CN1336433A - Prepn. and application of properly cooled proteinase with special flavor - Google Patents
Prepn. and application of properly cooled proteinase with special flavor Download PDFInfo
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- CN1336433A CN1336433A CN 01127405 CN01127405A CN1336433A CN 1336433 A CN1336433 A CN 1336433A CN 01127405 CN01127405 CN 01127405 CN 01127405 A CN01127405 A CN 01127405A CN 1336433 A CN1336433 A CN 1336433A
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- proteinase
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- special flavor
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Abstract
The present invention relates to prepn. of cold adaptive flavour protease and its application, use deep sea cold adaptive pseudomoas with high protease yield as bacteria strain, to prepare fermentation liquid through liquid submerged culture fermentation technology, then filer, low temp. ultrafiltration and concentration, add stabilizing agent sucrose to obtain product. The most suitable temp. of invented protease is 30-35 deg.c, and at below zero deg.C, it can still maintain 20-25% of its maximum enzyme activity, the most suitable pH is 8.0. Spray 1-2% of invented enzyme onto the surface of animal or poultry meat or seafood, then packaged and cold stored, the enzyme can party enzymolyze the protein, liberate free amino acid to improve the flavour and taste of meat.
Description
(1) technical field
The present invention relates to a kind of new properly cooled proteinase with special flavor preparation method and application thereof, belong to technical field of food biotechnology.
(2) background technology
For a long time, outlet fishery products of China and meat (especially meat of poultris) are always to freeze the form outlet of product, along with external human consumer, particularly Hong Kong of south east asia and China, Taiwan, demand to food freshness is more and more higher, add the fast development of fresh-keeping transport trade, the ratio of the bright product outlet of ice will be more and more higher.The bright product of so-called ice, be meant fishery products, livestock and poultry are butchered after processing as early as possible, after the precooling of strictness, cutting apart, pack, being cooled to the meat core temperature rapidly is-2 ℃～0 ℃, a kind of fishery products of the insulating container of packing into then, meat preservation product.General requirement is no more than 6～7 days from beginning to be worked into to enter the gastronomical process.Freeze product through quick-frozen (30 ℃) and low temperature (18 ℃); mainly be for the prolonged preservation convenience; but freezing process destroys very big to the color and luster and the fragrance of fishery products, meat itself; and the suitable precooling of process keeps-2 ℃～0 ℃ the bright product of ice; fishery products, the original color and luster of meat and fragrance have not only been kept; and process of cooling is that fishery products and meat surface form protective membrane; add that non-refrigerated low temperature environment has prevented microbial growth and procreation, it is putrid and deteriorated that fishery products, meat can not taken place in 7～10 days.Along with improving constantly of standard of living, people require more and more higher to flavours in food products, not only require food fresh, and require food to have good local flavor.Therefore, keeping how increasing the local flavor of the bright product of ice on the fresh basis of the bright product of ice such as fishery products and meat, the quality that improves product is that the bright product of ice need one of key issue that solves.Cold-adapted protease has good application prospects aspect the local flavor of the bright product of the ice that increases fishery products, meat.Because cold-adapted protease still can keep higher catalytic efficiency at 0 ℃, hydrolyzable protein discharges the amino acid with delicate flavour, increases the delicate flavour of fishery products and meat.In addition, studies show that the total free aminoacids that discharges can react with glucides such as glucose, sucrose in gastronomical process, form material, can significantly increase the local flavor of fishery products and meat product with fragrance.Therefore, cold-adapted protease the local flavor of fishery products and meat increase and increment aspect in have broad application prospects.
(3) summary of the invention
The invention provides a kind of preparation method and application thereof of the properly cooled proteinase with special flavor from the deep-sea, be used to improve the local flavor of the bright product of ice of fishery products and meat, improve the added value of the ice aquatic foods product of ocean water product and meat.
The preparation method of properly cooled proteinase with special flavor of the present invention comprises the steps:
1. the preparation of seed
Substratum: 2.5～3.5 parts of soybean cake powder, 2.5～3.5 parts of corns, 1.0～2.0 parts in wheat bran, Na
2HPO
40.3～0.4 part, KH
2PO
40.03 100 parts in～0.05 part and water are weight part.After sterilization, the cooling, (Pseudomonas sp.SM9913) is bacterial strain with the deep sea cold-adaptive microbe bacterium strain pseudomonas, connects the bacteria suspension of eggplant bottle bacterial classification, and inoculum size 5～7% weight percents ventilate, stir, and cultivate.
