CN101724616A - Preparation of freezing-point active protease for tuna freezing preservation, and freezing preservation method - Google Patents
Preparation of freezing-point active protease for tuna freezing preservation, and freezing preservation method Download PDFInfo
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- CN101724616A CN101724616A CN201010300284A CN201010300284A CN101724616A CN 101724616 A CN101724616 A CN 101724616A CN 201010300284 A CN201010300284 A CN 201010300284A CN 201010300284 A CN201010300284 A CN 201010300284A CN 101724616 A CN101724616 A CN 101724616A
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Abstract
The invention provides preparation of freezing-point active protease for tuna freezing preservation, and a freezing preservation method, which relates to a preparation method and application of freezing-point active protease. The invention solves the problem of poor extreme tolerance of freezing-point active protease for tuna freezing preservation prepared by the prior method. The preparation method comprises: 1, inoculating an optimization culture medium with an Antarctic sea ice bacterium Colwellia sp. NJ341 and obtaining culture solution through culture; and 2, obtaining the freezing-point active protease by subjecting the culture solution to centrifugation, dialysis and ultrafiltration membrane concentration. The freezing preservation method comprises the steps of adding the freezing-point active protease to sliced tuna, adding crushed ice and NaCl, sealing and cooling the obtained product. The freezing-point active protease obtained by the method is good in extreme tolerance, and the original color and flavor of tuna subjected to freezing preservation by use of the freezing-point active protease can kept better. On the basis of retaining freshness, the freezing-point active protease can also increase the flavor of chilled products and improve the quality of the chilled products.
Description
Technical field
The present invention relates to a kind of preparation method and application thereof of freezing point active protease.
Background technology
Tuna is as a kind of abyssal pelagic fishes, usually be of high nutritive value, pure natural, pollution-free and enjoy great prestige the world market, and the title of " ocean gold " arranged, meat is rich in quality protein, high intramuscular fat and homovitamin A, D, its amino acid is formed similar to the amino acid composition of needed by human, so utilization ratio is very high.For a long time, tuna is always to freeze the form outlet of product, along with external human consumer is more and more higher to the demand of meat product, to ice the meat food ratio increasing (Luo Yin etc., 2008) of bright product form outlet.The bright product of so-called ice, be meant that the sea-food tuna kills after, processing as early as possible, the process precooling, cut apart, pack after, be cooled to ℃ pack into the then a kind of fresh products (Zhao Yanning, 1998) of meat food of insulating container of meat core temperature-2～0 rapidly.The bright product of ice are compared with freezing product, have better kept original color and luster of sea-food and fragrance, have removed from and have frozen the product course of defrosting, have reduced the loss of nutritive components of meat.On the bright product of the ice basis of sea-food, how to increase the local flavor of ice aquatic foods product, improve the quality of the bright product of ice, be the key issue that needs solution.Because the freezing point active protease has the catalytic efficiency height under 0 ℃ and following temperature, therefore have wide application prospect in low temperature environment industry such as meat product and processing of aquatic products.But existing method prepares is used for tuna to ice the extreme tolerance of freezing point active protease of bright preservation poor, has limited its application at the bright product of tuna class ice greatly.
Summary of the invention
The present invention is for the problem of extreme tolerance difference that tuna is iced the freezing point active protease of bright preservation that is used for that solves that existing method prepares, and provide a kind of be used for tuna ice bright preservation the freezing point active protease preparation and ice the method for bright preservation.
