0.1 by using the hydrochloric acid solution, and standing for 1min;
adjusting the pH value to 4.5 +/-0.1 by using the sodium hydroxide solution;
adding 5mL of the beta-galactosidase-glucose oxidase solution, shaking uniformly, placing in a constant-temperature water bath kettle at 60 ℃ for shaking for 60min, rapidly cooling, adding water to a constant volume of 50mL, shaking uniformly, and filtering with a membrane to a sample bottle to serve as a liquid to be detected.
3. The method for detecting the content of oligomeric isomaltose in the formula milk powder of claim 1, wherein the chromatographic conditions comprise: amino chromatographic column, mobile phase, detector temperature, column temperature, flow rate, and sample size;
wherein the length of the amino chromatographic column is 250mm, the inner diameter is 4.6mm, and the film thickness is 5 μm; the mobile phase consists of 73% acetonitrile and 27% water by weight percentage; the detector temperature is 35 ℃, the column temperature is 35 ℃, the flow rate is 1.0mL/min, and the sample injection amount is 10 muL.
4. The method for detecting the content of oligomeric isomaltose in the formula milk powder of claim 1, wherein the concentration of the hydrochloric acid solution is 5.0mol/L or 0.1mol/L.
5. The method for detecting the content of oligomeric isomaltose in the formula milk powder of claim 1, wherein the concentration of the sodium hydroxide solution is 5.0mol/L and/or 0.1mol/L.
6. The method for detecting the content of oligomeric isomaltose in the formula milk powder of claim 1, wherein the pH value of the PBS buffer is 6.0, and the preparation process comprises the following steps: 1.1g of potassium dihydrogenphosphate and 0.3g of dipotassium hydrogenphosphate were weighed and made up to 50mL with water.
7. The method for detecting the content of isomaltose hypgather in the formula milk powder according to claim 1, wherein the preparation process of the beta-galactosidase-glucose oxidase solution is as follows: weighing 0.8g of beta-galactosidase and 0.5g of glucose oxidase in the same colorimetric tube, dissolving the beta-galactosidase and the glucose oxidase in 10mL of PBS buffer solution, and uniformly mixing to prepare the product for use.
8. The method for detecting the content of oligomeric isomaltose in the formula milk powder according to claim 7, wherein the activity of beta-galactosidase is not less than 240U/mg, and the activity of glucose oxidase is not less than 15U/mg.
9. The method for detecting the content of oligomeric isomaltose in the formula milk powder of claim 1, wherein the purity of the isomaltose standard substance, the panose standard substance and the isomaltotriose standard substance is greater than 95%.
10. The method for detecting the content of isomaltooligosaccharide in the formula milk powder of any one of claims 2-6, wherein the water, the water in the hydrochloric acid solution and the water in the sodium hydroxide solution are all primary water specified in GB/T6682.
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| CN116735772B (en) * | 2023-08-16 | 2023-10-24 | 天津医科大学总医院 | Adenosine content detection method and system for urine analysis |
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