CN105259284A - Method for measuring form of selenium in selenium-rich spiral seaweed - Google Patents
Method for measuring form of selenium in selenium-rich spiral seaweed Download PDFInfo
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Abstract
The invention discloses a method for measuring the form of selenium in selenium-rich spiral seaweed. Wall breaking treatment is carried out on a selenium-rich spiral seaweed sample through lysozyme, then the treated sample is subjected to enzymolysis in a substep continuous extracting mode, and after enzymatic hydrolysate obtained through enzymatic reactions at all steps passes through a film, the form of selenium is measured through an HPLC-HG-AFS upper computer. The method is used for measuring the form of selenium in the selenium-rich spiral seaweed sample, the extraction ratio is high, the form of selenium of the sample is stable, and the form of selenium cannot change in the reaction process.
Description
Technical field
The present invention relates to a kind of assay method for Se form in selenium enriched Spirulina, belong to the analytical test field of selenium.
Background technology
Selenium is the trace element of needed by human; it is the required composition of glutathione peroxidase; selenium has oxidation resistant effect; closely bound up with health, selenium has anticancer, cardioprotection, treating liver necrosis, control myopia and cataract, removing toxic substances, raising immunity, delays senility and strengthen the multiple pharmacological effect such as reproductive function.According to incompletely statistics, have more than 40 countries and regions to lack selenium in the world, and China there is the area of 72% to belong to scarce selenium area, therefore, finds and develop rich selenium product, to ensure that human body takes in sufficient selenium, just paid close attention to by various countries scientific worker.
Spirulina is a class unicellular lower eukaryote, prokaryotes, and the filamentous be made up of unicellular or many cells is cylindrical, in loose or closely well-regulated spiral rotary shape bend, shape as clockwork feed, so gain the name.Spirulina can directly eat for human body, nutritious, be rich in protein, protein content is up to 60%-70%, and it is fatty, content of cellulose is low, and containing miscellaneous vitamin, it is the food that cobalamin and content beta-carotene are the highest, in addition, in it or all food, absorbability irony content is the highest, also find that it contains and has anti-cancer simultaneously, the algae protein of effect for cancer, and the bioactivator of other a large amount of mineral elements and raising immunity of organisms, have and alleviate cancer radiation, the toxicity of chemotherapy, improve immunologic function, reduce cholesterol, the effects such as blood fat.Based on above-mentioned effect, spirulina is more and more subject to people's attention as the health-related food of one.Research shows, spirulina has stronger concentration effect to selenium, and selenium can be utilized to carry out fortification, makes the selenium enriched Spirulina product being rich in selenium, and make it possess the double effects of spirulina and selenium, development prospect is very wide simultaneously.
At present, all there is fairly large spirulina manually to cultivate both at home and abroad, but also mainly conceptual phase is rested on for the rich selenium cultivation of spirulina, market has no selenium enriched Spirulina product substantially.By to the document of selenium enriched Spirulina and patent investigation, find the research of current selenium enriched Spirulina, major part is only using total Se content as evaluation index, and the research be not distributed in what manner about selenium in selenium enriched Spirulina in frond, namely for the Se form distribution of selenium enriched Spirulina, there is not been reported.And there is certain toxicity due to selenium, Organic Selenium is higher than inorganic selenium security, and more easily absorb by human body, therefore the spirulina being rich in Organic Selenium is only safely and effectively for human body.Therefore, be necessary to measure the Se form in selenium enriched Spirulina, understand fully the distribution of its Se form.At present, for the assay method of Se form in selenium enriched Spirulina, there is not been reported specially, and existing Se form assay method is mainly for plant, aquatic products, yeast etc." in selenium-rich rice Organic Selenium, albumen selenium, polysaccharide selenium or RNA Determination of Selenium method " (number of patent application: 201310135560.3), micro-wave digestion-ICP-MS method directly measures the assay method of Organic Selenium in marine product, albumen selenium or polysaccharide selenium " patent such as (patent No.: ZL201210458397.1) then adopts and carries out extraction and isolation to sample; by containing after each Component seperation of selenium; then utilize machine ICP-MS on to measure the total Se content of each component, thus determine the content of the selenium