CN108118045A - The method that protease is extracted from squid viscera - Google Patents
The method that protease is extracted from squid viscera Download PDFInfo
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Abstract
The invention discloses the methods that protease is extracted from squid viscera, concretely comprise the following steps：Pretreatment：NaOH aqueous solutions ultrasonic wave is added in into squid viscera to impregnate, and isopropanol soak degreasing is used after being washed to neutrality, it is spare；Crude enzyme liquid extracts：NaCl solution is added in into pretreated squid viscera, homogeneous crushes, and is then ultrasonically treated, and is centrifuged after taking-up, and supernatant is crude enzyme liquid；Protease purification：Ammonium sulfate is added in into crude enzyme liquid, is stood, centrifugation, precipitation is dissolved with pH for 7 8Tris HCl buffer solutions, and dialysis, concentration, low temperature drying, crushing are to get squid viscera protease.It has the beneficial effect that：The method recovery rate of present invention extraction squid viscera protease is high, easy to operate, extraction rate is fast, will not destroy the ingredient of extract, can remove heavy metal cadmium in protease, extracts high proteinase activity, purity height, wide without fishy smell, use scope.
Description
Technical field
The present invention relates to fish products deep process technology field, more particularly, to the side that protease is extracted from squid viscera
Method.
Background technology
Squid, although traditionally they are referred to as fish, it is not fish in fact, but lives in the mollusk in ocean.
Squid belongs to Mollusca, Cephalopoda, and squid is generally called in squid section.Body colour is pale, and body cone, head is big, has filbert
Spot, front have 10 to touch foot, and Chang Chengqun cruises in ocean about 20 meters deep.Often it is active in shallow sea at the middle and upper levels, vertically
Moving range reaches over one hundred rice.Fine and tender taste, flavor is similar to abalone, but price is very low, is known as " abalones of the poor ".Westerner
Because squid epidermis is dark and variable, and squid is referred to as " devil fish "；Spaniard eats that squid is relatively more, they are processed into squid
Differently flavoured can, sleeve-fish sauce, squid loop etc.；American, which also begins to advocate in recent years, eats squid, they add squid
The form of work into similar abalone is sold；Squid is popular in Japan, it has also become essential aquatic products in Japanese daily life
Squid is sized generally to refrigerated products, dried product, rare delicacies product, salt preserved product, heating bactericide product by product, Japanese.Squid
The by-product generated in processing, such as skin, internal organ, eye and ink sac, all lose as discarded object, not only cause environmental pollution,
The added value of squid processing can not be improved.Squid whole body is all precious, takes the photograph the amino acid of bait in squid viscera containing promotion grass shrimp,
Crude fat（The content of unrighted acid is very high）, protein（It can be used to produce sleeve-fish sauce）；Prepared Chinese ink in ink sac is demonstrate,proved
It is real that there are antibacterial functions, there is function antitumor and that enhancing is immune.Squid viscera be generated in squid process it is main
Discarded object accounts for the 15% of squid weight in wet base, contains the nutriments such as abundant fat, protein.Fat accounts for internal organ weight in wet base
20-30%, protein account for the 20-25% of internal organ weight in wet base, in addition, also containing abundant protease in squid viscera, it can conduct
The high-quality source of protease is extracted, this also accelerates research of the researcher to marine animal endogenous protease so that sea
The research work of foreign animal protease is in the ascendant.
The prior art such as Authorization Notice No. is the Chinese invention patent of CN102392010B, discloses one kind with squid viscera
The protease and its extracting method that are extracted for raw material and application.Above-mentioned protease using it is fresh or frost squid viscera as
Raw material, squid viscera after crushing, are extracted with water or salt solution, by control salt solution concentration in extraction process,
Solid-to-liquid ratio, temperature, extraction time obtain the higher protease rate of recovery；Then sunk by isoelectric point precipitation and addition flocculant
The methods of shallow lake, removes foreign protein, improves the purity of enzyme；Last concentrated, addition stabilizer, preservative etc. become in liquid squid
Dirty protease enzyme preparation；Or solid squid viscera protease preparation is obtained through drying, crushing after addition filler.The present invention realizes
The increment of squid viscera is utilized, improves fish production added value, sleeve-fish product is reduced and produces pollution to environment, and make
Enzyme preparation of the standby obtained squid protease as the various food proteins of hydrolysis adds the kind of commercially available protein enzyme.But it uses
Contain heavy metal cadmium in the protease, strongly limit the use scope of protease, while pigment content is high in the protease, has
Fishy smell is unfavorable for the application of subsequent protease.
