CN103792298B - Method for detecting content of casein phosphopeptides in dairy product - Google Patents
Method for detecting content of casein phosphopeptides in dairy product Download PDFInfo
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Abstract
The invention provides a method for detecting the content of casein phosphopeptides in a dairy product. The method mainly comprises the steps of: purifying a casein phosphopeptides raw material by a BaCl2-ethanol technique; drawing a standard curve of the purified casein phosphopeptides raw material by HPLC (high performance liquid chromatography); purifying a dairy product sample by solid-phase extraction, and detecting the product by HPLC, thus obtaining the detection result. The detection method provided by the invention simplifies the operation steps, shortens the detection time, and especially can perform more effective and more accurate detection on dairy products with a low casein phosphopeptides content.
Description
Technical field
The present invention relates to the detection method of functional components in a kind of dairy products, particularly relate to the detection method of CPP content in a kind of dairy products, belong to detection technique field.
Background technology
CPP (casein phosphopeptides, be called for short CPP) be that raw material is through protease hydrolytic with milk casein, the natural physiological active peptide containing phosphoserine race obtained through separation and purification again, have in conjunction with calcium and the function promoting calcium uptake, so the short calcium uptake factor that is otherwise known as.
Domestic and international at present generally BaCl is adopted to the detection method of CPP content in CPP raw material
2-Ethanol Method, and in dairy products, the content of CPP adds by sincere completely.The detection method to CPP in dairy products of domestic report mainly comprises By Capillary Zone Electrophoresis, Liquid Chromatography-Mass Spectrometry and high performance liquid chromatography (HPLC).The interference of By Capillary Zone Electrophoresis because of rope more, be unsuitable for the detection of low content CPP; Liquid Chromatography-Mass Spectrometry testing cost is high, is unsuitable for the detection of workshop to product; Do not mention the purification process of dairy products in the document of the high performance liquid chromatography reported, and CPP standard items cannot bought on the market, therefore this detection method is difficult to repeat in actual applications.
During CPP content in current detection CPP finished product, first to use BaCl
2-Ethanol Method detects the content of CPP in CPP raw material, then calculates the CPP content in finished product with the addition of this CPP raw material, process operation complex steps, consuming time longer, generally need 2-3 talent to obtain testing result, and testing result error is comparatively large, is unsuitable for the requirement of modern production.
Summary of the invention
For solving the problems of the technologies described above, the object of the present invention is to provide the detection method of CPP content in a kind of dairy products.First this detection method carries out purifying to CPP raw material, it can be used as standard items, then detects purified CPP finished product, obtains testing result.This detection method simplifies operation steps, shortens detection time, the content detecting CPP in dairy products that can be accurate and effective.
For reaching above-mentioned purpose, the invention provides the detection method of CPP in a kind of dairy products, it comprises the following steps:
The purifying of A, CPP raw material
After being dissolved by CPP raw material, adjusted to ph is 4.6, and constant volume is the CPP material solution of 0.2g/mL, leaves standstill 30min;
By above-mentioned solution at 4 DEG C, the centrifugal 15min of 5000rpm, get supernatant, add BaCl
2make its concentration be 0.05M, after mixing, obtain one containing BaCl
2cPP material solution;
With above-mentioned containing BaCl
2cPP material solution: absolute ethyl alcohol=1: the volume ratio of 2, above-mentioned containing BaCl
2cPP material solution in add absolute ethyl alcohol, after mixing, at 4 DEG C of standing 12-16 hour;
By the solution after above-mentioned leaving standstill at 4 DEG C, the centrifugal 15min of 5000rpm, abandoning supernatant, with above-mentioned containing BaCl
2cPP material solution: H
2sO
4the volume ratio of=1: 0.4, adds the H that concentration is 0.125M in precipitation
2sO
4, then use Filter paper filtering;
Get filtrate and dry 60min at 60-70 DEG C, dry to constant weight at 105 DEG C afterwards, obtain the CPP raw material of purifying;
The drafting of B, typical curve
The CPP raw material ultrapure water of above-mentioned purifying is prepared the series standard working fluid that concentration is 0.1mg/100mL, 0.5mg/100mL, 1mg/100mL, 5mg/100mL, 10mg/100mL, 20mg/100mL, adopt high performance liquid chromatograph to carry out detection to this series standard working fluid to analyze, drawing standard curve;
The detection of C, CPP finished product
C1, Sample Purification on Single
C1.1, extraction
When sample is liquid diary product: take 20g sample, 50mL is settled to the trichloroacetic acid (trichloroacetic acid solution of mass concentration 1%) that concentration is 1%, ultrasonic 10min, leave standstill and filter to obtain liquid to be clean, wherein, persons skilled in the art can regulate and control according to the time of actual conditions to described stating step, and preferably, the described standing time is 10 minutes; The filter paper that described filtration step uses can for conventional qualitative filter paper;
When sample is solid-state dairy products: take 5g sample, 50mL is settled to after dissolving with the trichloroacetic acid that concentration is 1%, ultrasonic 10min, leave standstill and filter to obtain liquid to be clean, wherein, persons skilled in the art can regulate and control according to the time of actual conditions to described stating step, and preferably, the described standing time is 10 minutes; The filter paper that described filtration step uses can for conventional qualitative filter paper;
C1.2, purification
By solid-phase extraction column successively with 3mL methyl alcohol, the activation of 3mL water, the liquid to be clean getting proper volume is transferred in solid-phase extraction column, use 3mL water and 3mL methanol wash successively, be eluted in 10mL tool plug test tube with 6mL ammoniated methanol after discarding leacheate, whole solid phase extraction procedure flow velocity is no more than 1mL/min, eluent is dried up with nitrogen at 50 DEG C, obtains residue;
C2, detection and calculating
By the residue proper volume mobile phase A constant volume obtained after drying up, after crossing miillpore filter, adopt high performance liquid chromatograph to detect the supernatant obtained, go out the CPP content in sample according to the calculated by peak area of typical curve and sample.
