CN109613140B

CN109613140B - Method for detecting casein phosphopeptide in food

Info

Publication number: CN109613140B
Application number: CN201910012481.0A
Authority: CN
Inventors: 曾祥恒; 刘正冬
Original assignee: Yashili International Group Co ltd
Current assignee: Yashili International Group Co ltd
Priority date: 2019-01-07
Filing date: 2019-01-07
Publication date: 2021-10-08
Anticipated expiration: 2039-01-07
Also published as: CN109613140A

Abstract

The invention relates to a method for detecting casein phosphopeptide in food. Which comprises the following steps: preparation of a standard substance: dissolving 90% CPP serving as a standard substance I by adding a solvent to prepare a standard substance solution I; dissolving 20% CPP serving as a standard substance II by adding a solvent to prepare a standard substance solution II; sample pretreatment: dripping 40-60% hydrochloric acid aqueous solution into the aqueous solution of a sample with the concentration of 0.05-0.1 g/mL until precipitation is generated, adding water to a constant volume, centrifuging, and filtering to obtain a sample solution; and (3) determination: carrying out ultra performance liquid chromatography-mass spectrometry detection on the sample solution, and quantifying by an external standard method; the chromatographic conditions comprise: a chromatographic column: C18130A chromatography column; the mobile phase C is formic acid aqueous solution, and the mobile phase D is acetonitrile; gradient elution. The detection method can avoid the interference of other nutrients such as protein, vitamins and the like on the CPP detection result, and stably and accurately detect CPPs with different specifications in food.

Description

Method for detecting casein phosphopeptide in food

Technical Field

The invention relates to the field of detection and analysis, in particular to a method for detecting casein phosphopeptide in food.

Background

Casein phosphopeptide (CPP) is polypeptide with biological activity prepared by taking cow milk casein as a raw material and adopting a biological technology. The CPP molecule consists of twenty to thirty amino acid residues, wherein the CPP molecule comprises 4-7 clustered phosphoserine acyl groups, can be used in various nutritional and health-care foods, and can effectively promote the absorption and utilization of divalent mineral nutrients such as calcium, iron, zinc and the like by a human body.

Because CPP is a protein hydrolysis product, after CPP is added into milk powder, milk and other dairy products, the dairy products also contain a plurality of other proteins and vitamins, and the other proteins and vitamins can interfere the detection of CPP, so that the CPP cannot be stably and accurately detected, and the detection of CPP in food is difficult. In addition, when the CPP addition amount in the milk powder is low, the CPP can not be detected by the conventional HPLC, and meanwhile, the pretreatment speed is low, and abnormal results often occur. In addition, casein phosphopeptides added to food products generally have two specifications: the two CPPs have different compositions and bring certain difficulty for qualitatively detecting casein phosphopeptide in food, wherein the purity of the CPP is more than or equal to 20 percent and the purity of the CPP is more than or equal to 90 percent.

Disclosure of Invention

Based on the detection method, the interference of other proteins and vitamins on the CPP detection result can be avoided, CPPs of different specifications in the food can be stably and accurately detected, the detection limit is low, and the pretreatment is simple and rapid.

The specific technical scheme is as follows:

a method for detecting casein phosphopeptide in food comprises the following steps:

preparation of a standard substance: dissolving 90% CPP serving as a standard substance I by adding a solvent to prepare a standard substance solution I; dissolving 20% CPP serving as a standard substance II by adding a solvent to prepare a standard substance solution II;

sample pretreatment: dripping 40-60% hydrochloric acid aqueous solution into the aqueous solution of a sample with the concentration of 0.05-0.1 g/mL until precipitation is generated, adding water to a constant volume, centrifuging, filtering supernate, and taking filtrate as sample solution;

and (3) determination: carrying out ultra performance liquid chromatography-mass spectrometry detection on the sample solution, and quantifying by an external standard method;

the chromatographic conditions include: a chromatographic column: a C18 chromatography column; the mobile phase C is formic acid aqueous solution, and the mobile phase D is acetonitrile; gradient elution.