2. liquid submerged fermentation prepares local flavor cold-adapted protease preparation
Substratum: 3.5～4.0 parts of soybean cake powder, 2.0～2.5 parts of Semen Maydis powder, 2.0～3.5 parts of Rhizoma Dioscoreae powder, 2.0～2.5 parts in wheat bran, Na
2HPO
40.3～0.4 part, KH
2PO
40.04 100 parts in～0.05 part and water are weight part.Sterilization, seed 5～7% weight percents of inoculation above-mentioned steps preparation ventilate, stir temperature control, fermentation culture.
After above-mentioned steps, the facility in order to use can increase following post-processing step:
3. the aftertreatment of local flavor cold-adapted protease fermented liquid
Fermented liquid is centrifugal through 10000rpm, then with holding back the above ultra-filtration membrane of 5000 molecular weight, to 20～30% of original volume, adds 4～5% sucrose then as stablizer at 0～4 ℃ of ultrafiltration and concentration.
At last, quality inspection, be distributed into the properly cooled proteinase with special flavor preparation.
The substratum of above-mentioned steps 1 was 120～125 ℃ of sterilizations 30～35 minutes, and in cooling, the rearmounted fermentor tank of inoculation, sample-loading amount is 40～50% of a fermentor tank capacity, and under 10～12 ℃, ventilation 1:0.5～0.6 is stirred 320～330 rev/mins, cultivates 20～28 hours.Enzyme about 200～300 units per ml alive.
The sample-loading amount of the substratum of above-mentioned steps 2 is 40～50% of fermentor tank capacity, sterilization, and pH7.0～7.5, inoculation, in 200～220 rev/mins, control ventilation (V
Wind/ V
LiquidTime): 0-20 hour 1:0.5～0.6, under 20-50 hour 1:0.8～0.9 and the control culture temperature 10～12 ℃, fermented 43～50 hours.Every milliliter of enzyme work reaches 2000～2500 units.
The substratum of above-mentioned steps 1 adds soya-bean oil and makes defoamer for 0.3～0.35 part.
The substratum of above-mentioned steps 2 adds soya-bean oil and makes defoamer for 1～1.2 part.
The making of eggplant bottle slant strains can be by prior art in the above-mentioned steps 1.The invention provides following concrete operations step:
With 0.2～0.4 portion of extractum carnis, 0.8～1.2 part of peptone, 1.5～2 parts of agar, 0.4～0.6 part of NaCl is substratum, 95～100 parts in water, pH is 7.0～7.2, sterilization, on the test tube slant, line connects deep sea cold-adaptive microbe bacterium strain pseudomonas (Pseudomonas sp.SM9913) bacterial classification, cultivates 24～26 hours, and preserves in 4 ℃ for 12～15 ℃.Above-mentioned bacterial classification is moved into eggplant bottle inclined-plane (substratum is identical with the preservation inclined-plane), cultivated 24～26 hours for 12～15 ℃, wait to do bacteria suspension and use.
The application of properly cooled proteinase with special flavor of the present invention comprises that the protease preparation that uses aforesaid method to make is processed into the bright product of local flavor ice with meat, fish, shellfish.
Concrete technology is as follows:
Basic raw material: just butchered clean livestock and poultry meat, as pork, chicken etc., or the clean shellfish or handle clean fish of just having shelled, as, peeled shrimp, Bei Zhu, fish etc. take by weighing 98～100 parts of raw materials of fresh weight, then at its surface spray weight percent 1～2% above-mentioned properly cooled proteinase with special flavor, pack, vacuum packaging, cooling fast, secondary package, go into 0 ℃ of storehouse, become the bright product invention of local flavor ice product.
Excellent results of the present invention is: (1) utilization liquid submerged fermentation engineering and enzyme engineering technology are bacterial strain with the cold-adaptive microbe bacterium strain of high yield cold-adapted protease, at low temperatures (10～12 ℃) fermentative production properly cooled proteinase with special flavor preparation; (2) suitable cold enzyme keeps higher enzyme activity at low temperatures, but while less stable, fermented liquid is behind centrifugal, ultrafiltration and concentration, with sucrose properly cooled proteinase with special flavor is carried out stability protection, simultaneously, sucrose can be produced fragrance matter with the free amine group acid-respons that enzymolysis produces in the bright product gastronomical process of ice, increase the local flavor of meat; (3) this properly cooled proteinase with special flavor can be used for fishery products and meat the bright product of ice increase aquatic foods, thereby further improve the local flavor of fishery products and meat, raising value-added content of product.
