CN110105445A - The extracting method of myoglobins in a kind of beef - Google Patents
The extracting method of myoglobins in a kind of beef Download PDFInfo
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- CN110105445A CN110105445A CN201910349575.7A CN201910349575A CN110105445A CN 110105445 A CN110105445 A CN 110105445A CN 201910349575 A CN201910349575 A CN 201910349575A CN 110105445 A CN110105445 A CN 110105445A
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Abstract
The present invention provides a kind of extracting method of myoglobins in beef, this method is that Tris-HCl cocktail buffer, homogenate, standing, centrifugation are added into beef meat sample, and (NH is added4)2SO4It stands, after centrifugation, dialyses after adding Tris-HCl buffer b, afford myoglobins in beef with NaCl mixed solution in chromatographic column.The present invention optimizes Tris-HCl cocktail buffer pH value, extraction time, myoglobins are saltoutd, and liquid saturation degree improves the extracted amount of myoglobins；Contain glycerol and dithiothreitol (DTT) in Tris-HCl cocktail buffer of the present invention, glycerol maintains Myoglobin stability, is conducive to rapid precipitation and is precipitated, dithiothreitol (DTT) keeps the reproducibility and bioactivity of myoglobins, reinforcing body (NH4)2SO4It saltouts fractional precipitation, improves myoglobins purity, 4 DEG C carry out preventing myoglobins denaturation oxidation, dialysis can effectively remove remaining salt ion and his small protein, the purity of myoglobins is improved, gel permeation chromatography separation is finally carried out, does not destroy the structure and function of myoglobins.
Description
Technical field
The invention belongs to Protein Extraction technical fields, and in particular to the extracting method of myoglobins in a kind of beef.
Background technique
In beef myoglobins method for extraction and purification, Tris-HCl method is common myoglobins coarse extraction method, but
Existing myoglobins extractive technique, there is musculature ingredient is complex, myoglobins precipitates with being difficult to fast and stable；And
The problems such as (oxygen conjunction) myoglobins itself is easily oxidized, and is unfavorable for the preservation application of sample after purification, need to promote myoglobins and mention
Stability in pure procedure reduces the oxidation and denaturation loss of myoglobins.
Summary of the invention
Technical problem to be solved by the present invention lies in view of the above shortcomings of the prior art, providing, flesh in a kind of beef is red
The extracting method of albumen, pH value and extraction time, step of the invention by Tris-HCl cocktail buffer in Optimization Steps one
Myoglobins is saltoutd the saturation degree of liquid in two, effectively improves the extracted amount of myoglobins in beef；Tris- of the invention
Contain glycerol and dithiothreitol (DTT) in HCl cocktail buffer, glycerol forms lipoid membrane structure, effectively in muscle cell slurry
Myoglobin stability is maintained, the rapid precipitation for being more advantageous to it is precipitated, and dithiothreitol (DTT) avoids myoglobins portion
The oxidation for dividing sensitivity site, maintains its reproducibility and bioactivity, is then slowly uniformly slowly uniformly added into while stirring solid
Body (NH4)2SO4It saltouts, can be realized fractional precipitation, the purity of myoglobins can be effectively improved by making to saltout, under the conditions of 4 DEG C
Precipitating proteins can prevent temperature raising from leading to myoglobins denaturation oxidation, and the dialysis after saltouing can effectively remove remaining
(NH4)2SO4Salt ion and other small proteins, further increase the purity of myoglobins, finally carry out Gel filtration
Analysis, myoglobins is efficiently separated out, and will not destroy the structure and function of myoglobins.