In one embodiment, the gradient elution is specifically:

0-2min, the mobile phase C is 90%, and the mobile phase D is 10%;

2min-15min, wherein the mobile phase C is 90% → 60%, and the mobile phase D is 10% → 40%;

15min-16min, wherein the mobile phase C is 60% → 10%, and the mobile phase D is 40% → 90%;

16min-20min, wherein the mobile phase is C10%, and the mobile phase is D90%;

20min-21min, wherein the mobile phase C is 10% → 90%, and the mobile phase D is 90% → 10%;

21min-25min, the mobile phase C is 90%, and the mobile phase D is 10%.

In one embodiment, the aqueous formic acid solution has a concentration of 0.1% to 0.5% by volume.

In one embodiment, the C18 chromatography column is Waters CSH 130C 18.

In one embodiment, the chromatographic conditions further comprise: the flow rate is 0.3 plus or minus 0.2 mL/min; the column temperature is 35 +/-5 ℃; the sample amount is 1-5 μ L.

In one embodiment, the aqueous solution of the sample has a concentration of 0.08g/mL to 0.1 g/mL.

In one embodiment, the volume concentration of the hydrochloric acid aqueous solution is 48% -53%.

In one embodiment, before the constant volume, the method further comprises the step of adding methanol for defoaming.

Compared with the prior art, the invention has the following beneficial effects:

the method utilizes ultra-high performance liquid chromatography combined mass spectrometry (UPLC-MS) to detect the CPP in the food, optimizes chromatographic conditions, adopts a proper pretreatment method, ensures that the CPP is not interfered by other proteins and vitamins, has stable detection results, can distinguish CPP components with different specifications by adopting the detection method, and overcomes the problem that the CPP with different specifications is difficult to be detected qualitatively. Meanwhile, the pretreatment of the sample is simple and rapid. The recovery rate of the added standard of the detection method is 103 percent on average. The detection sensitivity and accuracy meet the detection requirement of the CPP in the food, and a new reference standard is provided for the detection of the CPP in the food.

Drawings

FIG. 1 is a liquid chromatogram of casein phosphopeptide standard I;

FIG. 2 is a full scan mass spectrum of casein phosphopeptide standard I;

FIG. 3 is a liquid chromatogram of casein phosphopeptide standard II;

FIG. 4 is a full scan mass spectrum of casein phosphopeptide standard II;

FIG. 5 is a liquid chromatogram of a casein phosphopeptide-free negative sample;

FIG. 6 is a full scan mass spectrum of a negative sample without casein phosphopeptide addition;

FIG. 7 is a liquid chromatogram of a milk powder sample to be tested;

FIG. 8 is a standard curve I of casein phosphopeptide standard I;

FIG. 9 is a standard curve II of casein phosphopeptide standard II.

Detailed Description

The method for detecting casein phosphopeptide in food according to the present invention will be described in further detail with reference to the following specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

1 generally specifies

Reagents and water used in the following embodiments, unless otherwise noted, refer to analytically pure reagents and tertiary water as specified in GB/T6682. The standard solutions used in the tests, the standard solutions for impurity measurement, the preparations and the products were prepared in accordance with the specifications of GB/T601, GB/T602 and GB/T603, unless otherwise noted. The solutions used in the tests are aqueous solutions unless any solvent is used for the preparation.

2 analytical procedure

2.1 reagents and materials

Water, which is first-grade water meeting the GB/T6682 regulation; acetonitrile is chromatographically pure; the methanol is chromatographically pure; formic acid, namely, chromatographic purity; hydrochloric acid, analytically pure; the casein phosphopeptide standard product I has a purity of more than or equal to 90.0% (namely 90% CPP raw material), and the content of effective substances in the raw material can be calculated according to a product quality inspection report (COA); casein phosphopeptide standard II with purity not less than 20.0% (20% CPP material)

2.2 instruments and devices

The ultra-high performance liquid chromatograph is provided with a mass spectrum detector QDA, a constant column temperature and a vacuum degassing system.

2.3 preparation of Standard solutions

2.3.1 weighing 100mg (accurate to 0.0001g) of the casein phosphopeptide standard product I, dissolving in a 100mL volumetric flask, adding 50mL of water and 10mL of methanol, mixing in a rotating manner, fully dissolving, and then adding water to a constant volume of 100mL (the stock solution is effective in refrigeration for 3 months) to obtain the casein phosphopeptide standard solution I (the standard solution is effective in refrigeration for 1 month).

