CN108148109A - A kind of preparation method of the preparation method of squid sexual gland saccharide complex and its ferrous complex of hydrolysate - Google Patents
A kind of preparation method of the preparation method of squid sexual gland saccharide complex and its ferrous complex of hydrolysate Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of the ferrous complex of the preparation method and its hydrolysate of squid sexual gland saccharide complex, by to freeze due to squid sexual gland carry out pretreatment and remove the grease that wherein contains, then it is extracted with sodium hydroxide/sodium borohydride mixed aqueous solution, squid sexual gland saccharide complex is obtained with two steps of purifying by slightly carrying, squid sexual gland saccharide complex under the action of enzyme is hydrolyzed and is complexed to obtain the ferrous complex of squid sexual gland saccharide complex hydrolysate with ferrous ion.The present invention has the advantages that：（1）A kind of squid sexual gland saccharide complex extraction process is simple（2）Extraction efficiency is higher；（3）The hydrolysate of squid sexual gland saccharide complex can be successfully generated complex compound with ferrous ion.
Description
Technical field
The present invention relates to a kind of preparation method of the metal ion complex of the preparation method and its hydrolysate of saccharide complex,
A kind of preparation method of the ferrous complex of preparation method and its hydrolysate more particularly, to squid sexual gland saccharide complex
Background technology
Squid, especially Peru squid, more embody its edible value, and rare medical value.Squid it is non-edible
The feed of low value is often dropped or is processed into part such as liver, sexual gland by-product.In recent years, someone is from squid skin
Albumen, nucleic acid, glycoprotein etc. are extracted in skin, liver, sexual gland etc., and activity research is carried out to it, there is certain achievement.Zhang Jian
If it waits the extraction glycoprotein from squid viscera and studies its immunocompetence.Wang Qian etc. optimizes squid spawn tangled gland with response phase method
The glycoprotein extraction process of (sexual gland).Refined collection of Liu etc. has studied the reducing blood lipid of squid spawn tangled gland (sexual gland) glycoprotein and non-specific exempts from
Epidemic disease function.
Molecule or ion are combined with metal ion, and the process for forming very stable new ion is just named complex reaction, raw
Into substance complex compound, also known as complex.Specific reaction principle is shown as：Two or more contain lone pair electrons
The molecule or ion of (or pi bond) make ligand, with the central atom with empty valence orbit or ions binding into structure list
Member.By bidentate and more than ligand participate in being formed the process of complex and become chelating.Chelating is one kind of complexing.With metal ion
The molecule or ion of complexing are known as complexing agent.Common complexing agent has aminocarboxylate, organic phosphonate, polyacrylic etc..
Polymeric complexing agent then has polysaccharide, polypeptide, nucleic acid etc..The complexing agent of super high molecular weight is generally resin, protein, glycoprotein
Deng.Complexing agent is commonly applied to dyeing industry, cleanness industry etc., and Review on Complexometry is also used in laboratory and makees catalyst.And network
The application for closing object is generally found from pharmaceuticals industry.There is theory to think at present, human small intestine to the absorption of metal ion be first make its with
Amino-acid complex, then cell is entered with this more stable state, chelate bonds are then disconnected under the action of cell liquid.But actually
Human body is relatively low to the absorptivity of minerals, can largely be expelled directly out external.If therefore in advance by metal ion and amino acid or
The chelatings such as polypeptide, absorptivity will improve.So more efficient mineral supplements can be obtained by chelating technology.It is and practical
In in the market there are many such drug.
Invention content
The present invention is to overcome squid sexual gland saccharide complex extraction process complexity in the prior art, and extraction efficiency is relatively low,
The problem of squid sexual gland saccharide complex is not complexed with ferrous ion provides a kind of squid sexual gland saccharide complex extraction process
Simply, extraction efficiency is higher, and a kind of squid sexual gland sugar that squid sexual gland saccharide complex successfully synthesizes complex compound with ferrous ion is answered
Close the preparation method of object and its preparation method of the ferrous complex of hydrolysate.