difference in sample in conjunction with component.But the method can only determine selenium with which kind of biomacromolecule is combined, and the kind obtaining each seleno-amino acids that can not be concrete; " a kind of assay method of selenium " (number of patent application: 201310658482.5) describe the method utilizing HPLC-HG-AFS to measure Se form, the method, by the total selenium in working sample and inorganic selenium content, then utilizes minusing to obtain the content of sample Organic Selenium.The method only simply utilizes the difference of total selenium and inorganic selenium to be defined as organic selenium content, not only result out of true, and cannot determine that concrete Organic Selenium is the selenium of which kind of form, (the number of patent application: 201110440135.6) then describe one and utilize the aqueous slkali of pH value 11 ~ 14 to the method for the extraction of Selenonic protein in Se-enriched yeast that " extracts the method for Selenonic protein from Se-enriched yeast ", but the highly basic class solution that this method utilizes can damage Se form, is unfavorable for the mensuration of Se form, " assay method of form of selenium in selenium-rich plant material " (number of patent application: 201110428248.4), " assay method of form of selenium in selenium-enriched edible fungi " (number of patent application: 201110428249.9), 201210215457.5) etc. " assay method of Selenium in Plants form " (number of patent application: the process assay method that patent describes plant Selenium In Some Selenium-rich Biological Samples form, these methods adopt utilizing Proteinase K and XIV to carry out enzymolysis after sample pre-treatments, by discharging of the selenium gentleness of component each in sample, then on sharp HPLC-HG-AFS or HPLC-ICP-MS, machine measures Se form.Inventor utilizes said method to carry out finding after Se form measures to selenium enriched Spirulina, the recall rate of form extraction ratio and instrument is all very low, Se form distribution in selenium enriched Spirulina cannot be obtained accurately, possible reason be spirulina belong to prokaryotes, its eucaryotic cell structure is all different with plant from selenoprotein structure in body, utilizes the method measuring Selenium in Plants form to be effectively separated the selenium of each form.
Summary of the invention
Object of the present invention will be set up one exactly and be specifically designed to Se form method for measuring in selenium enriched Spirulina, is intended to the blank filling up selenium enriched Spirulina Se form assay method, solves and utilizes other selenium to survey the not high problem that maybe cannot detect of morphological method extraction ratio.
Se form method for measuring in a kind of selenium enriched Spirulina, comprises the steps:
(1) pre-treatment of sample: the selenium enriched Spirulina dry sample ground of learning from else's experience, adds damping fluid and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the potpourri of step (1) through pre-treatment, adjust ph, to 5-7, then adds lysozyme wherein, enzymolysis;
(3) sample enzymolysis, one of them or its that comprise the following steps combination in any (as: combination such as abcd, acd, bcd) in order:
The pH value of the potpourri a. step (2) obtained is adjusted to 7.5, adds Proteinase K, enzymolysis,
B. the potpourri after step a enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, the sediment fraction after centrifugal is continued to add damping fluid, adjust ph to 7.5, adds proteinase XIV, enzymolysis,
C. the potpourri after previous step enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, continued to add damping fluid by the sediment fraction after centrifugal, adjust ph, to 1.5-2.5, adds pepsin, enzymolysis,
D. the potpourri after previous step enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, continued to add damping fluid by the sediment fraction after centrifugal, adjust ph to 7.0 ~ 8.0, add trypsase, enzymolysis,
Wherein, if step b is the first step in sample enzymolysis step, then step b is that the pH value of potpourri step (2) obtained is adjusted to 7.5, adds proteinase XIV, enzymolysis; If step c is the first step in sample enzymolysis step, the pH value of the potpourri that step (2) obtains is adjusted to 1.5-2.5 by step c, adds pepsin, enzymolysis; If steps d is the first step in sample enzymolysis step, the pH value of the potpourri that step (2) obtains is adjusted to 7.0 ~ 8.0 by steps d, adds trypsase, enzymolysis;
(4) sample separation: potpourri step (3) obtained after sample enzymolysis, carries out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured;
(5) upper machine measures: adopt the Se form in high performance liquid chromatography and By Hydride Generation-atomic Fluorescence Spectrometry coupling in machine working sample (to comprise: tetravalence selenium (Se to the enzymolysis liquid after crossing water system film in step (3) and (4)
4+), hexavalent selenium (Se
6+), selenocystine (SeCys
2), selenomethionine (SeMet), selenium methyl selenocystine (SeMeCys)).