2.3.2 weighing 100mg (accurate to 0.0001g) of the casein phosphopeptide standard substance II, dissolving in a 100mL volumetric flask, adding 50mL of water and 10mL of methanol, mixing in a rotating manner, fully dissolving, and then adding water to a constant volume of 100mL (the stock solution is effective in refrigeration for 3 months) to obtain the casein phosphopeptide standard solution II (the standard solution is effective in refrigeration for 1 month).

2.4 preparation of a Casein-free phosphopeptide negative sample solution

Taking 2.5g (accurate to 0.0001g) of base powder without casein phosphopeptide, dissolving in a 100mL volumetric flask, adding 50mL of water and 10mL of methanol, mixing in a rotating manner, fully dissolving, and then diluting to 100mL with water to obtain a casein phosphopeptide-free negative sample solution.

2.5 chromatographic conditions

The column was a Waters CSH 130C18(100mm) column, the mobile phase was as shown in Table 1, the flow rate was 0.3mL/min, and the column was eluted with a gradient, C was 0.1% aqueous formic acid, and D was acetonitrile.

The column temperature was 35 ℃. The sample injection amount is 1 muL.

TABLE 1

Time (min)	Flow (mL/min)	％A	％B	％C	％D	Curve
							Initial	0.300	0.0	0.0	90.0	10.0	Initial
2.00	0.300	0.0	0.0	90.0	10.0	6
							15.00	0.300	0.0	0.0	60.0	40.0	6
16.00	0.300	0.0	0.0	10.0	90.0	6
							20.00	0.300	0.0	0.0	10.0	90.0	6
21.00	0.300	0.0	0.0	90.0	10.0	1
							25.00	0.300	0.0	0.0	90.0	10.0	6

2.6 Mass Spectrometry Condition I

Mass spectrometer qda (waters), two-channel ion scan SIR, where one channel is set to SIR 651.9, the other channel is set to SIR 765.0, cone voltage: 15V.

2.7 determination

And (2) with reference to 2.5 chromatographic conditions and 2.6 mass spectrum conditions, injecting the standard solution I into a chromatograph for determination to obtain a liquid chromatogram of the standard substance I (see figure 1), and performing mass spectrum full scanning to obtain a mass spectrum of the standard substance I (see figure 2).

And (3) injecting the standard solution II into a chromatograph for measurement to obtain a liquid chromatogram of the standard product II (see figure 3), and obtaining a mass spectrum of the standard product II (see figure 4) after mass spectrum full scan.

And (3) injecting the prepared negative sample solution to obtain a liquid chromatogram of the negative sample without adding the casein phosphopeptide (see figure 5), and performing mass spectrum full scanning to obtain a mass spectrum of the negative sample without adding the casein phosphopeptide (see figure 6).

2.8 analysis

With the combination of the figures 1-6, the retention time of the main peak of the casein phosphopeptide standard product I is 7.679min, the response value of the characteristic ion m/z 651.9 is high, and the interference of other proteins and vitamins is avoided; the main peak retention time of the casein phosphopeptide standard product II is 7.1min, the response value of the characteristic ion m/z 765.0 is high, and the interference of other proteins and vitamins is avoided.

And (4) conclusion: in the casein phosphopeptide content determination test, the qualitative determination is carried out on the sample to be detected according to the retention time of the main peak of the chromatogram of the standard solution. Finding M/Z651.9 characteristic ions aiming at a CPP raw material with the specification of 90 percent; the m/z 765.0 characteristic ion was found for a specification of 20% CPP feedstock.

3 determination of Casein phosphopeptides in food products

3.1 weighing 100mg (to the nearest 0.0001g) of casein phosphopeptide standard I, dissolving in a 100mL volumetric flask, adding 50mL of water and 10mL of methanol, mixing by rotation, fully dissolving, and adding water to the volume of 100mL (the stock solution is effective in refrigerating for 3 months). Then the casein phosphopeptide standard solution I is respectively diluted into 2ug/mL, 10ug/mL, 20ug/mL, 100ug/mL and 1000ug/mL by water in sequence (the standard solution is effective after being refrigerated for 1 month).