To achieve these goals, the present invention uses following technical scheme：
A kind of preparation method of squid sexual gland saccharide complex, the preparation method include following steps：
(a) it pre-processes：The squid of freezing is taken, is allowed to naturally to thaw softening, take its sexual gland and is smashed to pieces in pureed, it will after freeze-drying
Its crush, successively cross 40 mesh sieve and 60 mesh sieve, obtain powdered squid sexual gland, it is carried out using organic solvent Soxhlet extraction into
Row degreasing, degreasing time are 10-18 hours, and organic solvent is evaporated after degreasing, obtains degreasing squid sexual gland powder；
(b) it slightly carries：The degreasing obtained in step (a) squid sexual gland powder is taken to be placed in sodium hydroxide/sodium borohydride aqueous solution to stir
The liquid ratio in mass of extraction, sodium hydroxide/sodium borohydride mixed aqueous solution and degreasing squid sexual gland powder is 10-40：1, it carries
It is 20-40 DEG C to take temperature, and extraction time is 2-5 hours, and 4000r/min is centrifuged 10 minutes after extraction, takes upper strata clear
Liquid dialyses 15-24 hours, the 1/10 of original volume is concentrated into after dialysis, it is compound to obtain squid sexual gland sugar in deionized water
Object crude extract；
(c) it purifies：The squid sexual gland saccharide complex crude extract obtained in step (b) is taken, then adds in squid sexual gland saccharide complex
The Sevag reagents of crude extract volume 1/5-1/2 are sufficiently stirred 10-30 minutes, supernatant liquor are taken to repeat purification step 1-3 times, most
The supernatant liquor once obtained afterwards is placed in a centrifuge to be centrifuged 5-20 minutes with the speed of 4000r/min, is taken supernatant liquor, is obtained
Squid sexual gland saccharide complex refined solution, obtains squid sexual gland saccharide complex after freeze-drying.
Carry out the mode degreasing of Soxhlet extraction in step (a) to squid sexual gland using organic solvent, it can be effectively by squid
Fat in piscinity gland removes, and sodium hydroxide enables to the squid sexual gland saccharide complex in squid sexual gland to be dissolved in step (b)
In water, and sodium borohydride has reproducibility, can reduce sugared chain break.Sevag reagents can make squid squid property in step (c)
Protein denaturation in gland saccharide complex and separation is so as to obtain more pure squid sexual gland from squid sexual gland saccharide complex
Saccharide complex.
Preferably, the organic solvent in the step is one kind in acetone, ethyl alcohol, isopropanol or ether.This is several
Kind organic solvent is higher to the dissolubility of fat, is conducive to the fat-extraction in squid sexual gland, obtains degreasing squid sexual gland
Powder.
Preferably, a concentration of 0.1-1.5mol/L of the sodium hydroxide in mixed aqueous solution in the step.
Preferably, a concentration of 0-0.4mol/L of the sodium borohydride in mixed aqueous solution in the step (b).
Preferably, molecular cut off is used to dialyse for the bag filter of 14000Da in the step (b).
Preferably, Sevag reagents are chloroform in the step (c): n-butanol volume ratio is 4: 1 mixed liquors.
A kind of preparation method of the ferrous complex of squid sexual gland saccharide complex hydrolysate, the preparation method are as follows：
(d) it hydrolyzes：10 parts of the squid obtained in claim 1 sexual gland saccharide complex is taken to be dissolved in 20-50 parts of water, heating water bath
Temperature is increased to 55 DEG C, then adds in 1.5-4.5 parts of papain, after being stirred to react 2-5 hours, boiling water bath 10-20 minutes
Enzyme deactivation is cooled to 4 DEG C, and 10000r/min is centrifuged 15-25 minutes, and supernatant is taken to filter, obtains enzymolysis liquid, is lyophilized, is obtained dry squid
Piscinity gland glycoprotein hydrolysate；
(e) the squid sexual gland glycoprotein hydrolysate in step (d) is taken to add in can be completely dissolved squid sexual gland glycoprotein water just
The distilled water of object amount is solved, the sulfuric acid of a concentration of 0.1-2.4mg/L is then added in squid sexual gland glycoprotein hydrolysate aqueous solution
Ferrous iron solution, in terms of ferrous ion, the mass ratio of squid sexual gland glycoprotein and ferrous ion is 20-200: 1, at 35-45 DEG C
Stirring complexing 0.5-10 hours, 8000r/min centrifugation 5min, takes supernatant liquor to measure light absorption value, calculates complexing rate, lower sediment
The ferrous complex of squid sexual gland saccharide complex hydrolysate is obtained after dialysis is freeze-dried.
The protein in squid sexual gland saccharide complex is hydrolyzed to obtain squid property using papain in step (d)
Gland glycoprotein hydrolysate so that protein therein will not have an impact the complexing of the ferrous ion of next step.
Preferably, the assay method of light absorption value is as follows in step (e)：Precision pipettes 500 μ l of supernatant liquor, adds 1ml salt
Sour azanol after 5min plus 5ml Phens, after shaking up plus 9.5ml ammonium acetates-glacial acetic acid buffer solution, moves to 50ml volumetric flasks, fixed
Hold, light absorption value is measured by blank group of distilled water at 510nm wavelength with 1cm cuvettes after 10min.
