CN106290626B - Organic selenium content method for measuring in a kind of selenium-rich peanuts or its product prepared - Google Patents
Organic selenium content method for measuring in a kind of selenium-rich peanuts or its product prepared Download PDFInfo
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- CN106290626B CN106290626B CN201610623681.6A CN201610623681A CN106290626B CN 106290626 B CN106290626 B CN 106290626B CN 201610623681 A CN201610623681 A CN 201610623681A CN 106290626 B CN106290626 B CN 106290626B
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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
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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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
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- G01N30/04—Preparation or injection of sample to be analysed
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- G01N30/14—Preparation by elimination of some components
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Abstract
The invention discloses organic selenium content methods for measuring in a kind of selenium-rich peanuts or its product prepared.It includes the following steps：1) sample to be tested is mixed with enzyme, obtains mixture；2) by mixture successively through ultrasonic wave assisted extraction, water bath with thermostatic control oscillation after, centrifuge, take supernatant and residue；Residue carries out the processing of mixture, obtains supernatant 1 and residue 1；3) take supernatant and supernatant 1 that HPLC ICP MS methods detection organic selenium content is respectively adopted；4) by organic selenium content summation in supernatant and supernatant 1 to get to organic selenium content in sample to be tested and Se-rich wheat reference substance.The present invention can more accurately measure organic selenium content in selenium-rich peanuts or its product.
Description
Technical field
The present invention relates to organic selenium content methods for measuring in a kind of selenium-rich peanuts or its product prepared, belong to food product
Matter analysis method field.
Background technology
Selenium (selenium) has effects that various biological, especially its tool as one of the essential trace elements of the human body
There are the physiological functions such as strengthen immunity, Chronic disease prevention and anticancer, is concerned.Body selemium nutrition passes through dietary int ake.And by
In the limitation (as China has 72% region for different degrees of Selenium deficiency area) of Se in Environment resource, most area natural food
Se content is relatively low.It is improved by agricultural measures, to improve crop Se content, it is biological selenium-enriched that agricultural product may be implemented.Nowadays, selenium-rich
Industry grows up under the support of government, and selenium-rich product has also occupied certain market.However in current selenium-rich industrial chain,
Most selenium-rich product only has restriction or the mark of total Se content, organic selenium content but to rarely have explanation.This allows for some and adds
Add the product of inorganic selenium (sodium selenite, sodium selenate etc.) to mix the genuine with the fictitious, restricts the sound development of industry.
Peanut is large grain and oil crop, and rich in protein is a kind of preferable selenium-rich crops.Have in selenium-rich peanuts
The measurement of machine selenium has to, by grease removing process, but in the process, necessarily introduce processing selenium loss.Organic Selenium is examined at present
Survey method is usually combined into standard specimen with selenium singulation, uses the method for recovery of standard addition to evaluate the recovery rate of selenium, such method is only
Can realize in sample free state Organic Selenium detect evaluation, and cannot in sample in the form of reference state existing for selenium compound
It is quantified, to influence to objectively respond to total organic selenium content in sample.
Invention content
The object of the present invention is to provide organic selenium content methods for measuring in a kind of selenium-rich peanuts or its product prepared, originally
Invention can measure free state Organic Selenium and reference state Organic Selenium, can measure the total amount of Organic Selenium in selenium-rich peanuts.
Organic selenium content method for measuring in selenium-rich peanuts provided by the invention or its product prepared, including walk as follows
Suddenly：
1) sample to be tested is mixed with enzyme, obtains mixture；
The sample to be tested includes selenium-rich peanuts or its product prepared；
2) by the mixture successively through ultrasonic wave assisted extraction, water bath with thermostatic control oscillation after, centrifuge, obtain supernatant
And residue；
After the residue carries out above-mentioned ultrasonic wave assisted extraction, water bath with thermostatic control oscillation, centrifuges, obtain 1 He of supernatant
Residue 1；
3) take the supernatant and the supernatant 1 that HPLC-ICP-MS methods detection organic selenium content is respectively adopted；
4) by organic selenium content summation in the supernatant and the supernatant 1 to get to organic in the sample to be tested
Se content.
