CN108489920A - The detection method of worm's ovum content and application in broken head - Google Patents
The detection method of worm's ovum content and application in broken head Download PDFInfo
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- CN108489920A CN108489920A CN201810715767.0A CN201810715767A CN108489920A CN 108489920 A CN108489920 A CN 108489920A CN 201810715767 A CN201810715767 A CN 201810715767A CN 108489920 A CN108489920 A CN 108489920A
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Abstract
The present invention provides a kind of detection method of worm's ovum content in broken head and application, it is related to detection method technical field, the detection method of worm's ovum content includes the following steps in the broken head：(a) broken head are soaked in distilled water, are separated by solid-liquid separation, obtain worm's ovum pregnant solution：(b) worm's ovum pregnant solution is collected and is purified, obtain worm's ovum refined solution；(c) chitinase is added in worm's ovum refined solution, obtains worm's ovum enzymolysis liquid；(d) dimethylaminobenzaldehyde is added in worm's ovum enzymolysis liquid and carries out chromogenic reaction, obtain chromophoric solution, measure the absorbance of chromophoric solution, obtain worm's ovum content in broken head, it alleviates currently without the method detected specifically for worm's ovum in broken head, the technical issues of causing the waste of resource, the detection method of worm's ovum content can accurately measure the content of worm's ovum in broken head in broken head provided by the invention, and the recycling for broken head provides foundation.
Description
Technical field
The present invention relates to detection method technical fields, detection method more particularly, to worm's ovum content in a kind of broken head and
Using.
Background technology
Vermicelli are the dried noodles with certain length cut into after loft dried with wheat flour addition salt, water.Vermicelli
Principal item have common liner, varied fine dried noodles, handcraft dried noodle etc., eat vermicelli have improve anaemia, strengthen immunity, balance
Nutrient absorption and other effects.
When being processed to vermicelli, needs to carry out preprocessing raw material and auxiliary material, knead dough, curing, tabletting, slitting, wet section, do
It is dry, cut-out, metering, packaging, etc. processes.In the production process of vermicelli, need to enter cut off operation after vermicelli drying, in vermicelli
Usually will produce some broken head during cut-out, broken head can continue to produce for vermicelli through collecting, but broken head compared with
Few, need to often collect can just reuse after a certain period of time, since broken head are exposed in air for a long time, may have by flour
The risk of the worm's ovums such as common pests red flour beetle, rice weevil pollution, therefore broken head need to carry out worm's ovum detection before reusing,
It can decide whether to can continue to use.
Since there is presently no the methods detected specifically for worm's ovum in broken head, cause most of broken head that can not continue
It recycles, causes the waste of resource.
In view of this, special propose the present invention.
Invention content
One of the objects of the present invention is to provide a kind of detection methods of worm's ovum content in broken head, do not have at present to alleviate
There is the method detected specifically for worm's ovum in broken head, causes most of broken head that can not continue to recycle, cause resource
The technical issues of waste.
The detection method of worm's ovum content, includes the following steps in broken head provided by the invention：
(a) broken head are soaked in distilled water, are separated by solid-liquid separation, obtain worm's ovum pregnant solution：
(b) worm's ovum pregnant solution is collected and is purified, obtain worm's ovum refined solution；
(c) chitinase is added in worm's ovum refined solution, obtains worm's ovum enzymolysis liquid；
(d) dimethylaminobenzaldehyde is added in worm's ovum enzymolysis liquid and carries out chromogenic reaction, obtain chromophoric solution, measure colour developing
The absorbance of solution obtains worm's ovum content in broken head.
Further, in step (a), in step (a), the liquid ratio of broken head and distilled water is (10-20):1；
Preferably, soaking time 3-5h；
Preferably, the distilled water that distilled water is 0-5 DEG C；
Preferably, it is preferable that first pass through standing and supernatant taken once to be separated by solid-liquid separation, then by centrifugation by supernatant into
The secondary separation of solid and liquid of row, obtains worm's ovum pregnant solution.
Further, in step (b), the collection and purifying of worm's ovum pregnant solution are carried out using density-gradient centrifugation method；
Preferably, the layering solution using percoll solution or Focill solution as density-gradient centrifugation method；
Preferably, the separating liquid using ficoll as density-gradient centrifugation method.