Preferably, the supernatant liquor in the step (e) passes through 0.45 μm of disposable filtering with microporous membrane.
Therefore, the present invention has the advantages that：(1) a kind of squid sexual gland saccharide complex extraction process simple (2) carries
Take yield higher；(3) hydrolysate of squid sexual gland saccharide complex can be successfully generated complex compound with ferrous ion.
Description of the drawings
Fig. 1 is Phen spectrophotometry standard ferrous ion absorbance-concentration standard curve.
Fig. 2 is the relationship of complexation time and complexing rate.
Fig. 3 is hydrolysate and the mass ratio of metal ion and the relationship of complexing rate.
Specific embodiment
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 preparation method of squid sexual gland saccharide complex, the preparation method include following steps
(a) it pre-processes：The squid of freezing is taken, is allowed to naturally to thaw softening, take its sexual gland and is smashed to pieces in pureed, it will after freeze-drying
It is crushed, and is crossed 40 mesh sieve and 60 mesh sieve successively, is obtained powdered squid sexual gland, carrying out Soxhlet extraction to it using acetone takes off
Fat, degreasing time are 10 hours, and acetone is evaporated after degreasing, obtains degreasing squid sexual gland powder；
(b) it slightly carries：The degreasing obtained in step (a) squid sexual gland powder is taken to be placed in sodium hydroxide/sodium borohydride aqueous solution to stir
The liquid ratio in mass of extraction, sodium hydroxide/sodium borohydride mixed aqueous solution and degreasing squid sexual gland powder is 10: 1, wherein hydrogen
A concentration of 0.1mol/L of the sodium oxide molybdena in mixed aqueous solution, a concentration of 0.1mol/L of the sodium borohydride in mixed aqueous solution,
Extracting temperature is 20 DEG C, and extraction time is 2 hours, and 4000r/min is centrifuged 10 minutes after extraction, takes supernatant liquor,
Molecular cut off is used to carry out dialysis 15 hours for the bag filter of 14000Da in deionized water, original is concentrated into after dialysis
The 1/10 of volume obtains squid sexual gland saccharide complex crude extract；
(c) it purifies：The squid sexual gland saccharide complex crude extract obtained in step (b) is taken, then adds in squid sexual gland saccharide complex
The Sevag reagents (chloroform: n-butanol volume ratio is 4: 1) of the mixed liquor of crude extract volume 1/5, are sufficiently stirred 10 minutes,
Supernatant liquor is taken to repeat purification step 1 time, the supernatant liquor obtained for the last time is placed in a centrifuge the speed with 4000r/min
Centrifugation 5 minutes, takes supernatant liquor, obtains squid sexual gland saccharide complex refined solution, it is compound that squid sexual gland sugar is obtained after freeze-drying
Object.
A kind of preparation method of the ferrous complex of squid sexual gland saccharide complex hydrolysate, the preparation method are as follows：
(d) it hydrolyzes：10 parts of the squid obtained in claim 1 sexual gland saccharide complex is taken to be dissolved in 20 parts of water, heating water bath liter
Then high-temperature adds in 1.5 parts of papain, after being stirred to react 2 hours, boiling water bath enzyme deactivation in 10 minutes is cooled to 4 to 55 DEG C
DEG C, 10000r/min is centrifuged 15 minutes, and supernatant is taken to filter, obtains enzymolysis liquid, is lyophilized, and obtains dry squid sexual gland glycoprotein hydrolysis
Object；
(e) the squid sexual gland glycoprotein hydrolysate in step (d) is taken to add in can be completely dissolved squid sexual gland glycoprotein water just
The distilled water of object amount is solved, the ferrous sulfate of a concentration of 0.1mg/L is then added in squid sexual gland glycoprotein hydrolysate aqueous solution
Solution, in terms of ferrous ion, the mass ratio of squid sexual gland glycoprotein and ferrous ion is 20: 1, the stirring complexing 0.5 at 35 DEG C
Hour, 8000r/min centrifugation 5min take supernatant liquor, extinction are then measured by 0.45 μm of disposable filtering with microporous membrane
Value, calculates complexing rate, and lower sediment obtains the ferrous complex of squid sexual gland saccharide complex hydrolysate after dialysis is freeze-dried.
