A method for detecting the content of isomaltooligosaccharide in formula milk powder

Abstract

The invention discloses a detection method for oligomeric isomaltosaccharide content in a formula milk powder, including: S1: preparing hydrochloric acid, sodium hydroxide solution, PBS buffer and beta-galactosidase-glucose oxidase solution; S2: weighing the standard of each sugar respectively, configuring it into standard working solutions of different concentrations; S3: using high performance liquid chromatography, obtaining the peak area values ​​of standard working solutions of different concentrations, and using the peak area value as the ordinate, using the concentration value of the corresponding standard working solution as the abscissa, establishing a standard curve; S4: weighing the sample to be tested, adding beta-galactosidase-glucose oxidase solution to configure it into liquid to be tested; S5: obtaining the peak area value of liquid to be tested by high performance liquid chromatography, checking the concentration of each sugar in the liquid to be tested in the corresponding standard curve according to the peak area value, and then obtaining the content of each sugar in the sample to be tested by conversion formula. The present invention achieves the purpose of accurately detecting the content of oligomeric isomaltosaccharide in formula milk powder.

Description

Technical Field

[0001] The invention relates to the technical field of chemical analysis, and in particular to a method for detecting the content of isomaltooligosaccharide in formula milk powder. Background Art

[0002] Isomaltooligosaccharide (IMO) is a starch sugar product made from starch or starchy substances through enzymatic conversion, refining, and concentration. The main components are oligosaccharides (IG2), panose (P), isomaltotriose (IG3), etc., which are linked to α-1,6-glycosidic bonds. n Isomaltooligosaccharide is a non-digestible oligosaccharide with many physiological functions, including promoting the growth of probiotics, regulating intestinal flora, improving constipation, synthesizing essential vitamins for the human body, promoting mineral absorption, improving the body's immunity, and preventing colorectal cancer. Therefore, it is widely used in food, health products, medicine and other industries.

[0003] At present, my country has no standard document for the detection of oligosaccharides in food, only GB / T 20881-2017 "Oligosaccharides", and this standard is only applicable to the detection of pure oligosaccharides (oligosaccharides syrup or sugar powder). For foods with more complex ingredients, there is no relevant national standard detection method. Because the main component of oligosaccharides is isomaltose, which is a disaccharide composed of two glucose molecules connected by an α-1,6 glycosidic bond, if the sample to be tested contains disaccharides other than isomaltose, such as lactose, it is impossible to use high-performance liquid chromatography to qualitatively and quantitatively determine oligosaccharides because its peak time overlaps with isomaltose. The corresponding high-performance liquid chromatograms of isomaltose and lactose are as follows: Figure 1 , Figure 2 As shown in the figure, the peak times of isomaltose and lactose are consistent, and they cannot be accurately identified and quantified.

[0004] Most formula milk powder and its related products contain lactose, so existing detection methods are unable to accurately determine the qualitative and quantitative content of formula milk powder. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention provides a method for detecting the content of isomaltooligosaccharide in formula milk powder, which is used to solve the technical problem that the existing detection method cannot perform accurate qualitative and quantitative determination on formula milk powder, thereby achieving the purpose of accurately detecting the content of isomaltooligosaccharide in formula milk powder.

[0006] To solve the above problems, the technical solution adopted by the present invention is as follows:

[0007] A method for detecting the content of isomaltooligosaccharide in formula milk powder comprises the following steps:

[0008] Step S1: preparing hydrochloric acid solution, sodium hydroxide solution, PBS buffer solution and β-galactosidase-glucose oxidase solution;

[0009] Step S2: Weighing the isomaltose standard, the panose standard and the isomaltotriose standard respectively, and preparing isomaltose standard working solutions, panose standard working solutions and isomaltotriose standard working solutions of different concentrations;

[0010] Step S3: using high performance liquid chromatography and according to certain chromatographic conditions, obtaining the peak area values ​​of isomaltose, panose and isomaltotriose in the standard working solution with different concentrations in the chromatogram, and establishing an isomaltose standard curve, a panose standard curve and an isomaltotriose standard curve with the peak area value as the ordinate and the concentration value of the corresponding standard working solution as the abscissa;

[0011] Step S4: weighing a sample to be tested, and adding the β-galactosidase-glucose oxidase solution to prepare a test solution;

[0012] Step S5: obtaining the peak area values ​​of isomaltose, panose and isomaltotriose in the test solution in the chromatogram by high performance liquid chromatography, and obtaining the concentrations of isomaltose, panose and isomaltotriose in the test solution in the corresponding standard curve according to the peak area values, and then obtaining the content of isomaltose, panose and isomaltotriose in the test sample by conversion formula.

