CN102224938B - Application of cold-adapted protease MCP-01 for tenderizing meat - Google Patents
Application of cold-adapted protease MCP-01 for tenderizing meat Download PDFInfo
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- CN102224938B CN102224938B CN201110121380A CN201110121380A CN102224938B CN 102224938 B CN102224938 B CN 102224938B CN 201110121380 A CN201110121380 A CN 201110121380A CN 201110121380 A CN201110121380 A CN 201110121380A CN 102224938 B CN102224938 B CN 102224938B
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Abstract
The invention relates to an application of cold-adapted protease MCP-01 for tenderizing meat, which belongs to the technical field of food biological technology. The present invention has the following technical schemes for tenderizing meat: (1) preparing cold-adapted protease MCP-01 to a cold-adapted protease MCP-01 solution with the concentration of 5 - 20 micrograms/ml; (2) washing the meat required to be tenderized, and immersing into the cold-adapted protease MCP-01 solution obtained in step (1), storing at the temperature of 0 - 8 DEG C for 18 - 30 hours. The protease is capable of specifically degrading collagen in connective tissue of meat under the temperature of 0 - 4 DEG C, which has good potential application in the process of tenderizing meat.
Description
Technical field
The present invention relates to the application of cold-adapted protease MCP-01 in tenderization, belong to the food biotechnology technical field.
Background technology
China is maximum in the world meat production state, but new and high technology is also weak in the application of China's meat products processing industry, and the meat products of intensive processing seldom.In the edible quality of meat, tender degree is one of organoleptic indicator of paying attention to the most of consumer.The soft delicacy of meat and the meat of succulence, the consumer relatively takes like a shot.The hardness of meat comprises two parts: changed actomyosin hardness and because the background hardness that the connective tissue variation causes that causes by muscle fibril.At present, the method for tenderization has a lot, but it seems from effect, adopts the method for protease tenderization the most advanced.Its meat protein of can under proper temperature, leniently degrading, thus tender degree improved.Method is easy, and the used time is short, and mouthfeel is unique, significantly improves business efficiency, avoids unnecessary waste.
Be used at present commercial tenderization enzyme several big type of vegetable protein enzyme (papain, bromelain, ficin), bacterial protein enzyme (the hear resistance protease of the alkali protease of hay bacillus, neutral proteinase, bacillus alcalophilus), animal protease (pancreatin, clostridiopetidase A) etc. have been arranged.These enzymes all have the ability of degrade collagen albumen or elastin laminin.High temperature protease during research at present and all tenderization enzymes of using are, the righttest enzyme of these protease live temperature all 50 ℃ or more than, it is very low to live at 0-4 ℃ of following enzyme, is therefore generally using more than 40 ℃.In addition, middle high temperature protease is more stable to ratio of specific heat, and this can make the tenderization enzyme after accomplishing tenderization, be difficult to deactivation.The right former protease of cold glue has higher enzyme under low temperature and room temperature lives, and can at room temperature carry out quick tenderization to meat products.And fit cold enzyme and have heat sensitivity, inactivation easily just under mesophilic condition, thus tendcrizing enzyme is inactivated after accomplishing tenderization easily.These characteristics of fitting the former protease of cold glue make it in tenderization, have good application potential.Therefore, fit the former protease of cold glue and have broad application prospects in aspect the tenderization of meat products, but still do not have the report of the former protease of right cold glue of tenderization function at present, also do not have the commercial cold meat Collagenase of fitting.
Publication number is: (application number: one Chinese patent application 200710013366.2) discloses Gene MCP 01 of a kind of cold-adapted protease and preparation method thereof to CN101045933A, belongs to biological technical field.This invention contains the 2676BP genomic DNA fragment of deep-sea cold-adapted protease Gene MCP 01; Wherein contain a open reading frame with 2508 nucleotides; This open reading frame promptly is the gene of encoding proteins enzyme MCP-01, and the righttest enzyme of the cold-adapted protease temperature of being expressed by the Gene MCP 01 of protease MCP-01 alive is about 35 ℃, and optimal pH is 9.0; Can be used for low temperature meat fresh-keeping, increase industries such as aquatic foods, the processing of dairy products, washing agent.But this application is only mentioned cold-adapted protease MCP-01 and is had fresh-keeping, as to increase aquatic foods effect, does not mention its effect aspect the low temperature tenderization.