In above-mentioned method, the residue and the residue 1 is taken to measure detection institute using micro-wave digestion and ICP-MS methods
State total Se content in residue；Total Se content detection is used for evaluating carrying for sample to be tested Organic Selenium in the residue and the residue 1
Take effect；
According to the common knowledge of this field because in a sample to be tested content of Organic Selenium and total selenium be it is fixed, such as
The content of total selenium is low in fruit residue or is not detected, and illustrates the method for the present invention to there is no errors in the parsing extraction of Organic Selenium；Such as
The content of total selenium is higher in fruit residue, and the content of the Organic Selenium measured through the invention is then relatively low, illustrates to use the method for the present invention
To there is error or error in the parsing extraction of Organic Selenium, then need again to be detected sample to be tested.
In the method for the invention, in order to calculate recovery of standard addition, specifically internal standard compound can be added in the sample to be tested；
Internal standard compound is specially at least one of selenomethionine, selenocysteine and methyl selenocystine；
Additive amount of the internal standard compound in sample to be tested is 0.1~20ppm, concretely 0.1~10ppm；
In the present invention, the quantitative of the organic selenium content uses HPLC-ICP-MS methods, particular by the reservation of mark product
Time determines that sample peak, the quantitative of Organic Selenium use external standard method, determined by standard curve.
In the present invention, the organic selenium content refers to selenomethionine, selenocysteine and methyl selenocystine
Content；
Total Se content includes Organic Selenium and inorganic selenium, refers specifically to sample after resolution,82Se contents.
In the present invention, the selenium-rich peanuts are selected from any one of following a-c：A, it natural selenium-rich planting base or plants
The artificial selenium-rich peanuts arrived；B, by increasing the artificial selenium-rich peanuts that selenium fertilizer is planted to soil and/or blade face；C, purchase in
Selenium-rich peanuts printed words and/or the selenium-rich peanuts in the place of production are clearly indicated in the market；
In above-mentioned method, product prepared by the selenium-rich peanuts includes selenium-rich peanuts benevolence, degreasing selenium-rich peanuts powder, selenium-rich
Peanut protein.
In above-mentioned method, it is to include the following steps to prepare product prepared by the selenium-rich peanuts：By the selenium-rich peanuts
Decladding is impregnated, undresses, is dried, and selenium-rich peanuts benevolence is obtained；After the selenium-rich peanuts benevolence is carried out precomminution, superelevation is used successively
Assisted Extraction degreasing, secondary water bath with thermostatic control extraction, solvent precipitation are pressed, degreasing selenium-rich peanuts powder is obtained；By degreasing selenium-rich peanuts powder
Alkali carries, acid precipitation, dry, crushing are carried out, selenium-rich peanuts albumen is obtained.
In above-mentioned method, the enzyme includes protease and lipase；
The dosage of the protease is 0.7~3U：Mg selenium-rich peanuts or its product prepared, the concretely protease
0.7：Mg selenium-rich peanuts or its product prepared；
The dosage of the lipase is 5~20U：Mg selenium-rich peanuts or its product prepared, the concretely lipase
5U：Mg selenium-rich peanuts or its product prepared.
In the present invention, 1 enzyme activity unit of the protease is referred to when 37 DEG C, pH value are 7.5, in 1 minute
The enzyme amount needed for 1 micromolar tyrosine can be converted；
1 enzyme activity unit of the lipase refers to, when 40 DEG C, pH value are 5.6,1 capable of being converted in 1 minute
Enzyme amount needed for micromolar free fatty (vinyl laurate is as substrate).
In above-mentioned method, in step 1), the sample to be tested is dissolved using hypo solution,
The concentration of the hypo solution can be 4~8mmol/L, concretely 5mmol/L；
Per g selenium-rich peanuts or in the product of its preparation, the dosage of the hypo solution can be 18~25mL, specifically
Can be to use hypo solution described in 4mL in selenium-rich peanuts or its product prepared described in every g.
In above-mentioned method, the power of the ultrasonic wave assisted extraction can be 400~500W, concretely 500W or 450
~500W；
The temperature of the ultrasonic wave assisted extraction can be 30~45 DEG C, concretely 37 DEG C or 35~40 DEG C；
The time of the ultrasonic wave assisted extraction can be 0.5~2h, concretely 1h or 0.8~1.5h.
In above-mentioned method, the temperature of the water bath with thermostatic control oscillation can be 30~45 DEG C, concretely 37 DEG C or 34~40
℃；
The time of the water bath with thermostatic control oscillation can be 12~20h, concretely 20h or 15~20h；
The rotating speed of the water bath with thermostatic control oscillation can be 140~200rpm, concretely 150rpm, 150~200rpm, 140
~150rpm or 140~180rpm.
In above-mentioned method, total Se content detection is organic in sample to be tested for evaluating in the residue and the residue 1
The extraction effect of selenium, evaluation result, recovery of standard addition are 80~120%, the Se-rich wheat powder reference substance Organic Selenium percentage
It is 50~64%, effect is preferable.
