CN108576366A - The preparation method of high foaming fowl orgotein powder - Google Patents
The preparation method of high foaming fowl orgotein powder Download PDFInfo
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- CN108576366A CN108576366A CN201810437374.8A CN201810437374A CN108576366A CN 108576366 A CN108576366 A CN 108576366A CN 201810437374 A CN201810437374 A CN 201810437374A CN 108576366 A CN108576366 A CN 108576366A
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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
- A23J1/001—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste
- A23J1/002—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from waste materials, e.g. kitchen waste from animal waste materials
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Abstract
The invention discloses a kind of preparation methods of high foaming fowl orgotein powder, including：Step 1: it is degreasing fowl liver to take poultry liver processing；Step 2: extracting degreasing fowl liver is placed in Tris HCl buffer solutions, it is ultrasonically treated, the condition of supersound process is ultrasonic power 1000W~1500W, the percentage that ultrasonic power accounts for ultrasonic wave general power is 5~9%, ultrasonic time is 4~10min, per ultrasound 2~4s, rests 1~3s；Step 3: obtaining fowl orgotein after extracting degreasing fowl liver impurity elimination；Step 4: fowl orgotein is scattered in phosphate buffer, modified fowl orgotein powder crude product is obtained by processing；Step 5: isolating and purifying modified fowl orgotein powder crude product, molecular weight is collected<The protolysate of 5000Da obtains high foaming fowl orgotein powder.1.27~2.12 times and 59%~69% has been respectively increased in the present invention modified fowl orgotein powder foaming characteristic and stability.
Description
Technical field
The invention belongs to fowl orgotein method of modifying technical fields, are related to a kind of preparation side of high foaming fowl orgotein powder
Method.
Background technology
Poultry liver is the vitals of storage nutriment in animal body has nutrition and health care containing abundant nutriment
Function is optimal one of good merchantable brand of enriching blood, and the important source material rich in good protein.It is known as three big cuisines of the world
One of (fertile foie gras, caviar, matsutake mushroom).Poultry liver quality is delicate, fat is fragrant unique, is a kind of high tonic, contains someone
Various nutrients needed for body, especially contain abundant protein, fat, vitamin, lecithin, nucleic acid, various enzymes and
The metallic elements such as calcium, phosphorus.However, fresh poultry liver is cheap, and due to there is special fishy smell, seldom liked by people
It is joyous edible.Simultaneously as edible physics and chemistry and the processing characteristics understanding to poultry liver are very few, poultry liver majority is with marketing fresh and work
For the animal feed utilization of resources, some is even discarded, only a small amount of fresh poultry liver be used to deep processing be sauce halogen liver and
Liver pat etc., therefore poultry liver is normally used as a kind of low value resource and is not fully utilized, and causes a large amount of high-quality protein foods
The waste of resource.
The foaming characteristic of protein usually indicates with foam gas amount, i.e., foamability (Foaming Capacity, FC) and
Foam stability (Foaming Stability, FS).Influence protein foaming characteristic factor can be divided into internal factor and processing,
The external factor such as treatment conditions.The molecule performance of protein can influence frothing capacity, the amphipathic increase interfacial interaction of molecule；
Local flexibility promotes protein in the expansion at interface；Reciprocation promotes the contact of interface vapor-liquid two phases；Electrification and polarity group
Character is divided to prevent bubble in close proximity to promotion aquation；Spatial position is also most important to the foaming characteristic of protein.It is most of
External factor determines that operability is subject to certain restrictions by the processing and storage condition of food system.Therefore, protein is influenced to rise
The internal factor of bubble property has operability, can reach certain purpose by modification.Currently, both at home and abroad mainly by Physical,
Chemical method and enzyme process improve protein frothing capacity, and wherein enzyme modification has that reaction condition is mild, equipment requirement is low, safety
The advantages that property is high and be widely adopted.
Invention content
It is excellent it is an object of the invention to solve at least the above and/or defect, and provide at least to will be described later
Point.
It is a still further object of the present invention to provide a kind of preparation methods of high foaming fowl orgotein powder.
For this purpose, technical solution provided by the invention is：
A kind of preparation method of high foaming fowl orgotein powder, including：
Step 1: it is degreasing fowl liver to take poultry liver processing；
Step 2: the degreasing fowl liver is taken to be placed in Tris-HCl buffer solutions, it is ultrasonically treated, the condition of supersound process
For ultrasonic power 1000W~1500W, the percentage that ultrasonic power accounts for ultrasonic wave general power is 5~9%, ultrasonic time is 4~
10min rests 1~3s per ultrasound 2~4s；
Step 3: supernatant is taken after taking the degreasing fowl liver Tris-HCl buffer by centrifugation after ultrasound, later by supernatant pH
4.3 are adjusted to, is centrifuged again, precipitation is collected, obtains fowl orgotein；
Step 4: fowl orgotein is scattered in phosphate buffer, is digested and dialysed successively later, after finally drying
Obtain modified fowl orgotein powder crude product；
Step 5: isolating and purifying modified fowl orgotein powder crude product using gel permeation chromatography, molecular weight is collected<5000Da's
Protolysate obtains high foaming fowl orgotein powder.
Preferably, in the preparation method of the high foaming fowl orgotein powder, in the step 1, fowl liver is taken, is pressed
Mass ratio 1：2~4 are added water, are homogenized 1~3min later, then add the isopropyl of volume 5~15% after fowl liver after accounting for homogenate
Alcohol carries out 8~12h of ungrease treatment, then is centrifuged, and taking precipitation to be placed under draught cupboard makes isopropanol volatilization 12~for 24 hours, obtains institute
State degreasing fowl liver.
Preferably, in the preparation method of the high foaming fowl orgotein powder, in the step 2, the Tris-
A concentration of 0.01~0.03M of HCl buffer solutions, pH7.5~8.5, the matter of the degreasing fowl liver and the Tris-HCl buffer solutions
It is 20~30 to measure volume ratio：600~800g/mL.
