CN106008704A - Method for producing lactoferrin and lactoperoxidase - Google Patents
Method for producing lactoferrin and lactoperoxidase Download PDFInfo
- Publication number
- CN106008704A CN106008704A CN201610637105.7A CN201610637105A CN106008704A CN 106008704 A CN106008704 A CN 106008704A CN 201610637105 A CN201610637105 A CN 201610637105A CN 106008704 A CN106008704 A CN 106008704A
- Authority
- CN
- China
- Prior art keywords
- lactoferrin
- lactoperoxidase
- emulsion
- solution
- eluting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides a method for producing lactoferrin and lactoperoxidase. The method comprises the following steps: an emulsion used as a raw material undergoes ion exchange treatment by using a macro-porous cation exchange resin column to make lactoferrin and lactoperoxidase in the emulsion be adsorbed to the resin column, wherein the temperature of the emulsion is not greater than 55DEG C, and the stay time of the emulsion in the resin column is controlled to be 1-6min; different concentrations of aqueous solutions of sodium chloride or potassium chloride are adopted to carry out gradient elution on the resin column, and a lactoperoxidase-containing first eluate and a lactoperoxidase-containing second eluate are collected; and the first eluate and the second eluate respectively undergo membrane filtration concentration to form first eluate concentrate and second eluate concentrate with the volumes being 1/(8-12) of original volumes, and the first eluate concentrate and the second eluate concentrate respectively undergo filter washing desalination in order to obtain lactoperoxidase concentrate and lactoferrin concentrate.
Description
Technical field
The present invention is about a kind of method producing lactoferrin and lactoperoxidase, specifically about one with Lac Bovis seu Bubali,
The emulsions such as whey are the method for raw material production lactoferrin and lactoperoxidase, belong to technical field of processing dairy products.
Background technology
Lactoferrin (Lactoferrin, LF), also known as " Lactotransferrin ", is a kind of nonheme iron associativity glycoprotein, belongs to
In transferrins family.Various mammal Ruzhongs all have lactoferrin, but its concentration is different because of species, breast in human milk
Ferritin levels is about 1.0～3.2mg/ml, and in Lac Bovis seu Bubali, lactoferrin content is 0.02～0.35mg/ml.Research shows,
Lactoferrin has broad-spectrum antiseptic and viral infection resisting function, it is possible to participate in body iron metabolism, promotes absorption and the utilization of ferrum,
Regulate the balance of internal ferrum, the generation of regulation medullary cell, promote the growth of cell, enhancing human body immunity resistance against diseases,
Suppression human body tumour cell, can more effectively treat disease with Multiple Classes of Antibiotics and antifungal preparation synergism.Breast in recent years
Ferritin is widely used to the fields such as food, medicine, health product and cosmetics.Apply the business in food service industry at present
Mostly industry lactoferrin formulations is to extract the milk surum after casein from Lac Bovis seu Bubali or separate and obtains, and main method includes: from
Sub-exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, metal chelate chromatography, ultrafiltration and immobilization list
Clonal antibody method etc..Wherein, outside deionization exchange chromatography, other chromatographic processes all have defect in various degree, example
Such as technical sophistication, high in cost of production, it is unfavorable for that industrialized production is implemented.And ion exchange chromatography is the most more satisfactory,
Its cost is relatively low, acid and alkali-resistance, and loading volume is big, is easier to accurate control condition, and applicable amplification carries out industrialized production, is
The current main stream approach producing lactoferrin.
Lactoperoxidase (lactoperoxidase, LP) is the secretion a kind of oxidoreductase in Ruzhong, mammal
Mammary gland, salivary gland, lachrymal gland and secretions thereof can find, be one of composition the most common in human milk and Lac Bovis seu Bubali.Breast
The biological significance of peroxidase is the natural defending system participating in constituting host versus invading micro-organism, the most also has
There are antibacterial activity, various carcinogen of degrading and protection zooblast by snperoxiaized effect.Lactoperoxidase at present
Production also mainly extract from Lac Bovis seu Bubali and obtain, correlation technique specifically include that milk separation → except casein → ultrafiltration from
The heart → dialysis and ion-exchange chromatography etc..
CN1557494A disclose a kind of prepare from cattle colostrums, milk or serum and related raw material thereof simultaneously lactoferrin,
Lactoperoxidase and the method for immunoglobulin, mainly comprise the processes of centrifugation defat, with 50,000～70,000D's
Supermicro filtration membrane filters lactose, mineral, small protein, with cationic exchange resin adsorption lactoferrin and breast peroxide
After compound enzyme, respectively with variable concentrations buffer salt solution eluting, lyophilization becomes powder, respectively obtains lactoferrin and breast mistake
Oxide enzyme, is 100 by the liquid molecular weight of chromatographic column, the supermicro filtration membrane of 000D separates, filter, concentrate after be dried
Cheng Fen, obtains immunoglobulin.
