CN102706707A - Standard sample of diarrhetic shellfish toxin and preparation method of standard sample - Google Patents
Standard sample of diarrhetic shellfish toxin and preparation method of standard sample Download PDFInfo
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Abstract
The invention discloses a standard sample of diarrhetic shellfish toxin and a preparation method of the standard sample. Marine mussels are taken as materials, and research on a key preparation technology of a quality control sample of the diarrhetic shellfish toxin is emphasized to conduct from the aspects of sample impurity-removal and homogenization, freeze-drying, sample sub-packaging, determination and the like; on the basis, homogenization and stability testing is carried out; and the content reference value of the quality control sample of the diarrhetic shellfish toxin is determined by determination by a value fixing method and statistical treatment on fixed value data, and the standard sample of the diarrhetic shellfish toxin for biological detection is developed. The preparation of the standard sample has important practical significance and application value in comprehensively and deeply researching ingredient detection of diarrhetic shellfish toxin and the preparation technology and a stability ensuring technology of the standard sample, actively developing detection on the standard sample of the shellfish toxin, and filling up the blank in the detection field.
Description
Technical field
The invention belongs to marine fishery detection technique field, be specifically related to standard model of diarrhoea property saxitoxin and preparation method thereof.
Background technology
Saxitoxin has become an important public health problem in recent years, and existing a plurality of countries limit the quantity of to shellfish poison in the food and control, and WHO has also issued about the harm of prevention shellfish poison, protected sanitarian draft.China is that the marine economy shellfish produces and big export country, produces about 1,100 ten thousand tons of shellfish per year, accounts for world's total amount 70%.During the nearly last ten years, take place frequently, had a strong impact on the marine aquaculture development and the export trade owing to reasons such as industrial pollution cause China's coastal waters red tide.European Union has taked close the border indefinite duration to the shellfish import of China after nineteen ninety-five, edible Chinese import mussel poisoning incident took place; States such as Korea S, Japan, the U.S. have also formulated the strict restriction measure to China import shellfish and Related product.Saxitoxin has become big public hazards that hinder China's inshore fishing economic development, and the safety of shellfish food is great public health problem, is a key factor of restriction China sea fishery economic development.
The kind of saxitoxin is a lot; That finds at present mainly contains paralytic shellfish poisoning (PSP) (paralytic shellfish poisoning; PSP), diarrhoea property saxitoxin (Diarrhetic shellfish poisons, DSP), nerve saxitoxin (NSP) and Toxin-Domoic Acid (ASP) four big classes.That China is at present common mainly is PSP and DSP.
1976, what taken place in the Miyagi Prefecture, Japan that edible Mytilus galloprovincialis (Mytilus edulis) causes was the mass food poisoning of cardinal symptom with diarrhoea.At that time; Detected the fat-soluble toxin that can kill small white mouse from the mid intestinal gland (midgiit gland) of this shellfish; For this toxin is distinguished with water-soluble toxin mutually; And prevent to obscure with other toxin, renamed as again in 1977 diarrhoea property saxitoxin (Diarrhetic shellfish poisons, DSP).DSP is that bivalve shellfish is venerated algae (Dinophysis) as bait the poisonous planktonic organism of picked-up, has accumulated poisonous substance at mid intestinal gland.Thereby, behind edible these quilt shellfishes of poisoning of people, will poison by food.In Japan; Be accredited as at first poisonous planktonic be obovate fin algae (Dinophysis tortii); Afterwards through the fin algae of gathering is all over the world identified that the result is in obovate fin algae, has gradually detected dinophysistoxin and two kinds of compositions of okadaic acid in sharp fin algae 7 kinds of fin algaes such as (D.acuminata).In Japan, gradually sharp fin algae and D.acuta, D.norvegica.And in China, then obovate fin algae all has distribution with sharp fin algae gradually.In China, the same with Japan, the generation of DSP is mainly yellow in the north, the shellfish place of production, the Bohai Sea.In shellfish kinds such as variegated clam, clam, scallop, oyster, mussel, Ark Shell, all detected DSP.From kind, the highest with the recall rate of mussel, next is followed successively by clam, scallop, variegated clam, Ark Shell; From toxic amount, still the highest with mussel, be up to 0.4MU/g, be variegated clam secondly, can reach 0.2MU/g; From the time, all detect for 4 ~ November, but being early summer many.