Preferably, in above-mentioned selenium enriched Spirulina, Se form method for measuring comprises the steps:
(1) pre-treatment of sample: the selenium enriched Spirulina dry sample of grinding of learning from else's experience, adds damping fluid and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the potpourri of step (1) through pre-treatment, adjust ph, to 5-7, then adds lysozyme wherein, enzymolysis;
(3) sample enzymolysis, comprises the following steps:
The pH value of the potpourri a. step (2) obtained is adjusted to 7.5, adds Proteinase K, enzymolysis,
B. the potpourri after step a enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, the sediment fraction after centrifugal is continued to add damping fluid, adjust ph to 7.5, adds proteinase XIV, enzymolysis,
C. the potpourri after previous step enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, continued to add damping fluid by the sediment fraction after centrifugal, adjust ph, to 1.5-2.5, adds pepsin, enzymolysis,
D. the potpourri after previous step enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, continued to add damping fluid by the sediment fraction after centrifugal, adjust ph to 7.0 ~ 8.0, add trypsase, enzymolysis,
(4) sample separation: potpourri step (3) obtained after sample enzymolysis, carries out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured;
(5) upper machine measures: to the Se form crossed in step (3) and (4) in the enzymolysis liquid employing high performance liquid chromatography after water system film and By Hydride Generation-atomic Fluorescence Spectrometry coupling in machine working sample.
Preferably, described damping fluid is phosphate buffer or Tris-HCl damping fluid, and concentration is 0.1-0.2mol/L.
Preferably, described in step (1), the mass volume ratio of selenium enriched Spirulina dry sample and damping fluid is 0.1 ~ 0.2g:5mL.
Preferably, in step (2), the mass ratio of lysozyme and described selenium enriched Spirulina dry sample is 100 ~ 200:10 ~ 50, and hydrolysis temperature is 30-50 DEG C, and enzymolysis time is 12 ~ 18h.
Preferably, in step (3), the mass ratio of Proteinase K and described selenium enriched Spirulina dry sample is 20 ~ 50:100 ~ 200,37 DEG C of constant temperature enzymolysis 18 ~ 24h.
Preferably, in step (3), the mass ratio of proteinase XIV and described selenium enriched Spirulina dry sample is 20 ~ 50:100 ~ 200,37 DEG C of constant temperature enzymolysis 18 ~ 24h.
Preferably, in step (3), the mass ratio of pepsin and described selenium enriched Spirulina dry sample is 20 ~ 50:100 ~ 200,37 DEG C of constant temperature enzymolysis 18 ~ 24h.
Preferably, in step (3), the mass ratio of trypsase and described selenium enriched Spirulina dry sample is 20 ~ 50:100 ~ 200,37 DEG C of constant temperature enzymolysis 18 ~ 24h.
Preferably, described selenium enriched Spirulina sample refers to the selenium enriched Spirulina obtained through Liquid Culture, and total Se content is at 10 ~ 1000mg/kg(DW) in scope.As: the selenium enriched Spirulinas such as rich selenium blunt top spirulina, rich selenium spirulina maxim, rich selenium Spirulina subaalsa,
Relative to the scheme of prior art, the invention has the advantages that:
(1) spirulina is prokaryotes, its cell wall structure is different from plant, cell membrane cannot be opened completely by the effect of cellulase, and through the process of lysozyme, the cell membrane of selenium enriched Spirulina can be fully opened, the materials such as intracellular Selenonic protein are released in extract, are conducive to follow-up leaching process;
(2) during enzymolysis, centrifuge tube adopts the mode of horizontal positioned, increases specific surface area, contributes to the generation of enzymatic reaction;
(3) in the present invention, enzymatic reaction adopts different enzymes to carry out substep extraction continuously to sample, get rid of the mutual interference between different enzyme, also the enzymolysis liquid after enzymolysis and reaction system are separated simultaneously, ensure that the stability of each free form selenium in sample after enzymolysis, be also conducive to next step enzymatic reaction simultaneously.
