WO2018040846A1 - 检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法 - Google Patents

检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法 Download PDF

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WO2018040846A1
WO2018040846A1 PCT/CN2017/095900 CN2017095900W WO2018040846A1 WO 2018040846 A1 WO2018040846 A1 WO 2018040846A1 CN 2017095900 W CN2017095900 W CN 2017095900W WO 2018040846 A1 WO2018040846 A1 WO 2018040846A1
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casein
milk
minutes
capillary
rinse
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PCT/CN2017/095900
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English (en)
French (fr)
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刘宇
陈伟
陈云
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内蒙古蒙牛乳业（集团）股份有限公司
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Priority to NZ735717A priority Critical patent/NZ735717B2/en
Priority to AU2017232143A priority patent/AU2017232143B2/en
Publication of WO2018040846A1 publication Critical patent/WO2018040846A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44704—Details; Accessories
    • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
    • G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44791—Microapparatus

Definitions

  • the invention relates to the field of food.
  • the invention relates to a method of detecting A1 ⁇ -casein and A2 ⁇ -casein in milk.
  • the protein in milk mainly includes whey protein and casein.
  • Casein is divided into ⁇ -casein, ⁇ -casein and ⁇ -casein. Among them, ⁇ -casein accounts for about 30% of the total protein.
  • the milk containing both A1 ⁇ -casein and A2 ⁇ -casein is A1/A2 milk
  • the milk containing only A2 ⁇ -casein is A2 milk
  • the milk containing only A1 ⁇ -casein is A1 milk.
  • A2 ⁇ -casein is a natural affinity milk protein that contributes to the long-term health of infants and young children.
  • the present invention aims to solve at least to some extent one of the technical problems existing in the prior art. To this end, the present invention proposes a method for detecting A1 ⁇ -casein and A2 ⁇ -casein in cow's milk. The method can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in the milk, and the operation is simple and rapid.
  • the current identification methods are mainly focused on genetic means. However, this method is complicated to operate and the detection cost is high.
  • the present invention proposes a method for detecting A1 ⁇ -casein and A2 ⁇ -casein in cow's milk.
  • the method comprises: (1) sequentially heating, centrifuging, freezing and thawing the milk to obtain a stock solution to be tested; (2) pretreating the stock solution to be tested to obtain a liquid to be tested, wherein the pretreatment comprises mixing the stock solution to be tested with the pretreatment liquid; and (3) The test solution is detected by capillary electrophoresis to determine whether the milk contains A1 ⁇ -casein and A2 ⁇ -casein.
  • capillary electrophoresis can determine whether or not A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, for example, only A1 ⁇ -casein or A2 ⁇ -casein is present, or both A1 ⁇ -casein and A2 ⁇ -casein are present.
  • the milk contains fat
  • the fat can be well adsorbed on the inner wall surface of the capillary, and the double electric layer cannot be formed inside the capillary.
  • the effective separation of the protein cannot be achieved, and the fat is also likely to cause capillary blockage and affect the separation.
  • the casein in milk is mainly in the form of micelles.
  • the method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, and the operation is simple and rapid.
  • the above method for detecting A1 ⁇ -casein and A2 ⁇ -casein in cow's milk may further have the following additional technical features:
  • the heating is to heat the milk to 40 to 70 °C. Thereby, the milk fat removal rate is increased.
  • the centrifugation is carried out at 1500 to 10000 r/min for 5 to 40 minutes. Thereby, milk fat is sufficiently removed.
  • the freezing is carried out at -30 to -40 ° C for 5 to 60 minutes. Thereby, the casein micelle structure is destroyed.
  • the pretreatment liquid contains: 30 mmol/L of sodium dihydrogen phosphate; 0.1% by mass of hydroxypropylmethylcellulose; 7 mol/L of urea; and 0.05% by mass of dithiothreitol
  • the pH of the pretreatment solution was adjusted to 8.0 using a 0.1 mol/L NaOH solution. Thereby, the casein micelle structure is further destroyed.
  • the detection of the test solution is performed using an uncoated capillary. Thereby, the detection cost is reduced.
  • the step (3) comprises: activating the uncoated capillary tube and coating the activated uncoated capillary with a dynamic coating reagent to obtain a capillary with a dynamic coating. And using the capillary with dynamic coating for detection. Thereby, it is possible to accurately identify whether or not the milk contains A1 ⁇ - Casein and A2 ⁇ -casein.
  • the dynamic coating reagent contains: 200 mmol/L of trishydroxymethylaminomethane; 60 mmol/L of pentamethylenediamine; 120 mmol/L of cetyl ammonium bromide; and 80 mmol/L
  • the polyethylene glycol was adjusted to pH 3.0 with a 0.1 mol/L HCl solution.
  • the activation treatment comprises: rinsing the inner wall of the uncoated capillary with chromatographic grade methanol for 5-20 minutes, then rinsing with deionized water for 5-20 minutes, followed by using 1-4 mol/L NaOH solution. Rinse for 5 to 20 minutes, let stand for 5 to 30 minutes, and rinse with deionized water for 5 to 20 minutes. Thereby, in order to facilitate subsequent coating treatment.
  • the coating treatment comprises: rinsing with 0.1-0.5 mol/L NaOH solution for 3-5 minutes, then rinsing with deionized water for 3-5 minutes, followed by rinsing with the dynamic coating reagent. 3 to 10 minutes, let stand for 5 to 10 minutes, and finally rinse with buffer solution for 3 to 5 minutes.
  • a capillary tube having a dynamic coating reagent on the inner wall can be obtained.
  • the detecting condition comprises: the detecting wavelength is 200-450 nm; the detecting temperature is 0-40 ° C, preferably 25 ° C; the injection mode is pressure injection or vacuum injection, preferably pressure injection, pressure 0 to 1 psi, more preferably 0.5 psi; injection time is 1 to 20 s; operating voltage is 5 to 30 kv, preferably 20 to 30 kv; detection frequency is 4 to 18 Hz; capillary length is 30 to 100 cm, preferably 60 cm; capillary inner diameter is 25 to 75 ⁇ m.
