CN109673811A - A kind of method that high concentration myosin maintains liquid at high temperature - Google Patents
A kind of method that high concentration myosin maintains liquid at high temperature Download PDFInfo
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- CN109673811A CN109673811A CN201910039240.5A CN201910039240A CN109673811A CN 109673811 A CN109673811 A CN 109673811A CN 201910039240 A CN201910039240 A CN 201910039240A CN 109673811 A CN109673811 A CN 109673811A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/04—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from fish or other sea animals
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/04—Animal proteins
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Abstract
The present invention relates to aquatic products quality-improving technical fields, and in particular to a kind of method that high concentration myosin maintains liquid under high temperature；Specific step is as follows: the selection back flesh of fish first, cleaning, peeling；It extracts and obtains myosin solution, measurement protein content is 10 ~ 20 mg/mL；Then arginine, lysine or histidine, final concentration of 10 ~ 100 mM are added into the myosin solution of extraction；Then two-period form heating is carried out, first 40 DEG C of 60 min of heating, then 90 DEG C of 30 min of heating realize that high concentration myosin maintains liquid under high temperature；Operation of the present invention is easy, lays a good foundation for the preparation of liquid food fish protein product, high financial profit has broad prospects.
Description
Technical field
The present invention relates to aquatic products quality-improving technical fields, and in particular to high concentration myosin maintains under a kind of high temperature
The method of liquid.
Background technique
Fribrillin is the main albumen for forming fish protein, and it is flesh that content is highest in fribrillin
Globulin accounts for about 50%-60%.The research of the flesh of fish is concentrated on during fish protein gel characteristic and its gel-forming at present
The variation of protein composition has studied the change of minced fillet mainly using analytical technologies such as CD, Raman spectrum, SEM, dynamic rheometers
Learn the difference of composition, protein composition and gel characteristic, research myosin denaturation aggregation, the difference of gelling characteristic and Ca2+With
The influence of Microbial transglutaminase (MTGase), to prepare better surimi products；
Common fish product has fish bean curd, fish ball, fish meat sausage etc., they all have high protein, low fat, Gao Ying
It supports, advantage in good taste, but also there is the general character of elasticity height, big, the resistance to chewing of hardness simultaneously, so more by vast young consumers
Favor, and it is edible to be not suitable for the bradymassesises crowd such as the elderly, children, patient；On the other hand, since albumen is in hot conditions
Under can occur to be denaturalized and assemble, the especially myosin of high concentration will form gel after high-temperature heating, and the exploitation of food is final
It needs by high-temperature sterilization, therefore limits the exploitation of liquid food fish protein product；There has been no liquid food fish proteins at present
The correlative study report of product, so, being prepared with for liquid food fish protein product is to be developed, and has broad prospects.