The content of the invention
It is an object of the invention to provide a kind of recovery rate is high, easy to operate, extraction rate is fast, will not destroy extract
Ingredient can remove heavy metal cadmium in protease, extract proteinase activity is high, purity is high, without fishy smell, use scope it is wide from
The method that protease is extracted in squid viscera.
The problem of present invention in above-mentioned technology for mentioning, the technical solution taken is：Albumen is extracted from squid viscera
The method of enzyme including pretreatment, crude enzyme liquid extraction, protease purification, concretely comprises the following steps：
Pretreatment：By tissues such as the squid viscera being collected into removal squid ink sac, squid skin, squid eyes, retain liver, pancreas,
The tissues such as intestines, are cut into small pieces, are 1 by solid-liquid ratio:7-9 (g/mL) adds in the NaOH that concentration is 1.2-1.6 ‰ into squid viscera
In aqueous solution, containing 0.6-0.8% activated carbons in NaOH aqueous solutions, surpass in the case where ultrasonic power is 80-100W, temperature is 2-5 DEG C
Sound wave impregnates 3-5h, is washed to neutrality, is finally 1 by solid-liquid ratio:It is the different of 8-12% that 20-24, which adds in concentration into squid viscera,
Propanol solution is 2-5 DEG C of soak degreasing 10-15h in temperature, is cleaned and drained with distilled water, spare, squid viscera protease is carrying
Before taking, because wherein containing many foreign proteins and fat, so pre-treatment is first carried out, to remove these foreign proteins, fat, color
Element, cadmium and fishy smell substance etc., reduce influence of such substance to protease, which is combined using activated carbon and ultrasonic wave and carried out
Decoloration, ultrasonic wave have strong peptizaiton and cavitation effect so that activated carbon can come into full contact with squid viscera, can make work
Property charcoal quick adsorption pigment, cadmium and fishy smell substance, the reaction effect of activated carbon and squid viscera can be significantly improved, improve activated carbon
Utilization rate, simplify following purification steps, while expand the purposes of visceral protein enzyme；
Crude enzyme liquid extracts：By solid-liquid ratio 1:It is 0.2-0.3%'s that 2-4 (g/mL), which adds in concentration into pretreated squid viscera,
NaCl solution, homogeneous crush 1-3min, are then ultrasonically treated 20- in the case where ultrasonic power is 80-100W, temperature is 30-40 DEG C
40min is so that squid viscera protease fully leaches, through rotating speed is 6000-8000r/min, temperature is 2-5 DEG C height after taking-up
15-25min is centrifuged in fast refrigerated centrifuge, supernatant collection is got up as crude enzyme liquid, which is aided in using ultrasonic wave
NaCl extracts visceral protein enzyme, and the thick liquid of protease of high activity can be effectively extracted from internal organ, and recovery rate is high, operation letter
List, extraction rate are fast and will not destroy the ingredient of extract, and the NaCl solution in the concentration range can make in squid viscera
Protease is fully dissolved into extracting solution, " salt is molten " phenomenon occurs, beneficial to the extraction of protease, while NaCl is to the structure of protease
The stabilization of elephant helps to maintain the activity of visceral protein enzyme, and the selection of the ultrasonic power is so that the activity of protease
It can keep optimal；
Protease purification：It is 50-60% that ammonium sulfate to saturation degree is added in into crude enzyme liquid, 1-3h is stood under the conditions of 2-5 DEG C, so
15-25min is centrifuged in the high speed freezing centrifuge that rotating speed is 6000-8000r/min, temperature is 2-5 DEG C afterwards, precipitates and is with pH
The volume ratio of the Tris-HCl buffer solutions dissolving of 7-8, Tris-HCl buffer solutions and former crude enzyme liquid is 1:0.18-0.23, so
Afterwards under the conditions of 2-5 DEG C, dialyse using distilled water as extracellular fluid dialysis under magnetic stirring apparatus 20-30h, during which replace distilled water 3-5
It is secondary, finally by bag filter liquid concentration, low temperature drying, crushing to get squid viscera protease, the parent in protease molecule
Water base group can be combined by ammonia key with water is formed on its surface hydration shell, at the same hydrophilic radical can dissociate make on molecular surface band it is same
Kind of charge, make protease molecule it is mutually exclusive keep apart be scattered in solution, ammonium sulfate can all be ionized into ion in water,
It is combined with the opposite charges particle in protein enzyme solution and has neutralized the electrical of protease, agglomerated protease and precipitation is precipitated, no
The structure of protease is destroyed, improves the purity of protease, which can be used for hydrolyzing various food proteins, and it is delicious to obtain flavor
Protein hydrolysate class flavouring.
