CN109164201A - The extraction purification and the method for inspection of selenomethionine in a kind of BEIQI MUSHROOM - Google Patents
The extraction purification and the method for inspection of selenomethionine in a kind of BEIQI MUSHROOM Download PDFInfo
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- CN109164201A CN109164201A CN201811252446.8A CN201811252446A CN109164201A CN 109164201 A CN109164201 A CN 109164201A CN 201811252446 A CN201811252446 A CN 201811252446A CN 109164201 A CN109164201 A CN 109164201A
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- 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
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Abstract
The invention discloses a kind of methods of the extraction purification and inspection of selenomethionine in BEIQI MUSHROOM, it is related to the extraction purification of amino acid and inspection field in BEIQI MUSHROOM, using BEIQI MUSHROOM as raw material, 5% nitric acid solution is added, solid-liquid ratio is 1:6, centrifugation, Tris-Hcl is added in deposit, ultrasonic treatment, enzymatic hydrolysis 36 hours, it is centrifuged 10min, take supernatant, ultrafiltration centrifugation filters, obtain crude extract, using n-butanol: acetic acid: water=7:1:2 is solvent, selenomethionine is isolated and purified through thin-layer chromatography expansion, use chromatography methanol and ultrapure water as mobile phase, sample molecule quality information is measured with mass spectrograph, with its functional group of infrared detection, and analyze its molecular structure, double ultraviolet specrophotometers measure content, polarimeter measures optical activity and specific rotatory power, fusing point is measured after solution evaporative crystallization.The sensitive amino acid selenomethionine of the survivable low content of the method for the present invention, extraction efficiency is high, high sensitivity, is the method for a kind of easy to operate, high-efficient extraction purification and detection.
Description
Technical field
The present invention relates to the extraction purification of amino acid in BEIQI MUSHROOM and field is examined, seleno egg in specially a kind of BEIQI MUSHROOM
The extraction purification and the method for inspection of propylhomoserin.
Background technique
BEIQI MUSHROOM, Agaricales Pleurotus oyster mushroom under Basidiomycota, utilizes Shanxi Province Hunyuan County 74km2The geography of selenium-enriched area
Resources advantage is trained for many years using the authentic positive astragali leftover pieces in west mountain Huashan Mountain and a variety of Chinese herbal medicines and crop by-product as compost
Pleurotus oyster mushroom made of educating is the novel edible bacterium of Shanxi Huiyuan selenium-enriched area exploitation.According to bioconcentration, BEIQI MUSHROOM
The pharmaceutical component for including Huiyuan Radix Astragali has a degree of enrichment to astragalin in growth and development process, detects in BEIQI MUSHROOM
To containing trace elements of selenium, in human body necessary 8, amino acid content is relatively high, and only lysine one just up to 1.185%,
Polysaccharide 9.688%, selenium 0.54ppm, zinc 77.77ppm, and contain multivitamin and inorganic salts substance, nutrition is far super
In common oyster mushroom, contain higher edible value and medical value；With degassing apocenosis, invigorating qi for strengthening superficies, keeps fit and healthy, protects liver
The multiple functions such as dirty, stimulating milk secretion, moisturizing.
Selenium is a kind of microelement necessary to human body, is improved the immunity of the human body, prevention of various diseases, there is weight in body
The physiological action wanted.Selenium is the required component part of glutathione peroxidase etc., while being the group of internal many protein again
At ingredient, resisting stress anti-oxidant to animal body, raising immunity etc. play an important role.In growing process, to
Inorganic selenium is added in environment can be converted the inorganic selenium in environment in plant tissue by the biological metabolism of plant itself
Selenocompound is the effective ways that human body selenium level is improved in selenium deficiency area by the selenium-supply of plant containing selenium.In plant
Selenium mainly exists in the form of organic selenium and inorganic selenium, and relative to inorganic selenium, the selenium compound of organic form is usual
Physiological function with higher and lower toxicity, such as selenoprotein, seleno-amino acids.The basic existence form of organic selenium is selenium egg
It is white.Selenium only with selenocysteine (selenocysteine, SeCys) and selenomethionine (selenomethionine,
SeMet) two kinds of forms are covalently bind in protein.Lysine (1.19g/100g) abundant and selenium in BEIQI MUSHROOM
(0.54ppm/100g) may be closed with the biology of pyrrolysine and L- selenocysteine or L- selenium-methyl selenium substituted aminothiopropionic
At there is close relationship, be expected to inhibit tumour, anti-oxidant, adjuvant therapy of cardiovascular disease, in terms of into
Row exploitation.
Since selenomethionine content is less, and other amino acid contents of homologue are then higher than selenomethionine content
Thousands of times, severe jamming is formed, therefore is always one of the problem of analysis worker to the analysis of selenomethionine.Existing use
PC, TLC technical appraisement selenocystine and selenomethionine, hydrogen bromide GC method measure selenomethionine, but due to seleno
Methionine is sensitive amino acid and content is often lower, when with HCl/water solution, in the presence of especially having a hydrocarbon, it is easy to
Destroyed, solvent noise has a larger impact to result, extraction purification it is cumbersome, result is unreliable the problems such as.
