CN103454270A - Detection method for sea cucumber polysaccharide - Google Patents

Abstract

The invention provides a detection method for sea cucumber polysaccharide. The detection method comprises the steps of (1) preparing sea cucumber test liquid by adopting an alkaline protease enzyme hydrolysis method; (2) preparing a sea cucumber polysaccharide standard curve by taking an azure I reagent as a dyeing agent and trepang polysaccharide as a standard substance; (3) measuring a test sample by taking the azure I reagent as a dyeing agent, calculating a mass value corresponding to the sea cucumber polysaccharide according to a standard curve equation, and calculating the concentration of the sea cucumber polysaccharide in the test liquid. The method disclosed by the invention is sensitive and accurate and can be used in daily product monitoring.

Description

A kind of detection method of sea cucumber polysaccharide

Technical field

The invention belongs to the Food Chemistry analysis field, be specifically related to a kind of detection method of sea cucumber polysaccharide.

Background technology

The exploitation in sea cucumber market begins to take shape, and product form is also varied, but for example,, in the detection of sea cucumber index components (sea cucumber polysaccharide), still there is no unified, sensitive detection method.

Summary of the invention

The object of the present invention is to provide a kind of detection method for sea cucumber polysaccharide.

The detection method of sea cucumber polysaccharide of the present invention comprises the steps:

(1) prepare the sea cucumber test liquid

Take the 2g fluid sample or pulverize after solid sample be placed in the plastic centrifuge tube of 500ml, add the 100ml deionized water, mix, the drying box of putting into 105 ℃ of constant temperature heats 1h, after taking-up is cooled to room temperature, add the 0.2ml alkali protease, shake up, enzymolysis 1h under room temperature, put into 100 ℃ of water-baths, after inactivator 10min, 12000r/min high speed centrifugation 30min, centrifugate is all poured in measuring device and measured volume, pipette the 0.2ml supernatant to the 5ml volumetric flask, deionized water is settled to scale;

(2) prepare the sea cucumber polysaccharide typical curve

Accurately taking the 250mg Azure I is dissolved in the 250ml deionized water, dissolving shakes up, obtain the Azure I storing solution of 1mg/ml, by Azure I storing solution and deionized water by volume 1:29 mix, adjusting makes its maximum absorbance A=2.1～2.2, be placed in refrigerator and cooled and hide preservation, obtain the Azure I test solution;

Accurately take 0.4mg sea stichopus japonicus mucopolysaccharide standard substance and be dissolved in the 2ml deionized water, dissolving shakes up, and is placed in refrigerator and cooled and hides preservation, obtains the standard solution of 0.2mg/ml;

In 7 10ml test tubes, add respectively standard solution 0,5,10,20,30,40,50 μ l, the test tube of liquor capacity less than 50 μ l is added into water, make the liquor capacity in every test tube be 50 μ l, then add 1ml Azure I test solution, mix and measure immediately its absorbance, it is contrast that the blank 50 μ l water of take add the absorbance of 1ml Azure I test solution, parallelly do three groups, get its arithmetic mean, do straight-line regression with its corresponding absorbance of extra large stichopus japonicus mucopolysaccharide quality μ g in standard solution, obtain typical curve;

(3) measure sample

Pipette sea cucumber test liquid 50 μ l in test tube, add 1ml Azure I test solution, mix and measure immediately its absorbance, it is contrast that the blank 50 μ l water of take add the absorbance of 1ml Azure I test solution, utilize the typical curve equation to calculate the corresponding mass number of sea cucumber polysaccharide, then calculate the concentration of sea cucumber polysaccharide in test liquid.

Principle of the present invention is as follows: the sea cucumber sample is after enzymolysis, centrifuging, enzymolysis liquid Azure I reagent dyeing, its absorbance (510nm) is directly proportional to the polysaccharide quality in the finite concentration scope, do contrast with the standard polysaccharide solution, just can adopt spectrophotometric method to record the content of polysaccharide in test sample.

Method of the present invention is sensitive, accurate, can be used in daily product surveillance.

Embodiment

Below in conjunction with embodiment, the present invention is described, but the present invention is not limited to these embodiment.

1. reagent and standard substance

(1) Azure I: biological stain

(2) standard substance: extra large stichopus japonicus mucopolysaccharide

(3) Azure I storing solution (1mg/ml): take Azure I 250mg(accurately to 0.01mg) be dissolved in the 250ml deionized water, dissolving shakes up, and puts into refrigerator and cooled and hides and preserve.

(4) Azure I test solution: the Azure I storing solution with deionized water in 1:29(V/V) ratio mixes, and regulates and makes its maximum absorbance between A=2.1～2.2, be placed in refrigerator and cooled and hide and preserve.

