CN102787110B - Technology of extracting squid visceral enzyme - Google Patents
Technology of extracting squid visceral enzyme Download PDFInfo
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- CN102787110B CN102787110B CN 201210217954 CN201210217954A CN102787110B CN 102787110 B CN102787110 B CN 102787110B CN 201210217954 CN201210217954 CN 201210217954 CN 201210217954 A CN201210217954 A CN 201210217954A CN 102787110 B CN102787110 B CN 102787110B
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Abstract
The invention provides a technology of extracting squid visceral enzyme. The technology comprises using fresh squid viscera as raw materials, draining water, adding acetone into the raw materials in a quality-volume ratio of 1 : 1-3, and processing into slurry by using a tissue homogenizer; filtering to get a filtrate which is then processed in a refrigerated centrifuge, getting precipitate and drying under natural conditions to obtain acetone dried powder; dissolving the acetone dried powder by using a 20 mM Tris-HCl solution with a pH value of 7.5 in a weight-volume ratio of 1 : 8-12 to a uniform state, processing by using the refrigerated centrifuge, re-dissolving the precipitate by using a 20 mM Tris-HCl solution with a pH value of 7.5, re-centrifuging, combining supernatant, and filtering to obtain the alkaline enzyme solution of the squid viscera. The technology uses processing waste of the squid as the raw materials, extracts visceral enzyme therefrom, prevents waste of proteinresource therein and reduces environment pollution. The invention is advantageous by simple technology, low production cost, high efficiency and energy saving, short production period and high extraction rate, and is suitable for industrial production. The prepared enzyme powder is high in activity, strong in stability, and stable in product quality, and has good economic benefits and wide application prospect.
Description
Technical field
The present invention relates to a kind of method of from the fish processing byproduct, extracting enzyme, relate in particular to a kind of extraction process of squid viscera enzyme.
Background technology
Squid is the invertebrates that lives in the ocean, annual global amount is very big, and squid has head, foot, internal organ and epidermis the like waste about 30%, wherein internal organ account for 15.92%, usually go out of use and the emergency burial processing, so both fully do not developed the squid added value, and caused the wherein significant wastage of protein resource, but also exist the hidden danger of environmental pollution.Along with the continuous development of squid processing industry, internal organ account for major part in the processing fent, contain abundant enzyme in the squid viscera, but also it are not carried out the research of extracting method so far.At present the utilization of squid viscera mainly being concentrated on utilizes proteolytic enzyme to extract fish oil from squid viscera and with its production " the molten slurry of squid ", this method utilizes the autolysis of endogenous proteinase that squid viscera is liquefied, to obtain protein hydrolyte, but have tangible bitter taste and stench flavor by the hydrolyzed solution that autolysis obtains, product special flavour is poor.Therefore, handle the squid waste effectively and carry out high-valued comprehensive utilization, have bigger economy and the value of environmental protection.
Summary of the invention
Technical problem to be solved by this invention is at the deficiencies in the prior art, provides that a kind of extracting method is reasonable in design, the extraction process of the squid viscera enzyme that the squid waste can be fully utilized.
Technical problem to be solved by this invention is to realize by following technical scheme.The present invention is a kind of extraction process of squid viscera enzyme, is characterized in: its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:1～3, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 5～15min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 5～15min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 5～15min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of alkaline enzyme: with acetone dry powder by weight volume ratio 1:8～12 usefulness pH be that 7.5 20mM Tris-HCl is dissolved into uniform state, through 0 ℃ of refrigerated centrifuge, behind the centrifugal 10～20min of 3000～4000r/min, obtain supernatant liquor, precipitation is 7.5 20mM Tris-HCl processing back recentrifuge again with pH, discards precipitation, and supernatant liquor merges, filter, obtain squid viscera alkalescence enzyme liquid.
Technical problem to be solved by this invention can also further realize by following technical scheme.The present invention also provides the extraction process of another kind of squid viscera enzyme, is characterized in: its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:1～3, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 5～15min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 5～15min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 5～15min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of acidicenzym: with acetone dry powder by weight volume ratio 1:8～12 usefulness 0.065M HCl be dissolved into uniform state, through 0 ℃ of refrigerated centrifuge, 3000～4000r/min, behind centrifugal 5～20min, get supernatant liquor, precipitation is handled the back recentrifuge with 0.065M HCl damping fluid again, discard precipitation, supernatant liquor merges, and filters, and namely gets squid viscera acidicenzym liquid.
