CN108218973A - A kind of extracting method of black sea cucumbers from East China Sea glycoprotein - Google Patents
A kind of extracting method of black sea cucumbers from East China Sea glycoprotein Download PDFInfo
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Abstract
The invention discloses a kind of extracting method of black sea cucumbers from East China Sea glycoprotein, by by dry black sea cucumbers from East China Sea carry out pre-treatment, degreasing, it is thick carry and purify, black sea cucumbers from East China Sea glycoprotein has successfully been extracted from black sea cucumbers from East China Sea.Papain is used to extract the glycoprotein in black sea cucumbers from East China Sea in the present invention, it can be discharged into aqueous solution by the way that the usual protein in black sea cucumbers from East China Sea is decomposed glycoprotein components, the reaction condition of this method is as mild as a dove, without violent reaction, enable to black sea cucumbers from East China Sea glycoprotein that can not be destroyed, so as to ensure the quality of black sea cucumbers from East China Sea glycoprotein finally obtained.Meanwhile enzymatic isolation method has very high efficiency, enables to protein fast decoupled, therefore improve the extraction efficiency and yield of final black sea cucumbers from East China Sea glycoprotein.
Description
Technical field
The present invention relates to a kind of extracting method of glycoprotein, more particularly, to a kind of extraction side of black sea cucumbers from East China Sea glycoprotein
Method.
Background technology
Black sea cucumbers from East China Sea is the animal of spine skin door Holothuroidea Molpadida Kao Can sections Acaudina also known as is fragrant ginseng, there is " sea kind
The laudatory title of potato ".Black sea cucumbers from East China Sea mainly originates in the East China Sea lefteyes such as China Hainan, Zhejiang, and resource is very abundant, civil to be often used in generation
For sea cucumber.It very nutritious, but it is fairly well-developed utilization as marine resources not yet, because of the side such as processing technology
Limitation existing for face, the exploitation of black sea cucumbers from East China Sea processing industry chain extremely fall behind, so even if fisherman is captured, can also put and return
Sea.
Black sea cucumbers from East China Sea not merely contains high protein, and amino acid necessary to a variety of human bodies, micro- and a variety of dimensions are raw outer,
And disease also can be cured, prevent the medicinal saponin(e and polysaccharose substance of disease.Have in black sea cucumbers from East China Sea body wall a large amount of
Acid mucopolysaccharide and glycoprotein, i.e., the glycoprotein containing uronic acid and amino acid residue are important in its body wall and enteron aisle
Constituent.Black sea cucumbers from East China Sea glycoprotein has blood pressure lowering, anticoagulation, and anticancer inhibits tumour, and anti-angiogenic rebirth improves body and exempts from
The bioactive functions such as epidemic disease function are a kind of very promising marine organisms medical products.
Instantly progress shows more commonly used extracting method there are two types of the polysaccharide in Holothuroidea animal, and one kind is alkali
Solution, another kind are enzymatic isolation methods.Wherein alkali extraction method is carried according to instable principle of the proteoglycan on cardohydrata-peptide linkage
Polysaccharide is taken, is easy to Walden inversion after alkali process and desulfurization phenomenon occurs.So enzyme hydrolysis method is instantly ideal carries
Method is taken, this method can ensure that it under the premise of protease hydrolytic, does not change the structure of polysaccharide chain, so discharge polysaccharide
It is very effective.Fang Chongbo compared alkaline hydrolysiss and enzymatic isolation method both methods, the results showed that, sea cucumber is extracted by basic hydrolysis
Polysaccharide may lead to the degradation of glycosyl, change so as to cause polysaccharide structures；And the latter, since the process ratio of reaction is less violent,
This method can be such that the glycoprotein molecule structure of black sea cucumbers from East China Sea is not destroyed, so as to ensure that recovery rate.These older generations study
Effective extraction for black sea cucumbers from East China Sea glycoprotein plays a good evocation.
Invention content
The present invention be in order to overcome in the prior art black sea cucumbers from East China Sea glycoprotein extraction efficiency it is low, be easy to cause black sea cucumbers from East China Sea sugar
The problem of degradation of albumen, reaction process is violent, black sea cucumbers from East China Sea glycoprotein extraction efficiency can be improved by providing one kind, not easily led to
The degradation of black sea cucumbers from East China Sea glycoprotein, a kind of extracting method of the mild black sea cucumbers from East China Sea glycoprotein of reaction process.