It is cumbersome to solve the extraction purification of selenomethionine and checked operation in BEIQI MUSHROOM, it is unreliable vulnerable to interference result
The problem of, the present invention provides it is a kind of it is easily operated, test that time-consuming short and extraction efficiency is high, seleno egg in relatively mild BEIQI MUSHROOM
The method of propylhomoserin extraction purification and detection.
Summary of the invention
The purpose of the present invention is to provide a kind of methods of selenomethionine extraction purification and inspection in measurement BEIQI MUSHROOM, use
To overcome above-mentioned technological deficiency.
The present invention is achieved by the following technical solution: a kind of side measuring selenomethionine content in BEIQI MUSHROOM
Method carries out coarse extraction to selenomethionine using enzymatic isolation method using BEIQI MUSHROOM as raw material, after thin layer chromatography isolates and purifies, benefit
With mass spectrum, ultra-red mass spectrum and ultraviolet detection standard items and test sample and content is measured, optical activity and specific rotatory power are measured, after crystallization
Measure its fusing point；Specifically: using BEIQI MUSHROOM as raw material, 5% nitric acid solution, solid-liquid ratio 1:6, centrifugation behaviour is added in crushing and screening
To make, Tris-Hcl is added in deposit, and ultrasonic treatment digests 36 hours, is centrifuged 10min, takes supernatant, and ultrafiltration centrifugation filters,
Obtain crude extract；Using n-butanol: acetic acid: water=7:1:2 isolates and purifies selenomethionine as solvent, through thin-layer chromatography expansion, uses
Chromatography methanol and ultrapure water measure sample molecule quality information as mobile phase, with mass spectrograph, with its functional group of infrared detection, and
Its molecular structure is analyzed, measures content using double ultraviolet specrophotometers, measures optical activity and specific rotatory power using polarimeter, to
Fusing point is measured after solution evaporative crystallization.
Further, detailed process are as follows:
Step a, using BEIQI MUSHROOM as raw material, 5% nitric acid solution, solid-liquid ratio 1:6 is added in crushing and screening, and centrifugally operated retains
The Tris-Hcl of 0.5mmol/L is added in deposit, and ultrasonic treatment sequentially adds trypsase, Proteinase K every 12h,
Oscillation enzymatic hydrolysis 36 hours at 37 DEG C；Then centrifugation 10min, takes supernatant, is centrifuged, is removed with the ultrafiltration that molecular cut off is 3KD
Protease and macromolecular substances are filtered with the filter membrane of 0.45um, obtain crude extract；
Step b, L- selenomethionine standard items add Chromatographic Pure Methanol to dissolve, and constant volume obtains the L- selenium of 1mg/mL after completely dissolution
For methionine standard solution, and places it in 2~4 DEG C of refrigerator and save backup；
Step c by chromatoplate heat-activated and after cooling down, draws crude extract and 25 μ L of standard solution, away from bottom edge 2cm respectively
Uniform point sample on horizontal line, origin diffusion diameter is no more than 3mm after sample-adding, puts again after dry primary；With n-butanol: acetic acid: water=7:
1:2 is BEIQI MUSHROOM test sample solvent, and after sealing, presaturation 20min is put into ascending development in chromatography cylinder, to be deployed dose of forward position
When away from the 0.8-1.2cm of chromatoplate top, taking-up is dried, and the chromatoplate after colour developing is put into thin-layer chromatography imaging system, Yu Zi
Chromatography is inspected under outer wavelength 365nm, indicates, the chromatoplate extended is sprayed with ninhydrin solution, is allowed to develop the color, in ultraviolet lamp
Lower label, comparing calculation Rf value, system is taken pictures, and is indicated；
Step d, uses chromatography methanol and ultrapure water as mobile phase, draws a certain amount of standard solution with microsyringe, into
Sample is analyzed spectrogram, is saved；Test sample chromatoplate is placed on TLC- mass spectrometer interface, irises out laser alignment to be measured
At spot centers, sampling is analyzed spectrogram, is saved；
Step e, opens Fourier infrared spectrograph, and testing background is drawn standard solution at point sample center with capillary, surveyed
Test agent carries out processing analysis to infrared spectrum, and the spot to be measured gently scraped on chromatoplate is placed in a beaker, and chromatography methanol is added,
Filter, take filtrate, be made prepare liquid, observe prepare liquid infrared detection method and standard items detect whether it is identical；
Prepare liquid is put into double ultraviolet specrophotometers by step f, and methanol is reference, and setting scanning range is 200~700nm,
Spectral scan obtains ultraviolet scanning atlas, maximum absorption peak；
L- selenomethionine standard items are configured to the solution of 0.05-0.25mg/mL, concentration gradient is set in maximum absorption peak
Under make each filter liquor concentration of a standard curve determination and light absorption value；
Step g takes prepare liquid to measure optical activity and specific rotatory power respectively, records data；
Step h will measure its fusing point after prepare liquid evaporative crystallization.