[0013] As a preferred embodiment of the present invention, in step S4, the configuration process of the test liquid is:

[0014] Accurately weigh 2-5g of the sample to be tested;

[0015] Add 20 ml of water at 45°C to 50°C to the sample to be tested, mix well, let stand for 10 minutes, and cool to room temperature;

[0016] The pH value was adjusted to 1.7±0.1 using the hydrochloric acid solution and allowed to stand for 1 min;

[0017] Using the sodium hydroxide solution, the pH value is adjusted to 4.5±0.1;

[0018] Add 5 mL of the β-galactosidase-glucose oxidase solution, shake well, place in a 60°C constant temperature water bath and oscillate for 60 min, cool quickly, dilute to 50 mL with water, shake well, filter through a membrane and transfer to a sample bottle as the test solution.

[0019] As a preferred embodiment of the present invention, the chromatographic conditions include: amino chromatographic column, mobile phase, detector temperature, column temperature, flow rate and injection volume;

[0020] Among them, the column length of the amino chromatographic column is 250mm, the inner diameter is 4.6mm, and the film thickness is 5μm; in terms of weight percentage, the mobile phase consists of 73% acetonitrile and 27% water; the detector temperature is 35°C, the column temperature is 35°C, the flow rate is 1.0mL / min, and the injection volume is 10μL.

[0021] As a preferred embodiment of the present invention, the concentration of the hydrochloric acid solution is 5.0 mol / L or 0.1 mol / L.

[0022] As a preferred embodiment of the present invention, the concentration of the sodium hydroxide solution is 5.0 mol / L and / or 0.1 mol / L.

[0023] As a preferred embodiment of the present invention, the pH value of the PBS buffer solution is 6.0, and the preparation process is as follows: weigh 1.1 g of potassium dihydrogen phosphate and 0.3 g of dipotassium hydrogen phosphate, and dilute to 50 mL with water.

[0024] As a preferred embodiment of the present invention, the preparation process of the β-galactosidase-glucose oxidase solution is: weigh 0.8g of β-galactosidase and 0.5g of glucose oxidase in the same colorimetric tube, dissolve them with 10mL of the PBS buffer, mix them evenly, and use them immediately after preparation.

[0025] As a preferred embodiment of the present invention, the activity of the β-galactosidase is ≥240 U / mg, and the activity of the glucose oxidase is ≥15 U / mg.

[0026] As a preferred embodiment of the present invention, the purity of the isomaltose standard, the panose standard and the isomaltotriose standard are all greater than 95%.

[0027] As a preferred embodiment of the present invention, the water, the water in the hydrochloric acid solution and the water in the sodium hydroxide solution are all first-grade water as specified in GB / T 6682.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) The detection method provided by the present invention utilizes β-galactosidase to enzymatically hydrolyze lactose in formula milk powder, thereby removing the interference of lactose, and utilizes high performance liquid chromatography to achieve accurate qualitative and quantitative detection of oligosaccharides in formula milk powder;

[0030] (2) The present invention establishes a unified sample pretreatment method for formula milk powder raw materials and its products, provides technical support for the detection of oligosaccharide content in formula milk powder, fills the gap in existing analytical technology, and promotes industry development and supervision.