Summary of the invention
In view of the above-mentioned problems, present invention seek to address that one of described problem；Present invention myosin caused by the arginine
Angle changing incision after high-temperature heating from colloid to liquid, according to myosin heat accumulation process medium fluid mechanics radius and thoroughly
The size for crossing rate objectively evaluates influence of the arginine to myosin heat accumulation behavior, and providing one kind makes high concentration myosin
The method of liquid is maintained after high-temperature heating
In order to achieve the goal above, the specific steps of the present invention are as follows:
(1) preparation of flesh of fish sample: variegated carp is rinsed well with clear water, chooses the back flesh of fish, and cleaning, peeling are chopped into meat
Rotten shape, it is spare；
(2) extraction of myosin: the reagent A of 10 times of volumes is added in the meat gruel that step (1) obtains, is existed with homogenizer
3~5min of homogeneous under 11000r/min reacts 15min at 4 DEG C, is centrifuged (3000 × g, 5min, 4 DEG C), taking precipitate, is added 5
The reagent B of times volume, and ATP is added into suspension, it is allowed to final concentration of 10mM, after completely dissolution, is placed under the conditions of 4 DEG C
90min is centrifuged (11000 × g, 13min, 4 DEG C), the 1mM saleratus of 5 times of volumes is added into supernatant, and in 4 DEG C of conditions
Lower placement 20min, then it is centrifuged (11000 × g, 13min, 4 DEG C), the reagent C of 2.5 times of volumes is added, by it 4 in taking precipitate
The 1mM saleratus and magnesium chloride for sequentially adding 5 times of volumes at DEG C after reaction 10min, make the final concentration of magnesium chloride in mixed liquor
For 10mM, and stood overnight under the conditions of 4 DEG C；Then it is centrifuged (11000 × g, 25min, 4 DEG C) again, obtains flesh ball egg
White particle is dissolved in 0.5M NaCl-20mM Tris-HCl (pH 7.0) buffer, is centrifuged (5000 × g, 10min, 4 DEG C),
Supernatant is myosin solution, is placed in spare in 4 DEG C of refrigerators；Wherein, reagent A: 0.1M potassium chloride, 20mM Tris are used
Salt acid for adjusting pH is to 7.5；Reagent B:0.45M potassium chloride, 0.2M magnesium acetate, 1mM EGTA, 20mM Tris, 5mM β-sulfydryl second
Alcohol, with Malaysia acid for adjusting pH to 6.8；Reagent C: 0.5M potassium chloride, 20mM Tris, 5mM beta -mercaptoethanol, with salt acid for adjusting pH
To 7.5；
(3) amino acid is added in the myosin solution obtained to step (2), then carries out two-period form heating, realized and add
Myosin after heat maintains liquid form.
Preferably, the protein content of myosin solution described in step (3) is 10~20mg/mL.
Preferably, final concentration of 10~100mM of amino acid described in step (3).
Preferably, amino acid described in step (3) is arginine, lysine or histidine.
Preferably, amino acid described in step (3) is arginine.
Preferably, the concrete operations of the heating of two-period form described in step (3) are as follows: first 40 DEG C of heating 60min, then 90 DEG C of heating
30min。
Hydrodynamic radius and transmitance measurement: instrument LitesizerTM500 particle size analyzers, choosing then diameter 1cm
Glass cuvette covers high temperature dwell protecting cover, carries out the measurement of hydrodynamic radius, setting heating rate is 1 DEG C/min, from 25 DEG C
90 DEG C are risen to, the variation of sample fluid mechanics radius and transmitance during record is done pair to be not added with the myosin of amino acid
According to.
Albumen after finally using the size of hydrodynamic radius and transmitance as judge high concentration myosin high-temperature heating
The standard of aggregation size, according to testing result compared with blank sample detection data, to evaluate the height of addition amino acid
Whether exist without biggish protein masses after concentration myosin high-temperature heating but is still monomer or oligomerization.
Beneficial effects of the present invention:
This method is as the result is shown: with not plus compared with the myosin of amino acid, when amino acid additive amount 10mM~
When changing between 100mM, high concentration myosin can still maintain liquid after being heated at high temperature, and hydrodynamic radius accordingly subtracts
Small and transmitance is increase accordingly, and the exploitation for liquid food fish protein product provides theoretical foundation, enriches the multiplicity of processed fish meat products
Change, and then push the development in aquatic products field, brings preferable economic benefit for society.
Detailed description of the invention
In Fig. 1, a is the result figure of control group, and b is the result figure for adding 10mM arginine group, and c is addition 50mM arginine
The result figure of group, d are the result figure for adding 100mM arginine group.
Fig. 2 is the hydrodynamic radius and transmitance figure of control group.
Fig. 3 is the hydrodynamic radius and transmitance figure for adding 10mM arginine group.
Fig. 4 is the hydrodynamic radius and transmitance figure for adding 50mM arginine group.
Fig. 5 is the hydrodynamic radius and transmitance figure for adding 100mM arginine group.