Compared with prior art the beneficial effects of the present invention are: use relatively mild enzymatic isolation method decomposing protein, keep away
Exempted from selenomethionine for sensitive amino acid and content it is often lower, when with HCl/water solution, especially with the presence of hydrocarbon
When, it is easy to the problem of by destroying, this method does not cause the loss of target substance, and it is easy to operate, the used time is short, avoid
By the interference of other high-content amino acid, extraction efficiency is greatly improved.Thin-layer chromatography is relative to paper chromatography, efficient liquid phase
Chromatography, high resolution gas chromatography and high-speed countercurrent chromatography maintain the features such as easy to operate, equipment is simple, colour developing is easy, simultaneously
Deployment rate is fast, generally only needs 15~20 minutes；Mixture is easily separated, and resolving power is generally higher than previous paper chromatography by 10~100
Times, it is not only suitable for the sample separation of only 0.01 μ g, and the sample that can be separated greater than 500mg is used as preparation, but also can make
It with the corrosivity color developing agent of such as concentrated sulfuric acid, concentrated hydrochloric acid etc, is able to achieve and selenomethionine is isolated and purified, so that seleno egg ammonia
Acid is precisely separating, and is not interfered by other high-content amino acid, and MS and IR combination characterize its mass-to-charge ratio and contained functional group
With analysis, accuracy is high, avoids interfering.TLC-MS-IR combination, which is able to achieve from the separation of microgram magnitude, to be analyzed, easily operated, examination
Test time-consuming short and extraction efficiency is high, in relatively mild BEIQI MUSHROOM selenomethionine extraction purification and detection method.Suitable for from
The preparative separation of substance is carried out in BEIQI MUSHROOM.
Detailed description of the invention
Fig. 1 is the flow chart of assay of the invention.
Fig. 2 is TLC image after the colour developing of F254 chromatoplate L- selenomethionine standard items, and 1 is L- selenomethionine institute in figure
In speckle displacement.
Fig. 3 is TLC image before F254 chromatoplate astragali mushroom extracting solution develops the color, in figure 1 where L- selenomethionine spot
Position.
Fig. 4 is TLC image after the colour developing of F254 chromatoplate astragali mushroom extracting solution, in figure 1 where L- selenomethionine spot
Position.
Fig. 5 be GF254 chromatoplate BEIQI MUSHROOM, astragali mushroom grain, positive astragali extracting solution develop the color before TLC figure, from left to right according to
Secondary is astragali mushroom extracting solution, astragali mushroom grain extracting solution and positive astragali extracting solution chromatographic band, and 2 where L- selenomethionine in figure
Speckle displacement.
Fig. 6 is that TLC schemes after GF254 chromatoplate L- selenomethionine standard items develop the color, and 2 where L- selenomethionine in figure
Speckle displacement.
Fig. 7 is the structural formula of L- selenomethionine.
Fig. 8 is the mass spectrum wave spectrogram of L- selenomethionine standard items.
Fig. 9 is the mass spectrum wave spectrogram 1 of astragali mushroom extracting solution.
Figure 10 is the mass spectrum wave spectrogram 2 of astragali mushroom extracting solution.
Figure 11 is astragali mushroom grain extracting solution mass spectrogram 1.
Figure 12 is astragali mushroom grain extracting solution mass spectrogram 2.
Figure 13 is positive astragali mushroom grain extracting solution mass spectrogram 1.
Figure 14 is positive astragali mushroom grain extracting solution mass spectrogram 2.
Figure 15 is L- selenomethionine solid etalon product infrared spectrum.
Figure 16 is L- selenomethionine liquid standard product infrared spectrum.
Figure 17 is the infrared spectrum of astragali mushroom extracting solution.
Figure 18 is positive the infrared spectrum of astragali extracting solution.
Figure 19 is astragali mushroom grain extracting solution infrared spectrum.
Figure 20 is L- selenomethionine standard items ultraviolet scanning atlas.
Figure 21 is that the measurement of selenomethionine UV scanning obtains standard curve.
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 selenomethionine content in measurement BEIQI MUSHROOM, using BEIQI MUSHROOM as raw material, using enzymatic isolation method to seleno
Methionine carries out coarse extraction and utilizes mass spectrum, ultra-red mass spectrum and ultraviolet detection standard items and confession after thin layer chromatography isolates and purifies
Test product simultaneously measures content, measures optical activity and specific rotatory power, its fusing point is measured after crystallization；Specifically: using BEIQI MUSHROOM as raw material, powder
5% nitric acid solution, solid-liquid ratio 1:6, centrifugally operated, deposit addition Tris-Hcl, ultrasonic treatment, enzyme is added in broken screening
Solution 36 hours is centrifuged 10min, takes supernatant, and ultrafiltration centrifugation filters, obtains crude extract；With n-butanol: acetic acid: water=7:1:2 is
Solvent isolates and purifies selenomethionine through thin-layer chromatography expansion, uses chromatography methanol and ultrapure water as mobile phase, use mass spectrograph
Sample molecule quality information is measured, with its functional group of infrared detection, and its molecular structure is analyzed, utilizes double ultraviolet specrophotometers
Content is measured, optical activity and specific rotatory power is measured using polarimeter, measures fusing point after solution evaporative crystallization.