[0031] The present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 - is a chromatogram of the isomaltose working solution of the present invention;

[0033] Figure 2 - is a chromatogram of the lactose working solution of the present invention;

[0034] Figure 3 - is a chromatogram of the standard working solution concentration 5 of an embodiment of the present invention;

[0035] Figure 4 - is a chromatogram of the first test liquid in Example 1 of the present invention;

[0036] Figure 5 - is a chromatogram of the second test liquid in Example 2 of the present invention;

[0037] Figure 6 - is a chromatogram of the third test liquid of Example 3 of the present invention;

[0038] Figure 7 - is a chromatogram of the test liquid of the formula milk powder not containing isomaltooligosaccharide according to the comparative example of the present invention;

[0039] Figure 8 - is a chromatogram of the test solution of formula milk powder containing isomaltooligosaccharide and added with β-galactosidase-glucose oxidase solution in the comparative example of the present invention. DETAILED DESCRIPTION

[0040] The method for detecting the content of isomaltooligosaccharide in formula milk powder provided by the present invention comprises the following steps:

[0041] Step S1: prepare hydrochloric acid solution, sodium hydroxide solution, PBS buffer solution and β-galactosidase-glucose oxidase solution.

[0042] Furthermore, the concentration of the hydrochloric acid solution includes 5.0 mol / L and 0.1 mol / L, and the preparation process is as follows:

[0043] Measure 45 mL of hydrochloric acid (analytical grade), dilute it to 100 mL with first-grade water specified in GB / T 6682, and obtain a hydrochloric acid solution with a concentration of 5.0 mol / L;

[0044] Measure 9 mL of hydrochloric acid (analytical grade), dilute it with first-grade water specified in GB / T 6682 and make up to 1000 mL to obtain a hydrochloric acid solution with a concentration of 0.1 mol / L.

[0045] A high concentration hydrochloric acid solution (5.0 mol / L) and a low concentration hydrochloric acid solution (0.1 mol / L) are used to adjust the pH value of the solution.

[0046] Further, the concentration of the sodium hydroxide solution includes 5.0 mol / L and 0.1 mol / L, and the preparation process is as follows:

[0047] Weigh 20 g of sodium hydroxide (analytical grade), add 50 ml of first-grade water specified in GB / T 6682, cool, and dilute to 100 mL with first-grade water specified in GB / T 6682 to obtain a sodium hydroxide solution with a concentration of 5.0 mol / L;

[0048] Weigh 0.4 g of sodium hydroxide (analytical grade), add 50 ml of first-grade water specified in GB / T 6682, cool, and dilute to 100 mL with first-grade water specified in GB / T 6682 to obtain a sodium hydroxide solution with a concentration of 0.1 mol / L.

[0049] A high concentration sodium hydroxide solution (5.0 mol / L) and a low concentration sodium hydroxide solution (0.1 mol / L) can be used to adjust the pH value of the solution.

[0050] Furthermore, the pH value of the PBS buffer solution is 6.0, and the preparation process is as follows: weigh 1.1 g of potassium dihydrogen phosphate (analytical grade) and 0.3 g of dipotassium hydrogen phosphate (analytical grade), and dilute to 50 mL with first-grade water specified in GB / T 6682.

[0051] Furthermore, the preparation process of β-galactosidase-glucose oxidase solution is as follows: weigh 0.8 g of β-galactosidase and 0.5 g of glucose oxidase in the same colorimetric tube, dissolve them with 10 mL of PBS buffer, mix them evenly, and use them immediately after preparation.

[0052] Furthermore, the activity of β-galactosidase is ≥240 U / mg, and the activity of glucose oxidase is ≥15 U / mg.

[0053] Step S2: Weigh the isomaltose standard, panose standard and isomaltotriose standard respectively, and prepare isomaltose standard working solution, panose standard working solution and isomaltotriose standard working solution of different concentrations.

[0054] Furthermore, the purity of the isomaltose standard, the panose standard and the isomaltotriose standard are all greater than 95%, or they are standard substances certified by the state and awarded with a standard substance certificate.

[0055] Step S3: Using high performance liquid chromatography and according to certain chromatographic conditions, obtain the peak area values ​​of isomaltose, panose and isomaltotriose in the standard working solution of different concentrations in the chromatogram, and establish an isomaltose standard curve, a panose standard curve and an isomaltotriose standard curve with the peak area value as the ordinate and the concentration value of the corresponding standard working solution as the abscissa.

[0056] Furthermore, the instruments used in the high performance liquid chromatography method include: a high performance liquid chromatograph, a mobile phase vacuum filtration and degassing device, a microporous filter membrane and an analytical balance.

[0057] Furthermore, the high performance liquid chromatograph is equipped with a differential refractive index detector and a column thermostat system, and the accuracy of the analytical balance is 0.1 mg.