Specific embodiment
Comparative example:
(1) cleaning, peeling of the flesh of fish: variegated carp is rinsed well with clear water, chooses the back flesh of fish, cleaning, peeling；
(2) extraction of myosin: fresh variegated carp is subjected to slaughter processing, the back flesh of fish is taken, and remove the peel, clean, is chopped into
The reagent A of 10 times of volumes is added after meat gruel shape, with homogenizer at 11000r/min 3~5min of homogeneous, react 15min at 4 DEG C,
It is centrifuged (3000 × g, 5min, 4 DEG C), the reagent B of 5 times of volumes is added in taking precipitate, and ATP is added into suspension, is allowed to eventually
Concentration is 10mM, after completely dissolution, above-mentioned mixed liquor is placed 90min at 4 DEG C, is centrifuged (11000 × g, 13min, 4 DEG C),
The 1mM saleratus of 5 times of volumes is added into supernatant, and places 20min at 4 DEG C, then be centrifuged (11000 × g, 13min, 4
DEG C), the reagent C of 2.5 times of volumes is added in taking precipitate, it is reacted to the 1mM that 5 times of volumes are sequentially added after 10min at 4 DEG C
Saleratus and magnesium chloride make the final concentration of 10mM of magnesium chloride in mixed liquor, and stand overnight under the conditions of 4 DEG C；Second day
It is centrifuged (11000 × g, 25min, 4 DEG C), obtains myosin particle, and it is made to be completely dissolved in 0.5M NaCl-20mM
In Tris-HCl (pH 7.0) buffer, refrigerated centrifuge (5000 × g, 10min, 4 DEG C), supernatant is myosin solution,
Protein content is 10-20mg/mL, is placed in spare in 4 DEG C of refrigerators；
Reagent A: 0.1M potassium chloride, 20mM Tris, with salt acid for adjusting pH to 7.5；Reagent B:0.45M potassium chloride, 0.2M second
Sour magnesium, 1mM EGTA, 20mM Tris, 5mM beta -mercaptoethanol, with Malaysia acid for adjusting pH to 6.8；Reagent C: 0.5M potassium chloride,
20mM Tris, 5mM beta -mercaptoethanol, with salt acid for adjusting pH to 7.5.
(3) preparation of myosin sample: by myosin solution that concentration is 10mg/mL, first 40 DEG C of heating 60min are again
90 DEG C of heating 30min, photograph to record test result；
(4) hydrodynamic radius and transmitance measurement: instrument LitesizerTM500 particle size analyzers select then diameter
The glass cuvette of 1cm covers high temperature dwell protecting cover, carries out the measurement of hydrodynamic radius, and setting heating rate is 1 DEG C/min,
90 DEG C are risen to from 25 DEG C, the variation of sample fluid mechanics radius and transmitance during record；
(5) egg after being finally heated at high temperature using the size of hydrodynamic radius and transmitance as judge high concentration myosin
The standard of white aggregation size.