  • the detecting wavelength is 200-450 nm
  • the detecting temperature is 0-40 ° C, preferably 25 ° C
  • the injection mode is pressure injection or vacuum injection, preferably pressure injection, pressure 0 to 1 psi, more preferably 0.5 psi
  • injection time is 1 to 20 s
  • operating voltage is 5 to 30 kv, preferably 20 to 30 kv
  • detection frequency is 4 to 18 Hz
  • capillary length is 30
  • FIG. 1 is a schematic flow chart showing a method for detecting A1 ⁇ -casein and A2 ⁇ -casein in cow's milk according to an embodiment of the present invention
  • FIG. 2 is a schematic flow chart showing a method for detecting A1 ⁇ -casein and A2 ⁇ -casein in cow's milk according to another embodiment of the present invention
  • 3 to 16 respectively show maps according to another embodiment of the present invention.
  • the present invention provides a method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk.
  • the method includes: heating, centrifuging, freezing, and thawing S100; pretreating S200; and detecting S300.
  • the method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk according to an embodiment of the present invention can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, and the operation is simple and rapid.
  • the sample to be tested is sequentially heated, centrifuged, frozen, and thawed to obtain a stock solution to be tested.
  • the method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, and the operation is simple and rapid.
  • the milk comprises: raw milk, pasteurized milk (pasteurized milk) or UHT milk (ultra-sterilized milk).
  • the heating is to heat the milk to 40 to 70 °C.
  • the inventors have found that under these conditions of heating, the milk fat can be better separated during centrifugation to increase the fat removal rate. At the same time, the effect on protein in milk is low. If the heating temperature is too high, the whey protein is easily denatured, resulting in low accuracy of the test results.
  • centrifugation is carried out at 1500 to 10000 r/min for 5 to 40 minutes.
  • the inventors have found that under the above-mentioned centrifugation conditions, the fat can be well separated from the milk and attached to the inner wall of the centrifuge tube, so that the fat can be effectively removed and prevented from adsorbing on the inner wall of the capillary, resulting in the formation of a double inside the capillary.
  • the effective separation of the protein is ultimately impossible, and the fat is also likely to cause capillary blockage and affect the separation.
  • the temperature of the heated milk is maintained during centrifugation.
  • the freezing is carried out at -30 to -40 ° C for 5 to 60 minutes.
  • the inventors have found that by freezing the milk after centrifugation to remove fat, and then performing subsequent thawing, after such treatment, the molecular force between casein can be destroyed, and the different ⁇ -casein configurations are completely separated for subsequent follow-up. Detection.
  • thawing is to restore the frozen product to room temperature.
  • the manner of restoring the room temperature is not strictly limited.
  • the frozen product may be allowed to stand at room temperature or may be heated to bring the frozen product to room temperature.
  • the stock solution to be tested is pretreated to obtain a liquid to be tested, wherein the pretreatment comprises mixing the stock solution to be tested with the pretreatment liquid.
  • the pretreatment comprises mixing the stock solution to be tested with the pretreatment liquid.
  • the pretreatment liquid contains: 30 mmol/L of sodium dihydrogen phosphate; 0.1% by mass of hydroxypropylmethylcellulose; 7 mol/L of urea; and 0.05% by mass of dithiothreitol.
  • the pH of the pretreatment solution was adjusted to 8.0 by a 0.1 mol/L NaOH solution.
  • the inventors have obtained a large amount of experiments to obtain the above-mentioned optimal pretreatment liquid, which can prevent protein adsorption on the inner wall of the capillary, simultaneously separate the casein micelles completely, and prevent the casein micelles from re-polymerizing together.
  • 1 mL of the stock solution to be tested is vortexed with 4 mL of the pretreatment liquid for 3 minutes, and allowed to stand at 4 ° C for 30 to 60 minutes to obtain a liquid to be tested.
  • test liquid is detected by capillary electrophoresis to determine whether or not the milk contains A1 ⁇ -casein and A2 ⁇ -casein.
  • capillary electrophoresis can accurately determine whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk. For example, first, capillary electrophoresis was used to determine A1 ⁇ -casein standards and A2 ⁇ -casein standards, and the peak positions of A1 ⁇ -casein and A2 ⁇ -casein were determined. Then, under the same detection conditions, capillary electrophoresis was used. If the test liquid is detected, if there is a characteristic peak at the peak position of A1 ⁇ -casein, it can be determined that the sample contains A1 ⁇ -casein; if a characteristic peak appears at the peak position of A2 ⁇ -casein, the sample can be determined.
  • the method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, and the operation is simple and rapid.
  • the test liquid is tested using an uncoated capillary.
  • Uncoated capillaries are inexpensive, have high re-use rates, and have low environmental requirements, which can reduce inspection costs.
  • step S300 includes:
  • S310 gives a capillary with a dynamic coating
  • the uncoated capillary is subjected to an activation treatment, and the activated uncoated capillary is subjected to a coating treatment using a dynamic coating reagent to obtain a capillary having a dynamic coating.
  • uncoated capillaries are inexpensive, have high re-use rates, and have low environmental requirements, which can reduce inspection costs.
  • the inventors have found that coating the capillary with the dynamic coating reagent can effectively prevent the adsorption of protein on the inner wall of the capillary to improve the accuracy of the detection result.
  • the dynamic coating reagent contains: 200 mmol/L of trishydroxymethylaminomethane; 60 mmol/L of pentanediamine; 120 mmol/L of cetyl ammonium bromide; and 80 mmol/L of poly Ethylene glycol, the pH of the dynamic coating reagent was adjusted to 3.0 using a 0.1 mol/L HCl solution.