The present invention is used for tuna and ices the preparation method of the freezing point active protease of bright preservation and carry out according to following steps:
One, Antarctic Sea Ice bacterium Colwellia sp.NJ341 is inoculated in and optimizes on the substratum, in temperature is that 7.5～8.5 ℃, rotating speed are that shaking culture 93～100h obtains nutrient solution under the condition of 180～220r/min, wherein optimize substratum and be made up of 4.8～5.2 parts casein, 3.6～3.9 parts Trisodium Citrate, 0.15～0.25 part tween-80,0.8～1.2 part yeast extract, 8～12 parts NaCl and 950～1050 parts Chen Haishui according to ratio of weight and number, the pH value is 7.5;
Two, nutrient solution centrifugal 10min under the 10000r/min condition, then with supernatant liquor and ammonium sulfate according to 2.5～3.5: 6.5～7.5 weight ratio is mixed, under 0～0.5 ℃ of condition, leave standstill 7.5～8.5h, centrifugal 30min under the 12000r/min condition then, abandoning supernatant, be that 7.5 Tris-HC1 damping fluid mixes according to 1: 0.8～1.2 weight ratio with the precipitation after centrifugal and 50mmol/L, pH, 7.5～the 8.5h that dialyses under 4 ℃ of conditions is promptly obtaining being used for the freezing point active protease that tuna is iced bright preservation under 4 ℃ of conditions then after ultra-filtration membrane concentrates.
Freezing point active protease of the present invention is iced the method for bright preservation and is carried out according to following operation to tuna: tuna is cleaned up, be cut into the thick fillet of 3cm～4cm, in fillet, add freezing point active protease of the present invention, the add-on of freezing point active protease is 210～220U/g flesh of fish, in the fillet of every gram, add simultaneously the trash ice of 0.5～3g and the NaCl of 0.02～0.04g, put into Plastic film Bag by the form of layer fish layer ice then, sealing, cooling fast, put into-2～0 ℃ of freezers, promptly realized the bright preservation of ice tuna.
The present invention makes the freezing point active protease that obtains, its optimum temperuture is 34～26 ℃, can keep relative vigor more than 30% 0～10 ℃ of condition, be that the vigor of enzyme remains unchanged under the condition of 0～2mol/L in NaCl concentration, the extreme better tolerance of freezing point active protease of the present invention, the tuna that uses freezing point active protease of the present invention to ice after the bright preservation has obvious tenderization phenomenon, freezing point active protease obvious suppression of the present invention the TvB Tun value of tuna, the lift velocity of pH value and total number of bacterial colony, reach fresh-keeping purpose, use freezing point active protease of the present invention to ice the tuna of bright preservation, compare with fresh tuna, L-glutamic acid and histidine content have all increased more than 1.5%, increase the local flavor of meat, increase bright effect obviously, the freshness date of using freezing point active protease of the present invention to ice the tuna of bright preservation reached more than 50 days, long fresh-keeping period, hence one can see that, the tuna that uses freezing point active protease of the present invention to ice bright preservation can better keep original color and luster and fragrance, removed from and frozen the product course of defrosting, reduced the loss of nutritive components of meat, on fresh-keeping basis, can increase the local flavor of the bright product of ice again, improve the quality of the bright product of ice.
Description of drawings
Fig. 1 is for making total volatile basic nitrogen content figure in the bright product of big order tuna ice after the freezing point active protease that obtains is iced bright preservation of embodiment ten;
Fig. 2 is for making total volatile basic nitrogen content figure in the bright product of yellowfin tuna ice after the freezing point active protease that obtains is iced bright preservation of embodiment ten;
Fig. 3 is for making pH variation diagram in the bright product of big order tuna ice after the freezing point active protease that obtains is iced bright preservation of embodiment ten;
The yellowfin tuna that the freezing point active protease that Fig. 4 obtains for embodiment ten making is iced after the bright preservation is iced pH variation diagram in the bright product;
The big order tuna that the freezing point active protease that Fig. 5 obtains with embodiment ten making is iced after the bright preservation is iced total count spirogram in the bright product;
The yellowfin tuna that the freezing point active protease that Fig. 6 obtains for embodiment ten making is iced after the bright preservation is iced total count spirogram in the bright product.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: present embodiment is used for tuna and ices the preparation method of the freezing point active protease of bright preservation and carry out according to following steps:
One, Antarctic Sea Ice bacterium Colwellia sp.NJ341 is inoculated in and optimizes on the substratum, in temperature is that 7.5～8.5 ℃, rotating speed are that shaking culture 93～100h obtains nutrient solution under the condition of 180～220r/min, wherein optimize substratum and be made up of 4.8～5.2 parts casein, 3.6～3.9 parts Trisodium Citrate, 0.15～0.25 part tween-80,0.8～1.2 part yeast extract, 8～12 parts NaCl and 950～1050 parts Chen Haishui according to ratio of weight and number, the pH value is 7.5;
Two, nutrient solution is centrifugal 10min under the 10000r/min condition, with supernatant liquor and mass concentration be then 65%～75% ammonium sulfate according to 2.5～3.5: 6.5～7.5 weight ratio is mixed, under 0～0.5 ℃ of condition, leave standstill 7.5～8.5h, centrifugal 30min under the 12000r/min condition then, abandoning supernatant, with precipitation after centrifugal and 50mmol/L, pH is that 7.5 Tris-HCl damping fluid mixes according to 1: 0.8～1.2 weight ratio, 7.5～the 8.5h that dialyses under 4 ℃ of conditions is promptly obtaining being used for the freezing point active protease that tuna is iced bright preservation under 4 ℃ of conditions then after ultra-filtration membrane concentrates.