Preferably, activated carbon is potassium permanganate modified activated carbon in pre-treatment step, its preparation method is：In 70-90
With deionized water cleaning active charcoal at DEG C, fine powder and pollutant are removed, it is then dry at 100-120 DEG C, it is 1 by solid-liquid ratio:
Activated carbon is added to the KMnO that concentration is 0.02-0.05mol/L by 4-64In solution, KMnO is added4Weight 0.33-0.45%'s
Camphorsulfonic acid stirs 5-8h at 20-30 DEG C, and it is constant to be then washed to filtrate pH value, dry to get activated carbon.Above-mentioned camphor sulphur
The weight ratio of L- camphorsulfonic acids and D- camphorsulfonic acids is 1 in acid:On the one hand 0.03-0.05, the addition of the camphorsulfonic acid can enhance
The polarity and hydrophily of activated carbon surface so that KMnO4Increase with the contact area of activated carbon, improve KMnO4Oxidation modification activity
The rate and uniformity of charcoal, the short time calcination of another aspect camphorsulfonic acid can open the aperture of activated carbon occlusion, improve hole
Footpath ratio further improves the absorption property of activated carbon, can adsorb pigment, cadmium and fishy smell substance in squid viscera so that
Protease purity is high, reduces the content of heavy metal cadmium in visceral protein enzyme；By KMnO4Activated carbon after oxidation modification, table
The acid oxygen-containing functional group in face increases, and with the pigment in squid viscera and cadmium complexing can occur for these functional groups, increases
To the adsorbance of pigment and cadmium；Manganese dioxide is also supported on activated carbon surface simultaneously, enhances its adsorption capacity, shows as good
Good decoloration performance and absorption property can reduce the dosage of activated carbon, shorten decoloration duration, while can remove cadmium and fishy smell object
Matter, and the activated carbon can recycle, and have no adverse effects to squid viscera protease, non-environmental-pollution, etching apparatus, uneconomical
It is applicable in, there is good prospect, from the viewpoint of environmental protection, modified activated carbon is a kind of environmentally friendly material.
Compared with prior art, the advantage of the invention is that：1）Extracting method of the present invention is simple, and extraction rate is fast, is easy to
Industrialized production, security higher will not destroy the ingredient of extract, and the recovery rate of squid viscera protease is high, can remove egg
Heavy metal cadmium in white enzyme, substantially increases the added value of squid viscera, and market development potential is big；2）What the extracting method obtained
Proteinase activity is high, purity is high, without fishy smell, and use scope is wide, available for various food proteins are hydrolyzed, obtains the delicious hydrolysis of flavor
Protide flavouring；3）The activated carbon that present invention decoloration uses has good decoloration performance and absorption property, can reduce work
Property charcoal dosage, shorten decoloration duration, while fishlike smell can be removed, and the activated carbon can recycle, to squid viscera albumen
Enzyme has no adverse effects, non-environmental-pollution, not etching apparatus, economic and practical, has good prospect, from the viewpoint of environmental protection,
Modified activated carbon is a kind of environmentally friendly material.
