CN107253990A - A kind of preparation method that ovotransferrins and its freeze-dried powder are extracted from egg - Google Patents
A kind of preparation method that ovotransferrins and its freeze-dried powder are extracted from egg Download PDFInfo
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- CN107253990A CN107253990A CN201710683086.6A CN201710683086A CN107253990A CN 107253990 A CN107253990 A CN 107253990A CN 201710683086 A CN201710683086 A CN 201710683086A CN 107253990 A CN107253990 A CN 107253990A
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- 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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Abstract
The invention discloses a kind of ovotransferrins that extracted from egg come the method for preparing freeze-dried powder, it comprises the following steps：（1）Prepare egg white dilution；（2）Separate ovomucin；（3）Adjust PH；（4）Ammonium sulfate precipitation；（5）Ion exchange is eluted；（6）Concentration, vacuum freeze drying, milled processed be made ovotransferrins freeze-dried powder.The present invention is separated using the method for ammonium sulfate precipitation and ion-exchange chromatography to the ovotransferrins in egg, to set up the technique that ovotransferrins is extracted from egg white, laid the foundation for its exploitation, its freeze-dried powder prepared is easy to store, transported, the freezing dry powder of the present invention can be added to varieties of food items as functional components, or natural antibacterial medicine or efficient iron supplementary is made, and for strengthening senile-resistant efficacy in cosmetics.In addition, it may also be used for the medicine for the treatment of Escherichia coli, Pseudomonas alba, streptococcus, staphylococcus aureus, Li bacillus and Chlamydia etc..
Description
Technical field
The present invention relates to egg white extractive technique field, and in particular to one kind extracts ovotransferrins to prepare jelly from egg
The method of dry powder.
Background technology
Ovotransferrins is the protein of single peptide chain, is a kind of important ferro element carrier in human body, many diseases are all
Relevant with the changes of contents of transferrins in human serum, such as hypoferric anemia, oxyhepatitis and protein lack.Ovum turns iron egg
Be in vain it is a kind of can transport Fe3+ iron-binding protein, be a kind of betaglobulin of non-heme combination iron, a molecule can be reversible
Ground combines two Fe3+, in combination with two CO3 2-Or HCO3 -, the combination of anion and Fe3+ combination are cooperateed with.Have
Research shows that tyrosine residue plays the effect of key in the cohesive process of transferrins and iron, and transferrin molecules are being combined
Iron ion front-end geometry is incomplete same：During uncombined iron ion, the molecule of transferrins is in relatively open, evacuation structure；Knot
Close after iron ion, the part of transferrins causes whole molecular structure to become even closer with the interphase interaction of iron ion.
Ovotransferrins (Ovotransferrin, OVT) is referred to as ovoconalbumin in the past, is a kind of glycoprotein, ovum turns iron
Albumen accounts for the 12% of egg total protein content, with antibacterial, antiviral, immunological regulation, anticancer and the effect such as anti-oxidant.Its
It can promote absorption of the human body to iron and nutriment, prevent anaemia.To the inhibitory action of Escherichia coli etc. with
Transferrins in serum is identical, while to Pseudomonas alba, streptococcus, staphylococcus aureus, Li bacillus and Chlamydia
Deng also there is inhibitory action, its antiviral performance is better than lactoferrin and serum transferrin, in addition, having to baby's chordapsus
Good curative effect.Therefore, ovotransferrins is extracted from the egg of abundance, is developed as natural preservative and antibacterial
Agent is applied to varieties of food items, or is added to varieties of food items as functional components, or natural antibacterial medicine is made or iron is efficiently mended
Agent, and for strengthening senile-resistant efficacy in cosmetics, its application and DEVELOPMENT PROSPECT are very wide.
The content of the invention
The present invention prepares freeze-dried powder for existing technical problem there is provided a kind of extraction ovotransferrins from egg
Method, to improve the ability that ovotransferrins is extracted in egg, with applied to varieties of food items.