CN1557950A discloses and a kind of extracts lactoferrin and lactoperoxidase from cattle colostrums, milk or serum
Method, its process specifically includes that employing centrifugation removes fat, and the staggered tangent plane filtering flow segregation apparatus of application filters
Lactose and small-molecule substance, application cationic exchange resin adsorption lactoferrin and lactoperoxidase, ooze ion with height and ooze
The lactoferrin of transparent liquid eluting absorption and lactoperoxidase, exchanged by ion or ultrafiltration removed deionization and concentrates, through cold
Lyophilizing is dry or vacuum drying is prepared as powder.The lactoferrin of the eluting absorption described in it and the height of lactoperoxidase ooze from
Sub-penetrating fluid is variable concentrations salt buffer: the buffer (about 0.2～0.5mol NaCl) of low concentration is only capable of eluting breast peroxide
Compound enzyme, the buffer (about 2.5mol NaCl) of high concentration can independent eluting lactoferrin, and the buffering of intermediate concentration
Liquid (about > 0.7mol NaCl) energy eluting lactoferrin and lactoperoxidase simultaneously.Additionally, eluent need to carry out desalination
Program, the method for desalination can be ion exchange, electrodialysis or ultrafiltration.
CN102348378A discloses a kind of method producing lactoperoxidase and Lactotransferrin, at least includes step:
A) use the raw material not processed at a temperature of higher than 50 DEG C, b) make this raw material acceptance process, to obtain lactenin
(LN) this LN or MBP solution or the solution of milk basic protein (MBP), c) is made to accept purification on cation exchange resin
Step, the acetate buffer balance of this resin pH4～9, and with containing the different buffer solution elution of different solute concentrations,
And d) collect containing purity more than 95%, do not contain polymer and there is no LPS, endotoxin and angiogenin
The fraction of Lactotransferrin.Wherein step c) includes at least four elution step, a step collecting impurity, one
Collect the step of lactoperoxidase, collect LPS, endotoxin, protease and the step of angiogenin and one for one
Collect the step of Lactotransferrin.Wherein also state that the application different buffer containing different NaCl concentration carry out eluting LN
Different molecular contained by.
Existing ion exchange chromatography produces lactoperoxidase and the method for Lactotransferrin, is mostly to need to remove in advance
Lactose, mineral and/or small protein in emulsion, balances pillar with balance liquid, and step is the most loaded down with trivial details, and
For Pu Bian, speed is slow, unfavorable industrialized production, or the response rate is the most very good.
Summary of the invention
It is an object of the present invention to provide the production breast ferrum egg that a kind of technique is simple, economic, efficient and product purity is higher
White and the method for lactoperoxidase.
Another object of the present invention is to provide the lactoferrin prepared according to the method described above and lactoperoxidase to produce
Product.
On the one hand, the invention provides the production lactoferrin that a kind of technique is simple, economic, efficient and product purity is higher
Method with lactoperoxidase.
According to production lactoferrin provided by the present invention and the method for lactoperoxidase, comprising:
With emulsion as raw material, utilize sulfonate functional groups large hole cation exchanger resin post to carry out ion-exchange treatment, make breast
Lactoferrin and lactoperoxidase in liquid adsorb on resin column；Wherein, described emulsion temperature≤55 DEG C, and control
The emulsion time of staying in resin column is 1～6 minute (10-60 column volume is per hour)；
Use the first eluting solution that resin column carries out eluting, collect eluent, obtain first containing lactoperoxidase
Eluent；Wherein, described first eluting solution is sodium chloride or the potassium chloride solution of concentration 1%～2.5%；
Use the second eluting solution that resin column carries out eluting, collect eluent, obtain the second eluting containing lactoferrin
Liquid；Wherein, described second eluting solution is sodium chloride or the potassium chloride solution of concentration 4.5%～12%；
First eluent, the second eluent are concentrated 8～12 times through membrane filtration respectively, filter wash desalination the most respectively, obtain breast mistake
Oxide enzyme concentrated solution and lactoferrin concentrated solution.
According to specific embodiments of the present invention, in the method producing lactoferrin and lactoperoxidase of the present invention, also
Can farther include: respectively lactoferrin concentrated solution and lactoperoxidase concentrated solution are carried out microfiltration is degerming or Pasteur kills
Bacterium, prepares aseptic lactoferrin concentrated solution and lactoperoxidase concentrated solution.
According to specific embodiments of the present invention, in the method producing lactoferrin and lactoperoxidase of the present invention, also
Can farther include: (aforementioned without sterilization or through sterilization to lactoferrin concentrated solution and lactoperoxidase concentrated solution respectively
Concentrated solution) be dried, prepare lactoferrin powder and lactoperoxidase enzyme powder；Wherein, described it is dried as lyophilization
Or low temperature spray drying.