Chen Haishui is the seawater of placing more than 15 days in the present embodiment step 3, has removed the part microorganism in the seawater and some meta-bolitess etc.
The molecular weight of ultra-filtration membrane is 50Kda in the present embodiment step 2.
Tuna being iced the method for bright preservation with embodiment freezing point active protease carries out according to following operation: tuna is cleaned up, be cut into the thick fillet of 3cm～4cm, the freezing point active protease that in fillet, adds present embodiment, the add-on of freezing point active protease is the 215U/g flesh of fish, in the fillet of every gram, add simultaneously the trash ice of 0.5～3g and the NaCl of 0.02～0.04g, put into Plastic film Bag by the form of layer fish layer ice then, sealing, cooling fast, put into 0 ℃～-2 ℃ freezers, promptly realized tuna is iced bright preservation.
Present embodiment is made the freezing point active protease that obtains, its optimum temperuture is 34～26 ℃, can keep relative vigor more than 30% 0～10 ℃ of condition, be that the vigor of enzyme remains unchanged under the condition of 0～2mol/L in NaCl concentration, the freezing point active protease better tolerance of present embodiment, use present embodiment freezing point active protease that the tuna of icing after the bright preservation is had obvious tenderization phenomenon, present embodiment freezing point active protease obvious suppression the TvB Tun value of tuna, the lift velocity of pH value and total number of bacterial colony, reach fresh-keeping purpose, use present embodiment freezing point active protease to ice the tuna of bright preservation, compare with fresh tuna, L-glutamic acid and histidine content have all increased more than 1.5%, increase the local flavor of meat, increase bright effect obviously, the freshness date of using present embodiment freezing point active protease to ice the tuna of bright preservation reaches more than 50 days long fresh-keeping period.
Embodiment two: what present embodiment and embodiment one were different is: optimize substratum in the step 1 and be made up of 5 parts casein, 3.75 parts Trisodium Citrate, 0.2 part tween-80,1 part yeast extract, 10 parts NaCl and 1000 parts Chen Haishui according to ratio of weight and number.Other step and parameter are identical with embodiment one.
Embodiment three: what present embodiment and embodiment one to two were different is: be that 8 ℃, rotating speed are that shaking culture 96h obtains nutrient solution under the condition of 200r/min in temperature in the step 1.Other step and parameter are identical with embodiment one to two.
Embodiment four: what present embodiment and embodiment one to three were different is: supernatant liquor and ammonium sulfate mix according to 3: 7 weight ratio in the step 2.Other step and parameter are identical with embodiment one to three.
Embodiment five: what present embodiment and embodiment one to four were different is: leave standstill 8h in the step 2 under 0 ℃ of condition.Other step and parameter are identical with embodiment one to four.
Embodiment six: what present embodiment and embodiment one to five were different is: 8h dialyses in the step 2.Other step and parameter are identical with embodiment one to five.
