CN109164191A - A kind of method of sulfur amino acid content in measurement BEIQI MUSHROOM - Google Patents
A kind of method of sulfur amino acid content in measurement BEIQI MUSHROOM Download PDFInfo
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- CN109164191A CN109164191A CN201811252440.0A CN201811252440A CN109164191A CN 109164191 A CN109164191 A CN 109164191A CN 201811252440 A CN201811252440 A CN 201811252440A CN 109164191 A CN109164191 A CN 109164191A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
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- G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
Abstract
The invention discloses a kind of methods of the assay of sulfur-containing amino acid in BEIQI MUSHROOM, it is related to the assay field of amino acid in BEIQI MUSHROOM, using BEIQI MUSHROOM as raw material, the sour water solution after performic oxidation is protected, ultrafiltration centrifugation, with 2, 4- dinitrofluorobenzene is that derivative reagent is derived, gained derivative sample liquid is mobile phase in acetonitrile-water and sodium acetate through high performance liquid chromatography, gradient elution, it is detected under 360nm wavelength, the derivative products of cysteic acid and methionine sulfone are separated, measure cystine standard items under various concentration gradient, the peak area of methionine standard items, draw standard curve, the content of cystine and methionine is measured respectively, obtain the content of sulfur-containing amino acid.The requirement of the method for the present invention instrument is low, it is not necessary to which stringent limited reactions condition, required sample size is few, and sample will not be destroyed, and can carry out the analysis of single component, is a kind of efficiently and accurately, simple and easy to operate, cheap measuring method.
Description
Technical field
The present invention relates in the extraction of amino acid in BEIQI MUSHROOM and assay field more particularly to a kind of measurement BEIQI MUSHROOM
The method of sulfur amino acid content.
Background technique
BEIQI MUSHROOM, Agaricales Pleurotus oyster mushroom under Basidiomycota, with west mountain Huashan Mountain orthodox school Radix Astragali leftover pieces and a variety of medium-height grass
Medicine and crop by-product are Pleurotus oyster mushroom made of compost is cultivated for many years, are the new of Shanxi Huiyuan selenium-enriched area exploitation
Type edible mushroom.Lysine (1.19g/100g) rich in and selenium (0.54ppm/100g) may rely ammonia with pyrroles in BEIQI MUSHROOM
The biosynthesis of acid and L- selenocysteine or L- selenium-methyl selenium substituted aminothiopropionic is expected to pressing down there is close relationship
Tumour processed, anti-oxidant, adjuvant therapy of cardiovascular disease, detoxication and toxicant eliminating function etc. are developed.According to the evaluation and test table of relevant departments
Bright, the content of amino acid accounts for 1/6th or so in BEIQI MUSHROOM, and wherein 8 kinds of ammonia necessary to human body are total to acid content and all compare relatively
Height is the edible mushroom of multipotential nutrition type, and nutrition is far more than common oyster mushroom, contains higher edible value and medicinal valence
Value.With degassing apocenosis, invigorating qi for strengthening superficies, keeps fit and healthy, protects the multiple functions such as liver, stimulating milk secretion, moisturizing.
Sulfur-containing amino acid is made of three kinds of cystine, methionine, cysteine amino acid, is human and animal's growth
One of necessary primary amino acid in the process.Most of sulfur-containing amino acid and many organic matters are combined into complexity in BEIQI MUSHROOM
Supramolecular structure, and being connected with peptide bond together in protein, thus need to by certain means by acid hydrolysis to swimming
Amorph.But-S- the key of sulfur-containing amino acid is easily oxidized destruction in conventional protein hydrolytic process, it is easy in generation
Oxidation reaction and influence subsequent analysis detection, and then quantitative analysis can not accurately be carried out.