At present, people also can't effectively control red tide and saxitoxin, and therefore prevention and forecast are the effective means that alleviates its harm.The most effectively prevention method that WHO recommends is that the control monitoring facilities is carried out at water source or results zone.In each link that shellfish produces, the variation of monitoring saxitoxin at any time is vital.A lot of detection methods have been set up, like bioassay method, immunoassay, cytotoxicity detection method and chemical analysis etc.Receive the restriction of various factors, the most effective now, also be still the mouse bioanalysis by the universally recognized shellfish poison detection method of various countries official.Also be to adopt mouse bioanalysis, this method to use a large amount of to contain multi-component PSP and the DSP standard items satisfy Quality Control and quantitative requirement in the national standard of China and inspection and quarantine industry standard.But DSP, PSP standard items all are the pure article of standard of single component, no matrix; Prices are rather stiff, and each component is up to about 6000 yuan/100 μ g, in addition; The shellfish poison standard items are extremely toxic substance, and all there are strict administrative provisions countries in the world to the entry and exit and the use of saxitoxin.Development saxitoxin material standard sample can satisfy the market demand that the saxitoxin standard model is constantly enlarged day by day; Solve the demand of the famine saxitoxin standard model of domestic and international seashell products test experience chamber; To improving food safety detection technology, technical solution barrier, ensuring food safety and play great function, also improve China's influence power in the world simultaneously.The biological detection of preparing is with diarrhoea property saxitoxin (Diarrhetic shellfish poisons; DSP) standard model can be used for quantitatively or qualitative detection diarrhoea property saxitoxin; Being suitable for each department such as food, fishing prison, environmental protection, quality inspection, fishery, health uses; Can promote aquaculture, processing and Trade Development, guarantee food security, will bring remarkable social benefit and economic benefit.
Summary of the invention
In view of domestic biological detection is used diarrhoea property saxitoxin (Diarrhetic shellfish poisons; DSP) standard model situation in short supply; Start with in several aspects such as our sampling, sample preparation, specimen preparation, sample packing, assay method; Study emphasis the research of the crucial technology of preparing of diarrhoea property saxitoxin quality-control sample, on this basis, carried out homogeneity and stability test; Through confirming of valued methods, and, confirm diarrhoea property saxitoxin quality-control sample content reference value, developed biological detection with diarrhoea property saxitoxin standard model to the statistical treatment and the uncertainty evaluation of value data.
Diarrhoea property saxitoxin (Diarrhetic shellfish poisons, DSP) method of application of standard model: be in the biological detection process, to use diarrhoea property saxitoxin (Diarrhetic shellfish poisons; DSP) standard model is as the positive criteria reference substance; Sample to be checked and positive control are done parallel experiment, the result who detects is compared, whether contain DSP in the confirmatory sample; Also can pass through the production standard curve, utilize formula to calculate the content of DSP in the testing sample again.