Due to the problem that CPP material purity is low, seldom have and use CPP raw material to detect finished product as standard items, but in view of obtaining CPP standard items on the market both at home and abroad at present, therefore, the present invention adopts BaCl
2-Ethanol Method has carried out purifying to CPP raw material, it can be used as the standard items detecting CPP finished product.In the purification step of the CPP raw material of above-mentioned detection method, hydrochloric acid can be adopted to adjust the pH value to 4.6 of CPP aqueous solution of raw material, make casein precipitate, wherein, the concentration of hydrochloric acid and addition can carry out conventional selection and regulation and control by persons skilled in the art under the prerequisite of carrying out smoothly reacting; In addition, constant volume is the CPP material solution of 0.2g/mL is conveniently follow-up calculating, but can not be also 0.2g/mL by the concentration constant volume of CPP material solution, as long as the CPP raw material of the purifying obtained under this concentration can prepare follow-up series standard working fluid smoothly; In the purification step of above-mentioned CPP raw material, adopt and centrifugally can remove impurity better twice, make the purity of the CPP standard items obtained higher.
In above-mentioned detection method, the high performance liquid chromatograph adopted and mobile phase thereof and chromatographic condition all can adopt for routine when detecting CPP content.Preferably, described high performance liquid chromatograph is chromatographic column is C
18250 × 4.6mm, the high performance liquid chromatograph of 5 μm.The mobile phase A adopted can be trifluoroacetic acid aqueous solution.
In above-mentioned detection method, the solid-phase extraction column adopted and solid-phase extraction device are conventional; Preferably, the solid-phase extraction column adopted is strong cat ion exchange column (SCX).According to the specific embodiment of the present invention, also not having peak to occur near mark peak on the high-efficient liquid phase chromatogram of the sample of procyanidin, on the high-efficient liquid phase chromatogram of the sample of procyanidin, then lack a lot of Interference Peaks.And, adopt solid phase extraction that dairy products sample is carried out purifying, detect again after purer CPP is extracted, can carry out more effectively, more accurately detecting to the dairy products of low CPP content.Therefore, the present invention adopts solid phase extraction techniques to carry out purifying to the dairy products sample containing CPP, effectively CPP can be separated with interfering component, reduce sample pretreatment process, simple to operate, save time, laborsaving, reach other materials reduced in sample and interference is caused to detection, and then improve the effect of the accuracy detected.
In above-mentioned detection method, preferably, the described aperture crossing miillpore filter is 0.2 μm.
In above-mentioned detection method, preferably, in the detecting step of described CPP finished product, the volume being transferred to the liquid to be clean in solid phase extraction column is identical with the volume of the mobile phase A for constant volume residue.
In above-mentioned detection method, preferably, the volume to be clean be transferred to described in solid phase extraction column is 6mL with the volume for the mobile phase A of constant volume residue.