To achieve these goals, the present invention uses following technical scheme：
A kind of extracting method of black sea cucumbers from East China Sea glycoprotein, the extracting method include the following steps：
(a) pre-treatment：Dry black sea cucumbers from East China Sea is taken, using cold water soak 12 hours, then removes the internal organ of black sea cucumbers from East China Sea and body table
The mud in face is placed at shady and cool drying and dries in the shade, is subsequently placed in baking oven and dries, the dry black sea cucumbers from East China Sea after drying is placed in omnipotent powder
It is crushed among broken machine, crosses 80 mesh sieve, obtain black sea cucumbers from East China Sea dry powder；
(b) degreasing：The extra large Sea Cucumber dry powder obtained in step (a) is taken, then places it in Soxhlet extractor, uses organic solvent
Carry out degreasing to it to it, degreasing time is 5-10 hours, organic solvent is evaporated after degreasing obtains degreasing black sea cucumbers from East China Sea and do
Powder；
(c) it slightly carries：Take 5 parts of the extra large Sea Cucumber dry powder obtained in step (b), then add in 120-160 parts of PBS buffer solutions and
0.05-0.55 parts of papains stir evenly latter water-bath 18-24 hours, are after reaction warming up to bath temperature
It 100 DEG C, is kept for 5-10 minutes, then takes out standing and be down to room temperature, be placed in a centrifuge centrifugation, collect gained after centrifuging
Supernatant obtains black sea cucumbers from East China Sea glycoprotein crude extract；
(d) essence carries：The black sea cucumbers from East China Sea liquid of extracting polysaccharide obtained in step (c) is taken, 95% alcoholic solution is added in thereto, stands
It is then centrifuged for detaching, takes the precipitation of centrifugation gained, and be dissolved in distilled water, be configured to saturated solution, then add
95% alcoholic solution precipitation purification obtains black sea cucumbers from East China Sea glycoprotein refining liquid after 2-3 times repeatedly；
(e) it decolourizes：100 parts of the black sea cucumbers from East China Sea glycoprotein refining liquid obtained in step (d) is removed, adds in 5 parts of activated carbon thereto, is surpassed
Sound aided bleaching 1 hour, filters to take filtrate, obtains colourless black sea cucumbers from East China Sea glycoprotein refining liquid；
(f) it isolates and purifies：The colourless black sea cucumbers from East China Sea glycoprotein refining liquid obtained in step (f) is taken to collect one by level-one filter membrane
Grade trapped fluid；With, again by two level filter membrane, collection two level passes through liquid and second entrapment liquid after pure water dilution level-one trapped fluid；With
Pure water dilutes two level by, again by three-level filter membrane, collecting three-level trapped fluid after liquid；By second entrapment liquid and three-level trapped fluid
Merging obtains black sea cucumbers from East China Sea glycoprotein purification liquid, is then concentrated under reduced pressure into 1/10th of original volume, is then centrifuged for being spray-dried,
Obtain black sea cucumbers from East China Sea glycoprotein powder.
Papain is used to extract the polysaccharide in black sea cucumbers from East China Sea in the present invention, by will be in black sea cucumbers from East China Sea
Breaks down proteins polysaccharide component is discharged into aqueous solution, the reaction condition of this method as mild as a dove, without violent
Reaction, enable to black sea cucumbers from East China Sea glycoprotein that can not be destroyed, so as to ensure the black sea cucumbers from East China Sea glycoprotein finally obtained
Quality.Meanwhile enzymatic isolation method has very high efficiency, enables to protein fast decoupled, therefore improve the final East Sea
The extraction efficiency and yield of Sea Cucumber glycoprotein.
Preferably, oven temperature is 48-55 DEG C in the step (a), drying time is 8-12 hours.Black sea cucumbers from East China Sea
It is dried before extraction, the yield of black sea cucumbers from East China Sea glycoprotein can be promoted.
Preferably, organic solvent is one kind in acetone, ethyl alcohol, isopropanol or ether in the step (b).These
Solvent can effectively extract the fat constituent in black sea cucumbers from East China Sea, and will not be reacted with black sea cucumbers from East China Sea glycoprotein or molten
Solution so that grease does not interfere with the quality of final black sea cucumbers from East China Sea glycoprotein, will not reduce the yield of black sea cucumbers from East China Sea glycoprotein.
Preferably, bath temperature when slightly being carried in the step (c) is 45-55 DEG C.Papain is in the temperature
In the range of there is highest reactivity, the breaks down proteins for enabling to non-glycoprotein are more thorough.
Preferably, the volume of 95% alcohol and black sea cucumbers from East China Sea glycoprotein crude extract ratio are 3-5 in the step (d)
∶1.Effectively black sea cucumbers from East China Sea glycoprotein can be precipitated out in the range of this volume ratio, improve extraction efficiency.
Preferably, ultrasonic power is 700-800W in the step (e).