In order to solve the above technical problems, the technical solution adopted by the present invention is that: the extraction side of myoglobins in a kind of beef
Method, method includes the following steps:
Step 1: slightly mentioning: beef longissimus dorsi muscle is taken, surface fascia and connective tissue are removed, chopping obtains beef meat sample, to
Beef meat sample be added concentration be 10mmol/L, the Tris-HCl cocktail buffer that pH value is 8.0~8.4, be then in revolving speed
After being homogenized 1min under conditions of 10000rpm, is stood under conditions of temperature is 4 DEG C and extract 1h~2h, be then in revolving speed
9600rpm, temperature filter after being centrifuged 10min under conditions of being 4 DEG C, obtain filtrate；Contain in the Tris-HCl cocktail buffer
There are the two of the glycerol and 1mmol/L that concentration is EDTA, 0.54mmol/L of Tris-HCl buffer a, 1mmol/L of 10mmol/L
Sulphur threitol；
Step 2: saltouing: being slowly uniformly added into solid (NH while stirring into filtrate obtained in step 14)2SO4, obtain
Saltout liquid to myoglobins, when myoglobins saltout the saturation degree of liquid be 85%~95% when, it is quiet under conditions of temperature is 4 DEG C
30min precipitating proteins are set, then 30min is centrifuged under conditions of revolving speed is 10000rpm, temperature is 4 DEG C, obtains flesh red eggs
White sediment；
Step 3: dialysis: into myoglobins sediment obtained in step 2, addition concentration is 10mmol/L, pH value is
After 8.5 Tris-HCl buffer b stirring and dissolving, it is placed in the bag filter that molecular cut off is 8kDa~14kDa, concentration is added
The Tris-HCl buffer c for being 8.5 for 10mmol/L, pH value is stirred under conditions of temperature is 0 DEG C~4 DEG C and is dialysed for 24 hours, thoroughly
The Tris-HCl buffer b is replaced 10 times during analysis, obtains high-purity myoglobins；
Step 4: gel permeation chromatography: by Sephadex G-75 chromatographic column with concentration be 1mmol/L, pH value is 8.5
After Tris-HCl buffer d balance, the NaCl mixed solution for being 0.1mol/L with concentration is to high-purity flesh obtained in step 3
Lactoferrin carries out linear elution, flow velocity 2mL/min, and continuous collection multitube eluent to elution finishes, and every pipe eluent exists
It is detected under the ultraviolet light that wavelength is 280nm, merges the maximum eluent of eluting peak absorption value, obtain flesh red eggs in beef
It is white；The NaCl mixed solution contains the Tris-HCl of the EDTA and 10mmol/L of NaCl, 1mmol/L that concentration is 0.1mol/L
Buffer e.
Preferably, the pH value of Tris-HCl cocktail buffer described in step 1 is 8.2, extraction time 1.5h；Step
Myoglobins described in two saltout liquid saturation degree be 90%.
Preferably, the pH value of Tris-HCl cocktail buffer described in step 1 is 8.15, extraction time 1.63h；Step
Myoglobins described in rapid two saltout liquid saturation degree be 90.93%.
Preferably, the quality and volume ratio of beef meat sample described in the step 1 and Tris-HCl cocktail buffer are
1g:5mL.
Preferably, the quality and volume ratio of myoglobins sediment described in step 3 and Tris-HCl buffer b are 1g:
2mL。
Preferably, the pH value of NaCl mixed solution described in step 4 is 8.4.
The trishydroxymethylaminomethane and hydrochloric acid mixture that Tris-HCl, that is, molar concentration rate in the present invention is 1:1.
Compared with the prior art, the present invention has the following advantages:
In pH value and extraction time, step 2 of the present invention invention by Tris-HCl cocktail buffer in Optimization Steps one
Myoglobins is saltoutd the saturation degree of liquid, and the extracted amount of myoglobins in beef is effectively improved；Tris-HCl of the invention is mixed
It closes and contains glycerol and dithiothreitol (DTT) in buffer, glycerol forms lipoid membrane structure, effectively maintain in muscle cell slurry
Myoglobin stability, the rapid precipitation for being more advantageous to it are precipitated, and dithiothreitol (DTT) avoids myoglobins part sensitivity
The oxidation in site maintains its reproducibility and bioactivity, is then slowly uniformly slowly uniformly added into solid (NH while stirring4)2SO4It saltouts, can be realized fractional precipitation, the purity of myoglobins, protein precipitation under the conditions of 4 DEG C can be effectively improved by making to saltout
Matter, can prevent temperature raising from leading to myoglobins denaturation oxidation, and the dialysis after saltouing can effectively remove remaining (NH4)2SO4
Salt ion and other small proteins, further increase the purity of myoglobins, finally carry out gel permeation chromatography, and flesh is red
Albumen efficiently separates out, and will not destroy the structure and function of myoglobins.