In the present invention, those skilled in the art can by the computing method of CPP content in above-mentioned detection method determination sample, these computing method specifically comprise the following steps: first bring the peak area of sample into typical curve, calculate machine concentration on CPP, again according to formula 1: CPP content (mg/100g)=[upper machine concentration (the mg/100mL) × 50mL of CPP] ÷ sample quality (g) in sample, calculates CPP content in sample.At this specifically be, in the testing process of above-mentioned CPP finished product, can comprise the liquid to be clean getting 6mL is transferred in solid phase extraction column, and the step of mobile phase A constant volume residue with 6mL, therefore, the concentration of the CPP finally recorded is consistent with the concentration of CPP in the sample after constant volume, so, in above-mentioned computing formula, save intermediate computations step.When the above-mentioned volume of liquid to be clean is different from the volume of the mobile phase A of constant volume, those skilled in the art can transform above-mentioned computing formula, to calculate CPP content in sample.
The present invention adopts BaCl
2-Ethanol Method carries out purifying to CPP raw material, obtain CPP standard items, so, when detecting dairy products finished product at every turn, high performance liquid chromatography only need be adopted to carry out detection and drawing standard curve to CPP standard items, then adopt high performance liquid chromatography to detect after carrying out procyanidin to sample, and utilize typical curve to obtain CPP content in finished product.Detection method of the present invention simplifies operation steps, shortens detection time, and each detection only needs just can obtain a result for 1 hour, and accuracy in detection is higher.
Accompanying drawing explanation
Fig. 1 is the CPP typical curve 1 in the detection method of embodiment 1.
Fig. 2 is the CPP typical curve 2 in the detection method of embodiment 2.
Fig. 3 is the CPP typical curve 3 in the detection method of embodiment 3.
Fig. 4 is the CPP typical curve 4 in the detection method of comparative example 1.
Fig. 5 is the chromatogram of CPP content detection in the sample in the detection method of embodiment 1.
Fig. 6 is the chromatogram of CPP content detection in the sample in the detection method of embodiment 2.
Fig. 7 is the chromatogram of CPP content detection in the sample in the detection method of embodiment 3.
Fig. 8 is the chromatogram of CPP content detection in the sample in the detection method of comparative example 1.
Embodiment
In following embodiment and comparative example, the high performance liquid chromatograph adopted is Agilentll00 liquid chromatograph, and its chromatographic condition is: chromatographic column is Diamonsil C
18250 × 4.6mm, 5 μm; Column temperature is 25 DEG C; Mobile phase A is trifluoroacetic acid aqueous solution, and Mobile phase B is the acetic acid aqueous solution of mass concentration 0.1%; The condition of gradient elution is: 0-25min mobile phase A is by 0% → 100%; Flow velocity is 1.00mL/min; Sample size is 20 μ L, and UV-detector wavelength is 280nm.The solid-phase extraction device model adopted: 24-port-visiprep, its manufacturer is supelco company; The model of the solid-phase extraction column adopted is 5982-3236, and its manufacturer is Agilent company.
Embodiment 1
The present embodiment provides the detection method of CPP in a kind of dairy products, and it comprises the following steps:
The purifying of A, CPP raw material
Take 10g CPP raw material in 50mL beaker, dissolve with pure water, then with hydrochloric acid, the pH value of this solution is adjusted to 4.6, the solution after adjusted to ph is shifted and is settled in 50mL centrifuge tube, leave standstill 30min;
By above-mentioned solution at 4 DEG C, the centrifugal 15min of 5000rpm, get supernatant, add BaCl
2make its concentration be 0.05M, after mixing, obtain about 25mL and contain BaCl
2cPP material solution;
Above-mentioned containing BaCl
2cPP material solution in add about 50mL absolute ethyl alcohol, after mixing, 4 DEG C of hold over night;
By the solution after above-mentioned leaving standstill at 4 DEG C, the centrifugal 15min of 5000rpm, abandoning supernatant, in precipitation, add about 10mL concentration is the H of 0.125M
2sO
4, then use Filter paper filtering;
Get filtrate and dry 60min at 60-70 DEG C, dry to constant weight at 105 DEG C afterwards, obtain the CPP raw material of purifying;
The drafting of B, typical curve
The CPP raw material ultrapure water of above-mentioned purifying is diluted to every 100 milliliters be equivalent to 0.1,0.5,1,5,10, the series standard working fluid of 20mg, high performance liquid chromatograph is adopted to detect this series standard working fluid, detect the collection of illustrative plates of gained carry out integration after peak area be respectively 47,210,450,2000,4100,7340, typical curve 1 is drawn according to the relation between the concentration of above-mentioned CPP series standard working fluid and the peak area of correspondence, as shown in Figure 1, the linearly dependent coefficient of gained typical curve 1 is 0.996, and the range of linearity is 0.1-20mg/100mL;
The detection of C, CPP finished product
C1, Sample Purification on Single
C1.1, extraction: take 20.9600g liquid milk sample, be settled to 50mL with the trichloroacetic acid of 1%, ultrasonic 10min, filter to obtain liquid to be clean after leaving standstill 10min.