In the extraction process of squid viscera enzyme of the present invention: alkalescence or the preservation under-20 ℃～-40 ℃ temperature condition of acidicenzym liquid of gained can be deposited in as enzyme liquid.Perhaps through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.Temperature condition the best of preservation is-23.5 ℃.
Compared with prior art, the inventive method is raw material with the sleeve-fish-processing waste, therefrom extracts visceral enzym, effectively utilizes the by product in the squid course of processing---internal organ, turns waste into wealth, and has avoided the wherein waste of protein resource, reduces the pollution to environment.This technology is simple, and production cost is low, and is energy-efficient, and gained enzyme powder of the present invention is active high, stability is strong, constant product quality, and technology is simple, and is with short production cycle, the extraction yield height, energy-efficient, be fit to suitability for industrialized production, good economic benefits and wide application prospect are arranged.
Embodiment
Below further describe concrete technical scheme of the present invention, so that those skilled in the art understands the present invention further, and do not constitute the restriction to its right.
Embodiment 1, a kind of extraction process of squid viscera enzyme, and its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:1, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 5min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 5min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 5min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of alkaline enzyme: with acetone dry powder by weight volume ratio 1:8 pH be that 7.5 20mM Tris-HCl is dissolved into uniform state, through 0 ℃ of refrigerated centrifuge, behind the centrifugal 10min of 3000r/min, obtain supernatant liquor, precipitation is 7.5 20mM Tris-HCl processing back recentrifuge again with pH, discards precipitation, and supernatant liquor merges, filter, obtain squid viscera alkalescence enzyme liquid.
Embodiment 2, a kind of extraction process of squid viscera enzyme, and its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:3, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 15min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 15min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 15min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of alkaline enzyme: with acetone dry powder by weight volume ratio 1:12 pH be that 7.5 20mM Tris-HCl is dissolved into uniform state, through 0 ℃ of refrigerated centrifuge, behind the centrifugal 20min of 4000r/min, obtain supernatant liquor, precipitation is 7.5 20mM Tris-HCl processing back recentrifuge again with pH, discards precipitation, and supernatant liquor merges, filter, obtain squid viscera alkalescence enzyme liquid.
Embodiment 3, a kind of extraction process of squid viscera enzyme, and its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:2, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 10min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 10min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 10min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of alkaline enzyme: with acetone dry powder by weight volume ratio 1:10 pH be that 7.5 20mM Tris-HCl is dissolved into uniform state, through 0 ℃ of refrigerated centrifuge, behind the centrifugal 15min of 3500r/min, obtain supernatant liquor, precipitation is 7.5 20mM Tris-HCl processing back recentrifuge again with pH, discards precipitation, and supernatant liquor merges, filter, obtain squid viscera alkalescence enzyme liquid.
Embodiment 4, in the extraction process of any one described squid viscera enzyme of embodiment 1-3: with alkaline enzyme liquid preservation under-20 ℃ temperature condition of gained, or through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.
Embodiment 5, in the extraction process of any one described squid viscera enzyme of embodiment 1-3: with alkaline enzyme liquid preservation under-40 ℃ temperature condition of gained, or through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.
Embodiment 6, in the extraction process of any one described squid viscera enzyme of embodiment 1-3: with alkaline enzyme liquid preservation under-23.5 ℃ temperature condition of gained, or through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.
Embodiment 7, a kind of extraction process of squid viscera enzyme, and its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:1, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 5min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 5min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 5min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of acidicenzym: with acetone dry powder by weight volume ratio 1:8 be dissolved into uniform state with 0.065M HCl, through 0 ℃ of refrigerated centrifuge, 3000r/min, behind the centrifugal 5min, get supernatant liquor, precipitation is handled the back recentrifuge with 0.065M HCl damping fluid again, discard precipitation, supernatant liquor merges, and filters, and namely gets squid viscera acidicenzym liquid.
Embodiment 8, a kind of extraction process of squid viscera enzyme, and its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:3, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 15min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 15min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 15min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of acidicenzym: with acetone dry powder by weight volume ratio 1:12 be dissolved into uniform state with 0.065M HCl, through 0 ℃ of refrigerated centrifuge, 4000r/min, behind the centrifugal 20min, get supernatant liquor, precipitation is handled the back recentrifuge with 0.065M HCl damping fluid again, discard precipitation, supernatant liquor merges, and filters, and namely gets squid viscera acidicenzym liquid.