Embodiment 1:
(1) cleaning, peeling of the flesh of fish: variegated carp is rinsed well with clear water, chooses the back flesh of fish, cleaning, peeling；
(2) extraction of myosin: fresh variegated carp is subjected to slaughter processing, the back flesh of fish is taken, and remove the peel, clean, is chopped into
The reagent A of 10 times of volumes is added after meat gruel shape, with homogenizer at 11000r/min 3~5min of homogeneous, react 15min at 4 DEG C,
It is centrifuged (3000 × g, 5min, 4 DEG C), the reagent B of 5 times of volumes is added in taking precipitate, and ATP is added into suspension, is allowed to eventually
Concentration is 10mM, after completely dissolution, above-mentioned mixed liquor is placed 90min at 4 DEG C, is centrifuged (11000 × g, 13min, 4 DEG C),
The 1mM saleratus of 5 times of volumes is added into supernatant, and places 20min at 4 DEG C, then be centrifuged (11000 × g, 13min, 4
DEG C), the reagent C of 2.5 times of volumes is added in taking precipitate, it is reacted to the 1mM that 5 times of volumes are sequentially added after 10min at 4 DEG C
Saleratus and magnesium chloride make the final concentration of 10mM of magnesium chloride in mixed liquor, and stand overnight under the conditions of 4 DEG C.Second day
It is centrifuged (11000 × g, 25min, 4 DEG C), obtains myosin particle, and it is made to be completely dissolved in 0.5M NaCl-20mM
In Tris-HCl (pH 7.0) buffer, refrigerated centrifuge (5000 × g, 10min, 4 DEG C), supernatant is myosin solution,
Protein content is 10-20mg/mL, is placed in spare in 4 DEG C of refrigerators；
Reagent A: 0.1M potassium chloride, 20mM Tris, with salt acid for adjusting pH to 7.5；
Reagent B:0.45M potassium chloride, 0.2M magnesium acetate, 1mM EGTA, 20mM Tris, 5mM beta -mercaptoethanol use Malaysia
Acid for adjusting pH is to 6.8；
Reagent C: 0.5M potassium chloride, 20mM Tris, 5mM beta -mercaptoethanol, with salt acid for adjusting pH to 7.5；
(3) arginine is added into the myosin solution that concentration is 10mg/mL, so that arginic final concentration of
10mM；By each sample of above-mentioned preparation, first 40 DEG C of heating 60min again tie up by 90 DEG C of heating 30min, the myosin after realizing heating
Hold liquid form；
(4) hydrodynamic radius and transmitance measurement: instrument LitesizerTM500 particle size analyzers select then diameter
The glass cuvette of 1cm covers high temperature dwell protecting cover, carries out the measurement of hydrodynamic radius, and setting heating rate is 1 DEG C/min,
90 DEG C are risen to from 25 DEG C, the variation of sample fluid mechanics radius and transmitance during record.
(5) egg after being finally heated at high temperature using the size of hydrodynamic radius and transmitance as judge high concentration myosin
The standard of white aggregation size, according to testing result compared with blank sample detection data, so that it is arginic to evaluate addition
Whether exist without biggish protein masses after high concentration myosin high-temperature heating but is still monomer or oligomer.
Embodiment 2:
(1) cleaning, peeling of the flesh of fish: variegated carp is rinsed well with clear water, chooses the back flesh of fish, cleaning, peeling；
(2) extraction of myosin: fresh variegated carp is subjected to slaughter processing, the back flesh of fish is taken, and remove the peel, clean, is chopped into
The reagent A of 10 times of volumes is added after meat gruel shape, with homogenizer at 11000r/min 3~5min of homogeneous, react 15min at 4 DEG C,
It is centrifuged (3000 × g, 5min, 4 DEG C), the reagent B of 5 times of volumes is added in taking precipitate, and ATP is added into suspension, is allowed to eventually
Concentration is 10mM, after completely dissolution, above-mentioned mixed liquor is placed 90min at 4 DEG C, is centrifuged (11000 × g, 13min, 4 DEG C),
The 1mM saleratus of 5 times of volumes is added into supernatant, and places 20min at 4 DEG C, then be centrifuged (11000 × g, 13min, 4
DEG C), the reagent C of 2.5 times of volumes is added in taking precipitate, it is reacted to the 1mM that 5 times of volumes are sequentially added after 10min at 4 DEG C
Saleratus and magnesium chloride make the final concentration of 10mM of magnesium chloride in mixed liquor, and stand overnight under the conditions of 4 DEG C；Second day
It is centrifuged (11000 × g, 25min, 4 DEG C), obtains myosin particle, and it is made to be completely dissolved in 0.5M NaCl-20mM
In Tris-HCl (pH 7.0) buffer, refrigerated centrifuge (5000 × g, 10min, 4 DEG C), supernatant is myosin solution,