Common method for hydrolysis is that basic hydrolysis is that sample is hydrolyzed at 110 DEG C with KOH, NaOH etc., but a few amino acids
It is easy racemization, and the amino acid such as cysteine can be destroyed.Proteolytic is to utilize hydrolase by the peptide bond water of protein
Solution is broken apart, and generates small peptide or amino acid, but hydrolysis time is long, and needs several enzyme synergistic effects that could hydrolyze
Entirely, otherwise hydrolysis is not thorough, and Contents of Amino Acids is caused severe deviations occur.There are also some measuring methods, such as: reduction derives
Method, oxidizing process etc., but there are some problems for majority, and can only determine the one of which in two kinds of sulfur-containing amino acid.
For solve BEIQI MUSHROOM in sulfur-containing amino acid assay vulnerable to interference, result is unreliable, experimental condition is complicated,
Problem at high cost, the present invention provides it is a kind of it is easily operated, require simple and assay accuracy high experimental facilities
The content assaying method of sulfur-containing amino acid in BEIQI MUSHROOM.
Summary of the invention
It is above-mentioned to overcome the purpose of the present invention is to provide a kind of method of sulfur amino acid content in measurement BEIQI MUSHROOM
Technological deficiency.
The present invention is achieved by the following technical solution: a kind of side measuring sulfur amino acid content in BEIQI MUSHROOM
Method, using BEIQI MUSHROOM as raw material, oxydrolysis carries out column front derivation after ultrafiltration centrifugal treating, and derivative passes through gradient elution, root
The content of sulfur-containing amino acid is measured according to the peak area of standard items, wherein using BEIQI MUSHROOM as raw material, crushing and screening, by performic acid
Oxidation protection carries out sour water solution using the hydrochloric acid solution of 6mol/L, and ultrafiltration centrifugation takes supernatant to mix with derivative buffer solution,
Derived by derivative reagent of 2,4-dinitrofluorobenzene, gained derivative sample liquid is 1:1 in volume ratio through high performance liquid chromatography
Acetonitrile-water and 0.05mol/L sodium acetate be mobile phase, gradient elution detects under 360nm wavelength, derivative to cysteic acid
Product and methionine sulfone derivative products are separated, and cystine standard items, methionine standard items under various concentration gradient are measured
Peak area draws standard curve, measures the content of cystine and methionine respectively, obtain the content of sulfur-containing amino acid.
Further, detailed process are as follows:
Step a, using BEIQI MUSHROOM as raw material, it is spare to be stored in -4 DEG C of refrigerators for crushing and screening；
Step b, concentration is that 88% formic acid solution and 30% hydrogenperoxide steam generator are mixed to form performic acid by 9:1 by volume
Hydrogenperoxide steam generator；40 ~ 60mg of BEIQI MUSHROOM is weighed, is placed in a beaker, adds performic acid hydrogenperoxide steam generator, solid-liquid ratio 1:
40, which is put into ice-water bath and be put in refrigerator together and reacts 16h, Sodium Metabisulfite solution terminator, shake is added
It swings and shakes up, stand, it is spare to obtain oxidation solution；
6mol/L hydrochloric acid solution is added into above-mentioned oxidation solution by step c, seals test tube, hydrolyzes under 110 DEG C of forced air drying environment
22-24h；Solution after taking partial oxidation to hydrolyze carries out 10 times of dilutions, scans, obtains on dual-beam ultraviolet-uisible spectrophotometer
The ultraviolet spectrogram of sample liquid after oxydrolysis；
Step d, centrifugal filtration is dry, is dissolved out the amino acid sample in test tube with ultrapure water, constant volume, 4000r/min centrifugation
5min takes supernatant to mix with derivative buffer solution, and it is stand-by to obtain sample liquid for the constant volume in volumetric flask；
Step e is kept in dark place after sample liquid ultrafiltration using 2,4-dinitrofluorobenzene as derivative reagent, and derivative reagent, thermostatted water is added
1h is reacted in bath, is down to room temperature to it after reaction, with equilibrating buffer constant volume, is sufficiently shaken up, and stand for standby use takes part
Sample liquid after derivative scans on dual-beam ultraviolet-uisible spectrophotometer, obtains the ultraviolet spectrogram of derivative sample liquid；
Step f configures the cysteic acid and methionine sulfone standard solution of various concentration gradient, and the peak for measuring standard items absorbs
Standard curve is made according to concentration and the linear relationship of absorption area in area；
Derivative sample liquid is flowing in the sodium acetate of acetonitrile-water and 0.05mol/L that volume ratio is 1:1 through high performance liquid chromatography
Phase detects under 360nm wavelength, gradient elution, carries out to cysteic acid derivative products in sample liquid and methionine sulfone derivative products
It separates, the appearance time and peak area of cysteic acid derivative products and methionine sulfone derivative products in analyzing liquid sample, in conjunction with mark
Directrix curve measures the content of cystine and methionine respectively, obtains the content of sulfur-containing amino acid.