According to specific embodiments of the present invention, in the method for the present invention, described raw emulsion includes that all lactoferrin come
The emulsion in source, such as human milk, Lac Bovis seu Bubali, Lac caprae seu ovis, Os Equi, Lac Cameli etc., can be colostrum, rich milk, skimmed milk or breast
Clear liquid (comprises condensed whey liquid).Preferably, these emulsions process without 55 DEG C of temperatures above.
According to specific embodiments of the present invention, in the method for the present invention, described emulsion materials is handed over utilizing macroporous cation
Change before resin column carries out ion-exchange treatment, it is not necessary to remove lactose, mineral and/or small protein.
According to specific embodiments of the present invention, in the method for the present invention, carry out utilizing large hole cation exchanger resin post
Without using buffer to balance pillar before ion-exchange treatment, i.e. the method is not necessarily comprised in and utilizes macroporous cation to exchange
Resin column uses the process of buffer balance pillar before carrying out ion-exchange treatment.
According to specific embodiments of the present invention, in the method for the present invention, the temperature of described raw emulsion is preferably 42 DEG C
～55 DEG C, this process at a temperature of emulsion can be full-cream emulsion can also be separated milk, and can effectively prevent lactoferrin
Heated denaturalization, can significantly shorten the emulsion time of staying in resin column simultaneously, improves production efficiency；It addition, such as
Fruit processes skimmed milk, then need not after thermal debinding carry out heat exchange process again, can be with energy efficient.
According to specific embodiments of the present invention, in the method for the present invention, the distribution of described raw emulsion particle diameter is less than 10 microns
(D90 particle diameter), such as 2～8 microns, it is ensured that effectively carrying out of ion exchange, filters simultaneously by preferably less than 8 microns
The optional pore diameter range of device is relatively big, and operating cost also can reduce.
According to specific embodiments of the present invention, in the method for the present invention, described large hole cation exchanger resin is a class sun
Ion exchange resin.The preferably functional group of strong cation-exchanging resin has a sulfonic acid base class, such as sulfonic group, sulfoethyl,
Propyl sulfonic acid etc..It is furthermore preferred that the particle diameter of strong cation macropore exchanger resin used by the present invention is micro-at 200 microns～350
Between meter.
According to specific embodiments of the present invention, the emulsion of the present invention time of staying in ion exchange resin column is
1～6 minute, preferably 1～2 minute, both can ensure the adsorption rate of lactoferrin and lactoperoxidase, breast ferrum can be reduced again
Albumen and the heated time of lactoperoxidase, shorten processing extraction time, improves production efficiency.
According to specific embodiments of the present invention, in the method for the present invention, elution process uses gradient elution, and first
Step gradient elution solution is the sodium chloride or potassium chloride solution or buffer solution comprising low concentration, second step gradient elution
Solution comprises high concentration sodium chloride or potassium chloride solution or buffer solution.Sodium chloride according to the present invention the first eluting solution
Or the concentration of potassium chloride is 1%～2.5%, the sodium chloride of second step gradient elution solution or the concentration of potassium chloride are
4.5%～12%；Or it is the dibastic sodium phosphate buffer solution comprising this concentration sodium chloride or potassium chloride, thus can well
Eluting separation lactoperoxidase and lactoferrin, and solution salt content is relatively low, sewage disposal difficulty is relatively low.
According to specific embodiments of the present invention, in the method for the present invention, membrane filtration processes is used to be received by the first and second eluting
Liquid collecting carries out concentrating and desalination respectively.Membrane aperture used by first step membrane filtration concentration process is 30kDa～50kDa, during concentration
Not only can guarantee that concentration concentration but also good flux can be kept, improve work efficiency.Feed liquid is concentrated 10 times, and then filter wash takes off
Salt, to electrical conductivity less than 2mS/cm.Owing to lactoferrin and lactoperoxidase belong to high molecular weight protein, at low ion
In strength solution, along with the increase of protein concentration, feed liquid can become sticky and then be prone to occur aggregate and precipitate, and then blocking fenestra,
Therefore the multiple concentrated was difficult to too high, suitably.Desalination make feed liquid ionic strength significantly reduce, work as feed liquid
Viscosity too high, the removal efficiency of ion can significantly reduce, and compared with feed liquid desalting efficiency during low viscosity, removing is mutually the most on year-on-year basis
The salinity of example, then need to consume substantial amounts of water resource, and therefore ion removal efficiency is moderate, it is not necessary to ion is emphasized fall
Low to ultimate attainment；On the other hand albumen precipitation can be caused in LISS so that produce and cannot be carried out, thus de-
After salt, the ionic strength of feed liquid is difficult to too low.Therefore the present invention selects feed liquid to concentrate 10 times, feed liquid solid content after desalination
About 10%～25%, electrical conductivity is less than 2mS/cm, the most i.e. can ensure that the quality of product, can ensure that again equipment
Efficient normal operation, the most energy-saving and cost-reducing.
