CN102392010B - Protease extracted by using squid viscera as raw materials and extraction method and application thereof - Google Patents
Protease extracted by using squid viscera as raw materials and extraction method and application thereof Download PDFInfo
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- CN102392010B CN102392010B CN 201110396162 CN201110396162A CN102392010B CN 102392010 B CN102392010 B CN 102392010B CN 201110396162 CN201110396162 CN 201110396162 CN 201110396162 A CN201110396162 A CN 201110396162A CN 102392010 B CN102392010 B CN 102392010B
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Abstract
The invention discloses protease extracted by using squid viscera as raw materials and an extraction method and an application thereof. According to the invention, fresh or freezed squid viscera are used as raw materials; the squid viscera are crushed, and extracted by water or a table salt solution; the concentration of the table salt solution, the solid-liquid ratio, the temperature, and the extraction time are controlled during the extraction so as to obtain a high protease recovery rate; then protein is removed by methods of isoelectric point precipitation and flocculant addition precipitation so as to increase the protease purity; finally a liquid squid viscera protease enzyme preparation is obtained by concentration, stabilizer and preservative addition, and the like; or a solid squid viscera protease preparation is obtained by filler addition, drying and crushing. The invention realizes the application of squid viscera with increased value, increases the added value of fishery production, and reduces environment pollution caused by squid product production; the prepared squid protease can be used as an enzyme preparation for hydrolyzing various food protein; and the variety of commercial protease is increased.
Description
Technical field
The invention belongs to the aquatic products field, relate to the waste of aquatic comprehensive utilization, particularly a kind of is that raw material extracts proteolytic enzyme and process for extracting and the application that obtains with the squid viscera.
Background technology
In general, the processing byproduct of squid compositions such as remaining internal organ, skin, fin and prepared Chinese ink after processing obtains edible portion accounts for 30% of overall proportion.The composition of processing byproduct consists of: moisture 63%, crude fat 22.5%, crude protein 13.5%, other 1%.To these wastes, domestic some be processed into squid and dissolve slurry, some is by as the rubbish emergency burial.This is still to the huge waste of fishing resources, but also exists the hidden danger of environmental pollution.Along with the development of modern marine processing industry and aquaculture, the comprehensive utilization of squid waste more and more comes into one's own.
The utilization of relevant squid viscera, more existing both at home and abroad correlative studys, as Yuan Yahui etc. utilize squid viscera produce seafood delights plain (utilize squid viscera to produce the plain research of seafood delights. " fishery modernization " the 01st phase in 2002); Xuwei etc. to change with the biogenic amine in the sleeve-fish-processing waste fermentation squid soy sauce process done some researchs (sleeve-fish-processing waste less salt petis speed is made technology and biochemical characteristic research. Chinese Marine University's doctorate paper); Y; Z; LEE etal (1982) has reported that squid muscle and internal organ (mainly being liver and pancreas) contain abundant active higher cathepsin C, and this enzyme has two peptidyl transferases and two peptidohydrolase vigor, utilizes its hydrolysis squid albumen can improve deliciousness flavor (the Lee YZ of hydrolyzate; Simpson BK, HaardNF.supplementation Of squid fermentation with proteolytic enzymes.Journal of the 06th phase of FoodBiochemistry1982); Scout's cutting edge of a knife or a sword (2001) proposes squid viscera can adopt self enzymatic hydrolysis and fermentation method, can be used as the raw material of making petis (processing of strengthening the squid resource is studied with comprehensive utilization technique. the 04th phase of " oceanographic research " calendar year 2001).Up to now, there is not the technology of utilizing squid viscera to extract proteolytic enzyme to occur both at home and abroad as yet.
Summary of the invention
The shortcoming that primary and foremost purpose of the present invention is to overcome prior art is with not enough, and providing a kind of is the method that raw material extracts proteolytic enzyme with the squid viscera.Realized increment utilization, can improve the fish production added value, but also can avoid environmental pollution squid viscera.
Another object of the present invention is to provide the squid that obtains through aforesaid method proteolytic enzyme, increased the kind of commercially available protein enzyme.
A purpose more of the present invention is to provide the application of described squid proteolytic enzyme.