Preferably, in the preparation method of the high foaming fowl orgotein powder, in the step 4, the enzymolysis
Specific steps include：First by fowl orgotein phosphate buffer in 80~90 DEG C of 25~45min of humid heat treatment of temperature, later by it
PH is adjusted to 8.0~8.5, be cooled to room temperature be added with the mass volume ratio of the fowl orgotein phosphate buffer be 0.04~
0.06% alkali protease, and 35~45min of water-bath is carried out in 45~55 DEG C, it is put into boiling water bath inactivation after reaction
10~20min.
Preferably, in the preparation method of the high foaming fowl orgotein powder, in the step 4, the dialysis
Specific steps include：Supernatant is removed into the fowl orgotein phosphate buffer centrifugation after enzymolysis first, is later placed in supernatant
It analyses in bag, 4 DEG C of 24~48h of dialysis, wherein replacing deionized water every 2h, finally carries out dialyzate in -55~-60 DEG C true
24~46h of vacuum freecing-dry collects powder and obtains the modified fowl orgotein powder crude product.
Preferably, in the preparation method of the high foaming fowl orgotein powder, in the step 4, the phosphoric acid is slow
The mass ratio of a concentration of 0.01M of fliud flushing, the fowl orgotein and the phosphate buffer is 6~10%.
Preferably, in the preparation method of the high foaming fowl orgotein powder, in the step 5, using described solidifying
The specific steps that glue filtration chromatography isolates and purifies modified fowl orgotein powder crude product include：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water,
It is swollen 4h in 100 DEG C of water-baths later, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method,
It drains, it is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times,
It drains, the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 is used to impregnate later, after ultrasound degassing, be once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl liver egg
A concentration of 0.01M of white phosphorus acid buffer is adopted later by the sephadex Sephadex G-25 gel columns on the sample
It is eluted with pH 8.0Tris-HCl buffer solutions, using DHL-A constant flow pumps, automatic collection is used in combination in coutroi velocity 0.3mL/min
Device is collected, and often pipe collects 3mL, and collection liquid Coomassie Brilliant Blue tracing detection is cross with pipe number until there is no albumen to detect
Coordinate, absorbance are ordinate, draw elution curve, merge the eluent of each eluting peak, with pH 8.0Tris-HCl buffer solutions
Dialyse 72h, and the high bubble fowl orgotein is obtained after dry.
Preferably, in the preparation method of the high foaming fowl orgotein powder, in the step 2, at the ultrasound
It manages and is carried out under vacuum condition, and in the supersound process, also carried out microwave cooperating processing, the 0- of the supersound process
In 2 minutes, the microwave power of the microwave cooperating processing is 450~550W, described micro- in the 2-4 minutes of the supersound process
The microwave power of wave collaboration processing is 350~450W, the 4th minute of the supersound process and after, the microwave cooperating processing
Microwave treatment be 200~250W.
Preferably, in the preparation method of the high foaming fowl orgotein powder, in the step 2, at the ultrasound
The condition of reason is ultrasonic power 1200W, ultrasonic time 5min, per ultrasound 3s, rests 2s.
The present invention includes at least following advantageous effect：
The fowl orgotein that the method for the present invention is prepared, compared with conventional liver protein, foaming characteristic and foaming stability
It respectively obtains conspicuousness and improves (P<0.05), this is because the mechanical effect and cavitation that are ultrasonically treated change liver protein
Structure, more hydrophobic groups and hydrophilic radical exposure, the surface-active with bigger, to reduce the surface of water
Power shows that ultrasonic effect has the function of improving liver protein foaming characteristic and foaming stability foaming characteristic and foaming stability result
Show.Meanwhile the fluorescence intensity of fowl orgotein that method of the invention is prepared significantly improves, and fluorescence intensity is different
The variation tendency of degree, this is because after sonicated, liver protein internal structure stretches, and destroys its intermolecular work
Firmly, part trp residue is caused to expose, to improve its fluorescence intensity, this shows to lead to house using ultrasound assisted extraction
Fowl liver protein secondary structure occurred conformation changes, this may be related with the shearing force generated when being ultrasonically treated.
Compared with unmodified fowl orgotein powder, modified rises the high foaming fowl orgotein powder that the present invention is prepared
1.27~2.12 times and 69%~59% has been respectively increased in bubble property and stability.
Part is illustrated to embody by further advantage, target and the feature of the present invention by following, and part will also be by this
The research and practice of invention and be understood by the person skilled in the art.
Description of the drawings
Fig. 1 is the fluorescence pattern of the liver protein of the present invention in one embodiment；
Fig. 2 is the circular dichroism spectrogram of the liver protein of the present invention in one embodiment.
Specific implementation mode
Present invention will be described in further detail below with reference to the accompanying drawings, to enable those skilled in the art with reference to specification text
Word can be implemented according to this.
It should be appreciated that such as " having ", "comprising" and " comprising " term used herein do not allot one or more
The presence or addition of a other elements or combinations thereof.