In the present invention, the condition of liquid chromatogram is specially Agilent Zorbax RX-C8 chromatographic columns；0.1% 5 fluorine propionic acid
Acid anhydride, 2% methanol, pH 1.98 are mobile phase；The flow velocity of 0.9mL/min.
In order to evaluate the dissociation, extraction efficiency of the method for the present invention, using the method for the present invention Se-rich wheat powder standard substance
It is detected instead of sample to be tested；It, can be with known to the content of total Se content and Organic Selenium is in selenium wheat flour standard substance
It is compared with the result that sample to be tested measures, with the accuracy of the evaluation present invention；Total Se content in selenium wheat flour standard substance
For 17.23 ± 0.91 μ g/g, Organic Selenium (selenomethionine) content is 27.4 ± 2.6 μ g/g；The experiment proved that the method for the present invention
There is accuracy to organic selenium content detection in sample to be tested.
The invention has the advantages that lacking present situation for product Organic Selenium data information in selenium-rich industrial chain, a kind of richness is established
The method that organic selenium content is analyzed in selenium peanut and product.Influence of the different pre-treatments to Organic Selenium recovery rate is explored, it is excellent
The chromatographic condition of Organic Selenium measurement is changed, it is determined that the assay method of defatting peanut powder or albumen powder Organic Selenium；In combination with flower
Raw skimmed milk or albumen powder the selenium loss analysis in preparation process are realized and are determined to selenium-rich peanuts raw material organic selenium content.This hair
Free state Organic Selenium can be measured in bright and measures reference state Organic Selenium, compensate for the deficiency measured to single free selenium, it is real
Now to the objective quantitative of sample Organic Selenium.The present invention uses the enzyme extracting method of protease and lipase, has modified existing water
It carries, the method for different buffer solution or only proteolysis, improves the efficiency that reference state selenium is dissociated into free state；Avoid Organic Selenium
Oxidation, recovery rate higher；Shorten extraction time and avoids the too long Organic Selenium Morphological Transitions brought of sonication times and influence
To extraction effect；The preferable separation for realizing Organic Selenium and quantitative etc., extractive technique are efficiently dissociated in the Organic Selenium of sample, are extracted
Good amendment is played in terms of the transformation of reduction Se form, loss.In addition, in selenium-rich peanuts the measurement of Organic Selenium have to through
Grease removing process is crossed, but in the process, necessarily introduces processing selenium loss, existing method can not be to the Organic Selenium of peanut raw material
Quantitative, selenium damaed cordition in being processed in the preparation process of the invention that can be detected from selenium-rich peanuts raw material to defatted peanut realizes
Selenium-rich peanuts raw material organic selenium content is determined.
Description of the drawings
Fig. 1 is Organic Selenium standard items HPLC-ICP-MS chromatography peak figures in the embodiment of the present invention.
Fig. 2 be in from selenium-rich peanuts raw material to selenium-rich peanuts albumen powder preparation process selenium loss figure (82Hundred flowers of Se μ g/
It is raw).
Fig. 3 is distribution of the selenium in peanut different parts.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
In following embodiments, Se-rich wheat powder standard substance- BC210a is bought in the Beijing LGC Agency；
Protease is available commercially from sigma companies, catalog number 96888；Lipase is available commercially from sigma companies, product mesh
Record number is p5147.
Embodiment 1,
1) preparation of sample：Selenium-rich peanuts raw material (total Se content is 0.513mg/kg) is selected from national public welfare industry (agriculture
Industry) special project project (number：201303106) the Boshan Shandong Province planting base under subsidizing；By after the selenium-rich peanuts decladding of selection
5min is impregnated in ultra-pure water, undresses, dry, and obtains selenium-rich peanuts benevolence；Selenium-rich peanuts benevolence is subjected to precomminution, obtains selenium-rich peanuts
Broken progress super-pressure secondary solvent extraction, selenium-rich peanuts powder and No. 6 solvent naphtha solid-liquid ratios 1:6g/mL, ultra high pressure treatment pressure are
300MPa, extraction time 10min, turbid solution water bath with thermostatic control shaking table 6h, 50 DEG C, centrifugation 4000r/min, 20min, which are obtained, once to be soaked
Carry grouts, grouts carry out water bath with thermostatic control extraction, centrifuge secondary extraction grouts, precipitation obtain selenium-rich peanuts skimmed milk.