Preferably, centrifugal spray drying intake air temperature is 180 DEG C in the step (f), drop temperature is 40 DEG C.
Preferably, the molecular cut off of level-one filter membrane, two level filter membrane and three-level filter membrane is respectively in the step (f)
5000~10000,50000~80000 and 20000~40000.
Therefore, the present invention has the advantages that：(1) black sea cucumbers from East China Sea glycoprotein extraction efficiency is high；(2) east is not easily led to
The degradation of extra large Sea Cucumber glycoprotein；(3) reaction process is mild.
Description of the drawings
Fig. 1 is the glucose standard curve of the present invention.
Fig. 2 is the protein content standard curve of the present invention.
Fig. 3 is sulfate content standard curve of the present invention.
Technical scheme of the present invention is made below by specific embodiment to further describe explanation.
If the raw material that use is said without specified otherwise, in the embodiment of the present invention is raw material commonly used in the art, implement
Method employed in example is the conventional method of this field.
Embodiment 1
A kind of extracting method of black sea cucumbers from East China Sea glycoprotein, the extracting method include the following steps：
(a) pre-treatment：Dry black sea cucumbers from East China Sea is taken, using cold water soak 12 hours, then removes the internal organ of black sea cucumbers from East China Sea and body table
The mud in face is placed at shady and cool drying and dries in the shade, is subsequently placed in 48 DEG C of baking ovens and dries, and drying time is 12 hours, after drying
Dry black sea cucumbers from East China Sea be placed in Universalpulverizer among crush, cross 80 mesh sieve, obtain black sea cucumbers from East China Sea dry powder；
(b) degreasing：The extra large Sea Cucumber dry powder obtained in step (a) is taken, is then placed it in Soxhlet extractor, using acetone to it
Degreasing is carried out to it, degreasing time is 5 hours, and acetone obtains degreasing black sea cucumbers from East China Sea dry powder after degreasing；
(c) it slightly carries：5 parts of the extra large Sea Cucumber dry powder obtained in step (b) is taken, then adds in 120 parts of PBS buffer solutions and 0.05 part
Papain, stirs evenly rear 45 DEG C of water-baths 18 hours, and bath temperature is warming up to 100 DEG C after reaction, keeps 5
Minute, it then takes out standing and is down to room temperature, be placed in a centrifuge centrifugation, collect the supernatant of gained after centrifugation, obtain east
Extra large Sea Cucumber glycoprotein crude extract；
(d) essence carries：The black sea cucumbers from East China Sea liquid of extracting polysaccharide obtained in step (c) is taken, 95% alcoholic solution is added in thereto, stands
It is then centrifuged for detaching, takes the precipitation of centrifugation gained, and be dissolved in distilled water, be configured to saturated solution, then add
95% alcoholic solution precipitation purification obtains black sea cucumbers from East China Sea glycoprotein refining liquid after 2 times repeatedly；
(e) it decolourizes：100 parts of the black sea cucumbers from East China Sea glycoprotein refining liquid obtained in step (d) is removed, adds in 5 parts of activated carbon thereto,
700W ultrasonic wave addeds decolourize 1 hour, filter to take filtrate, obtain colourless black sea cucumbers from East China Sea glycoprotein refining liquid；
(f) it isolates and purifies：It by molecular cut off is 5000 to take the colourless black sea cucumbers from East China Sea glycoprotein refining liquid that is obtained in step (f)
~10000 level-one filter membrane collects level-one trapped fluid；It is by molecular cut off again after diluting level-one trapped fluid with pure water
50000~80000 two level filter membrane collects two level and passes through liquid and second entrapment liquid；With pure water dilute two level by after liquid again
It is 20000~40000 three-level filter membranes by molecular cut off, collects three-level trapped fluid；By second entrapment liquid and three-level trapped fluid
Merging obtains black sea cucumbers from East China Sea glycoprotein purification liquid, is then concentrated under reduced pressure into 1/10th of original volume, is then centrifuged for being spray-dried,
Obtain black sea cucumbers from East China Sea glycoprotein powder.