3.2 weighing 100mg (to the nearest 0.0001g) of casein phosphopeptide standard II, dissolving in a 100mL volumetric flask, adding 50mL of water and 10mL of methanol, mixing by rotation, fully dissolving, and adding water to the volume of 100mL (the stock solution is effective in refrigerating for 3 months). Then the casein phosphopeptide standard solution II is respectively diluted into 2ug/mL, 10ug/mL, 20ug/mL, 100ug/mL and 1000ug/mL by water in sequence (the standard solution is effective after being refrigerated for 1 month).

3.3 weigh 6 test milk powder samples 2.5g (to the nearest 0.0001g) into a 50mL volumetric flask. Adding about 30mL of water, rotating and shaking for dissolving, adding 1-3 drops of hydrochloric acid solution with the volume concentration of 50% to generate precipitates (different formulas can be pre-tested firstly, the best precipitation effect is determined to be several drops), adding water for constant volume (if foams are inconvenient to observe for constant volume, several drops of methanol can be added), rotating and uniformly mixing, pouring into a 50mL centrifuge tube, standing for 10min, rotating and separating for 1min at 6000 rpm, taking clarified solution, filtering, taking filtrate, obtaining 6 parts of milk powder sample solution to be tested, and processing on a machine.

3.4 chromatographic conditions

Reference 2.5 chromatographic conditions

3.5 Mass Spectrometry conditions

3.6 determination

And (3) injecting standard solutions I with different concentrations, standard solutions II with different concentrations and 6 parts of milk powder sample solutions to be detected into a chromatograph for determination according to 3.4 chromatographic conditions and 3.5 mass spectrum conditions.

3.7 analysis

And (3) qualitative analysis: the above measurement results in a liquid chromatogram of 1 part of the milk powder to be measured (see fig. 7). And (4) determining the chromatographic peak of the component to be detected in the sample solution according to the retention time of the standard substance I and the retention time of the standard substance II. In fig. 7, the retention time of the main peak is 7.659, which is consistent with the retention time of the main peak of 90% CPP raw material, and it is shown that the milk powder to be tested contains 90% CPP component, which is consistent with the formula design.

Quantitative analysis: a standard curve I was prepared based on the peak areas and concentrations of the standard solutions I of different concentrations (see FIG. 8). A standard curve II was prepared based on the peak areas and concentrations of different concentrations of the standard solution II (see FIG. 9). And calculating the content of the component to be detected through a corresponding standard curve according to the peak area of the sample solution to be detected.

The content w1(mg/100g) of the component to be measured (casein phosphopeptide CPP) in the sample was calculated according to the formula (A.1):

w1＝C/1000×50/W×100…………………………(A.1)

in the formula:

c-concentration of CPP in the solution to be tested, unit is microgram per milliliter (ug/mL);

w-sample weight in grams (g).

The test results are based on the arithmetic mean of the results of the replicates. The relative standard deviation of the results of two independent measurements obtained under reproducible conditions is not more than 5%.

The test results of 6 samples of the milk powder to be tested are shown in table 2.

TABLE 2

	Retention time	Peak area	Content (wt.)
				Sample to be tested-1	7.658	262432	49.705
Sample to be tested-1	7.658	247045	47.139
				Sample to be tested-1	7.651	243347	47.153
Sample to be tested-1	7.659	236902	45.793
				Sample to be tested-1	7.661	246443	46.334
Sample to be tested-1	7.654	238127	44.914
				Mean value of	7.657	245716	46.840
Std.Dev.	0.004	9194	1.641
				％RSD	0.0	3.7	3.5

4 method verification report

4.1 qualitative test

In a casein phosphopeptide content determination test, the main peak retention time of a chromatogram of a milk powder sample solution to be determined is consistent with that of a standard solution chromatogram, and other components are free of interference and meet requirements.

4.2 precision of the instrument

The sample injection of the intermediate concentration standard solution is repeated for 5 times, and the RSD is 0.7 percent and less than 2.0 percent, which meets the requirement.