To achieve the above object, the present invention provides following technical scheme：One kind extracts ovotransferrins to make from egg
The method of standby freeze-dried powder, it is characterised in that it comprises the following steps：
（1）Prepare egg white dilution：Fresh Egg is cleaned, is placed at 2-3 DEG C and refrigerates 3-6h, then the egg of refrigeration is carried out
Yolk and egg white are separated, egg white solution is taken, the water added in egg white solution equal to egg white volume is diluted, and obtains egg white dilution
Refrigerated in liquid, the refrigerator for being placed in 3 DEG C at least one hour；
（2）Separate ovomucin：The HCL that 3mol/L is added in egg white dilution adjusts PH to 5.8, and uses 300-450r/min
Mixer be stirred 5h so that ovomucin precipitate, afterwards using centrifuge, remove precipitation, obtain supernatant；
（3）Adjust PH：By step（2）2mol/L NaOH solution is added in obtained supernatant, PH is to 7.5 for regulation, is used in combination
600-800r/min mixer stirring 0.5-1h, then, be placed in it is cold in 3 DEG C of refrigerator put 2h, afterwards with centrifuge, go
Except precipitation, supernatant is obtained；
（4）Ammonium sulfate precipitation：In the step（3）Supernatant in add the ammonium sulfate of 40% saturation degree, stand 2h at 4 DEG C,
Then 5000r/min centrifuge 30min is utilized；Then, supernatant is taken to add the ammonium sulfate of 85% saturation degree, at 4 DEG C
Lower standing 1h, then utilizes 5500r/min centrifuge 20min；Precipitation distilled water dissolves, dialysis desalting；Dialysis
Egg white solution adjust PH to 8.2 with 3mol/L NaOH solution, then in centrifugation 20min, take supernatant；
（5）Using Q-Sepharose Fast Flow ion exchange columns, exchanged with PH for 8.2 HCL buffer solutions ion balance
Post, then, by step（4）Obtained supernatant is added in ion exchange column with 2.5ml/min flow velocity, then respectively with containing
0.01mol/L, 0.02mol/L, 0.03mol/L, 0.05mol/L, 0.1mol/L NaCL solution are carried out with 1Ml/min flow velocity
Stepwise elution, and collect the eluent containing ovotransferrins；
（6）The eluent of ovotransferrins is concentrated, vacuum freeze drying, milled processed, that is, be made ovotransferrins freeze
Dry powder.
Further, preferably, in described step（4）With step（5）Between be additionally provided with microfiltration step, described is micro-
It is that described step is removed using filter membrane to filter step（4）The impurity of 30-80 micrometer ranges in obtained supernatant, during micro-filtration
Operating pressure is 0.1MPa.
Further, preferably, being additionally provided with ultrafiltration step after described microfiltration step, ultrafiltration step uses 0.01-
0.02 micron of milipore filter carries out hyperfiltration treatment, and operating pressure during ultrafiltration is 0.2MPa.
Further, preferably, described step（2）Middle is 6500r/min using centrifugal rotating speed, during centrifugation
Between be 40min, described step（3）Middle is 8000r/min using centrifugal rotating speed, and centrifugation time is 20min.
Further, preferably, in ultrafiltration, ultrafiltration while stirring, the speed of stirring is 80r/min.
Further, preferably, in described step（6）In, the cryogenic temperature of vacuum freeze drying is -40 DEG C, freezing
Time is 36h.
Compared with prior art, the beneficial effects of the invention are as follows：
The present invention is separated using the method for ammonium sulfate precipitation and ion-exchange chromatography to the ovotransferrins in egg, with
Phase sets up the technique that ovotransferrins is extracted from egg white, is that its exploitation lays the foundation, and its freeze-dried powder prepared is easy to storage
Hide, transport, also allow for being applied to natural preservative and antiseptic applied to varieties of food items, freezing dry powder of the invention can also
It is added to varieties of food items as functional components, or natural antibacterial medicine or efficient iron supplementary is made, and for cosmetics
Middle enhancing senile-resistant efficacy, its application prospect is very wide.In addition, it can be also used for preparing to treatment Escherichia coli, false list
The medicine of born of the same parents bacillus, streptococcus, staphylococcus aureus, Li bacillus and Chlamydia etc..
