CN109609480A - The extracting method of protein in a kind of egg white - Google Patents
The extracting method of protein in a kind of egg white Download PDFInfo
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- CN109609480A CN109609480A CN201910053091.8A CN201910053091A CN109609480A CN 109609480 A CN109609480 A CN 109609480A CN 201910053091 A CN201910053091 A CN 201910053091A CN 109609480 A CN109609480 A CN 109609480A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2462—Lysozyme (3.2.1.17)
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/465—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from birds
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/77—Ovalbumin
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01017—Lysozyme (3.2.1.17)
Abstract
The present invention provides a kind of extracting methods of protein in egg white, the main method using the absorption of salt precipitation binding resin once isolates ovomucin, ovotransferrins, ovalbumin, ovoinhibitor, ovomucoid and lysozyme totally six kinds of albumen from egg white, this method simple process, extracting cycle is short, not only avoid toxic reagent residue problem, production efficiency is substantially increased simultaneously, it is easy to accomplish industrialization.The present invention substantially increases the added value of egg processing, produces significant economic benefit and social benefit.
Description
Technical field
The present invention relates to Protein Extraction technical fields, and in particular to the extracting method of protein in a kind of egg white.
Background technique
China is Yang Qin big country, and the yield of birds, beasts and eggs accounts for first place in the world, and egg is as animal most commonly seen in human lives
Property one of food and the mankind obtain the important sources of nutrition.In addition, egg also has a variety of industrialization properties, such as blister
The characteristics such as property, emulsibility and gelation, are widely used in multiple fields such as medicine, food industry, daily chemical industries.And these are special
Property and the various protein being rich in egg are closely bound up, such as the highest ovalbumin of abundance accounts for about the total egg of egg white in egg white
Bai Hanliang is 54%, there is preferable foaming characteristic, gelation and emulsibility；The ovotransferrins that content is 12% has very strong
Chelates ferric ions ability, have exploitation for mend iron product potentiality；Although and lysozyme content only 3.4%, due to having
Stronger antibacterial action has been widely used in the anti-corrosion of food；Same content is only 3.5% ovomucin, research shows that its
With antiviral, antibacterial, immunological regulation and reduce the multiple biological activities such as cholesterol.Therefore, with different function characteristic
Either in market, still its demand is very huge in scientific research for albumen.
With deepening continuously for the Physiology and biochemistry activity research to albumen various in egg, more and more researchs are focused on
Different types of albumen is successively separated from egg, develops the physiological activity object with compared with high added value to realize
Matter.But for current present Research, the research for extracting single albumen is the most universal, and combines about multiple protein in egg and mention
The research taken is actually rare.General combined extracting albumen main method is by pH or salt precipitation albumen, again first
Further make protein purification using chromatographic purification methods.Patent No. 201210083362.2 is using first with polyethylene glycol
8000 fractional precipitation albumen, then lysozyme, albumen have been further separated out by Q Sepharose FF anion-exchange chromatography
Albumen, ovotransferrins and with flavine binding protein.2010, wu etc. disclosed two step NaCl saltings out method precipitating ovum and sticks egg
It is white, ovalbumin, ovotransferrins, lysozyme and riboflavin combination egg have been isolated in conjunction with anion cation-exchange chromatography
It is white.BingFu in 2013 etc. separates ovalbumin, lysozyme and ovotransferrins with ion-exchange chromatography using ammonium sulfate precipitation
Three kinds of protein.
It can be seen that the chromatographic separation technology that protein isolate uses makes it in industrial production from above-mentioned patent and document
In application it is very limited, the foundation for extracting egg white multiple protein method extensive at present is also immature, one is extracting
It joined the toxic reagents such as trichloroacetic acid in journey, be not only easy to be denaturalized protein irreversibility, but also since toxic reagent is residual
It stays and existing safety problem is also unfavorable for it in the application of food industry.Second, a variety of eggs of most researchers combined separation
The purity for needing to use different chromatogram arrangements to improve various albumen when white, but since chromatogram arrangement applied sample amount is low, production effect
Rate is low, causes albumen preparation cost too high, is unfavorable for industrialized production.