Diarrhoea property saxitoxin standard model, its preparation method comprises the steps:
One. sample decon and homogenate
Get the scallop of diarrhoea property saxitoxin tests positive, the shell appearance is thoroughly cleaned, cut off closed shell flesh with clear water; Open shell; With inner silt and other impurity removed of double distilled water drip washing, closed shell flesh and the tissue that is connected gummed portion are separated, take out mid intestinal gland; Pick broken shell foreign material, place sieve draining 5min; Homogenate is 5 minutes in frozen water, operates light and slow as far as possible; Make the sample of treating freeze-drying;
Two. the freeze-drying program
1. the above-mentioned sample of treating freeze-drying is put in-70 ℃ pre-freeze 4h at least;
2. refrigerating process: closed the hothouse door of freeze drier, beginning hothouse refrigeration; When the hothouse temperature is reduced to-35 ℃, begin to cool down the trap refrigeration; When the cooling pit cryogenic temperature is reduced to-40 ℃, the pre-freeze product that 1. step obtains is put into hothouse rapidly, closed the hothouse door; Start vacuum pump and begin to vacuumize, the vacuum meter displayed value begins to descend; When treating that vacuum tightness is reduced to 0.5Torr, finish refrigerating process;
3. sample drying:, improve sample drying speed to the hothouse heating; The temperature of hothouse is set to 15 ℃; When vacuum tightness was reduced to 0.1Torr or more hanged down, the expression sample was dry good; Close vacuum pump, hothouse communicated with ambient atmosphere, treat inside and outside air pressure balance after, open the hothouse door, take out sample, in the sealing bag of packing into rapidly;
4. the sample that 3. step is made carries out the bowl mill fine grinding, crosses 40 mesh sieves after the fine grinding; The sample that obtains after sieving is carried out freeze drying once more, and cryodesiccated program repeats above-mentioned steps 2. ~ 3. once, removes moisture and gets standard samples;
5. packing: between constant temperature and humidity, sterile working,, be sub-packed in 2 hours the brown cillin bottle of clean 5ml of 160 ℃ of dry heat treatment the capping sealing, dress freeze-dried powder 1.0g in every bottle with the freeze-dried powder that 4. step obtains; Sample after bottled is sticked on the uniqueness sign.
Through above-mentioned steps one and two preparation methods; It is a raw material with the ocean mussel; Start with through several aspects such as sample decon and homogenate, freeze-drying program, sample packing, assay methods; Study emphasis the research of the crucial technology of preparing of diarrhoea property saxitoxin quality-control sample, on this basis, carried out homogeneity and stability test; Through confirming of valued methods, and, confirm diarrhoea property saxitoxin quality-control sample content reference value, developed biological detection with diarrhoea property saxitoxin standard model to the statistical treatment of value data.
Character of innovation of the present invention is:
The preparation of this standard model is accomplished; To the deep comprehensively technology of preparing of researching and solving saxitoxin composition detection standard model and stability assurance technology; Actively develop the development of China's saxitoxin examination criteria sample; Replenish the blank of this field of measurement, have very important practical sense and using value.
Description of drawings
Fig. 1. diarrhoea property saxitoxin standard model preparation technology process flow diagram.
Embodiment
Following non-limiting example can make those of ordinary skill in the art more fully understand the present invention, but does not limit the present invention in any way.
Mortar, 100 mesh standard sieves, detectable used in the present embodiment all prepares through conventional method, or buys acquisition in commodity approach.
Embodiment 1
1 material choice:
Adopt the mussel of river deer island, Dalian natural marine site growth, with the mouse bioanalysis with enzyme-linked immunosorbent assay detection suffer from diarrhoea the property saxitoxin (Diarrhetic shellfish poisons, DSP)
[1-3]Content.
2. the diarrhoea property saxitoxin in the raw material is measured:
By SN/T 1996-2007 assay method the DSP in the above-mentioned collection mussel is confirmed experiment.With clear water the shell appearance is thoroughly cleaned, cut off closed shell flesh, open shell, with inner silt and other exotics removed of double distilled water drip washing.Closed shell flesh and the tissue that is connected gummed portion are separated, carefully take out mid intestinal gland.Collect about 200g and place sieve draining 5min (meat is piled up), detect foreign material such as broken shell.
Get the 10g mid intestinal gland, add 5 times of volume 90% methanol solutions, homogeneous 5min, the centrifugal 10min of 3000g collects supernatant, and residue is with the 90% methanol solution extracting of 2 times of volumes, and the centrifugal 10min of 3000g collects supernatant.Merge twice supernatant; (its testing process is conventional standard test operation to carry out ELISA; React reaction reagent, reaction system and response parameter settings such as used damping fluid, all belong to those skilled in the art's common practise), it is subsequent use to get the scallop that DSP is positive in the testing result.