Embodiment
Below in conjunction with the embodiment of the present invention, the technical scheme in the embodiment of the present invention is clearly and completely described,
Obviously, described embodiment is only a part of embodiment of the invention, rather than whole embodiments.Based in the present invention
Embodiment, the every other embodiment that those of ordinary skill in the art are obtained under the premise of creative work is not made, all
Belong to the scope of protection of the invention.
Embodiment one
A kind of ovotransferrins that extracted from egg is come the method for preparing freeze-dried powder, and it comprises the following steps：
（1）Prepare egg white dilution：Fresh Egg is cleaned, is placed at 2 DEG C and refrigerates 4h, then the egg of refrigeration is separated
Yolk and egg white, take egg white solution, and the water added in egg white solution equal to egg white volume is diluted, and obtains egg white dilution, and
It is placed in 3 DEG C of refrigerator and refrigerates 3 hours；
（2）Separate ovomucin：The HCL that 3mol/L is added in egg white dilution adjusts PH to 5.8, and stirring with 300r/min
The machine of mixing is stirred 5h so that ovomucin is precipitated, afterwards using centrifuge, and centrifugal rotating speed is 6500r/
Min, centrifugation time is 40min, removes precipitation, obtains supernatant；
（3）Adjust PH：By step（2）2mol/L NaOH solution is added in obtained supernatant, PH is to 7.5 for regulation, is used in combination
600r/min mixer stirring 0.6h, then, be placed in it is cold in 3 DEG C of refrigerator put 2h, afterwards with centrifuge, centrifuge from
The rotating speed of the heart is 8000r/min, and centrifugation time is 20min, removes precipitation, obtains supernatant；
（4）Ammonium sulfate precipitation：In the step（3）Supernatant in add the ammonium sulfate of 40% saturation degree, stand 2h at 4 DEG C,
Then 5000r/min centrifuge 30min is utilized；Then, supernatant is taken to add the ammonium sulfate of 85% saturation degree, at 4 DEG C
Lower standing 1h, then utilizes 5500r/min centrifuge 20min；Precipitation distilled water dissolves, dialysis desalting；Dialysis
Egg white solution adjust PH to 8.2 with 3mol/L NaOH solution, then in centrifugation 20min, take supernatant；
（5）Using Q-Sepharose Fast Flow ion exchange columns, exchanged with PH for 8.2 HCL buffer solutions ion balance
Post, then, by step（4）Obtained supernatant is added in ion exchange column with 2.5ml/min flow velocity, then respectively with containing
0.01mol/L, 0.02mol/L, 0.03mol/L, 0.05mol/L, 0.1mol/L NaCL solution are carried out with 1Ml/min flow velocity
Stepwise elution, and collect the eluent containing ovotransferrins；
（6）The eluent of ovotransferrins is concentrated, vacuum freeze drying, the cryogenic temperature of vacuum freeze drying is -40
DEG C, cooling time is 36h, afterwards milled processed, that is, ovotransferrins freeze-dried powder is made.