According to specific embodiments of the present invention, in the method for the present invention, lyophilization can be carried out by concentrating solution further,
Obtain the purity lactoferrin lyophilized powder more than 90% and the purity lactoperoxidase lyophilized powder more than 90%, and two hatching egg
In white lead, effective ingredient accounts for more than the 95% of albumen, beneficially industrial applications and accumulating.
According to specific embodiments of the present invention, in the method for the present invention, cold nebulization can be carried out by concentrating solution further
It is dried, obtains the purity lactoferrin spray powder more than 90% and the purity lactoperoxidase spray powder more than 90%, and
In two kinds of egg albumen powders, effective ingredient accounts for more than the 95% of total protein content, beneficially industrial applications and accumulating respectively.
According to specific embodiments of the present invention, in the method for the present invention, also further concentrated solution can be used membrane filtration
Degerming or 72～75 DEG C, pasteurize in 15 seconds, it is prepared as the sterile liquid bag of 10%～25%, it is simple to the nothing of fluid product
Add after bacterium.
On the other hand, present invention also offers the lactoferrin prepared according to the described method of the present invention and breast peroxidating
Thing enzyme product.The response rate is up to more than 80%, and the lactoferrin product produced according to the present invention is pale pink, and granule is uniform,
Purity reaches more than 90%, and effective ingredient accounts for more than the 95% of albumen, has good biocidal property and non-oxidizability.According to
The lactoperoxidase enzyme product that the present invention produces is front brown or lilac, and purity reaches more than 90%, and effective ingredient accounts for egg
White more than 95%, has good enzymatic activity.
Accompanying drawing explanation
Fig. 1 is the schematic flow sheet of the method for the present invention.
Fig. 2 is lactoferrin purity detecting spectrogram in embodiment 1.
Fig. 3 is lactoperoxidase purity detecting spectrogram in embodiment 1.
Detailed description of the invention
In order to be more clearly understood that the essence of the present invention, below by specific embodiment and coordinate accompanying drawing to further describe
The present invention, but the present invention is not therefore subject to any restriction.
The detection method used in each embodiment:
1. bacteriostasis rate detection method
(1) prepared by bacterium solution: be inoculated in by escherichia coli in 10mL peptone water, cultivates activation 16h (water in 37 DEG C
Bath shaking table and incubator), the first generation is activated bacterium solution it is transferred in new peptone water according to inoculation is a certain amount of, live
Change cultivation its concentration of adjustment and reach 106～107CFU/ml, then dilute 10 times standby.
(2) prepared by lactoferrin solution: dissolves lactoferrin powder, prepares certain density lactoferrin solution, with 0.2 μm
Needle-based membrane filtration lactoferrin solution, is diluted to same concentrations by sterile liquid sample packet simultaneously.
(3) plate count method: add 0.4mL in 8.6mL peptone water and configured the lactoferrin solution of concentration,
Add 1mL 10 again5～106Escherichia coli bacteria liquid, cultivate 24h sampling carry out plate count, do simultaneously without breast ferrum egg
White blank sample.
2. oxidation resistance (FRAP) property method
FRAP reagent: 40mL 0.3M acetate buffer solution (pH 3.6), 4mL 20mM FeCl3 solution, 4mL 10mM
TPTZ (2,4,6-tris (2-pyridine-s-triazine)) mixes.All reagent are preheated to 37 DEG C before testing, at 96 hole enzyme marks
Plate is tested, mixes 20 μ L pure water, 10 μ L breast iron samples and 150 μ L FRAP reagent, immediately at 37 DEG C
Insulation, for ascorbic acid, measures light absorption value during 5min under 593nm, and for breast iron sample, every 5min measures
Once, until 24h.The antioxidation score value (FRAP value) of sample is worth to relative to the FRAP of ascorbic acid.Should
It is worth the highest, shows that non-oxidizability is the strongest.
3. enzyme activity method detection
3.1 solution preparations
The hydrogen peroxide of 100 microlitres 30% is settled to 10mL with pure water, is made into 0.3% hydrogen peroxide solution.
0.5mg/mL ABTS solution, weighs 0.0125g ABTS in 25mL volumetric flask, with the citric acid of pH5.5
Sodium-citrate buffer solution constant volume.
Sample pre-treatments: the LP solution obtained by purification dilutes certain multiple.