Embodiment seven: present embodiment freezing point active protease is iced the method for bright preservation and is carried out according to following operation to tuna: tuna is cleaned up, be cut into the thick fillet of 3cm～4cm, in fillet, add freezing point active protease of the present invention, the add-on of freezing point active protease is 210～220U/g flesh of fish, in the fillet of every gram, add simultaneously the trash ice of 0.5～3g and the NaCl of 0.02～0.04g, put into Plastic film Bag by the form of layer fish layer ice then, sealing, cooling fast, put into-2～0 ℃ of freezers, promptly realized the bright preservation of ice tuna.
Embodiment eight: present embodiment and embodiment seven are different is that the add-on of freezing point active protease is the 215U/g flesh of fish.Other step and parameter are identical with embodiment seven.
Embodiment nine: what present embodiment was different with embodiment seven or eight is to add the trash ice of 2g and the NaCl of 5g in the fillet of every gram.Other step and parameter are identical with embodiment seven or eight.
Embodiment ten: present embodiment is used for tuna and ices the preparation method of the freezing point active protease of bright preservation and carry out according to following steps:
One, Antarctic Sea Ice bacterium Colwellia sp.NJ341 is inoculated in and optimizes on the substratum, in temperature is that 7.5～8.5 ℃, rotating speed are that shaking culture 93～100h obtains nutrient solution under the condition of 180～220r/min, wherein optimize substratum and be made up of 4.8～5.2 parts casein, 3.6～3.9 parts Trisodium Citrate, 0.15～0.25 part tween 80,0.8～1.2 part yeast extract, 8～12 parts NaCl and 950～1050 parts Chen Haishui according to ratio of weight and number, the pH value is 7.5;
Two, nutrient solution centrifugal 10min under the 10000r/min condition, then supernatant liquor and ammonium sulfate are mixed according to 3: 7 weight ratio, under 0～0.5 ℃ of condition, leave standstill 7.5～8.5h, centrifugal 30min under the 12000r/min condition then, abandoning supernatant, be that 7.5 Tris HCl damping fluid mixes according to 1: 1 weight ratio with the precipitation after centrifugal and 50mmol/L, pH, 7.5～the 8.5h that dialyses under 4 ℃ of conditions is promptly obtaining being used for the freezing point active protease that tuna is iced bright preservation under 4 ℃ of conditions then after ultra-filtration membrane concentrates.
Chen Haishui is the seawater of placing more than 15 days in the present embodiment step 3, has removed the part microorganism in the seawater and some meta-bolitess etc.
The molecular weight of ultra-filtration membrane is 50Kda in the present embodiment step 2.
Tuna being iced the method for bright preservation with embodiment freezing point active protease carries out according to following operation: big order tuna or yellowfin tuna are cleaned up, be cut into the thick fillet of 3cm～4cm, the freezing point active protease that in fillet, adds present embodiment, the add-on of freezing point active protease is the 215U/g flesh of fish, the trash ice of while 0.2g in the fillet of every gram and the mass concentration of 0.03g are 4% NaCl, put into Plastic film Bag by the form of layer fish layer ice then, sealing, cooling fast, put into 0 ℃～-2 ℃ freezers, promptly realized the bright preservation of ice (having obtained the bright product of tuna ice) tuna.
Present embodiment is made the freezing point active protease that obtains, its optimum temperuture is 34～26 ℃, can keep relative vigor more than 30% 0～10 ℃ of condition, the vigor of enzyme remains unchanged under 0～2MNaCl concentration, the freezing point active protease better tolerance of present embodiment uses the bright product of tuna ice of present embodiment freezing point active protease that obvious tenderization phenomenon is arranged.