Further, in continuous mode, sodium acetate is mobile phase A, and acetonitrile-water is Mobile phase B, pH 6.8, gradient elution
Program is 0~0.4min, 84%A, 16%B；0.4~4min, 70%A, 30%B；4~9.4min, 64%A, 36%B；9.4~
17min, 56%A, 44%B；17~28min, 36%A, 64%B；28~34min, 0%A, 100%B；34~36min, 0%A,
100%B；36~38min, 84%A, 16%B.
The beneficial effects of the present invention are: column front derivation high performance liquid chromatographies compared with prior art tries by derivative
Agent and amino acid effect carry out the separation determination of chromatography according to characteristic after its transformation thus after generating specific substance, before column
Requirement of the derivatization method to instrument is also very low, it may not be necessary to stringent limitation derivative reaction condition, when can permit longer reaction
Between and use various forms of reactors, derivative by-product can reduce interference, and arbitrarily selected derivatization conditions make to derive effect
Reach best.Efficient, accurate, the easy and cheap analysis suitable for amino acid of high performance liquid chromatography, and its needs
Sample size is few, can be recycled, sample is not destroyed after chromatographic column, can carry out the analysis of single component.High performance liquid chromatography skill
Derivative method is efficient before art column, easy to operate, and experimental cost is lower, and measurement result is accurate, and method is relatively new, is suitable for
Many object Quality Research, are suitble to the testing requirements of sulfur-containing amino acid in BEIQI MUSHROOM.
Detailed description of the invention
Fig. 1 is the flow chart of assay of the invention.
Fig. 2 is methionine sulfone standard curve.
Fig. 3 is cysteic acid standard curve.
Fig. 4 is the ultraviolet scanning atlas of sample liquid after BEIQI MUSHROOM oxydrolysis.
Fig. 5 is positive the ultraviolet scanning atlas of sample liquid after astragali oxydrolysis.
Fig. 6 is the ultraviolet scanning atlas of sample liquid after BEIQI MUSHROOM is derivative.
Fig. 7 be positive astragali it is derivative after sample liquid ultraviolet scanning atlas.
Fig. 8 is the high-efficient liquid phase chromatogram of BEIQI MUSHROOM sample liquid.
Fig. 9 is positive the high-efficient liquid phase chromatogram of astragali stilbene sample liquid.
Specific embodiment
Below in conjunction with attached drawing, the forgoing and additional technical features and advantages are described in more detail.
A kind of method of sulfur amino acid content in measurement BEIQI MUSHROOM, using BEIQI MUSHROOM as raw material, oxydrolysis, ultrafiltration centrifugation
Column front derivation is carried out after processing, derivative passes through gradient elution, measures containing for sulfur-containing amino acid according to the peak area of standard items
Amount, wherein using BEIQI MUSHROOM as raw material, crushing and screening is protected by performic oxidation, carries out acid using the hydrochloric acid solution of 6mol/L
Hydrolysis, ultrafiltration centrifugation take supernatant to mix with derivative buffer solution, are derived by derivative reagent of 2,4-dinitrofluorobenzene,
Gained derivative sample liquid through high performance liquid chromatography volume ratio be 1:1 acetonitrile-water and 0.05mol/L sodium acetate be mobile phase,
Gradient elution is detected under 360nm wavelength, is separated to cysteic acid derivative products and methionine sulfone derivative products, is measured
The peak area of cystine standard items, methionine standard items under various concentration gradient, draw standard curve, measure respectively cystine and
The content of methionine obtains the content of sulfur-containing amino acid.