3.2 operating procedure
Take the ABTS solution that 2mL configures to be positioned in cuvette, 37 DEG C of baking ovens or water-bath be incubated 10min,
Rapidly join lactoperoxidase enzymatic solution (after dilution) and 2 microlitre hydrogen peroxide solutions, the mix homogeneously of 0.1mL afterwards
Put into the most rapidly in spectrophotometer, under 412nm wavelength, measure absorbance, simultaneously timing, 1min and 2min time-division
Other reading.
3.3 calculate
Formula is as follows:
4. purity detecting
4.1 experiment materials: lactoferrin；Tris；Sodium hydroxide (analytical pure)；Sodium chloride (analytical pure)；Hydrochloric acid (point
Analyse pure)；Ethanol (analytical pure)；Cation exchange resin.
4.2 experimental apparatus: chromatograph；Analysis of milk composition instrument (FT120)
4.3 experimental techniques:
Raw milk is stored after fetching at 4 DEG C, detects on the same day or next day, carries out ungrease treatment before detection,
Carry out two times centrifugal under conditions of 10000 revs/min, after abjection fat, carry out loading.
Raw milk breast iron content testing process
Solution A: 0.05M Tris-HCl pH value of solution=7；
B solution: salinity is 1M Tris-HCl-NaCl pH value of solution=7.
Embodiment 1
With reference to technological process shown in Fig. 1, prepare lactoferrin and lactoperoxidase according to following steps:
Take the emulsion (skimmed milk) that fat content is less than 0.2%, particle diameter is less than 8 microns, temperature between 4 DEG C～10 DEG C,
Utilize ion exchange resin that this emulsion is carried out ion-exchange treatment, in order to make lactoferrin and lactoperoxidase be adsorbed onto
On ion exchange resin, milk retention time in resin column is 2min, then emulsion is ejected with water.
First step eluting uses the sodium chloride solution of the 1% of about 4 column volumes to carry out eluting, collects eluent, obtains breast mistake
Oxide enzyme saline solution；
Second step eluting uses the sodium chloride solution of the 4.5% of about 4 column volumes to carry out eluting, collects eluent, obtains breast
Ferritin saline solution；
The lactoferrin saline solution afforded and lactoperoxidase saline solution are utilized respectively the film that aperture is 30kDa incite somebody to action
Feed liquid concentrates 10 times, then filter wash desalting processing, in order to obtain lactoferrin and the lactoperoxidase of purification, and the two takes off
After salt, the solid content of feed liquid is 10%, electrical conductivity 1.5mS/cm.
Lyophilization: above-mentioned lactoferrin and lactoperoxidase are carried out lyophilization respectively and obtains lyophilized protein powder；
Or select liquid bag person, above-mentioned lactoferrin and lactoperoxidase are carried out respectively membrane filtration degerming or 72～75 DEG C,
Pasteurize in 15 seconds, is prepared as sterile milk ferritin or lactoperoxidase liquid bag.
Fig. 2 is lactoferrin purity detecting spectrogram in the present embodiment.Fig. 3 is lactoperoxidase purity detecting in the present embodiment
Spectrogram.
Embodiment 2
With reference to technological process shown in Fig. 1, prepare lactoferrin and lactoperoxidase according to following steps:
Taking the emulsion that fat content is less than 0.5%, particle diameter is less than 8 microns, temperature, between 15 DEG C～20 DEG C, utilizes ion to hand over
Change resin and this emulsion is carried out ion-exchange treatment, in order to make lactoferrin and lactoperoxidase be adsorbed onto amberlite
On fat, milk retention time in resin column is 2min, then emulsion is ejected with water.
First step eluting uses the Klorvess Liquid of the 1.2% of about 4 column volumes to carry out eluting, obtains lactoperoxidase salt
Solution；
Second step eluting uses the Klorvess Liquid of the 5% of about 4 column volumes to carry out eluting, obtains lactoferrin saline solution；
The lactoferrin saline solution afforded and lactoperoxidase saline solution are utilized respectively the film that aperture is 50kDa incite somebody to action
Feed liquid concentrates 10 times, then filter wash desalting processing, in order to obtain lactoferrin and the lactoperoxidase of purification, and the two takes off
After salt, the solid content of feed liquid is 12%, and electrical conductivity is less than 1.8mS/cm.
Lyophilization or low temperature spray drying or membrane filtration are degerming: above-mentioned lactoferrin and lactoperoxidase are carried out respectively
Lyophilization obtains lyophilized protein powder；Or above-mentioned lactoferrin and lactoperoxidase are carried out low temperature spray drying respectively
Obtain the egg albumen powder of spray drying；Or above-mentioned lactoferrin and lactoperoxidase are carried out respectively membrane filtration degerming or
72～75 DEG C, pasteurize in 15 seconds, it is prepared as sterile milk ferritin or lactoperoxidase liquid bag.