C1.2, purification: by solid-phase extraction column successively with 3mL methyl alcohol, the activation of 3mL water, the liquid to be clean getting 6mL is transferred in solid-phase extraction column, use 3mL water and 3mL methanol wash successively, be eluted in 10mL tool plug test tube with 6mL ammoniated methanol after discarding leacheate, whole solid phase extraction procedure flow velocity is no more than 1mL/min, eluent is dried up with nitrogen at 50 DEG C, obtains residue;
C2, result calculate
By residue 6mL mobile phase A constant volume obtained above, after crossing miillpore filter, high performance liquid chromatograph is adopted to detect the supernatant obtained, the chromatogram obtained as shown in Figure 5, the peak area of this CPP sample is 850, and being inserted in the content that typical curve 1 and formula 1 calculate CPP in this liquid milk is 4.83mg/100g.
Embodiment 2
The present embodiment provides the detection method of CPP in a kind of dairy products, it adopts the CPP raw material of the obtained purifying of embodiment 1 to detect as standard items, the linearly dependent coefficient of the typical curve 2 (as shown in Figure 2) obtained is 0.998, and the range of linearity is 0.1-20mg/100mL; The detecting step of the CPP finished product of the present embodiment is basic identical with embodiment 1, difference is that taking 20.5370g liquid milk sample detects, the chromatogram obtained as shown in Figure 6, the peak area of this CPP sample is 98, and being inserted in the content that typical curve 2 and formula 1 calculate CPP in this liquid milk is 0.12mg/100g.
Embodiment 3
The present embodiment provides the detection method of CPP in a kind of dairy products, it adopts the CPP raw material of the obtained purifying of embodiment 1 to detect as standard items, the linearly dependent coefficient of the typical curve 3 (as shown in Figure 3) obtained is 0.999, and the range of linearity is 0.1-20mg/100mL; The detecting step of the CPP finished product of the present embodiment is basic identical with embodiment 1, difference is that taking 5.3450g powdered milk sample detects, the chromatogram obtained as shown in Figure 7, the peak area of this CPP sample is 1300, and being inserted in the content that typical curve 3 and formula 1 calculate CPP in this milk powder is 148.0mg/100g.
Comparative example 1
This comparative example provides the detection method of CPP in a kind of dairy products, it adopts the CPP raw material of the obtained purifying of embodiment 1 to detect as standard items, the linearly dependent coefficient of the typical curve 4 (as shown in Figure 4) obtained is 0.997, and the range of linearity is 0.1-20mg/100mL; The detection of the CPP finished product of this comparative example comprises the following steps: take 5.0650g powdered milk sample and detect, be settled to 50mL with the trichloroacetic acid of 1%, ultrasonic 10min, filters and obtain supernatant after leaving standstill 10min; High performance liquid chromatograph is adopted to detect described supernatant.As shown in Figure 8, the peak area of this CPP sample is 1050 to the chromatogram obtained, and being inserted in the content that typical curve 4 calculates CPP in this milk powder is 123.5mg/100g.
Observe Fig. 5 to Fig. 8 can find: on the high-efficient liquid phase chromatogram of the sample of procyanidin, near mark peak, also do not having peak to occur (as shown in Figure 8), a lot of Interference Peaks has then been lacked (as Fig. 5 on the high-efficient liquid phase chromatogram of the sample of procyanidin, shown in Fig. 6 and Fig. 7), especially in the unconspicuous situation in mark peak, very serious impact is there is in Interference Peaks on test result, therefore, adopt Solid-Phase Extraction to carry out purifying to testing sample and effectively can reduce other materials in sample to detecting the interference caused, and then improve the accuracy detected.
The accuracy validation of detection method
For determining that detection method of the present invention detects the accuracy of CPP content in dairy products, prepared the CPP finished product of different target concentration, detect the content of CPP in finished product by the method for embodiment 2, testing result is as shown in table 1.
Wherein, the preparation method of the CPP finished product of described different target concentration comprises the following steps:
First calculate the amount of the CPP raw material needed for CPP finished product of different target concentration;
Add CPP raw material after 2-3kg milk (not containing CPP) is heated to 65-70 DEG C, stir 20min;
Dosage is settled to milk;
Carry out filling after ultra high temperature sterilization (UHTS), prepare the CPP finished product of different target concentration.
In the preparation method of the CPP finished product of above-mentioned different target concentration, it should be noted that, in CPP raw material, the content of CPP is that producer provides (BaCl
2-Ethanol Method), in this confirmatory experiment, the CPP content of the CPP raw material adopted is 17% (percentage by weight), and the present invention calculates the amount of the CPP raw material needed for the CPP finished product of different target concentration by this content meter.