  • the inventors have found through a large number of experiments that the types of dynamic coating reagents used to detect different proteins are different, and the reagents do not have universal applicability. Therefore, the inventors have screened a large number of experimentally to obtain the optimal composition of the reagents,
  • the dynamic coating reagent can effectively inhibit protein adsorption, improve experimental repeatability and capillary column utilization.
  • A1 ⁇ -casein and A2 ⁇ -casein can be effectively separated, so that the detection results are highly accurate. Excessive or too low concentrations of reagent components can affect the detection.
  • the pH of the dynamic coating reagent is adjusted to 3.0 to ensure consistency with the electrophoresis buffer system, so that the proteins are positively charged and the separation efficiency is improved.
  • the method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk according to an embodiment of the present invention can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, and the operation is simple and rapid.
  • the activation treatment comprises: rinsing the inner wall of the uncoated capillary with chromatographic grade methanol for 5-20 minutes, then rinsing the inner wall of the uncoated capillary with deionized water for 5-20 minutes, followed by 1-4 mol/L NaOH.
  • the solution was washed with the inner wall of the uncoated capillary for 5 to 20 minutes, allowed to stand for 5 to 30 minutes, and finally the inner wall of the uncoated capillary was rinsed with deionized water for 5 to 20 minutes. Flushing with methanol is designed to remove lipids from the inner walls of the capillary to prevent subsequent effects and subsequent detection of these lipids.
  • the rinsing with NaOH is intended to activate the silicon hydroxy group on the inner wall of the capillary to charge it.
  • the method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk according to an embodiment of the present invention can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, and the operation is simple and rapid.
  • the coating treatment comprises: rinsing the activated uncoated capillary inner wall with a 0.1-0.5 mol/L NaOH solution for 3 to 5 minutes, and then rinsing the activated uncoated capillary inner wall with deionized water. After 3 to 5 minutes, the activated uncoated capillary inner wall is washed with a dynamic coating reagent for 3 to 10 minutes, allowed to stand for 5 to 10 minutes, and finally the activated uncoated capillary inner wall is washed with a buffer solution for 3 to 5 minutes. Specifically, all of the above coating treatment steps were repeated 2 to 5 times.
  • the method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk according to an embodiment of the present invention can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, and the operation is simple and rapid.
  • the detection is carried out using a capillary having a dynamic coating.
  • the detection conditions include: the detection wavelength is 200-450 nm; the detection temperature is 0-40 ° C, preferably 25 ° C; the injection mode is pressure injection or vacuum injection, preferably pressure injection, pressure is 0 ⁇ 1 psi, more preferably 0.5 psi;
  • the injection time is 1 to 20 s; the working voltage is 5 to 30 kV, preferably 20 to 30 kV; the detection frequency is 4 to 18 Hz; the capillary length is 30 to 100 cm, preferably 60 cm; and the capillary inner diameter is 25 to 75 ⁇ m.
  • the inventors have optimized the experimental conditions to obtain optimal detection conditions, and the method can effectively separate and detect A1 ⁇ -casein and/or A2 ⁇ -casein. If the length of the capillary is too long, the detection time is too long, and the detection efficiency is low. When the voltage is too small, the detection time is too long, the detection efficiency is low, and the voltage time is too long, a certain amount of Joule heat will be generated, which will affect the detection result.
  • the method for detecting A1 ⁇ -casein and A2 ⁇ -casein in milk according to an embodiment of the present invention can accurately detect whether A1 ⁇ -casein and A2 ⁇ -casein are contained in milk, and the operation is simple and rapid.
  • the raw milk is tested for the presence of A1 ⁇ -casein and A2 ⁇ -casein according to the following method:
  • Preparation of dynamic coating reagent accurately prepare 100mL dynamic coating reagent with deionized water, so that the concentration of each component is 200mmol/L of trishydroxymethylaminomethane, 60mmol/L of pentamethylenediamine, 120mmol of cetylammonium bromide /L, polyethylene glycol 80 mmol/L, and the pH was adjusted to 3.0 with a 0.1 mol/L HCl solution.
  • Dynamic coating rinse the inner wall of the capillary with 0.1mol/L NaOH solution for 5min, rinse with deionized water for 5min, rinse the dynamic coating reagent for 10 minutes, let stand for 10 minutes, rinse the buffer solution for 5 minutes, and then cycle 3 times.
  • the detection device is a high-performance capillary electrophoresis instrument equipped with an ultraviolet light detector.
  • detection wavelength is 214nm
  • detection temperature is 25 °C
  • injection mode is pressure injection
  • pressure is 0.5 psi
  • injection time is 10s
  • working voltage is 30kv
  • detection frequency is 8Hz.
  • the A1 ⁇ -casein, A2 ⁇ -casein standard and the test solution are sequentially injected to obtain the A1 ⁇ -casein standard map (Fig. 3), the A2 ⁇ -casein standard map (Fig. 4) and the test solution.
  • the full map of the protein (Fig. 5), and then compare the peak time of the A1 ⁇ -casein and A2 ⁇ -casein standards with the full spectrum of the protein to be tested, and then initially determine that the milk contains both A1 ⁇ -casein and A2 ⁇ -case. protein.
  • This raw milk contains both A1 ⁇ -casein and A2 ⁇ -casein, which is A1/A2 type raw milk (ie ordinary milk).
  • An uncoated quartz capillary column (Bechman Coulter) with an inner diameter of 50 ⁇ m and a total length of 800 mm and an effective length of 700 mm was cut out, rinsed with methanol for 10 min, rinsed with deionized water for 10 min, and washed with 1 mol/L NaOH solution for 10 min. Leave for 10 min, then rinse with deionized water for 10 min.
  • Preparation of dynamic coating reagent accurately prepare 100mL dynamic coating reagent with deionized water, so that the concentration of each component is 200mmol/L of trishydroxymethylaminomethane, 60mmol/L of pentamethylenediamine, 120mmol of cetylammonium bromide /L, polyethylene glycol 80 mmol/L, and the pH was adjusted to 3.0 with a 0.1 mol/L HCl solution.