Embodiment 3
With reference to technological process shown in Fig. 1, prepare lactoferrin and lactoperoxidase according to following steps:
Taking emulsion full-cream, that particle diameter is less than 8 microns, temperature, between 52 DEG C～55 DEG C, utilizes ion exchange resin to this breast
Liquid carries out ion-exchange treatment, in order to making lactoferrin and lactoperoxidase be adsorbed onto on ion exchange resin, milk exists
Retention time in resin column is 1min, then emulsion is ejected with water.
First step eluting uses the sodium chloride solution of the 2.5% of about 4 column volumes to carry out eluting, obtains lactoperoxidase salt
Solution；
Second step eluting uses the sodium chloride solution of the 10% of about 4 column volumes to carry out eluting, obtains lactoferrin saline solution；
The lactoferrin saline solution afforded and lactoperoxidase saline solution are utilized respectively the film that aperture is 50kDa incite somebody to action
Feed liquid concentrates 10 times, then filter wash desalting processing, in order to obtain lactoferrin and the lactoperoxidase of purification, and the two takes off
After salt, the solid content of feed liquid is 15%, and electrical conductivity is less than 1.2mS/cm.
Lyophilization or low temperature spray drying or membrane filtration are degerming: above-mentioned lactoferrin and lactoperoxidase are carried out respectively
Lyophilization obtains lyophilized protein powder；Or above-mentioned lactoferrin and lactoperoxidase are carried out low temperature spray drying respectively
Obtain the egg albumen powder of spray drying；Or above-mentioned lactoferrin and lactoperoxidase are carried out respectively membrane filtration degerming or
72～75 DEG C, pasteurize in 15 seconds, it is prepared as sterile milk ferritin or lactoperoxidase liquid bag.
Embodiment 4
With reference to technological process shown in Fig. 1, prepare lactoferrin and lactoperoxidase according to following steps:
Take fat content be 0.8%, particle diameter less than the emulsion of 8 microns, temperature, between 52 DEG C～55 DEG C, utilizes ion to exchange
Resin carries out ion-exchange treatment to this emulsion, in order to make lactoferrin and lactoperoxidase be adsorbed onto ion exchange resin
On, milk retention time in resin column is 1min, then emulsion is ejected with water.
First step eluting uses the sodium chloride solution of the 2.5% of about 4 column volumes to carry out eluting, obtains lactoperoxidase salt
Solution；
Second step eluting uses the sodium chloride solution of the 12% of about 4 column volumes to carry out eluting, obtains lactoferrin saline solution；
The lactoferrin saline solution afforded and lactoperoxidase saline solution are utilized respectively the film that aperture is 50kDa incite somebody to action
Feed liquid concentrates 10 times, then filter wash desalting processing, in order to obtain lactoferrin and the lactoperoxidase of purification, and the two takes off
After salt, the solid content of feed liquid is 18%, and electrical conductivity is less than 1.01mS/cm.
Lyophilization or low temperature spray drying or membrane filtration are degerming: above-mentioned lactoferrin and lactoperoxidase are carried out respectively
Lyophilization obtains lyophilized protein powder；Or above-mentioned lactoferrin and lactoperoxidase are carried out low temperature spray drying respectively
Obtain the egg albumen powder of spray drying；Or above-mentioned lactoferrin and lactoperoxidase are carried out respectively membrane filtration degerming or
72～75 DEG C, pasteurize in 15 seconds, it is prepared as sterile milk ferritin or lactoperoxidase liquid bag.
Embodiment 5
With commercially available lactoferrin for comparison, use plate count method measuring and calculating bacteriostasis rate, the simultaneously breast to embodiment 1～4
Ferritin sample carries out purity, bacteriostatic activity and non-oxidizability detection, result such as following table:
Table 1 lactoferrin specificity analysis result
Note: result above is 3 meansigma methodss measured, and antioxidation value is the highest, represents non-oxidizability the strongest.
Table 2 lactoperoxidase specificity analysis result
Note: result above is 3 meansigma methodss measured.
Claims (10)
1. the method producing lactoferrin and lactoperoxidase, the method includes:
With emulsion as raw material, utilize sulfonate functional groups large hole cation exchanger resin post to carry out ion-exchange treatment, make
Lactoferrin and lactoperoxidase in emulsion adsorb on resin column；Wherein, described emulsion temperature≤55 DEG C, and
The control emulsion time of staying in resin column is 1～6 minute；
Use the first eluting solution that resin column carries out eluting, collect eluent, obtain the containing lactoperoxidase
One eluent；Wherein, described first eluting solution is sodium chloride or the potassium chloride solution of concentration 1%～2.5%；
Use the second eluting solution that resin column carries out eluting, collect eluent, obtain washing containing the second of lactoferrin
De-liquid；Wherein, described first eluting solution is sodium chloride or the potassium chloride solution of concentration 4.5%～12%；
First eluent, the second eluent are concentrated 8～12 times through membrane filtration respectively, filter wash desalination the most respectively, obtain breast
Peroxidase concentrated solution and lactoferrin concentrated solution.