Invention is further described in detail with reference to the accompanying drawings and examples.
Detailed description of the invention
Fig. 1 is 1 myoglobins chromatography elution curves of the embodiment of the present invention.
Fig. 2 is the beef that the Tris-HCl cocktail buffer of the different pH value of the embodiment of the present invention 1 and comparative example 1 is extracted
Middle myoglobin content.
Fig. 3 be the different saturation of the embodiment of the present invention 1 and comparative example 2 myoglobins saltout liquid extraction beef in
Myoglobin content.
Fig. 4 is that myoglobins contains in the beef of the different extraction times extractions of the embodiment of the present invention 1 and comparative example 3
Amount.
Fig. 5 is the pH and extraction time interactive response song of the Tris-HCl cocktail buffer of the embodiment of the present invention 4
Face.
Fig. 6 is that the myoglobins of the embodiment of the present invention 4 is saltoutd the saturation degree and extraction time interactive response song of liquid
Face.
Fig. 7 be the Tris-HCl cocktail buffer of the embodiment of the present invention 4 pH and myoglobins saltout liquid saturation degree hand over
The response surface design of interaction.
Specific embodiment
Embodiment 1
The extracting method of myoglobins in the beef of the present embodiment, method includes the following steps:
Step 1: slightly mentioning: beef longissimus dorsi muscle is taken, surface fascia and connective tissue are removed, chopping obtains beef meat sample, to
100g beef meat sample be added 500mL concentration be 10mmol/L, the Tris-HCl cocktail buffer that pH value is 8.2, then turn
After speed is homogenized 1min under conditions of being 10000rpm, is stood under conditions of temperature is 4 DEG C and extract 1.5h, be then in revolving speed
9600rpm, temperature filter after being centrifuged 10min under conditions of being 4 DEG C, obtain filtrate；Contain in the Tris-HCl cocktail buffer
There are the two of the glycerol and 1mmol/L that concentration is EDTA, 0.54mmol/L of Tris-HCl buffer a, 1mmol/L of 10mmol/L
Sulphur threitol；
Step 2: saltouing: being slowly uniformly added into solid (NH while stirring into filtrate obtained in step 14)2SO4, obtain
Saltout liquid to myoglobins, when myoglobins saltout the saturation degree of liquid be 90% when, stood under conditions of temperature is 4 DEG C
Then 30min precipitating proteins are centrifuged 30min under conditions of revolving speed is 10000rpm, temperature is 4 DEG C, obtain myoglobins
Sediment；
Step 3: dialysis: the concentration that 36mL is added into myoglobins sediment obtained in 18g step 2 is
After 10mmol/L, the Tris-HCl buffer b stirring and dissolving that pH value is 8.5, being placed in molecular cut off is the saturating of 8kDa~14kDa
It analyses in bag, the Tris-HCl buffer c that addition concentration is 10mmol/L, pH value is 8.5 is under conditions of temperature is 0 DEG C~4 DEG C
It stirs and dialyses for 24 hours, the b of Tris-HCl buffer described in dialysis procedure is replaced 10 times, obtains high-purity myoglobins；
Step 4: gel permeation chromatography: by Sephadex G-75 chromatographic column with concentration be 1mmol/L, pH value is 8.5
After Tris-HCl buffer d balance, the NaCl mixed solution for being 0.1mol/L with the concentration that pH value is 8.4 in step 3 to obtaining
High-purity myoglobins carry out linear elution, flow velocity 2mL/min is continuous to collect multitube eluent to elution and finish, every pipe
Eluent is detected in the case where wavelength is the ultraviolet light of 280nm, is merged the maximum eluent of eluting peak absorption value, is obtained beef
Middle myoglobins；The NaCl mixed solution contains the EDTA's and 10mmol/L of NaCl, 1mmol/L that concentration is 0.1mol/L
Tris-HCl buffer e.