  • Dynamic coating rinse the inner wall of the capillary with 0.1mol/L NaOH solution for 5min, rinse with deionized water for 5min, rinse with dynamic coating reagent for 10 minutes, let stand for 10 minutes, rinse the buffer solution for 5 minutes, and then cycle 4 times.
  • the detection device is a high-performance capillary electrophoresis instrument equipped with an ultraviolet light detector.
  • detection wavelength is 214nm
  • detection temperature is 25 °C
  • injection mode is pressure injection
  • pressure is 0.5 psi
  • injection time is 10s
  • working voltage is 30kv
  • detection frequency is 8Hz.
  • the A1 ⁇ -casein, A2 ⁇ -casein standard and the test solution are sequentially injected to obtain the A1 ⁇ -casein standard map (Fig. 3), the A2 ⁇ -casein standard map (Fig. 4) and the test solution.
  • the full map of the protein (Fig. 8), and then the peak time of the A1 ⁇ -casein and A2 ⁇ -casein standards is compared with the full spectrum of the protein to be tested, and it is preliminarily determined that only A2 ⁇ -casein is contained in the milk.
  • the UHT milk was tested for the presence of A1 ⁇ -casein and A2 ⁇ -casein according to the method of Example 1, except that in step 1, the UHT pre-treatment was as follows:
  • UHT milk contains both A1 ⁇ -casein and A2 ⁇ -casein, which is type A1/A2 pasteurized milk (see Figure 11).
  • the separation map is shown in Fig. 13.
  • the raw milk which has not been centrifuged cannot completely separate A1 ⁇ -casein and A2 ⁇ -casein, which is prone to misjudgment.

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Abstract

一种检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法，包括：(1)将待测样品依次进行加热、离心、冷冻及解冻，以便得到待测储备液；(2)将待测储备液进行预处理，以便得到待测液，其中，预处理包括将待测储备液与预处理液进行混合；以及(3)利用毛细管电泳法对待测液进行检测，以便确定牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白。

Description

检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法 技术领域
本发明涉及食品领域。具体地，本发明涉及检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法。
背景技术
牛奶中蛋白质主要有乳清蛋白和酪蛋白两大类，酪蛋白又分为β-酪蛋白、α-酪蛋白，κ-酪蛋白三种，其中β-酪蛋白约占蛋白总量的30％，β-酪蛋白有两种主要的变异型，即A1β-酪蛋白和A2β-酪蛋白。既含有A1β-酪蛋白又含有A2β-酪蛋白的牛乳为A1/A2型牛奶，只含有A2β-酪蛋白的牛奶为A2型牛奶，只含有A1β-酪蛋白的牛奶为A1型牛奶。
母乳中的β-酪蛋白与A2β-酪蛋白在结构和消化特点方面更为接近，而与A1型则相对差异较大。但是，目前发表的数据显示，若由于缺乏母乳而需使用配方奶喂养的婴儿，如采用不含A1β-酪蛋白的配方奶以及对年龄稍大的婴儿采用不含A1β-酪蛋白的奶制品可能有助于降低多种不良作用或反应的风险，由此可见，A2β-酪蛋白是自然亲和的牛奶蛋白质，有助于婴幼儿的长远健康。
由于不是所有的奶牛都能产出只含有纯净A2β-酪蛋白而不含有A1β-酪蛋白的牛奶，现今，西方的奶牛中只有约30％的奶牛是纯正的A2奶牛，由其所产的牛奶只含有100％纯净的A2β-酪蛋白。
然而，对于牛奶中A1β-酪蛋白和A2β-酪蛋白的检测方法仍有待研究。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题之一。为此，本发明提出了一种检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法。利用该方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
需要说明的是，本发明是基于发明人的下列发现而完成的：
目前的鉴定手段主要集中于基因手段。然而，该方法操作复杂，检测成本较高。