The present invention provides a kind of preparation method of high foaming fowl orgotein powder, includes the following steps：
Step 1: it is degreasing fowl liver to take poultry liver processing；
Step 2: the degreasing fowl liver is taken to be placed in Tris-HCl buffer solutions, can preferably soluble protein, and make egg
It is unlikely to deform, is ultrasonically treated in vain, the condition of supersound process is ultrasonic power 1000W~1500W, and ultrasonic power accounts for ultrasonic wave
The percentage of general power is 5~9%, and ultrasonic time is 4~10min, per ultrasound 2~4s, rests 1~3s；Ultrasonic wave extraction is
The mechanical effect having using ultrasonic wave, cavitation effect and fuel factor, by increasing the movement velocity of medium molecule, increasing medium
Penetration power to extract biological effective components.Being ultrasonically treated has following effect：1) mechanical effect：The biography of ultrasonic wave in the medium
Broadcasting can make medium particle generate vibration in its communication space, and to strengthen the diffusion of medium, propagate, here it is ultrasonic waves
Mechanical effect.Ultrasonic wave generates a kind of radiation pressure in communication process, is propagated along sound wave direction, has very strong destruction to material
Effect can make cell metaplasia, phytoprotein denaturation；Meanwhile it can also give medium and suspended substance and be added with different
Speed, and the movement velocity of medium molecule is much larger than the movement velocity of suspended substance molecule.It is this to generate friction between the two
Frictional force can make biomolecule depolymerization, and the active ingredient on cell wall is made quickly to be dissolved among solvent.2) cavitation effect：It is logical
In the case of often, media interior has more or less dissolved some microbubbles, these bubbles generate vibration under the action of ultrasonic wave,
When acoustic pressure reaches certain value, bubble increases due to orientation diffusion (rectieddiffvsion), forms resonant cavity, then dashes forward
It is so closed, here it is the cavitation effects of ultrasonic wave.This bubble can generate the pressure of thousands of a atmospheric pressure when being closed around it
Power forms micro laser wave, it can cause plant cell wall and entire organism to rupture, and entire rupture process is completed in moment,
Be conducive to the dissolution of active ingredient.3) fuel factor：As other physical waves, the communication process of ultrasonic wave in the medium is also one
In the communication process of medium, acoustic energy is constantly absorbed by the particle of medium for the propagation of a energy and diffusion process, i.e. ultrasonic wave,
The energy absorbed is wholly or largely transformed into thermal energy by medium, so as to cause the liter of medium itself and material tissue temperature
Height increases the solution rate of material active ingredient.The raising of drug entities internal temperature caused by this absorption acoustic energy
It is moment, therefore the bioactivity for the ingredient being extracted can be made to remain unchanged.It is destroyed carefully in the present invention by being ultrasonically treated
After birth promotes being fully dissolved out for intracellular reactive protein ingredient, is acted on using low frequency ultrasound penetration, is reducing active egg
White structure promotes albumen dissolution while destroying risk.
Step 3: supernatant is taken after taking the degreasing fowl liver Tris-HCl buffer by centrifugation after ultrasound, later by supernatant pH
4.3 are adjusted to, is centrifuged again, precipitation is collected, obtains fowl orgotein；It is adjusted to the isoelectric point of fowl orgotein, for cleaning.
Step 4: fowl orgotein is scattered in phosphate buffer, is digested and dialysed successively later, after finally drying
Obtain modified fowl orgotein powder crude product；Albumen is modified.The effect of enzymolysis is that high molecular weight protein is made to be degraded to small molecule egg
In vain, dialyse is to further remove the small molecule salt in deproteinized, keep albumen powder purer.
Step 5: isolating and purifying modified fowl orgotein powder crude product using gel permeation chromatography, molecular weight is collected<5000Da's
Protolysate obtains high foaming fowl orgotein powder.
In one of present invention embodiment, preferably, in the step 1, fowl liver, in mass ratio 1 are taken：2~
4 are added water, are homogenized 1~3min later, and the isopropanol for then adding volume 5~15% after fowl liver after accounting for homogenate carries out at degreasing
8~12h is managed, then is centrifuged, taking precipitation to be placed under draught cupboard makes isopropanol volatilization 12~for 24 hours, obtains the degreasing fowl liver.
In one of present invention embodiment, preferably, in the step 2, the Tris-HCl buffer solutions
The mass volume ratio of a concentration of 0.01~0.03M, pH7.5~8.5, the degreasing fowl liver and the Tris-HCl buffer solutions is 20
~30：600~800g/mL.
In one of present invention embodiment, preferably, in the step 4, the specific steps packet of the enzymolysis
It includes：Its pH is adjusted to by fowl orgotein phosphate buffer later in 80~90 DEG C of 25~45min of humid heat treatment of temperature first
8.0~8.5, it is cooled to room temperature and the alkali for being 0.04~0.06% with the mass volume ratio of the fowl orgotein phosphate buffer is added
Property protease, and in 45~55 DEG C carry out 35~45min of water-bath, be put into after reaction boiling water bath inactivate 10~20min.
In one of present invention embodiment, preferably, in the step 4, the specific steps packet of the dialysis
It includes：Supernatant is removed into the fowl orgotein phosphate buffer centrifugation after enzymolysis first, supernatant is placed in bag filter later, 4 DEG C
Dialyse 24~48h, wherein replacing deionized water every 2h, dialyzate is finally carried out vacuum freeze drying in -55~-60 DEG C
24~46h collects powder and obtains the modified fowl orgotein powder crude product.
In one of present invention embodiment, preferably, in the step 4, the concentration of the phosphate buffer
For 0.01M, the mass ratio of the fowl orgotein and the phosphate buffer is 6~10%.
In one of present invention embodiment, preferably, in the step 5, using the gel permeation chromatography
The specific steps for isolating and purifying modified fowl orgotein powder crude product include：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water,
It is swollen 4h in 100 DEG C of water-baths later, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method,
It drains, it is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times,
It drains, the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 is used to impregnate later, after ultrasound degassing, be once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl liver egg
A concentration of 0.01M of white phosphorus acid buffer is adopted later by the sephadex Sephadex G-25 gel columns on the sample
It is eluted with pH 8.0Tris-HCl buffer solutions, using DHL-A constant flow pumps, automatic collection is used in combination in coutroi velocity 0.3mL/min
Device is collected, and often pipe collects 3mL, and collection liquid Coomassie Brilliant Blue tracing detection is cross with pipe number until there is no albumen to detect
Coordinate, absorbance are ordinate, draw elution curve, merge the eluent of each eluting peak, with pH 8.0Tris-HCl buffer solutions
Dialyse 72h, and the high bubble fowl orgotein is obtained after dry.
In one of present invention embodiment, preferably, in the step 2, the supersound process is in vacuum item
It is carried out under part, and in the supersound process, has also carried out microwave cooperating processing, in 0-2 minutes of the supersound process, institute
The microwave power for stating microwave cooperating processing is 450~550W, in the 2-4 minutes of the supersound process, the microwave cooperating processing
Microwave power be 350~450W, the 4th minute of the supersound process and after, the microwave treatment of the microwave cooperating processing
For 200~250W.When being ultrasonically treated, handled by microwave cooperating, microwave treatment can keep the form and structure of albumen, keep away
Exempt from its structure to be destroyed, and, it may have bactericidal effect prevents albumen to be contaminated in extraction process.With ultrasound into
The extension of row and time, gradually reduces microwave power, gradually " tenderness " with the active force to albumen, is protecting its structure and form
While, also further promote the rupture of cell wall, promotes albumen dissolution, saves extraction time, improve extraction efficiency.