Introduce that toxic reagent, extraction efficiency be high, Yi Jikao during various albumen for extracting from egg at present
The problems such as considering the cost of industrialized production, it is necessary to develop a kind of extracting method efficiently, green, economic.
Summary of the invention
In view of above-mentioned deficiencies of the prior art, the present invention provides a kind of extracting methods of protein in egg white, special
Sign is, comprising the following steps:
(1) it extracts lysozyme: taking egg white 3000mL, distilled water is added, adjust PH, resin is added, stir, stand,
Centrifugation, obtains the first sediment and the first supernatant, the first sediment is rinsed using distilled water and buffer, eluent is washed
De-, centrifugation, obtains lysozyme；
(2) it extracts ovomucin: taking step (1) first supernatant, calcium chloride solution is added, adjust PH, centrifugation obtains
To the second sediment and the second supernatant, second sediment is taken, distilled water is added, it is homogeneous, PH is adjusted, centrifugation obtains ovum
Mucin；
(3) it extracts ovotransferrins: taking step (2) second supernatant, ammonium sulfate and citric acid is added, adjusts for the first time
PH is saved, is stood, centrifugation, is obtained third sediment and third supernatant, take the third sediment, add to lauryl sodium sulfate
In solution, centrifugation obtains the 4th sediment and the 4th supernatant, and the 4th sediment is poured into distilled water, second of adjusting
PH, standing, third time adjust PH, and centrifugation obtains ovotransferrins；
(4) it extracts ovomucoid: taking step (3) the third supernatant and the 4th supernatant, ammonium sulfate is added, it is quiet
It sets, be centrifuged, obtain the 5th supernatant, heat the 5th supernatant, centrifugation takes supernatant i.e. the 6th supernatant, is added anhydrous
Ethyl alcohol, centrifugation obtain the 5th sediment and ovomucoid；
(5) it extracts ovalbumin: taking step (4) the 5th sediment, distilled water is added, adjust PH, centrifugation obtains ovum
Albumin and the 7th supernatant；
(6) it extracts ovoinhibitor: taking step (5) the 7th supernatant, concentrating and desalinating adjusts PH, and it is molten that ammonium sulfate is added
Liquid, stirring, centrifugation, obtaining supernatant is ovomucoid, and obtaining sediment is ovoinhibitor.
Step (1) egg white, distilled water and resin ratio are 50mL:50mL:5-20g, and the resin is FPC3500 type
Resin；The speed of agitator is 3500r/min, mixing time 10-15min.The buffer is the glycine hydrogen that PH is 9.3
Sodium hydroxide solution, the eluent are the mixed liquors of glycine sodium hydroxide and sodium chloride；The glycine sodium hydroxide solution
For 0.05-0.2mol, the sodium chloride solution is 0.1-1.0mol.
The concentration of step (2) described calcium chloride solution is 0.2-0.5mol/L；The volume of calcium chloride solution, the first supernatant
Than for 1:1.
The quality of step (3) described ammonium sulfate is 137-275g, and the quality of citric acid is 82.5-137.5g；Described 12
The amount of the substance of sodium alkyl sulfate solution is 1-30mmol, volume 800-1200mL.
Step (4) the ammonium sulfate quality is 93-155g, and dehydrated alcohol volume is 1500-4500mL；The heating temperature
It is 50-70 DEG C, it is preferable that the heating temperature is 55 DEG C, heating time 15-30min.
Step (6) ammonium sulfate is saturated solution.
Step (1) the adjusting pH value is 9.0-9.5；
Step (2) the adjusting pH value is 6.0；
It is 4.5 that step (3) first time, which adjusts pH value, and it is 8.0-10.0, the third that described second, which adjusts pH value,
Secondary adjusting pH value is 4.0-6.0；
Step (5) the adjusting pH value is 7.0；
Step (6) the adjusting pH value is 4.0.
The temperature of the standing is 4 DEG C, and time of repose is 12 hours.