Embodiment 2
A kind of extracting method of black sea cucumbers from East China Sea glycoprotein, the extracting method include the following steps：
(a) pre-treatment：Dry black sea cucumbers from East China Sea is taken, using cold water soak 12 hours, then removes the internal organ of black sea cucumbers from East China Sea and body table
The mud in face is placed at shady and cool drying and dries in the shade, is subsequently placed in 55 DEG C of baking ovens and dries, and drying time is 8 hours, after drying
Dry black sea cucumbers from East China Sea crushes among being placed in Universalpulverizer, crosses 80 mesh sieve, obtains black sea cucumbers from East China Sea dry powder；
(b) degreasing：The extra large Sea Cucumber dry powder obtained in step (a) is taken, is then placed it in Soxhlet extractor, using ethyl alcohol to it
Degreasing is carried out to it, degreasing time is 10 hours, and ethyl alcohol obtains degreasing black sea cucumbers from East China Sea dry powder after degreasing；
(c) it slightly carries：5 parts of the extra large Sea Cucumber dry powder obtained in step (b) is taken, then adds in 160 parts of PBS buffer solutions and 0.55 part
Papain, stirs evenly rear 55 DEG C of water-baths 24 hours, and bath temperature is warming up to 100 DEG C after reaction, is kept
It 10 minutes, then takes out standing and is down to room temperature, be placed in a centrifuge centrifugation, collect the supernatant of gained after centrifugation, obtain
Black sea cucumbers from East China Sea glycoprotein crude extract；
(d) essence carries：The black sea cucumbers from East China Sea liquid of extracting polysaccharide obtained in step (c) is taken, 95% alcoholic solution is added in thereto, stands
It is then centrifuged for detaching, takes the precipitation of centrifugation gained, and be dissolved in distilled water, be configured to saturated solution, then add
95% alcoholic solution precipitation purification obtains black sea cucumbers from East China Sea glycoprotein refining liquid after 3 times repeatedly；
(e) it decolourizes：100 parts of the black sea cucumbers from East China Sea glycoprotein refining liquid obtained in step (d) is removed, adds in 5 parts of activated carbon thereto,
800W ultrasonic wave addeds decolourize 1 hour, filter to take filtrate, obtain colourless black sea cucumbers from East China Sea glycoprotein refining liquid；
(f) it isolates and purifies：It by molecular cut off is 5000 to take the colourless black sea cucumbers from East China Sea glycoprotein refining liquid that is obtained in step (f)
~10000 level-one filter membrane collects level-one trapped fluid；It is by molecular cut off again after diluting level-one trapped fluid with pure water
50000~80000 two level filter membrane collects two level and passes through liquid and second entrapment liquid；With pure water dilute two level by after liquid again
It is 20000~40000 three-level filter membranes by molecular cut off, collects three-level trapped fluid；By second entrapment liquid and three-level trapped fluid
Merging obtains black sea cucumbers from East China Sea glycoprotein purification liquid, is then concentrated under reduced pressure into 1/10th of original volume, is then centrifuged for being spray-dried,
Obtain black sea cucumbers from East China Sea glycoprotein powder.
Embodiment 3
A kind of extracting method of black sea cucumbers from East China Sea glycoprotein, the extracting method include the following steps：
(a) pre-treatment：Dry black sea cucumbers from East China Sea is taken, using cold water soak 12 hours, then removes the internal organ of black sea cucumbers from East China Sea and body table
The mud in face is placed at shady and cool drying and dries in the shade, is subsequently placed in 50 DEG C of baking ovens and dries, and drying time is 10 hours, after drying
Dry black sea cucumbers from East China Sea be placed in Universalpulverizer among crush, cross 80 mesh sieve, obtain black sea cucumbers from East China Sea dry powder；
(b) degreasing：The extra large Sea Cucumber dry powder obtained in step (a) is taken, then places it in Soxhlet extractor, uses isopropanol pair
It carries out it degreasing, and degreasing time is 8 hours, and isopropanol obtains degreasing black sea cucumbers from East China Sea dry powder after degreasing；
(c) it slightly carries：5 parts of the extra large Sea Cucumber dry powder obtained in step (b) is taken, then adds in 150 parts of PBS buffer solutions and 0.5 part
Papain, stirs evenly rear 50 DEG C of water-baths 20 hours, and bath temperature is warming up to 100 DEG C after reaction, keeps 8
Minute, it then takes out standing and is down to room temperature, be placed in a centrifuge centrifugation, collect the supernatant of gained after centrifugation, obtain east
Extra large Sea Cucumber glycoprotein crude extract；
(d) essence carries：The black sea cucumbers from East China Sea liquid of extracting polysaccharide obtained in step (c) is taken, 95% alcoholic solution is added in thereto, stands