Table 1
As shown in Table 1, adopt detection method of the present invention to detect the CPP content in the CPP finished product of different target concentration, the recovery of this detection method is 95.0-110%.The recovery weighs the important indicator of detection method accuracy, it is generally acknowledged that the recovery reaches 80-120%, just illustrate that this detection method meets testing requirement.And the recovery of detection method of the present invention is 95.0-110%, prove that this detection method meets the requirement detected completely.Meanwhile, be that 110% detection limit demonstrating this detection method can reach 0.1mg/100mL in the recovery of typical curve minimum point 0.1mg/100mL, accuracy is very high.
Claims (5)
1. the detection method of CPP content in dairy products, it comprises the following steps:
The purifying of A, CPP raw material
After being dissolved by CPP raw material, adjusted to ph is 4.6, and constant volume is the CPP material solution of 0.2g/mL, leaves standstill 30min;
By above-mentioned solution at 4 DEG C, the centrifugal 15min of 5000rpm, get supernatant, add BaCl
2make its concentration be 0.05M, after mixing, obtain one containing BaCl
2cPP material solution;
With above-mentioned containing BaCl
2cPP material solution: the volume ratio of absolute ethyl alcohol=1:2, above-mentioned containing BaCl
2cPP material solution in add absolute ethyl alcohol, after mixing, at 4 DEG C of standing 12-16 hour;
By the solution after above-mentioned leaving standstill at 4 DEG C, the centrifugal 15min of 5000rpm, abandoning supernatant, with above-mentioned containing BaCl
2cPP material solution: H
2sO
4the volume ratio of=1:0.4, adds the H that concentration is 0.125M in precipitation
2sO
4, then use Filter paper filtering;
Get filtrate and dry 60min at 60-70 DEG C, dry to constant weight at 105 DEG C afterwards, obtain the CPP raw material of purifying;
The drafting of B, typical curve
The CPP raw material ultrapure water of above-mentioned purifying is prepared the series standard working fluid that concentration is 0.1mg/100mL, 0.5mg/100mL, 1mg/100mL, 5mg/100mL, 10mg/100mL, 20mg/100mL, adopt high performance liquid chromatograph to carry out detection to this series standard working fluid to analyze, drawing standard curve;
The detection of C, CPP finished product
A, Sample Purification on Single
A1, extraction
When sample is liquid diary product: take 20g sample, be settled to 50mL with the trichloroacetic acid that concentration is 1%, ultrasonic 10min, leave standstill and filter to obtain liquid to be clean;
When sample is solid-state dairy products: take 5g sample, is settled to 50mL, ultrasonic 10min after dissolving with the trichloroacetic acid that concentration is 1%, leaves standstill and filter to obtain liquid to be clean;
A2, purification
By solid-phase extraction column successively with 3mL methyl alcohol, the activation of 3mL water, the liquid to be clean getting proper volume is transferred in solid phase extraction column, use 3mL water and 3mL methanol wash successively, be eluted in 10mL tool plug test tube with 6mL ammoniated methanol after discarding leacheate, whole solid phase extraction procedure flow velocity is no more than 1mL/min, eluent is dried up with nitrogen at 50 DEG C, obtains residue;
B, detection and calculating
By the residue proper volume mobile phase A constant volume obtained after drying up, the mobile phase A adopted is trifluoroacetic acid aqueous solution, after crossing miillpore filter, adopt high performance liquid chromatograph to detect the supernatant obtained, go out the CPP content in finished product according to the calculated by peak area of typical curve and sample.
2. detection method as claimed in claim 1, wherein, described solid-phase extraction column is strong cat ion exchange column.
3. detection method as claimed in claim 1, wherein, the aperture of described miillpore filter is 0.2 μm.
4. detection method as claimed in claim 1, wherein, in the detecting step of described CPP finished product, the volume being transferred to the liquid to be clean in solid phase extraction column is identical with the volume of the mobile phase A for constant volume residue.
5. detection method as claimed in claim 4, wherein, described in the volume to be clean be transferred in solid phase extraction column be 6mL with the volume for the mobile phase A of constant volume residue.
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Non-Patent Citations (2)
| Title |
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| HPLC法测定保健食品中酪蛋白磷酸肽;田随安等;《中国卫生检验杂志》;20070228;第17卷(第2期);273，306 * |
| 冯凤琴等.高效液相色谱法测定奶粉中酪蛋白磷酸肽(CPP)含量.《中国食品学报》.2006,第6卷(第3期),128-132. * |
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