Embodiment 2
A kind of preparation method of squid sexual gland saccharide complex, the preparation method include following steps
(a) it pre-processes：The squid of freezing is taken, is allowed to naturally to thaw softening, take its sexual gland and is smashed to pieces in pureed, it will after freeze-drying
It is crushed, and is crossed 40 mesh sieve and 60 mesh sieve successively, is obtained powdered squid sexual gland, carrying out Soxhlet extraction to it using ethyl alcohol takes off
Fat, degreasing time are 18 hours, and ethyl alcohol is evaporated after degreasing, obtains degreasing squid sexual gland powder；
(b) it slightly carries：The degreasing obtained in step (a) squid sexual gland powder is taken to be placed in sodium hydroxide/sodium borohydride aqueous solution to stir
The liquid ratio in mass of extraction, sodium hydroxide/sodium borohydride mixed aqueous solution and degreasing squid sexual gland powder is 40: 1, wherein hydrogen
A concentration of 1.5mol/L of the sodium oxide molybdena in mixed aqueous solution, a concentration of 0.4mol/L of the sodium borohydride in mixed aqueous solution,
Extracting temperature is 40 DEG C, and extraction time is 5 hours, and 4000r/min is centrifuged 10 minutes after extraction, takes supernatant liquor,
Molecular cut off is used to carry out dialysis 24 hours for the bag filter of 14000Da in deionized water, original is concentrated into after dialysis
The 1/10 of volume obtains squid sexual gland saccharide complex crude extract；
(c) it purifies：The squid sexual gland saccharide complex crude extract obtained in step (b) is taken, then adds in squid sexual gland saccharide complex
The Sevag reagents (chloroform: n-butanol volume ratio is 4: 1) of the mixed liquor of crude extract volume 1/2, are sufficiently stirred 30 minutes,
Supernatant liquor is taken to repeat purification step 3 times, the supernatant liquor obtained for the last time is placed in a centrifuge the speed with 4000r/min
Centrifugation 20 minutes, takes supernatant liquor, obtains squid sexual gland saccharide complex refined solution, it is compound that squid sexual gland sugar is obtained after freeze-drying
Object.
A kind of preparation method of the ferrous complex of squid sexual gland saccharide complex hydrolysate, the preparation method are as follows：
(d) it hydrolyzes：10 parts of the squid obtained in claim 1 sexual gland saccharide complex is taken to be dissolved in 50 parts of water, heating water bath liter
Then high-temperature adds in 4.5 parts of papain, after being stirred to react 5 hours, boiling water bath enzyme deactivation in 20 minutes is cooled to 4 to 55 DEG C
DEG C, 10000r/min is centrifuged 25 minutes, and supernatant is taken to filter, obtains enzymolysis liquid, is lyophilized, and obtains dry squid sexual gland glycoprotein hydrolysis
Object；
(e) the squid sexual gland glycoprotein hydrolysate in step (d) is taken to add in can be completely dissolved squid sexual gland glycoprotein water just
The distilled water of object amount is solved, the ferrous sulfate of a concentration of 2.4mg/L is then added in squid sexual gland glycoprotein hydrolysate aqueous solution
Solution, in terms of ferrous ion, the mass ratio of squid sexual gland glycoprotein and ferrous ion is 200: 1, the stirring complexing 10 at 45 DEG C
Hour, 8000r/min centrifugation 5min take supernatant liquor, extinction are then measured by 0.45 μm of disposable filtering with microporous membrane
Value, calculates complexing rate, and lower sediment obtains the ferrous complex of squid sexual gland saccharide complex hydrolysate after dialysis is freeze-dried.
Embodiment 3
A kind of preparation method of squid sexual gland saccharide complex, it is characterized in that, the preparation method includes following steps
(a) it pre-processes：The squid of freezing is taken, is allowed to naturally to thaw softening, take its sexual gland and is smashed to pieces in pureed, it will after freeze-drying
It is crushed, and is crossed 40 mesh sieve and 60 mesh sieve successively, is obtained powdered squid sexual gland, carrying out Soxhlet extraction to it using acetone takes off
Fat, degreasing time are 12 hours, and acetone is evaporated after degreasing, obtains degreasing squid sexual gland powder；
(b) it slightly carries：The degreasing obtained in step (a) squid sexual gland powder is taken to be placed in sodium hydroxide/sodium borohydride aqueous solution to stir
The liquid ratio in mass of extraction, sodium hydroxide/sodium borohydride mixed aqueous solution and degreasing squid sexual gland powder is 20: 1, wherein hydrogen
A concentration of 0.37mol/L of the sodium oxide molybdena in mixed aqueous solution, a concentration of 0.1mol/L of the sodium borohydride in mixed aqueous solution,
Extracting temperature is 25 DEG C, and extraction time is 3.5 hours, and 4000r/min is centrifuged 10 minutes after extraction, takes upper strata clear
Liquid uses molecular cut off to carry out dialysis 15-24 hours for the bag filter of 14000Da in deionized water, dense after dialysis
The 1/10 of original volume is reduced to, obtains squid sexual gland saccharide complex crude extract；
(c) it purifies：The squid sexual gland saccharide complex crude extract obtained in step (b) is taken, then adds in squid sexual gland saccharide complex
The Sevag reagents (chloroform: n-butanol volume ratio is 4: 1) of the mixed liquor of crude extract volume 1/4, are sufficiently stirred 15 minutes,
Supernatant liquor is taken to repeat purification step 2 times, the supernatant liquor obtained for the last time is placed in a centrifuge the speed with 4000r/min
Centrifugation 10 minutes, takes supernatant liquor, obtains squid sexual gland saccharide complex refined solution, it is compound that squid sexual gland sugar is obtained after freeze-drying
Object.