The object of the invention is realized through following technical proposals: a kind of is the method that raw material extracts proteolytic enzyme with the squid viscera; Be to be raw material with fresh or freezing squid viscera; Squid viscera is after fragmentation; Water or salt solution extract, and salt solution concentration, solid-to-liquid ratio, temperature, extraction time obtain the higher proteolytic enzyme recovery in the leaching process through controlling; Remove foreign protein through isoelectric point precipitation and methods such as adding the flocculation agent deposition then, improve the purity of enzyme; Become liquid squid viscera proteolytic enzyme zymin after concentrate, add stablizer, sanitas etc.; Perhaps add filler after dry, pulverizing gets solid squid viscera protease preparation.Specifically comprise following steps:
(1) broken squid viscera;
(2) in breaked squid viscera, add entry or salt solution and extract, remove supernatant fat, obtain extracting solution;
(3) extracting solution is removed deposition, obtain crude enzyme liquid;
(4) the pH value with crude enzyme liquid is adjusted to 4.0～5.0, leaves standstill and remove deposition, obtains supernatant;
(5) in the supernatant that step (4) obtains, add flocculation agent deposition foreign protein; Remove deposition, obtain supernatant;
(6) supernatant in the step (5) adds stablizer and sanitas after concentrating, and obtains liquid squid viscera proteolytic enzyme zymin; Perhaps add filler after dry, pulverize, the squid viscera protease preparation of solid-like.
Squid viscera described in the step (1) is fresh or cold storage, can not use drying to cross;
Fragmentation described in the step (1) is preferably the using-system stamp mill and smashs to pieces to the mud shape;
The concentration of the salt solution described in the step (2) is below 1.0% (mass percentage concentration), is preferably 0.3～0.5% (mass percentage concentration);
Squid viscera described in the step (2) and described salt solution are preferably by solid-to-liquid ratio (g/ml) 1: 0.5～10 proportionings, preferably by 1: 1.5～1: 2 proportioning of solid-to-liquid ratio;
The condition optimization of the extraction described in the step (2) is to extract 15～120min in 4～40 ℃; More preferably extract 60min in 20 ℃;
The precipitation mode of removing described in step (3), (4) and (5) is centrifugal or filtration;
Described centrifugal condition optimization is more than the 3000r/min, more than the centrifugation time 10min;
Described filtration can be adopted industry to go up various filters commonly used and filter;
PH value described in the step (4) is preferably 4.5, and transferring the reagent of pH is various edible acids;
Flocculation agent described in the step (5) is preferably the various common flocculation agents of ferric sulfate, Tai-Ace S 150, iron trichloride, alum or the like, and the concentration of flocculation agent is 1% (mass volume ratio g/ml), and the ratio of interpolation is crude enzyme liquid and flocculation agent 3: 1～10: 1 (V/V); The preferred ferric sulfate that adopts, adding proportion is 4: 1 (V/V) or Tai-Ace S 150, adding proportion is 5: 1 (V/V);
Flocculation agent described in the step (5) leaves standstill 30～60min after adding, and the foreign protein flocculation sediment is got off, and centrifugal or mistake elimination deposition is isolated supernatant, is the protein enzyme solution of extraction;
Spissated method described in the step (6) can adopt methods such as vacuum decompression concentrates, thin film concentration, freeze concentration, ultrafiltration and concentration;
Stablizer in the step (6), sanitas, filler etc. all adopt zymin industry material commonly used;
Described stablizer can adopt trehalose, XG 550, polyalcohols material; Described polyvalent alcohol is preferably N.F,USP MANNITOL;
Described sanitas adopts phenylformic acid and its esters, Sorbic Acid and its esters, parabens etc.;
Described filler can adopt materials such as lime carbonate, calcium sulfate, sal epsom, Mierocrystalline cellulose, starch.
Exsiccant method described in the step (6) can adopt various drying meanss such as spraying drying, forced air drying, fluidised bed drying, roller drying, vacuum-drying.
A kind of squid viscera protease preparation obtains through method for preparing.
The squid viscera proteolytic enzyme that the present invention extracts can be used for the various food proteins of hydrolysis, like the flesh of fish, crab meat, chicken, beef, pork, Sunlover 10 or the like.For example; Can produce a large amount of crab pin the like waste in the crab meat products production, crab meat residual in the crab pin can't utilize at present, as in advance the crab pin being pulverized; Adopt residual crab meat in this squid viscera protease hydrolysis crab pin then, can obtain the X 1000 class seasonings of the delicious crab meat local flavor of local flavor.In addition, residual a small amount of meat above hydrolysis ox bone, the chicken bone etc. also can obtain the X 1000 class seasonings of corresponding beef-flavouring, chicken flavor.
