CN107389852A - A kind of method that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method - Google Patents
A kind of method that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method Download PDFInfo
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- CN107389852A CN107389852A CN201710408710.1A CN201710408710A CN107389852A CN 107389852 A CN107389852 A CN 107389852A CN 201710408710 A CN201710408710 A CN 201710408710A CN 107389852 A CN107389852 A CN 107389852A
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Abstract
The present invention relates to a kind of method that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method, including：(1) the Bivalve biology of normal life state is gathered, soft tissue sample is made after cleaning；(2) enzymolysis liquid is prepared；Soft tissue sample is placed in enzymolysis liquid and cleared up, obtains soft tissue digestion solution；Vacuum filter is carried out to soft tissue digestion solution, is dried after the completion of filtering, is counted by stereoscope, micro- plastic content is calculated.The present invention will not be had an impact to the physicochemical property and form of micro- plastics, can carry out accurate quantitative analysis to micro- plastics in Bivalve biologic soft tissue, and science reference is provided for micro- plastic pollution situation in target organism living environment.
Description
Technical field
It is more particularly to a kind of to be detected by enzymatic isolation method in Bivalve biologic soft tissue the invention belongs to micro- plastics detection field
The method of micro- plastic content.
Background technology
Plastics are because its high stability, persistence and corrosion resistance are used widely, and substantial amounts of plastics are used and discharged
The pollution of getting worse, especially small size plastic garbage are brought to environment.Micro- plastics (Microplastics, MPs) refer to grain
Plastic grain of the footpath less than 5 μm, fragment, fiber etc., are moulded from industrial raw plastic material, personal care articles or by big particle diameter
Material degraded.Invertebrate (shell-fish, bivalve software class, crinosity link class), vertebrate (seabird, ocean fish) or even the food in one's mouth
Newborn animal (whale) can take in MPs, and MPs can be delivered to high nutrition level by food chain from low nutrition level, so as to life
State environment has a negative impact.
MPs be widely present and potential hazard causes mondial research boom, and Bivalve biology is as sea
The common biological group of midocean, because it is migrated, scope is small, and the height of its internal MPs content is often used for by some scientific research institutions
Object of reference as MPs pollution levels in specified sea areas.MPs quantitative analysis method main flow is that Bivalve soft tissue disappears
Solution, extracts MPs therein, then draws MPs contents by quantifying analysis method.What the resolution of wherein soft tissue was mainly applied is
Acid-base method, Bivalve soft tissue is cleared up by the aqueous solution of strong acid or highly basic, this method cost is relatively low, and material is easy
, but danger coefficient is higher in use for its strong acid or highly basic, and existing research finds that part MPs is in strong acid
Or physicochemical character can change among highly basic, or even MPs resolution phenomenons occur so that MPs quantizing process is affected, knot
Fruit error is larger.Also there is the experiment cleared up using protease to soft tissue in existing research, but using albumen
Enzyme K is as resolution substrate, and this protease is usually used in the extraction research of DNA in scientific research, and its price is higher, configures complex operation,
These all turn into the obstacle that enzyme resolution mode is not used widely.
The content of the invention
The technical problems to be solved by the invention are to provide micro- in a kind of detection Bivalve biologic soft tissue by enzymatic isolation method
The method of plastic content, this method will not have an impact to the physicochemical property and form of micro- plastics, can be soft to Bivalve biology
Micro- plastics in tissue carry out accurate quantitative analysis, and science ginseng is provided for micro- plastic pollution situation in target organism living environment
Examine.
A kind of method that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method of the present invention, including：
(1) the Bivalve biology of normal life state is gathered, soft tissue sample is made after cleaning；
(2) enzymolysis liquid is prepared；It is 8.4uhb/ml-16.8uhb/ml enzymolysis liquids that 2-10g soft tissue sample is placed in into vigor
Middle 50-60 DEG C of resolution 12-24h, obtains soft tissue digestion solution；Vacuum filter is carried out to soft tissue digestion solution, done after the completion of filtering
It is dry, counted by stereoscope, micro- plastic content is calculated.
It is ripe individual no less than 1 year that Bivalve biology in the step (1) is growth year.
Bivalve biology in the step (1) freezes under -20 DEG C of environment in advance, is thawed at room temperature at least before use
2 hours.
The enzyme used in enzymolysis liquid in the step (2) is corolase7089 (No. CAS:9001-92-7) (technical grade).
Vacuum filter in the step (2) uses 0.8 μm of nitrocellulose or 1.6 μm of glass fiber filters.
Drying temperature in the step (2) is 60-80 DEG C, drying time 24-36h.