Protein content is 10-20mg/mL, is placed in spare in 4 DEG C of refrigerators；
Reagent A: 0.1M potassium chloride, 20mM Tris, with salt acid for adjusting pH to 7.5；
Reagent B:0.45M potassium chloride, 0.2M magnesium acetate, 1mM EGTA, 20mM Tris, 5mM beta -mercaptoethanol use Malaysia
Acid for adjusting pH is to 6.8；
Reagent C: 0.5M potassium chloride, 20mM Tris, 5mM beta -mercaptoethanol, with salt acid for adjusting pH to 7.5；
(3) smart ammonia myosin-arginine sample preparation: is added into the myosin solution that concentration is 10mg/mL
Acid, so that arginic final concentration of 50mM；By first 40 DEG C of heating 60min, the 90 DEG C of heating 30min again of each sample of above-mentioned preparation,
Myosin after realizing heating maintains liquid form；
(4) hydrodynamic radius and transmitance measurement: instrument LitesizerTM500 particle size analyzers select then diameter
The glass cuvette of 1cm covers high temperature dwell protecting cover, carries out the measurement of hydrodynamic radius, and setting heating rate is 1 DEG C/min,
90 DEG C are risen to from 25 DEG C, the variation of sample fluid mechanics radius during record；
(5) egg after being finally heated at high temperature using the size of hydrodynamic radius and transmitance as judge high concentration myosin
The standard of white aggregation size, according to testing result compared with blank sample detection data, so that it is arginic to evaluate addition
Whether exist without biggish protein masses after high concentration myosin high-temperature heating but is still monomer or oligomer.
Embodiment 3:
(1) cleaning, peeling of the flesh of fish: variegated carp is rinsed well with clear water, chooses the back flesh of fish, cleaning, peeling；
(2) extraction of myosin: fresh variegated carp is subjected to slaughter processing, the back flesh of fish is taken, and remove the peel, clean, is chopped into
The reagent A of 10 times of volumes is added after meat gruel shape, with homogenizer at 11000r/min 3~5min of homogeneous, react 15min at 4 DEG C,
It is centrifuged (3000 × g, 5min, 4 DEG C), the reagent B of 5 times of volumes is added in taking precipitate, and ATP is added into suspension, is allowed to eventually
Concentration is 10mM, after completely dissolution, above-mentioned mixed liquor is placed 90min at 4 DEG C, is centrifuged (11000 × g, 13min, 4 DEG C),
The 1mM saleratus of 5 times of volumes is added into supernatant, and places 20min at 4 DEG C, then be centrifuged (11000 × g, 13min, 4
DEG C), the reagent C of 2.5 times of volumes is added in taking precipitate, it is reacted to the 1mM that 5 times of volumes are sequentially added after 10min at 4 DEG C
Saleratus and magnesium chloride make the final concentration of 10mM of magnesium chloride in mixed liquor, and stand overnight under the conditions of 4 DEG C；Second day
It is centrifuged (11000 × g, 25min, 4 DEG C), obtains myosin particle, and it is made to be completely dissolved in 0.5M NaCl-20mM
In Tris-HCl (pH 7.0) buffer, refrigerated centrifuge (5000 × g, 10min, 4 DEG C), supernatant is myosin solution,
Protein content is 10-20mg/mL, is placed in spare in 4 DEG C of refrigerators；
Reagent A: 0.1M potassium chloride, 20mM Tris, with salt acid for adjusting pH to 7.5；
Reagent B:0.45M potassium chloride, 0.2M magnesium acetate, 1mM EGTA, 20mM Tris, 5mM beta -mercaptoethanol use Malaysia
Acid for adjusting pH is to 6.8；
Reagent C: 0.5M potassium chloride, 20mM Tris, 5mM beta -mercaptoethanol, with salt acid for adjusting pH to 7.5；
(3) arginine is added into the myosin solution that concentration is 10mg/mL, so that arginic final concentration of
100mM；By each sample of above-mentioned preparation, first 40 DEG C of heating 60min again tie up by 90 DEG C of heating 30min, the myosin after realizing heating
Hold liquid form；
(4) hydrodynamic radius and transmitance measurement: instrument LitesizerTM500 particle size analyzers select then diameter
The glass cuvette of 1cm covers high temperature dwell protecting cover, carries out the measurement of hydrodynamic radius, and setting heating rate is 1 DEG C/min,
90 DEG C are risen to from 25 DEG C, the variation of sample fluid mechanics radius and transmitance during record；
(5) egg after being finally heated at high temperature using the size of hydrodynamic radius and transmitance as judge high concentration myosin
The standard of white aggregation size, according to testing result compared with blank sample detection data, so that it is arginic to evaluate addition
Whether exist without biggish protein masses after high concentration myosin high-temperature heating but is still monomer or oligomer.