Further, the detection method of worm's ovum content further includes step (p) in the broken head, and step (p) is set to step
Suddenly between (b) and step (c), step (p) is to be crushed worm's ovum refined solution；
Preferably, the broken of worm's ovum refined solution is carried out using ultrasound cracking or liquid nitrogen grinding.
Further, in step (c), first chitinase is added in worm's ovum refined solution, adds hydrochloric acid, adjusts pH value
It is 3, is separated by solid-liquid separation, obtains worm's ovum enzymolysis liquid；
Preferably, it is separated by solid-liquid separation using centrifugation；
Preferably, centrifugal speed 2500-3500r/min, centrifugation time 3-8min；
It is further preferred that centrifugal speed is 3000r/min, centrifugation time 5min.
Further, in step (d), sodium carbonate liquor is first added in worm's ovum enzymolysis liquid and is uniformly mixed, is placed in boiling water
It is cooling after bath heating, it adds glacial acetic acid solution and is uniformly mixed, dimethylaminobenzaldehyde reagent is then added and is uniformly mixed, most
After be added acetic acid, carry out chromogenic reaction；
Preferably, the time heated in boiling water bath is 3-8min, preferably 5min.
Further, the detection method of worm's ovum content further includes step (s) in the broken head, and step (s) is set to step
Suddenly before (a), step (s) is to draw the standard curve of worm's ovum concentration and absorbance.
Further, it in step (b), after first worm's ovum pregnant solution is dialysed, then is collected and purifies.
Further, molecular cut off is used to dialyse for the bag filter of 8000-10000.
The second object of the present invention is that the detection method for providing worm's ovum content in above-mentioned broken head recycles profit in broken head
Application in.
The detection method of worm's ovum content can accurately measure containing for worm's ovum in broken head in broken head provided by the invention
Amount, provides foundation, while detection method provided by the invention is easy to operate for the recycling of broken head, is concealment in food
Pest detection provides a kind of new way.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
According to an aspect of the present invention, the present invention provides a kind of detection methods of worm's ovum content in broken head, including
Following steps：
(a) broken head are soaked in distilled water, are separated by solid-liquid separation, obtain worm's ovum pregnant solution：
(b) worm's ovum pregnant solution is collected and is purified, obtain worm's ovum refined solution；
(c) chitinase is added in worm's ovum refined solution, obtains worm's ovum enzymolysis liquid；
(d) dimethylaminobenzaldehyde is added in worm's ovum enzymolysis liquid and carries out chromogenic reaction, obtain chromophoric solution, measure colour developing
Worm's ovum content in broken head is calculated according to the absorbance of chromophoric solution in the absorbance of solution.
The detection method of worm's ovum content can accurately measure containing for worm's ovum in broken head in broken head provided by the invention
Amount, provides foundation, while detection method provided by the invention is easy to operate for the recycling of broken head, is concealment in food
Pest detection provides a kind of new way.
In a kind of preferred embodiment of the present invention, in step (a), the liquid ratio of broken head and distilled water is (10-20):
1。
In the typical but non-limiting embodiment of the present invention, the liquid material of broken head and distilled water is such as 10:1、
11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1 or 20:1.
By the way that the liquid ratio of broken head and distilled water is set as (10-20)：1 so that it is complete that broken head can be distilled water
Complete immersion.
In the preferred embodiment of the present invention, soaking time 3-5h.
In the preferred embodiment of the present invention, soaking time is as being 3,3.5,4,4.5 or 5h.
By the way that broken head are impregnated 3-5h in distilled water, so that the abundant water swelling of broken head, is dispersed in distilled water
In so that the worm's ovum in broken head is extracted into solution completely.
In the preferred embodiment of the present invention, the distilled water that distilled water is 0-5 DEG C.
By using 0-5 DEG C of distilled water, avoids causing broken head that corruption occurs because temperature is excessively high, influence measurement result
Accuracy.
In the preferred embodiment of the present invention, it first passes through standing and carries out primary separation of solid and liquid acquirement supernatant, then
Supernatant is subjected to secondary separation of solid and liquid by centrifuging, obtains worm's ovum pregnant solution.