Specific embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
The method that protease is extracted from squid viscera, including pretreatment, crude enzyme liquid extraction, protease purification, specific steps
For：
1）Pretreatment：By tissues such as the squid viscera being collected into removal squid ink sac, squid skin, squid eyes, retain liver, pancreas
The tissues such as gland, intestines, are cut into small pieces, are 1 by solid-liquid ratio:It is water-soluble that 9 (g/mL) add in the NaOH that concentration is 1.2 ‰ into squid viscera
In liquid, containing 0.8% activated carbon in NaOH aqueous solutions, in the case where ultrasonic power is 80W, temperature is 5 DEG C, ultrasonic wave impregnates 3h, water
Neutrality is washed till, is finally 1 by solid-liquid ratio:24 add in the aqueous isopropanol that concentration is 8% into squid viscera, are 5 DEG C of leachings in temperature
Degreasing 10h is steeped, is cleaned and drained with distilled water, spare, activated carbon is potassium permanganate modified activated carbon in above-mentioned pre-treatment step,
Preparation method is：With deionized water cleaning active charcoal at 70 DEG C, fine powder and pollutant are removed, it is then dry at 120 DEG C, it presses
Solid-liquid ratio is 1:4 are added to activated carbon the KMnO that concentration is 0.05mol/L4In solution, KMnO is added4The camphor tree of weight 0.33%
Brain sulfonic acid stirs 5h at 30 DEG C, and it is constant to be then washed to filtrate pH value, dry to get activated carbon.L- camphor trees in above-mentioned camphorsulfonic acid
The weight ratio of brain sulfonic acid and D- camphorsulfonic acids is 1:0.05；
2）Crude enzyme liquid extracts：By solid-liquid ratio 1:4 (g/mL) add in the NaCl that concentration is 0.2% into pretreated squid viscera
Solution, homogeneous crush 3min, and 20min is then ultrasonically treated in the case where ultrasonic power is 80W, temperature is 40 DEG C so that squid viscera
Protease fully leaches, and through rotating speed is 8000r/min after taking-up, centrifuges 25min in the high speed freezing centrifuge that temperature is 2 DEG C,
Supernatant collection is got up as crude enzyme liquid；
3）Protease purification：It is 60% that ammonium sulfate to saturation degree is added in into crude enzyme liquid, and 3h is stood under the conditions of 2 DEG C, is then being turned
Speed is 8000r/min, 25min is centrifuged in the high speed freezing centrifuge that temperature is 2 DEG C, and precipitation is buffered with the Tris-HCl that pH is 7
Solution dissolves, and the volume ratio of Tris-HCl buffer solutions and former crude enzyme liquid is 1:0.23, then under the conditions of 2 DEG C, with distilled water
It dialyses for extracellular fluid dialysis under magnetic stirring apparatus 30h, during which replaces distilled water 3 times, finally by the liquid concentration, low in bag filter
Temperature is dry, crushes to get squid viscera protease.
Embodiment 2：
The method that protease is extracted from squid viscera, including pretreatment, crude enzyme liquid extraction, protease purification, specific steps
For：
1）Pretreatment：By tissues such as the squid viscera being collected into removal squid ink sac, squid skin, squid eyes, retain liver, pancreas
The tissues such as gland, intestines, are cut into small pieces, are 1 by solid-liquid ratio:It is water-soluble that 7 (g/mL) add in the NaOH that concentration is 1.6 ‰ into squid viscera
In liquid, containing 0.6% activated carbon in NaOH aqueous solutions, in the case where ultrasonic power is 100W, temperature is 2 DEG C, ultrasonic wave impregnates 5h, water
Neutrality is washed till, is finally 1 by solid-liquid ratio:20 add in the aqueous isopropanol that concentration is 8% into squid viscera, are 5 DEG C of leachings in temperature
Degreasing 10h is steeped, is cleaned and drained with distilled water, spare, activated carbon is potassium permanganate modified activated carbon in above-mentioned pre-treatment step,
Preparation method is：With deionized water cleaning active charcoal at 90 DEG C, fine powder and pollutant are removed, it is then dry at 100 DEG C, it presses
Solid-liquid ratio is 1:6 are added to activated carbon the KMnO that concentration is 0.02mol/L4In solution, KMnO is added4The camphor tree of weight 0.45%
Brain sulfonic acid stirs 8h at 20 DEG C, and it is constant to be then washed to filtrate pH value, dry to get activated carbon.L- camphor trees in above-mentioned camphorsulfonic acid
The weight ratio of brain sulfonic acid and D- camphorsulfonic acids is 1:0.03；
2）Crude enzyme liquid extracts：By solid-liquid ratio 1:2 (g/mL) add in the NaCl that concentration is 0.3% into pretreated squid viscera
Solution, homogeneous crush 1min, and 40min is then ultrasonically treated in the case where ultrasonic power is 100W, temperature is 30 DEG C so that in squid
Dirty protease fully leaches, and through rotating speed is 6000-r/min after taking-up, is centrifuged in the high speed freezing centrifuge that temperature is 5 DEG C
15min gets up supernatant collection as crude enzyme liquid；
3）Protease purification：It is 50% that ammonium sulfate to saturation degree is added in into crude enzyme liquid, and 1h is stood under the conditions of 5 DEG C, is then being turned
Speed is 6000r/min, 15min is centrifuged in the high speed freezing centrifuge that temperature is 5 DEG C, and precipitation is buffered with the Tris-HCl that pH is 8
Solution dissolves, and the volume ratio of Tris-HCl buffer solutions and former crude enzyme liquid is 1:0.18, then under the conditions of 5 DEG C, with distilled water
It dialyses for extracellular fluid dialysis under magnetic stirring apparatus 20h, during which replaces distilled water 5 times, finally by the liquid concentration, low in bag filter
Temperature is dry, crushes to get squid viscera protease.