The present invention has following advantage and effect with respect to prior art:
(1) the present invention is that raw material extracts proteolytic enzyme with the squid viscera, has realized the increment utilization to squid viscera, can improve the fish production added value.
(2) the present invention can reduce the pollution of squid production of articles to environment.
(3) the squid viscera proteolytic enzyme for preparing of the present invention reaches 9400U/g than enzyme work and (is defined under 37 ℃, the condition of pH 7.0; The enzyme amount that the caseinhydrolysate PM produces 1.0 μ g tyrosine is a unit of activity); Extraction yield is 80%; It can be as the zymin of the various food proteins of hydrolysis, thereby has increased the kind of commercially available protein enzyme.
(4) the squid viscera proteolytic enzyme that extracts of the present invention is better at pH3～7 scope internal stabilities, with this proteolytic enzyme behind 30 ℃ of pH3 condition held 5h, proteolytic enzyme become 7500U/g than enzyme work by 9400U/g, visible comparatively stable.
(5) the squid viscera proteolytic enzyme of the present invention's extraction has certain thermotolerance, and is more stable at proteinase activity below 60 ℃.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail, but embodiment of the present invention is not limited thereto.
Embodiment 1
Present embodiment utilizes squid viscera to extract proteolytic enzyme, and its concrete steps are following:
(1) gets the fresh squid viscera of 100g, blend to the mud shape with refiner.
(2) add 150mL 0.5% (mass percentage concentration) NaCl solution and extract at normal temperatures, extraction time is 60min, obtains extracting solution.
(3) the centrifugal 10min of 3000r/min removes the deposition of extracting solution and the grease on surface, gets supernatant.
(4) use alimentary acetic acid to regulate supernatant pH value and be about 4.5, centrifugal, condition is the same, removes deposition, takes supernatant.
(5) step (4) being obtained supernatant and 1% (mass percentage concentration) ferrum sulfuricum oxydatum solutum is that 4: 1 mixed staticly settles 30min after evenly by volume, and the centrifugal 10min of 3000r/min goes deposition, and supernatant is protein enzyme solution.
(6) in above-mentioned protein enzyme solution, add the 1.5g trehalose; Adopt vacuum concentration; After being concentrated into volume and being reduced to about 1/3; Add 10g starch, vacuum-drying again, pulverize then, promptly get solid squid viscera protease preparation (remove the trehalose of salt and interpolation, the solid squid viscera proteolytic enzyme that the quality of starch obtains is 6.2g).
" enzyme and enzyme engineering and application thereof " (Beijing: Chemical Industry Press that the mensuration employing Sun Jun society of enzyme activity etc. writes; 2006) determined by ultraviolet spectrophotometry in the book (under 37 ℃, the condition of pH 7.0, the enzyme amount that the caseinhydrolysate PM produces 1.0 μ g tyrosine is a unit of activity).It is converted, and the ratio enzyme when not containing composition such as stablizer, filler is lived and is 9400U/g.
Embodiment 2
(1) gets the fresh squid viscera of 100g, blend to the mud shape with refiner.
(2) add 200mL 0.3% (mass percentage concentration) NaCl solution and extract at normal temperatures, extraction time is 60min, obtains extracting solution.
(3) the centrifugal 15min of 3000r/min removes the deposition of extracting solution and the grease on surface, gets supernatant.
The pH value of the supernatant that (4) step (3) is obtained with alimentary acetic acid is adjusted to about 4.5, and centrifugal, the same step of condition (3) is removed deposition, takes supernatant.
(5) step (4) being obtained supernatant and 1% (mass percentage concentration) alum liquor is that 5: 1 mixed staticly settles 60min after evenly by volume, centrifugal, the same step of condition (3), and the gained supernatant is protein enzyme solution.
(6) in above-mentioned protein enzyme solution, add 2g N.F,USP MANNITOL; Adopt vacuum concentration; After being concentrated into volume and being reduced to about 1/3; Add the 10g cellulose powder, vacuum-drying again, pulverize then, promptly get solid squid viscera protease preparation (remove the N.F,USP MANNITOL of salt and interpolation, the solid squid viscera proteolytic enzyme that the quality of cellulose powder obtains is 6.0g).
" enzyme and enzyme engineering and application thereof " (Beijing: Chemical Industry Press that the mensuration employing Sun Jun society of enzyme activity etc. writes; 2006) determined by ultraviolet spectrophotometry in the book (be defined under 37 ℃, the condition of pH 7.0, the enzyme amount that the caseinhydrolysate PM produces 1.0 μ g tyrosine is a unit of activity).It is converted, and the ratio enzyme when not containing composition such as stablizer, filler is lived and is 9200U/g.