It takes supernatant to detach worm's ovum pregnant solution with broken head by standing, by centrifuging supernatant, makes
Broken head particle precipitation in supernatant, take the supernatant after centrifugation to get to worm's ovum pregnant solution.
By carrying out secondary separation of solid and liquid, the particle substance in worm's ovum pregnant solution is removed, avoids influencing measurement
Accuracy.
In the preferred embodiment of the present invention, be stirred while broken head impregnate, the speed of stirring
Degree is 50-200r/min.
In the preferred embodiment of the present invention, mixing speed as 50,60,80,100,120,150,180 or
200r/min。
In the preferred embodiment of the present invention, in step (b), it is rich that worm's ovum is carried out using density-gradient centrifugation method
The collection and purifying of liquid collecting.
By the way that worm's ovum pregnant solution to be collected and be purified using density gradient method, the impurity substances in broken head are gone
It removes, while worm's ovum pregnant solution can also be made further to concentrate, to improve the accuracy of subsequent detection.
The collection and purifying of worm's ovum pregnant solution are carried out using density gradient method so that more complete, the enrichment of impurity removal
It is better, to ensure the follow-up accuracy for carrying out worm's ovum detection.
In the preferred embodiment of the present invention, using percoll solution or Focill solution as density gradient
The layering solution of centrifugal process.
In the preferred embodiment of the present invention, it when using percoll solution or Focil as layering liquid, uses
Discontinuous density gradient centrifuges.
In the preferred embodiment of the present invention, discontinuous density is carried out as layering liquid using percoll solution
Gradient centrifugation carries out the collection and purifying of worm's ovum pregnant solution, specifically comprises the following steps：
(1) discontinuous density gradient Percoll layers of preparation：Using 10%, 30%, 50%, 70% Percoll solution
As layering solution, the Percoll solution vacuum outgas of various concentration to bubble-free is generated, the Percoll that will be successively superimposed
Liquid is steadily flowed down along tube wall, is formed interface, is successively placed from high density to low-density with long needle applicator, by Percoll liquid
It is put into syringe, tube wall is close on small pinhead inclined-plane, it is made slowly to flow down naturally；
(2) density gradient centrifugation is carried out：Appropriate volume worm's ovum pregnant solution is added in separating liquid surface.It is in relative centrifugal force
20~25min of centrifugation time under the conditions of 400g.Centrifuge acceleration time 23min, deceleration time 2-3min.
(3) worm's ovum is collected：Worm's ovum enrichment region can form misty band due to index of refraction difference after centrifugation, when institute is to be separated
Worm's ovum at two layers of interface, can successively remove the worm's ovum that interface locations are collected after Percoll liquid；
(4) worm's ovum elutes:Worm's ovum containing Percoll liquid is fully cleaned with distilled water, 10000r/min centrifuges 15-
Precipitation is dissolved in 1-2ml distilled water to get to worm's ovum refined solution by 20min.
In the preferred embodiment of the present invention, the collection of worm's ovum pregnant solution is carried out as separating liquid using ficoll
And purifying, specifically comprise the following steps：15% ficoll solution is prepared as separating liquid, be added appropriate volume worm's ovum pregnant solution in
Separating liquid surface, under the conditions of 4 DEG C, 12000r/min centrifuges 15-20min, collects interface mist band, fully clear with distilled water
It washes, 10000r/min centrifuges 15-20min, and precipitation is dissolved in 1-2ml distilled water to get to worm's ovum refined solution.
In the preferred embodiment of the present invention, further include step (p), step (p) is set to step (b) and step
(c) between, step (p) is to be crushed worm's ovum refined solution.
Contain chitin in worm's ovum, by the way that worm's ovum refined solution is crushed, to accelerate the separation of the chitin in worm's ovum,
Help to improve the accuracy of detection method.
In present invention further optimization embodiment, worm's ovum refined solution is carried out using ultrasound cracking or liquid nitrogen grinding
It is broken.
In the preferred embodiment of the present invention, in step (c), worm's ovum refined solution first is added in chitinase
In, hydrochloric acid is added, it is 3 to adjust pH value, is separated by solid-liquid separation, obtains worm's ovum enzymolysis liquid.