Protein extracting method set forth in the present invention can once be isolated from egg white ovomucin, ovotransferrins,
Ovalbumin, ovoinhibitor, ovomucoid and lysozyme totally six kinds of albumen, compared to protein extraction side in existing egg white
Method, reagent used is simple, at low cost, and process flow is easy, and time cost is low, and the big rule of multiple protein in egg white may be implemented
Mould industrialized production substantially increases the utilization efficiency of raw material, can dramatically increase economic benefit；Do not corrode in extraction process
Property substance and poisonous and harmful substance involve in, and six proposed kind albumen all has good safety；This method is for the first time
Ovoinhibitor albumen is isolated using combined extraction method.Since the content of ovoinhibitor is few, the 1.5% of egg white is only accounted for, generally
Only affinity chromatography could extract a small amount of ovoinhibitor, and therefore, this method is whether in experimental field or actual production
There is good application value.
Detailed description of the invention
Fig. 1 is the yield and comparison or purity figure of the present invention with six kinds of albumen in prior art combined extracting egg white；
Fig. 2 is the electrophoretogram of egg white of the present invention and various extracts, wherein 1 test object is egg white, 2 test object
It is lysozyme, 3 test object is ovomucin, and 4 test object is ovotransferrins, and 5 test object is ovomucin,
6 test object is ovalbumin, and 7 test object is ovoinhibitor.
Fig. 3 is the process flow diagram of six kinds of albumen in combined extracting egg white of the present invention；
Specific embodiment
Preparation method of the present invention includes egg white pretreatment and then isolates six hatching eggs by different extraction steps
It is white, it is characterised in that following steps, such as Fig. 3:
(1) the egg white 3000mL of Fresh Egg, homogeneous after mixing with isometric distilled water, using 3500r/min's are taken
Egg white is uniformly mixed by revolving speed, homogeneous 15min, and adjusting mixed liquor PH is 9.3, and resin 1200g is added, is placed under 4 DEG C of environment
12h is stirred, is then centrifuged for, after obtaining the first sediment and the first supernatant, first washes away the first sediment surface with distilled water
Egg white, then surface foreign protein is washed away with the 0.1mol Glycine-NaOH buffer that pH is 9.30, then with 16L's
Lysozyme in resin is eluted out and is collected by 0.1mol Glycine-NaOH (sodium chloride containing 0.5mol) eluent.
(2) fresh albumen for the not lysozyme isolated is diluted and is stirred with isometric 0.5mol calcium chloride solution
15min, adjusting pH is 6.0, stands 12h, is then centrifuged, obtains the second sediment and the second supernatant, then second is sunk
Starch is added into 1500mL distilled water, homogeneous and adjust pH be 6.0 after, the precipitating being centrifuged again is ovomucin.
(3) the second supernatant is taken, ammonium sulfate 165g, citric acid solid 110g is added, stirring, adjusting pH value of solution is 4.5, and
It is centrifuged after placing 12 hours under the conditions of 4 DEG C, obtains third sediment and third supernatant；Take third sediment in 200mL,
In the sodium dodecyl sulfate solution of 20mmol, it is sufficiently stirred and is centrifuged, the 800mL that the new precipitating of gained is suspended in again,
It in 20mmol lauryl sodium sulfate and is centrifuged, will be centrifuged twice after obtained supernatant mixing for use, and be denoted as the 4th supernatant
Liquid；And resulting 4th sediment adds in distilled water, and it is 12 small to placement under the conditions of 8.0,4 DEG C that pH value is adjusted after being sufficiently stirred
When, then be centrifuged after pH value is adjusted to 6.0 to get ovotransferrins is arrived；
(4) third supernatant and the 4th supernatant are taken, ammonium sulfate 105g is added, and place 12 hours under the conditions of 4 DEG C, from
5th supernatant heating water bath 30min (temperature is 55 DEG C) is centrifuged to the 5th supernatant by gains in depth of comprehension afterwards, discards precipitating, gained the
Six supernatants continue to be slowly added to 1800mL or so dehydrated alcohol to ethyl alcohol final concentration to reach percent by volume to be 20%, obtain
After suspension centrifugation, precipitating is suspended in 20% ethanol solution of 1500mL again and is centrifuged, the supernatant being centrifuged twice
Liquid is ovomucoid；The 5th sediment obtained simultaneously is added into the distilled water of 2000mL, and centrifugation (repeats after mixing evenly
The step 1 all over), collection is precipitated as ovalbumin, and merging is centrifuged resulting supernatant twice, adjust pH value to after 7.0 from
The heart, obtains the 7th supernatant, and gained is precipitated as ovalbumin；
(5) the 7th supernatant is taken, after carrying out desalination, concentration, solution ph is adjusted and is 4.0 and 20% saturation sulphur is added
Acid ammonium solution is centrifuged after stirring, and gained is precipitated as ovoinhibitor, and gained supernatant is ovomucoid；
Six kinds of protein yields (such as Fig. 1 and Fig. 2) that the present invention extracts are followed successively by, lysozyme 95.40%, ovomucin
90.31%, ovotransferrins 92.06%, ovomucoid 91.76%, ovalbumin 92.18%, ovoinhibitor 51%, and make
Analyzing measurement purity with Bole's gel imaging system (model is * GEL-EQ) is respectively lysozyme 97.15%, ovomucin
91.50%, ovotransferrins 92.3%, ovomucoid 96.7%, ovalbumin 95.9%, ovoinhibitor 91.9%.