Comparative Examples 1
(1) gets the fresh squid viscera of 100g, blend to the mud shape with refiner.
(2) add 200mL 2.0% (mass percentage concentration) NaCl solution and extract at normal temperatures, extraction time is 60min, obtains extracting solution.
(3) the centrifugal 10min of 3000r/min removes the deposition of extracting solution and the grease on surface, gets supernatant.
(4) use alimentary acetic acid to regulate supernatant pH value and be about 4.5, centrifugal, condition is the same, removes deposition, takes supernatant.
(5) step (4) being obtained supernatant and 1% (mass percentage concentration) ferrum sulfuricum oxydatum solutum is that 4: 1 mixed staticly settles 30min after evenly by volume, and the centrifugal 10min of 3000r/min goes deposition, and supernatant is protein enzyme solution.
(6) in above-mentioned protein enzyme solution, add the 1.5g trehalose; Adopt vacuum concentration, after being concentrated into volume and being reduced to about 1/3, add 10g starch; Vacuum-drying again, pulverizing then promptly get solid squid viscera protease preparation 8.5g (removing the trehalose of salt and interpolation, the quality of starch).
" enzyme and enzyme engineering and application thereof " (Beijing: Chemical Industry Press that the mensuration employing Sun Jun society of enzyme activity etc. writes; 2006) determined by ultraviolet spectrophotometry in the book (be defined under 37 ℃, the condition of pH 7.0, the enzyme amount that the caseinhydrolysate PM produces 1.0 μ g tyrosine is a unit of activity).It is converted, and the ratio enzyme when not containing composition such as stablizer, filler is lived and is 2200U/g.
It is thus clear that sodium chloride concentration is too high, the vigor of the squid viscera proteolytic enzyme that extracts is far below the ratio vigor of gained proteolytic enzyme in embodiment 1 and 2.
Comparative Examples 2
(1) gets the fresh squid viscera of 100g, blend to the mud shape with refiner.
(2) add 150mL 0.5% (mass percentage concentration) NaCl solution and extract at normal temperatures, extraction time is 60min, obtains extracting solution.
(3) the centrifugal 10min of 3000r/min removes the deposition of extracting solution and the grease on surface, gets supernatant.
(4) use alimentary acetic acid to regulate supernatant pH value and be about 4.5, centrifugal, condition is the same, removes deposition, takes supernatant.
(5) step (4) obtains directly adding in the supernatant 1.5g trehalose; Adopt vacuum concentration, after being concentrated into volume and being reduced to about 1/3, add 10g starch; Vacuum-drying again, pulverizing then promptly get solid squid viscera protease preparation 9.1g (removing the trehalose of salt and interpolation, the quality of starch).
" enzyme and enzyme engineering and application thereof " (Beijing: Chemical Industry Press that the mensuration employing Sun Jun society of enzyme activity etc. writes; 2006) determined by ultraviolet spectrophotometry in the book (be defined under 37 ℃, the condition of pH 7.0, the enzyme amount that the caseinhydrolysate PM produces 1.0 μ g tyrosine is a unit of activity).It is converted, and the ratio enzyme when not containing composition such as stablizer, filler is lived and is 6500U/g.
It is thus clear that without flocculation agent, the vigor of the squid viscera proteolytic enzyme that extracts is starkly lower than the ratio vigor of gained proteolytic enzyme in embodiment 1 and 2.
The foregoing description is a preferred implementation of the present invention; But embodiment of the present invention is not restricted to the described embodiments; Other any do not deviate from change, the modification done under spirit of the present invention and the principle, substitutes, combination, simplify; All should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (8)
1. one kind is the method that raw material extracts proteolytic enzyme with the squid viscera, it is characterized in that comprising following steps:
(1) broken squid viscera;
(2) in breaked squid viscera, add entry or salt solution and extract, remove supernatant fat, obtain extracting solution;
(3) extracting solution is removed deposition, obtain crude enzyme liquid;
(4) the pH value with crude enzyme liquid is adjusted to 4.5, leaves standstill and remove deposition, obtains supernatant;
(5) in the supernatant that step (4) obtains, add flocculation agent deposition foreign protein; Remove deposition, obtain supernatant;
(6) supernatant in the step (5) adds stablizer and sanitas after concentrating, and obtains liquid squid viscera proteolytic enzyme zymin; Perhaps add filler after dry, pulverize, the squid viscera protease preparation of solid-like;
The concentration of the salt solution described in the step (2) is that mass percent is below 1.0%;
Squid viscera described in the step (2) and described salt solution are pressed mass volume ratio 1:0.5～10 proportionings;
The condition of the extraction described in the step (2) is to extract 15～120 min in 4～40 ℃;
Flocculation agent described in the step (5) is at least a in ferric sulfate, Tai-Ace S 150, iron trichloride or the alum;
The concentration of described flocculation agent is mass volume ratio 1%, and the amount of interpolation is crude enzyme liquid and flocculation agent 3:1～10:1 calculating by volume;
Stablizer described in the step (6) is trehalose, XG 550 or polyalcohols material;
Sanitas described in the step (6) is phenylformic acid and its esters, Sorbic Acid and its esters or parabens;
Filler described in the step (6) is lime carbonate, calcium sulfate, sal epsom, Mierocrystalline cellulose or starch.
