CN105602918A - Method for extracting lysozyme from egg white or whole egg juice - Google Patents
Method for extracting lysozyme from egg white or whole egg juice Download PDFInfo
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- CN105602918A CN105602918A CN201610164753.5A CN201610164753A CN105602918A CN 105602918 A CN105602918 A CN 105602918A CN 201610164753 A CN201610164753 A CN 201610164753A CN 105602918 A CN105602918 A CN 105602918A
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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
- 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)
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Abstract
The invention discloses a method for extracting lysozyme from egg white or whole egg juice, and belongs to the technical field of biology. The method comprises the following steps: (1) adding 1-10-fold water to the egg white or whole egg juice, mixing evenly, and filtering out impurities to obtain a diluent; (2) carrying out ultrafiltration on the diluent with an ultrafiltration membrane to obtain a thin liquid and a thick liquid; (3) adding resin to the thin liquid for adsorption, wherein the weight ratio of the thin liquid to the resin is (1:3) to (1:8), and the adsorbed resin is obtained; (4) eluting the adsorbed resin with a 0.001-0.5mol/L NaCl water solution to obtain an eluant; (5) desalting the eluant with the ultrafiltration membrane to obtain ultrafiltrate; and (6) drying the ultrafiltrate to obtain the lysozyme. The method is simple and fast in process, mild in condition, and free of complicated equipment; high-purity and high-bioactivity lysozyme can be obtained; and the defects of an existing lysozyme preparation technology can be overcome.
Description
Technical field
The present invention relates to a kind of method of extracting lysozyme from egg or full egg liquid, belong to biological skillArt field.
Background technology
Lysozyme is a kind of alkaline protein, and it is distributed widely in animal, plant and microorganism, is feedingIn the histocytes such as blood plasma, tracheae, enteron aisle, stomach, kidney and the liver of breast animal, all have expressed. LysozymeOwing to thering is antibiotic property, and without toxicity, side effect, can be used as potential Substitutes For Antibiotic and apply to foodThe fields such as product, bioengineering, medical treatment and animal and fowl fodder.
The preparation method of lysozyme mainly contains direct crystallization method, ion-exchange, ultrafiltration, poly-electricity at presentSeparate the matter precipitation method, affinity chromatography etc. Direct crystallization method easy and simple to handle, but the production cycle is long, yieldLow, purity and activity are also low, are not suitable for suitability for industrialized production; Ultrafiltration simple to operate, free from admixture is introduced,But extracting method is extensive, poor product quality, and milipore filter easily stops up; The life of polyelectrolyte precipitation methodThe product cycle is long, and the reagent of introducing in leaching process has unsafe factor, and product is not suitable for food medicineIn field; Affinity chromatography can be realized separating of object and other impurity fast, but solution viscosityWhen large, easily cause the obstruction of chromatographic column. Industrial production mainly adopts base exchange method at present, by sunThe steps such as ion exchange resin absorption, high eluting salt, desalting and dewatering and atomization drying obtain comparatively desirable yieldAnd purity. Because lysozyme eluate solution amount is large, salt content is high, conventional ultrafiltration apparatus burden is heavy, superFilter membrane is because of the vulnerable to pollution that works long hours, and be greatly affected the service life of film; In while egg whiteMost of protein is destroyed, causes the following process application that cannot carry out albumen.
In egg, contain the multiple bioactive protein that has, as the antibacterial activity of lysozyme, ovum turn ironThe characteristic of protein combination iron, the antiviral activity of ovomucin, and Ovoflavoprotein syncaryon flavineActivity, these important biologically actives have caused the strong interest of vast researcher. Said methodAll be unfavorable for being divided into other activated protein such as ovalbumin, ovotransferrins from preparing in egg.
Summary of the invention
The object of the invention is to solve the deficiencies in the prior art, provide a kind of from egg or full egg liquidExtract the method for lysozyme. Method of the present invention technique simple and fast used, mild condition, without complexityEquipment, can obtain the lysozyme of high-purity and high bioactivity, can overcome existing lysozyme and prepare workThe shortcoming of skill, and in preparing lysozyme, can also obtain other activated protein of higher degree.