Refering to Figure 1, detailed process are as follows:
Step a, using BEIQI MUSHROOM as raw material, 5% nitric acid solution, solid-liquid ratio 1:6 is added in crushing and screening, and centrifugally operated retains
The Tris-Hcl of 0.5mmol/L is added in deposit, and ultrasonic treatment sequentially adds trypsase, Proteinase K every 12h,
Oscillation enzymatic hydrolysis 36 hours at 37 DEG C；Then centrifugation 10min, takes supernatant, is centrifuged, is removed with the ultrafiltration that molecular cut off is 3KD
Protease and macromolecular substances are filtered with the filter membrane of 0.45um, obtain crude extract；
Step b, L- selenomethionine standard items add Chromatographic Pure Methanol to dissolve, and constant volume obtains the L- selenium of 1mg/mL after completely dissolution
For methionine standard solution, and places it in 2~4 DEG C of refrigerator and save backup；
Step c by chromatoplate heat-activated and after cooling down, draws crude extract and 25 μ L of standard solution, away from bottom edge 2cm respectively
Uniform point sample on horizontal line, origin diffusion diameter is no more than 3mm after sample-adding, puts again after dry primary；With n-butanol: acetic acid: water=7:
1:2 is BEIQI MUSHROOM test sample solvent, and after sealing, presaturation 20min is put into ascending development in chromatography cylinder, to be deployed dose of forward position
When away from the 0.8-1.2cm of chromatoplate top, taking-up is dried, and the chromatoplate after colour developing is put into thin-layer chromatography imaging system, Yu Zi
Chromatography is inspected under outer wavelength 365nm, indicates, the chromatoplate extended is sprayed with ninhydrin solution, is allowed to develop the color, in ultraviolet lamp
Lower label, comparing calculation Rf value, system is taken pictures, and is indicated；
Step d, uses chromatography methanol and ultrapure water as mobile phase, draws a certain amount of standard solution with microsyringe, into
Sample is analyzed spectrogram, is saved；Test sample chromatoplate is placed on TLC- mass spectrometer interface, irises out laser alignment to be measured
At spot centers, sampling is analyzed spectrogram, is saved；
Step e, opens Fourier infrared spectrograph, and testing background is drawn standard solution at point sample center with capillary, surveyed
Test agent carries out processing analysis to infrared spectrum, and the spot to be measured gently scraped on chromatoplate is placed in a beaker, and chromatography methanol is added,
Filter, take filtrate, be made prepare liquid, observe prepare liquid infrared detection method and standard items detect whether it is identical；
Prepare liquid is put into double ultraviolet specrophotometers by step f, and methanol is reference, and setting scanning range is 200~700nm,
Spectral scan obtains ultraviolet scanning atlas, maximum absorption peak；
L- selenomethionine standard items are configured to the solution of 0.05-0.25mg/mL, concentration gradient is set in maximum absorption peak
Under make each filter liquor concentration of a standard curve determination and light absorption value；
Step g corrects polarimeter, pours into standard items, prepare liquid respectively, and shaping modes measure optical activity and specific rotatory power, record
Data.
Step h will measure its fusing point after prepare liquid evaporative crystallization.
It is illustrated below by embodiment.
Embodiment:
Step a, the coarse extraction of selenomethionine:
It by BEIQI MUSHROOM, astragali mushroom grain, the drying of positive astragali, is crushed with high-speed multifunctional pulverizer, crosses the screening of 80 meshes, be packed into glass
It is saved in glass vessel, accurately weighs 0.5g and be respectively placed in the plastic centrifuge tube of 4mL, be added in 5% nitric acid solution of 3mL, stood
Cover the violent shake of bottle cap；It is centrifuged 5min with 12000r/min, removes supernatant, twice with pure water, retains sediment
Matter is spare, and deposit is transferred in 250mL conical flask, and 30mLTris-Hcl(0.5mmol/L, PH=7.2 are added), ultrasound
5min is handled, 15mg trypsase, 15mg Proteinase K are sequentially added every 12h, to shake 100~180r/min of speed at 37 DEG C
Oscillation enzymatic hydrolysis 36 hours；10min is then centrifuged with 4000r/min, takes supernatant, is 3KD ultrafiltration centrifugation with molecular cut off, removes
Deproteinized enzyme and macromolecular substances are filtered with the filter membrane of 0.45um, obtain crude extract.
The preparation of step b, L- selenomethionine standard items:
The L- selenomethionine standard items of accurate weighing 10mg add Chromatographic Pure Methanol to dissolve in weighing bottle, and transfer is in ultrasound
In clean 10mL volumetric flask, constant volume obtains the L- selenomethionine standard solution of 1mg/mL after completely dissolution, and is set
It is saved backup in 2~4 DEG C of refrigerator.
Step c, thin-layer chromatography isolate and purify selenomethionine:
Point sample: after the chromatoplate cooling after activation, L- selenomethionine solution is drawn with capillary, away from bottom edge 2cm horizontal line
Upper uniform point sample, origin diffusion diameter is no more than 3mm after sample-adding every time, puts again after dry primary.
The spotting methods of product to be tested and the spotting methods of standard items are identical.