3. the preparation of diarrhoea property saxitoxin standard model:
One sample decon and homogenate
Get the scallop of diarrhoea property saxitoxin tests positive, the shell appearance is thoroughly cleaned, cut off closed shell flesh with clear water; Open shell; With inner silt and other impurity removed of double distilled water drip washing, closed shell flesh and the tissue that is connected gummed portion are separated, take out mid intestinal gland; Pick broken shell foreign material, place sieve draining 5min; Homogenate is 5 minutes in frozen water, operates light and slow as far as possible; Make the sample of treating freeze-drying;
Two freeze-drying programs
1. the above-mentioned sample of treating freeze-drying is put in-70 ℃ pre-freeze 4h at least;
Potpourri after the homogenate is put in the rectangle stainless steel pallet, puts in-70 ℃ of ultra low temperature freezers pre-freeze 4h (in order to practice thrift freeze-drying time, the pre-freeze of adopting the method for the outer pre-freeze of case to carry out product is done) at least.Carry out following operation subsequently:
2. refrigerating process: closed the hothouse door of freeze drier, beginning hothouse refrigeration; When the hothouse temperature is reduced to-35 ℃, begin to cool down the trap refrigeration; When the cooling pit cryogenic temperature is reduced to-40 ℃, the pre-freeze product that 1. step obtains is put into hothouse rapidly, closed the hothouse door; Start vacuum pump and begin to vacuumize, the vacuum meter displayed value begins to descend; When treating that vacuum tightness is reduced to 0.5Torr, finish refrigerating process;
3. sample drying:, improve sample drying speed to the hothouse heating; The temperature of hothouse is set to 15 ℃; When vacuum tightness was reduced to 0.1Torr or more hanged down, the expression sample was dry good; Close vacuum pump, hothouse communicated with ambient atmosphere, treat inside and outside air pressure balance after, open the hothouse door, take out sample, in the sealing bag of packing into rapidly; Carry out the preparation of gross sample according to above-mentioned technology;
4. the sample that 3. step is made carries out the bowl mill fine grinding, crosses 40 mesh sieves after the fine grinding; The sample that obtains after sieving is carried out freeze drying once more, and cryodesiccated program repeats above-mentioned steps 2. ~ 3. once, removes moisture and gets standard samples;
5. packing: between constant temperature and humidity, sterile working,, be sub-packed in 2 hours the brown cillin bottle of clean 5ml of 160 ℃ of dry heat treatment the capping sealing, dress freeze-dried powder 1.0g in every bottle with the freeze-dried powder that 4. step obtains; Sample after bottled is sticked on the uniqueness sign.
This biological detection has prepared packing 400 bottles altogether with diarrhoea property saxitoxin standard model.
Diarrhoea property saxitoxin constituent analysis in 3 standard models
For confirming that above-mentioned sample made freeze-dried powder sample after freeze drying contains diarrhoea property saxitoxin (Diarrhetic shellfish poisons really; DSP) composition; We choose the freeze-dried powder sample that branch installs, and entrust Shandong Entry-Exit Inspection and Quarantine Bureau and Qingdao Marine University to adopt LC-MS to analyze the diarrhoea property saxitoxin composition in this freeze-dried powder sample.Analysis result shows: contain diarrhoea property saxitoxin component in this sample really.
Assay method:
Accurately take by weighing 1g (being accurate to 0.1g) freeze-dried powder and move into centrifuge tube, add 6mL 80% methanol solution, vortex mixed 5min; The centrifugal 10min of 3000g collects supernatant, and residue adds 2mL 80% methanol solution once more; Vortex mixed 5min, the centrifugal 10min of 3000g collects supernatant.Merge twice supernatant, the supernatant of collecting is settled to 10mL with 80% methanol solution.Filter extract as detecting stoste S1 with the 0.45um filter, subsequent use.