It is then centrifuged for detaching, takes the precipitation of centrifugation gained, and be dissolved in distilled water, be configured to saturated solution, then add
95% alcoholic solution precipitation purification obtains black sea cucumbers from East China Sea glycoprotein refining liquid after 3 times repeatedly；
(e) it decolourizes：100 parts of the black sea cucumbers from East China Sea glycoprotein refining liquid obtained in step (d) is removed, adds in 5 parts of activated carbon thereto,
750W ultrasonic wave addeds decolourize 1 hour, filter to take filtrate, obtain colourless black sea cucumbers from East China Sea glycoprotein refining liquid；
(f) it isolates and purifies：It by molecular cut off is 5000 to take the colourless black sea cucumbers from East China Sea glycoprotein refining liquid that is obtained in step (f)
~10000 level-one filter membrane collects level-one trapped fluid；It is by molecular cut off again after diluting level-one trapped fluid with pure water
50000~80000 two level filter membrane collects two level and passes through liquid and second entrapment liquid；With pure water dilute two level by after liquid again
It is 20000~40000 three-level filter membranes by molecular cut off, collects three-level trapped fluid；By second entrapment liquid and three-level trapped fluid
Merging obtains black sea cucumbers from East China Sea glycoprotein purification liquid, is then concentrated under reduced pressure into 1/10th of original volume, is then centrifuged for being spray-dried,
Obtain black sea cucumbers from East China Sea glycoprotein powder.Embodiment 4
A kind of extracting method of black sea cucumbers from East China Sea glycoprotein, the extracting method include the following steps：
(a) pre-treatment：Dry black sea cucumbers from East China Sea is taken, using cold water soak 12 hours, then removes the internal organ of black sea cucumbers from East China Sea and body table
The mud in face is placed at shady and cool drying and dries in the shade, is subsequently placed in 52 DEG C of baking ovens and dries, and drying time is 9 hours, after drying
Dry black sea cucumbers from East China Sea crushes among being placed in Universalpulverizer, crosses 80 mesh sieve, obtains black sea cucumbers from East China Sea dry powder；
(b) degreasing：The extra large Sea Cucumber dry powder obtained in step (a) is taken, is then placed it in Soxhlet extractor, using ether to it
Degreasing is carried out to it, degreasing time is 6 hours, and ether obtains degreasing black sea cucumbers from East China Sea dry powder after degreasing；
(c) it slightly carries：5 parts of the extra large Sea Cucumber dry powder obtained in step (b) is taken, then adds in 140 parts of PBS buffer solutions and 0.3 part
Papain, stirs evenly rear 48 DEG C of water-baths 22 hours, and bath temperature is warming up to 100 DEG C after reaction, keeps 8
Minute, it then takes out standing and is down to room temperature, be placed in a centrifuge centrifugation, collect the supernatant of gained after centrifugation, obtain east
Extra large Sea Cucumber glycoprotein crude extract；
(d) essence carries：The black sea cucumbers from East China Sea liquid of extracting polysaccharide obtained in step (c) is taken, 95% alcoholic solution is added in thereto, stands
It is then centrifuged for detaching, takes the precipitation of centrifugation gained, and be dissolved in distilled water, be configured to saturated solution, then add
95% alcoholic solution precipitation purification obtains black sea cucumbers from East China Sea glycoprotein refining liquid after 3 times repeatedly；
(e) it decolourizes：100 parts of the black sea cucumbers from East China Sea glycoprotein refining liquid obtained in step (d) is removed, adds in 5 parts of activated carbon thereto,
800W ultrasonic wave addeds decolourize 1 hour, filter to take filtrate, obtain colourless black sea cucumbers from East China Sea glycoprotein refining liquid；
(f) it isolates and purifies：It by molecular cut off is 5000 to take the colourless black sea cucumbers from East China Sea glycoprotein refining liquid that is obtained in step (f)
~10000 level-one filter membrane collects level-one trapped fluid；It is by molecular cut off again after diluting level-one trapped fluid with pure water
50000~80000 two level filter membrane collects two level and passes through liquid and second entrapment liquid；With pure water dilute two level by after liquid again
It is 20000~40000 three-level filter membranes by molecular cut off, collects three-level trapped fluid；By second entrapment liquid and three-level trapped fluid
Merging obtains black sea cucumbers from East China Sea glycoprotein purification liquid, is then concentrated under reduced pressure into 1/10th of original volume, is then centrifuged for being spray-dried,
Obtain black sea cucumbers from East China Sea glycoprotein powder.