The technical scheme that the present invention solves the problems of the technologies described above is as follows: a kind of from egg or full egg liquidThe method of extracting lysozyme, comprises the steps:
(1) egg or full egg liquid add 1～10 times of weight water, mix, and filter, except impurity eliminationMatter, obtains dilution;
(2) dilution milipore filter ultrafiltration step (1) being obtained, obtains thin liquid and dope;
(3) in the thin liquid obtaining in step (2), add resin to adsorb, described thin liquid and resinPart by weight is 1:3～1:8, obtains the resin after absorption;
(4) the resin 0.001～0.5mol/LNaCl after absorption step (3) being obtained is water-solubleLiquid carries out wash-out, obtains eluent;
(5) milipore filter ultrafiltration desalination for eluent step (4) being obtained, obtains ultrafiltrate;
(6) ultrafiltrate step (5) being obtained obtains described lysozyme after being dried.
On the basis of technique scheme, the present invention can also do following improvement.
Further, described in step (1), be filtered into filter-cloth filtering or filtered through gauze. Impurity, is mainly eggLipid component in Huang is egg or the full egg liquid insoluble granule occurring after mixing that adds water.
Further, the molecular cut off of the described milipore filter of step (2) is 15000～30000Da.
Further, the described dope of step (2) obtains macromolecule protein after drying.
Further, described macromolecule protein be used for preparing ovalbumin, ovotransferrins, ovomucin withAnd Ovoflavoprotein.
Further, the described resin of step (3) is cationic ion-exchange resin or macroporous absorbent resin.
Further, the model of described cationic ion-exchange resin be 001*7,732, AmberliteIR-120,Dowex-50、Lewatit-100、DiaionSK-1、AllassionCS、DuoliteC-20、SDB-3In one, described macroporous absorbent resin be AB-8, D101, D3520, X-5, NKA-II, NKA-9,One or more in S-8, XDA-1, H-20, H-30, H-40.
Further, the molecular cut off of the described milipore filter of step (5) is 2000～10000Da.
Further, step (6) is described is dried, low-temperature vacuum drying dry for spraying or freeze drying.
Above-mentioned steps (1), adopts water to replace acid solution or salting liquid to carry out egg or egg liquid dilution,Reduce cost, simultaneously reduced again the salt content in dilution system, reduced subsequent technique desalinationPressure, is also conducive to the recovery of other albumen in egg or egg liquid.
Above-mentioned steps (2), adopts ultrafiltration, is to prepare high molecular weight protein in order to separate, and has both improved warpJi benefit, has reduced again the viscosity of lysozyme extract, and follow-up absorption is more easily carried out.
Above-mentioned steps (4), adopts NaCl solution to substitute traditional ammonium sulfate and carries out wash-out, both fallsLow cost, has increased again the security of lysozyme product simultaneously, makes lysozyme product can be used for foodAnd pharmaceuticals industry.
Above-mentioned steps (5), adopts ultrafiltration, is for desalination.
The invention has the beneficial effects as follows:
1. the present invention can prepare lysozyme and other large molecular activity albumen simultaneously, has both been applicable to eggClearly, be also applicable to full egg liquid. The lysozyme product that the present invention prepares has high-purity and high biologicalActivity, and technique is simple, and mild condition, without special installation.
2. the present invention adopts water to replace acid solution or salting liquid to carry out the dilution of egg liquid, has reduced cost, simultaneouslyReduce again the salt content in dilution, reduced the pressure of subsequent technique desalination, be also conducive in egg liquidThe recovery of other albumen. Before step of the present invention (3) ion-exchange, do not need to regulate the pH of feed liquid, wholeIndividual exchange process does not need phosphate.
3. the present invention adopts the alternative traditional ammonium sulfate of NaCl solution to carry out wash-out, has both reduced one-tenthThis, increased again the security of lysozyme product simultaneously, makes lysozyme product can be used for food medicine rowIndustry.
4. the present invention adopts two times of ultrafiltration technique, both can obtain highly active large molecular activity albumen andHighly active lysozyme, has reduced again ultrafiltration pressure simultaneously, and operating cost is reduced.
Brief description of the drawings
Fig. 1 is process chart of the present invention.
Detailed description of the invention
Below in conjunction with accompanying drawing, principle of the present invention and feature are described, example is only for explaining thisInvention, is not intended to limit scope of the present invention.