In one of present invention embodiment, preferably, in the step 2, the condition of the supersound process is
Ultrasonic power 1200W, ultrasonic time 5min rest 2s per ultrasound 3s.
To make those skilled in the art more fully understand technical scheme of the present invention, following embodiment is now provided and is said
It is bright：
Embodiment 1
A kind of preparation method of high foaming fowl orgotein powder, includes the following steps：
A certain amount of fowl liver is taken, by 1：3 ratios add water, high-speed homogenization 2min to be put into the large beaker of 1000mL, are added 10%
Isopropanol ungrease treatment 10h after standing overnight, centrifuges 20min under the conditions of 5000g, and top layer is oil layer, and liver is obtained after centrifugation
Oil takes out precipitation and is laid in hit-gain trust disk, and being placed under draught cupboard makes isopropanol volatilization 18h, finally collects degreasing fowl liver.Weigh one
Quantitative degreasing fowl liver (25g fowl hepar siccatum), is put into a certain amount of Tris-HCl (0.02M, pH8.0,700mL) buffer solution, in ultrasound
It is extracted certain time in wave extraction apparatus, ultrasonic 200W, ultrasonic power accounts for the percentage 7% of ultrasonic wave general power, and ultrasonic 5min surpasses
Sound 3s, rests 2s, then stirring in water bath 30min.20min is centrifuged with 5000g, supernatant is taken to be adjusted to isoelectric point, pH4.3, then with
5000g centrifuges 20min, collects precipitation, washes 2~3 times.
Fowl orgotein is scattered in concentration 0.01M phosphate buffers, albumen a concentration of 8%, in 85 DEG C of humid heat treatments
After 35min, pH is adjusted to 8.25.It is cooled to room temperature addition 0.05% (w/v) alkali protease, 50 DEG C of water-baths 35~
45min.It is put into boiling water bath inactivation 15min after reaction, 20min is centrifuged with 5000g, supernatant is taken to be placed in bag filter, 4 DEG C
Dialyse 36h, and deionized water is replaced every 2h；For dialyzate in -57 DEG C of vacuum freeze drying 35h, it is modified fowl liver to collect powder
Albumen powder crude product.Modified fowl orgotein powder crude product is isolated and purified using gel permeation chromatography again, collects molecular weight<The egg of 5000Da
White hydrolysate obtains high foaming fowl orgotein powder.It is thick that modified fowl orgotein powder is isolated and purified using the gel permeation chromatography
The specific steps of product include：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water,
It is swollen 4h in 100 DEG C of water-baths later, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method,
It drains, it is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times,
It drains, the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 is used to impregnate later, after ultrasound degassing, be once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl liver egg
A concentration of 0.01M of white phosphorus acid buffer is adopted later by the sephadex Sephadex G-25 gel columns on the sample
It is eluted with pH 8.0Tris-HCl buffer solutions, using DHL-A constant flow pumps, automatic collection is used in combination in coutroi velocity 0.3mL/min
Device is collected, and often pipe collects 3mL, and collection liquid Coomassie Brilliant Blue tracing detection is cross with pipe number until there is no albumen to detect
Coordinate, absorbance are ordinate, draw elution curve, merge the eluent of each eluting peak, with pH 8.0Tris-HCl buffer solutions
Dialyse 72h, and the high bubble fowl orgotein is obtained after dry.
Measure the foaming characteristic and bubble stability of albumen powder.Foaming characteristic and foaming stability the result shows that, and it is unmodified
Fowl orgotein powder compares, and 2.12 times and 69% has been respectively increased in modified foaming characteristic and stability.
Embodiment 2
A kind of preparation method of high foaming fowl orgotein powder, includes the following steps：
A certain amount of fowl liver is taken, by 1：2 ratios add water, high-speed homogenization 1min to be put into the large beaker of 1000mL, are added 10%
Isopropanol ungrease treatment 8h after standing overnight, centrifuges 15min under the conditions of 4000g, and top layer is oil layer, and liver is obtained after centrifugation
Oil takes out precipitation and is laid in hit-gain trust disk, and being placed under draught cupboard makes isopropanol volatilization 12h, finally collects degreasing fowl liver.Weigh one
Quantitative degreasing fowl liver (20g fowl hepar siccatum), is put into Tris-HCl (0.02M, pH7.5,600mL) buffer solution, in ultrasonic wave extraction
It is extracted in instrument certain time, ultrasonic power 1000W, ultrasonic power accounts for the percentage 5% of ultrasonic wave general power, and ultrasonic 4min surpasses
Sound 2s, rests 1s, then stirring in water bath 20min.15min is centrifuged with 4000g, supernatant is taken to be adjusted to isoelectric point, pH4.3, then with
4000g centrifuges 15min, collects precipitation, washes 2~3 times.
Fowl orgotein is scattered in concentration 0.01M phosphate buffers, albumen a concentration of 6%, in 80 DEG C of humid heat treatments
After 25min, pH is adjusted to 8.0.It is cooled to room temperature addition 0.04% (w/v) alkali protease, 45 DEG C of water-bath 35min.Instead
It is put into boiling water bath inactivation 10min after answering, 15~25min is centrifuged with 4000g, supernatant is taken to be placed in bag filter, 4 DEG C of dialysis
For 24 hours, deionized water is replaced every 2h；For 24 hours in -55 DEG C of vacuum freeze dryings, it is modified fowl orgotein to collect powder to dialyzate
Powder crude product.Modified fowl orgotein powder crude product is isolated and purified using gel permeation chromatography again, collects molecular weight<The albumen water of 5000Da
Object is solved, high foaming fowl orgotein powder is obtained.Modified fowl orgotein powder crude product is isolated and purified using the gel permeation chromatography
Specific steps include：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water,
It is swollen 4h in 100 DEG C of water-baths later, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method,
It drains, it is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times,
It drains, the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 is used to impregnate later, after ultrasound degassing, be once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl liver egg
A concentration of 0.01M of white phosphorus acid buffer is adopted later by the sephadex Sephadex G-25 gel columns on the sample
It is eluted with pH 8.0Tris-HCl buffer solutions, using DHL-A constant flow pumps, automatic collection is used in combination in coutroi velocity 0.3mL/min
Device is collected, and often pipe collects 3mL, and collection liquid Coomassie Brilliant Blue tracing detection is cross with pipe number until there is no albumen to detect
Coordinate, absorbance are ordinate, draw elution curve, merge the eluent of each eluting peak, with pH 8.0Tris-HCl buffer solutions
Dialyse 72h, and the high bubble fowl orgotein is obtained after dry.