A kind of preparation method of the ferrous complex of squid sexual gland saccharide complex hydrolysate, the preparation method are as follows：
(d) it hydrolyzes：10 parts of the squid obtained in claim 1 sexual gland saccharide complex is taken to be dissolved in 30 parts of water, heating water bath liter
Then high-temperature adds in 3 parts of papain, after being stirred to react 4 hours, boiling water bath enzyme deactivation in 15 minutes is cooled to 4 to 55 DEG C
DEG C, 10000r/min is centrifuged 20 minutes, and supernatant is taken to filter, obtains enzymolysis liquid, is lyophilized, and obtains dry squid sexual gland glycoprotein hydrolysis
Object；(e) the squid sexual gland glycoprotein hydrolysate in step (d) is taken to add in can be completely dissolved the hydrolysis of squid sexual gland glycoprotein just
Then the distilled water of object amount adds in the ferrous sulfate of a concentration of 1.483mg/L in squid sexual gland glycoprotein hydrolysate aqueous solution
Solution, in terms of ferrous ion, the mass ratio of squid sexual gland glycoprotein and ferrous ion is 100: 1, and stirring complexing 5 is small at 37 DEG C
When, 8000r/min centrifugation 5min take supernatant liquor, light absorption value are then measured by 0.45 μm of disposable filtering with microporous membrane,
Complexing rate is calculated, lower sediment obtains the ferrous complex of squid sexual gland saccharide complex hydrolysate after dialysis is freeze-dried.
Embodiment 4
A kind of preparation method of squid sexual gland saccharide complex, the preparation method include following steps
(a) it pre-processes：The squid of freezing is taken, is allowed to naturally to thaw softening, take its sexual gland and is smashed to pieces in pureed, it will after freeze-drying
It is crushed, and is crossed 40 mesh sieve and 60 mesh sieve successively, is obtained powdered squid sexual gland, Soxhlet extraction progress is carried out to it using isopropanol
Degreasing, degreasing time are 15 hours, and isopropanol is evaporated after degreasing, obtains degreasing squid sexual gland powder；
(b) it slightly carries：The degreasing obtained in step (a) squid sexual gland powder is taken to be placed in sodium hydroxide/sodium borohydride aqueous solution to stir
The liquid ratio in mass of extraction, sodium hydroxide/sodium borohydride mixed aqueous solution and degreasing squid sexual gland powder is 30: 1, wherein hydrogen
A concentration of 1mol/L of the sodium oxide molybdena in mixed aqueous solution, a concentration of 0.3mol/L of the sodium borohydride in mixed aqueous solution, carries
It is 30 DEG C to take temperature, and extraction time is 3 hours, and 4000r/min is centrifuged 10 minutes after extraction, takes supernatant liquor,
Molecular cut off is used to carry out dialysis 18 hours for the bag filter of 14000Da in deionized water, substance is concentrated into after dialysis
Long-pending 1/10 obtains squid sexual gland saccharide complex crude extract；
(c) it purifies：The squid sexual gland saccharide complex crude extract obtained in step (b) is taken, then adds in squid sexual gland saccharide complex
The Sevag reagents (chloroform: n-butanol volume ratio is 4: 1) of the mixed liquor of crude extract volume 1/5-1/2, are sufficiently stirred 15 points
Clock takes supernatant liquor to repeat purification step 2 times, and the supernatant liquor obtained for the last time is placed in a centrifuge with 4000r/min's
Speed centrifuges 15 minutes, takes supernatant liquor, obtains squid sexual gland saccharide complex refined solution, and squid sexual gland sugar is obtained after freeze-drying
Compound.