有鉴于此，发明人经过大量实验发现，通过采用毛细管电泳法对牛奶进行检测，从而能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
为此，本发明提出了一种检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法。根据本发明的实施例，所述方法包括：(1)将所述牛乳依次进行加热、离心、冷冻及解冻，以便得到 待测储备液；(2)将所述待测储备液进行预处理，以便得到待测液，其中，所述预处理包括将所述待测储备液与预处理液进行混合；以及(3)利用毛细管电泳法对所述待测液进行检测，以便确定所述牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白。
发明人发现，利用毛细管电泳法能够确定牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，例如仅存在A1β-酪蛋白或A2β-酪蛋白，或者同时存在A1β-酪蛋白和A2β-酪蛋白。然而，由于牛奶中含有脂肪，脂肪可以很好的吸附于毛细管内壁表面而导致毛细管内部无法形成双电层，最终无法实现蛋白的有效分离，同时脂肪还容易导致毛细管阻塞而影响分离。此外，牛奶中的酪蛋白主要是以胶束的形式存在的，若直接进行毛细管电泳，各类型的酪蛋白之间无法分离，导致检测失败。为此，发明人经过大量实验发现，先将牛奶进行离心处理，以充分除去脂肪，避免脂肪对后续检测的影响。进一步地，离心之前，将牛奶进行加热，除脂肪效果较好。接着，通过将离心后的牛奶进行冷冻及解冻，以破坏酪蛋白胶束结构，从而游离出β-酪蛋白，以便于后续检测。进一步地，利用预处理液使酪蛋白胶束进一步分离，以充分游离出β-酪蛋白。由此，根据本发明实施例检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
根据本发明的实施例，上述检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法还可以具有下列附加技术特征：
根据本发明的实施例，所述加热是将所述牛乳加热至40～70℃。由此，以提高乳脂肪去除率。
根据本发明的实施例，所述离心是在1500～10000r/min下进行5～40分钟。由此，以充分除去乳脂肪。
根据本发明的实施例，所述冷冻是在-30～-40℃下进行5～60分钟。由此，以破坏酪蛋白胶束结构。
根据本发明的实施例，所述预处理液含有：30mmol/L的磷酸二氢钠；0.1质量％的羟丙基甲基纤维素；7mol/L的尿素；以及0.05质量％二硫苏糖醇，利用0.1mol/L的NaOH溶液调节所述预处理液的pH值至8.0。由此，以进一步破坏酪蛋白胶束结构。
根据本发明的实施例，利用未涂层毛细管对所述待测液进行所述检测。由此，以降低检测成本。
根据本发明的实施例，步骤(3)包括：将所述未涂层毛细管进行活化处理，并利用动态涂层试剂将活化后的未涂层毛细管进行涂层处理，得到具有动态涂层的毛细管；以及利用所述具有动态涂层的毛细管进行检测。由此，能够准确地鉴定出牛乳中是否含有A1β- 酪蛋白及A2β-酪蛋白。
根据本发明的实施例，所述动态涂层试剂含有：200mmol/L的三羟甲基氨基甲烷；60mmol/L的戊二胺；120mmol/L的十六烷基溴化铵；以及80mmol/L的聚乙二醇，利用0.1mol/L的HCl溶液调节所述动态涂层试剂的pH值至3.0。由此，能够准确地鉴定出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白。
根据本发明的实施例，所述活化处理包括：用色谱级甲醇冲洗所述未涂层毛细管内壁5-20分钟，再用去离子水冲洗5-20分钟，接着用1～4mol/L NaOH溶液冲洗5～20分钟，静置5～30分钟，最后用去离子水冲洗5～20分钟。由此，以便于后续涂层处理。
根据本发明的实施例，所述涂层处理包括：用0.1～0.5mol/L的NaOH溶液冲洗3～5分钟，再用去离子水冲洗3～5分钟，接着用所述动态涂层试剂冲洗3～10分钟，静置5～10分钟，最后用缓冲溶液冲洗3～5分钟。由此，能够得到内壁具有动态涂层试剂的毛细管。
根据本发明的实施例，所述检测条件包括：检测波长为200～450nm；检测温度为0～40℃，优选25℃；进样方式为压力进样或真空进样，优选压力进样，压力为0～1psi，更优选0.5psi；进样时间为1～20s；工作电压为5～30kv，优选20～30kv；检测频率为4～18Hz；毛细管长度为30～100cm，优选60cm；毛细管内径为25～75μm。由此，能够准确地检测牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法的流程示意图；
图2显示了根据本发明另一个实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法的流程示意图；以及
图3～16分别显示了根据本发明另一个实施例的图谱。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
本发明提出了一种检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法。根据本发明的实施例，参见图1，该方法包括：加热、离心、冷冻及解冻S100；预处理S200；以及检测S300。由此，根据本发明实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
下面将详细描述检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法。
S100加热、离心、冷冻及解冻
在该步骤中，将待测样品依次进行加热、离心、冷冻及解冻，以便得到待测储备液。
由于牛奶中含有脂肪，脂肪可以很好的吸附于毛细管内壁表面而导致毛细管内部无法形成双电层，最终无法实现蛋白的有效分离，同时脂肪还容易导致毛细管阻塞而影响分离。为此，发明人经过大量实验发现，先将牛奶进行离心处理，以充分除去脂肪，避免脂肪对后续检测的影响。进一步地，离心之前，将牛奶进行加热，除脂肪效果较好。接着，通过将离心后的牛奶进行冷冻及解冻，以破坏酪蛋白胶束结构，以便于后续检测。由此，根据本发明实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