Expansion: using n-butanol: acetic acid: water=7:1:2 is BEIQI MUSHROOM test sample solvent, and after sealing, presaturation 20min makes
Solvent steam is uniformly distributed in chromatography cylinder, the silica gel column chromatography plate after point sample is put into chromatography cylinder with about 75 ° of inclination angles, uplink
Expansion, solvent do not submerge point sample spot, dissolve in solvent to prevent desired substance, and forward position to be deployed is away from silica gel column chromatography plate top
When holding at 1cm, takes out chromatoplate and dry.
It inspects: chromatoplate being put into thin-layer chromatography imaging system, chromatoplate is inspected under ultraviolet wavelength 365nm, system
It takes pictures, and indicates.
Colour developing: the chromatoplate extended is sprayed with ninhydrin solution, is allowed to develop the color, is drawn in the UV lamp with pencil to be measured
It runs away in product chromatoplate each substance, comparing calculation Rf value finds out the position of selenomethionine in product to be tested lamellae, and system is clapped
According to, and indicate.
Step d, Mass Spectrometer Method:
The Mass Spectrometer Method of L- selenomethionine standard items: use chromatography methanol and ultrapure water as mobile phase, by Expressions matter
Spectrometer and software opening are at working condition, are inserted with the L- selenomethionine standard solution that microsyringe draws 10 μ l
Enter at injection port, get to Load grades of beginning sample introductions, into complete sample after return Inject grades.Point is opened spectrogram interface and is carried out to spectrogram
It analyzes and saves.
The Mass Spectrometer Method of prepare liquid: it uses chromatography methanol and ultrapure water as mobile phase, mass spectrograph and software is made to be in work
Test sample chromatoplate is placed on TLC- mass spectrometer interface by state, at the L- selenomethionine spot centers for irising out laser alignment,
It is sampled with software operation, point opens spectrogram interface and spectrogram is analyzed and saved.
Step e, infrared detection:
The infrared detection of L- selenomethionine standard items: it opens Fourier infrared spectrograph and selects corresponding working interface, with wiping
Mirror paper is wiped clean at point sample center, testing background, draws a small amount of L- selenomethionine standard solution in point sample with capillary
At center, test sample, the infrared spectrum occurred to spectrogram interface carries out processing analysis.
The infrared detection of prepare liquid: the L- selenomethionine spot to be measured drawn gently scraped on chromatoplate with pocket knife is placed in burning
In cup, the Rf value of the L- selenomethionine spot to be measured drawn is identical as L- selenomethionine standard items.It is added in beaker
10ml chromatography methanol, filters to take filtrate, and prepare liquid is made, and opens Fourier infrared spectrograph and selects corresponding working interface, uses
Lens wiping paper is wiped clean at point sample center, testing background, draws a small amount of prepare liquid at point sample center with capillary, test specimens
Product, the infrared spectrum occurred to spectrogram interface carry out processing analysis.
Step f, ultraviolet assay:
Resulting BEIQI MUSHROOM prepare liquid is placed in quartz colorimetric utensil, using methanol as reference, is put into double ultraviolet specrophotometers
It is that spectral scan is carried out within the scope of 200~700nm that scanning range, which is arranged, obtains ultraviolet scanning atlas, obtains maximum absorption peak.
By L- selenomethionine standard items be configured to 0.05mg/mL, 0.1mg/mL, 0.15mg/mL, 0.2mg/mL,
0.25mg/mL is made a standard curve under maximum absorption peak with this concentration gradient, it is dense to measure each filtrate on this basis
Degree and light absorption value.
Step g, polarimeter carry out physical characterization detection:
Polarimeter is corrected with methanol, pours into standard items, prepare liquid respectively in polarimeter, shaping modes measure optical activity and than rotation
Luminosity records data.
Step h, the measurement of fusing point；
By the sample stainless steel electric hot plate heating crystalline after measurement optical activity and specific rotatory power, by prepare liquid evaporating completely knot
Crystalline substance is placed in melting point apparatus and measures fusing point respectively, and measurement takes its average value three times.
Interpretation of result:
1. selenomethionine thin-layer chromatographic analysis:
By L- selenomethionine standard solution, astragali mushroom extracting solution, 60 F254 (5 of model TLC Silica gel is used
X7.5CM lamellae expansion), using n-butanol: acetic acid: water=7:1:2 observes map after expansion as solvent, reuses indenes three
It is observed after ketone colour developing, the map before colour developing is shot under the ultraviolet light of 365nm, and the map after colour developing is shot under white light.
By Fig. 2-4 it is found that the spot that is marked out of horizontal line 1 is co-located.It is computed the marked spot of Fig. 2, i.e. L- seleno egg
Propylhomoserin standard solution, Rf value are 0.368, Fig. 3,4 marked spots, i.e. astragali mushroom extracting solution, and RF value is 0.386, are tentatively recognized
Spot by marking in three width figures is same substance, is selenomethionine, can further detect.