In the preferred embodiment of the present invention, by the way that chitinase to be added in worm's ovum refined solution, so that worm's ovum
In chitin under the action of chitinase, generate N-acetyl-glucosamine.
In the preferred embodiment of the present invention, the pH value of worm's ovum enzymolysis liquid is adjusted by the way that hydrochloric acid is added, so that after
Continuous progress chromogenic reaction is more sensitive and accurate.
In present invention further optimization embodiment, it is separated by solid-liquid separation using centrifugation.
It is separated by solid-liquid separation by centrifugation, to remove the impurity particle in worm's ovum enzymolysis liquid, avoids impurity particle from influencing aobvious
The accuracy of colour response.
In the preferred embodiment of the present invention, centrifugal speed 2500-3500r/min, centrifugation time 3-
8min。
In the typical but non-limiting embodiment of the present invention, the speed of centrifugation as 2500,2600,2700,
2800,2900,3000,3100,3200,3300,3400 or 3500r/min；Centrifugation time is as being 3,4,5,6,7 or 8min.
In the preferred embodiment of the present invention, in step (d), it is molten that sodium carbonate is first added in worm's ovum enzymolysis liquid
Liquid is uniformly mixed, cooling after being placed in boiling water bath heating, is added glacial acetic acid solution and is uniformly mixed, Dimethylaminobenzene is then added
Formaldehyde formulations are uniformly mixed, and are eventually adding acetic acid, carry out chromogenic reaction.
It is uniformly mixed by the way that sodium carbonate liquor is added in worm's ovum enzymolysis liquid, is placed in boiling water bath and heats, to promote N-
Acetylglucosamine generates chitosan, in order to subsequently carry out chromogenic reaction.
By the way that glacial acetic acid is added so that reaction solution is acidic environment, to promote the progress of chromogenic reaction, by adding
It is more complete further to promote chromogenic reaction to carry out to enter acetic acid, to further increase the accuracy of detection method.
In the preferred embodiment of the present invention, the detection method of worm's ovum content further includes step (s) in broken head,
Step (s) is set to before step (a), and step (s) is to draw the standard curve of worm's ovum concentration and absorbance.
By first preparing the standard curve of worm's ovum content and absorbance, with according to the standard curve of worm's ovum content and absorbance
And the absorbance of chromophoric solution, worm's ovum content in broken head is calculated.
In the preferred embodiment of the present invention, the preparation method of the standard curve of worm's ovum concentration and absorbance includes
Following steps：
(A) the worm's ovum pregnant solution of different worm's ovum concentration is provided, and is ground respectively broken；
(B) the worm's ovum pregnant solution for grinding broken different worm's ovum concentration is separately added into chitinase to digest, is obtained
To the worm's ovum enzymolysis liquid of different worm's ovum concentration；
(C) it will be separately added into dimethylaminobenzaldehyde in the worm's ovum enzymolysis liquid of different worm's ovum concentration, obtains different colour developings
Different chromophoric solutions is measured absorbance by solution respectively, with a concentration of abscissa of worm's ovum, using absorbance as ordinate, is drawn
The standard curve of worm's ovum concentration and absorbance.
It should be noted that the concrete operations of the step of when prepared by above-mentioned standard curve (B) and step (C) are suitable for this hair
Step (c) in the detection method of bright offer and (d), details are not described herein.
In the preferred embodiment of the present invention, it in step (b), after first worm's ovum pregnant solution is dialysed, then carries out
It collects and purifies.
Dialysis is that the principle of water (or buffer solution) is diffused by small molecule semi-permeable membrane, and small molecule and macromolecular are separated
A kind of separating and purifying technology, to reach the further collection and purifying of worm's ovum pregnant solution.
In present invention further optimization embodiment, molecular cut off is used to be carried out for the bag filter of 8000-10000
Dialysis.
It dialyses to worm's ovum pregnant solution by using the bag filter that molecular cut off is 8000-10000, so that moisture
Son diffuses to outside from the inside of bag filter, has the function that non-heated concentrating and purifying worm's ovum pregnant solution.
According to the second aspect of the invention, the present invention provides the detection methods of worm's ovum content in above-mentioned broken head broken
Application in head recycling.