Embodiment 4:
(1) cleaning, peeling of the flesh of fish: variegated carp is rinsed well with clear water, chooses the back flesh of fish, cleaning, peeling；
(2) extraction of myosin: fresh variegated carp is subjected to slaughter processing, the back flesh of fish is taken, and remove the peel, clean, is chopped into
The reagent A of 10 times of volumes is added after meat gruel shape, with homogenizer at 11000r/min 3~5min of homogeneous, react 15min at 4 DEG C,
It is centrifuged (3000 × g, 5min, 4 DEG C), the reagent B of 5 times of volumes is added in taking precipitate, and ATP is added into suspension, is allowed to eventually
Concentration is 10mM, after completely dissolution, above-mentioned mixed liquor is placed 90min at 4 DEG C, is centrifuged (11000 × g, 13min, 4 DEG C),
The 1mM saleratus of 5 times of volumes is added into supernatant, and places 20min at 4 DEG C, then be centrifuged (11000 × g, 13min, 4
DEG C), the reagent C of 2.5 times of volumes is added in taking precipitate, it is reacted to the 1mM that 5 times of volumes are sequentially added after 10min at 4 DEG C
Saleratus and magnesium chloride make the final concentration of 10mM of magnesium chloride in mixed liquor, and stand overnight under the conditions of 4 DEG C.Second day
It is centrifuged (11000 × g, 25min, 4 DEG C), obtains myosin particle, and it is made to be completely dissolved in 0.5M NaCl-20mM
In Tris-HCl (pH 7.0) buffer, refrigerated centrifuge (5000 × g, 10min, 4 DEG C), supernatant is myosin solution,
Protein content is 10-20mg/mL, is placed in spare in 4 DEG C of refrigerators；
Reagent A: 0.1M potassium chloride, 20mM Tris, with salt acid for adjusting pH to 7.5；
Reagent B:0.45M potassium chloride, 0.2M magnesium acetate, 1mM EGTA, 20mM Tris, 5mM beta -mercaptoethanol use Malaysia
Acid for adjusting pH is to 6.8；
Reagent C: 0.5M potassium chloride, 20mM Tris, 5mM beta -mercaptoethanol, with salt acid for adjusting pH to 7.5；
(3) lysine is added into the myosin solution that concentration is 15mg/mL, so that lysine is final concentration of
50mM；By each sample of above-mentioned preparation, first 40 DEG C of heating 60min again tie up by 90 DEG C of heating 30min, the myosin after realizing heating
Hold liquid form；
(4) hydrodynamic radius and transmitance measurement: instrument LitesizerTM500 particle size analyzers select then diameter
The glass cuvette of 1cm covers high temperature dwell protecting cover, carries out the measurement of hydrodynamic radius, and setting heating rate is 1 DEG C/min,
90 DEG C are risen to from 25 DEG C, the variation of sample fluid mechanics radius and transmitance during record；
(5) egg after being finally heated at high temperature using the size of hydrodynamic radius and transmitance as judge high concentration myosin
The standard of white aggregation size, according to testing result compared with blank sample detection data, to evaluate addition lysine
Whether exist without biggish protein masses after high concentration myosin high-temperature heating but is still monomer or oligomer.