Computational methods in the step (2) are：Obtain that micro- plastics count number is subtracted into sky after micro- plastics count number
Micro- plastics quantity in white control, obtain actual micro- plastic content；Divided by soft tissue weight, micro- plastic content in per unit weight is obtained,
Formula is (count number value-blank value)/soft tissue weight；Multiple parallel data for repeating to obtain in testing are carried out whole
Reason, the larger data of deviation are removed, obtain average value as final micro- plastic content.
Operating personnel wear the unartificial woven material clothes of cotton in preparation process.
In resolution and drying process, the container for holding sample is sealed.
Beneficial effect
Mainly handled at present in terms of processing early stage to the micro- plastic monitoring of biologic soft tissue in a manner of acid, alkali, but acid, alkali
MPs detections can be caused to occur larger influence part MPs physicochemical property, soft tissue ferment treatment mode has research display use
Proteinase K is tested, but Proteinase K price is higher, needs buffer solution to handle in use, and operation is not easy, because
This does not have widely used on a large scale by industry.
Corolase7089 used in the present invention is a kind of technical grade neutral proteinase, and it can be in no buffer
In the case of directly used in water, all there is higher activity in the range of pH6-9, and price is than egg that scientific research institution uses
White enzyme is cheaply a lot, is industrially widely used, is widely applied in protein such as food, feed, cosmetics, nutrient and healthcare products
In hydrolysis process work.By the present invention in that solves the high and difficult behaviour of cost of ferment treatment mode with protease corolase7089
The problem of making, and the physicochemical property and form of micro- plastics will not be had an impact, can be to micro- in Bivalve biologic soft tissue
Plastics carry out accurate quantitative analysis, and science reference is provided for micro- plastic pollution situation in target organism living environment.
Brief description of the drawings
Fig. 1 is the process chart of the present invention.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.In addition, it is to be understood that after the content of the invention lectured has been read, people in the art
Member can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Scope.
Embodiment 1
(1) sample is gathered：In the stable coastline marine site collection of environmental aspect normal life state, 1 year growth period with
Mussel sample upper, apparent size form is similar, collecting quantity are no less than 20 pieces, are placed in equipped with being preserved in low-temperature (low temperature) vessel, take back
Laboratory is further processed.
(2) soft tissue collection and processing：Collection mussel is placed in freezing processing at least 1h under -20 degrees Celsius of environment, then
Frozen samples are thawed at room temperature, thawing time is no more than 2h.Then whole soft tissues are taken out, using ultra-pure water to soft group
Knit and simply cleaned, reach the effect for removing bulky grain silt, then select the complete disease-free, inorganization of state to lack
Lose, without bulky grain magazine, weight about in 2-5g or so 5 pieces of soft tissue sample, record data of weighing (is accurate to 0.01g), so
After be placed in 250ml triangular pyramidal bottles and cleared up, and prepare a blank control sample, blank control is used as reference using pure water
Thing.
(3) enzymolysis liquid is configured：Configuration vigor is 8.4uhb/ml-16.8uhb/ml enzymolysis liquid 100ml.
(4) soft tissue is cleared up：Soft tissue sample is placed in enzymolysis liquid, each conical flask places a complete soft tissue
Sample, and covered conical flask bottleneck with aluminium foil, to prevent extraneous micro- plastics from entering in container, all processes all should be ultra-clean
Carried out in workbench, operating personnel should wear the unartificial woven material lab-gown of cotton and be operated, and be kept in all conditions
Resolution (continuing gentle agitation, 50-60 DEG C, 12h) is completed under steady state, obtains soft tissue digestion solution.
(5) vacuum filter：After treating soft tissue resolution completely, using 1.6 μm of glass fiber filters, to soft tissue digestion solution
Carry out vacuum filter.
(6) filter membrane is dried：Filter membrane is positioned in plastic culture dish, and culture dish, which is covered, prevents introduced contaminants from entering
Enter, then dried 24 hours in 60 DEG C of baking ovens.
(7) count：Data statistics is carried out to micro- plastics in filter membrane by stereoscope (60-300 times of lower observation), obtained
To micro- plastics count number.
(8) data preparation：, will be multiple according to the micro- plastic content of formula=(count number value-blank value)/soft tissue weight
The parallel data for repeating to obtain in testing are arranged, and remove the larger data of deviation, obtain average value as final micro- plastics
Content.
As a result it is as follows：
In all operating process of the present embodiment, operating personnel need to wear the unartificial woven material clothes of cotton.
In all operating process of the present embodiment, Polythene Bag, aluminium-foil paper etc. should be selected to carry out closed and sealing to sample
Preserve.