Measure the foaming characteristic and bubble stability of albumen powder.Foaming characteristic and foaming stability the result shows that, and it is unmodified
Fowl orgotein powder compares, and 1.27 times and 59% has been respectively increased in modified foaming characteristic and stability.
Embodiment 3
A kind of preparation method of high foaming fowl orgotein powder, includes the following steps：
A certain amount of fowl liver is taken, by 1：4 ratios add water, high-speed homogenization 3min to be put into the large beaker of 1000mL, are added 10%
Isopropanol ungrease treatment 12h after standing overnight, centrifuges 25min under the conditions of 6000g, and top layer is oil layer, and liver is obtained after centrifugation
Oil takes out precipitation and is laid in hit-gain trust disk, and being placed under draught cupboard makes isopropanol volatilization for 24 hours, finally collects degreasing fowl liver.Weigh one
Quantitative degreasing fowl liver (30g fowl hepar siccatum), is put into Tris-HCl (0.02M, pH8.5,800mL) buffer solution, in ultrasonic wave extraction
Certain time is extracted in instrument, and (1500W, ultrasonic power account for the percentage 9% of ultrasonic wave general power, and ultrasonic 10min, ultrasonic 4s stop
Have a rest 3s, then stirring in water bath 40min).Centrifuge 25min with 6000g, supernatant taken to be adjusted to isoelectric point, pH4.3, then with 6000g from
Heart 25min collects precipitation, washes 2~3 times.
Fowl orgotein is scattered in concentration 0.01M phosphate buffers, albumen a concentration of 10%, in 90 DEG C of humid heat treatments
After 45min, pH is adjusted to 8.5.It is cooled to room temperature addition 0.06% (w/v) alkali protease, 55 DEG C of water-bath 45min.Instead
It is put into boiling water bath inactivation 20min after answering, 25min is centrifuged with 6000g, supernatant is taken to be placed in bag filter, 4 DEG C of dialysis 48h,
Deionized water is replaced every 2h；For dialyzate in -60 DEG C of vacuum freeze drying 46h, it is that modified fowl orgotein powder is thick to collect powder
Product.Modified fowl orgotein powder crude product is isolated and purified using gel permeation chromatography again, collects molecular weight<The proteolysis of 5000Da
Object obtains high foaming fowl orgotein powder.The tool of modified fowl orgotein powder crude product is isolated and purified using the gel permeation chromatography
Body step includes：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water,
It is swollen 4h in 100 DEG C of water-baths later, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method,
It drains, it is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times,
It drains, the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 is used to impregnate later, after ultrasound degassing, be once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl liver egg
A concentration of 0.01M of white phosphorus acid buffer is adopted later by the sephadex Sephadex G-25 gel columns on the sample
It is eluted with pH 8.0Tris-HCl buffer solutions, using DHL-A constant flow pumps, automatic collection is used in combination in coutroi velocity 0.3mL/min
Device is collected, and often pipe collects 3mL, and collection liquid Coomassie Brilliant Blue tracing detection is cross with pipe number until there is no albumen to detect
Coordinate, absorbance are ordinate, draw elution curve, merge the eluent of each eluting peak, with pH 8.0Tris-HCl buffer solutions
Dialyse 72h, and the high bubble fowl orgotein is obtained after dry.
Embodiment 4
A kind of preparation method of high foaming fowl orgotein powder, includes the following steps：
A certain amount of fowl liver is taken, by 1：3 ratios add water, high-speed homogenization 2min to be put into the large beaker of 1000mL, are added 10%
Isopropanol ungrease treatment 9h after standing overnight, centrifuges 22min under the conditions of 5500g, and top layer is oil layer, and liver is obtained after centrifugation
Oil takes out precipitation and is laid in hit-gain trust disk, and being placed under draught cupboard makes isopropanol volatilization 20h, finally collects degreasing fowl liver.Weigh one
Quantitative degreasing fowl liver (28g fowl hepar siccatum), is put into Tris-HCl (0.02M, pH7.8,750mL) buffer solution, in ultrasonic wave extraction
It being extracted in instrument certain time, ultrasonic power 1100W, ultrasonic power accounts for the percentage 5~9% of ultrasonic wave general power, ultrasonic 6min,
Ultrasonic 2s, rests 1s, then stirring in water bath 25min.The supersound process carries out under vacuum condition, and in the supersound process
In, also carry out microwave cooperating processing, in 0-2 minutes of the supersound process, the microwave power of the microwave cooperating processing
For 500W, in the 2-4 minutes of the supersound process, the microwave power of the microwave cooperating processing is 400W, the supersound process
The 4th minute and after, the microwave treatment of microwave cooperating processing is 225W.22min is centrifuged with 5500g, takes supernatant tune
To isoelectric point, pH4.3, then 22min is centrifuged with 5500g, collects precipitation, wash 2~3 times.