(4) the present invention is adopted to measure selenium enriched Spirulina sample, extraction rate reached is to 50-80%, and adopt traditional plant sample Morphometric Methods, without broken wall, just directly utilize Proteinase K and proteinase XIV to carry out enzymolysis processing to sample merely, and do not carry out stepwise discretization, its extraction ratio is less than 20%, the present invention on this basis, improves nearly 4 times by the highest for extraction ratio.
(5) each step reaction mild condition in the present invention, the steps such as the enzymolysis of the ultrasonic extraction pretreatment process that experiment adopts and each step all can not change the Se form in sample body, Se form can not be caused to change, more can reflect the distribution of Se form in sample really.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described, can better understand the present invention and can be implemented, but illustrated embodiment is not as a limitation of the invention to make those skilled in the art.
Embodiment 1: rich selenium blunt top spirulina sample Se form measures embodiment
The total Se content of rich selenium blunt top spirulina selected is 1003mg/kg(DW).
(1) pre-treatment of sample: the selenium enriched Spirulina dry sample through grinding getting 0.1g, adds 5ml0.1mol/L phosphate buffer and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the above-mentioned sample system adjust ph through pre-treatment to 5-7, then add the lysozyme of 50mg wherein, then centrifuge tube is placed horizontally in 30 ~ 50 DEG C of constant temperature oscillation shaking tables and cultivates 12-18h.
(3) sample enzymolysis: the pH value of above-mentioned reaction system is adjusted to 7.5, then adds the Proteinase K of 5mg, then centrifuge tube is placed horizontally at 8-12h in 50 DEG C of constant temperature oscillation shaking tables.
(4) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph to 7.5, adds the proteinase XIV of 5mg, then centrifuge tube is placed horizontally at 8-12h in 37 DEG C of constant temperature oscillation shaking tables.
(5) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph, to 1.5-2.5, adds the pepsin of 50mg, then centrifuge tube is placed horizontally at 18-24h in 37 DEG C of constant temperature oscillation shaking tables.
(6) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph, to 7.0-8.0, adds the trypsase of 50mg, then centrifuge tube is placed horizontally at 18-24h in 37 DEG C of constant temperature oscillation shaking tables.
(7) sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured.
(8) upper machine measures: to the Se form crossed in above-mentioned four steps in the enzymolysis liquid employing high performance liquid chromatography after film and By Hydride Generation-atomic Fluorescence Spectrometry (HPLC-HG-AFS) coupling in machine working sample.
Implementation result:
Through morphometric analysis, selenium enriched Spirulina form is based on selenocysteine (SeCys), and Organic Selenium ratio reaches 65%, and Se form extraction ratio is 50%.
Embodiment 2: rich selenium blunt top spirulina sample Se form measures embodiment
The total Se content of rich selenium blunt top spirulina selected is 12mg/kg(DW).
(1) pre-treatment of sample: the selenium enriched Spirulina dry sample through grinding getting 0.2g, adds 5ml0.2mol/LTris-HCl damping fluid and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the above-mentioned sample system adjust ph through pre-treatment to 5-7, then add the lysozyme of 10mg wherein, then centrifuge tube is placed horizontally in 30 ~ 50 DEG C of constant temperature oscillation shaking tables and cultivates 12-18h.
(3) sample enzymolysis: the pH value of above-mentioned reaction system is adjusted to 7.5, then adds the Proteinase K of 1mg, then centrifuge tube is placed horizontally at 8-12h in 50 DEG C of constant temperature oscillation shaking tables.
(4) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph to 7.5, adds the proteinase XIV of 1mg, then centrifuge tube is placed horizontally at 8-12h in 37 DEG C of constant temperature oscillation shaking tables.
(5) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph, to 1.5-2.5, adds the pepsin of 20mg, then centrifuge tube is placed horizontally at 18-24h in 37 DEG C of constant temperature oscillation shaking tables.
(6) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph, to 7.0-8.0, adds the trypsase of 20mg, then centrifuge tube is placed horizontally at 18-24h in 37 DEG C of constant temperature oscillation shaking tables.
(7) sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured.