By astragali mushroom extracting solution, astragali mushroom grain extracting solution, positive astragali extracting solution, model GF254(Qingdao Haiyang is used
10x10cm) silica gel column chromatography plate, L- selenomethionine standard solution use model GF254(Qingdao Haiyang 7.5x2.5cm) silicon
The expansion of glue chromatoplate, in Fig. 5, the 1st, 2 group is astragali mushroom extracting solution from left to right；3rd, 4 group is astragali mushroom grain extracting solution；
5th, the 6 group of astragali extracting solution that is positive, in figure 2 where L- selenomethionine speckle displacement, it has been observed that at 2 mark of horizontal line
BEIQI MUSHROOM and positive astragali have the same spot at same position, are unfolded with reference to L- selenomethionine standard solution in Fig. 6
Speckle displacement afterwards is nearly at sustained height with the speckle contrast through developing the color.The Rf value for being computed the marked spot of Fig. 5 is
In 0.587 and 0.575, Fig. 6 the Rf value of spot be 0.588, so it was initially believed that astragali mushroom extracting solution and positive astragali extracting solution all
Selenomethionine is proposed, does not extract selenomethionine in astragali mushroom grain.
2. selenomethionine Mass Spectrometer Method is analyzed:
The Mass Spectrometer Method of astragali mushroom extracting solution is analyzed:
Mass spectrogram is measured using ESI ion source, and ion mode is double ion mode.L- selenomethionine relative molecular mass is
196.6.Base peak is 195.9m/z in map, and with reference to Fig. 7, selenomethionine contains amino and carboxyl, bombards state in high temperature
Under be easy to lose hydrogen ion in-COOH, therefore in map base peak mass-to-charge ratio value lower than relative molecular mass 1, be normal
Losses of ions.Fig. 9,10 are all the mass spectrogram of astragali mushroom extracting solution, the measurement that is identical, but being intercepted of selected BACKGROUND Time section
Period is different, and the two fundamental peak is identical more further to prove that this kind of structure of matter is that electric shock bangs the structure from after rather than idol
So gained.With reference to Fig. 8-10, the base peak occurred in the mass spectrogram for testing the astragali mushroom extracting solution measured twice is respectively
195.9m/z and 160m/z, it is identical as the base peak of standard items map of L- selenomethionine, it is believed that its be selenomethionine
Peak value, other peak values be possible to for by other react obtained by substance peaks may also be impurity peak.
The Mass Spectrometer Method of astragali mushroom grain extracting solution is analyzed:
Figure 11,12 are observed, the peak measured is more and miscellaneous, and from 400m/z to 700m/z etc., compares and find with Fig. 8, astragali mushroom
The mass spectrogram of grain extracting solution does not have the peak value of 196m/z or so, it is believed that not detecting the peak value of selenomethionine, thin layer
It is consistent to detect obtained result.
The Mass Spectrometer Method analysis of positive astragali extracting solution:
Figure 13,14 are observed, compares and finds with Fig. 8, the base peak of the map of positive astragali extracting solution and the standard items of L- selenomethionine
Base peak it is identical, be the peak value of 196m/z or so, it is believed that this section of peak value is the peak value of selenomethionine.
3. selenomethionine ultra-red mass spectrum tests and analyzes:
Shown in ultra-red mass spectrum detection figure be using measured by ATR, be saturated c h bond absorption peak be located at 2800~
3000cm-1, Figure 15 is observed, is found in 2800~3000cm-1Only one obvious absorption peak in range, and 600~
1600cm-1In the range of there are many obvious absorption peak.As can be seen from FIG. 7, characteristic peak should be carboxyl, amino and selenium
Shown peak value, comparison discovery is in 697cm-1There is absorption peak at place, and preliminary judgement contains C-Se key, in 2918cm-1Place has a bright
Aobvious absorption peak can tentatively be judged as caused by the stretching vibration of c h bond, in 1219cm-1There is apparent absorption peak that can tentatively sentence
Break in carboxylic acid C-O key, in 1344cm-1C-N key that place has absorption peak to be tentatively judged as in amino, in 1566cm-1Place has
Absorption peak is tentatively judged as the N-H key in amino；In addition to this it is located at 1604cm-1The absorption peak at place may be c h bond bending vibration
Caused by dynamic, peak value described above is the characteristic peaks of selenomethionine.
Observe Figure 15,16, selenomethionine liquid standard product be liquid is dissolved into using chromatography methanol, therefore with seleno egg
Propylhomoserin solid etalon product map is compared to there is biggish difference, in 3200~3400cm-1There is a very big wide absorption peak in range,
It is judged as the peak-OH.In 1645cm-1There is obvious absorption peak, can determine whether to be caused by N-H deformation vibration, in 1014cm-1
The absorption peak at place can determine whether as caused by C-N key stretching vibration.
Figure 17-19 is observed, compares discovery with L- selenomethionine liquid standard product map and solid etalon product map
The tendency of entire map is roughly the same, and the characteristic absorption peak of Figure 16-18 is substantially similar, in 1640cm-1、1340cm-1、
1030cm-1And 700cm-1There is absorption peak in left and right；There is no similar absorption peak in Figure 19, so can more determine from astragali
Selenomethionine has been extracted in mushroom and Radix Astragali, and does not find selenomethionine in astragali mushroom grain.