Fowl orgotein is scattered in concentration 0.01M phosphate buffers, albumen a concentration of 8%, in 88 DEG C of humid heat treatments
After 40min, pH is adjusted to 8.2.It is cooled to room temperature addition 0.05% (w/v) alkali protease, 458 DEG C of water-bath 40min.Instead
It is put into boiling water bath inactivation 18min after answering, 22min is centrifuged with 5500g, supernatant is taken to be placed in bag filter, 4 DEG C of dialysis 40h,
Deionized water is replaced every 2h；For dialyzate in -58 DEG C of vacuum freeze drying 40h, it is that modified fowl orgotein powder is thick to collect powder
Product.Modified fowl orgotein powder crude product is isolated and purified using gel permeation chromatography again, collects molecular weight<The proteolysis of 5000Da
Object obtains high foaming fowl orgotein powder.The tool of modified fowl orgotein powder crude product is isolated and purified using the gel permeation chromatography
Body step includes：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water,
It is swollen 4h in 100 DEG C of water-baths later, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method,
It drains, it is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times,
It drains, the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 is used to impregnate later, after ultrasound degassing, be once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl liver egg
A concentration of 0.01M of white phosphorus acid buffer is adopted later by the sephadex Sephadex G-25 gel columns on the sample
It is eluted with pH 8.0Tris-HCl buffer solutions, using DHL-A constant flow pumps, automatic collection is used in combination in coutroi velocity 0.3mL/min
Device is collected, and often pipe collects 3mL, and collection liquid Coomassie Brilliant Blue tracing detection is cross with pipe number until there is no albumen to detect
Coordinate, absorbance are ordinate, draw elution curve, merge the eluent of each eluting peak, with pH 8.0Tris-HCl buffer solutions
Dialyse 72h, and the high bubble fowl orgotein is obtained after dry.
Embodiment 5
A kind of preparation method of high foaming fowl orgotein powder, includes the following steps：
A certain amount of fowl liver is taken, by 1：4 ratios add water, high-speed homogenization 3min to be put into the large beaker of 1000mL, are added 10%
Isopropanol 8~12h of ungrease treatment after standing overnight, centrifuges 25min, top layer is oil layer, after centrifugation under the conditions of 4500g
Liver oil, take out precipitation be laid in hit-gain trust disk, be placed under draught cupboard make isopropanol volatilization 12~for 24 hours, finally collect degreasing fowl
Liver.A certain amount of degreasing fowl liver (22g fowl hepar siccatum) is weighed, is put into Tris-HCl (0.02M, pH8.2,650mL) buffer solution, super
It being extracted certain time in sound wave extraction apparatus, ultrasonic power 1300W, ultrasonic power accounts for the percentage 5~9% of ultrasonic wave general power,
Ultrasonic 8min, ultrasonic 4s, rest 3s, then stirring in water bath 38min).The supersound process carries out under vacuum condition, and in institute
It states in supersound process, has also carried out microwave cooperating processing, in 0-2 minutes of the supersound process, the microwave cooperating processing
Microwave power be 550W, in 2-4 minutes of the supersound process, the microwave power of microwave cooperating processing is 450W, institute
State the 4th minute of supersound process and after, the microwave treatment of the microwave cooperating processing is 250W.25min is centrifuged with 4500g,
It takes supernatant to be adjusted to isoelectric point, pH4.3, then 25min is centrifuged with 4500g, collect precipitation, wash 2~3 times.
Fowl orgotein is scattered in concentration 0.01M phosphate buffers, albumen a concentration of 7%, in 83 DEG C of humid heat treatments
After 28min, pH is adjusted to 8.2.It is cooled to room temperature addition 0.06% (w/v) alkali protease, 55 DEG C of water-bath 35min.Instead
It is put into boiling water bath inactivation 12min after answering, 25min is centrifuged with 4500g, supernatant is taken to be placed in bag filter, 4 DEG C of dialysis 42h,
Deionized water is replaced every 2h；For dialyzate in -59 DEG C of vacuum freeze drying 41h, it is that modified fowl orgotein powder is thick to collect powder
Product.Modified fowl orgotein powder crude product is isolated and purified using gel permeation chromatography again, collects molecular weight<The proteolysis of 5000Da
Object obtains high foaming fowl orgotein powder.The tool of modified fowl orgotein powder crude product is isolated and purified using the gel permeation chromatography
Body step includes：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water,
It is swollen 4h in 100 DEG C of water-baths later, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method,
It drains, it is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times,
It drains, the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 is used to impregnate later, after ultrasound degassing, be once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl liver egg
A concentration of 0.01M of white phosphorus acid buffer is adopted later by the sephadex Sephadex G-25 gel columns on the sample
It is eluted with pH 8.0Tris-HCl buffer solutions, using DHL-A constant flow pumps, automatic collection is used in combination in coutroi velocity 0.3mL/min
Device is collected, and often pipe collects 3mL, and collection liquid Coomassie Brilliant Blue tracing detection is cross with pipe number until there is no albumen to detect
Coordinate, absorbance are ordinate, draw elution curve, merge the eluent of each eluting peak, with pH 8.0Tris-HCl buffer solutions
Dialyse 72h, and the high bubble fowl orgotein is obtained after dry.
Embodiment 6
A kind of preparation method of high foaming fowl orgotein powder, includes the following steps：
A certain amount of fowl liver is taken, by 1：2 ratios add water, high-speed homogenization 1min to be put into the large beaker of 1000mL, are added 10%
Isopropanol ungrease treatment 8h after standing overnight, centrifuges 15min under the conditions of 4000g, and top layer is oil layer, and liver is obtained after centrifugation
Oil, take out precipitation be laid in hit-gain trust disk, be placed under draught cupboard make isopropanol volatilization 12~for 24 hours, finally collect degreasing fowl liver.Claim
A certain amount of degreasing fowl liver (30g fowl hepar siccatum) is taken, is put into Tris-HCl (0.02M, pH7.5,600mL) buffer solution, in ultrasonic wave
It is extracted in extraction apparatus certain time, ultrasonic power 1200W, ultrasonic power accounts for the percentage 5~9% of ultrasonic wave general power, ultrasound
5min, ultrasonic 3s, rest 2s, then stirring in water bath 30min.The supersound process carries out under vacuum condition, and described super
In sonication, also carried out microwave cooperating processing, in 0-2 minutes of the supersound process, the microwave cooperating processing it is micro-
Wave power is 450W, and in the 2-4 minutes of the supersound process, the microwave power of the microwave cooperating processing is 350W, described super
The 4th minute of sonication and after, the microwave treatment of the microwave cooperating processing is 200.25min is centrifuged with 4000g, takes supernatant
Liquid is adjusted to isoelectric point, pH4.3, then centrifuges 15min with 4000g, collects precipitation, washes 2~3 times.