Embodiment 9, a kind of extraction process of squid viscera enzyme, and its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:2, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 10min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 10min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 10min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of acidicenzym: with acetone dry powder by weight volume ratio 1:10 be dissolved into uniform state with 0.065M HCl, through 0 ℃ of refrigerated centrifuge, 3500r/min, behind the centrifugal 12min, get supernatant liquor, precipitation is handled the back recentrifuge with 0.065M HCl damping fluid again, discard precipitation, supernatant liquor merges, and filters, and namely gets squid viscera acidicenzym liquid.
Embodiment 10, in the extraction process of any one described squid viscera enzyme of embodiment 7-9: with acidicenzym liquid preservation under-20 ℃ temperature condition of gained, or through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.
Embodiment 11, in the extraction process of any one described squid viscera enzyme of embodiment 7-9: with acidicenzym liquid preservation under-40 ℃ temperature condition of gained, or through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.
Embodiment 12, in the extraction process of any one described squid viscera enzyme of embodiment 7-9: with acidicenzym liquid preservation under-23.5 ℃ temperature condition of gained, or through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.
Enzyme is a kind of protein of energy catalytic reaction process, and the present invention verifies that by the mensuration of enzyme activity the material that extracts is enzyme.
Proteolytic enzyme is that the hydrolyzed casein substrate adds trichoroacetic acid(TCA) then and stops enzyme reaction, and unhydrolysed casein precipitation is removed under 7 the condition at 20 ℃, pH, and filtrate has absorption to UV-light, available determined by ultraviolet spectrophotometry.Calculate its enzyme activity according to absorbancy.The definition of enzyme unit alive: every 1mL crude enzyme liquid, under certain temperature and pH value condition, it is an enzyme activity unit that the 1min hydrolyzed casein produces 1ug tyrosine, represents with (u/mL).
1. draw L-tyrosine typical curve
(1) get 0.05g L-tyrosine and be dissolved in the pure water, be settled to 25ml, as standardized solution 1, its concentration is 2mg/ml;
(2) standardized solution 1 is diluted one times as standardized solution 2, its concentration is 1mg/ml;
(3) standardized solution 2 is diluted one times as standardized solution 3, its concentration is 0.5mg/ml;
(4) standardized solution 3 is diluted one times as standardized solution 4, its concentration is 0.25mg/ml;
(5) again standardized solution 4 is diluted one times as standardized solution 5, its concentration is 0.125mg/ml;
(6) get standardized solution 1,2,3,4,5 each 1.4ml, add 0.65ml 22.5% trichoroacetic acid(TCA) solution respectively, survey its absorbancy in 290nm.
(7) be ordinate zou according to the absorbance A that records, the concentration c of standardized solution is that X-coordinate is made a typical curve.
2. the mensuration of enzyme activity
(1) alkaline enzyme liquid vitality test
Earlier casein solution is put into 40 ± 0.2 ℃ of waters bath with thermostatic control, preheating 5min, draw the alkaline enzyme liquid 2ml of an amount of dilution in test tube, put into 20 ℃ of waters bath with thermostatic control after adding casein 2ml shakes up and react 20min, the concussion of adding 4ml 22.5% trichoroacetic acid(TCA) solution shakes up and is placed on 10min in the ice-water bath, take out centrifugal 15 min of 6000r/min under the room temperature condition, get supernatant liquid filtering, 290nm surveys its absorbancy.
Blank is earlier enzyme-added liquid again behind casein and the trichoroacetic acid(TCA) mixing 10min, then with the common centrifugal survey absorbance of reaction solution, with the distilled water calibration instrument.
Produce 1 μ g tyrosine at 20 ℃ of following every milliliter of enzyme liquid per minute caseinhydrolysates, be defined as 1 protease activity unit of force.
X＝A×K×8/2×1/20×n×E＝1/5×A×K×n×E
In the formula: X---the enzyme activity of sample, (u/mL);
The mean light absorbency of A---sample solution;
K---extinction constant;
8---the cumulative volume of reaction reagent, mL;
2---draw enzyme liquid 2.00mL;
1/10---reaction times 10min, in 1min;
N---extension rate;
The reduction factor of E---ultraviolet method and folin's methods (neutral, Sumizyme MP coefficient is 0.50).
Gained is the result represent to integer.
Gained alkalescence enzyme liquid vigor is not less than 12u/mL.