Embodiment 5
A kind of extracting method of black sea cucumbers from East China Sea glycoprotein, the extracting method include the following steps：
(a) pre-treatment：Dry black sea cucumbers from East China Sea is taken, using cold water soak 12 hours, then removes the internal organ of black sea cucumbers from East China Sea and body table
The mud in face is placed at shady and cool drying and dries in the shade, is subsequently placed in 55 DEG C of baking ovens and dries, and drying time is 12 hours, after drying
Dry black sea cucumbers from East China Sea be placed in Universalpulverizer among crush, cross 80 mesh sieve, obtain black sea cucumbers from East China Sea dry powder；
(b) degreasing：The extra large Sea Cucumber dry powder obtained in step (a) is taken, is then placed it in Soxhlet extractor, using acetone to it
Degreasing is carried out to it, degreasing time is 5 hours, and acetone obtains degreasing black sea cucumbers from East China Sea dry powder after degreasing；
(c) it slightly carries：5 parts of the extra large Sea Cucumber dry powder obtained in step (b) is taken, then adds in 160 parts of PBS buffer solutions and 0.15 part
Papain, stirs evenly rear 50 DEG C of water-baths 18 hours, and bath temperature is warming up to 100 DEG C after reaction, keeps 5
Minute, it then takes out standing and is down to room temperature, be placed in a centrifuge centrifugation, collect the supernatant of gained after centrifugation, obtain east
Extra large Sea Cucumber glycoprotein crude extract；
(d) essence carries：The black sea cucumbers from East China Sea liquid of extracting polysaccharide obtained in step (c) is taken, 95% alcoholic solution is added in thereto, stands
It is then centrifuged for detaching, takes the precipitation of centrifugation gained, and be dissolved in distilled water, be configured to saturated solution, then add
95% alcoholic solution precipitation purification obtains black sea cucumbers from East China Sea glycoprotein refining liquid after 3 times repeatedly；
(e) it decolourizes：100 parts of the black sea cucumbers from East China Sea glycoprotein refining liquid obtained in step (d) is removed, adds in 5 parts of activated carbon thereto,
800W ultrasonic wave addeds decolourize 1 hour, filter to take filtrate, obtain colourless black sea cucumbers from East China Sea glycoprotein refining liquid；
(f) it isolates and purifies：It by molecular cut off is 5000 to take the colourless black sea cucumbers from East China Sea glycoprotein refining liquid that is obtained in step (f)
~10000 level-one filter membrane collects level-one trapped fluid；It is by molecular cut off again after diluting level-one trapped fluid with pure water
50000~80000 two level filter membrane collects two level and passes through liquid and second entrapment liquid；With pure water dilute two level by after liquid again
It is 20000~40000 three-level filter membranes by molecular cut off, collects three-level trapped fluid；By second entrapment liquid and three-level trapped fluid
Merging obtains black sea cucumbers from East China Sea glycoprotein purification liquid, is then concentrated under reduced pressure into 1/10th of original volume, is then centrifuged for being spray-dried,
Obtain black sea cucumbers from East China Sea glycoprotein powder.
The black sea cucumbers from East China Sea glycoprotein physicochemical property test obtained in embodiment 1-5.
Total sugar content measures
Sulfuric acid-phynol method：
Extracted black sea cucumbers from East China Sea glycoprotein powder 1mg is weighed using the electronic balance of high precision, with distillation water dissolution after appearance
Measuring bottle (10ml, 25 DEG C) is settled to 10ml, in addition, in a clean and dry test tube, the East Sea crow of 1ml is taken with liquid-transfering gun
Join glycoprotein solution to place wherein, and add in distilled water 1ml, small commotio cordis mixing.Phenol is put among water-bath and is added
Heat fusing, precision weigh the phenol of 5mg, then carry out constant volume with volumetric flask (100ml, 25 DEG C), since phenol compares in room temperature
Easily solidification, is heated so placing into water-bath, is dissolved after heating with supersonic wave cleaning machine, is made all in volumetric flask
Phenol is completely dissolved, and obtains the phenol solution of clear, and the solution in the phenol solution of gained and test tube is put into ventilation
It is operated in cupboard, the phenol solution after 1ml constant volumes is added in first into test tube, then add in the dense sulphur of 5ml into test tube again
Acid, careful quick oscillation is uniform, and test tube mouth is sealed with preservative film, is positioned in 100 DEG C of water-bath and heats 15 minutes.It is operating
During ultraviolet specrophotometer can be carried out preheating 20 minutes, by the wavelength tune of ultraviolet specrophotometer after preheating
Start to measure to 490nm, record each absorption values, finally gone out accordingly according to the regression equation calculation that computer software obtains
Total sugar content.
The drafting of standard curve：
1.00mg/mL glucose standards solutions 0mL, 0.2mL, 0.4mL are accurately critically inhaled, 0.6mL, 0.8mL, 1mL, which is added in, to pass through
In dried tool plug test tube (10mL), next operation is same as above the absorption values warp described in sulfuric acid-phynol method, measured
Corresponding standard curve is drawn out after summarizing.