Summary of the invention
The present invention is directed to the deficiency of prior art, cold-adapted protease MCP-01 is provided the application in tenderization.This cold-adapted protease MCP-01 can carry out tenderization to meat in the meat preservation cold storage procedure or under the normal temperature, improve the mouthfeel of meat.
The application of cold-adapted protease MCP-01 in the meat products tenderization.
Above-mentioned application, step is following:
(1) cold-adapted protease MCP-01 is mixed with the cold-adapted protease MCP-01 solution that concentration is 5～20 μ g/ml;
(2) after the meat that will treat tenderization is cleaned, immerse in the cold-adapted protease MCP-01 solution that step (1) makes 0～8 ℃ of preservation 18～30 hours.
The cold-adapted protease MCP-01 solution concentration of said step (1) is 10～15 μ g/ml.
In the said step (2) 0～4 ℃ of preservation 20～25 hours.
Above-mentioned preservation temperature is 4 ℃.
Meat in the said step (2) is the animal tissue of containing the various human consumptions of collagen.
The preparation of above-mentioned cold-adapted protease MCP-01 can make through following bacterial strain:
Deep sea cold-adaptive microbe bacterium strain Pseudoalteromonas strain (Pseudoalteromonas) SM9913 was preserved in Chinese typical culture collection center, China on December 7th, 2010. Wuhan. and Wuhan University.Culture presevation CCTCC NO:M2010336.
Above-mentioned cold-adapted protease MCP-01 above-mentioned bacterial strains capable of using makes through following method:
Get deep sea cold-adaptive microbe bacterium strain Pseudoalteromonas strain SM9913 and after seed culture, be inoculated in the liquid fermentation medium, 10～15 ℃ of concussions were cultivated 55～70 hours, first centrifugal after; It is 50～55wt% that supernatant adds ammonium sulfate powder to final concentration, and after the secondary centrifuging, deposition is dissolved among the Tris-HCl; Three times centrifugal after, get supernatant, 2～8 ℃ of following Macrogol 2000 0 ultrafiltration concentrate; Concentrate through column chromatography for separation, reclaims the peak nose part at enzyme peak alive under 2～8 ℃, promptly get cold-adapted protease MCP-01.
Said seed culture be in solid medium 12～15 ℃ cultivate after 48～70 hours; 12～15 ℃ of concussions were cultivated 20～30 hours in fluid nutrient medium again; The solid medium component is following, is weight portion:
1 part of peptone, 0.5 part, 1.5 parts agar of dusty yeast, 100 parts of artificial seawaters, pH are 7.0～7.2;
The fluid nutrient medium component is following, is weight portion:
1 part of peptone, 0.5 part, 1.5 parts agar of dusty yeast, 100 parts of artificial seawaters, pH are 7.0～7.2.
Said first centrifugal condition is: 10000g, and 2～8 ℃ are centrifugal 15 minutes; The secondary centrifuging condition is: 8000g, and 2～8 ℃ are centrifugal 10 minutes; Three times centrifugal condition is: 10000g, 2～8 ℃ centrifugal 20 minutes.
The concentration of said Tris-HCl is 20mmol/l, and pH 8.5; Said chromatographic column is a Sephardex G100 chromatographic column.
Said liquid fermentation medium component is following, is weight portion:
3.5～4.0 parts in soya-bean cake powder, 2.0～2.5 parts of corn flour, 2.0～2.5 parts in wheat bran, Na
2HPO
40.3～0.4 part, KH
2100 parts in P040.04～0.05 part, water.
Excellent results of the present invention is:
(1) cold-adapted protease MCP-01 according to the invention still can keep 12.5% of enzyme activity under the optimum temperature in the time of 0 ℃, under (0-4 ℃) and room temperature under the deepfreeze condition, can improve the tender degree of meat products; This protease can have good application potential at the collagen in 0～4 ℃ of following specificity degraded meat connective tissue in the tenderization process.
(2) heat endurance of cold-adapted protease MCP-01 is relatively poor, under middle hot conditions, is easy to inactivation, thereby enzyme is inactivated after accomplishing tenderization easily; This enzyme is along with the raising of temperature, and self-dissolving speed increases, thus after meat is cooked processing, this enzyme meeting complete deactivation, thereby can not cause pollution to meat.