[0058] Furthermore, the above-mentioned chromatographic conditions include: amino chromatographic column, mobile phase, detector temperature, column temperature, flow rate and injection volume.

[0059] Furthermore, the amino chromatographic column has a column length of 250 mm, an inner diameter of 4.6 mm, and a film thickness of 5 μm, or a chromatographic column with equivalent performance.

[0060] Furthermore, the mobile phase consisted of 73% acetonitrile and 27% water, and the acetonitrile was of chromatographic grade.

[0061] Furthermore, the detector temperature was 35°C, the column temperature was 35°C, the flow rate was 1.0 mL / min, and the injection volume was 10 μL.

[0062] Step S4: weigh the sample to be tested, and add the β-galactosidase-glucose oxidase solution to prepare a test solution.

[0063] Furthermore, the configuration process of the test liquid is as follows:

[0064] Accurately weigh 2-5g of the sample (accurate to 0.0001g, the content of various sugars should be within the linear range of the standard curve, otherwise the sample amount can be appropriately increased or decreased or diluted) into a 100mL beaker, add 20ml of 45℃-50℃ water (first-grade water specified in GB / T6682), mix well, let stand for 10 minutes, and cool to room temperature;

[0065] The pH value of the above solution was adjusted to 1.7±0.1 with hydrochloric acid solution, left for 1 min, and then the pH value was adjusted to 4.5±0.1 with sodium hydroxide solution;

[0066] Place the beaker containing the sample in an ultrasonic oscillator for 10 minutes, then transfer the solution in the beaker to a 50mL centrifuge tube, rinse the beaker with water (first-grade water specified in GB / T 6682), combine the washing solution into a 50mL centrifuge tube, add 5mL of β-galactosidase-glucose oxidase, shake well, place in a 60℃ constant temperature water bath and oscillate for 60 minutes. After rapid cooling, transfer to a 50mL volumetric flask, dilute to 50mL with water (first-grade water specified in GB / T 6682), shake well, filter through a membrane into a sample bottle, and use as the test solution.

[0067] Step S5: obtaining the peak area values ​​of isomaltose, panose, and isomaltotriose in the test solution in the chromatogram by high performance liquid chromatography, and finding the concentrations of isomaltose, panose, and isomaltotriose in the test solution in the corresponding standard curve according to the peak area values, and then obtaining the content of isomaltose, panose, and isomaltotriose in the test sample by the conversion formula.

[0068] Furthermore, the content of each sugar in the sample to be tested is calculated according to the following conversion formula:

[0069]

[0070] Wherein, X is the content of each sugar (isomaltose, panose, isomaltotriose) in the sample to be tested, in grams per hundred grams (g / 100g); c is the concentration of each sugar in the test solution, in milligrams per milliliter (mg / mL); V is the fixed volume, in milliliters (mL); d is the dilution factor, m is the mass of the sample, in grams (g); 100 is the conversion factor, and 1000 is the conversion factor.

[0071] The principles upon which the present invention is based are as follows:

[0072] Lactose is a disaccharide in which D-glucose and D-galactose are bonded by a β-1,4 bond, also known as 1,4-galactoside glucose. Lactase is a digestive enzyme in the human body. It is the most important digestive enzyme in infants and young children whose main source of nutrition is breast milk / milk powder. It is widely present in various animals, plants and microorganisms, including β-galactosidase, acid β-galactosidase and isoβ-galactosidase. The latter two enzymes rarely work on lactose. The main function of lactase is to digest lactose and hydrolyze lactose into galactose and glucose that can be absorbed by the human body. Therefore, the present invention uses β-galactosidase to hydrolyze lactose in formula milk powder in a targeted manner to remove the interference of lactose, and then cooperates with other technical means such as high performance liquid chromatography to achieve accurate determination of the content of oligosaccharides in formula milk powder.

[0073] The following examples are provided to further illustrate the present invention, but the scope of the present invention is not limited thereto.