(5) standard solution (0.2mg/ml): accurately take extra large stichopus japonicus mucopolysaccharide standard substance 0.4mg(accurately to 0.0001g) be dissolved in the 2ml deionized water, shake up after dissolving, be placed in refrigerator and cooled and hide and preserve.

2. instrument

(1) ultraviolet spectrophotometer (UV)

(2) cuvette: 1mm * 1cm or 1cm * 1cm

(3) test tube: 10ml

(4) analytical balance: sensibility reciprocal 0.1mg, 0.01mg

(5) micro quantitative determination pin: 100 μ l, 50 μ l, 25 μ l

(6) hydro-extractor: the 500ml maximum (top) speed is 12000r/min

(7) water-bath

(8) thermostatic drying chamber

3. sample preparation

3.1 sample is pulverized to (except liquid), takes 2g(and be accurate to 0.001g) be placed in the plastic centrifuge tube of 500ml, add the 100ml deionized water, mix, the drying box of putting into 105 ℃ of constant temperature heats 1h.Taking-up is cooled to room temperature, adds the 0.2ml alkali protease, shakes up, and enzymolysis 1h under room temperature, put into 100 ℃ of water-baths, after inactivator 10min, and 12000r/min high speed centrifugation 30min.Centrifugal clear liquid is all poured in measuring device and measured volume V.

3.2 pipette the 0.2ml supernatant to the 5ml volumetric flask, deionized water is settled to scale.

4. determination step

4.1 the preparation of typical curve

In 7 10ml test tubes, add respectively standard solution 0,5,10,20,30,40,50 μ l, in the test tube of liquor capacity less than 50 μ l, add into water, make the liquor capacity in every test tube identical.Then add 1ml Azure I test solution, mix and measure immediately its absorbance, it is contrast that the blank 50 μ l water of take add the absorbance of 1ml Azure I test solution, parallelly do three groups, get its arithmetic mean, do straight-line regression with its corresponding absorbance of polysaccharide quality in standard solution (μ g), related coefficient is not less than 0.99.

4.2 the mensuration of sample

Pipette sample 50 μ l in test tube, add 1ml Azure I test solution, mix and read immediately absorbance, it is contrast that the blank 50 μ l water of take add the absorbance of 1ml Azure I test solution.Utilize regression equation calculation to go out corresponding mass number, then the content of sea cucumber mucopolysaccharide in calculation sample.

Claims (1)

1. the detection method of a sea cucumber polysaccharide, is characterized in that, comprises the steps:

(1) prepare the sea cucumber test liquid

Take the 2g fluid sample or pulverize after solid sample be placed in the plastic centrifuge tube of 500ml, add the 100ml deionized water, mix, the drying box of putting into 105 ℃ of constant temperature heats 1h, after taking-up is cooled to room temperature, add the 0.2ml alkali protease, shake up, enzymolysis 1h under room temperature, put into 100 ℃ of water-baths, after inactivator 10min, 12000r/min high speed centrifugation 30min, centrifugate is all poured in measuring device and measured volume, pipette the 0.2ml supernatant to the 5ml volumetric flask, deionized water is settled to scale;

(2) prepare the sea cucumber polysaccharide typical curve

Accurately taking the 250mg Azure I is dissolved in the 250ml deionized water, dissolving shakes up, obtain the Azure I storing solution of 1mg/ml, by Azure I storing solution and deionized water by volume 1:29 mix, adjusting makes its maximum absorbance A=2.1～2.2, be placed in refrigerator and cooled and hide preservation, obtain the Azure I test solution;

Accurately take 0.4mg sea stichopus japonicus mucopolysaccharide standard substance and be dissolved in the 2ml deionized water, dissolving shakes up, and is placed in refrigerator and cooled and hides preservation, obtains the standard solution of 0.2mg/ml;

In 7 10ml test tubes, add respectively standard solution 0,5,10,20,30,40,50 μ l, the test tube of liquor capacity less than 50 μ l is added into water, make the liquor capacity in every test tube be 50 μ l, then add 1ml Azure I test solution, mix and measure immediately its absorbance, it is contrast that the blank 50 μ l water of take add the absorbance of 1ml Azure I test solution, parallelly do three groups, get its arithmetic mean, do straight-line regression with its corresponding absorbance of extra large stichopus japonicus mucopolysaccharide quality μ g in standard solution, obtain typical curve;

(3) measure sample

Pipette sea cucumber test liquid 50 μ l in test tube, add 1ml Azure I test solution, mix and measure immediately its absorbance, it is contrast that the blank 50 μ l water of take add the absorbance of 1ml Azure I test solution, utilize the typical curve equation to calculate the corresponding mass number of sea cucumber polysaccharide, then calculate the concentration of sea cucumber polysaccharide in test liquid.