2) Organic Selenium extracts：The extracting method of different condition is as shown in table 1.According to the analysis of table 2, E9 groups are finally chosen
Method extracts Organic Selenium.Weigh sample and enzyme mixing：Weighing sample, (selenium-rich peanuts skimmed milk, control defatting peanut powder, selenium-rich are small
Wheat reference substance) 0.2000g, protease dosage be 0.7U：Mg samples, lipase 5U：Mg samples are in 10mL light-proof centrifuge tubes；
(basic, normal, high mark-on group is that selenomethionine is made an addition to control flower to mixing in 5mmol/L hypo solution 4mL solution
The hypo solution of raw skimmed milk so that the initial Se content of solution is 0.1ppb, 1ppb and 10ppb respectively)；Ultrasonic wave is auxiliary
Help extraction：500W、37℃、1h；Water bath with thermostatic control is vibrated：37℃、150rpm、20h；Mixture centrifuges：8000rpm、20min、4
DEG C, take supernatant；Precipitation repetition dissolving, ultrasonic wave assisted extraction, water bath with thermostatic control are vibrated, centrifuge, take supernatant.Above-mentioned protease
1 enzyme activity unit refer to 37 DEG C, pH value be 7.5 when, can be converted in 1 minute needed for 1 micromolar tyrosine
Enzyme amount；1 enzyme activity unit of above-mentioned lipase refer to 40 DEG C, pH value be 5.6 when, 1 can be converted in 1 minute and micro- is rubbed
Enzyme amount needed for your free fatty (vinyl laurate is as substrate).
Extracting method under 1 Organic Selenium different condition of table
The recovery of standard addition of 2 Se content of table (Se μ g/g) and form selenium
In table 2, selenomethionine is made an addition to control defatting peanut powder (total selenium is not detected) by basic, normal, high respectively represent
Hypo solution so that amount (i.e. additive amount) of the solution containing Organic Selenium is 0.1ppb, 1ppb and 10ppb respectively；Skimmed milk
Represent selenium-rich peanuts skimmed milk；
Reference substance refers to Se-rich wheat powder reference substance.
It is unlisted in table since methyl selenocystine, selenocysteine are not detected in selenium-rich peanuts sample.
3) assay：The detection of HPLC-ICP-MS methods will be respectively adopted for gained supernatant twice, the total Se content of residue is adopted
With micro-wave digestion, ICP-MS methods measure, and instrument actual conditions parameter is as shown in table 3.Organic Selenium HPLC-ICP-MS chromatography peak figures
As shown in Figure 1, three peaks in Fig. 1 from left to right：1、SeCys2(tret=2.67 ± 0.02min)；2、MeSeCys(tret
=4.58 ± 0.02min)；3, SeMet (tret=10.50 ± 0.04min).
It is as shown in table 4 that HPLC-ICP-MS measures performance.By above-mentioned the result shows that, the Organic Selenium of selenium-rich peanuts skimmed milk
Mainly exist with selenomethionine, content (in terms of selenium) is 0.73 μ g/g, accounts for the 69% of total Se content.
3 HPLC-ICP-MS method conditional parameters of table
4 HPLC-ICP-MS analytical performances of table
Embodiment 2,
Step 1) is only obtained selenium-rich peanuts skimmed milk and adds deionized water, material and water feed liquid by the step of according to example 1
Than 1:12g/mL adjusts pH to 9.5,40 DEG C of water bath with thermostatic control stirring extraction 1h with NaOH (a concentration of 1mol/L).8) after extracting
Solution is filtered by vacuum, and filtrate is collected, and pH to 4.7 is adjusted with HCl (a concentration of 1mol/L)；Centrifugation：5000r/min、
15min takes precipitation；Gained precipitation is freeze-dried, carries out ultramicro grinding with the broken machine of low-temperature submicron powder, 150 mesh sieve is crossed, obtains
To selenium-rich peanuts albumen.The result shows that the protein content of selenium-rich peanuts albumen powder is 91%, measured by 3) containing in embodiment 1
Fixed method detection, obtains its Organic Selenium and mainly exists with selenomethionine, content (in terms of selenium) is 0.80 μ g/g, accounts for total selenium
The 76% of content.