Embodiment two
A kind of ovotransferrins that extracted from egg is come the method for preparing freeze-dried powder, and it comprises the following steps：
（1）Prepare egg white dilution：Fresh Egg is cleaned, is placed at 3 DEG C and refrigerates 6h, then the egg of refrigeration is separated
Yolk and egg white, take egg white solution, and the water added in egg white solution equal to egg white volume is diluted, and obtains egg white dilution, and
It is placed in 3 DEG C of refrigerator and refrigerates 2 hours；
（2）Separate ovomucin：The HCL that 3mol/L is added in egg white dilution adjusts PH to 5.8, and stirring with 350r/min
The machine of mixing is stirred 5h so that ovomucin is precipitated, afterwards using centrifuge, and centrifugal rotating speed is 6500r/
Min, centrifugation time is 40min, removes precipitation, obtains supernatant；
（3）Adjust PH：By step（2）2mol/L NaOH solution is added in obtained supernatant, PH is to 7.5 for regulation, is used in combination
800r/min mixer stirring 1h, then, be placed in it is cold in 3 DEG C of refrigerator put 2h, afterwards with centrifuge, centrifuge
Rotating speed be 8000r/min, centrifugation time is 20min, remove precipitation, obtain supernatant；
（4）Ammonium sulfate precipitation：In the step（3）Supernatant in add the ammonium sulfate of 40% saturation degree, stand 2h at 4 DEG C,
Then 5000r/min centrifuge 30min is utilized；Then, supernatant is taken to add the ammonium sulfate of 85% saturation degree, at 4 DEG C
Lower standing 1h, then utilizes 5500r/min centrifuge 20min；Precipitation distilled water dissolves, dialysis desalting；Dialysis
Egg white solution adjust PH to 8.2 with 3mol/L NaOH solution, then in centrifugation 20min, take supernatant；
（5）Microfiltration step, described step is removed using filter membrane（4）The impurity of 30-80 micrometer ranges in obtained supernatant,
Operating pressure during micro-filtration is 0.1Mpa, obtains micro-filtrate；
（6）Using Q-Sepharose Fast Flow ion exchange columns, exchanged with PH for 8.2 HCL buffer solutions ion balance
Post, then, by step（5）Obtained micro-filtrate is added in ion exchange column with 2.5ml/min flow velocity, then respectively with containing
0.01mol/L, 0.02mol/L, 0.03mol/L, 0.05mol/L, 0.1mol/L NaCL solution are carried out with 1Ml/min flow velocity
Stepwise elution, and collect the eluent containing ovotransferrins；
（7）The eluent of ovotransferrins is concentrated, vacuum freeze drying, the cryogenic temperature of vacuum freeze drying is -40
DEG C, cooling time is 36h, afterwards milled processed, that is, ovotransferrins freeze-dried powder is made.
Embodiment three
A kind of ovotransferrins that extracted from egg is come the method for preparing freeze-dried powder, and it comprises the following steps：
（1）Prepare egg white dilution：Fresh Egg is cleaned, is placed at 3 DEG C and refrigerates 5h, then the egg of refrigeration is separated
Yolk and egg white, take egg white solution, and the water added in egg white solution equal to egg white volume is diluted, and obtains egg white dilution, and
It is placed in 3 DEG C of refrigerator and refrigerates 1 hour；
（2）Separate ovomucin：The HCL that 3mol/L is added in egg white dilution adjusts PH to 5.8, and stirring with 450r/min
The machine of mixing is stirred 5h so that ovomucin is precipitated, afterwards using centrifuge, and centrifugal rotating speed is 6500r/
Min, centrifugation time is 40min, removes precipitation, obtains supernatant；
（3）Adjust PH：By step（2）2mol/L NaOH solution is added in obtained supernatant, PH is to 7.5 for regulation, is used in combination
800r/min mixer stirring 1h, then, be placed in it is cold in 3 DEG C of refrigerator put 2h, afterwards with centrifuge, centrifuge
Rotating speed be 8000r/min, centrifugation time is 20min, remove precipitation, obtain supernatant；
（4）Ammonium sulfate precipitation：In the step（3）Supernatant in add the ammonium sulfate of 40% saturation degree, stand 2h at 4 DEG C,
Then 5000r/min centrifuge 30min is utilized；Then, supernatant is taken to add the ammonium sulfate of 85% saturation degree, at 4 DEG C
Lower standing 1h, then utilizes 5500r/min centrifuge 20min；Precipitation distilled water dissolves, dialysis desalting；Dialysis
Egg white solution adjust PH to 8.2 with 3mol/L NaOH solution, then in centrifugation 20min, take supernatant；
（5）Microfiltration step, described step is removed using filter membrane（4）The impurity of 30-80 micrometer ranges in obtained supernatant,