(4) embodiment
Embodiment 1. 1. the preparation method of properly cooled proteinase with special flavor
A. slant strains (solid)
With 0.4 part of weight part extractum carnis, 0.9 part of peptone, 2 parts in agar, NaCl is substratum for 0.6 part, and 98 parts in water, pH are 7.1, sterilization, line connects deep sea cold-adaptive microbe bacterium strain pseudomonas (Pseudomonas sp.SM9913) bacterial classification, 15 ℃ of cultivations, cultivated 4 ℃ of storages 25 hours.Above-mentioned bacterial classification is moved into eggplant inclined-plane (substratum is identical with the preservation inclined-plane), cultivated 25 hours for 15 ℃, wait to do bacteria suspension and use.
B. seed culture
Substratum: 3.5 parts of soybean cake powder, 3.0 parts of Semen Maydis powder, 1 part in wheat bran, Na
2HPO
40.3 part, KH
2PO
40.03 part, add soya-bean oil and make defoamer for 0.3 part.Sterilized 30 minutes for 120 ℃, after the cooling, connect the bacteria suspension of above-mentioned eggplant bottle bacterial classification, in 6%, 150 liter of fermentor tank of inoculum size, 70 liters of sample-loading amounts, under 15 ℃, ventilation 1:0.6 stirs 330 rev/mins, cultivates 20 hours.
C. the fermentation of protease preparation (liquid submerged fermentation)
1000 liters of fermentor tanks are adorned 450 liters of substratum, inoculation 6% liquid seeds, and in 210 rev/mins, in the control ventilation, 0-20 hour 1:0.6 under 12 ℃ of 20-50 hour 1:0.8 and the control culture temperature, fermented 50 hours.The composition of substratum is weight part: 4 parts of soybean cake powder, 2 parts of Semen Maydis powder, 3.0 parts of Rhizoma Dioscoreae powder, 2 parts in wheat bran, Na
2HPO
40.4 part, KH
2PO
40.03 part, add soya-bean oil and make defoamer for 1.2 parts.
D. the aftertreatment of properly cooled proteinase with special flavor
Fermented liquid is centrifugal through 10000rpm, then with holding back the ultra-filtration membrane of 5000 molecular weight, to 20% of original volume, adds 4% sucrose then as stablizer at 2 ℃ of ultrafiltration and concentration.
At last, through quality inspection, be distributed into properly cooled proteinase with special flavor invention product.Two. be applied to the processing of the bright product of local flavor ice
Take by weighing 100 portions of chicken of fresh weight, then at the above-mentioned properly cooled proteinase with special flavor of its surface spray 2% weight percent, pack, vacuum packaging, cooling fast, the secondary loading packing is gone into 0 ℃ of storehouse, becomes the bright product invention of local flavor ice product.
Embodiment 2.
One. the preparation method of properly cooled proteinase with special flavor
A, B, C are with embodiment 1.
D, fermented liquid are centrifugal through 10000rpm, then with holding back the above ultra-filtration membrane of 5200 molecular weight, to 30% of original volume, add 5% sucrose then as stablizer at 3 ℃ of ultrafiltration and concentration.
At last, through quality inspection, be distributed into properly cooled proteinase with special flavor invention product.
Two. be applied to the processing of the bright product of local flavor ice
Take by weighing 98 portions of ocean fishes of fresh weight, then at the above-mentioned local flavor cold-adapted protease of its surface spray 1.5% weight percent, pack, vacuum packaging, cooling fast, the secondary loading packing is gone into 0 ℃ of storehouse, becomes the bright product invention of local flavor ice product.Embodiment 3. is as described in the embodiment 1, and different is
One. the preparation method of properly cooled proteinase with special flavor
A. slant strains (solid) is a substratum for 0.5 part with 0.3 part of weight part extractum carnis, 1.0 parts of peptones, 1.5 parts in agar, NaCl, 100 parts in water, pH is 7.0, sterilization, on the test tube slant, line connects deep sea cold-adaptive microbe bacterium strain pseudomonas (Pseudomonassp.SM9913) bacterial classification, 15 ℃ of cultivations, cultivated 4 ℃ of storages 25 hours.Above-mentioned bacterial classification is moved into eggplant inclined-plane (substratum is identical with the preservation inclined-plane), cultivated 25 hours for 15 ℃, wait to do bacteria suspension and use.