[0074] Preparation of standard solution

[0075] Use an analytical balance (precision of 0.1 mg) to weigh appropriate amounts of isomaltose standard, panose standard and isomaltotriose standard to prepare six standard working solutions of different concentrations, as shown in Table 1:

[0076] Table 1 Standard working solution concentration table

[0077]

[0078]

[0079] The above six standard working solutions of different concentrations were detected by high performance liquid chromatography, and the chromatographic conditions were as follows:

[0080] a) Amino chromatography column: column length 250mm, inner diameter 4.6mm, film thickness 5μm;

[0081] b) Mobile phase: acetonitrile: water = 73:27 (by weight ratio);

[0082] c) Detector temperature: 35°C;

[0083] d) Column temperature: 35°C;

[0084] e) Flow rate: 1.0 mL / min;

[0085] f) Injection volume: 10 μL.

[0086] The test results are shown in Table 2:

[0087] Table 2 Peak area of ​​standard working solution

[0088] Isomaltose peak area Panose peak area Isomaltotriose peak area Concentration 1 25103.402768 12535.318406 5623.319866 Concentration 2 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

[0089] Among them, the chromatogram of the standard working solution with a concentration of 5 is as follows: Figure 3 shown.

[0090] With the concentration values ​​of the above six concentrations of standard working solutions as the horizontal coordinate and the peak area value as the vertical coordinate, a linear equation was established and the corresponding correlation coefficient was obtained, as shown in Table 3:

[0091] Table 3 Linear equations and correlation coefficients of isomaltose, panose and isomaltotriose

[0092] Compound Linear equations Correlation coefficient Isomaltose Y=58945X-14098 0.999900 Pan Sugar Y=64240X-7585.2 0.999700 Isomaltotriose Y=39002X-3251.5 0.999650

[0093] It can be seen from the correlation coefficients in Table 3 that isomaltose, isomaltotriose and panose have a good linear relationship.

[0094] Example 1

[0095] Weigh 2g formula milk powder into a 100ml beaker, add 20ml of 45℃ water, mix well, let stand for 10min, and cool to room temperature; adjust the pH value to 1.6 with hydrochloric acid solution and let stand for 1min; adjust the pH value to 4.4 with sodium hydroxide solution; put the beaker into an ultrasonic oscillator for 10min, transfer the solution in the beaker to a 50mL centrifuge tube, rinse the beaker with water (first-grade water specified in GB / T 6682), combine the washing liquid into a 50mL centrifuge tube, add 5mL β-galactosidase-glucose oxidase, shake well, place in a 60℃ constant temperature water bath and shake for 60min, quickly cool, transfer to a 50mL volumetric flask, and dilute to 50mL with water (first-grade water specified in GB / T 6682), shake well, filter the membrane into a sample bottle, and use it as the first test solution. The first test solution is subjected to chromatographic analysis on a high performance liquid chromatograph to obtain a chromatogram of the first test solution, as shown in Figure 4 shown.

[0096] Example 2

[0097] Weigh 3g formula milk powder into a 100ml beaker, add 20ml of 47℃ water, mix well, let stand for 10min, and cool to room temperature; adjust the pH value to 1.7 with hydrochloric acid solution and let stand for 1min; adjust the pH value to 4.5 with sodium hydroxide solution; put the beaker into an ultrasonic oscillator for 10min, transfer the solution in the beaker to a 50mL centrifuge tube, rinse the beaker with water (first-grade water specified in GB / T 6682), combine the washing liquid into a 50mL centrifuge tube, add 5mL β-galactosidase-glucose oxidase, shake well, place in a 60℃ constant temperature water bath and shake for 60min, quickly cool, transfer to a 50mL volumetric flask, and dilute to 50mL with water (first-grade water specified in GB / T 6682), shake well, filter the membrane into a sample bottle, and use it as the second test solution. The second test solution is subjected to chromatographic analysis on a high performance liquid chromatograph to obtain a chromatogram of the second test solution, as shown in Figure 5 shown.