Operating pressure during micro-filtration is 0.1Mpa, obtains micro-filtrate；
（6）Ultrafiltration step, ultrafiltration step is using 0.01-0.02 microns of milipore filter to step（5）Obtained micro-filtrate is surpassed
Filter is handled, and operating pressure during ultrafiltration is 0.2Mpa, and is required in ultrafiltration, ultrafiltration while stirring, and the speed of stirring is 80r/
Min, obtains ultrafiltrate；
（7）Using Q-Sepharose Fast Flow ion exchange columns, exchanged with PH for 8.2 HCL buffer solutions ion balance
Post, then, by step（6）Obtained ultrafiltrate is added in ion exchange column with 2.5ml/min flow velocity, then respectively with containing
0.01mol/L, 0.02mol/L, 0.03mol/L, 0.05mol/L, 0.1mol/L NaCL solution are carried out with 1Ml/min flow velocity
Stepwise elution, and collect the eluent containing ovotransferrins；
（8）The eluent of ovotransferrins is concentrated, vacuum freeze drying, the cryogenic temperature of vacuum freeze drying is -40
DEG C, cooling time is 36h, afterwards milled processed, that is, ovotransferrins freeze-dried powder is made.
The present invention is divided the ovotransferrins in egg using the method for ammonium sulfate precipitation and ion-exchange chromatography
From, to set up from egg white extract ovotransferrins technique, be that its exploitations lays the foundation, its preparation freeze-dried powder just
In storage, transport, also allow for being applied to natural preservative and antiseptic applied to varieties of food items, freezing dry powder of the invention
Varieties of food items can be added to as functional components, or natural antibacterial medicine or efficient iron supplementary is made, and for changing
Strengthen senile-resistant efficacy in cosmetic, its application prospect is very wide.In addition, its can be also used for prepare to treatment Escherichia coli,
The medicine of Pseudomonas alba, streptococcus, staphylococcus aureus, Li bacillus and Chlamydia etc..
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
A variety of changes, modification can be carried out to these embodiments, replace without departing from the principles and spirit of the present invention by understanding
And modification, the scope of the present invention is defined by the appended.
Claims (6)
1. a kind of method for extracting ovotransferrins from egg to prepare freeze-dried powder, it is characterised in that it comprises the following steps：
（1）Prepare egg white dilution：Fresh Egg is cleaned, is placed at 2-3 DEG C and refrigerates 3-6h, then the egg of refrigeration is carried out
Yolk and egg white are separated, egg white solution is taken, the water added in egg white solution equal to egg white volume is diluted, and obtains egg white dilution
Refrigerated in liquid, the refrigerator for being placed in 3 DEG C at least one hour；
（2）Separate ovomucin：The HCL that 3mol/L is added in egg white dilution adjusts PH to 5.8, and uses 300-450r/min
Mixer be stirred 5h so that ovomucin precipitate, afterwards using centrifuge, remove precipitation, obtain supernatant；
（3）Adjust PH：By step（2）2mol/L NaOH solution is added in obtained supernatant, PH is to 7.5 for regulation, is used in combination
600-800r/min mixer stirring 0.5-1h, then, be placed in it is cold in 3 DEG C of refrigerator put 2h, afterwards with centrifuge, go
Except precipitation, supernatant is obtained；
（4）Ammonium sulfate precipitation：In the step（3）Supernatant in add the ammonium sulfate of 40% saturation degree, stand 2h at 4 DEG C,
Then 5000r/min centrifuge 30min is utilized；Then, supernatant is taken to add the ammonium sulfate of 85% saturation degree, at 4 DEG C
Lower standing 1h, then utilizes 5500r/min centrifuge 20min；Precipitation distilled water dissolves, dialysis desalting；Dialysis
Egg white solution adjust PH to 8.2 with 3mol/L NaOH solution, then in centrifugation 20min, take supernatant；
（5）Using Q-Sepharose Fast Flow ion exchange columns, exchanged with PH for 8.2 HCL buffer solutions ion balance
Post, then, by step（4）Obtained supernatant is added in ion exchange column with 2.5ml/min flow velocity, then respectively with containing
0.01mol/L, 0.02mol/L, 0.03mol/L, 0.05mol/L, 0.1mol/L NaCL solution are carried out with 1Ml/min flow velocity
Stepwise elution, and collect the eluent containing ovotransferrins；
（6）The eluent of ovotransferrins is concentrated, vacuum freeze drying, milled processed, that is, be made ovotransferrins freeze
Dry powder.