B. seed culture
Substratum: 3.0 parts of soybean cake powder, 3.0 parts of Semen Maydis powder, 1.5 parts in wheat bran, Na
2HPO
40.35 part, KH
2PO
40.04 part, add soya-bean oil and make defoamer for 0.3 part.Sterilized 30 minutes for 120 ℃, after the cooling, connect the bacteria suspension of eggplant bottle bacterial classification, in 7%, 150 liter of fermentor tank of inoculum size, 70 liters of sample-loading amounts, under 15 ℃, ventilation 1:0.6 stirs 330 rev/mins, cultivates 20 hours.
C. the fermentation of protease preparation (liquid submerged fermentation)
1000 liters of fermentor tanks are adorned 450 liters of substratum, inoculation 6% liquid seeds, and in 210 rev/mins, in the control ventilation, 0-20 hour 1:0.5,12 ℃ of 20-50 hour 1:0.9 and control culture temperature were fermented 48 hours.The composition of substratum is weight part: 3.5 parts of soybean cake powder, 2.2 parts of Semen Maydis powder, 3.5 parts of Rhizoma Dioscoreae powder, 2.2 parts in wheat bran, Na
2HPO
40.3 part, KH
2PO
40.04 100 parts in part, water add soya-bean oil and make defoamer for 1.1 parts.
D. the aftertreatment of properly cooled proteinase with special flavor
Fermented liquid is centrifugal through 10000rpm, then with holding back the ultra-filtration membrane of 5000 molecular weight, to 20% of original volume, adds 4% sucrose then as stablizer at 2 ℃ of ultrafiltration and concentration.
The outward appearance of the properly cooled proteinase with special flavor liquid of the embodiment of the invention is yellow, the fermented product peat-reek is arranged, liquid.Innovation part of the present invention is: 12～15 ℃ of the optimum growth temperatures of this product bacterial strain uses therefor deep sea cold-adaptive microbe bacterium strain pseudomonas (Pseudomonas sp.SM9913), the suitableeest product enzyme temperature is 10～12 ℃, live 30～35 ℃ of temperature of the suitableeest enzyme of the local flavor cold-adapted protease that is produced still can keep 25% of enzyme activity under the optimum temperuture in the time of 0 ℃.Have good application prospects increasing of the bright product of the ice of fishery products and meat (0～4 ℃) aspect bright, can significantly increase the local flavor of the bright product of ice, improve the quality of the bright product of ice, thereby the added value of aquatic foods product is iced in increase.
The invention is not restricted to the foregoing description.
Claims (9)
1. the preparation method of a properly cooled proteinase with special flavor comprises the steps:
(1) preparation of seed
Substratum: 2.5～3.5 parts of soybean cake powder, 2.5～3.5 parts of corns, 1.0～2.0 parts in wheat bran, Na
2HPO
40.3～0.4 part, KH
2PO
40.03 100 parts in～0.05 part and water are weight part, after sterilization, the cooling, are bacterial strain with deep sea cold-adaptive microbe bacterium strain pseudomonas Pseudomonas sp.SM9913, connect the bacteria suspension of eggplant bottle bacterial classification, inoculum size 5～7% weight percents ventilate, stir, and cultivate;
(2) liquid submerged fermentation prepares the properly cooled proteinase with special flavor preparation
Substratum: 3.5～4.0 parts of soybean cake powder, 2.0～2.5 parts of Semen Maydis powder, 2.0～3.5 parts of Rhizoma Dioscoreae powder, 2.0～2.5 parts in wheat bran, Na
2HPO
40.3～0.4 part, KH
2PO
40.04 100 parts in～0.05 part and water are weight part, sterilization, and seed 5～7% weight percents of inoculation above-mentioned steps preparation ventilate, stir temperature control, fermentation culture.
2. the preparation method of properly cooled proteinase with special flavor as claimed in claim 1, it is characterized in that, the post-processing step that also comprises protease fermented liquid is as follows: fermented liquid is centrifugal through 10000rpm, then with holding back the above ultra-filtration membrane of 5000 molecular weight, to 20～30% of original volume, add 4～5% sucrose then at 0～4 ℃ of ultrafiltration and concentration as stablizer.
3. the preparation method of properly cooled proteinase with special flavor as claimed in claim 1 or 2, it is characterized in that, the substratum of described step (1) was 120～125 ℃ of sterilizations 30～35 minutes, in cooling, the rearmounted fermentor tank of inoculation, sample-loading amount is 40～50% of a fermentor tank capacity, under 10～12 ℃, and ventilation 1:0.5～0.6, stir 320～330 rev/mins, cultivated 20～28 hours.