[0098] Example 3

[0099] Weigh 5g formula milk powder into a 100ml beaker, add 20ml of 50℃ water, mix well, let stand for 10min, and cool to room temperature; adjust the pH value to 1.8 with hydrochloric acid solution and let stand for 1min; adjust the pH value to 4.6 with sodium hydroxide solution; put the beaker into an ultrasonic oscillator for 10min, transfer the solution in the beaker to a 50mL centrifuge tube, rinse the beaker with water (first-grade water specified in GB / T 6682), combine the washing liquid into a 50mL centrifuge tube, add 5mL β-galactosidase-glucose oxidase, shake well, place in a 60℃ constant temperature water bath and shake for 60min, quickly cool, transfer to a 50mL volumetric flask, and dilute to 50mL with water (first-grade water specified in GB / T 6682), shake well, filter the membrane into a sample bottle, and use it as the third test solution. The third test solution is subjected to chromatographic analysis on a high performance liquid chromatograph to obtain a chromatogram of the third test solution, as shown in Figure 6 shown.

[0100] Comparative Example

[0101] To verify that the amount of β-galactosidase-glucose oxidase solution added in the claims can completely remove the interference of lactose, a formula milk powder without isomaltooligosaccharide was taken and β-galactosidase-glucose oxidase solution was added. The other aspects were consistent with Example 1. The chromatogram obtained was as follows: Figure 7 By comparison Figure 2 and Figure 7 It can be seen that at the lactose peak time, Figure 7 The absence of chromatographic peaks indicates that the lactose in the test solution has been completely removed.

[0102] Another formula milk powder containing oligosaccharide is taken, and the β-galactosidase-glucose oxidase solution is not added for treatment. The other aspects are consistent with Example 1. The chromatogram obtained is as follows Figure 8 As shown. Figure 4 and Figure 8 It can be seen that the peak order is isomaltose, panose, and isomaltotriose. By comparing the contents, it can be seen that Figure 8 The peak area value of isomaltose in Figure 4 The peak area value of isomaltose in the formula milk powder indicated that β-galactosidase-glucose oxidase could effectively remove lactose from the formula milk powder.

[0103] Depend on Figures 4 to 6 The peak area values ​​of Examples 1 to 3 can be obtained, as shown in Table 4:

[0104] Table 4 Peak area values ​​of Examples 1 to 3

[0105] Isomaltose peak area Panose peak area Isomaltotriose peak area Example 1 46615.35 23378.48 10984.23 Example 2 77856.21 38731.84 17848.58 Example 3 145380 70161 35419

[0106] According to the linear equations of isomaltose, panose and isomaltotriose, the concentrations of isomaltose peak, panose and isomaltotriose in the test solutions of Examples 1 to 3 were obtained, as shown in Table 5:

[0107] Table 5 Concentrations of isomaltose, panose and isomaltotriose in Examples 1 to 3

[0108]

[0109] According to the concentrations of isomaltose, panose and isomaltotriose, the contents of isomaltose, panose and isomaltotriose in the formula milk powders of Examples 1 to 3 are calculated using the above conversion formula, as shown in Table 6:

[0110] Table 6 Contents of isomaltose, panose and isomaltotriose in formula milk powders of Examples 1 to 3

[0111] Isomaltose(g / 100g) Pantothenic acid (g / 100g) Isomaltotriose (g / 100g) Embodiment 1 (formulated milk powder) 2.7 1.2 0.9 Embodiment 2 (formulated milk powder) 2.6 1.2 0.9 Embodiment 3 (formulated milk powder) 2.6 1.2 0.9

[0112] In order to verify the feasibility and accuracy of the present invention, three concentration gradient standard solutions were added based on Example 3, and the experiment was repeated 6 times for each concentration. The recovery rate of spiked recovery was tested according to the above method. The results are shown in Table 7, and the results of the method detection limit are shown in Table 8:

[0113] Table 7 Spiked recovery

[0114]

[0115]

[0116] Table 8 Method detection limit

[0117] Isomaltose Pan Sugar Isomaltotriose Detection limit (g / 100g) 0.6 0.5 0.5

[0118] Note: The method detection limit is the minimum concentration of the analyte that can be detected with a reasonable confidence level by a given analytical method under specific conditions; the present invention compares the measurement signals of the analyte sample with a known low concentration with the blank sample to determine the minimum concentration for reliable detection, and the selected signal-to-noise ratio is 3:1.

[0119] It can be seen from Tables 7 and 8 that the spiked recovery rate of the target compound of the present invention reaches 98.3%-100.2%, and the method detection limit reaches 0.5-0.6g / 100g, indicating that the detection method provided by the present invention has good recovery rate and high accuracy.