Method the most according to claim 1, the method also includes:
Respectively lactoferrin concentrated solution and lactoperoxidase concentrated solution are carried out microfiltration is degerming or pasteurize, prepares nothing
The lactoferrin concentrated solution of bacterium and lactoperoxidase concentrated solution.
Method the most according to claim 1 and 2, the method also includes:
Respectively lactoferrin concentrated solution and lactoperoxidase concentrated solution are dried, prepare lactoferrin powder and breast mistake
Oxide enzyme powder；Wherein, described it is dried as lyophilization or low temperature spray drying.
Method the most according to claim 1, wherein, described raw emulsion includes containing lactoferrin and breast peroxide
One or more in the human milk of compound enzyme, Lac Bovis seu Bubali, Lac caprae seu ovis, Os Equi, Lac Cameli；Can be colostrum, rich milk, take off
Fat breast or whey；Preferably, these emulsions process without 55 DEG C of temperatures above；It is highly preferred that these emulsions exist
Before utilizing large hole cation exchanger resin post to carry out ion-exchange treatment, it is not necessary to remove lactose, mineral and/or little
Molecule protein.
Method the most according to claim 1, wherein, the temperature of described raw emulsion is 42 DEG C～55 DEG C, emulsion
Particle diameter distribution is less than 10 microns, preferably less than 8 microns.
Method the most according to claim 1, the method is carried out not included in utilizing large hole cation exchanger resin post
The process of buffer balance pillar is used before ion-exchange treatment.
Method the most according to claim 1, wherein, the described emulsion time of staying in resin column is 1～2min.
Method the most according to claim 1, wherein, the membrane aperture used by described membrane filtration concentration process is
30kDa～50kDa.
Method the most according to claim 1, wherein, in described filter wash desalination processes, desalination is less than 2 to electrical conductivity
mS/cm。
10. the lactoferrin product prepared according to method described in any one of claim 1～9 or lactoperoxidase
Product；Preferably, protein content more than 90% in lactoferrin product, effective ingredient accounts for more than the 95% of albumen；Breast
Protein content more than 90% in peroxide enzyme product, effective ingredient accounts for more than the 95% of albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610637105.7A CN106008704A (en) | 2016-08-05 | 2016-08-05 | Method for producing lactoferrin and lactoperoxidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610637105.7A CN106008704A (en) | 2016-08-05 | 2016-08-05 | Method for producing lactoferrin and lactoperoxidase |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106008704A true CN106008704A (en) | 2016-10-12 |
Family
ID=57134565
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610637105.7A Pending CN106008704A (en) | 2016-08-05 | 2016-08-05 | Method for producing lactoferrin and lactoperoxidase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106008704A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107328767A (en) * | 2017-05-25 | 2017-11-07 | 新希望乳业股份有限公司 | The quick determination method of peroxidase in a kind of guaranteed milk |
| CN112655768A (en) * | 2020-12-16 | 2021-04-16 | 黑龙江飞鹤乳业有限公司 | Preparation method of milk powder containing lactoferrin |
| CN112996806A (en) * | 2018-11-06 | 2021-06-18 | 安赫尔工程建模有限责任公司 | Method for preparing high purity lactoferrin and lactoperoxidase from milk, colostrum and acid whey or sweet whey |
| CN113105542A (en) * | 2021-04-15 | 2021-07-13 | 黑龙江飞鹤乳业有限公司 | Preparation method of lactoferrin |
| US11109604B2 (en) | 2019-05-09 | 2021-09-07 | Memtec LLC | Dairy processing systems and methods |
| CN114145344A (en) * | 2021-12-10 | 2022-03-08 | 光明乳业股份有限公司 | Liquid dairy product and preparation method thereof |
| IT202100001208A1 (en) * | 2021-01-22 | 2022-07-22 | Grinlux S R L | METHOD FOR PREPARING WHEY PROTEIN POWDERS |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101117351A (en) * | 2007-04-30 | 2008-02-06 | 北京济普霖生物技术有限公司 | Method for purifying restructuring lactoferrin from transgenic cow's milk |
| CN103709246A (en) * | 2013-11-25 | 2014-04-09 | 北京济福霖生物技术有限公司 | Purification method for lactoferrin and lactoperoxidase |
| CN104593340A (en) * | 2015-01-15 | 2015-05-06 | 浙江工业大学 | Method for separating lactoperoxidase from bovine whey |
| EP2873329A1 (en) * | 2013-11-15 | 2015-05-20 | Univerzita Palackeho | Process of whey protein separation from milk medium and apparatus for its implementation |