Fowl orgotein is scattered in concentration 0.01M phosphate buffers, albumen a concentration of 6%, in 80 DEG C of humid heat treatments
After 25min, pH is adjusted to 8.0.It is cooled to room temperature addition 0.04% (w/v) alkali protease, 45 DEG C of water-bath 35min.Instead
It is put into boiling water bath inactivation 10min after answering, 15min is centrifuged with 4000g, supernatant is taken to be placed in bag filter, 4 DEG C are dialysed for 24 hours,
Deionized water is replaced every 2h；For 24 hours in -55 DEG C of vacuum freeze dryings, it is that modified fowl orgotein powder is thick to collect powder to dialyzate
Product.Modified fowl orgotein powder crude product is isolated and purified using gel permeation chromatography again, collects molecular weight<The proteolysis of 5000Da
Object obtains high foaming fowl orgotein powder.The tool of modified fowl orgotein powder crude product is isolated and purified using the gel permeation chromatography
Body step includes：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water,
It is swollen 4h in 100 DEG C of water-baths later, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method,
It drains, it is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times,
It drains, the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 is used to impregnate later, after ultrasound degassing, be once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl liver egg
A concentration of 0.01M of white phosphorus acid buffer is adopted later by the sephadex Sephadex G-25 gel columns on the sample
It is eluted with pH 8.0Tris-HCl buffer solutions, using DHL-A constant flow pumps, automatic collection is used in combination in coutroi velocity 0.3mL/min
Device is collected, and often pipe collects 3mL, and collection liquid Coomassie Brilliant Blue tracing detection is cross with pipe number until there is no albumen to detect
Coordinate, absorbance are ordinate, draw elution curve, merge the eluent of each eluting peak, with pH 8.0Tris-HCl buffer solutions
Dialyse 72h, and the high bubble fowl orgotein is obtained after dry.
Compliance test result
1. the foaming characteristic of poultry liver albumen and the measurement of foaming stability
Prepare the poultry liver of 0.1%, 0.5%, 1.0% and 2.0% 20mL routine water-bath extractions and ultrasound assisted extraction
In dirty protein solution to 100mL graduated cylinders, the volume (V of solution at this time is measured0).Then by solution under high-shear homogenizer with
12000g measures the sample volume (V in graduated cylinder at this time to being immediately transferred in graduated cylinder after 30sT), it stands 60 minutes and measures this
When graduated cylinder in sample volume (Vt).Shown in foaming characteristic (FC) and foaming stability (FS) calculation formula such as formula (1) and formula (2)：
In formula：VTIt is the total volume after homogeneous；V0It is the initial volume before homogeneous；Vt is after standing 60min at room temperature
Total volume.
2. foaming characteristic and foaming stability value
The foaming characteristic and foam stability of 1 poultry liver albumen of table
Note：Different lowercase letters have significant difference, P between the two under same protein concentration in two groups of samples<
0.05。
Albumen foaming characteristic plays an important roll in the food industry, in extensive applications such as beverage, coffees.It can by table 1
Know, compared with conventional liver protein, ultrasonic liver protein foaming characteristic and foaming stability respectively obtain conspicuousness and improve (P<
0.05), it may be possible to since the mechanical effect and cavitation of supersound process change liver protein structure, more hydrophobicity bases
Group and hydrophilic radical exposure, the surface-active with bigger show that ultrasonic effect has and carry to reduce the surface tension of water
The effect of high liver protein foaming characteristic and foaming stability.
3. two collection of illustrative plates of the fluorescence of liver protein and circle
Fluorescence spectrum is that characterization protein molecule conceives changed another means, by measuring part energy in protein
The amino acid residue (tryptophan, tyrosine, phenylalanine) of fluorescence is generated to speculate the variation tendency of its albumen conception.By Fig. 1
It is found that at 330~340nm of wavelength, the fluorescence intensity highest of conventional liver protein and ultrasonic liver protein.With conventional liver egg
It compares, is significantly improved by using the liver protein fluorescence intensity of assisted extraction, and fluorescence intensity has different degrees of change in vain
Change trend, this may be since after sonicated, liver protein internal structure stretches, and destroys its intermolecular interaction
Power causes part trp residue to expose, to improve its fluorescence intensity.Show to lead to poultry liver using ultrasound assisted extraction
Dirty Protein secondary structure occurred conformation variation, this may be related with the shearing force generated when being ultrasonically treated.
The secondary structure of 2 poultry liver albumen of table
Fig. 2 is control group and ultrasound group liver protein circular dichroism figure.It is calculated each using circular dichroism spectrometer program
Liver protein Secondary Structure Content is shown in Table 2.Far-ultraviolet region (190-240nm) circular dichroism of CD spectrum is mainly by the electronics of peptide bond
Transition causes, and reflects the conformation of peptide chain main chain, is usually used in secondary protein structure analysis.Secondary protein structure is different, production
Raw two bands of a spectrum peak intensity of circle and position are different, therefore can be believed according to far ultraviolet CD spectrum analysis Secondary structures
Breath.
In two spectrum of circle there are three characteristic peak, 192nm locates to bear there are two a posivtive spike and the places 208 and 222nm alpha-helix
Peak.The variation for the liver protein secondary structure reacted in figure and table, it may be possible to cavitation effect of ultrasonic wave etc. and placement process
Middle active site of protein structure change, makes the beta sheet of alpha-helix Unfolding Time-varying Linear Systems, protein structure is caused to change so that liver
Dirty protein binding interaction changes, its conformation is caused to change.This may be the dissolubility and foaming characteristic of albumen after ultrasound
The reason of increase module number described herein and treatment scale be for simplify the present invention explanation.To the raised of the present invention
The application of the preparation method of bubble property fowl orgotein, modifications and variations will be readily apparent to persons skilled in the art.
As described above, according to the present invention, as a result of ultrasonic, enzymolysis, the fowl liver egg obtained according to the present invention
White powder, compared with unmodified fowl orgotein powder, modified foaming characteristic and stability be respectively increased 1.27~2.12 times and
59%~69%.
Although the embodiments of the present invention have been disclosed as above, but its is not only in the description and the implementation listed
With it can be fully applied to various fields suitable for the present invention, for those skilled in the art, can be easily
Realize other modification, therefore without departing from the general concept defined in the claims and the equivalent scope, the present invention is simultaneously unlimited
In specific details and legend shown and described herein.
