阅读说明：本技术 一种母乳α-淀粉酶活性检测方法 (Method for detecting activity of alpha-amylase of breast milk ) 是由 李达 康学军 于 2020-06-30 设计创作，主要内容包括：本发明公开一种母乳α-淀粉酶活性检测方法,包括如下步骤：将新鲜母乳样本与饱和硫酸铵溶液混合,充分震荡摇匀后,离心取清液,然后运用碘-淀粉比色法实现母乳α-淀粉酶活性测定。本方法能在实现澄清母乳样本的同时,不损伤α-淀粉酶活性的目的,且不用大比例稀释待测母乳,使得分光光度法能够准确检测母乳α-淀粉酶活性值。(The invention discloses a method for detecting the activity of alpha-amylase of breast milk, which comprises the following steps: mixing a fresh breast milk sample with a saturated ammonium sulfate solution, fully shaking and shaking uniformly, centrifuging to obtain a clear solution, and then measuring the activity of the alpha-amylase of the breast milk by using an iodine-starch colorimetric method. The method can realize the purpose of clarifying a breast milk sample without damaging the alpha-amylase activity, and does not dilute the breast milk to be detected in a large proportion, so that the spectrophotometry can accurately detect the alpha-amylase activity value of the breast milk.)

1. A method for detecting the activity of alpha-amylase in breast milk is characterized by comprising the following steps: mixing a fresh breast milk sample with a saturated ammonium sulfate solution, fully shaking and shaking uniformly, centrifuging to obtain a clear solution, and then measuring the activity of the alpha-amylase of the breast milk by using an iodine-starch colorimetric method.

2. The method of claim 1, wherein the volume ratio of fresh breast milk to saturated ammonium sulfate solution is 1:1 to 1: 5.

3. The method of claim 1, wherein the oscillation condition is: shaking the vortex apparatus for 1-5 min.

4. The method according to claim 1, wherein the centrifugation condition is centrifugation for 2 to 10min, and the set rotation speed is 2000 to 10000 r/min.

5. The method of claim 1, comprising the steps of:

(1) 1ml of fresh breast milk is sucked into a plastic test tube with the specification of 4ml, and then 1ml of saturated ammonium sulfate solution is added;

(2) placing the plastic test tube on a vortex instrument, shaking for 1min, and mixing thoroughly;

(3) placing the plastic test tube into a centrifuge for centrifuging for 5min, and setting the rotating speed to be 5000 r/min;

(4) taking 100 mu l of centrifuged clear liquid to be placed in a glass cuvette, wherein the glass cuvette contains 2ml of starch solution with the concentration of 0.8 mg/ml;

(5) dripping iodine/potassium iodide solution under the condition of keeping out of the sun, and reacting for 5-20 min;

(6) quickly putting the sample into a visible spectrophotometer, and reading an absorbance value Abs;

(7) substituting the absorbance value Abs into a calculation formula to obtain the activity value of the breast milk alpha-amylase.

6. The method according to claim 5, characterized in that the expression for the activity value AMY of breast milk alpha-amylase, calculated on the basis of the addition of 2ml of a starch solution at a concentration of 0.8mg/ml, is (in U/L):

wherein the content of the first and second substances,representing the hydrolysis degree of the starch in the tube, the size range is 0-1,meaning that under the experimental conditions, amylase in 100. mu.l of the centrifuged supernatant hydrolyses 0.4mg of starch within 15 minutes, giving an activity of 800 units.

Technical Field

The invention relates to a method for detecting the activity of alpha-amylase in breast milk, in particular to a pretreatment method for detecting the alpha-amylase with extremely low content of active ingredients in the breast milk.

Background

Neither a natural substrate starch detection method nor a method for determining a substrate (such as reducing maltose) based on molecular composition based on emulsion characteristics of natural breast milk can effectively detect alpha-amylase activity of breast milk based on spectrophotometry, which requires sample pretreatment of breast milk before detection.

At present, domestic and domestic pretreatment methods include a method of firstly centrifuging and defatting a breast milk sample at a high speed, diluting the breast milk sample by a specific reagent (such as phosphate buffer solution) at a specific pH value in a certain proportion to prepare a breast milk sample to be detected, and the like. The method has the problems that the breast milk cannot be clarified completely, and a large error exists when the activity detection is carried out based on the spectrophotometry.

Disclosure of Invention

The purpose of the invention is as follows: in order to solve the technical problems in the prior art, the invention provides a method for detecting the activity of alpha-amylase of breast milk, in particular a pretreatment method for detecting the alpha-amylase with extremely low content of active ingredients in the breast milk.

The technical scheme is as follows: in order to achieve the purpose, the technical scheme of the invention is as follows: a method for detecting the activity of alpha-amylase in breast milk is characterized by comprising the following steps: mixing a fresh breast milk sample with a saturated ammonium sulfate solution, fully shaking and shaking uniformly, centrifuging to obtain a clear solution, and then measuring the activity of the alpha-amylase of the breast milk by using an iodine-starch colorimetric method.

Preferably, the volume ratio of the fresh breast milk to the saturated ammonium sulfate solution is 1: 1-1: 5, and further preferably, the volume ratio of the fresh breast milk to the saturated ammonium sulfate solution is 1: 1.