Refering to Figure 1, detailed process are as follows:
Step a, using BEIQI MUSHROOM as raw material, it is spare to be stored in -4 DEG C of refrigerators for crushing and screening；
Step b, concentration is that 88% formic acid solution and 30% hydrogenperoxide steam generator are mixed to form performic acid by 9:1 by volume
Hydrogenperoxide steam generator；40 ~ 60mg of BEIQI MUSHROOM is weighed, is placed in a beaker, adds performic acid hydrogenperoxide steam generator, solid-liquid ratio 1:
40, which is put into ice-water bath and be put in refrigerator together and reacts 16h, Sodium Metabisulfite solution terminator, shake is added
It swings and shakes up, stand, it is spare to obtain oxidation solution；
6mol/L hydrochloric acid solution is added into above-mentioned oxidation solution by step c, seals test tube, hydrolyzes under 110 DEG C of forced air drying environment
22-24h；Solution after taking partial oxidation to hydrolyze carries out 10 times of dilutions, scans, obtains on dual-beam ultraviolet-uisible spectrophotometer
The ultraviolet spectrogram of sample liquid after oxydrolysis；
Step d, centrifugal filtration is dry, is dissolved out the amino acid sample in test tube with ultrapure water, constant volume, 4000r/min centrifugation
5min takes supernatant to mix with derivative buffer solution, and it is stand-by to obtain sample liquid for the constant volume in volumetric flask；
Step e is kept in dark place after sample liquid ultrafiltration using 2,4-dinitrofluorobenzene as derivative reagent, and derivative reagent, thermostatted water is added
1h is reacted in bath, is down to room temperature to it after reaction, with equilibrating buffer constant volume, is sufficiently shaken up, and stand for standby use takes part
Sample liquid after derivative scans on dual-beam ultraviolet-uisible spectrophotometer, obtains the ultraviolet spectrogram of derivative sample liquid；
Step f configures the cysteic acid and methionine sulfone standard solution of various concentration gradient, and the peak for measuring standard items absorbs
Standard curve is made according to concentration and the linear relationship of absorption area in area；
Derivative sample liquid is flowing in the sodium acetate of acetonitrile-water and 0.05mol/L that volume ratio is 1:1 through high performance liquid chromatography
Phase detects under 360nm wavelength, gradient elution, carries out to cysteic acid derivative products in sample liquid and methionine sulfone derivative products
It separates, the appearance time and peak area of cysteic acid derivative products and methionine sulfone derivative products in analyzing liquid sample, in conjunction with mark
Directrix curve measures the content of cystine and methionine respectively, obtains the content of sulfur-containing amino acid.
It is specifically illustrated below by embodiment.
Embodiment
Step a, pretreatment:
It by BEIQI MUSHROOM and positive astragali, is placed in ventilation and dries in the shade to material embrittlement, be put into drying in electric drying oven with forced convection
(40-50 DEG C) is crushed with high-speed multifunctional pulverizer, is crossed the screening of 80 meshes, is fitted into glassware and seals, be stored in -4 DEG C of ice
Case is spare.
Step b, performic oxidation:
88% formic acid solution that volume ratio is 9: 1 is mixed with 30% hydrogenperoxide steam generator, is put into ice water after reacting 1h under room temperature
Cooling down 30 minutes, ready-to-use in bath, and performic acid hydrogenperoxide steam generator is made.BEIQI MUSHROOM, positive astragali are accurately weighed respectively
Each 50mg of sample, is respectively placed in beaker, each to add performic acid solutions 2mL, is put into ice-water bath and is put in refrigerator together and reacts
16h carries out oxidation protection, chooses Sodium Metabisulfite solution as oxide termination agent, the agent of 0.5mL oxide termination is added, by it
Concussion shakes up, and stands a moment, it is spare to obtain oxidation solution.