Enzyme linked immunosorbent assay (ELISA); Stoste is carried out the dilution of suitable multiple with sample buffer; The sample liquid S2 that draws 100 μ l titers, dilution draws 50 μ l diarrhoea property saxitoxin enzyme labeling thing to each microwell plate in microwell plate, draw 50 μ l antibody-solutions to each microwell plate; Mixing, the room temperature lucifuge is hatched 60min.After hatching end, pour out the solution in the microwell plate, add 300 μ l to every hole and wash the plate damping fluid, wash plate 3 times.Pour out liquid, on thieving paper, pat, water is clapped done as far as possible.Every hole adds 150 μ l substrate solutions, and the room temperature lucifuge is hatched 30min.Every hole adds 50 μ l reaction terminating liquids, in 10min, measures and write down the absorbance of each micropore solution 450nm wavelength.Calculate the concentration C of DSP in the S2 solution, μ g/g through typical curve.
Press following formula result of calculation:
X=C * 10mL * extension rate/freeze-dried powder weight g
In the formula: the content of DSP in the every g sample of X-, unit: μ g/g;
DSP content in the sample liquid S2 solution of C-dilution, μ g/g;
10mL refers to the sample preparation volume.
Embodiment 2 homogeneitys
For checking that (Diarrhetic shellfish poisons, the DSP) homogeneity of standard model are randomly drawed 15 bottles of samples to the diarrhoea property saxitoxin for preparing, and every bottle of sample is done 2 repeatability and detected.Adopt the enzyme linked immunosorbent assay test among the SN/T 1996-2007, the DSP content of analytical standard sample is represented with μ g/g.All test portions are tested under repeated condition with random order, promptly in same laboratory, use identical method of testing and instrument test within a short period of time by identical personnel.
The data statistics formula is following:
Be the homogeneity of check sample, extract i sample, each sample is tested j time under repeat condition.
The testing mean of each sample
The population mean
of whole sample tests
Testing total number of times
The sample room quadratic sum
All square
Quadratic sum in the sample
All square
Degree of freedom f
1=m-1
f
2=N-m
Statistic
The uniformity of diarrhoea property saxitoxin standard sample the results are shown in Table 1, and its Evaluation for Uniformity the results are shown in Table 2.Above result can know: as α=0.05, f
1=14, f
2=15, look into the F distribution table, the F critical value is 1.47, because of F than 1.47<f critical value 2.42 shows in the sample and the sample room there was no significant difference, and interpret sample is uniformly, can satisfy requirement of experiment.
Table 1 diarrhoea property saxitoxin standard model uniformity result
| Sample number | Test result μ g/g | Test result μ g/g | Mean value |
| 1 | 9.35 | 8.97 | 9.16 |
| 2 | 8.78 | 8.48 | 8.63 |
| 3 | 9.87 | 8.89 | 9.38 |
| 4 | 8.85 | 9.69 | 9.27 |
| 5 | 8.69 | 7.98 | 8.34 |
| 6 | 9.50 | 8.49 | 8.99 |
| 7 | 8.75 | 8.67 | 8.71 |
| 8 | 8.27 | 7.99 | 8.13 |
| 9 | 8.87 | 8.02 | 8.45 |
| 10 | 9.32 | 9.55 | 9.44 |
| 11 | 7.91 | 8.76 | 8.34 |
Table 2 diarrhoea property saxitoxin standard model Evaluation for Uniformity result
Embodiment 3 stability
The selected packaged sample that is used for stability test has been gone through long-time preservation under-18 ~-20 ℃ of cryogenic conditions.The sampling principle of dredging according to close back before the time interval, respectively the 0th, 2,4,6,9,12,18 and 24 months picked at random be used for the packaged sample of stability test.Choose 3 samples tests at every turn.Adopt the enzyme linked immunosorbent assay test among the SN/T 1996-2007, diarrhoea property saxitoxin (DSP) content of analytical standard sample is represented with μ g/g.In whole stability tests, used personnel, instrument, method of testing and laboratory are all identical with uniformity test.In lucifuge, sealing ,-20 ℃ of following temperature are stored, and are stable in two years.Test result is seen table 3,4.
Table 3 diarrhoea property saxitoxin stability test result
Table 4 diarrhoea property saxitoxin standard model estimation of stability result
In the stability test, slope can be used computes:
In the formula:
Intercept is by computes:
The standard deviation of the point on the straight line can be by computes:
Get its square root s=0.33287, the uncertainty relevant with slope used computes:
Degree of freedom is that the student of n-2 and p=0.95 (95% confidence level) the t-factor that distributes equals 2.45.