根据本发明的实施例，牛乳包括：生牛乳、巴氏乳(经过巴氏杀菌处理的乳品)或UHT乳(经过超高温杀菌处理的乳品)。
根据本发明的实施例，加热是将牛乳加热至40～70℃。发明人发现，在此条件下加热，乳脂肪在离心过程中能更好的被分离，以提高脂肪的去除率。同时，对于牛奶中蛋白质影响较低。若加热温度过高，容易导致乳清蛋白变性，导致检测结果的准确性较低。
根据本发明的实施例，离心是在1500～10000r/min下进行5～40分钟。发明人发现，在上述离心处理条件下，脂肪能够很好的从牛奶中分离出来，贴附在离心管内壁上方，从而能够有效地除去脂肪，防止其吸附于毛细管内壁上导致毛细管内部无法形成双电层，最终无法实现蛋白的有效分离，同时脂肪还容易导致毛细管阻塞而影响分离。具体地，离心过程中保持加热后的牛乳的温度。
根据本发明的实施例，冷冻是在-30～-40℃下进行5～60分钟。发明人发现，通过将离心除去脂肪后的牛奶进行冷冻，再进行后续的解冻，如此处理后，能够破坏酪蛋白之间的分子作用力，使不同β-酪蛋白构型彻底分开，以便于后续检测。
根据本发明的实施例，解冻是将冷冻后的产物恢复至室温。具体地，对于恢复室温的方式不作严格限定，例如，可以将冷冻后的产物于室温条件下静置，也可以进行加热，使冷冻后的产物达到室温。
S200预处理
在该步骤中，将待测储备液进行预处理，以便得到待测液，其中，预处理包括将待测储备液与预处理液进行混合。发明人发现，通过将待测储备液与预处理液混合，能够进一步破坏酪蛋白胶束结构，使β-酪蛋白游离，防止重新聚合，以便于后续检测，保证检测结果的准确性。
根据本发明的实施例，预处理液含有：30mmol/L的磷酸二氢钠；0.1质量％的羟丙基甲基纤维素；7mol/L的尿素；以及0.05质量％二硫苏糖醇，利用0.1mol/L的NaOH溶液调节预处理液的pH值至8.0。发明人经过大量实验得到上述最优预处理液，该预处理液能够防止蛋白在毛细管内壁吸附，同时使酪蛋白胶束彻底分离，且防止各酪蛋白胶束重新聚合在一起。
根据本发明的实施例，将1mL待测储备液与4mL预处理液旋涡振荡3分钟，于4℃下静置30～60分钟，得到待测液。
S300检测
在该步骤中，利用毛细管电泳法对待测液进行检测，以便确定牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白。
发明人发现，利用毛细管电泳法能够准确地确定牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白。例如，首先，分别利用毛细管电泳测定A1β-酪蛋白标准品以及A2β-酪蛋白标准品，能够确定A1β-酪蛋白和A2β-酪蛋白的出峰位置；然后，在相同检测条件下，利用毛细管电泳对待测液进行检测，若在A1β-酪蛋白的出峰位置出现具有特征峰，则可确定样品中含有A1β-酪蛋白；若在A2β-酪蛋白的出峰位置出现特征峰，则可确定样品中含有A2β-酪蛋白；若在A1β-酪蛋白和A2β-酪蛋白的出峰位置均出现相应的特征峰，则可确定样品中同时含有A1β-酪蛋白和A2β-酪蛋白。由此，根据本发明实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
根据本发明的实施例，利用未涂层毛细管对待测液进行检测。未涂层毛细管价格低廉，重复使用率高，对环境要求低，可以降低检测成本。
根据本发明的实施例，参见图2，步骤S300包括：
S310得到具有动态涂层的毛细管
在该步骤中，将未涂层毛细管进行活化处理，并利用动态涂层试剂将活化后的未涂层毛细管进行涂层处理，得到具有动态涂层的毛细管。
由于未涂层的毛细管内壁的硅羟基在被氢氧化钠活化后容易吸附蛋白质，无法形成双电层而导致样品无法彻底分离，同时出现不同程度的电渗流而导致毛细管电泳的基线不平。 但是，未涂层毛细管价格低廉，重复使用率高，对环境要求低，可以降低检测成本。
发明人发现，利用该动态涂层试剂对毛细管进行涂层处理，可以有效地防止蛋白质在毛细管内壁的吸附，以提高检测结果的准确性。
根据本发明的实施例，动态涂层试剂含有：200mmol/L的三羟甲基氨基甲烷；60mmol/L的戊二胺；120mmol/L的十六烷基溴化铵；以及80mmol/L的聚乙二醇，利用0.1mol/L的HCl溶液调节动态涂层试剂的pH值至3.0。
发明人经过大量实验发现，检测不同蛋白质所用的动态涂层试剂的种类有所不同，该试剂并不具有普遍适用性，由此，发明人经过大量实验筛选得到该试剂的最优组成成分，具有该动态涂层试剂能够有效的抑制蛋白质吸附，提高实验重复性及毛细管柱利用率。同时，能够有效地分离A1β-酪蛋白及A2β-酪蛋白，使得检测结果准确性较高。试剂组分浓度过高或者过低都会对检测造成影响。此外，将动态涂层试剂的pH值调节至3.0，以保证其与电泳缓冲液体系的一致性，使得蛋白质均带正电荷，提高分离效率。由此，根据本发明实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
根据本发明的实施例，活化处理包括：用色谱级甲醇冲洗未涂层毛细管内壁5～20分钟，再用去离子水冲洗未涂层毛细管内壁5～20分钟，接着用1～4mol/L NaOH溶液冲洗未涂层毛细管内壁5～20分钟，静置5～30分钟，最后用去离子水冲洗未涂层毛细管内壁5～20分钟。利用甲醇进行冲洗，目的是去除毛细管内壁的脂类物质，以防止这些脂类物质影响后续操作以及最终的检测结果。利用NaOH进行冲洗，目的是为了活化毛细管内壁硅羟基，使其带电。由此，根据本发明实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
根据本发明的实施例，涂层处理包括：用0.1～0.5mol/L的NaOH溶液冲洗活化后的未涂层毛细管内壁3～5分钟，再用去离子水冲洗活化后的未涂层毛细管内壁3～5分钟，接着用动态涂层试剂冲洗活化后的未涂层毛细管内壁3～10分钟，静置5～10分钟，最后用缓冲溶液冲洗活化后的未涂层毛细管内壁3～5分钟。具体地，重复上述所有涂层处理步骤2～5次。由此，根据本发明实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
S320检测
在该步骤中，利用具有动态涂层的毛细管进行检测。