The trishydroxymethylaminomethane and hydrochloric acid mixture that Tris-HCl, that is, molar concentration rate in the present embodiment is 1:1.
Fig. 1 is the myoglobins chromatography elution curves of the present embodiment, as shown in Figure 1, after gel permeation chromatography, in wavelength
To there is 2 main absorption peaks at 280nm, the 2nd peak absorption value is maximum, and corresponding eluent shows pale red brown, the 2nd main suction
Receive myoglobins in the i.e. corresponding beef in peak, calculate in the beef that extracts of the present embodiment the content of myoglobins be 1.62mg/mL.
Comparative example 1
For the extracting method of myoglobins with embodiment 1, difference is Tris- in step 1 in the beef of this comparative example
The pH value of HCl cocktail buffer is respectively 7.8,8.0,8.4 and 8.6, extracts obtain myoglobins CK1, myoglobins CK2 respectively
With myoglobins myoglobins CK3.
Fig. 2 is flesh red eggs in the beef of the Tris-HCl cocktail buffer extraction of the different pH value of embodiment 1 and comparative example 1
Bai Hanliang, as shown in Figure 2, myoglobin content are mixed with the increase of the pH of Tris-HCl cocktail buffer in Tris-HCl
When the pH value of buffer is 8.2, the content of the middle myoglobins of extraction is up to 1.62mg/mL, and Tris-HCl mixing buffering
The pH of liquid is continued growing, and myoglobin content is decreased obviously, the myoglobins CK3 and CK1 extracted when pH is 8.6 and pH is 7.8
Content it is lower, respectively 1.40mg/mL and 1.51mg/mL.Illustrate slightly to propose middle Tris-HCl cocktail buffer selection properly
PH help to improve the content of myoglobins in the beef finally extracted.
Comparative example 2
The extracting method of myoglobins with embodiment 1, saltout by myoglobins described in step 2 in the beef of this comparative example
The saturation degree of liquid is respectively 80%, 85%, 95% and 100%, extracts obtain myoglobins CK4, myoglobins CK5, flesh respectively
Lactoferrin CK6 and myoglobins myoglobins CK7.
Fig. 3 be the different saturation of embodiment 1 and comparative example 2 myoglobins saltout liquid extraction beef in myoglobins
Content, from the figure 3, it may be seen that as myoglobins is saltoutd the increasing of the saturation degree of liquid, the myoglobin content extracted is presented first
Downward trend after rising, at 90%, myoglobin content reaches maximum value 1.62mg/mL, on the whole, in myoglobins
The saturation degree of liquid of saltouing is in 85%~95% range, and the content of the myoglobins of extraction is relatively high, and myoglobins is saltoutd liquid
The content of myoglobins CK4 and CK6 extracted when being 80% and 100% of saturation degree it is lower, respectively 1.46mg/mL and
1.45mg/mL。
Comparative example 3
The extracting method of myoglobins is with embodiment 1 in the beef of this comparative example, the difference of extraction time described in step 1
For 0.5h, 1h, 2h and 2.5h, extracts obtain myoglobins CK8, myoglobins CK9, myoglobins CK10 and myoglobins respectively
CK11。
Fig. 4 is myoglobin content in the beef of the different extraction times extractions of embodiment 1 and comparative example 3, can by Fig. 4
Know, myoglobin content extends with extraction time, stepped up in 0-1.5h, at the extraction between be 1.5h when myoglobins
Content reaches maximum value 1.62mg/mL, then continues to extend extraction time, and myoglobin content is decreased obviously, at the extraction between
Within the scope of 1-2h, the content of the myoglobins of extraction is relatively high, the myoglobins that extraction time extracts when being 0.5h and 2.5h
The content of CK8 and CK11 is lower, respectively 1.49mg/mL and 1.46mg/mL.