4. ultraviolet assay analysis:
Figure 20 is observed it is found that L- selenomethionine standard items have maximum absorption band at 214nm, through looking into, L- selenomethionine exists
220nm or so has maximum absorption band, and measured result is consistent with it.Concentration ladder is set within the scope of 0.05000-0.2500mg/mL
Degree, UV scanning measurement obtain standard curve, as shown in figure 21.Seleno egg in the concentration of selenomethionine and Radix Astragali in BEIQI MUSHROOM
The concentration of propylhomoserin is respectively 0.04mg/mL, 0.07mg/mL, and it is respectively 0.394 and 0.737 that measurement, which obtains Abs, and corresponding standard is bent
Line computation, the content of selenomethionine is 0.24% in BEIQI MUSHROOM, and selenomethionine content is 0.42% in positive astragali.
5. the optical activity of selenomethionine, specific rotatory power are analyzed:
Reference table 1-3 only mentions astragali mushroom extracting solution and Radix Astragali because not extracting selenomethionine from astragali bacterium grain
Take liquid to measure, the selenomethionine standard items measured and BEIQI MUSHROOM prepare liquid, positive astragali prepare liquid are almost the same, thus into
The prepare liquid isolated and purified in one step confirmation BEIQI MUSHROOM, positive astragali is selenomethionine.
1 L- selenomethionine standard items optical activity of table, specific rotatory power
| Number | 1 | 2 | 3 | 4 | 5 | 6 |
| Optical activity | 18.286 | 18.652 | 18.952 | 19.064 | 19.216 | 18.626 |
| Specific rotation | 17.539 | 17.267 | 18.531 | 19.056 | 16.998 | 18.925 |
Selenomethionine optical activity, specific rotatory power in 2 astragali mushroom extracting solution of table
| Number | 1 | 2 | 3 | 4 | 5 | 6 |
| Optical activity | 17.825 | 18.506 | 18.793 | 19.064 | 17.967 | 17.859 |
| Specific rotation | 18.576 | 17.624 | 17.965 | 19.026 | 18.764 | 18.361 |
Selenomethionine optical activity, specific rotatory power in the positive astragali extracting solution of table 3
| Number | 1 | 2 | 3 | 4 | 5 | 6 |
| Optical activity | 18.206 | 19.652 | 17.952 | 18.065 | 18.692 | 19.426 |
| Specific rotation | 17.439 | 17.853 | 19.420 | 17.646 | 18.543 | 18.624 |
6. the melting point analysis of selenomethionine:
By the sample stainless steel electric hot plate heating crystalline after measurement optical activity and specific rotatory power, it is placed in melting point apparatus and measures respectively
Fusing point, measurement take its average value three times.Crystalline melt point through measuring astragali mushroom extracting solution is respectively 268,265,266, average value
About 266；The crystalline melt point of Radix Astragali extractive solution is respectively 266,265,266, and average value is about 266；L- selenomethionine standard
The crystalline melt point of product is 265,267,267, and average value is about 266.
Conclusion: the present invention, using astragali mushroom grain, positive astragali as control, is tentatively judged from north using BEIQI MUSHROOM as raw material
Stilbene mushroom, astragali bacterium, the method that selenomethionine is extracted in Radix Astragali are as follows: dust technology, which is added, is denaturalized protein precipitation, then uses
15mg trypsase, 15mg Proteinase K vibrate enzymolysis protein matter 36h, obtain crude extract through centrifugal filtration, isolate and purify through thin layer
BEIQI MUSHROOM obtains four groups of clearly spots afterwards, and all purple after ninhydrin develops the color, have in positive astragali one group it is relatively clear
Spot has no clear spot in astragali mushroom grain, sends out after the speckle contrast being unfolded with the standard items of L- selenomethionine
In existing BEIQI MUSHROOM, positive astragali and the standard items of L- selenomethionine have the spot positioned at sustained height, and the Rf for being computed three is big
It causes identical.It is dissolved after spot obtained is snapped out with pocket knife with methanol, the solution of acquisition is detected with mass spectrograph, preliminary analysis institute
Obtain one group of spot in the closest expansion forward position of mass spectrum graph discovery BEIQI MUSHROOM, the mark of the spot in positive astragali and L- selenomethionine
Quasi- quality spectrogram has identical mass-to-charge ratio peak value (196 or so), and does not have in astragali bacterium grain, then by each prepare liquid infrared spectroscopy
Instrument detection, finally again with melting point apparatus and polarimeter detection characterization.In addition the provable object extracted from BEIQI MUSHROOM and positive astragali
Matter is selenomethionine, does not propose selenomethionine from astragali bacterium grain.It calculates and measures through content, selenomethionine in BEIQI MUSHROOM
Content is 0.24%, and selenomethionine content is 0.42% in positive astragali.Compare selenomethionine in the two content discovery BEIQI MUSHROOM
Content is few, it follows that conclusion subtracts with the content that positive astragali etc. is selenomethionine during leftover bits and pieces is trained BEIQI MUSHROOM
It is few.