Step c, sour water solution:
18mL hydrochloric acid solution (6mol/L) is added into above-mentioned oxidation solution, seals test tube, is placed in 110 DEG C of electric drying oven with forced convections
22-24h is hydrolyzed, the solution after taking partial oxidation to hydrolyze carries out 10 times of dilutions, sweeps on dual-beam ultraviolet-uisible spectrophotometer
It retouches, obtains the ultraviolet spectrogram of sample liquid after oxydrolysis.
Step d, centrifugal filtration:
Borax 7.630g, boric acid 1.240g are accurately weighed, ultrapure water dissolution is added, is settled to 500mL, is stood after sufficiently shaking up,
It is spare to obtain derivative buffer solution；Solution after oxydrolysis is dried, with ultrapure water by the amino acid sample in test tube
Dissolution, and it is settled to 25mL, 4000r/min is centrifuged 5min in supercentrifuge；Supernatant and derivative buffering after taking centrifugation
Solution constant volume in 25mL volumetric flask, it is stand-by to obtain sample liquid.
Step e, column front derivation:
Disodium hydrogen phosphate 3.580g, potassium dihydrogen phosphate 0.910g are accurately weighed, ultrapure water dissolution is added, is settled to 500mL, sufficiently
It is stood after shaking up, it is spare to be balanced buffer solution.1mL2 is taken, 4- dinitrofluorobenzene is dissolved in acetonitrile solution, and constant volume is in 100mL
It is spare to obtain derivative reagent for volumetric flask.It takes 10mL sample liquid to be placed in brown volumetric flask through organic membrane filtration to be kept in dark place,
5mL derivative reagent is added, reacts 1h in 60 DEG C of waters bath with thermostatic control, is down to room temperature to it after reaction, uses equilibrating buffer
Constant volume sufficiently shakes up, and stands a moment, spare.Sample liquid after taking part derivative, sweeps on dual-beam ultraviolet-uisible spectrophotometer
It retouches, obtains the ultraviolet spectrogram of derivative sample liquid.
Step f, high performance liquid chromatography detection:
It is that 1 μ g/mL, 5 μ g/mL, 10 μ g/mL, the cysteic acid of 15 μ g/mL and methionine sulfone standard items are molten that concentration, which is respectively configured,
Standard curve is made according to concentration and the linear relationship of absorption area with the peak absorption area of liquid phase measurement standard items in liquid.
With the sodium acetate of electronic balance precise 4.063g and the acetic acid of 0.027g in the volumetric flask of 500mL, add super
Pure water constant volume sufficiently shakes up to scale, is filtered with high-pressure vacuum pump, and ultrasonic degassing after twenty minutes, obtains the mobile phase A for liquid phase
For the sodium acetate solution of 0.05mol/L.
0.45 μm of organic filter membrane is impregnated with methanol, 0.20 μm of moisture film is impregnated with ultrapure water, and soaking time is for 24 hours.By color
It composes methanol, acetonitrile and ultrapure water to be filtered three times with high-pressure vacuum pump respectively, is put into ultrasonic vibration instrument ultrasonic degassing after twenty minutes
Access high performance liquid chromatograph.Check and open power supply, mobile phase it is normal after, successively open autosampler, column oven, high pressure
Pump, UV detector, chromatographic work station, with methanol: ultrapure water=9:1 cleaning robot 30min or more, until pressure level-off
It can sample introduction.
Chromatographiccondition is as follows:
Chromatographic column: C18,4 μm, the U.S. 4.6mm × 150mm(Agilent).Mobile phase A is 0.05mol/L sodium acetate, Mobile phase B
For acetonitrile-water (1:1, v/v)；pH6.8；Gradient elution program: 0~0.4min, 84%A, 16%B；0.4~4min, 70%A,
30%B；4~9.4min, 64%A, 36%B；9.4~17min, 56%A, 44%B；17~28min, 36%A, 64%B；28~
34min, 0%A, 100%B；34~36min, 0%A, 100%B；36~38min, 84%A, 16%B.Detection wavelength is
360nm, flow velocity 1.2mL/min, sample volume are 20 μ L, and column temperature is 31 DEG C.Observe and record map.