Application No. is the extracting methods of CN201210083362.2 to obtain lysozyme, ovomucin, ovotransferrins, albumen
Albumen, purity are followed successively by 91.84%, 82.40%, 94.55% and 96.45%, gained yield 29.97%-88.63% it
Between, and the claimed available six kinds of protein of extracting method of the present invention, and the yield of corresponding protein improves
15.3%-152.13%, lysozyme purity improve 5.78%, and ovomucin purity improves 11.04%.
The method that the present invention mainly uses salt precipitation binding resin to adsorb has isolated six kinds of albumen in egg white, the party
Method simple process, extracting cycle is short, not only avoids toxic reagent residue problem, while substantially increasing production efficiency, is easy to
Realize industrialization.The present invention substantially increases the added value of egg processing, produces significant economic benefit and social benefit.
These are only the preferred embodiment of the present invention, is not intended to restrict the invention, for those skilled in the art
For member, the invention may be variously modified and varied.All within the spirits and principles of the present invention, it is made it is any modification,
Equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
Claims (10)
1. the extracting method of protein in a kind of egg white, which comprises the following steps:
(1) it extracts lysozyme: taking egg white 3000mL, distilled water is added, adjust PH, resin is added, stirring is stood, centrifugation,
Obtain the first sediment and the first supernatant, by the first sediment using distilled water and buffer flushing, elution, from
The heart obtains lysozyme；
(2) it extracts ovomucin: taking step (1) first supernatant, calcium chloride solution is added, adjust PH, centrifugation obtains the
Two sediments and the second supernatant take second sediment, and distilled water is added, homogeneous, adjust PH, and centrifugation obtains ovum and sticks egg
It is white；
(3) it extracts ovotransferrins: taking step (2) second supernatant, ammonium sulfate and citric acid is added, adjusts for the first time
PH stands, centrifugation, obtains third sediment and third supernatant, take the third sediment, it is molten to add to lauryl sodium sulfate
In liquid, centrifugation obtains the 4th sediment and the 4th supernatant, and the 4th sediment is poured into distilled water, second of adjusting
PH, standing, third time adjust PH, and centrifugation obtains ovotransferrins；
(4) extract ovomucoid: taking step (3) the third supernatant and the 4th supernatant, ammonium sulfate is added, stand, from
The heart, obtains the 5th supernatant, heats the 5th supernatant, and centrifugation takes supernatant i.e. the 6th supernatant, be added dehydrated alcohol,
Centrifugation, obtains the 5th sediment and ovomucoid；
(5) it extracts ovalbumin: taking step (4) the 5th sediment, distilled water is added, adjust PH, centrifugation obtains albumen egg
White and the 7th supernatant；
(6) it extracts ovoinhibitor: taking step (5) the 7th supernatant, concentrating and desalinating adjusts PH, and ammonium sulfate is added, stirs
It mixes, be centrifuged, obtaining supernatant is ovomucoid, and obtaining sediment is ovoinhibitor.