A kind of preparation method of the ferrous complex of squid sexual gland saccharide complex hydrolysate, the preparation method are as follows：
(d) it hydrolyzes：10 parts of the squid obtained in claim 1 sexual gland saccharide complex is taken to be dissolved in 20-50 parts of water, heating water bath
Temperature is increased to 55 DEG C, then adds in 1.5-4.5 parts of papain, after being stirred to react 2-5 hours, boiling water bath 10-20 minutes
Enzyme deactivation is cooled to 4 DEG C, and 10000r/min is centrifuged 15-25 minutes, and supernatant is taken to filter, obtains enzymolysis liquid, is lyophilized, is obtained dry squid
Piscinity gland glycoprotein hydrolysate；
(e) the squid sexual gland glycoprotein hydrolysate in step (d) is taken to add in can be completely dissolved squid sexual gland glycoprotein water just
The distilled water of object amount is solved, the ferrous sulfate that a concentration of 2mg/L is then added in squid sexual gland glycoprotein hydrolysate aqueous solution is molten
Liquid, in terms of ferrous ion, the mass ratio of squid sexual gland glycoprotein and ferrous ion is 120: 1, and stirring complexing 8 is small at 40 DEG C
When, 8000r/min centrifugation 5min take supernatant liquor, light absorption value are then measured by 0.45 μm of disposable filtering with microporous membrane,
Complexing rate is calculated, lower sediment obtains the ferrous complex of squid sexual gland saccharide complex hydrolysate after dialysis is freeze-dried.
Embodiment 5
A kind of preparation method of squid sexual gland saccharide complex, the preparation method include following steps
(a) it pre-processes：The squid of freezing is taken, is allowed to naturally to thaw softening, take its sexual gland and is smashed to pieces in pureed, it will after freeze-drying
It is crushed, and is crossed 40 mesh sieve and 60 mesh sieve successively, is obtained powdered squid sexual gland, carrying out Soxhlet extraction to it using ether takes off
Fat, degreasing time are 10 hours, and ether is evaporated after degreasing, obtains degreasing squid sexual gland powder；
(b) it slightly carries：The degreasing obtained in step (a) squid sexual gland powder is taken to be placed in sodium hydroxide/sodium borohydride aqueous solution to stir
The liquid ratio in mass of extraction, sodium hydroxide/sodium borohydride mixed aqueous solution and degreasing squid sexual gland powder is 35: 1, wherein hydrogen
A concentration of 1.2mol/L of the sodium oxide molybdena in mixed aqueous solution, a concentration of 0.2mol/L of the sodium borohydride in mixed aqueous solution,
Extracting temperature is 35 DEG C, and extraction time is 2 hours, and 4000r/min is centrifuged 10 minutes after extraction, takes supernatant liquor,
Molecular cut off is used to carry out dialysis 16 hours for the bag filter of 14000Da in deionized water, original is concentrated into after dialysis
The 1/10 of volume obtains squid sexual gland saccharide complex crude extract；
(c) it purifies：The squid sexual gland saccharide complex crude extract obtained in step (b) is taken, then adds in squid sexual gland saccharide complex
The Sevag reagents (chloroform: n-butanol volume ratio is 4: 1) of the mixed liquor of crude extract volume 1/5, are sufficiently stirred 30 minutes,
Supernatant liquor is taken to repeat purification step 3 times, the supernatant liquor obtained for the last time is placed in a centrifuge the speed with 4000r/min
Centrifugation 20 minutes, takes supernatant liquor, obtains squid sexual gland saccharide complex refined solution, it is compound that squid sexual gland sugar is obtained after freeze-drying
Object.
A kind of preparation method of the ferrous complex of squid sexual gland saccharide complex hydrolysate, the preparation method are as follows：
(d) it hydrolyzes：10 parts of the squid obtained in claim 1 sexual gland saccharide complex is taken to be dissolved in 45 parts of water, heating water bath liter
Then high-temperature adds in 3 parts of papain, after being stirred to react 2 hours, boiling water bath enzyme deactivation in 10 minutes is cooled to 4 to 55 DEG C
DEG C, 10000r/min is centrifuged 15 minutes, and supernatant is taken to filter, obtains enzymolysis liquid, is lyophilized, and obtains dry squid sexual gland glycoprotein hydrolysis
Object；
(e) the squid sexual gland glycoprotein hydrolysate in step (d) is taken to add in can be completely dissolved squid sexual gland glycoprotein water just
The distilled water of object amount is solved, the ferrous sulfate of a concentration of 2.4mg/L is then added in squid sexual gland glycoprotein hydrolysate aqueous solution
Solution, in terms of ferrous ion, the mass ratio of squid sexual gland glycoprotein and ferrous ion is 50: 1, the stirring complexing 0.5 at 35 DEG C
Hour, 8000r/min centrifugation 5min take supernatant liquor, extinction are then measured by 0.45 μm of disposable filtering with microporous membrane
Value, calculates complexing rate, and lower sediment obtains the ferrous complex of squid sexual gland saccharide complex hydrolysate after dialysis is freeze-dried.