(2) acidicenzym vitality test
Earlier bovine hemoglobin solution is put into 40 ± 0.2 ℃ of waters bath with thermostatic control, preheating 5min, draw the enzyme liquid 2ml of an amount of dilution in test tube, put into 30 ℃ of waters bath with thermostatic control after adding bovine hemoglobin 2ml shakes up and react 20min, the concussion of adding 4ml 22.5% trichoroacetic acid(TCA) solution shakes up and is placed on 10min in the ice-water bath, take out centrifugal 15 min of 6000r/min under the room temperature condition, get supernatant liquid filtering, 275nm surveys its absorbancy.
Blank is earlier enzyme-added liquid again behind bovine hemoglobin and the trichoroacetic acid(TCA) mixing 10min, then with the common centrifugal survey absorbance of reaction solution, with the distilled water calibration instrument.
Produce 1 μ g tyrosine at 20 ℃ of following every milliliter of enzyme liquid per minute caseinhydrolysates, be defined as 1 protease activity unit of force.
X＝A×K×8/2×1/20×n×E＝1/5×A×K×n×E
In the formula: X---the enzyme activity of sample, (u/mL);
The mean light absorbency of A---sample solution;
K---extinction constant;
8---the cumulative volume of reaction reagent, mL;
2---draw enzyme liquid 2.00mL;
1/20---reaction times 20min, in 1min;
N---extension rate
The reduction factor of E---ultraviolet method and folin's methods (the aspartic protease coefficient is 0.77).
Gained acidicenzym liquid vigor is not less than 12u/mL.
Claims (6)
1. the extracting method of a squid viscera enzyme, it is characterized in that: its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:1～3, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 5～15min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 5～15min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 5～15min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of alkaline enzyme: with acetone dry powder by weight volume ratio 1:8～12 usefulness pH be that 7.5 20mM Tris-HCl is dissolved into uniform state, through 0 ℃ of refrigerated centrifuge, behind the centrifugal 10～20min of 3000～4000r/min, obtain supernatant liquor, precipitation is 7.5 20mM Tris-HCl processing back recentrifuge again with pH, discards precipitation, and supernatant liquor merges, filter, obtain squid viscera alkalescence enzyme liquid.
2. the extracting method of squid viscera enzyme according to claim 1 is characterized in that: with alkaline enzyme liquid preservation under-20 ℃～-40 ℃ temperature condition of gained, or through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.
3. the extracting method of squid viscera enzyme according to claim 2, it is characterized in that: the temperature condition of preservation is-23.5 ℃.
4. the extracting method of a squid viscera enzyme, it is characterized in that: its step is as follows:
(1) is raw material with fresh squid viscera, after water is drained, in raw material, adds acetone by mass volume ratio 1:1～3, be processed into pulpous state with tissue refiner again; Filter, gained filtrate is through 0 ℃ of refrigerated centrifuge, 3000r/min, centrifugal 5～15min, abandoning supernatant, precipitation acetone solution, solution is again through 0 ℃ in whizzer, 3000r/min, centrifugal 5～15min, abandoning supernatant, precipitation is used acetone solution, and gained solution is through 0 ℃ in whizzer, 3000r/min, centrifugal 5～15min, abandoning supernatant, taking-up is deposited under the natural condition air-dry, gets acetone dry powder;
(2) extraction of acidicenzym: with acetone dry powder by weight volume ratio 1:8～12 usefulness 0.065M HCl be dissolved into uniform state, through 0 ℃ of refrigerated centrifuge, 3000～4000r/min, behind centrifugal 5～20min, get supernatant liquor, precipitation is handled the back recentrifuge with 0.065M HCl damping fluid again, discard precipitation, supernatant liquor merges, and filters, and namely gets squid viscera acidicenzym liquid.
5. the extracting method of squid viscera enzyme according to claim 4 is characterized in that: with acidicenzym liquid preservation under-20 ℃～-40 ℃ temperature condition of gained, or through lyophilize, namely obtain highly active squid viscera enzyme enzyme powder raw product.
6. the extracting method of squid viscera enzyme according to claim 5, it is characterized in that: the temperature condition of preservation is-23.5 ℃.
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| Title |
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| 张俊杰等.鱿鱼鱼皮酶溶性胶原蛋白部分生化性质研究.《食品与发酵工业》.2009,第35卷(第12期),57-60. |
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Denomination of invention: Technology of extracting squid visceral enzyme Effective date of registration: 20191119 Granted publication date: 20131009 Pledgee: Bank of China Limited by Share Ltd Lianyungang branch Pledgor: Jiangsu Tianfulai Group Co.,Ltd. Registration number: Y2019320000292 |
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