(3) cold-adapted protease MCP-01 can keep the fresh color and luster and the retentiveness of meat products preferably.
Description of drawings
The Capillary Electrophoresis of the cold-adapted protease MCP-01 of Fig. 1, purifying detects figure;
Fig. 2, temperature are to the influence of the soluble collagen vigor of cold-adapted protease MCP-01 degraded ox tendon;
Fig. 3, cold-adapted protease MCP-01 handle the block diagram to the influence of ox chest muscle shearing force;
Fig. 4, different tendcrizing enzymes are handled the block diagram to the influence of ox chest muscle percentage of water loss;
Wherein: contrast, do not add any protease; MCP-01, interpolation cold-adapted protease MCP-01; Bromelain, interpolation bromelain; Papain, interpolation papain;
Fig. 5, different tendcrizing enzymes are handled the photo to the influence of ox chest muscle color and luster;
Wherein: Control, do not add any protease; MCP-01, interpolation cold-adapted protease MCP-01; Bromelain, interpolation bromelain; Papain, interpolation papain.
The specific embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further specified, but be not limited thereto.
The Pseudoalteromonas of deep sea cold-adaptive microbe bacterium strain described in embodiment strain (Pseudoalteromonas) SM9913 was preserved in Chinese typical culture collection center, China on December 7th, 2010. Wuhan. and Wuhan University, culture presevation CCTCC NO:M2010336.
The artificial seawater element is available from the plain factory of Chinese Marine University seawater, and elastin laminin is available from Sigma company, and the soluble collagen of fish is for adopting method (Song, the E. of Song etc.; Kim, S.Y., Chun, T.; Byun, H.-J., and Lee; Y.M. (2006) Biomaterials 27 2951-2961) extracts from fish-skin, and the soluble collagen of ox tendon is available from WorthingtonBiochemical Co.; Ox acid is dissolved type i collagen albumen available from Sigma company, and ox II collagen type is available from Sigma company, and ox IV collagen type is available from Sigma company.
Embodiment 1
Deep sea cold-adaptive microbe bacterium strain Pseudoalteromonas strain SM9913 prepares the method for cold-adapted protease MCP-01, and step is following:
1. the preparation of liquid seeds
With 1 part of peptone, 0.5 part, 1.5 parts agar of dusty yeast, 100 parts of artificial seawaters, pH are 7.0～7.2, (being weight portion) preparation solid medium.Medium sterilization, after the cooling, streak inoculation deep sea cold-adaptive microbe bacterium strain Pseudoalteromonas strain (Pseudoalteromonas) SM9913 on the test tube slant cultivated 48～70 hours for 12～15 ℃.Above-mentioned bacterial classification inoculation is equipped with in the 500ml triangular flask of 150ml fluid nutrient medium (the same solid medium of filling a prescription does not add agar), 12～15 ℃, 200 change concussion cultivated 24 hours, as liquid seeds.
2. liquid deep layer fermenting prepares the cold-adapted protease preparation
With 3.5～4.0 parts in soya-bean cake powder, 2.0～2.5 parts of corn flour, 2.0～2.5 parts in wheat bran, Na
2HPO
40.3～0.4 part, KH
2PO
40.04～0.05 part with 100 parts in water, be weight portion, the obtaining liq fermentation medium.Medium sterilization, the liquid seeds by 1 preparation of 5～7% percentage by weight inoculation steps ventilates, stirs, controls rotating speed 200rpm, cultivates 60 hours for 15 ℃.
3. the separation and purification of cold-adapted protease MCP-01
With 4 ℃ of above-mentioned enzymatic production culture mediums, 10, centrifugal 15 minutes of 000g, supernatant slowly stir adding ammonium sulfate powder in ice bath be 55wt% to final concentration, centrifugal 10 minutes of 8000g.Deposition is dissolved in pH 8.5, and among the 20mmol/l Tris-HCl, in 4 ℃, centrifugal 20 minutes of 10000g gets supernatant, is loaded on the ultrafiltration bag, and 4 ℃ concentrate with Macrogol 2000 0 down, and concentrate reclaims the peak nose part at enzyme peak alive through Sephardex G100 column chromatography for separation.All operations are all being carried out below 15 ℃.The protease that separates detects purity (like Fig. 1) with Capillary Electrophoresis, called after cold-adapted protease MCP-01.