Fig. 1 is for making total volatile basic nitrogen content figure in the bright product of big order tuna ice after the freezing point active protease obtain is iced bright preservation, among Fig. 1 of present embodiment
Expression is made the bright product of tuna ice with embodiment freezing point active protease
Expression is without the tuna of any processing; Fig. 2 is for making total volatile basic nitrogen content figure in the bright product of yellowfin tuna ice after the freezing point active protease obtain is iced bright preservation, among Fig. 2 of present embodiment
Expression is made the bright product of tuna ice with embodiment freezing point active protease,
Expression is without the tuna of any processing; Fig. 3 is for making pH variation diagram in the bright product of big order tuna ice after the freezing point active protease obtain is iced bright preservation, among Fig. 3 of present embodiment
Expression is made the bright product of tuna ice with embodiment freezing point active protease,
Expression is without the tuna of any processing; Fig. 4 is for making pH variation diagram in the bright product of yellowfin tuna ice after the freezing point active protease obtain is iced bright preservation, among Fig. 4 of present embodiment
Expression is made the bright product of tuna ice with embodiment freezing point active protease,
Expression is without the tuna of any processing; Fig. 5 is for making total count spirogram in the bright product of big order tuna ice after the freezing point active protease obtain is iced bright preservation, among Fig. 5 of present embodiment
Expression is made the bright product of tuna ice with embodiment freezing point active protease,
Expression is without the tuna of any processing; Fig. 6 is for making total count spirogram in the bright product of yellowfin tuna ice after the freezing point active protease obtain is iced bright preservation, among Fig. 6 of present embodiment
Expression is made the bright product of tuna ice with embodiment freezing point active protease,
Expression is without the tuna of any processing
From Fig. 1～6 as can be seen, big order tuna and yellowfin tuna are after the freezing point protease treatment, can use present embodiment to make the freezing point active protease that obtains and realize the purpose that tuna is fresh-keeping than the lift velocity of obvious suppression TvB Tun value, pH value and total number of bacterial colony.
The freezing point active protease is as shown in table 1 to big order tuna and the influence of yellowfin tuna enzymolysis.
Table 1
Annotate: the freezing point active protease that " freezing point active protease " expression obtains with the present embodiment making is iced the bright product of yellowfin tuna ice after the bright preservation, and " contrast " expression is without the tuna of any processing.
Data from table 1 are found out, present embodiment is made in the bright product of yellowfin tuna ice that obtain after the freezing point active protease is iced bright preservation and has been discharged more total free aminoacids, L-glutamic acid and histidine content significantly increase, increase the local flavor of meat, play and increase bright effect, making the bright product freshness date of yellowfin tuna ice that obtains after the freezing point active protease is iced bright preservation through present embodiment is 50 days, long fresh-keeping period.
Use present embodiment to make better original color and luster and the fragrance of keeping of tuna that the freezing point active protease that obtains is iced bright preservation, removed from and frozen the product course of defrosting, reduced the loss of nutritive components of meat, on fresh-keeping basis, can increase the local flavor of the bright product of ice again, improve the quality of the bright product of ice.
Claims (9)
1. one kind is used for the preparation method that tuna is iced the freezing point active protease of bright preservation, it is characterized in that being used for tuna and ices the preparation method of the freezing point active protease of bright preservation and carry out according to following steps:
One, Antarctic Sea Ice bacterium Colwellia sp.NJ341 is inoculated in and optimizes on the substratum, in temperature is that 7.5～8.5 ℃, rotating speed are that shaking culture 93～100h obtains nutrient solution under the condition of 180～220r/min, wherein optimize substratum and be made up of 4.8～5.2 parts casein, 3.6～3.9 parts Trisodium Citrate, 0.15～0.25 part tween-80,0.8～1.2 part yeast extract, 8～12 parts NaCl and 950～1050 parts Chen Haishui according to ratio of weight and number, the pH value is 7.5;
Two, nutrient solution centrifugal 10min under the 10000r/min condition, then with supernatant liquor and ammonium sulfate according to 2.5～3.5: 6.5～7.5 weight ratio is mixed, under 0～0.5 ℃ of condition, leave standstill 7.5～8.5h, centrifugal 30min under the 12000r/min condition then, abandoning supernatant, be that 7.5 Tris-HC1 damping fluid mixes according to 1: 0.8～1.2 weight ratio with the precipitation after centrifugal and 50mmol/L, pH, 7.5～the 8.5h that dialyses under 4 ℃ of conditions is promptly obtaining being used for the freezing point active protease that tuna is iced bright preservation under 4 ℃ of conditions then after ultra-filtration membrane concentrates.