Embodiment 5:
(1) cleaning, peeling of the flesh of fish: variegated carp is rinsed well with clear water, chooses the back flesh of fish, cleaning, peeling；
(2) extraction of myosin: fresh variegated carp is subjected to slaughter processing, the back flesh of fish is taken, and remove the peel, clean, is chopped into
The reagent A of 10 times of volumes is added after meat gruel shape, with homogenizer at 11000r/min 3~5min of homogeneous, react 15min at 4 DEG C,
It is centrifuged (3000 × g, 5min, 4 DEG C), the reagent B of 5 times of volumes is added in taking precipitate, and ATP is added into suspension, is allowed to eventually
Concentration is 10mM, after completely dissolution, above-mentioned mixed liquor is placed 90min at 4 DEG C, is centrifuged (11000 × g, 13min, 4 DEG C),
The 1mM saleratus of 5 times of volumes is added into supernatant, and places 20min at 4 DEG C, then be centrifuged (11000 × g, 13min, 4
DEG C), the reagent C of 2.5 times of volumes is added in taking precipitate, it is reacted to the 1mM that 5 times of volumes are sequentially added after 10min at 4 DEG C
Saleratus and magnesium chloride make the final concentration of 10mM of magnesium chloride in mixed liquor, and stand overnight under the conditions of 4 DEG C.Second day
It is centrifuged (11000 × g, 25min, 4 DEG C), obtains myosin particle, and it is made to be completely dissolved in 0.5M NaCl-20mM
In Tris-HCl (pH 7.0) buffer, refrigerated centrifuge (5000 × g, 10min, 4 DEG C), supernatant is myosin solution,
Protein content is 10-20mg/mL, is placed in spare in 4 DEG C of refrigerators.
Reagent A: 0.1M potassium chloride, 20mM Tris, with salt acid for adjusting pH to 7.5.
Reagent B:0.45M potassium chloride, 0.2M magnesium acetate, 1mM EGTA, 20mM Tris, 5mM beta -mercaptoethanol use Malaysia
Acid for adjusting pH is to 6.8.
Reagent C: 0.5M potassium chloride, 20mM Tris, 5mM beta -mercaptoethanol, with salt acid for adjusting pH to 7.5.
(3) histidine is added into the myosin solution that concentration is 20mg/mL, so that histidine is final concentration of
100mM, by each sample of above-mentioned preparation, first 40 DEG C of heating 60min again tie up by 90 DEG C of heating 30min, the myosin after realizing heating
Hold liquid form；
(4) hydrodynamic radius and transmitance measurement: instrument LitesizerTM500 particle size analyzers select then diameter
The glass cuvette of 1cm covers high temperature dwell protecting cover, carries out the measurement of hydrodynamic radius, and setting heating rate is 1 DEG C/min,
90 DEG C are risen to from 25 DEG C, the variation of sample fluid mechanics radius and transmitance during record；
(5) egg after being finally heated at high temperature using the size of hydrodynamic radius and transmitance as judge high concentration myosin
The standard of white aggregation size, according to testing result compared with blank sample detection data, to evaluate addition histidine
Whether exist without biggish protein masses after high concentration myosin high-temperature heating but is still monomer or oligomer.
In Fig. 1, a is the result figure of control group, and b is the result figure for adding 10mM arginine group, and c is addition 50mM arginine
The result figure of group, d are the result figure for adding 100mM arginine group；It can be seen that with arginic addition, solution becomes
Clarification, obtains the conclusion that arginine can make high concentration myosin maintain liquid after high-temperature heating
Fig. 2 is the hydrodynamic radius and transmitance figure of control group, and wherein a is hydrodynamic radius curve；B is transmitance
Curve；It can be seen that myosin hydrodynamic radius constantly increases, and transmitance accordingly reduces, and obtains myosin and exists
It produces in heating process and largely assembles.