4. the property testing of cold-adapted protease MCP-01
4.1 substrate specificity
(1) protein concentration of the cold-adapted protease MCP-01 of Bradford method determination step 3 preparations.(the Bradford method is this area conventional techniques, also can with reference to the data http://wenku.baidu.com/view/7b3eb1d4195f312b3169a521.html in the following network address).
(2) measure the degradation efficiency of enzyme to soluble collagen, soluble collagen comprises soluble collagen of fish and the soluble collagen of ox tendon.Enzyme unit definition alive produces the needed enzyme amount of L-leucine of 1nmol for the substrate of per hour degrading.Concrete steps are: take by weighing the soluble collagen of 5mg, adding 1ml concentration is the MCP-01 enzyme liquid of 10 μ g/ml, places 40 ℃ of water-baths to react 5h.Constantly rock reactant liquor in the reaction, to guarantee fully contacting of soluble substrate and enzyme liquid.
(3) measure enzyme to the soluble substrate degradation efficiency, enzyme unit definition alive is the needed enzyme amount of L-leucine that per minute degraded substrate produces 1nmol.Concrete steps are: (contain 0.36mM CaCl with 50mM Tris-HCl
2, pH 7.5) and the different solvable substrate solution of preparation, comprise the molten type i collagen albumen of ox acid, ox II collagen type, ox IV collagen type, place 40 ℃ of water-baths to react 30min.
(4) after above-mentioned steps (2) (3) reaction finishes, 10, the centrifugal 10min of 000g.Get supernatant 20 μ l and add 100 μ l ninhydrin-sodium citrate solutions, boiling water bath 20min colour developing, the cooling back adds 500 μ l normal propyl alcohols, measures light absorption value in the 600nm place.
(5) measure the degradation efficiency of enzyme to the elastin laminin substrate, the elastin laminin that takes by weighing 5mg adds the MCP-01 enzyme liquid that 250 μ l concentration are 10 μ g/ml, places 40 ℃ of water-bath reactions 60 minutes, and 10, the centrifugal 10min cessation reaction of 000g; Get supernatant and measure light absorption value in the 595nm place.
(6) result show cold-adapted protease MCP-01 can specificity degraded all kinds collagen, and elastin laminin is not had degradation (result is as shown in table 1).
The temperature measuring 4.2 the righttest enzyme is lived
With the soluble collagen of ox tendon is substrate, and 50mM Tris-HCl (contains 0.36mM CaCl
2, pH 7.5) and be buffer system, under 0,10,20,30,40,50,55,60,70,80 ℃ of condition, measure the enzyme of Collagenase MCP-01 respectively and live, to confirm the optimal reactive temperature of enzyme.Measuring enzyme concrete grammar alive is:
Take by weighing the soluble collagen of ox tendon of 5mg, adding 1ml concentration is the cold-adapted protease MCP-01 enzyme liquid of 10 μ g/ml, and reaction is 5 hours under the different temperatures, 10, and the centrifugal 10min of 000g.Get supernatant 20 μ l and add 100 μ l ninhydrin-sodium citrate solutions, boiling water bath 20min develops the color, and the cooling back adds 500 μ l normal propyl alcohols, measures light absorption value in the 600nm place, and the enzyme activity that defines under the highest enzyme temperature alive is 100%.The result shows 60 ℃ of the righttest enzyme of this enzyme temperature alive, under 0 ℃, has still kept 12.4% relative activity, significantly descends as shown in Figure 2 at 70 ℃ of enzyme activities.
Embodiment 2
The application of cold-adapted protease MCP-01 in the meat products tenderization, step is following:
(I) the cold-adapted protease MCP-01 that step 3 is made is mixed with the cold-adapted protease MCP-01 solution that concentration is 10 μ g/ml and 100 μ g/ml;
(II) ox chest muscle meat is cleaned after, immerse in the cold-adapted protease MCP-01 solution that step (I) makes 4 ℃ of preservations 20 hours.