The oscillation conditions are as follows: shaking on a vortex apparatus for 1-5 min, and in a preferred embodiment shaking on a vortex apparatus for 1 min.

Preferably, the centrifugation condition is centrifugation for 2-10 min, and the set rotation speed is 2000-10000 r/min, and in a preferred embodiment, the centrifugation condition is centrifugation for 5min and the rotation speed is 5000 r/min.

In a preferred embodiment, the determination is carried out as follows:

(1) 1ml of fresh breast milk is sucked into a plastic test tube with the specification of 4ml, and then 1ml of saturated ammonium sulfate solution is added;

(2) placing the plastic test tube on a vortex instrument, shaking for 1min, and mixing thoroughly;

(3) placing the plastic test tube into a centrifuge for centrifugation for 5 minutes, and setting the rotating speed to be 5000 r/min;

(4) taking 100 mu l of centrifuged clear liquid to be placed in a glass cuvette, wherein the glass cuvette contains 2ml of starch solution with the concentration of 0.8 mg/ml;

(5) and (3) dropwise adding an iodine/potassium iodide solution under the condition of keeping out of the sun, reacting for 5-20 min, preferably, after reacting for 5-20 min under the condition of keeping the temperature at 37 ℃ (keeping out of the sun), dropwise adding an iodine/potassium iodide solution with the concentration of 50mmol/L, wherein the volume ratio of the solution to the starch solution in the glass cuvette is 1: 10-1: 50, more preferably, in a ratio of 1: 20;

(6) quickly putting the sample into a visible spectrophotometer, and reading an absorbance value Abs;

(7) substituting the absorbance value Abs into a calculation formula to obtain the activity value of the breast milk alpha-amylase.

Wherein, the calculation formula of the breast milk alpha-amylase activity value AMY is (unit U/L) based on the addition of 2ml of starch solution with the concentration of 0.8 mg/ml:

wherein the content of the first and second substances,

representing the hydrolysis degree of the starch in the tube, the size range is 0-1,

meaning that under the experimental conditions, amylase in 100. mu.l of the centrifuged supernatant hydrolyses 0.4mg of starch within 15 minutes, giving an activity of 800 units.

Has the advantages that: compared with the prior art, the invention has the following advantages:

(1) the invention unexpectedly discovers that the method can realize the purpose of clarifying a breast milk sample without damaging the activity of alpha-amylase by adopting a saturated ammonium sulfate solution as a reaction reagent, and does not dilute the breast milk to be detected in a large proportion (the activity of the alpha-amylase of the breast milk is extremely low), so that the spectrophotometry can accurately detect the activity value of the alpha-amylase of the breast milk;

(2) the pretreatment method can effectively coagulate fat in breast milk, can effectively prevent experimental error caused by the fact that the fat can not enter the breast milk sample again in the centrifugal defatting process, and also improves operation feasibility;

(3) because the pretreatment method is to add the volume of the breast milk sample into the breast milk sample for reaction, the breast milk to be detected does not need to be diluted in a large proportion (the activity of alpha-amylase of the breast milk is extremely low), and the accuracy of a spectrophotometry detection result is ensured;

(4) because the pretreatment method can completely clarify the breast milk, the experimental error caused by the fact that the proportional dilution method can not completely clarify the breast milk can be effectively avoided, and the accuracy of the spectrophotometry detection result is ensured.

Drawings

FIG. 1 is a graph showing the clarifying coagulation effect of the method of the present invention on breast milk, wherein the left graph A shows 1ml of pure milk +1ml of saturated ammonium sulfate, and the right graph B shows 1ml of deionized water +1ml of saturated ammonium sulfate.

FIG. 2 is a graph showing the effect of the method of the present invention on the verification of the activity of alpha-amylase in breast milk, where the experimental group C is (1ml of saturated ammonium sulfate +1ml of alpha-amylase labeling solution with different gradients), the control group D (1ml of deionized water +1ml of alpha-amylase labeling solution with different gradients) is prepared by adding 100ul of starch solution to each group, after sufficient reaction, adding iodine/potassium iodide solution to show blue change, and the absorbance values Abs detected by the spectrometer are the same for the mixed solution 723 with different gradients.

Detailed Description

The invention provides a method for detecting the activity of alpha-amylase of breast milk.

The specific operation of each step is as follows:

(1) taking out frozen fresh breast milk from a freezing layer of a refrigerator, and unfreezing the breast milk under the condition of keeping out of the sun at room temperature (25 ℃);

(2) 1ml of fresh breast milk is sucked into a plastic test tube with the specification of 4ml by a hand-held pipette, and then 1ml of saturated ammonium sulfate is added;

(3) placing the plastic test tube on a vortex instrument, shaking for 1min, and mixing thoroughly;

(4) placing the plastic test tube into a centrifuge for centrifugation for 5 minutes, and setting the rotating speed to be 5000 r/min;

(5) taking 100ul of the centrifuged clear solution to a glass cuvette (containing 2ml of starch solution with the concentration of 0.8 mg/ml);

(6) dripping 100ul of iodine/potassium iodide solution with the concentration of 50mmol/L, and avoiding light in the process;

(7) quickly putting into 723 visible spectrophotometer, and reading absorbance value Abs;

(8) substituting the absorbance value Abs into a calculation formula to obtain the activity value of the breast milk alpha-amylase.

The present invention will be described in detail below with reference to specific examples.