(8) upper machine measures: to the Se form crossed in above-mentioned four steps in the enzymolysis liquid employing high performance liquid chromatography after film and By Hydride Generation-atomic Fluorescence Spectrometry (HPLC-HG-AFS) coupling in machine working sample.
Implementation result:
Through morphometric analysis, rich selenium blunt top spirulina form is based on selenocysteine (SeCys), and Organic Selenium ratio reaches 95%, and Se form extraction ratio is 80%.
Embodiment 3: rich selenium Spirulina subaalsa sample Se form measures embodiment
The total Se content of rich selenium Spirulina subaalsa selected is 117mg/kg(DW).
(1) pre-treatment of sample: the rich selenium Spirulina subaalsa dry sample through grinding getting 0.2g, adds 5ml0.1mol/L phosphate buffer and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the above-mentioned sample system adjust ph through pre-treatment to 5-7, then add the lysozyme of 25mg wherein, then centrifuge tube is placed horizontally in 30 ~ 50 DEG C of constant temperature oscillation shaking tables and cultivates 12-18h.
(3) sample enzymolysis: the pH value of above-mentioned reaction system is adjusted to 7.5, then adds the Proteinase K of 2mg, then centrifuge tube is placed horizontally at 8-12h in 50 DEG C of constant temperature oscillation shaking tables.
(4) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph to 7.5, adds the proteinase XIV of 2mg, then centrifuge tube is placed horizontally at 8-12h in 37 DEG C of constant temperature oscillation shaking tables.
(5) sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured.
(6) upper machine measures: to the Se form crossed in above-mentioned two steps in the enzymolysis liquid employing high performance liquid chromatography after film and By Hydride Generation-atomic Fluorescence Spectrometry (HPLC-HG-AFS) coupling in machine working sample.
Implementation result:
Through morphometric analysis, rich selenium Spirulina subaalsa form is based on selenocysteine (SeCys), and Organic Selenium ratio reaches 76%, and Se form extraction ratio is 73%.
Embodiment 4: rich selenium Spirulina subaalsa sample Se form measures embodiment
The total Se content of rich selenium Spirulina subaalsa selected is 651mg/kg(DW).
(1) pre-treatment of sample: the rich selenium Spirulina subaalsa dry sample through grinding getting 0.2g, adds 5ml0.2mol/LTris-HCl damping fluid and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the above-mentioned sample system adjust ph through pre-treatment to 5-7, then add the lysozyme of 30mg wherein, then centrifuge tube is placed horizontally in 30 ~ 50 DEG C of constant temperature oscillation shaking tables and cultivates 12-18h.
(3) sample enzymolysis: the pH value of above-mentioned reaction system is adjusted to 7.5, adds the proteinase XIV of 5mg, then centrifuge tube is placed horizontally at 8-12h in 37 DEG C of constant temperature oscillation shaking tables.
(4) sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured.
(5) upper machine measures: to the above-mentioned Se form crossed in the enzymolysis liquid employing high performance liquid chromatography after film and By Hydride Generation-atomic Fluorescence Spectrometry (HPLC-HG-AFS) coupling in machine working sample.
Implementation result:
Through morphometric analysis, rich selenium Spirulina subaalsa form is based on selenocysteine (SeCys), and Organic Selenium ratio reaches 86%, and Se form extraction ratio is 53%.
Embodiment 5: rich selenium spirulina maxim sample Se form measures embodiment
The total Se content of rich selenium blunt top spirulina selected is 355mg/kg(DW).
(1) pre-treatment of sample: the rich selenium spirulina maxim dry sample through grinding getting 0.2g, adds 5ml0.1mol/L phosphate buffer and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the above-mentioned sample system adjust ph through pre-treatment to 5-7, then add the lysozyme of 45mg wherein, then centrifuge tube is placed horizontally in 30 ~ 50 DEG C of constant temperature oscillation shaking tables and cultivates 12-18h.
(3) sample enzymolysis: the pH value of above-mentioned reaction system is adjusted to 1.5-2.5, adds the pepsin of 45mg, then centrifuge tube is placed horizontally at 18-24h in 37 DEG C of constant temperature oscillation shaking tables.
(4) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph, to 7.0-8.0, adds the trypsase of 45mg, then centrifuge tube is placed horizontally at 18-24h in 37 DEG C of constant temperature oscillation shaking tables.