Embodiment 1
As shown in Figure 1, the method for extracting lysozyme from egg or full egg liquid of the present embodiment, bagDraw together following steps:
(1) egg or full egg liquid add 1 times of weight water, mix, and with filter-cloth filtering, removeLipid component in yolk, obtains dilution;
(2) the milipore filter ultrafiltration that dilution molecular cut off step (1) being obtained is 20000Da,Obtain thin liquid and dope, described dope obtains macromolecule protein after drying, and described macromolecule protein is used forPrepare ovalbumin and ovotransferrins;
(3) in the thin liquid obtaining in step (2), adding model is that the cationic ion-exchange resin of 001*7 entersRow absorption, the part by weight of described thin liquid and resin is 1:3, obtains the resin after absorption;
(4) the resin 0.001mol/LNaCl aqueous solution after absorption step (3) being obtained carries outWash-out, obtains eluent;
(5) the milipore filter ultrafiltration that eluent molecular cut off step (4) being obtained is 5000DaDesalination, obtains ultrafiltrate;
(6), after ultrafiltrate spraying step (5) being obtained is dried, obtain described lysozyme.
Embodiment 2
As shown in Figure 1, the method for extracting lysozyme from egg or full egg liquid of the present embodiment, bagDraw together following steps:
(1) egg or full egg liquid add 5 times of weight water, mix, and with filter-cloth filtering, removeLipid component in yolk, obtains dilution;
(2) the milipore filter ultrafiltration that dilution molecular cut off step (1) being obtained is 30000Da,Obtain thin liquid and dope, described dope obtains macromolecule protein after drying, and described macromolecule protein is used forPrepare ovalbumin, ovotransferrins, ovomucin and Ovoflavoprotein;
(3) in the thin liquid obtaining in step (2), add model be AmberliteIR-120 sun fromSub-exchange resin adsorbs, and the part by weight of described thin liquid and resin is 1:5, obtains the tree after absorptionFat;
(4) the resin 0.2mol/LNaCl aqueous solution after absorption step (3) being obtained is washedDe-, obtain eluent;
(5) the milipore filter ultrafiltration that eluent molecular cut off step (4) being obtained is 10000DaDesalination, obtains ultrafiltrate;
(6), after ultrafiltrate low-temperature vacuum drying step (5) being obtained, obtain described lysozyme.
Embodiment 3
As shown in Figure 1, the method for extracting lysozyme from egg or full egg liquid of the present embodiment, bagDraw together following steps:
(1) egg or full egg liquid add 10 times of weight water, mix, and with filter-cloth filtering, removeLipid component in yolk, obtains dilution;
(2) the milipore filter ultrafiltration that dilution molecular cut off step (1) being obtained is 15000Da,Obtain thin liquid and dope, described dope obtains macromolecule protein after drying, and described macromolecule protein is used forPrepare ovalbumin, ovotransferrins, ovomucin and Ovoflavoprotein;
(3) in the thin liquid obtaining in step (2), adding model is that the macroporous absorbent resin of D101 carries outAbsorption, the part by weight of described thin liquid and resin is 1:8, obtains the resin after absorption;
(4) the resin 0.001～0.5mol/LNaCl after absorption step (3) being obtained is water-solubleLiquid carries out wash-out, obtains eluent;
(5) the milipore filter ultrafiltration that eluent molecular cut off step (4) being obtained is 2000DaDesalination, obtains ultrafiltrate;
(6), after ultrafiltrate freeze drying step (5) being obtained, obtain described lysozyme.
The foregoing is only preferred embodiment of the present invention, in order to limit the present invention, not all in the present inventionSpirit and principle within, any amendment of doing, be equal to replacement, improvement etc., all should be included in thisWithin bright protection domain.
Claims (9)
1. a method of extracting lysozyme from egg or full egg liquid, is characterized in that, comprisesFollowing steps:
(1) add the water of 1～10 times of weight to egg or full egg liquid, mix, filter, removeDecontamination, obtains dilution;
(2) dilution milipore filter ultrafiltration step (1) being obtained, obtains thin liquid and dope;
(3) in the thin liquid obtaining in step (2), add resin to adsorb, described thin liquid and resinPart by weight is 1:3～1:8, obtains the resin after absorption;
(4) the resin 0.001～0.5mol/LNaCl after absorption step (3) being obtained is water-solubleLiquid carries out wash-out, obtains eluent;
(5) milipore filter ultrafiltration desalination for eluent step (4) being obtained, obtains ultrafiltrate;
(6) ultrafiltrate step (5) being obtained obtains described lysozyme after being dried.