Claims (9)
1. a kind of method that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method, including：
(1) the Bivalve biology of normal life state is gathered, soft tissue sample is made after cleaning；
(2) enzymolysis liquid is prepared；2-10g soft tissue sample is placed in vigor as in 8.4uhb/ml-16.8uhb/ml enzymolysis liquids
50-60 DEG C of resolution 12-24h, obtains soft tissue digestion solution；Vacuum filter is carried out to soft tissue digestion solution, dried after the completion of filtering,
Counted by stereoscope, micro- plastic content is calculated.
A kind of 2. side that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method according to claim 1
Method, it is characterised in that：It is ripe individual no less than 1 year that Bivalve biology in the step (1) is growth year.
A kind of 3. side that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method according to claim 1
Method, it is characterised in that：Bivalve biology in the step (1) freezes under -20 DEG C of environment in advance, is solved at room temperature before use
Freeze at least 2 hours.
A kind of 4. side that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method according to claim 1
Method, it is characterised in that：The enzyme used in enzymolysis liquid in the step (2) is corolase7089.
A kind of 5. side that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method according to claim 1
Method, it is characterised in that：Vacuum filter in the step (2) is using 0.8 μm of nitrocellulose or 1.6 μm of glass fibre filters
Film.
A kind of 6. side that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method according to claim 1
Method, it is characterised in that：Drying temperature in the step (2) is 60-80 DEG C, drying time 24-36h.
A kind of 7. side that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method according to claim 1
Method, it is characterised in that：Computational methods in the step (2) are：Obtain micro- plastics count number after micro- plastics count number
Micro- plastics quantity in blank control is subtracted, obtains actual micro- plastic content；Divided by soft tissue weight, obtain micro- plastics in per unit weight
Content, formula are (count number value-blank value)/soft tissue weight；Multiple parallel data for repeating to obtain in testing are entered
Row arranges, and removes the larger data of deviation, obtains average value as final micro- plastic content.
A kind of 8. side that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method according to claim 1
Method, it is characterised in that：Operating personnel wear the unartificial woven material clothes of cotton in preparation process.
A kind of 9. side that micro- plastic content in Bivalve biologic soft tissue is detected by enzymatic isolation method according to claim 1
Method, it is characterised in that：In resolution and drying process, the container for holding sample is sealed.
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Cited By (5)
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|---|---|---|---|---|
| CN107449655A (en) * | 2017-08-15 | 2017-12-08 | 浙江工业大学 | The pre-treating method of micro- plastics in a kind of identification marine product |
| CN109238949A (en) * | 2018-09-19 | 2019-01-18 | 浙江大学 | A method of micro- plastic density distribution in detection marine organisms soft tissue |
| CN111504741A (en) * | 2020-04-22 | 2020-08-07 | 山东师范大学 | Method for detecting micro-plastics in fish body |
| CN112798569A (en) * | 2021-02-04 | 2021-05-14 | 斡得霈克(武汉)医疗科技有限责任公司 | Method for measuring trace elements in blood by atomic absorption fluorescence spectrometry |
| CN114252427A (en) * | 2021-12-10 | 2022-03-29 | 暨南大学 | Method for effectively analyzing micro-plastics in grease-containing food sample |
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| CN105651748A (en) * | 2016-01-18 | 2016-06-08 | 南京大学 | Method for quantitatively analyzing enrichment and distribution of micro plastics in aquatic organisms |
| CN105675566A (en) * | 2016-01-25 | 2016-06-15 | 南京大学 | Method for quantitatively analyzing enrichment and distribution of micro-plastic in mammal body |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107449655A (en) * | 2017-08-15 | 2017-12-08 | 浙江工业大学 | The pre-treating method of micro- plastics in a kind of identification marine product |
| CN109238949A (en) * | 2018-09-19 | 2019-01-18 | 浙江大学 | A method of micro- plastic density distribution in detection marine organisms soft tissue |
| CN111504741A (en) * | 2020-04-22 | 2020-08-07 | 山东师范大学 | Method for detecting micro-plastics in fish body |
| CN111504741B (en) * | 2020-04-22 | 2023-04-18 | 山东师范大学 | Method for detecting micro-plastics in fish body |
| CN112798569A (en) * | 2021-02-04 | 2021-05-14 | 斡得霈克(武汉)医疗科技有限责任公司 | Method for measuring trace elements in blood by atomic absorption fluorescence spectrometry |
| CN114252427A (en) * | 2021-12-10 | 2022-03-29 | 暨南大学 | Method for effectively analyzing micro-plastics in grease-containing food sample |
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