The experimental data of embodiment 1-5
Relationship between 1 squid sexual gland saccharide complex yield of table and each factor
| Factor | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
| Extracting temperature (DEG C) | 20 | 40 | 25 | 30 | 35 |
| Naoh concentration (mol/L) | 0.1 | 1.5 | 0.37 | 1 | 1.2 |
| Sodium borohydride concentration (mol/L) | 0.1 | 0.4 | 0.1 | 0.3 | 0.2 |
| Extraction time (h) | 2 | 5 | 3.5 | 3 | 2 |
| Liquid ratio | 10∶1 | 40∶1 | 20∶1 | 30∶1 | 35∶1 |
| Squid sexual gland saccharide complex yield (%) | 9.10 | 10.30 | 13.18 | 10.90 | 10.60 |
The assay method of light absorption value is as follows：Precision pipettes 500 μ l of supernatant liquor, adds 1ml hydroxylamine hydrochlorides, adds 5ml adjacent luxuriant and rich with fragrance after 5min
Sieve quinoline after shaking up plus 9.5ml ammonium acetates-glacial acetic acid buffer solution, moves to 50ml volumetric flasks, constant volume, and 1cm cuvettes are used after 10min
Light absorption value is measured by blank group of distilled water at 510nm wavelength, is compared with standard ferrous ion absorbance-concentration chart
Calculate complexing rate.
Ferrous standard curve
Phen spectrophotometry standard ferrous ion absorbance-concentration chart is obtained, it is as follows：
2 Phen spectrophotometry standard ferrous ion absorbance of table-concentration
| Serial number | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Taken amount/μ 1 | 0 | 25 | 50 | 100 | 200 | 400 | 600 |
| Ferrous iron concentration/(mg/L) | 0 | 0.1 | 0.2 | 0.4 | 0.8 | 1.6 | 2.4 |
| Absorbance A | 0.091 | 0.119 | 0.139 | 0.198 | 0.281 | 0.469 | 0.656 |
Complexation time and the chart of the relationship of complexing rate are obtained, it is as follows：
3 complexation time of table and the relationship of complexing rate
| Complexation time/h | 0.5 | 1 | 3.5 | 18 |
| Absorbance A | 0.539 | 0.485 | 0.442 | 0.463 |
| Ferrous iron concentration/(mg/L) | 1.897 | 1.667 | 1.483 | 1.572 |
| Complexing rate | 0.39% | 12.51% | 22.16% | 17.45% |
The mass ratio of hydrolysate and metal ion and the chart of the relationship of complexing rate, it is as follows：
The mass ratio of 4 hydrolysate of table and metal ion and the relationship of complexing rate
| Mass ratio | 20∶1 | 50∶1 | 100∶1 | 200∶1 |
| Absorbance A | 0.441 | 0.328 | 0.223 | 0.214 |
| Ferrous iron concentration/(mg/L) | 1.478 | 0.995 | 0.546 | 0.508 |
| Complexing rate | 22.38% | 45.26% | 69.95% | 72.06% |
Claims (8)
1. a kind of preparation method of squid sexual gland saccharide complex, it is characterized in that, the preparation method includes following steps：
（a）Pretreatment：The squid of freezing is taken, is allowed to naturally to thaw softening, take its sexual gland and is smashed to pieces in pureed, it will after freeze-drying
Its crush, successively cross 40 mesh sieve and 60 mesh sieve, obtain powdered squid sexual gland, it is carried out using organic solvent Soxhlet extraction into
Row degreasing, degreasing time are 10-18 hours, and organic solvent is evaporated after degreasing, obtains degreasing squid sexual gland powder；
（b）Slightly carry：Take step（a）In obtained degreasing squid sexual gland powder be placed in sodium hydroxide/sodium borohydride aqueous solution and stir
The liquid ratio in mass of extraction, sodium hydroxide/sodium borohydride mixed aqueous solution and degreasing squid sexual gland powder is 10-40:1, it carries
It is 20-40 DEG C to take temperature, and extraction time is 2-5 hours, and 4000r/min is centrifuged 10 minutes after extraction, takes upper strata clear
Liquid dialyses 15-24 hours, the 1/10 of original volume is concentrated into after dialysis, it is compound to obtain squid sexual gland sugar in deionized water
Object crude extract；
（c）Purifying：Take step（b）In obtained squid sexual gland saccharide complex crude extract, then add in squid sexual gland saccharide complex
The Sevag reagents of crude extract volume 1/5-1/2 are sufficiently stirred 10-30 minutes, supernatant liquor are taken to repeat purification step 1-3 times, most
The supernatant liquor once obtained afterwards is placed in a centrifuge to be centrifuged 5-20 minutes with the speed of 4000r/min, is taken supernatant liquor, is obtained
Squid sexual gland saccharide complex refined solution, obtains squid sexual gland saccharide complex after freeze-drying.