The present invention be using BEIQI MUSHROOM as raw material, using astragali mushroom grain, positive astragali as control, tentatively judge from BEIQI MUSHROOM,
The method of selenomethionine is extracted in astragali bacterium, Radix Astragali are as follows: dust technology, which is added, is denaturalized protein precipitation, then uses 15mg pancreas egg
White enzyme, 15mg Proteinase K vibrate enzymolysis protein matter 36h, obtain crude extract through centrifugal filtration, gained crude extract is through thin-layer chromatography point
It from purifying, scrapes deblurring and prepare liquid is made, detected using mass spectrograph, infrared spectrometer detection, ultraviolet detection use automatic rotation
Light instrument, melting point apparatus etc. are tested.It can guarantee that selenomethionine is not in BEIQI MUSHROOM using the method for originally isolating and purifying with examining
What is be destroyed isolates and purifies, and the method recovery rate that can confirm that the present invention uses through the method for the present invention detection is high, and separating effect is preferable,
Purity is high realizes the separation preparation for analyzing gram quantity grade from microgram magnitude, and being that one kind is effective extracts selenium in BEIQI MUSHROOM
For the method for methionine.
Claims (2)
1. a kind of method of selenomethionine content in measurement BEIQI MUSHROOM, which is characterized in that using BEIQI MUSHROOM as raw material, using enzymatic hydrolysis
Method carries out coarse extraction to selenomethionine and utilizes mass spectrum, ultra-red mass spectrum and ultraviolet detection mark after thin layer chromatography isolates and purifies
Quasi- product and test sample simultaneously measure content, measure optical activity and specific rotatory power, its fusing point is measured after crystallization；Specifically: with BEIQI MUSHROOM
For raw material, 5% nitric acid solution is added in crushing and screening, and solid-liquid ratio 1:6, centrifugally operated, Tris-Hcl is added in deposit, super
Sonication digests 36 hours, is centrifuged 10min, takes supernatant, and ultrafiltration centrifugation filters, obtains crude extract；With n-butanol: acetic acid:
Water=7:1:2 is solvent, isolates and purifies selenomethionine through thin-layer chromatography expansion, uses chromatography methanol and ultrapure water as flowing
Phase measures sample molecule quality information with mass spectrograph, with its functional group of infrared detection, and analyzes its molecular structure, utilize double purples
Outer spectrophotometric determination content measures optical activity and specific rotatory power using polarimeter, measures fusing point after solution evaporative crystallization.
2. the method for selenomethionine 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, 5% nitric acid solution, solid-liquid ratio 1:6 is added in crushing and screening, and centrifugally operated retains
The Tris-Hcl of 0.5mmol/L is added in deposit, and ultrasonic treatment sequentially adds trypsase, Proteinase K every 12h,
Oscillation enzymatic hydrolysis 36 hours at 37 DEG C；Then centrifugation 10min, takes supernatant, is centrifuged, is removed with the ultrafiltration that molecular cut off is 3KD
Protease and macromolecular substances are filtered with the filter membrane of 0.45um, obtain crude extract；
Step b, L- selenomethionine standard items add Chromatographic Pure Methanol to dissolve, and constant volume obtains the L- selenium of 1mg/mL after completely dissolution
For methionine standard solution, and places it in 2~4 DEG C of refrigerator and save backup；
Step c by chromatoplate heat-activated and after cooling down, draws crude extract and 25 μ L of standard solution, away from bottom edge 2cm respectively
Uniform point sample on horizontal line, origin diffusion diameter is no more than 3mm after sample-adding, puts again after dry primary；With n-butanol: acetic acid: water=7:
1:2 is BEIQI MUSHROOM test sample solvent, and after sealing, presaturation 20min is put into ascending development in chromatography cylinder, to be deployed dose of forward position
When away from the 0.8-1.2cm of chromatoplate top, taking-up is dried, and the chromatoplate after colour developing is put into thin-layer chromatography imaging system, Yu Zi
Chromatography is inspected under outer wavelength 365nm, indicates, the chromatoplate extended is sprayed with ninhydrin solution, is allowed to develop the color, in ultraviolet lamp
Lower label, comparing calculation Rf value, system is taken pictures, and is indicated；
Step d, uses chromatography methanol and ultrapure water as mobile phase, draws a certain amount of standard solution with microsyringe, into
Sample is analyzed spectrogram, is saved；Test sample chromatoplate is placed on TLC- mass spectrometer interface, irises out laser alignment to be measured
At spot centers, sampling is analyzed spectrogram, is saved；
Step e, opens Fourier infrared spectrograph, and testing background is drawn standard solution at point sample center with capillary, surveyed
Test agent carries out processing analysis to infrared spectrum, and the spot to be measured gently scraped on chromatoplate is placed in a beaker, and chromatography methanol is added,
Filter, take filtrate, be made prepare liquid, observe prepare liquid infrared detection method and standard items detect whether it is identical；
Prepare liquid is put into double ultraviolet specrophotometers by step f, and methanol is reference, and setting scanning range is 200~700nm,
Spectral scan obtains ultraviolet scanning atlas, maximum absorption peak；
L- selenomethionine standard items are configured to the solution of 0.05-0.25mg/mL, concentration gradient is set in maximum absorption peak
Under make each filter liquor concentration of a standard curve determination and light absorption value；
Step g takes prepare liquid to measure optical activity and specific rotatory power respectively, records data；
Step h will measure its fusing point after prepare liquid evaporative crystallization.
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| CN114166790A (en) * | 2021-11-16 | 2022-03-11 | 华中农业大学 | Intermediate infrared rapid batch detection method for content of free methionine in milk |
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