Embodiment 2
The extracting method of myoglobins in the beef of the present embodiment, method includes the following steps:
Step 1: slightly mentioning: beef longissimus dorsi muscle is taken, surface fascia and connective tissue are removed, chopping obtains beef meat sample, to
100g beef meat sample be added 500mL concentration be 10mmol/L, the Tris-HCl cocktail buffer that pH value is 8.0, then turn
After speed is homogenized 1min under conditions of being 10000rpm, is stood under conditions of temperature is 4 DEG C and extract 1h, be then in revolving speed
9600rpm, temperature filter after being centrifuged 10min under conditions of being 4 DEG C, obtain filtrate；Contain in the Tris-HCl cocktail buffer
There are the two of the glycerol and 1mmol/L that concentration is EDTA, 0.54mmol/L of Tris-HCl buffer a, 1mmol/L of 10mmol/L
Sulphur threitol；
Step 2: saltouing: being slowly uniformly added into solid (NH while stirring into filtrate obtained in step 14)2SO4, obtain
Saltout liquid to myoglobins, when myoglobins saltout the saturation degree of liquid be 85% when, stood under conditions of temperature is 4 DEG C
Then 30min precipitating proteins are centrifuged 30min under conditions of revolving speed is 10000rpm, temperature is 4 DEG C, obtain myoglobins
Sediment；
Step 3: dialysis: the concentration that 36mL is added into myoglobins sediment obtained in 18g step 2 is
After 10mmol/L, the Tris-HCl buffer b stirring and dissolving that pH value is 8.5, being placed in molecular cut off is the saturating of 8kDa~14kDa
It analyses in bag, the Tris-HCl buffer c that addition concentration is 10mmol/L, pH value is 8.5 is under conditions of temperature is 0 DEG C~4 DEG C
It stirs and dialyses for 24 hours, the b of Tris-HCl buffer described in dialysis procedure is replaced 10 times, obtains high-purity myoglobins；
Step 4: gel permeation chromatography: by Sephadex G-75 chromatographic column with concentration be 1mmol/L, pH value is 8.5
After Tris-HCl buffer d balance, the NaCl mixed solution for being 0.1mol/L with the concentration that pH value is 8.4 in step 3 to obtaining
High-purity myoglobins carry out linear elution, flow velocity 2mL/min is continuous to collect multitube eluent to elution and finish, every pipe
Eluent is detected in the case where wavelength is the ultraviolet light of 280nm, is merged the maximum eluent of eluting peak absorption value, is obtained beef
Middle myoglobins；The NaCl mixed solution contains the EDTA's and 10mmol/L of NaCl, 1mmol/L that concentration is 0.1mol/L
Tris-HCl buffer e.
The trishydroxymethylaminomethane and hydrochloric acid mixture that Tris-HCl, that is, molar concentration rate in the present embodiment is 1:1.
The content of myoglobins is 1.58mg/mL in the beef that the present embodiment extracts.
Embodiment 3
The extracting method of myoglobins in the beef of the present embodiment, method includes the following steps:
Step 1: slightly mentioning: beef longissimus dorsi muscle is taken, surface fascia and connective tissue are removed, chopping obtains beef meat sample, to
100g beef meat sample be added 500mL concentration be 10mmol/L, the Tris-HCl cocktail buffer that pH value is 8.4, then turn
After speed is homogenized 1min under conditions of being 10000rpm, is stood under conditions of temperature is 4 DEG C and extract 2h, be then in revolving speed
9600rpm, temperature filter after being centrifuged 10min under conditions of being 4 DEG C, obtain filtrate；Contain in the Tris-HCl cocktail buffer
There are the two of the glycerol and 1mmol/L that concentration is EDTA, 0.54mmol/L of Tris-HCl buffer a, 1mmol/L of 10mmol/L
Sulphur threitol；
Step 2: saltouing: being slowly uniformly added into solid (NH while stirring into filtrate obtained in step 14)2SO4, obtain
Saltout liquid to myoglobins, when myoglobins saltout the saturation degree of liquid be 95% when, stood under conditions of temperature is 4 DEG C
Then 30min precipitating proteins are centrifuged 30min under conditions of revolving speed is 10000rpm, temperature is 4 DEG C, obtain myoglobins
Sediment；
Step 3: dialysis: the concentration that 36mL is added into myoglobins sediment obtained in 18g step 2 is
After 10mmol/L, the Tris-HCl buffer b stirring and dissolving that pH value is 8.5, being placed in molecular cut off is the saturating of 8kDa~14kDa
It analyses in bag, the Tris-HCl buffer c that addition concentration is 10mmol/L, pH value is 8.5 is under conditions of temperature is 0 DEG C~4 DEG C
It stirs and dialyses for 24 hours, the b of Tris-HCl buffer described in dialysis procedure is replaced 10 times, obtains high-purity myoglobins；
Step 4: gel permeation chromatography: by Sephadex G-75 chromatographic column with concentration be 1mmol/L, pH value is 8.5
After Tris-HCl buffer d balance, the NaCl mixed solution for being 0.1mol/L with the concentration that pH value is 8.4 in step 3 to obtaining
High-purity myoglobins carry out linear elution, flow velocity 2mL/min is continuous to collect multitube eluent to elution and finish, every pipe
Eluent is detected in the case where wavelength is the ultraviolet light of 280nm, is merged the maximum eluent of eluting peak absorption value, is obtained beef
Middle myoglobins；The NaCl mixed solution contains the EDTA's and 10mmol/L of NaCl, 1mmol/L that concentration is 0.1mol/L
Tris-HCl buffer e.