2. a kind of preparation method of squid sexual gland saccharide complex according to claim 1, it is characterized in that, the step
（a）In organic solvent be one kind in acetone, ethyl alcohol, isopropanol or ether.
3. a kind of preparation method of squid sexual gland saccharide complex according to claim 1, it is characterized in that, the step
（b）A concentration of 0.1-1.5mol/L of the middle sodium hydroxide in mixed aqueous solution.
4. a kind of preparation method of squid sexual gland saccharide complex according to claim 1 or 3, it is characterized in that, the step
Suddenly（b）A concentration of 0-0.4mol/L of the middle sodium borohydride in mixed aqueous solution.
5. a kind of preparation method of squid sexual gland saccharide complex according to claim 1, it is characterized in that, the step
（b）It is middle that molecular cut off is used to dialyse for the bag filter of 14000Da.
6. a kind of preparation method of squid sexual gland saccharide complex according to claim 1, it is characterized in that, the step
（c）Middle Sevag reagents are chloroform:N-butanol volume ratio is 4:1 mixed liquor.
A kind of 7. preparation method of the ferrous complex of squid sexual gland saccharide complex hydrolysate, which is characterized in that the preparation
Method is as follows：
（d）Hydrolysis：10 parts of the squid obtained in claim 1 sexual gland saccharide complex is taken to be dissolved in 20-50 parts of water, heating water bath
Temperature is increased to 55 DEG C, then adds in 1.5-4.5 parts of papain, after being stirred to react 2-5 hours, boiling water bath 10-20 minutes
Enzyme deactivation is cooled to 4 DEG C, and 10000r/min is centrifuged 15-25 minutes, and supernatant is taken to filter, obtains enzymolysis liquid, is lyophilized, is obtained dry squid
Piscinity gland glycoprotein hydrolysate；
（e）Take step（d）In squid sexual gland glycoprotein hydrolysate addition can be completely dissolved squid sexual gland glycoprotein water just
The distilled water of object amount is solved, the sulfuric acid of a concentration of 0.1-2.4mg/L is then added in squid sexual gland glycoprotein hydrolysate aqueous solution
Ferrous iron solution, in terms of ferrous ion, the mass ratio of squid sexual gland glycoprotein and ferrous ion is 20-200:1, at 35-45 DEG C
Stirring complexing 0.5-10 hours, 8000r/min centrifugation 5min, takes supernatant liquor to measure light absorption value, calculates complexing rate, lower sediment
The ferrous complex of squid sexual gland saccharide complex hydrolysate is obtained after dialysis is freeze-dried.
8. a kind of preparation method of the ferrous complex of squid sexual gland saccharide complex hydrolysate according to claim 7,
It is characterized in, the step（e）In supernatant liquor pass through 0.45 μm of disposable filtering with microporous membrane.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923201A (en) * | 2014-04-01 | 2014-07-16 | 宁波大学 | Preparation method of hippocampus active glycoprotein |
| CN105348370A (en) * | 2015-11-24 | 2016-02-24 | 荣成广润水产食品有限公司 | Method for extracting purified glycoprotein from internal organs of squid |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103923201A (en) * | 2014-04-01 | 2014-07-16 | 宁波大学 | Preparation method of hippocampus active glycoprotein |
| CN105348370A (en) * | 2015-11-24 | 2016-02-24 | 荣成广润水产食品有限公司 | Method for extracting purified glycoprotein from internal organs of squid |
Non-Patent Citations (2)
| Title |
|---|
| 宋荪阳 等: "虾夷扇贝性腺糖蛋白的制备及抗氧化活性研究", 《中国食品科学技术学会第七届年会论文摘要集》 * |
| 王倩 等: "响应面法优化鱿鱼缠卵腺糖蛋白提取工艺研究", 《食品工业技术》 * |
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