Embodiment 3：
The method that protease is extracted from squid viscera, including pretreatment, crude enzyme liquid extraction, protease purification, specific steps
For：
1）Pretreatment：By tissues such as the squid viscera being collected into removal squid ink sac, squid skin, squid eyes, retain liver, pancreas
The tissues such as gland, intestines, are cut into small pieces, are 1 by solid-liquid ratio:It is water-soluble that 8 (g/mL) add in the NaOH that concentration is 1.4 ‰ into squid viscera
In liquid, containing 0.7% activated carbon in NaOH aqueous solutions, in the case where ultrasonic power is 90W, temperature is 4 DEG C, ultrasonic wave impregnates 4h, water
Neutrality is washed till, is finally 1 by solid-liquid ratio:22 add in the aqueous isopropanol that concentration is 10% into squid viscera, are 4 DEG C in temperature
Soak degreasing 12h, is cleaned with distilled water and drained, spare, and activated carbon is potassium permanganate modified activated carbon in above-mentioned pre-treatment step,
Its preparation method is：With deionized water cleaning active charcoal at 80 DEG C, fine powder and pollutant are removed, it is then dry at 110 DEG C,
It is 1 by solid-liquid ratio:5 are added to activated carbon the KMnO that concentration is 0.04mol/L4In solution, KMnO is added4Weight 0.4%
Camphorsulfonic acid stirs 6h at 25 DEG C, and it is constant to be then washed to filtrate pH value, dry to get activated carbon.L- in above-mentioned camphorsulfonic acid
The weight ratio of camphorsulfonic acid and D- camphorsulfonic acids is 1:0.04；
2）Crude enzyme liquid extracts：By solid-liquid ratio 1:3 (g/mL) add in the NaCl that concentration is 0.25% into pretreated squid viscera
Solution, homogeneous crush 2min, and 30min is then ultrasonically treated in the case where ultrasonic power is 90W, temperature is 35 DEG C so that squid viscera
Protease fully leaches, and through rotating speed is 7000r/min after taking-up, centrifuges 20min in the high speed freezing centrifuge that temperature is 4 DEG C,
Supernatant collection is got up as crude enzyme liquid；
3）Protease purification：It is 55% that ammonium sulfate to saturation degree is added in into crude enzyme liquid, and 2h is stood under the conditions of 4 DEG C, is then being turned
Speed is 7000r/min, centrifuges 20min in the high speed freezing centrifuge that temperature is 4 DEG C, precipitates and is delayed with the Tris-HCl that pH is 7.5
It rushes solution to dissolve, the volume ratio of Tris-HCl buffer solutions and former crude enzyme liquid is 1:0.2, then under the conditions of 4 DEG C, with distilled water
It dialyses for 24 hours under magnetic stirring apparatus for extracellular fluid dialysis, during which replaces distilled water 4 times, finally by the liquid concentration, low in bag filter
Temperature is dry, crushes to get squid viscera protease.