Broken head worm's ovum detection method of content provided by the invention can worm's ovum content in the broken head of Accurate Determining, in order to
Technical staff carries out classification recycling according to the difference of worm's ovum content, as being entirely free of worm's ovum in broken head, then can continue on for hanging
The preparation in face then can be used for the preparation of feed if worm's ovum content is relatively low, if worm's ovum content in broken head is higher, then be only used for
Fertilizer fermentation uses.
Technical solution provided by the invention is further described with reference to embodiment and comparative example.
Embodiment 1
The detection method for present embodiments providing worm's ovum content in a kind of broken head, includes the following steps：
(A) standard curve of worm's ovum concentration and absorbance is drawn
(A1) the worm's ovum pregnant solution of different worm's ovum concentration is prepared：It takes 20 adults to be put into 1kg flour, after 1 month, crosses and screen out
Adult is gone, flour is collected, by flour according to liquid ratio 10:1 is added in 4 DEG C of distilled water, impregnates 4h under the conditions of 4 DEG C, then exists
It is stirred under the conditions of rotating speed 100r/min, until flour is fully absorbed water, expands, is dispersed in water；Then stop stirring, stand 30-
40min takes supernatant, 8min is centrifuged under the conditions of 800r/min, makes flour particle precipitation remaining in supernatant, takes supernatant,
Obtain worm's ovum pregnant solution；It takes the above-mentioned worm's ovum pregnant solutions of lmL to be counted under the microscope by traditional dyeing method, determines that the worm's ovum is enriched with
Liquid worm's ovum concentration.Then by the worm's ovum pregnant solution be each configured to a concentration of 5/mL of worm's ovum, 15/mL, 25/mL, 30/
The worm's ovum pregnant solution of mL, 45/mL,
(A2) the worm's ovum enzymolysis liquid of different worm's ovum concentration is prepared：By the worm's ovum pregnant solution of different worm's ovum concentration, pass through respectively
After grinding is broken, then it is separately added into the chitinase of phase homogenous quantities, is then separately added into hydrochloric acid again, adjusts pH to 3, then distinguish
3000r/min is centrifuged 5 minutes, is discarded precipitation, is taken supernatant, obtains the worm's ovum enzymolysis liquid of different worm's ovum concentration；
(A2) chromogenic reaction is carried out：The worm's ovum enzymolysis liquid of 1mL difference worm's ovum concentration is taken to be placed in test tube respectively, then every
0.1mL sodium carbonate liquors (concentration 0.5mmol/L) are added in a test tube, each test tube is respectively placed in boiling water bath and is heated
5min, it is cooling, then respectively then addition glacial acetic acid solution is separately added into paradime thylaminobenzaldehyde solution (concentration again to 2mL
5%) 1mL is eventually adding acetic acid solution (concentration 90%) 7mL, respectively mixing, stands, and respectively obtains the aobvious of different colour developing depth
Color solution；
(A3) standard curve is drawn：The chromophoric solution of different colour developing depth is respectively put into spectrophotometer and carries out extinction
Degree measures, and with a concentration of abscissa of worm's ovum, using absorbance as ordinate, draws standard curve.
(B) worm's ovum pregnant solution is extracted from broken head
By broken head according to liquid ratio 10:1 is added in 4 DEG C of distilled water, 4h is impregnated under the conditions of 4 DEG C, then in rotating speed
It is stirred under the conditions of 100r/min, until broken head are fully absorbed water, expand, are dispersed in water；Then stop stirring, stand 30-
40min takes supernatant, 8min is centrifuged under the conditions of 800r/min, makes flour particle precipitation remaining in supernatant, takes supernatant,
Obtain worm's ovum pregnant solution；
(C) worm's ovum pregnant solution purifies
15% ficoll solution is prepared as separating liquid, appropriate volume worm's ovum pregnant solution is added in separating liquid surface, 4 DEG C of items
Under part, 12000r/min centrifuges 20min, collects interface mist band, is fully cleaned with distilled water, 10000r/min centrifugations
Precipitation is dissolved in 2ml distilled water to get to worm's ovum refined solution by 20min.