(5) sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured.
(6) upper machine measures: to the Se form crossed in above-mentioned two each and every one steps in the enzymolysis liquid employing high performance liquid chromatography after film and By Hydride Generation-atomic Fluorescence Spectrometry (HPLC-HG-AFS) coupling in machine working sample.
Implementation result:
Through morphometric analysis, rich selenium spirulina maxim form is based on selenocysteine (SeCys), and Organic Selenium ratio reaches 85%, and Se form extraction ratio is 72%.
Embodiment 6: rich selenium spirulina maxim sample Se form measures embodiment
The total Se content of rich selenium spirulina maxim selected is 987mg/kg(DW).
(1) pre-treatment of sample: the selenium enriched Spirulina dry sample through grinding getting 0.1g, adds 5ml0.2mol/LTris-HCl damping fluid and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the above-mentioned sample system adjust ph through pre-treatment to 5-7, then add the lysozyme of 50mg wherein, then centrifuge tube is placed horizontally in 30 ~ 50 DEG C of constant temperature oscillation shaking tables and cultivates 12-18h.
(3) sample enzymolysis: the pH value of above-mentioned reaction system is adjusted to 7.5, then adds the proteinase XIV of 3mg, then centrifuge tube is placed horizontally at 8-12h in 37 DEG C of constant temperature oscillation shaking tables.
(4) enzymolysis again after sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, sediment fraction after centrifugal is continued to add 5ml damping fluid, adjust ph to 7.5, adds the trypsase of 45mg, then centrifuge tube is placed horizontally at 18-24h in 37 DEG C of constant temperature oscillation shaking tables.
(5) sample separation: the sample after above-mentioned enzymolysis is carried out centrifuging in high-speed refrigerated centrifuge, rotating speed is 10000rpm, centrifugation time 15-30min, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured.
(6) upper machine measures: to the Se form crossed in above-mentioned two steps in the enzymolysis liquid employing high performance liquid chromatography after film and By Hydride Generation-atomic Fluorescence Spectrometry (HPLC-HG-AFS) coupling in machine working sample.
Implementation result:
Through morphometric analysis, rich selenium spirulina maxim form is based on selenocysteine (SeCys), and Organic Selenium ratio reaches 82%, and Se form extraction ratio is 77%.
The above embodiment is only that protection scope of the present invention is not limited thereto in order to absolutely prove the preferred embodiment that the present invention lifts.The equivalent alternative or conversion that those skilled in the art do on basis of the present invention, all within protection scope of the present invention.Protection scope of the present invention is as the criterion with claims.
Claims (10)
1. a Se form method for measuring in selenium enriched Spirulina, is characterized in that, comprise the steps:
(1) pre-treatment of sample: the selenium enriched Spirulina dry sample ground of learning from else's experience, adds damping fluid and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the potpourri of step (1) through pre-treatment, adjust ph, to 5-7, then adds lysozyme wherein, enzymolysis;
(3) sample enzymolysis, one of them or its that comprise the following steps combination in any in order:
The pH value of the potpourri a. step (2) obtained is adjusted to 7.5, adds Proteinase K, enzymolysis,
B. the potpourri after step a enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, the sediment fraction after centrifugal is continued to add damping fluid, adjust ph to 7.5, adds proteinase XIV, enzymolysis,
C. the potpourri after previous step enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, continued to add damping fluid by the sediment fraction after centrifugal, adjust ph, to 1.5-2.5, adds pepsin, enzymolysis,
D. the potpourri after previous step enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, continued to add damping fluid by the sediment fraction after centrifugal, adjust ph to 7.0 ~ 8.0, add trypsase, enzymolysis,
Wherein, if step b is the first step in sample enzymolysis step, then step b is that the pH value of potpourri step (2) obtained is adjusted to 7.5, adds proteinase XIV, enzymolysis; If step c is the first step in sample enzymolysis step, the pH value of the potpourri that step (2) obtains is adjusted to 1.5-2.5 by step c, adds pepsin, enzymolysis; If steps d is the first step in sample enzymolysis step, the pH value of the potpourri that step (2) obtains is adjusted to 7.0 ~ 8.0 by steps d, adds trypsase, enzymolysis;
(4) sample separation: potpourri step (3) obtained after sample enzymolysis, carries out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured;
(5) upper machine measures: to the Se form crossed in step (3) and (4) in the enzymolysis liquid employing high performance liquid chromatography after water system film and By Hydride Generation-atomic Fluorescence Spectrometry coupling in machine working sample.