Embodiment 4：
The method that protease is extracted from squid viscera, including pretreatment, crude enzyme liquid extraction, protease purification, specific steps
For：
1）Pretreatment：By tissues such as the squid viscera being collected into removal squid ink sac, squid skin, squid eyes, retain liver, pancreas
The tissues such as gland, intestines, are cut into small pieces, are 1 by solid-liquid ratio:It is water-soluble that 8 (g/mL) add in the NaOH that concentration is 1.4 ‰ into squid viscera
In liquid, containing 0.7% activated carbon in NaOH aqueous solutions, in the case where ultrasonic power is 90W, temperature is 4 DEG C, ultrasonic wave impregnates 4h, water
Neutrality is washed till, is finally 1 by solid-liquid ratio:22 add in the aqueous isopropanol that concentration is 10% into squid viscera, are 4 DEG C in temperature
Soak degreasing 12h, is cleaned with distilled water and drained, spare, and activated carbon is potassium permanganate modified activated carbon in above-mentioned pre-treatment step,
Its preparation method is：With deionized water cleaning active charcoal at 80 DEG C, fine powder and pollutant are removed, it is then dry at 110 DEG C,
It is 1 by solid-liquid ratio:5 are added to activated carbon the KMnO that concentration is 0.04mol/L4In solution, KMnO is added4Weight 0.4%
Camphorsulfonic acid stirs 6h at 25 DEG C, and it is constant to be then washed to filtrate pH value, dry to get activated carbon.L- in above-mentioned camphorsulfonic acid
The weight ratio of camphorsulfonic acid and D- camphorsulfonic acids is 1:0.04；
2）Crude enzyme liquid extracts：By solid-liquid ratio 1:3 (g/mL) add in the NaCl that concentration is 0.25% into pretreated squid viscera
Solution, homogeneous crush 2min, and 30min is then ultrasonically treated in the case where ultrasonic power is 90W, temperature is 35 DEG C so that squid viscera
Protease fully leaches, and through rotating speed is 7000r/min after taking-up, centrifuges 20min in the high speed freezing centrifuge that temperature is 4 DEG C,
Supernatant collection is got up as crude enzyme liquid；
3）Protease purification：It is 40% that ammonium sulfate to saturation degree is added in into crude enzyme liquid, adds ammonium sulfate weight 0.72-
8.82 ‰ acetyl spiramycin stands 2h under the conditions of 4 DEG C, then in the high speed that rotating speed is 7000r/min, temperature is 4 DEG C
Centrifuge 20min in refrigerated centrifuge, the precipitation Tris-HCl buffer solutions that pH is 7.5 dissolve, Tris-HCl buffer solutions and
The volume ratio of former crude enzyme liquid is 1:0.2, then under the conditions of 4 DEG C, dialyse using distilled water as extracellular fluid dialysis under magnetic stirring apparatus
For 24 hours, during which replace distilled water 4 times, finally by bag filter liquid concentration, low temperature drying, crushing is to get squid viscera albumen
Enzyme, acetyl spiramycin can be catalyzed ammonium sulfate and quickly be ionized into ion in water, and then can be quickly so that protease precipitate is quick
It is precipitated, reduces flocculated protein and to the inhibition of ammonium sulfate ion moving line, improve the rate and purity of purifying,
The hydration shell of protein surface can be destroyed simultaneously, protein is made to be bound to each other to form Precipitation, so that protease is molten
Solubility in liquid is reduced and is precipitated, and can reduce the dosage of ammonium sulfate, the final purity for improving protease.
Routine operation in the operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail in embodiment described above, it should be understood that the above is only
For specific embodiments of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (8)
1. extracting the method for protease from squid viscera, including pretreatment, crude enzyme liquid extraction, protease purification, feature exists
In：The pre-treatment step is：By tissues such as the squid viscera being collected into removal squid ink sac, squid skin, squid eyes, retain
The tissues such as liver, pancreas, intestines, are cut into small pieces, are added in into squid viscera in NaOH aqueous solutions, and ultrasonic wave impregnates, in being washed to
Property, aqueous isopropanol soak degreasing is eventually adding, is cleaned and drained with distilled water, it is spare.
2. the method according to claim 1 that protease is extracted from squid viscera, it is characterised in that：The pretreatment
The concentration of NaOH aqueous solutions is 1.2-1.6 ‰ in step, and the solid-liquid ratio of squid viscera and NaOH aqueous solutions is 1:7-9 (g/mL),
The ultrasonic power of immersion is 80-100W, temperature is 2-5 DEG C, time 3-5h.
3. the method according to claim 1 that protease is extracted from squid viscera, it is characterised in that：The pretreatment
Containing 0.4-0.6% activated carbons in NaOH aqueous solutions in step, the activated carbon is potassium permanganate modified activated carbon.