2. a kind of side that extracts lysozyme from egg or full egg liquid according to claim 1Method, is characterized in that, step is filtered into filter-cloth filtering or filtered through gauze described in (1).
3. a kind of side that extracts lysozyme from egg or full egg liquid according to claim 1Method, is characterized in that, the molecular cut off of the described milipore filter of step (2) is 15000～30000Da.
4. a kind of side that extracts lysozyme from egg or full egg liquid according to claim 1Method, is characterized in that, the described dope of step (2) obtains macromolecule protein after drying.
5. a kind of side that extracts lysozyme from egg or full egg liquid according to claim 4Method, is characterized in that, described macromolecule protein is used for preparing ovalbumin, ovotransferrins, the sticky egg of ovumWhite and Ovoflavoprotein.
6. a kind of side that extracts lysozyme from egg or full egg liquid according to claim 1Method, is characterized in that, the described resin of step (3) is cationic ion-exchange resin or macroporous absorbent resin.
7. a kind of side that extracts lysozyme from egg or full egg liquid according to claim 6Method, is characterized in that, the model of described cationic ion-exchange resin is 001*7,732, AmberliteIR-120、Dowex-50、Lewatit-100、DiaionSK-1、AllassionCS、DuoliteC-20、One in SDB-3, described macroporous absorbent resin be AB-8, D101, D3520, X-5, NKA-II,One or more in NKA-9, S-8, XDA-1, H-20, H-30, H-40.
8. a kind of side that extracts lysozyme from egg or full egg liquid according to claim 1Method, is characterized in that, the molecular cut off of the described milipore filter of step (5) is 2000～10000Da.
9. a kind of side that extracts lysozyme from egg or full egg liquid according to claim 1Method, is characterized in that, step (6) is described is dried, low-temperature vacuum drying or freezing dry dry for sprayingDry.
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| CN201610164753.5A CN105602918A (en) | 2016-03-22 | 2016-03-22 | Method for extracting lysozyme from egg white or whole egg juice |
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| CN201610164753.5A CN105602918A (en) | 2016-03-22 | 2016-03-22 | Method for extracting lysozyme from egg white or whole egg juice |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496318A (en) * | 2016-12-01 | 2017-03-15 | 成都大学 | A kind of method of combined extracting CLU and ovomacroglobulin from fowls albumen |
| CN107916191A (en) * | 2017-12-29 | 2018-04-17 | 烟台智祺化学技术有限公司 | A kind of production method for not adding sulfur dioxide organic wine |
| CN107964467A (en) * | 2017-12-29 | 2018-04-27 | 烟台智祺化学技术有限公司 | A kind of production method for not adding sulfur dioxide Organic grape fruit wine |
| CN107987995A (en) * | 2017-12-29 | 2018-05-04 | 烟台智祺化学技术有限公司 | A kind of production method of Organic grape fruit wine |
| CN108059535A (en) * | 2017-12-29 | 2018-05-22 | 梁涵 | A kind of production method of ecological fertilizer |
| CN108220341A (en) * | 2017-12-29 | 2018-06-29 | 梁涵 | A kind of preparation method of ecology seaweed biostimulant |
| 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 |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106496318A (en) * | 2016-12-01 | 2017-03-15 | 成都大学 | A kind of method of combined extracting CLU and ovomacroglobulin from fowls albumen |
| CN106496318B (en) * | 2016-12-01 | 2019-10-01 | 成都大学 | A method of combined extracting cluster protein and ovomacroglobulin from fowls albumen |
| CN107916191A (en) * | 2017-12-29 | 2018-04-17 | 烟台智祺化学技术有限公司 | A kind of production method for not adding sulfur dioxide organic wine |
| CN107964467A (en) * | 2017-12-29 | 2018-04-27 | 烟台智祺化学技术有限公司 | A kind of production method for not adding sulfur dioxide Organic grape fruit wine |
| CN107987995A (en) * | 2017-12-29 | 2018-05-04 | 烟台智祺化学技术有限公司 | A kind of production method of Organic grape fruit wine |
| CN108059535A (en) * | 2017-12-29 | 2018-05-22 | 梁涵 | A kind of production method of ecological fertilizer |
| CN108220341A (en) * | 2017-12-29 | 2018-06-29 | 梁涵 | A kind of preparation method of ecology seaweed biostimulant |
| 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 |
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