4. the preparation method of properly cooled proteinase with special flavor as claimed in claim 1 or 2 is characterized in that, the sample-loading amount of the substratum of described step (2) is 40～50% of a fermentor tank capacity, sterilization, and pH7.0～7.5, inoculation, in 200～220 rev/mins, control ventilation (V
Wind/ V
LiquidTime): 0-20 hour 1:0.5～0.6, under 20-50 hour 1:0.8～0.9 and the control culture temperature 10～12 ℃, fermented 43～50 hours.
5. the preparation method of properly cooled proteinase with special flavor as claimed in claim 1 or 2 is characterized in that, described step (1) substratum adds soya-bean oil and makes defoamer for 0.3～0.35 part.
6. the preparation method of properly cooled proteinase with special flavor as claimed in claim 1 or 2 is characterized in that, described step (1) substratum adds soya-bean oil and makes defoamer for 1～1.2 part.
7. the preparation method of properly cooled proteinase with special flavor as claimed in claim 1 or 2, it is characterized in that, the making of eggplant bottle slant strains in the described step (1), the concrete operations step: with 0.2～0.4 portion of extractum carnis, 0.8～1.2 part of peptone, 1.5～2 parts of agar, 0.4～0.6 part of NaCl is substratum, 95～100 parts in water, pH is 7.0～7.2, sterilization, on the test tube slant, line connects deep sea cold-adaptive microbe bacterium strain pseudomonas Pseudomonas sp.SM9913 bacterial classification, cultivated 24～26 hours, and preserved for 12～15 ℃ in 4 ℃; Above-mentioned bacterial classification is moved into eggplant bottle inclined-plane, cultivated 24～26 hours for 12～15 ℃, wait to do bacteria suspension and use.
8.～and the application of kind of properly cooled proteinase with special flavor, comprise that the protease preparation that uses claim 1 to make is processed into the bright product of local flavor ice with meat, fish, shellfish.
9. the application of properly cooled proteinase with special flavor as claimed in claim 8, it is characterized in that, basic raw material: just butchered clean livestock and poultry meat, firm shell clean shellfish or handle clean fish, taken by weighing 98～100 parts of raw materials of fresh weight, then at its surface spray weight percent 1～2% above-mentioned properly cooled proteinase with special flavor, pack, vacuum packaging, cooling fast, secondary package, go into 0 ℃ of storehouse, become the bright product of local flavor ice.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011274050A CN1141388C (en) | 2001-09-13 | 2001-09-13 | Prepn. and application of properly cooled proteinase with special flavor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB011274050A CN1141388C (en) | 2001-09-13 | 2001-09-13 | Prepn. and application of properly cooled proteinase with special flavor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1336433A true CN1336433A (en) | 2002-02-20 |
| CN1141388C CN1141388C (en) | 2004-03-10 |
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ID=4667363
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB011274050A Expired - Fee Related CN1141388C (en) | 2001-09-13 | 2001-09-13 | Prepn. and application of properly cooled proteinase with special flavor |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100343386C (en) * | 2005-01-12 | 2007-10-17 | 中国水产科学研究院黄海水产研究所 | Hypothermal alkaline metal protease of marine microorganism and its enzyme producing strain |
| CN102168018A (en) * | 2010-12-01 | 2011-08-31 | 山东大学 | Trichoderma sp. K9301 strain and application thereof in preparation of Trichoderma sp. protease |
| CN102321689A (en) * | 2011-08-18 | 2012-01-18 | 山东福洋生物科技有限公司 | Semicontinuous fermentation technology of gluconate |
-
2001
- 2001-09-13 CN CNB011274050A patent/CN1141388C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100343386C (en) * | 2005-01-12 | 2007-10-17 | 中国水产科学研究院黄海水产研究所 | Hypothermal alkaline metal protease of marine microorganism and its enzyme producing strain |
| CN102168018A (en) * | 2010-12-01 | 2011-08-31 | 山东大学 | Trichoderma sp. K9301 strain and application thereof in preparation of Trichoderma sp. protease |
| CN102168018B (en) * | 2010-12-01 | 2012-11-07 | 山东大学 | Trichoderma sp. K9301 strain and application thereof in preparation of Trichoderma sp. protease |
| CN102321689A (en) * | 2011-08-18 | 2012-01-18 | 山东福洋生物科技有限公司 | Semicontinuous fermentation technology of gluconate |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1141388C (en) | 2004-03-10 |
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