(D) worm's ovum enzymolysis liquid is prepared
After worm's ovum refined solution is crushed by grinding, chitinase is added, is then separately added into hydrochloric acid again, adjusts pH to 3, then
3000r/min is centrifuged 5 minutes, discards precipitation, takes supernatant to get to the worm's ovum enzymolysis liquid of various concentration.
(E) absorbance is measured
It takes the worm's ovum enzymolysis liquid of 1mL various concentrations to be placed in test tube, 0.1mL sodium carbonate liquors is then added in test tube
Test tube is respectively placed in boiling water bath and heats 5min by (concentration 0.5mmol/L), cooling, then adds glacial acetic acid solution to 2mL, so
After add paradime thylaminobenzaldehyde solution (concentration 5%) 1mL, be eventually adding acetic acid solution (concentration 90%) 7mL, mixing,
It stands, obtains chromophoric solution, measure the absorbance of chromophoric solution.
(F) worm's ovum content in broken head is calculated
According to the absorbance of the standard curve and chromophoric solution of worm's ovum content and absorbance, worm in chromophoric solution is calculated
Worm's ovum content in broken head is calculated according to worm's ovum concentration in chromophoric solution in ovum concentration.
Embodiment 2
Present embodiments provide a kind of detection method of worm's ovum content in broken head, the difference of the present embodiment and embodiment 1
Place is that the liquid ratio of broken head and distilled water is 20 in step (B):1.
Embodiment 3
Present embodiments provide a kind of detection method of worm's ovum content in broken head, the difference of the present embodiment and embodiment 1
Place is that the liquid ratio of broken head and distilled water is 15 in step (B):1.
Embodiment 4
Present embodiments provide a kind of detection method of worm's ovum content in broken head, the difference of the present embodiment and embodiment 3
Place is, rich as layering liquid progress discontinuous density gradient centrifugation progress worm's ovum using percoll solution in step (C)
The collection and purifying of liquid collecting, specifically comprise the following steps：
(C1) discontinuous density gradient Percoll layers of preparation：Percoll using 10%, 30%, 50%, 70% is molten
Liquid generates the Percoll solution vacuum outgas of various concentration to bubble-free, as layering solution by what is be successively superimposed
Percoll liquid is steadily flowed down along tube wall, is formed interface, is successively placed from high density to low-density with long needle applicator, will
Percoll liquid is put into syringe, and tube wall is close on small pinhead inclined-plane, it is made slowly to flow down naturally；
(C2) density gradient centrifugation is carried out：Appropriate volume worm's ovum pregnant solution is added in separating liquid surface.In relative centrifugal force
For centrifugation time 25min under the conditions of 400g.Centrifuge acceleration time 23min, deceleration time 3min.
(C3) worm's ovum is collected：Worm's ovum enrichment region can form misty band due to index of refraction difference after centrifugation, when institute is to be separated
Worm's ovum at two layers of interface, can successively remove the worm's ovum that interface locations are collected after Percoll liquid；
(C4) worm's ovum elutes:Worm's ovum containing Percoll liquid is fully cleaned with distilled water, 10000r/min centrifugations
Precipitation is dissolved in 2ml distilled water to get to worm's ovum refined solution by 20min.
Comparative example 1
This comparative example provides a kind of detection method of worm's ovum content in broken head, the difference of the present embodiment and embodiment 3
Place is, does not carry out step (C).
Verify example
Insect-taking ovum is dissolved in 4 DEG C of distilled water, is configured to the worm's ovum solution of a concentration of 10/mL, worm's ovum solution is pressed respectively
Step (C)-(F) carries out the detection of worm's ovum in the inspection side method provided according to embodiment 1-4,；Then according to the worm's ovum concentration of measurement
Calculate accuracy in detection, accuracy in detection=(measuring worm's ovum concentration-practical worm's ovum concentration) × 100%/practical worm's ovum concentration, knot
Fruit is as shown in table 1.
Egg detecting method accuracy data table in 1 broken head of table
| Group | Accuracy (%) |
| Embodiment 1 | 95.6 |
| Embodiment 2 | 96.2 |
| Embodiment 3 | 97.1 |
| Embodiment 4 | 95.6 |
| Comparative example 1 | 62.3 |
The detection method that embodiment 1-4 provides worm's ovum in broken head is can be seen that from the accuracy of embodiment 1-4 in table 1
Accuracy reach 95% or more, this illustrates that the detection method of worm's ovum content in the broken head that embodiment 1-4 is provided can be accurate
The content of worm's ovum, can provide foundation for the recycling of broken head in the true broken head of measurement.