根据本发明的实施例，检测条件包括：检测波长为200～450nm；检测温度为0～40℃，优选25℃；进样方式为压力进样或真空进样，优选压力进样，压力为0～1psi，更优选0.5psi； 进样时间为1～20s；工作电压为5～30kv，优选20～30kv；检测频率为4～18Hz；毛细管长度为30～100cm，优选60cm；毛细管内径为25～75μm。
发明人经过大量实验优化得到最优检测条件，利用该方法能够有效地分离检出A1β-酪蛋白和/或A2β-酪蛋白。毛细管长度过长，使检测时间过长，检测效率较低；电压过小，检测时间过长，检测效率较低，电压时间过长，将产生一定的焦耳热，对检测结果造成影响。由此，根据本发明实施例的检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法能够准确地检测出牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，且操作简便、快速。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
在该实施例中，按照下列方法检测生牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白：
1、生牛乳前处理：
取30mL待测生牛乳，置于50mL离心管中，加热至60℃，于4000r/min条件下，离心15min，除去上层乳脂肪；然后置于-40℃冰箱中冷冻20min后；取出恢复至室温状态，制得待测储备液。
2、预处理液的配制：
用去离子水配置100ml样品缓冲液，使得各成分浓度分别为磷酸二氢钠浓度为30mmol/L、羟丙基甲基纤维素0.1质量％、尿素7mol/L、二硫苏糖醇(DTT)0.05质量％，用0.1mol/L的NaOH溶液调节pH值至8.0。
3、电泳缓冲溶液的配制：
用去离子水准确配制100ml缓冲溶液，使得各成分浓度达到如下：磷酸二氢钠60mmol/L，尿素7mol/L，十六烷基溴化铵120mmol/L，羟丙基甲基纤维素0.1质量％，乙二胺四乙酸二钠80mmol/L，用0.5mol/L磷酸溶液调节pH至3.0。
4、待测液的配制：
取1mL待测储备液，添加4mL预处理液，漩涡震荡3min，于4℃静置60min，制得待测液。
5、未涂层毛细管活化：
截取内径为50μm，总长度为700mm，有效长度为600mm的未涂层石英毛细管柱(美国Bechman Coulter)，用色谱级甲醇冲洗10min，去离子水冲洗10min，1mol/L的NaOH溶 液冲洗10min，静置10min，然后用去离子水冲洗10min。
6、动态涂层处理：
①动态涂层试剂的配制：用去离子水准确配制100mL动态涂层试剂，使得各成分浓度为三羟甲基氨基甲烷200mmol/L，戊二胺60mmol/L，十六烷基溴化铵120mmol/L，聚乙二醇80mmol/L，用0.1mol/L的HCl溶液调节pH值至3.0。
②动态涂层：用0.1mol/L的NaOH溶液冲洗毛细管内壁5min，去离子水冲洗5min，动态涂层试剂冲洗10分钟，静置10分钟，缓冲溶液冲洗5分钟，如此循环3次即可。
7、A1β-酪蛋白、A2β-酪蛋白标准品、待测液的测定：
①检测设备为配备紫外光检测器的高效毛细管电泳仪。
②检测设备参数的确定：检测波长为214nm，检测温度为25℃，进样方式为压力进样，压力为0.5psi，进样时间为10s，工作电压为30kv；检测频率为8Hz。
③将配置好的A1β-酪蛋白、A2β-酪蛋白标准品以及待测液依次进样，得到A1β-酪蛋白标准图(图3)、A2β-酪蛋白标准图(图4)以及待测液的蛋白全图谱(图5)，然后将A1β-酪蛋白、A2β-酪蛋白标准品出峰时间与待测液蛋白全图谱对比，即可初步确定牛乳中同时含有A1β-酪蛋白和A2β-酪蛋白。
④内标法进一步准确定性β-酪蛋白：取待测液，分别加入A1β-酪蛋白、A2β-酪蛋白标准品，重复上述分离步骤，获得加标准品的全蛋白图谱(图6)、(图7)。
可以看出，图5中，生牛乳中35分钟左右出现两个峰，其中左侧峰低于右侧峰，而图6中，含有A1β-酪蛋白标准品的生牛乳在35分钟左右出现两个峰中，左侧峰高于右侧峰，由此说明左侧峰为A1β-酪蛋白对应峰。同理，图7中右侧峰为A2β-酪蛋白。
⑤生牛乳的鉴定：此生牛乳同时含有A1β-酪蛋白和A2β-酪蛋白，为A1/A2型生牛乳(即普通牛奶)。
实施例2
在该实施例中，按照下列方法检测巴氏乳中是否含有A1β-酪蛋白及A2β-酪蛋白：
1、巴氏乳前处理：
取20mL待测巴氏乳，置于50mL离心管中，加热至50℃，于3000r/min条件下，离心20min，除去上层乳脂肪；然后置于-40℃冰箱中冷冻10min后；取出恢复至室温状态，制得待测储备液。
2、预处理液的配制：
用去离子水配置100ml样品缓冲液，使得各成分浓度分别为磷酸二氢钠浓度30mmol/L、 羟丙基甲基纤维素0.1质量％、尿素7mol/L、二硫苏糖醇(DTT)0.05质量％，用0.1mol/L的NaOH溶液调节pH值至8.0。
3、电泳缓冲溶液的配制：
用去离子水准确配制100mL缓冲溶液，使得各成分浓度达到如下：磷酸二氢钠60mmol/L，尿素7mol/L，十六烷基溴化铵120mmol/L，羟丙基甲基纤维素0.1质量％，乙二胺四乙酸二钠80mmol/L，用0.5mol/L磷酸溶液调节pH至3.0。
4、待测液的配制：
取1mL待测储备液，添加4mL预处理液，漩涡震荡3min，于4℃静置60min，制得待测液。
5、未涂层毛细管活化：
截取内径为50μm，总长度为800mm，有效长度为700mm的未涂层石英毛细管柱(美国Bechman Coulter)，用色谱级甲醇冲洗10min，去离子水冲洗10min，1mol/L的NaOH溶液冲洗10min，静置10min，然后用去离子水冲洗10min。
6、动态涂层处理：
①动态涂层试剂的配制：用去离子水准确配制100mL动态涂层试剂，使得各成分浓度为三羟甲基氨基甲烷200mmol/L，戊二胺60mmol/L，十六烷基溴化铵120mmol/L，聚乙二醇80mmol/L，用0.1mol/L的HCl溶液调节pH值至3.0。
②动态涂层：用0.1mol/L的NaOH溶液冲洗毛细管内壁5min，去离子水冲洗5min，动态涂层试剂冲洗10分钟，静置10分钟，缓冲溶液冲洗5分钟，如此循环4次即可。
7、A1β-酪蛋白、A2β-酪蛋白标准品、待测液的测定：
①检测设备为配备紫外光检测器的高效毛细管电泳仪。
②检测设备参数的确定：检测波长为214nm，检测温度为25℃，进样方式为压力进样，压力为0.5psi，进样时间为10s，工作电压为30kv；检测频率为8Hz。
③将配置好的A1β-酪蛋白、A2β-酪蛋白标准品以及待测液依次进样，得到A1β-酪蛋白标准图(图3)、A2β-酪蛋白标准图(图4)以及待测液的蛋白全图谱(图8)，然后将A1β-酪蛋白、A2β-酪蛋白标准品出峰时间与待测液蛋白全图谱对比，即可初步确定牛乳中只含有A2β-酪蛋白。
④内标法进一步准确定性β-酪蛋白：取待测液，分别加入A1β-酪蛋白、A2β-酪蛋白标准品，重复上述分离步骤，获得加标准品的全蛋白图谱(图9)、(图10)。
可以看出，图10中，生牛乳中35分钟左右出现一个特征峰，说明此峰为A2β-酪蛋白对应峰。
⑤巴氏乳的鉴定：此巴氏杀菌乳只含有A2β-酪蛋白，为A2型牛奶。
实施例3
按照实施例1的方法检测UHT乳中是否含有A1β-酪蛋白及A2β-酪蛋白，区别在于，步骤1中，UHT乳前处理操作如下：
取25mL待测UHT乳，置于50mL离心管中，加热至60℃，于6000r/min条件下，离心30min，除去上层乳脂肪；然后置于-40℃冰箱中冷冻10min后；取出恢复至室温状态，制得待测储备液。
UHT乳的鉴定：此UHT乳同时含有A1β-酪蛋白和A2β-酪蛋白，为A1/A2型巴氏杀菌乳(见图11)。
对比例1
在该对比例中，按照实施例1的方法检测生牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，区别在于，不含步骤6。
分离图谱如图12所示，可见动态涂层分离效果明显好于未涂层裸管。
对比例2
在该对比例中，按照实施例1的方法检测生牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，区别在于，步骤1中，生牛乳不进行离心处理，直接进行冷冻处理。
分离图谱如图13所示，未经离心处理的生牛乳无法完全分离出A1β-酪蛋白和A2β-酪蛋白，从而容易出现误判。
对比例3
在该对比例中，按照实施例1的方法检测生牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，区别在于，步骤1中，生牛乳未进行冷冻处理。
结果如图14所示，可以看出，未经冷冻处理的生牛乳无法完全分离出A1β-酪蛋白和A2β-酪蛋白，从而容易出现误判。
对比例4
在该对比例中，按照实施例1的方法检测生牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，区别在于，预处理液中不含有尿素。
结果如图15所示。可以看出，利用不含尿素的预处理液进行处理，无法完全分离出A1β-酪蛋白和A2β-酪蛋白，从而容易造成误判。
对比例5
在该对比例中，按照实施例1的方法检测生牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白，区别在于，动态涂层试剂中不含有聚乙二醇。
结果如图16所示。可以看出，利用不含聚乙二醇的动态涂层试剂进行涂层，并利用所得到的具有动态涂层的毛细管进行检测，无法完全分离出A1β-酪蛋白和A2β-酪蛋白，从而容易造成误判。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (10)

  1. 一种检测牛乳中A1β-酪蛋白及A2β-酪蛋白的方法，其特征在于，包括：
    (1)将所述牛乳依次进行加热、离心、冷冻及解冻，以便得到待测储备液；
    (2)将所述待测储备液进行预处理，以便得到待测液，其中，所述预处理包括将所述待测储备液与预处理液进行混合；以及
    (3)利用毛细管电泳法对所述待测液进行检测，以便确定所述牛乳中是否含有A1β-酪蛋白及A2β-酪蛋白。
  2. 根据权利要求1所述方法，其特征在于，所述加热是将所述牛乳加热至40～70℃。
  3. 根据权利要求1所述方法，其特征在于，所述离心是在1500～10000r/min下进行5～40分钟。
  4. 根据权利要求1所述方法，其特征在于，所述冷冻是在-30～-40℃下进行5～60分钟。
  5. 根据权利要求1所述方法，其特征在于，所述预处理液含有：
    30mmol/L的磷酸二氢钠；
    0.1质量％的羟丙基甲基纤维素；
    7mol/L的尿素；以及
    0.05质量％二硫苏糖醇，
    利用0.1mol/L的NaOH溶液调节所述预处理液的pH值至8.0。
  6. 根据权利要求1所述方法，其特征在于，利用未涂层毛细管对所述待测液进行所述检测。
  7. 根据权利要求6所述方法，其特征在于，步骤(3)包括：
    将所述未涂层毛细管进行活化处理，并利用动态涂层试剂将活化后的未涂层毛细管进行涂层处理，得到具有动态涂层的毛细管；以及
    利用所述具有动态涂层的毛细管进行检测。
  8. 根据权利要求7所述方法，其特征在于，所述动态涂层试剂含有：
    200mmol/L的三羟甲基氨基甲烷；
    60mmol/L的戊二胺；
    120mmol/L的十六烷基溴化铵；以及
    80mmol/L的聚乙二醇，
    利用0.1mol/L的HCl溶液调节所述动态涂层试剂的pH值至3.0。
  9. 根据权利要求7所述方法，其特征在于，
    所述活化处理包括：
    用色谱级甲醇冲洗所述未涂层毛细管内壁5～20分钟，再用去离子水冲洗5～20分钟，接着用1～4mol/L NaOH溶液冲洗5～20分钟，静置5～30分钟，最后用去离子水冲洗5～20分钟，
    所述涂层处理包括：
    用0.1～0.5mol/L的NaOH溶液冲洗所述活化后的未涂层毛细管内壁3～5分钟，再用去离子水冲洗3～5分钟，接着用所述动态涂层试剂冲洗3～10分钟，静置5～10分钟，最后用缓冲溶液冲洗3～5分钟。
  10. 根据权利要求1所述方法，其特征在于，所述检测条件包括：
    检测波长为200～450nm；
    检测温度为0～40℃，优选25℃；
    进样方式为压力进样或真空进样，优选压力进样，压力为0～1psi，更优选0.5psi；
    进样时间为1～20s；
    工作电压为5～30kv，优选20～30kv；
    检测频率为4～18Hz；
    毛细管长度为30～100cm，优选60cm；
    毛细管内径为25～75μm。
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