4. the method according to claim 3 that protease is extracted from squid viscera, it is characterised in that：The activated carbon
Preparation method be：It is dry with deionized water cleaning active charcoal at 70-90 DEG C, it is 1 by solid-liquid ratio:4-6 adds in activated carbon
To the KMnO that concentration is 0.02-0.05mol/L4In solution, camphorsulfonic acid is added, 5-8h is stirred at 20-30 DEG C, washes, do
It is dry to get activated carbon.
5. the method according to claim 4 that protease is extracted from squid viscera, it is characterised in that：The camphor sulphur
The additive amount of acid is KMnO4The 0.33-0.45% of weight, the weight of L- camphorsulfonic acids and D- camphorsulfonic acids in the camphorsulfonic acid
Than for 1:0.03-0.05.
6. the method according to claim 1 that protease is extracted from squid viscera, it is characterised in that：The pretreatment
The concentration of aqueous isopropanol is 8-12% in step, and the solid-liquid ratio of squid skin and aqueous isopropanol is 1:20-24, soak degreasing temperature
It spends for 2-5 DEG C, time 10-15h.
7. the method according to claim 1 that protease is extracted from squid viscera, it is characterised in that：The pretreatment
Crude enzyme liquid extraction step is in step：By solid-liquid ratio 1:2-4 (g/mL) adds in concentration into pretreated squid viscera
The NaCl solution of 0.2-0.3%, homogeneous crush 1-3min, then surpass in the case where ultrasonic power is 80-100W, temperature is 30-40 DEG C
Sonication 20-40min through rotating speed is 6000-8000r/min after taking-up, temperature is so that squid viscera protease fully leaches
15-25min is centrifuged in 2-5 DEG C of high speed freezing centrifuge, supernatant collection is got up as crude enzyme liquid.
8. the method according to claim 1 that protease is extracted from squid viscera, it is characterised in that：The pretreatment
Protease purification step is in step：It is 50-60% that ammonium sulfate to saturation degree is added in into crude enzyme liquid, is stood under the conditions of 2-5 DEG C
Then 1-3h centrifuges 15-25min in the high speed freezing centrifuge that rotating speed is 6000-8000r/min, temperature is 2-5 DEG C, sink
It forms sediment and is dissolved with the pH Tris-HCl buffer solutions for being 7-8, the volume ratio of Tris-HCl buffer solutions and former crude enzyme liquid is 1:0.18-
0.23, it then under the conditions of 2-5 DEG C, dialyses using distilled water as extracellular fluid dialysis under magnetic stirring apparatus 20-30h, during which replaces and steam
Distilled water 3-5 times, finally by bag filter liquid concentration, low temperature drying, crushing is to get squid viscera protease.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110564800A (en) * | 2019-09-25 | 2019-12-13 | 浙江海洋大学 | Preparation method of squid skin protein Maillard peptide liquid with antioxidant activity |
| CN112827210A (en) * | 2020-12-24 | 2021-05-25 | 四川德博尔制药有限公司 | Preparation method of clear pancreas extracting solution |
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| CN102392010A (en) * | 2011-12-02 | 2012-03-28 | 华南农业大学 | Protease extracted by using squid viscera as raw materials and extraction method and application thereof |
| CN106906199A (en) * | 2016-12-30 | 2017-06-30 | 浙江海洋大学 | A kind of fast purifying squid viscera albumen enzyme method and application |
| CN108060155A (en) * | 2017-12-29 | 2018-05-22 | 舟山富晟食品科技有限公司 | The protease extracted from squid viscera |
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| CN102392010A (en) * | 2011-12-02 | 2012-03-28 | 华南农业大学 | Protease extracted by using squid viscera as raw materials and extraction method and application thereof |
| CN106906199A (en) * | 2016-12-30 | 2017-06-30 | 浙江海洋大学 | A kind of fast purifying squid viscera albumen enzyme method and application |
| CN108060155A (en) * | 2017-12-29 | 2018-05-22 | 舟山富晟食品科技有限公司 | The protease extracted from squid viscera |
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| CN110564800A (en) * | 2019-09-25 | 2019-12-13 | 浙江海洋大学 | Preparation method of squid skin protein Maillard peptide liquid with antioxidant activity |
| CN110564800B (en) * | 2019-09-25 | 2023-06-06 | 浙江海洋大学 | Preparation method of squid skin protein Maillard peptide liquid with antioxidant activity |
| CN112827210A (en) * | 2020-12-24 | 2021-05-25 | 四川德博尔制药有限公司 | Preparation method of clear pancreas extracting solution |
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