The trishydroxymethylaminomethane and hydrochloric acid mixture that Tris-HCl, that is, molar concentration rate in the present embodiment is 1:1.
The content of myoglobins is 1.57mg/mL in the beef that the present embodiment extracts.
Embodiment 4
The present embodiment is the response surface optimization experimental design to the extracting method of myoglobins in beef
Using myoglobin content in beef as response, the pH value of Tris-HCl cocktail buffer is 8.0 in step 1
Extraction time is 1h~2h (being denoted as A) in~8.4 (being denoted as B), step 1, the saltout saturation degree of liquid of myoglobins is in step 2
Within the scope of the single factor experiment of 85%~95% (being denoted as C), rotation group in center is further completed by Design-expert software
Close test.Experimental factor level design, test result and variance analysis are shown in Table 1~3.Fig. 5~7 are respectively B and A, C and A, B and C
Interactive response surface design.
1 center combination experimental design factor level of table
2 center combination experimental design of table and result
3 center combination of table tests variance analysis
Center rotation combination test the results of analysis of variance shows that: model terms P < 0.01 illustrates to myoglobins in beef
The relationship between correlative factor of the optimization of extracting method, gained regression equation is extremely significant.Quasi- item P=0.8556 > 0.05 is lost,
Coefficient of multiple correlation R2With correction multiple correlation coefficient Adj.R2Respectively 0.9745 and 0.9417, the two difference is less.In addition, red to flesh
Protein content prediction of result carries out overall estimation, coefficient of multiple correlation R2With prediction multiple correlation coefficient Pred.R2Respectively 0.9745 He
0.9012, illustrate that the model has relatively reasonable degree of fitting.
Meanwhile influence of two factors of pH value to myoglobin content of extraction time, Tris-HCl cocktail buffer are aobvious
It writes (p < 0.05)；Quadratic term, the myoglobins of three factors saltout liquid saturation degree and its interaction with extraction time, to flesh
Lactoferrin content influences extremely significant (p < 0.01).
Myoglobins optimum extraction parameter is by completing regression equation maximizing, maximum response Y (myoglobins
Content) reach 1.62mg/mL, at this time corresponding variable factors are as follows: and the pH value of Tris-HCl cocktail buffer is 8.15, myoglobins
Saltout liquid saturation degree be 90.93%, extraction time 1.63h, then using predict resulting optimum extraction condition repeat into
Row 3 times tests, myoglobins average content is 1.65mg/mL in final beef.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way.It is all according to invention skill
Art any simple modification, change and equivalence change substantially to the above embodiments, still fall within technical solution of the present invention
Protection scope in.