Determining the protein quantity
Folin- phenol reagent process (LowryShi methods)：
The configuration of reagent：Na is weighed first2CO3Then 1g weighs sodium hydroxide 0.2g again, sequentially add to dry volumetric flask
In (50ml, 25 DEG C), then addition distilled water progress constant volume is spare to make, labeled as A.Next anhydrous cupric sulfate is weighed
Then 0.125g weighs tartaric acid 0.25g again, added in also according to sequence in the volumetric flask of 25ml dryings, adds in distilled water and determines
It is spare after appearance, labeled as B.It is carefully pipetted with liquid-transfering gun among solution A 1ml above is added to B solution, last shaken well.
Precision weighs black sea cucumbers from East China Sea glycoprotein dry powder 5mg, adds in the distilled water of 1ml thereto, black sea cucumbers from East China Sea glycoprotein sample is prepared
It is spare to make into the solution of 5mg/ml.
The drafting of standard curve
Accurate precision pipettes bovine serum albumin(BSA) (BSA) standard solution (matching while using) 0.1mL, 0.2mL of 0.5mg/mL,
0.3mL, 0.4mL, 0.5mL are added in into the tool plug test tube (10ml) dried, and are then sequentially added distilled water to volume and are
0.5mL, it is 0.5mL that blank control group set in addition adds in distilled water to volume equally in through dried tool plug test tube,
Next Folin- phenol reagent A2.5mL are respectively added in this 6 test tubes, careful shaking is uniformly mixed, and is then stood at room temperature
Next 10min in each tool plug test tube adds Folin- phenol reagent B0.25mL, and quickly to shake mixing successively again, together
Sample places a hour at room temperature, next is adjusted to measure at 750nm by the wavelength of ultraviolet specrophotometer, obtains each
Absorption values under a concentration will obtain standard curve after all data preparations.
The measure of sulfate content
Barium sulfate turbidimetry
Standard solution is prepared：By potassium sulfate drying to constant weight, precision weighs a certain amount of substance, is configured to a concentration of 0.6mg/
The potassium sulfate standard solution of mL.The preparation of 1mol/L hydrochloric acid solutions：The concentrated hydrochloric acid of 8.3ml is measured with the graduated cylinder of 10ml, in capacity
In bottle (100ml, 25 DEG C) after constant volume to get.The preparation of gelatin solution：Precision weighs the drying gelatin of 1.25g, is dissolved in
In the distilled water of 250ml, and mixing is quickly shaken to get labeled as A.The preparation of barium chloride gelatin solution：Precision weighs 0.5g
Dry BaCl2Powder, be adequately dissolve among the prepared solution As of 100ml to get.
The black sea cucumbers from East China Sea glycoprotein powder of precision weighing 2mg, after being poured into ampoule bottle, 1.0mol/L HCl are pipetted
1.0mL is melting sealed to ampoule bottle, places it in 105 DEG C of environment and stands 10 hours, its cooling is treated after taking-up, with rotation
Evaporimeter is dissolved after being spin-dried for the distilled water of 2.0ml.The solution of 320 μ L is pipetted using liquid-transfering gun precision, is surveyed at 360 nm
Its fixed absorption values, finally substitute into the calibration curve equation hereinafter obtained, calculate sulfate radical content therein.
The drafting of standard curve
0,120,160,200,240,280,320 μ L of standard liquor are drawn in 10mL test tubes with liquid-transfering gun precision, add water to it
Volume is 1000 μ L, and the HCl 1.0mL of 1mol/L are drawn with liquid-transfering gun, make itself and BaCl2Gelatin solution 0.5mL is equably mixed
It closes, labeled as A.At the same time, by the identical standard solution of gradient, liquor capacity is added water to as 1000 μ L, is drawn with liquid-transfering gun
Dilute HCl 1.0mL, make it uniformly be mixed with 0.5mL gelatin solutions, labeled as B.Later A, two solution of B is individually placed to rack for test tube
On stand about 20 minutes, while the measure wavelength for adjusting ultraviolet-uisible spectrophotometer measures its extinction to 360 nanometers
Number of degrees value, and using standard solution concentration gradient as abscissa, with A, the difference of the absorbance of two solution of B is ordinate, draws mark
Directrix curve obtains regression equation.
The black sea cucumbers from East China Sea glycoprotein physicochemical data obtained in embodiment 1-5
Shown in table 1 is black sea cucumbers from East China Sea glycoprotein total sugar content, protein content and the sulfate content obtained in embodiment 1-5
Data summarization.