With methanol after measurement: liquid rinse whole system of the ultrapure water volume ratio for 1:9, flow velocity 1mL/min,
Cleaning 1 hour, wherein ultrapure water is to clean this and test the salt seen in system, and a small amount of methanol plays protection chromatography
The effect of column.Again with the methanol of 9:1: ultrapure water cleans again, flow velocity 1mL/min, and chromatography is closed in termination of pumping after cleaning 1 hour
Work station, then autosampler, column oven, high-pressure pump, UV detector are successively closed, finally shut down.
Cystine and methionine content are analyzed according to standard curve.
Interpretation of result:
1. the standard curve of sulfur-containing amino acid standard items is analyzed:
Methionine sulfone standard items, peak area of the cysteic acid standard items under various concentration are measured respectively, can obtain methionine
Standard curve such as Fig. 2, cysteic acid standard items such as Fig. 3.Related coefficient is respectively 0.9998 and 0.9999, illustrates the color of liquid phase
The mass concentration regression of spectral peak area and substance is good.
2. sample liquid UV scanning interpretation of result after oxydrolysis:
To after oxydrolysis BEIQI MUSHROOM sample liquid and positive astragali sample liquid be diluted, dilute 10 times by obtaining after constantly testing
The effect that is scanned on dual-beam ultraviolet-uisible spectrophotometer of sample liquid it is relatively good, then detect ultraviolet spectrogram.Observation
Fig. 4, Fig. 5, discovery scanning spectra structure is much like, and the two contains identical substance, can tentatively infer cysteic acid and egg
The ultraviolet absorption peak of propylhomoserin sulfone is about in 197nm or so.
3. sample liquid UV scanning interpretation of result after column front derivation:
UV scanning is carried out to the sample liquid after derivative, obtains the ultraviolet spectrogram of derivative sample liquid.As shown in Figure 6, Figure 7, BEIQI MUSHROOM
With occur two wave crests, respectively 192nm, 238nm and 193nm, 237nm in the derivative map of positive astragali sample liquid, and sulphur
Base alanine, methionine sulfone derivative products are at 240nm with the presence of absorption peak.Substance at analysis 192nm is without having reacted
Sample liquid, the substance at 238nm is derivative.Tentatively it may infer that containing derivative.
4. efficient liquid phase Analysis of test results after column front derivation:
The sulfur amino acid content of BEIQI MUSHROOM is analyzed:
It is shown according to Fig. 8, component 5 is cysteic acid derivative products, peak area 3832.91797mAU*s, with reference to Guang in Fig. 2
The linear relationship of propylhomoserin standard items, can obtain cystine concentration in BEIQI MUSHROOM is 26.016 μ g/mL, and separating degree R is 18.54, sample liquid
Middle cysteic acid derivative products are kept completely separate, and the 65.8153% of total concentration is accounted for.
Component 7 is methionine sulfone derivative products, peak area 1241.46899mAU*s, with reference to Fig. 3 methionine sulfone standard items
Linear relationship, can obtain in BEIQI MUSHROOM methionine concentration is 11.282 μ g/mL, and separating degree R is 20.24, methionine sulfone in sample liquid
Derivative products are kept completely separate, and the 21.3273% of total concentration is accounted for.
The sulfur amino acid content analysis of positive astragali:
It is shown according to Fig. 9, component 6 is cysteic acid derivative products, peak area 2248.14380mAU*s, with reference in Fig. 2
Linear relationship, can obtain cystine concentration in positive astragali is 15.279 μ g/mL, and separating degree R is 1.05, and cysteic acid spreads out in sample liquid
Production object is kept completely separate, and the 41.8804% of total concentration is accounted for.