Claims (6)
1. the extracting method of myoglobins in a kind of beef, which is characterized in that method includes the following steps:
Step 1: slightly mentioning: taking beef longissimus dorsi muscle, remove surface fascia and connective tissue, chopping obtains beef meat sample, to beef
Meat sample be added concentration be 10mmol/L, the Tris-HCl cocktail buffer that pH value is 8.0~8.4, be then in revolving speed
After being homogenized 1min under conditions of 10000rpm, is stood under conditions of temperature is 4 DEG C and extract 1h~2h, be then in revolving speed
9600rpm, temperature filter after being centrifuged 10min under conditions of being 4 DEG C, obtain filtrate；Contain in the Tris-HCl cocktail buffer
There are the two of the glycerol and 1mmol/L that concentration is EDTA, 0.54mmol/L of Tris-HCl buffer a, 1mmol/L of 10mmol/L
Sulphur threitol；
Step 2: saltouing: being slowly uniformly added into solid (NH while stirring into filtrate obtained in step 14)2SO4, obtain flesh
Lactoferrin is saltoutd liquid, when myoglobins saltout the saturation degree of liquid be 85%~95% when, stood under conditions of temperature is 4 DEG C
Then 30min precipitating proteins are centrifuged 30min under conditions of revolving speed is 10000rpm, temperature is 4 DEG C, obtain myoglobins
Sediment；
Step 3: dialysis: into myoglobins sediment obtained in step 2 be added concentration be 10mmol/L, pH value 8.5
Tris-HCl buffer b stirring and dissolving after, be placed in molecular cut off be 8kDa~14kDa bag filter in, be added concentration be
The Tris-HCl buffer c that 10mmol/L, pH value are 8.5 is stirred under conditions of temperature is 0 DEG C~4 DEG C and is dialysed for 24 hours, dialysis
The Tris-HCl buffer b is replaced 10 times in the process, obtains high-purity myoglobins；
Step 4: gel permeation chromatography: by Sephadex G-75 chromatographic column with concentration being 1mmol/L, the Tris- that pH value is 8.5
After HCl buffer d balance, the NaCl mixed solution for being 0.1mol/L with concentration is to high-purity myoglobins obtained in step 3
Linear elution, flow velocity 2mL/min are carried out, continuous collection multitube eluent to elution finishes, and every pipe eluent is in wavelength
It is detected under the ultraviolet light of 280nm, merges the maximum eluent of eluting peak absorption value, obtain myoglobins in beef；It is described
NaCl mixed solution contains the Tris-HCl buffer of the EDTA and 10mmol/L of NaCl, 1mmol/L that concentration is 0.1mol/L
e。
2. the extracting method of myoglobins in a kind of beef according to claim 1, which is characterized in that described in step 1
The pH value of Tris-HCl cocktail buffer is 8.2, extraction time 1.5h；Myoglobins described in step 2 is saltoutd the saturation of liquid
Degree is 90%.
3. the extracting method of myoglobins in a kind of beef according to claim 1, which is characterized in that described in step 1
The pH value of Tris-HCl cocktail buffer is 8.15, extraction time 1.63h；Myoglobins described in step 2 is saltoutd the full of liquid
It is 90.93% with degree.
4. the extracting method of myoglobins in a kind of beef according to claim 1, which is characterized in that in the step 1
The quality and volume ratio of the beef meat sample and Tris-HCl cocktail buffer are 1g:5mL.
5. the extracting method of myoglobins in a kind of beef according to claim 1, which is characterized in that described in step 3
The quality and volume ratio of myoglobins sediment and Tris-HCl buffer b are 1g:2mL.
6. the extracting method of myoglobins in a kind of beef according to claim 1, which is characterized in that described in step 4
The pH value of NaCl mixed solution is 8.4.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150120A (en) * | 2021-05-21 | 2021-07-23 | 江南大学 | Method for separating and purifying porcine myoglobin in fermentation liquor |
| CN114773454A (en) * | 2022-04-27 | 2022-07-22 | 广州蕊特生物科技有限公司 | Extraction and purification method for extracting myoglobin from horse heart |
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2019
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113150120A (en) * | 2021-05-21 | 2021-07-23 | 江南大学 | Method for separating and purifying porcine myoglobin in fermentation liquor |
| CN114773454A (en) * | 2022-04-27 | 2022-07-22 | 广州蕊特生物科技有限公司 | Extraction and purification method for extracting myoglobin from horse heart |
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Application publication date: 20190809 |