2. a kind of preparation method that tuna is iced the freezing point active protease of bright preservation that is used for according to claim 1 is characterized in that optimizing in the step 1 substratum and is made up of 5 parts casein, 3.75 parts Trisodium Citrate, 0.2 part tween 80,1 part yeast extract, 10 parts NaCl and 1000 parts Chen Haishui according to ratio of weight and number.
3. a kind of preparation method that tuna is iced the freezing point active protease of bright preservation that is used for according to claim 1 and 2 is characterized in that in the step 1 in temperature being that 8 ℃, rotating speed are that shaking culture 96h obtains nutrient solution under the condition of 200r/min.
4. a kind of preparation method that tuna is iced the freezing point active protease of bright preservation that is used for according to claim 3 is characterized in that supernatant liquor and ammonium sulfate mix according to 3: 7 weight ratio in the step 2.
5. according to claim 1,2 or 4 described a kind of preparation methods that tuna is iced the freezing point active protease of bright preservation that are used for, it is characterized in that under 0 ℃ of condition, leaving standstill 8h in the step 2.
6. a kind of preparation method that tuna is iced the freezing point active protease of bright preservation that is used for according to claim 1 is characterized in that the 8h that dialyses in the step 2.
7. the freezing point active protease is iced the method for bright preservation to tuna, it is characterized in that the freezing point active protease ices the method for bright preservation and carry out according to following operation to tuna: tuna is cleaned up, be cut into the thick fillet of 3cm～4cm, in fillet, add freezing point active protease of the present invention, the add-on of freezing point active protease is 210～220U/g flesh of fish, in the fillet of every gram, add simultaneously the trash ice of 0.5～3g and the NaCl of 0.02～0.04g, put into Plastic film Bag by the form of layer fish layer ice then, sealing, cooling fast, put into-2～0 ℃ of freezers, promptly realized the bright preservation of ice tuna.
8. freezing point active protease according to claim 7 is iced the method for bright preservation to tuna, and the add-on that it is characterized in that the freezing point active protease is the 215U/g flesh of fish.
9. according to claim 7 or 8 described freezing point active proteases tuna is iced the method for bright preservation, it is characterized in that in the fillet of every gram, adding the trash ice of 2g and the NaCl of 0.03g.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9073760B2 (en) | 2010-12-24 | 2015-07-07 | Shoei Chemical Inc. | Manufacturing method and manufacturing device for multiple oxide |
| CN106754501A (en) * | 2016-12-15 | 2017-05-31 | 国家海洋局第三海洋研究所 | The thermophilic cold novel bacterial of Colville Bordetella and its cultural method and application |
| CN109601599A (en) * | 2019-02-21 | 2019-04-12 | 福州宏东食品有限公司 | A kind of freeze preservation method of tuna |
| CN110623060A (en) * | 2019-10-31 | 2019-12-31 | 福州大学 | Application of protease K in fresh-keeping of fresh fish products |
-
2010
- 2010-01-14 CN CN201010300284A patent/CN101724616A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9073760B2 (en) | 2010-12-24 | 2015-07-07 | Shoei Chemical Inc. | Manufacturing method and manufacturing device for multiple oxide |
| CN106754501A (en) * | 2016-12-15 | 2017-05-31 | 国家海洋局第三海洋研究所 | The thermophilic cold novel bacterial of Colville Bordetella and its cultural method and application |
| CN106754501B (en) * | 2016-12-15 | 2019-07-09 | 自然资源部第三海洋研究所 | The thermophilic cold novel bacterial of Colville Bordetella and its cultural method and application |
| CN109601599A (en) * | 2019-02-21 | 2019-04-12 | 福州宏东食品有限公司 | A kind of freeze preservation method of tuna |
| CN110623060A (en) * | 2019-10-31 | 2019-12-31 | 福州大学 | Application of protease K in fresh-keeping of fresh fish products |
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Open date: 20100609 |