2. the extracting method of protein in egg white according to claim 1, which is characterized in that step (1) egg white,
Distilled water and resin ratio are 50mL:50mL:5-20g.
3. the extracting method of protein in egg white according to claim 1, which is characterized in that
Step (1) resin is FPC3500 type resin；
The speed of agitator is 3500r/min, mixing time 10-15min；.
4. the extracting method of protein in egg white according to claim 1, which is characterized in that the buffer is that PH is
9.3 glycine sodium hydroxide solution, the eluent are the mixed liquors of glycine sodium hydroxide and sodium chloride,
Optionally, the glycine sodium hydroxide solution is 0.05-0.2mol, and the sodium chloride solution is 0.1-1.0mol.
5. the extracting method of protein in egg white according to claim 1, which is characterized in that step (2) described chlorination
The concentration of calcium solution is 0.2-0.5mol/L；
Optionally, calcium chloride solution, the first supernatant volume ratio be 1:1.
6. the extracting method of protein in egg white according to claim 1, which is characterized in that step (3) described sulfuric acid
The quality of ammonium is 137-275g, and the quality of citric acid is 82.5-137.5g；The amount of the substance of the sodium dodecyl sulfate solution
For 1-30mmol, volume 800-1200mL.
7. the extracting method of protein in egg white according to claim 1, which is characterized in that step (4) described sulfuric acid
Ammonium quality is 93-155g, and dehydrated alcohol volume is 1500-4500mL；The heating temperature is 50-70 DEG C, it is preferable that described to add
Hot temperature is 55 DEG C, heating time 15-30min.
8. the extracting method of protein in egg white according to claim 1, which is characterized in that step (6) described sulfuric acid
Ammonium salt solution is saturated solution.
9. the extracting method of protein in egg white according to claim 1, which is characterized in that
Step (1) the adjusting pH value is 9.0-9.5；
Step (2) the adjusting pH value is 6.0；
It is 4.5 that step (3) first time, which adjusts pH value, and it is 8.0-10.0, the third time tune that described second, which adjusts pH value,
Section pH value is 4.0-6.0；
Step (5) the adjusting pH value is 7.0；
Step (6) the adjusting pH value is 4.0.
10. the extracting method of protein in egg white according to claim 1, which is characterized in that the temperature of the standing
It is 4 DEG C, time of repose is 12 hours.
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Cited By (5)
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| CN110204609A (en) * | 2019-05-31 | 2019-09-06 | 华中农业大学 | The method and its albumen iron product of ovotransferrins are extracted in a kind of industrialization |
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| CN111848721A (en) * | 2020-07-29 | 2020-10-30 | 中国人民解放军联勤保障部队第九二〇医院 | Special extracting solution for chicken protein and extracting method thereof |
| CN112898408A (en) * | 2021-01-19 | 2021-06-04 | 南开大学 | Method and device for separating ovalbumin from albumen |
| CN113214385A (en) * | 2021-05-26 | 2021-08-06 | 杭州佰倍优生物科技有限公司 | Enzymolysis ovalbumin composition for improving hypoalbuminemia and immunity |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110204609A (en) * | 2019-05-31 | 2019-09-06 | 华中农业大学 | The method and its albumen iron product of ovotransferrins are extracted in a kind of industrialization |
| CN110204609B (en) * | 2019-05-31 | 2022-01-07 | 华中农业大学 | Industrial extraction method of ovotransferrin and protein iron product thereof |
| CN110563837A (en) * | 2019-08-01 | 2019-12-13 | 华中农业大学 | Method for efficiently extracting ovomucoid from egg white |
| CN111848721A (en) * | 2020-07-29 | 2020-10-30 | 中国人民解放军联勤保障部队第九二〇医院 | Special extracting solution for chicken protein and extracting method thereof |
| CN112898408A (en) * | 2021-01-19 | 2021-06-04 | 南开大学 | Method and device for separating ovalbumin from albumen |
| CN113214385A (en) * | 2021-05-26 | 2021-08-06 | 杭州佰倍优生物科技有限公司 | Enzymolysis ovalbumin composition for improving hypoalbuminemia and immunity |
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