2. Se form method for measuring in selenium enriched Spirulina according to claim 1, is characterized in that, comprise the steps:
(1) pre-treatment of sample: the selenium enriched Spirulina dry sample of grinding of learning from else's experience, adds damping fluid and carry out ultrasonic extraction;
(2) sample broken wall enzymolysis: to the potpourri of step (1) through pre-treatment, adjust ph, to 5-7, then adds lysozyme wherein, enzymolysis;
(3) sample enzymolysis, comprises the following steps:
The pH value of the potpourri a. step (2) obtained is adjusted to 7.5, adds Proteinase K, enzymolysis,
B. the potpourri after step a enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, the sediment fraction after centrifugal is continued to add damping fluid, adjust ph to 7.5, adds proteinase XIV, enzymolysis,
C. the potpourri after previous step enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, continued to add damping fluid by the sediment fraction after centrifugal, adjust ph, to 1.5-2.5, adds pepsin, enzymolysis,
D. the potpourri after previous step enzymolysis is carried out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured, continued to add damping fluid by the sediment fraction after centrifugal, adjust ph to 7.0 ~ 8.0, add trypsase, enzymolysis,
(4) sample separation: potpourri step (3) obtained after sample enzymolysis, carries out centrifuging, get centrifugal after supernatant to cross 0.22 μm of water system film to be measured;
(5) upper machine measures: to the Se form crossed in step (3) and (4) in the enzymolysis liquid employing high performance liquid chromatography after water system film and By Hydride Generation-atomic Fluorescence Spectrometry coupling in machine working sample.
3. Se form method for measuring in selenium enriched Spirulina according to claim 1 and 2, is characterized in that, described damping fluid is phosphate buffer or Tris-HCl damping fluid, and concentration is 0.1-0.2mol/L.
4. Se form method for measuring in selenium enriched Spirulina according to claim 1 and 2, is characterized in that, described in step (1), the mass volume ratio of selenium enriched Spirulina dry sample and damping fluid is 0.1 ~ 0.2g:5mL.
5. in the selenium enriched Spirulina described in claim 1 or 2, Se form method for measuring, is characterized in that, in step (2), the mass ratio of lysozyme and described selenium enriched Spirulina dry sample is 100 ~ 200:10 ~ 50, and hydrolysis temperature is 30-50 DEG C, and enzymolysis time is 12 ~ 18h.
6. Se form method for measuring in selenium enriched Spirulina according to claim 1 and 2, is characterized in that, in step (3), the mass ratio of Proteinase K and described selenium enriched Spirulina dry sample is 20 ~ 50:100 ~ 200,37 DEG C of constant temperature enzymolysis 18 ~ 24h.
7. Se form method for measuring in selenium enriched Spirulina according to claim 1 and 2, is characterized in that, in step (3), the mass ratio of proteinase XIV and described selenium enriched Spirulina dry sample is 20 ~ 50:100 ~ 200,37 DEG C of constant temperature enzymolysis 18 ~ 24h.
8. Se form method for measuring in selenium enriched Spirulina according to claim 1 and 2, is characterized in that, in step (3), the mass ratio of pepsin and described selenium enriched Spirulina dry sample is 20 ~ 50:100 ~ 200,37 DEG C of constant temperature enzymolysis 18 ~ 24h.
9. Se form method for measuring in selenium enriched Spirulina according to claim 1 and 2, is characterized in that, in step (3), the mass ratio of trypsase and described selenium enriched Spirulina dry sample is 20 ~ 50:100 ~ 200,37 DEG C of constant temperature enzymolysis 18 ~ 24h.
10. Se form method for measuring in selenium enriched Spirulina according to claim 1 and 2, is characterized in that, described selenium enriched Spirulina sample refers to the selenium enriched Spirulina obtained through Liquid Culture, and total Se content is at 10 ~ 1000mg/kg(DW) in scope.
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