Fig. 3 is the hydrodynamic radius and transmitance figure for adding 10mM arginine group, and wherein a is that hydrodynamic radius is bent
Line；B is transmittance curve；By the way that, it is found that hydrodynamic radius reduces, transmitance increases, and obtaining arginine can be with Fig. 2 comparison
Inhibit the conclusion of myosin heat accumulation.
Fig. 4 is the hydrodynamic radius and transmitance figure for adding 50mM arginine group, and wherein a is that hydrodynamic radius is bent
Line；B is transmittance curve；By the way that, it is found that hydrodynamic radius reduces, transmitance increases, and obtaining arginine can be with Fig. 2 comparison
Inhibit the conclusion of myosin heat accumulation.
Fig. 5 is the hydrodynamic radius and transmitance figure for adding 100mM arginine group, and wherein a is that hydrodynamic radius is bent
Line；B is transmittance curve；By the way that, it is found that hydrodynamic radius reduces, transmitance increases, and obtaining arginine can be with Fig. 2 comparison
Inhibit the conclusion of myosin heat accumulation, and the radius base of the hydrodynamic radius of final myosin and single myosin
This is consistent.
Illustrate: above embodiments are only to illustrate the present invention and not limit the technical scheme described by the invention；Therefore,
Although this specification is referring to above-mentioned each embodiment, the present invention has been described in detail, the common skill of this field
Art personnel should be appreciated that and still can modify to the present invention or equivalent replacement；And all do not depart from spirit of the invention and
The technical solution and its improvement of range, should all cover in scope of the presently claimed invention.
Claims (6)
1. a kind of method that high concentration myosin maintains liquid under high temperature, which comprises the following steps:
(1) preparation of flesh of fish sample；
(2) extraction that myosin is carried out with the flesh of fish sample that step (1) obtains, obtains myosin solution；
(3) amino acid is added in the myosin solution obtained to step (2), then carries out two-period form heating, after realizing heating
Myosin maintain liquid form.
2. the method that high concentration myosin maintains liquid under high temperature according to claim 1, which is characterized in that step
(3) protein content of myosin solution described in is 10 ~ 20 mg/mL.
3. the method that high concentration myosin maintains liquid under high temperature according to claim 1, which is characterized in that step
(3) final concentration of 10 ~ 100 mM of amino acid described in.
4. the method that high concentration myosin maintains liquid under high temperature according to claim 1 or 3, which is characterized in that step
Suddenly amino acid described in (3) is arginine, lysine or histidine.
5. the method that high concentration myosin maintains liquid under high temperature according to claim 4, which is characterized in that described
Amino acid is arginine.
6. the method that high concentration myosin maintains liquid under high temperature according to claim 1, which is characterized in that step
(3) concrete operations of the heating of two-period form described in are as follows: first 40 DEG C of 60 min of heating, then 90 DEG C of 30 min of heating.
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| CN110506873A (en) * | 2019-07-26 | 2019-11-29 | 江苏大学 | A method of animal protein juice drinks are prepared using compound amino acid |
| CN111227256A (en) * | 2020-02-28 | 2020-06-05 | 江苏大学 | Preparation method of fish protein stable oil-in-water emulsion |
| CN111358005A (en) * | 2020-02-28 | 2020-07-03 | 江苏大学 | Method for improving stability of Pickering emulsion based on arginine modification |
| CN112369586A (en) * | 2020-10-16 | 2021-02-19 | 江苏大学 | Method for improving stability and quality of fish head soup based on amino acid and compound phosphate |
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| CN111227256A (en) * | 2020-02-28 | 2020-06-05 | 江苏大学 | Preparation method of fish protein stable oil-in-water emulsion |
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| CN112369586A (en) * | 2020-10-16 | 2021-02-19 | 江苏大学 | Method for improving stability and quality of fish head soup based on amino acid and compound phosphate |
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