2. according to claim 1 is the method that raw material extracts proteolytic enzyme with the squid viscera, and it is characterized in that: the squid viscera described in the step (l) is fresh or cold storage;
The using-system stamp mill that is broken for described in the step (1) is smashed to pieces to the mud shape.
3. according to claim 1 is the method that raw material extracts proteolytic enzyme with the squid viscera, and it is characterized in that: the concentration of the salt solution described in the step (2) is mass percent 0.3～0.5%;
Squid viscera described in the step (2) and described salt solution are pressed mass volume ratio 1:1.5～1:2 proportioning;
The condition of the extraction described in the step (2) is to extract 60 min in 20 ℃.
4. according to claim 1 is the method that raw material extracts proteolytic enzyme with the squid viscera, it is characterized in that: the precipitation mode of removing described in step (3), (4) and (5) is centrifugal or filtration;
Described flocculation agent leaves standstill 30～60min after adding, and the foreign protein flocculation sediment is got off.
5. according to claim 1 is the method that raw material extracts proteolytic enzyme with the squid viscera, it is characterized in that: the spissated method described in the step (6) is that vacuum decompression concentrates, thin film concentration, freeze concentration or ultrafiltration and concentration;
Exsiccant method described in the step (6) is spraying drying, forced air drying, fluidised bed drying, roller drying or vacuum-drying.
6. squid viscera protease preparation prepares through each described method that with the squid viscera is raw material extracts proteolytic enzyme of claim 1～5.
7. the application of the described squid viscera protease preparation of claim 6 in the hydrolysis food protein.
8. the application of squid viscera protease preparation according to claim 7 in the hydrolysis food protein is characterized in that: described food protein is the flesh of fish, crab meat, chicken, beef, pork or Sunlover 10.
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| CN102864134A (en) * | 2012-06-28 | 2013-01-09 | 江苏天福莱集团有限公司 | Extraction technology for squid viscera enzyme |
| CN103549121B (en) * | 2013-11-13 | 2016-01-20 | 朱勤 | The method of protein peptide is prepared with squid viscera enzyme enzymolysis sea low value little fish |
| CN105039292A (en) * | 2015-08-30 | 2015-11-11 | 常州思宇环保材料科技有限公司 | Preparation and application of compound protease for promoting meat curing |
| 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 |
| CN107828766A (en) * | 2017-09-30 | 2018-03-23 | 金华市川璞农业科技有限公司 | The preparation method of catfish fish guts protease |
| 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 |
| CN108118045A (en) * | 2018-01-16 | 2018-06-05 | 舟山昌国海洋科技有限公司 | The method that protease is extracted from squid viscera |
| CN108740839A (en) * | 2018-06-09 | 2018-11-06 | 浙江亿丰海洋生物制品有限公司 | A kind of method of squid leftover bits and pieces extraction nutrients |
| CN110477261A (en) * | 2019-08-05 | 2019-11-22 | 武汉市农业科学院 | It is a kind of for the compound degreasing liquid of fish guts protein extraction and its application |
| 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 |
| CN112806543A (en) * | 2020-11-23 | 2021-05-18 | 浙江工商大学 | Flavored cuttlefish paste and preparation method thereof |
| CN112375161B (en) * | 2020-12-13 | 2022-06-28 | 浙江省农业科学院 | Method for preparing beta-chitin by utilizing squid cartilage |
| 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 |
| CN114532463B (en) * | 2022-03-08 | 2023-12-22 | 渤海水产股份有限公司 | Seaweed compound, preparation method and application thereof, and fish-farming feed |
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