2. a kind of ovotransferrins that extracted from egg according to claim 1 is come the method for preparing freeze-dried powder, its feature
It is：In described step（4）With step（5）Between be additionally provided with microfiltration step, described microfiltration step is to be gone using filter membrane
Except described step（4）The impurity of 30-80 micrometer ranges in obtained supernatant, operating pressure during micro-filtration is 0.1MPa.
3. a kind of ovotransferrins that extracted from egg according to claim 2 is come the method for preparing freeze-dried powder, its feature
It is：Ultrafiltration step is additionally provided with after described microfiltration step, ultrafiltration step is carried out using 0.01-0.02 microns of milipore filter
Hyperfiltration treatment, operating pressure during ultrafiltration is 0.2MPa.
4. a kind of according to claim 1-3 any one extracts ovotransferrins to prepare the side of freeze-dried powder from egg
The preparation method of method, it is characterised in that：Described step（2）Middle is 6500r/min using centrifugal rotating speed, during centrifugation
Between be 40min, described step（3）Middle is 8000r/min using centrifugal rotating speed, and centrifugation time is 20min.
5. a kind of ovotransferrins that extracted from egg according to claim 3 is come the method for preparing freeze-dried powder, its feature
It is：In ultrafiltration, ultrafiltration while stirring, the speed of stirring is 80r/min.
6. a kind of ovotransferrins that extracted from egg according to claim 4 is come the method for preparing freeze-dried powder, its feature
It is：In described step（6）In, the cryogenic temperature of vacuum freeze drying is -40 DEG C, and cooling time is 36h.
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| CN201710683086.6A CN107253990A (en) | 2017-08-11 | 2017-08-11 | A kind of preparation method that ovotransferrins and its freeze-dried powder are extracted from egg |
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| CN201710683086.6A CN107253990A (en) | 2017-08-11 | 2017-08-11 | A kind of preparation method that ovotransferrins and its freeze-dried powder are extracted from egg |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109609480A (en) * | 2019-01-21 | 2019-04-12 | 华中农业大学 | The extracting method of protein in a kind of egg white |
| 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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| CN102796193A (en) * | 2012-07-23 | 2012-11-28 | 华中农业大学 | Method for extracting ovotransferrin from egg white |
| CN102875669A (en) * | 2012-10-29 | 2013-01-16 | 天津商业大学 | Method for separating and extracting ovotransferrin |
| US20130260437A1 (en) * | 2012-03-27 | 2013-10-03 | Huazhong Agriculture University | Method of co-extracting multiple proteins from chicken egg white |
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2017
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| US20130260437A1 (en) * | 2012-03-27 | 2013-10-03 | Huazhong Agriculture University | Method of co-extracting multiple proteins from chicken egg white |
| CN102796193A (en) * | 2012-07-23 | 2012-11-28 | 华中农业大学 | Method for extracting ovotransferrin from egg white |
| CN102875669A (en) * | 2012-10-29 | 2013-01-16 | 天津商业大学 | Method for separating and extracting ovotransferrin |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609480A (en) * | 2019-01-21 | 2019-04-12 | 华中农业大学 | The extracting method of protein in a kind of egg white |
| 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 |
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Application publication date: 20171017 |