Component 9 is methionine sulfone derivative products, and peak area 1764.52527mAU*s can with reference to linear relationship in Fig. 3
Obtaining methionine concentration in positive astragali is 15.961 μ g/mL, and separating degree R is 18.75, and methionine sulfone derivative products have obtained in sample liquid
It is fully separating, account for the 32.9218% of total concentration.
Conclusion:
The present invention makees oxidant with performic acid, makees hydrolytic reagent with hydrochloric acid, BEIQI MUSHROOM, positive astragali is handled using oxydrolysis, by egg
Methionine and cysteine difference oxidation protection in white matter are stable methionine sulfone and cysteic acid, obtain sample,
Then 60min is reacted in the water-bath of 60 DEG C of dark place with 2,4-dinitrofluorobenzene, gained derivative sample liquid exists through high performance liquid chromatography
The acetonitrile-water and 0.05mol/L sodium acetate that volume ratio is 1:1 are mobile phase, are detected under 360nm wavelength, gradient elution, to sample liquid
Middle cysteic acid derivative products and methionine sulfone derivative products are separated, and obtain cysteic acid by the analysis to sample liquid
The appearance time and peak area of derivative products and methionine sulfone derivative products, the standard curve of combined standard product acquire BEIQI MUSHROOM
The 26.016 μ g/mL of content of middle cystine, the content of methionine are 11.282 μ g/mL, the content of cystine in positive astragali
15.279 μ g/mL, the content of methionine are 15.961 μ g/mL.Comparison is it is found that BEIQI MUSHROOM is made as with the leftover bits and pieces of positive astragali
The edible mushroom that ingredient is cultivated, cystine is higher than positive astragali, but methionine content is lower than positive astragali.Generally speaking, northern
Sulfur amino acid content in stilbene mushroom is higher than positive astragali.Bioconcentration of the BEIQI MUSHROOM on sulfur-containing amino acid is embodied, north
Stilbene mushroom includes the aminoacid ingredient of positive astragali, has a degree of enrichment to sulfur-containing amino acid in growth and development process.
The method of the present invention confirms that BEIQI MUSHROOM can play one to the sulfur-containing amino acid in culture medium in growth and development process
Fixed enrichment.From this, BEIQI MUSHROOM has higher biological conversion to act on, as economical edible mushroom, possess high
Nutritive value and medical value, be worth extensively cultivation.
The present invention has used oxydrolysis, 2,4- dinitrofluorobenzene as derivative reagent and has carried out column front derivation auxiliary to sulfur-bearing
The purifying of amino acid with separate, easy to operate, the requirement to instrument is also very low, have wide applicability, have it is very outstanding
Advantage, therefore can be widely used.Derivative method is realized before high-efficient liquid phase chromatogram technology column contains in BEIQI MUSHROOM
The measurement of sulphur amino acid.It can be shown that derivative method is efficient before high-efficient liquid phase chromatogram technology column, easily by embodiment analysis
Operation, experimental cost is lower, and measurement result is accurate, and method is novel, in accordance with the testing requirements of sulfur-containing amino acid in BEIQI MUSHROOM, is applicable in
In many object Quality Research, should be promoted energetically.
Claims (4)
1. a kind of method of sulfur amino acid content in measurement BEIQI MUSHROOM, which is characterized in that using BEIQI MUSHROOM as raw material, aoxidize water
It solves, column front derivation is carried out after ultrafiltration centrifugal treating, derivative passes through gradient elution, measures sulfur-bearing according to the peak area of standard items
The content of amino acid, wherein using BEIQI MUSHROOM as raw material, crushing and screening is protected by performic oxidation, using the hydrochloric acid of 6mol/L
Solution carries out sour water solution, and ultrafiltration centrifugation takes supernatant to mix with derivative buffer solution, using 2,4-dinitrofluorobenzene as derivative reagent
Derived, gained derivative sample liquid is through high performance liquid chromatography in the acetonitrile-water and 0.05mol/L sodium acetate that volume ratio is 1:1
For mobile phase, gradient elution is detected under 360nm wavelength, is carried out to cysteic acid derivative products and methionine sulfone derivative products
Separation measures the peak area of cystine standard items, methionine standard items under various concentration gradient, draws standard curve, surveys respectively
The content for obtaining cystine and methionine, obtains the content of sulfur-containing amino acid.