The comparison of embodiment 1-4 and comparative example 1 can be seen that by being purified to worm's ovum pregnant solution from table 1, can
Significantly improve the accuracy of worm's ovum assay.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, it will be understood by those of ordinary skill in the art that：Its according to
So can with technical scheme described in the above embodiments is modified, either to which part or all technical features into
Row equivalent replacement；And these modifications or replacements, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (10)
1. the detection method of worm's ovum content in a kind of broken head, which is characterized in that include the following steps：
(a) broken head are soaked in distilled water, are separated by solid-liquid separation, obtain worm's ovum pregnant solution：
(b) worm's ovum pregnant solution is collected and is purified, obtain worm's ovum refined solution；
(c) chitinase is added in worm's ovum refined solution, obtains worm's ovum enzymolysis liquid；
(d) dimethylaminobenzaldehyde is added in worm's ovum enzymolysis liquid and carries out chromogenic reaction, obtain chromophoric solution, measure chromophoric solution
Absorbance, obtain worm's ovum content in broken head.
2. the detection method of worm's ovum content in broken head according to claim 1, which is characterized in that broken in step (a)
The liquid ratio of head and distilled water is (10-20):1；
Preferably, soaking time 3-5h；
Preferably, the distilled water that distilled water is 0-5 DEG C；
Preferably, first passing through standing takes supernatant to be once separated by solid-liquid separation, then supernatant is carried out secondary solid-liquid by centrifuging
Separation, obtains worm's ovum pregnant solution.
3. the detection method of worm's ovum content in broken head according to claim 1, which is characterized in that in step (b), adopt
The collection and purifying of worm's ovum pregnant solution are carried out with density-gradient centrifugation method；
Preferably, the layering solution using percoll solution or Focill solution as density-gradient centrifugation method；
Preferably, the separating liquid using ficoll as density-gradient centrifugation method.
4. the detection method of worm's ovum content in broken head according to claim 1, which is characterized in that further include step (p),
Step (p) is set between step (b) and step (c), and step (p) is to be crushed worm's ovum refined solution；
Preferably, the broken of worm's ovum refined solution is carried out using ultrasound cracking or liquid nitrogen grinding.
5. the detection method of worm's ovum content in broken head according to claim 1, which is characterized in that in step (c), first
Chitinase is added in worm's ovum refined solution, hydrochloric acid is added, it is 3 to adjust pH value, is separated by solid-liquid separation, obtains worm's ovum enzymolysis liquid；
Preferably, it is separated by solid-liquid separation using centrifugation；
Preferably, centrifugal speed 2500-3500r/min, centrifugation time 3-8min；
It is further preferred that centrifugal speed is 3000r/min, centrifugation time 5min.
6. the detection method of worm's ovum content in broken head according to claim 1, which is characterized in that in step (d), first
Sodium carbonate liquor is added in worm's ovum enzymolysis liquid to be uniformly mixed, it is cooling after being placed in boiling water bath heating, it is mixed to add glacial acetic acid solution
It closes uniformly, dimethylaminobenzaldehyde reagent is then added and is uniformly mixed, is eventually adding acetic acid, carries out chromogenic reaction；
Preferably, the time heated in boiling water bath is 3-8min, preferably 5min.
7. the detection method of worm's ovum content in broken head according to claim 1, which is characterized in that further include step (s),
Step (s) is set to before step (a), and step (s) is to draw the standard curve of worm's ovum concentration and absorbance.
8. the detection method of worm's ovum content in broken head according to claim 1, which is characterized in that in step (b), first
After worm's ovum pregnant solution is dialysed, then it is collected and purifies.
9. the detection method of worm's ovum content in broken head according to claim 8, which is characterized in that use molecular cut off
It dialyses for the bag filter of 8000-10000.
10. being recycled in broken head according to the detection method of worm's ovum content in the broken head of claim 1-9 any one of them
In application.
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