Embodiment 3,
The step of according to example 1, only increases step 4) and measures selenium-rich peanuts raw material to selenium-rich peanuts albumen powder preparation process
The loss of middle selenium, as shown in Fig. 2, the result shows that, the selenium in peanut is mainly distributed across in skimmed milk, peanut oil in conjunction in Fig. 3
Without selenium, a certain amount of selenium is contained in peanut coat, but quality is small shared by peanut coat (be less than 1%), and main component be fiber,
Glucosides etc.；So selenium loss is not related to selenomethionine loss in the step of removal peanut coat and peanut oil；Dry, shattering process
Belong to hot-working, the Morphological Transitions of selenomethionine can be caused, causes volatilization loss, therefore the selenium of dry and broken link is lost
It is lost as seleno-amino acids.The 128 μ g of Se content of every hundred selenium-rich peanuts raw materials, wherein selenomethionine (in terms of selenium) are 83
μ g then account for the 65% of total Se content.
Claims (8)
1. organic selenium content method for measuring in a kind of selenium-rich peanuts or its product prepared, includes the following steps：It 1) will be to be measured
Sample is mixed with enzyme, obtains mixture；
The sample to be tested includes selenium-rich peanuts or its product prepared；
In step 1), the sample to be tested is dissolved using hypo solution；
2) it by the mixture successively after ultrasonic wave assisted extraction, water bath with thermostatic control oscillation, centrifuges, obtains supernatant and residual
Slag；
After the residue carries out above-mentioned ultrasonic wave assisted extraction, water bath with thermostatic control oscillation, centrifuges, obtain supernatant 1 and residue
1；
3) take the supernatant and the supernatant 1 that HPLC-ICP-MS methods detection organic selenium content is respectively adopted；
4) organic selenium content summation in the supernatant and the supernatant 1 is contained to get to Organic Selenium in the sample to be tested
Amount.
2. according to the method described in claim 1, it is characterized in that：Take the residue and the residue 1 using micro-wave digestion and
ICP-MS methods, which measure, detects total Se content in the residue.
3. method according to claim 1 or 2, it is characterised in that：Product prepared by the selenium-rich peanuts includes selenium-rich flower
Raw benevolence, degreasing selenium-rich peanuts powder, selenium-rich peanuts albumen.
4. method according to claim 1 or 2, it is characterised in that：It is to include to prepare product prepared by the selenium-rich peanuts
Following steps：It by the selenium-rich peanuts decladding, impregnates, undress, dry, obtain selenium-rich peanuts benevolence；By the selenium-rich peanuts benevolence into
After row precomminution, the degreasing of super-pressure Assisted Extraction, secondary water bath with thermostatic control extraction, solvent precipitation are used successively, obtains degreasing selenium-rich
Peanut powder；Degreasing selenium-rich peanuts powder is subjected to alkali carries, acid precipitation, dry, crushing, obtains selenium-rich peanuts albumen.
5. method according to claim 1 or 2, it is characterised in that：The enzyme includes protease and lipase；
The dosage of the protease is 0.7~3U：Mg selenium-rich peanuts or its product prepared；
The dosage of the lipase is 5~20U：Mg selenium-rich peanuts or its product prepared.
6. method according to claim 1 or 2, it is characterised in that：In step 1), the concentration of the hypo solution
For 4~8mmol/L；
Per g selenium-rich peanuts or in the product of its preparation, the dosage of the hypo solution is 18~25mL.
7. method according to claim 1 or 2, it is characterised in that：The power of the ultrasonic wave assisted extraction be 400~
500W；
The temperature of the ultrasonic wave assisted extraction is 30~45 DEG C；
The time of the ultrasonic wave assisted extraction is 0.5~2h.
8. method according to claim 1 or 2, it is characterised in that：In above-mentioned method, the temperature of the water bath with thermostatic control oscillation
Degree is 30~45 DEG C；
The time of the water bath with thermostatic control oscillation is 12~20h；
The rotating speed of the water bath with thermostatic control oscillation is 140~200rpm.
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| CN111948182A (en) * | 2020-07-22 | 2020-11-17 | 广西壮族自治区农业科学院 | Selenium-enriched peanut and method for measuring content of organic selenium in selenium-enriched peanut product |
| CN112649546A (en) * | 2021-01-11 | 2021-04-13 | 南京恒宝田功能农业产业研究院有限公司 | Pretreatment method for selenium form determination of low-selenium high-fat plant |
| WO2022147837A1 (en) * | 2021-01-11 | 2022-07-14 | 南京恒宝田功能农业产业研究院有限公司 | Pretreatment method for determining selenium form in low-selenium high-fat plant |
| CN114705767A (en) * | 2022-03-01 | 2022-07-05 | 海南省食品检验检测中心(海南省实验动物中心) | Analysis and detection method for selenium form in rice |
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