2. the method for sulfur amino acid content in a kind of measurement BEIQI MUSHROOM according to claim 1, which is characterized in that the tool
Body process are as follows:
Step a, using BEIQI MUSHROOM as raw material, it is spare to be stored in -4 DEG C of refrigerators for crushing and screening；
Step b, concentration is that 88% formic acid solution and 30% hydrogenperoxide steam generator are mixed to form performic acid by 9:1 by volume
Hydrogenperoxide steam generator；40 ~ 60mg of BEIQI MUSHROOM is weighed, is placed in a beaker, adds performic acid hydrogenperoxide steam generator, solid-liquid ratio 1:
40, which is put into ice-water bath and be put in refrigerator together and reacts 16h, Sodium Metabisulfite solution terminator, shake is added
It swings and shakes up, stand, it is spare to obtain oxidation solution；
6mol/L hydrochloric acid solution is added into above-mentioned oxidation solution by step c, seals test tube, hydrolyzes under 110 DEG C of forced air drying environment
22-24h；Solution after taking partial oxidation to hydrolyze carries out 10 times of dilutions, scans, obtains on dual-beam ultraviolet-uisible spectrophotometer
The ultraviolet spectrogram of sample liquid after oxydrolysis；
Step d, centrifugal filtration is dry, is dissolved out the amino acid sample in test tube with ultrapure water, constant volume, 4000r/min centrifugation
5min takes supernatant to mix with derivative buffer solution, and it is stand-by to obtain sample liquid for the constant volume in volumetric flask；
Step e is kept in dark place after sample liquid ultrafiltration using 2,4-dinitrofluorobenzene as derivative reagent, and derivative reagent, thermostatted water is added
1h is reacted in bath, is down to room temperature to it after reaction, with equilibrating buffer constant volume, is sufficiently shaken up, and stand for standby use takes part
Sample liquid after derivative scans on dual-beam ultraviolet-uisible spectrophotometer, obtains the ultraviolet spectrogram of derivative sample liquid；
Step f configures the cysteic acid and methionine sulfone standard solution of various concentration gradient, and the peak for measuring standard items absorbs
Standard curve is made according to concentration and the linear relationship of absorption area in area；
Derivative sample liquid is flowing in the sodium acetate of acetonitrile-water and 0.05mol/L that volume ratio is 1:1 through high performance liquid chromatography
Phase detects under 360nm wavelength, gradient elution, carries out to cysteic acid derivative products in sample liquid and methionine sulfone derivative products
It separates, the appearance time and peak area of cysteic acid derivative products and methionine sulfone derivative products in analyzing liquid sample, in conjunction with mark
Directrix curve measures the content of cystine and methionine respectively, obtains the content of sulfur-containing amino acid.
3. the method for sulfur amino acid content in a kind of measurement BEIQI MUSHROOM according to claim 2, which is characterized in that described
In step b, BEIQI MUSHROOM 50mg is weighed, is placed in a beaker, add 2mL performic acid hydrogenperoxide steam generator, is put into ice-water bath and together
It is put in refrigerator and reacts 16h, 0.5mL Sodium Metabisulfite solution terminator is added, concussion shakes up, and stands, it is standby to obtain oxidation solution
With.
4. the method for sulfur amino acid content in a kind of measurement BEIQI MUSHROOM according to claim 2, which is characterized in that institute
State: sodium acetate is mobile phase A, and acetonitrile-water is Mobile phase B, pH 6.8, and the gradient elution program is 0~0.4min, 84%
A, 16%B；0.4~4min, 70%A, 30%B；4~9.4min, 64%A, 36%B；9.4~17min, 56%A, 44%B；17
~28min, 36%A, 64%B；28~34min, 0%A, 100%B；34~36min, 0%A, 100%B；36~38min,
84%A, 16%B.
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