Because | b
1|＜t
0.95, n-2S (b
1)
So slope is inapparent.Thereby do not observe instability.
Embodiment 4 standard values and uncertainty thereof
Adopt enzyme linked immunosorbent assay
[1]Diarrhoea property saxitoxin standard model is carried out detection by quantitative.
Adopt enzyme linked immunosorbent assay that diarrhoea property saxitoxin standard model is carried out definite value.To process homogeneity and the qualified sample of stability test; Randomly draw 40 bottles; Be distributed to the laboratory that 8 families participate in the definite value test respectively; Select and appoint by each laboratory respectively to have the laboratory technician who enriches operating experience, according to of the detection test of unified test routine to sample diarrhoea property saxitoxin DSP content.8 breadboard value data adopt Xia Piluo-Weir gram (Shapiro-Wilk) method of inspection to investigate its normality.Meet under the prerequisite of normality approximate; Confirm that through the check of Grubb ' s method all there is not exceptional value in each laboratory again; Check each laboratory value data whether to wait precision with Cochran (Cochran), the result shows all data all through check, and each lab investigation precision is consistent.The value of sample finally settles the standard.The result is as shown in table 5.
Requirement according to GB/T 15000-94 is added up data, calculates the standard value and the fiducial interval of DSP standard model, and the result sees table 6.
Table 5 diarrhoea property saxitoxin standard model definite value is summary sheet as a result
Table 6 diarrhoea property saxitoxin standard model definite value is statistical analysis table as a result
Definite value result adopts the mode of standard value ± expanded uncertainty to represent: i.e. 8.83 ± 0.59 μ g/g, k=2.
The biological detection of preparing through the foregoing description with diarrhoea property saxitoxin (Diarrhetic shellfish poisons, DSP) the standard model stability, uniformity is good, precision is high; Can be used for quantitative or qualitative detection diarrhoea property saxitoxin; Being suitable for each department such as food, fishing prison, environmental protection, quality inspection, fishery, health uses; Can promote aquaculture, processing and Trade Development, guarantee food security, will bring remarkable social benefit and economic benefit.
List of references
1.SN/T diarrhoea property saxitoxin method of inspection enzyme linked immunosorbent assay in the 1996-2007 shellfish.
2.GB/T the mensuration of diarrhoea property saxitoxin in the 5009.212-2008 shellfish.
3.SN/T diarrhoea property saxitoxin method of inspection mouse bioanalysis in the 2131.2-2010 shellfish.
4.Aune,T.,Stabell,O.,Knordstoga,B.,Jjotta,K.,“Oral?Toxicity?in?Mice?of?Algal?Toxins?from?the?Diarrheic?Shellfish?toxin(DST)Complex?and?Associated?Toxins”Journal?of?Natural?Toxins?1998,7,141-158.
5.GB/T the mensuration of paralytic shellfish poisoning (PSP) in the 5009.213-2008 shellfish.
6.AOAC?officail?method?958.08?Paralytic?Shellfish?Poison:Biological?method.
7.Aune,T.,Stabell,O.,Knordstoga,B.,Jjotta,K.,“Oral?Toxicity?in?Mice?of?Algal?Toxins?from?the?Diarrheic?Shellfish?toxin(DST)Complex?and?Associated?Toxins”Journal?of?Natural?Toxins?1998,7,141-158.
8.Taleb,H.,Vale,P.,Jaime,E.,Blaghen,M.,“Study?of?paralytic?shellfish?poisoning?toxin?profile?in?shellfish?from?the?Mediterranean?shore?of?Morocco”,Toxicon?2001,39(12),1855-1861.
9.Wu?Y，Ho?AY，Qian?PY，Leung?KS,Cai?Z,Lin?JM.Determination?of?paralytic?shellfish?toxins?in?dinoflagellate?Alexandrium?tamarense?by?using?isotachophoresis/capillary?electrophoresis.J?Sep?Sci.2006?Feb;29(3):399-404.