The result show add cold-adapted protease MCP-01 4 ℃ down 20 hours ox chest muscle meat shearing forces of effect significantly descend, and the control group ox chest muscle meat shearing force of not adding cold-adapted protease descends not obvious.(like Fig. 3).
Ox chest muscle meat was handled 20 hours down at 4 ℃ with bromelain and papain with same concentrations respectively, measured percentage of water loss then.The definition percentage of water loss is:
Can know that with papain and bromelain contrast that commercialization is used the retentiveness of meat products is better, color and luster is also fresher, and result such as Fig. 4 are shown in Figure 5.
Cold-adapted protease MCP-01 of the present invention, can various each position of animal fresh or stored under refrigeration of tenderization can be edible the meat tissue of containing collagen.
Interpretation of result
Separate the cold-adapted protease MCP-01 that obtains from a strain deep sea cold-adaptive microbe bacterium strain Pseudoalteromonas strain (Pseudoalteromonas sp.SM9913); The about 60kDa of molecular weight (Fig. 1); Has very high collagen degrading activity, the various solvable and insoluble collagens (table 1) of can specificity degrading.Optimum temperature (Fig. 2) through measuring MCP-01 degrade collagen albumen shows that cold-adapted protease MCP-01 is the former protease of right cold glue, has very high collagen activity at low temperatures, and high temperature is unstable down.We have studied tenderization ability and the tenderization mechanism of MCP-01 to meat products, and compare with present commercial papain and bromelain.The result shows that cold-adapted protease MCP-01 can significantly reduce meat shearing force (Fig. 3) under 4 ℃, compare with bromelain with papain, and can keep preferably meat products fresh color and luster and water content (Fig. 4, Fig. 5).These results show that MCP-01 might be developed to a kind of novel cold tenderization enzyme of fitting, and is used for the low temperature tenderization of meat products, in the tenderization of meat products low temperature, has using value.
Table 1MCP-01 is to the catalytic degradation efficient of different collagen substrates
Claims (2)
1. the application of cold-adapted protease MCP-01 in the meat products tenderization is characterized in that step is following:
(1) cold-adapted protease MCP-01 is mixed with the cold-adapted protease MCP-01 solution that concentration is 5～20 μ g/ml;
(2) after the meat that will treat tenderization is cleaned, immerse in the cold-adapted protease MCP-01 solution that step (1) makes 0～8 ℃ of preservation 18～30 hours.
2. application as claimed in claim 1 is characterized in that, the cold-adapted protease MCP-01 solution concentration of said step (1) is 10～15 μ g/ml.
3, application as claimed in claim 1 is characterized in that, in the said step (2) 0～4 ℃ of preservation 20～25 hours.
4, application as claimed in claim 3 is characterized in that, above-mentioned preservation temperature is 4 ℃.
5, application as claimed in claim 1 is characterized in that, the meat in the said step (2) is the animal tissue of containing the various human consumptions of collagen.
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Families Citing this family (5)
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| CN102719421B (en) * | 2012-06-01 | 2013-06-19 | 中国科学院南海海洋研究所 | Marine bacteria cold-adapted protease and encoding gene and application thereof |
| CN102994601B (en) * | 2012-12-13 | 2014-01-29 | 山东大学 | Method for preparing collagen small peptide by utilizing marine collagenase MCP-01 |
| CN105349605A (en) * | 2015-12-04 | 2016-02-24 | 山东大学 | Method for efficiently preparing low-molecular-weight fish skin collagen peptide through enzyme method |
| CN105524965A (en) * | 2016-02-26 | 2016-04-27 | 福建华灿制药有限公司 | Extraction method of bovine tendon collagen |
| CN112322533B (en) * | 2020-11-09 | 2022-05-03 | 山东大学 | Strain for producing efficient collagenase and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1368854A (en) * | 1999-07-22 | 2002-09-11 | 诺沃奇梅兹北美公司 | Meat tenderization |
| CN101045933A (en) * | 2007-03-12 | 2007-10-03 | 山东大学 | Cryophilous proteinase gene mcp01 and its prepn process |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1368854A (en) * | 1999-07-22 | 2002-09-11 | 诺沃奇梅兹北美公司 | Meat tenderization |
| CN101045933A (en) * | 2007-03-12 | 2007-10-03 | 山东大学 | Cryophilous proteinase gene mcp01 and its prepn process |
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