4.3 Linear

The standard curve has good linearity, the detection limit is 1ug/mL-1000ug/mL, and R is 0.999993, which meets the requirement.

4.5 range

Under the condition of good linearity, the concentration of the standard substance is 1ug/mL-1000ug/mL, which meets the requirement.

4.6 repeatability (method precision)

6 uniform samples in the same pot are repeatedly processed for analysis, the precision is good, and the RSD is 3.5 percent and 4.0 percent, which meets the requirement.

4.7 lowest limit of quantitation

According to the calculation of the signal-to-noise ratio of 10 times, the noise is 140, and the quantitative limit of the method is 0.68ug/ml, which is equivalent to 1.36mg/100g, and meets the requirement.

4.8 recovery

By carrying out marking recovery verification on the base powder, respectively marking 3 concentration gradients of 50%, 100% and 150% of the target value of the finished product CPP, and analyzing 3 samples of each concentration, the recovery rate is 9, the average recovery rate is 103.6%, and the recovery rate meets the requirement of 90-110%. As shown in table 3:

TABLE 3

4.9 durability

The mobile phase and the gradient are changed within a certain range, and the qualitative and the quantitative are not influenced.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A method for detecting casein phosphopeptide in milk powder is characterized by comprising the following steps:

preparation of a standard substance: taking 90% casein phosphopeptide as a standard substance I, adding formic acid aqueous solution for dissolving, and preparing a standard substance solution I; taking 20% casein phosphopeptide as a standard substance II, adding formic acid aqueous solution for dissolving, and preparing a standard substance solution II;

sample pretreatment: dripping 40-60% hydrochloric acid aqueous solution into the aqueous solution of a sample with the concentration of 0.05-0.1 g/mL until precipitation is generated, adding water to a constant volume, centrifuging, and filtering to obtain a sample solution;

the chromatographic conditions include: a chromatographic column: waters CSH 130C18, 100 mm; the mobile phase C is formic acid aqueous solution with volume concentration of 0.1-0.5%, and the mobile phase D is acetonitrile; gradient elution; the flow rate is 0.3 plus or minus 0.2 mL/min; the column temperature is 35 +/-5 ℃; the sample amount is 1-5 muL;

the gradient elution is specifically as follows:

0-2min, the mobile phase C is 90%, and the mobile phase D is 10%;

16min-20min, wherein the mobile phase is C10%, and the mobile phase is D90%;

21min-25min, wherein the mobile phase is C90%, and the mobile phase is D10%;

the mass spectrum conditions are as follows: mass spectrometer QDA, dual channel ion scan SIR, characteristic ion of 90% casein phosphopeptide M/Z651.9, channel set SIR 651.9, characteristic ion of 20% casein phosphopeptide M/Z765.0, channel set SIR 765.0, cone voltage: 15V.

2. The detection method according to claim 1, wherein the concentration by volume of the aqueous formic acid solution is 0.1%.

3. The detection method according to claim 1, wherein the flow rate is 0.3 mL/min; the column temperature was 35 ℃; the amount of sample was 1. mu.L.

4. The detection method according to claim 1, wherein the preparation method of the standard solution I comprises the following steps: weighing 100mg of standard substance I, dissolving in a 100mL volumetric flask, adding 50mL of water and 10mL of methanol, rotating, mixing, fully dissolving, and diluting to 100mL with water to obtain a standard substance solution I.

5. The detection method according to claim 1, wherein the preparation method of the standard solution II comprises the following steps: weighing 100mg of a standard substance II, dissolving in a 100mL volumetric flask, adding 50mL of water and 10mL of methanol, rotating and mixing, fully dissolving, and then diluting to 100mL with water to obtain a standard solution II.

6. The detection method according to any one of claims 1 to 3, wherein the concentration of the aqueous solution of the sample is 0.08g/mL to 0.1 g/mL.

7. The assay of any one of claims 1 to 3, wherein the aqueous hydrochloric acid solution has a concentration of 48% to 53% by volume.

8. The detection method according to any one of claims 1 to 3, wherein the concentration by volume of the aqueous hydrochloric acid solution is 50%.

9. The detection method according to any one of claims 1 to 3, further comprising a step of adding methanol to remove bubbles before the constant volume measurement.