10.Gawley?RE,Mao?H,Haque?MM,Thorne?JB,Pharr?JS.Visible?fluorescence?chemosensor?for?saxitoxin.J?Org?Chem.2007,72(6):2187-91.
11.Humpage?AR,Ledreux?A,Fanok?S,Bernard?C,Briand?JF,Eaglesham?G，Papageorgiou?J,Nicholson?B,Steffensen?D.Application?of?the?neuroblastoma?assay?for?paralytic?shellfish?poisons?to?neurotoxic?freshwater?cyanobacteria:interlaboratory?calibration?and?comparison?with?other?methods?of?analysis.Environ?Toxicol?Chem.2007,26(7):1512-9.
| 12 | 8.79 | 9.34 | 9.07 |
| 13 | 9.14 | 7.98 | 8.56 |
| 14 | 8.68 | 8.77 | 8.73 |
| 15 | 9.68 | 8.86 | 9.27 |
Claims (1)
1. diarrhoea property saxitoxin standard model is characterized in that the preparation method comprises:
One. sample decon and homogenate
Get the scallop of diarrhoea property saxitoxin tests positive, the shell appearance is thoroughly cleaned, cut off closed shell flesh with clear water; Open shell; With inner silt and the impurity removed of double distilled water drip washing, closed shell flesh and the tissue that is connected gummed portion are separated, take out mid intestinal gland; Pick broken shell foreign material, place sieve draining 5min; Homogenate is 5 minutes in frozen water, operates light and slow as far as possible; Make the sample of treating freeze-drying;
Two. the freeze-drying program
1. the above-mentioned sample of treating freeze-drying is put in-70 ℃ pre-freeze 4h at least;
2. refrigerating process: closed the hothouse door of freeze drier, beginning hothouse refrigeration; When the hothouse temperature is reduced to-35 ℃, begin to cool down the trap refrigeration; When the cooling pit cryogenic temperature is reduced to-40 ℃, the pre-freeze product that 1. step obtains is put into hothouse rapidly, closed the hothouse door; Start vacuum pump and begin to vacuumize, the vacuum meter displayed value begins to descend; When treating that vacuum tightness is reduced to 0.5Torr, finish refrigerating process;
3. sample drying:, improve sample drying speed to the hothouse heating; The temperature of hothouse is set to 15 ℃; When vacuum tightness was reduced to 0.1Torr or more hanged down, the expression sample was dry good; Close vacuum pump, hothouse communicated with ambient atmosphere, treat inside and outside air pressure balance after, open the hothouse door, take out sample, in the sealing bag of packing into rapidly;
4. the sample that 3. step is made carries out the bowl mill fine grinding, crosses 40 mesh sieves after the fine grinding; The sample that obtains after sieving is carried out freeze drying once more, and cryodesiccated program repeats above-mentioned steps 2. ~ 3. once, removes moisture and gets standard samples; The sealing of packing 1.0g/ bottle is preserved.
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| CN105571918A (en) * | 2016-01-18 | 2016-05-11 | 中国农业科学院茶叶研究所 | Sample pre-treatment method for precise quantification detection of bioactive substances |
| CN106644642A (en) * | 2016-12-29 | 2017-05-10 | 青岛水务集团有限公司科技中心 | Preparing method of organic principle standard sample in urban sludge and prepared sample |
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| CN105571918B (en) * | 2016-01-18 | 2018-04-10 | 中国农业科学院茶叶研究所 | A kind of sample-pretreating method for the detection of bioactive substance accurate quantification |
| CN106644642A (en) * | 2016-12-29 | 2017-05-10 | 青岛水务集团有限公司科技中心 | Preparing method of organic principle standard sample in urban sludge and prepared sample |
| CN106644642B (en) * | 2016-12-29 | 2019-07-02 | 青岛水务集团有限公司科技中心 | The preparation method of organic principle standard sample and standard sample obtained in city sludge |
| CN115362963A (en) * | 2022-09-09 | 2022-11-22 | 青岛普瑞邦生物工程有限公司 | Production method of diarrhea shellfish poison quality control sample of Mytilus edulis and quality control sample |
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