| CN104926936A (en) * | 2015-06-26 | 2015-09-23 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for preparing lactoferrin |
-
2016
- 2016-08-05 CN CN201610637105.7A patent/CN106008704A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101117351A (en) * | 2007-04-30 | 2008-02-06 | 北京济普霖生物技术有限公司 | Method for purifying restructuring lactoferrin from transgenic cow's milk |
| EP2873329A1 (en) * | 2013-11-15 | 2015-05-20 | Univerzita Palackeho | Process of whey protein separation from milk medium and apparatus for its implementation |
| CN103709246A (en) * | 2013-11-25 | 2014-04-09 | 北京济福霖生物技术有限公司 | Purification method for lactoferrin and lactoperoxidase |
| CN104593340A (en) * | 2015-01-15 | 2015-05-06 | 浙江工业大学 | Method for separating lactoperoxidase from bovine whey |
| CN104926936A (en) * | 2015-06-26 | 2015-09-23 | 内蒙古蒙牛乳业（集团）股份有限公司 | Method for preparing lactoferrin |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107328767A (en) * | 2017-05-25 | 2017-11-07 | 新希望乳业股份有限公司 | The quick determination method of peroxidase in a kind of guaranteed milk |
| CN112996806A (en) * | 2018-11-06 | 2021-06-18 | 安赫尔工程建模有限责任公司 | Method for preparing high purity lactoferrin and lactoperoxidase from milk, colostrum and acid whey or sweet whey |
| US11109604B2 (en) | 2019-05-09 | 2021-09-07 | Memtec LLC | Dairy processing systems and methods |
| US11793211B2 (en) | 2019-05-09 | 2023-10-24 | Memtec LLC | Dairy processing systems and methods |
| CN112655768A (en) * | 2020-12-16 | 2021-04-16 | 黑龙江飞鹤乳业有限公司 | Preparation method of milk powder containing lactoferrin |
| IT202100001208A1 (en) * | 2021-01-22 | 2022-07-22 | Grinlux S R L | METHOD FOR PREPARING WHEY PROTEIN POWDERS |
| CN113105542A (en) * | 2021-04-15 | 2021-07-13 | 黑龙江飞鹤乳业有限公司 | Preparation method of lactoferrin |
| CN114145344A (en) * | 2021-12-10 | 2022-03-08 | 光明乳业股份有限公司 | Liquid dairy product and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106008704A (en) | Method for producing lactoferrin and lactoperoxidase | |
| Francis et al. | Extraction from cheese whey by cation-exchange chromatography of factors that stimulate the growth of mammalian cells | |
| CN105586379B (en) | A kind of preparation method with the collagen active peptide for inhibiting cancer cell multiplication effect | |
| CN104672328B (en) | A kind of production method of Human Antithrombin Ⅲ | |
| CN103709246B (en) | The purification process of a kind of lactoferrin and lactoperoxidase | |
| CN104031965B (en) | A kind of preparation method of kefir albumen derived antimicrobial peptide | |
| CN103275191B (en) | Method for largely and quick extracting tachyplesin peptide | |
| CN107080753A (en) | A kind of cosmetic formulation of human umbilical cord mesenchymal stem cells source excretion body | |
| LU501670B1 (en) | A Method for as1-Casein Separation | |
| CN101603038A (en) | A kind of preparation method of N,O-Diacetylmuramidase | |
| CN105648008A (en) | Feeding silk antibacterial peptide preparation and preparation method thereof | |
| CN112996806A (en) | Method for preparing high purity lactoferrin and lactoperoxidase from milk, colostrum and acid whey or sweet whey | |
| CN114014926B (en) | Method for simply and rapidly preparing high-purity immunoglobulin G1 and G2 from bovine coloctrum | |
| CN104450639B (en) | A kind of method using ultrafiltration auxiliary aqueous two phase extraction technique extraction lactoperoxidase | |
| RU2634859C1 (en) | Method for lactoferrin isolation and purification from dairy raw materials | |
| RU2390253C1 (en) | Method of lactoferrin obtaining from dairy raw materials | |
| CN104327175B (en) | A kind of method for separating antibacterial peptide | |
| KR20150036683A (en) | Novel fermented milk product and method for producing the same | |
| Taylor et al. | Quantity and carbohydrate content of glycomacropeptide fractions isolated from raw and heat-treated milk | |
| CN106222221A (en) | Prepare the purification process of recombined human granulocyte stimulating factors stock solution | |
| RU2416243C2 (en) | Method of extracting low molecular peptides | |
| CN107778332A (en) | Sialic acid oligosaccharide and its manufacture method, hair tonic and skeletal muscle form accelerator | |
| CN104513305B (en) | A kind of purification process of lactalbumin | |
| CN103864915A (en) | Method for purifying recombinant human interferon alpha2b and kit | |
| Tomar et al. | Evaluation of in vitro anti-microbial activity of goat urine peptides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination |