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Method for detecting A2-beta-casein in milk

A detection method, casein technology, applied in biochemical equipment and methods, measuring devices, biochemical instruments, etc., can solve the problems of time-consuming, high cost, cumbersome steps of separation and purification of casein, etc., and achieve simple purification process and low cost , to overcome the effect of long separation time

Active Publication Date: 2018-08-17
NEW HOPE SHUANGXI DAIRY SUZHOU CO LTD +1
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  • Summary
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Problems solved by technology

[0003] Since the cow's gene determines whether the β-casein in the milk produced is type A1 or A2, the type of β-casein in milk is mainly determined by the method of genetic identification of cows, which cannot determine the genotype of unknown cows. Types of beta-casein in milk samples, especially commercially available milk products
The existing casein analysis and detection methods can only detect αs-casein, β-casein and κ-casein by using high-performance liquid chromatography, gel electrophoresis or capillary electrophoresis, but cannot determine β-casein. Type A1 or A2 of casein, and the steps of separating and purifying casein in the pretreatment are cumbersome, time-consuming and expensive

Method used

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  • Method for detecting A2-beta-casein in milk
  • Method for detecting A2-beta-casein in milk
  • Method for detecting A2-beta-casein in milk

Examples

Experimental program
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Effect test

Embodiment 1

[0052] 1) Pre-treatment and detection of standard products: Weigh 1 mg of the purchased Sigma brand αs-casein, β-casein, and κ-casein standard products into three 1.5mL centrifuge tubes, add 500 μL buffer (from 6mol / L guanidine hydrochloride, 0.1mol / L Bis-Tris, 5.37mmol / L sodium citrate dihydrate), vortexed, centrifuged at 6000r / min for 15s, left at room temperature for 1h; then centrifuged at 6000r / min for 10min , after centrifugation, add 490 μL of 4.5 mol / L guanidine hydrochloride and 10 μL of β-mercaptoethanol, vortex and mix; finally filter with 0.45 μm PTFE membrane, and the filtrate can be used for detection by high performance liquid chromatography;

[0053] The chromatographic peaks of αs-casein, β-casein, and κ-casein standards are shown in the accompanying drawings in turn. figure 1 , figure 2 with image 3 , it can be clearly seen from the figure that the peak time of αs-casein is 38-39min, the peak time of κ-casein is 20-21min, and the peak time of β-casein i...

Embodiment 2

[0069] 1) Standard pretreatment and detection: Weigh 1 mg of the purchased Sigma brand αs-casein, β-casein, and κ-casein standards into three 1.5mL centrifuge tubes, add 600 μL buffer (from 7mol / L urea, 0.5% β-mercaptoethanol, 17.5mmol / L Bis-Tris), centrifuged at 6000r / min for 10min; removed 500μL of supernatant and transferred to another 1.5mL centrifuge tube, and added 500μL of the aforementioned buffer solution, After vortex mixing, filter with a 0.45 μm PTFE membrane, and the filtrate can be used for detection by high-performance liquid chromatography to obtain chromatographic peaks of αs-casein, β-casein, and κ-casein standards;

[0070] 2) Pretreatment and detection of type A1 or A2 milk: pipette 200 μL of skimmed or non-skimmed milk into a 1.5 mL centrifuge tube, add 600 μL of buffer (consisting of 7mol / L urea, 0.5% β-mercaptoethanol, 17.5mmol / L L Bis-Tris composition) was centrifuged at 6000r / min for 10min; 500μL of the supernatant was removed and transferred to anoth...

Embodiment 3

[0081] 1) Pre-treatment and detection of standard products: Weigh 1 mg of the purchased Sigma brand αs-casein, β-casein, and κ-casein standard products into three 1.5mL centrifuge tubes, add 800 μL buffer (from 7mol / L urea, 0.5% β-mercaptoethanol, 17.5mmol / L Bis-Tris), centrifuged at 6000r / min for 10min, took off the supernatant, and filtered it with a 0.45μm PTFE membrane, and the filtrate could be used for high-performance liquid chromatography detection. Obtain the chromatograms of αs-casein, β-casein, and κ-casein standard products;

[0082] 2) Pretreatment and detection of type A1 or A2 milk: pipette 200 μL of skimmed or non-skimmed milk into a 1.5 mL centrifuge tube, add 800 μL of buffer (consisting of 7mol / L urea, 0.5% β-mercaptoethanol, 17.5mmol / L L Bis-Tris), centrifuge at 6000r / min for 10min, remove the supernatant, and filter it with a 0.45μm PTFE filter membrane, the filtrate can be used for detection by high performance liquid chromatography, and the chromatograp...

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Abstract

The invention discloses a method for detecting A2-beta-casein in milk. The method comprises the following steps: respectively detecting the high performance liquid chromatograms of milks identified asA1 type and A2 type by genotype, comparing the high performance liquid chromatograms of the milks with the high performance liquid chromatograms of alpha s-casein, beta-casein and kappa-casein standard substances to determine the shape and appearance time of the chromatographic peak of each of A1-beta-casein and A2-beta-casein, and qualitatively detecting that whether beta-casein in unknown milkis A2-beta-casein or not; and successfully separating A2-beta-casein from the A2 type milk, measuring a corresponding relationship between the peak area and the concentration of the A2-beta-casein in a high performance liquid chromatogram, and quantitatively detecting the content of the A2-beta-casein in the unknown milk. The detection method is simple and convenient, and is suitable for large-scale promotion and application.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a method for detecting A2-β-casein in milk. Background technique [0002] Casein is the protein with the highest content in milk, accounting for about 80% of milk protein content. According to the amino acid composition and electrophoretic behavior, casein can be divided into αs-casein, β-casein and κ-casein, of which β-casein accounts for about 30-35% of the total bovine casein. There are many variants of β-casein, the most common ones are A1-β-casein and A2-β-casein, the difference between the two is that there is a variation in the 67th amino acid chain, the former is histidine, the latter Those are proline. Studies have found that A1-β-casein will be hydrolyzed in vivo to produce β-casomorphin (BCM-7), which can lead to ischemic heart disease, atherosclerosis, type I diabetes, and sudden neonatal death syndrome and some mental diseases, so dairy products containing A2-β...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02
CPCG01N30/02C12M45/05G01N30/06G01N30/88G01N2030/8831
Inventor 范光彩刘虎传郭建谭莲英钱成林宋艳梅夏忠悦林永裕何佳燕朱菲史寒琴
Owner NEW HOPE SHUANGXI DAIRY SUZHOU CO LTD
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