The black sea cucumbers from East China Sea glycoprotein physicochemical data obtained in 1 embodiment 1-5 of table
| Project | Total sugar content (%) | Protein content (%) | Sulfate content (%) |
| Embodiment 1 | 40.3 | 35.2 | 20.14 |
| Embodiment 2 | 42.7 | 33.5 | 21.98 |
| Embodiment 3 | 43.9 | 36.27 | 19.45 |
| Embodiment 4 | 40.5 | 31.28 | 16.23 |
| Embodiment 5 | 41.8 | 35.76 | 20.44 |
Claims (8)
1. a kind of extracting method of black sea cucumbers from East China Sea glycoprotein, it is characterized in that, the extracting method includes the following steps：
（a）Pre-treatment：Dry black sea cucumbers from East China Sea is taken, using cold water soak 12 hours, then removes the internal organ of black sea cucumbers from East China Sea and body table
The mud in face is placed at shady and cool drying and dries in the shade, is subsequently placed in baking oven and dries, the dry black sea cucumbers from East China Sea after drying is placed in omnipotent powder
It is crushed among broken machine, crosses 80 mesh sieve, obtain black sea cucumbers from East China Sea dry powder；
（b）Degreasing：Take step（a）In obtained extra large Sea Cucumber dry powder, then place it in Soxhlet extractor, use organic solvent
Carry out degreasing to it to it, degreasing time is 5-10 hours, organic solvent is evaporated after degreasing obtains degreasing black sea cucumbers from East China Sea and do
Powder；
（c）Slightly carry：Take step（b）In obtained 5 parts of extra large Sea Cucumber dry powder, then add in 120-160 parts of PBS buffer solutions and
0.05-0.55 parts of papains stir evenly latter water-bath 18-24 hours, are after reaction warming up to bath temperature
It 100 DEG C, is kept for 5-10 minutes, then takes out standing and be down to room temperature, be placed in a centrifuge centrifugation, collect gained after centrifuging
Supernatant obtains black sea cucumbers from East China Sea glycoprotein crude extract；
（d）Essence carries：Take step（c）In obtained black sea cucumbers from East China Sea liquid of extracting polysaccharide, add in thereto 95% alcoholic solution, stand
It is then centrifuged for detaching, takes the precipitation of centrifugation gained, and be dissolved in distilled water, be configured to saturated solution, then add 95%
Alcoholic solution precipitation purification, obtain black sea cucumbers from East China Sea glycoprotein refining liquid after 2-3 times repeatedly；
（e）Decoloration：Go step（d）In obtained 100 parts of black sea cucumbers from East China Sea glycoprotein refining liquid, add in 5 parts of activated carbon thereto, surpass
Sound aided bleaching 1 hour, filters to take filtrate, obtains colourless black sea cucumbers from East China Sea glycoprotein refining liquid；
（f）It isolates and purifies：Take step（e）In obtained colourless black sea cucumbers from East China Sea glycoprotein refining liquid lead to level-one filter membrane, collect level-one
Trapped fluid；With, again by two level filter membrane, collection two level passes through liquid and second entrapment liquid after pure water dilution level-one trapped fluid；With pure
Water purification dilutes two level by, again by three-level filter membrane, collecting three-level trapped fluid after liquid；Second entrapment liquid and three-level trapped fluid are closed
And black sea cucumbers from East China Sea glycoprotein purification liquid is obtained, 1/10th of original volume is then concentrated under reduced pressure into, is then centrifuged for being spray-dried, obtain
To black sea cucumbers from East China Sea glycoprotein powder.
2. a kind of extracting method of black sea cucumbers from East China Sea glycoprotein according to claim 1, it is characterized in that, the step（a）
Middle oven temperature is 48-55 DEG C, and drying time is 8-12 hours.
3. a kind of extracting method of black sea cucumbers from East China Sea glycoprotein according to claim 1, it is characterized in that, the step（b）
Middle organic solvent is one kind in acetone, ethyl alcohol, isopropanol or ether.
4. a kind of extracting method of black sea cucumbers from East China Sea glycoprotein according to claim 1, it is characterized in that, the step（c）
In bath temperature when slightly carrying be 45-55 DEG C.
5. the extracting method of a kind of black sea cucumbers from East China Sea glycoprotein stated according to claim 1, it is characterized in that, the step（d）In
The volume of 95% alcohol is 3-5 with black sea cucumbers from East China Sea glycoprotein crude extract ratio:1.
6. a kind of extracting method of black sea cucumbers from East China Sea glycoprotein according to claim 1, it is characterized in that, the step（e）
Middle ultrasonic power is 700-800W.
7. a kind of extracting method of black sea cucumbers from East China Sea glycoprotein according to claim 1, it is characterized in that, the step（f）
Middle centrifugal spray drying intake air temperature is 180 DEG C, drop temperature is 40 DEG C.
8. a kind of extracting method of black sea cucumbers from East China Sea glycoprotein according to claim 1, it is characterized in that, the step（f）
The molecular cut off of middle level-one filter membrane, two level filter membrane and three-level filter membrane is respectively 5000~10000,50000~80000 and
20000~40000.
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