[0120] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A method for detecting the content of isomaltooligosaccharide in formula milk powder, characterized in that: The following steps are involved: Step S1: preparing hydrochloric acid solution, sodium hydroxide solution, PBS buffer solution and β-galactosidase-glucose oxidase solution; Step S2: Weighing the isomaltose standard, the panose standard and the isomaltotriose standard respectively, and preparing isomaltose standard working solutions, panose standard working solutions and isomaltotriose standard working solutions of different concentrations; Step S3: using high performance liquid chromatography and according to certain chromatographic conditions, obtaining the peak area values ​​of isomaltose, panose and isomaltotriose in the standard working solution of different concentrations in the chromatogram, and establishing an isomaltose standard curve, a panose standard curve and an isomaltotriose standard curve with the peak area value as the ordinate and the concentration value of the corresponding standard working solution as the abscissa, wherein the chromatographic conditions include an amino chromatographic column and a mobile phase, wherein the amino chromatographic column has a column length of 250 mm, an inner diameter of 4.6 mm, and a film thickness of 5 μm, and the mobile phase is composed of 73% acetonitrile and 27% water by weight percentage, and the high performance liquid chromatograph used in the high performance liquid chromatography is equipped with a differential refractive index detector; Step S4: weighing the sample to be tested, adding the β-galactosidase-glucose oxidase solution to prepare the test solution, wherein the preparation process of the test solution includes: adjusting the pH value to 1.7±0.1 with the hydrochloric acid solution, leaving it for 1 min, adjusting the pH value to 4.5±0.1 with the sodium hydroxide solution, adding 5 mL of the β-galactosidase-glucose oxidase solution, shaking well, placing it in a 60°C constant temperature water bath for 60 min, cooling it quickly, and making it up to 50 mL with water, shaking well, filtering it through a membrane into a sample bottle, and using it as the test solution; Step S5: obtaining the peak area values ​​of isomaltose, panose and isomaltotriose in the test solution in the chromatogram by high performance liquid chromatography, and obtaining the concentrations of isomaltose, panose and isomaltotriose in the test solution in the corresponding standard curve according to the peak area values, and then obtaining the content of isomaltose, panose and isomaltotriose in the test sample by conversion formula.

2. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to claim 1, characterized in that: In step S4, the configuration process of the test liquid further includes: Accurately weigh 2-5g of the sample to be tested; Add 20 ml of water at 45° C. to 50° C. to the sample to be tested, mix well, let stand for 10 minutes, and cool to room temperature.

3. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to claim 1, characterized in that: The chromatographic conditions also include: detector temperature, column temperature, flow rate and injection volume; Wherein, the detector temperature is 35°C, the column temperature is 35°C, the flow rate is 1.0 mL / min, and the injection volume is 10 μL.

4. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to claim 1, characterized in that: The concentration of the hydrochloric acid solution is 5.0 mol / L or 0.1 mol / L.

5. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to claim 1, characterized in that: The concentration of the sodium hydroxide solution is 5.0 mol / L and / or 0.1 mol / L.

6. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to claim 1, characterized in that: The pH value of the PBS buffer is 6.0, the preparation process is as follows: weigh 1.1 g of potassium dihydrogen phosphate and 0.3 g of dipotassium hydrogen phosphate, and dilute to 50 mL with water.

7. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to claim 1, characterized in that: The preparation process of the β-galactosidase-glucose oxidase solution is as follows: weigh 0.8 g of β-galactosidase and 0.5 g of glucose oxidase in the same colorimetric tube, dissolve them in 10 mL of the PBS buffer, mix them evenly, and use them immediately after preparation.

8. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to claim 7, characterized in that: The activity of the β-galactosidase is ≥240 U / mg, and the activity of the glucose oxidase is ≥15 U / mg.

9. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to claim 1, characterized in that: The purity of the isomaltose standard, the panose standard and the isomaltotriose standard are all greater than 95%.

10. The method for detecting the content of isomaltooligosaccharide in formula milk powder according to any one of claims 2 to 6, characterized in that: The water, the water in the hydrochloric acid solution and the water in the sodium hydroxide solution are all first-grade water as specified in GB / T 6682.

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