Claims (9)
1. a kind of preparation method of high foaming fowl orgotein powder, which is characterized in that include the following steps：
Step 1: it is degreasing fowl liver to take poultry liver processing；
Step 2: the degreasing fowl liver is taken to be placed in Tris-HCl buffer solutions, it is ultrasonically treated, the condition of supersound process is super
Acoustical power 1000W~1500W, the percentage that ultrasonic power accounts for ultrasonic wave general power are 5~9%, and ultrasonic time is 4~10min,
Per ultrasound 2~4s, 1~3s is rested；
Step 3: taking supernatant after taking the degreasing fowl liver Tris-HCl buffer by centrifugation after ultrasound, supernatant pH is adjusted later
It to 4.3, centrifuges again, collects precipitation, obtain fowl orgotein；
Step 4: fowl orgotein is scattered in phosphate buffer, is digested and dialysed successively later, is obtained after finally drying
Modified fowl orgotein powder crude product；
Step 5: isolating and purifying modified fowl orgotein powder crude product using gel permeation chromatography, molecular weight is collected<The albumen of 5000Da
Hydrolysate obtains high foaming fowl orgotein powder.
2. the preparation method of high foaming fowl orgotein powder as described in claim 1, which is characterized in that in the step 1,
Take fowl liver, in mass ratio 1：2~4 are added water, are homogenized 1~3min later, then add account for after homogenate volume 5 after fowl liver~
15% isopropanol carries out 8~12h of ungrease treatment, then is centrifuged, take precipitation be placed under draught cupboard make isopropanol volatilization 12~
For 24 hours, the degreasing fowl liver is obtained.
3. the preparation method of high foaming fowl orgotein powder as described in claim 1, which is characterized in that in the step 2,
A concentration of 0.01~0.03M of the Tris-HCl buffer solutions, pH7.5~8.5, the degreasing fowl liver are slow with the Tris-HCl
The mass volume ratio of fliud flushing is 20~30：600~800g/mL.
4. the preparation method of high foaming fowl orgotein powder as described in claim 1, which is characterized in that in the step 4,
The specific steps of the enzymolysis include：First by fowl orgotein phosphate buffer in 80~90 DEG C of humid heat treatments 25 of temperature~
Its pH is adjusted to 8.0~8.5 by 45min later, is cooled to room temperature the mass body being added with the fowl orgotein phosphate buffer
Product carries out 35~45min of water-bath than the alkali protease for 0.04~0.06%, and in 45~55 DEG C, puts after reaction
Enter boiling water bath and inactivates 10~20min.
5. the preparation method of high foaming fowl orgotein powder as claimed in claim 4, which is characterized in that in the step 4,
The specific steps of the dialysis include：Supernatant is removed into the fowl orgotein phosphate buffer centrifugation after enzymolysis first, it later will be upper
Clear liquid is placed in bag filter, 4 DEG C of 24~48h of dialysis, wherein replace deionized water every 2h, finally by dialyzate in -55~-
60 DEG C of progress 24~46h of vacuum freeze drying, collect powder and obtain the modified fowl orgotein powder crude product.
6. the preparation method of high foaming fowl orgotein powder as described in claim 1, which is characterized in that in the step 4,
The mass ratio of a concentration of 0.01M of the phosphate buffer, the fowl orgotein and the phosphate buffer is 6~10%.
7. the preparation method of high foaming fowl orgotein powder as described in claim 1, which is characterized in that in the step 5,
The specific steps that modified fowl orgotein powder crude product is isolated and purified using the gel permeation chromatography include：
5.1) pretreatment of Sephadex G-25：Sephadex Sephadex G-25 are soaked in 48h in distilled water, later
It is swollen 4h in 100 DEG C of water-baths, after so that it is fully swollen, adds distilled water to wash repeatedly and is inclined suspended particulate with suspension method, drained,
It is spare；
5.2) column and balance are filled：The sephadex Sephadex G-25 pre-processed are washed with distilled water three times, are taken out
It is dry, it uses the Tris-HCl buffer solutions of 0.02mol/L pH 8.0 to impregnate later, after ultrasound degassing, is once added to using glass bar
In 1.6 × 50cm chromatographic columns, while post jamb is tapped, keeps filling settlement uniformly and close, so that pillar height is reached 45cm, then with the phosphorus
Acid buffer dynamic settling for 24 hours, makes filler fully be swollen, and obtains sephadex Sephadex G-25 gel columns；
5.3) the modified fowl orgotein is dissolved in the phosphate buffer of 0.01M as sample, the modified fowl orgotein phosphorus
A concentration of 0.01M of acid buffer, later by the sephadex Sephadex G-25 gel columns on the sample, using pH
8.0 Tris-HCl buffer solutions are eluted, and using DHL-A constant flow pumps, coutroi velocity 0.3mL/min is used in combination automatic collector to receive
Collection, often pipe collect 3mL, collection liquid Coomassie Brilliant Blue tracing detection, until there is no albumen to detect, with pipe number for horizontal seat
Mark, absorbance is ordinate, draws elution curve, merges the eluent of each eluting peak, saturating with pH 8.0Tris-HCl buffer solutions
72h is analysed, high bubble fowl orgotein is obtained after dry.
8. the preparation method of high foaming fowl orgotein powder as described in claim 1, which is characterized in that in the step 2,
The supersound process carries out under vacuum condition, and in the supersound process, has also carried out microwave cooperating processing, the ultrasound
In 0-2 minutes of processing, the microwave power of the microwave cooperating processing is 450~550W, the 2-4 minutes of the supersound process
Interior, the microwave power of microwave cooperating processing is 350~450W, the 4th minute of the supersound process and after, the microwave
The microwave treatment of collaboration processing is 200~250W.
9. the preparation method of high foaming fowl orgotein powder as described in claim 1, which is characterized in that in the step 2,
The condition of the supersound process is ultrasonic power 1200W, ultrasonic time 5min, per ultrasound 3s, rests 2s.
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| CN112279886A (en) * | 2020-09-23 | 2021-01-29 | 南昌大学 | Method for improving foaming performance of protein isolate of jackfruit seeds by high-pressure steam assisted high-intensity ultrasound |
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