CN103467597A - Method for removing endotoxin in lactoferrin product - Google Patents
Method for removing endotoxin in lactoferrin product Download PDFInfo
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- CN103467597A CN103467597A CN2013104142011A CN201310414201A CN103467597A CN 103467597 A CN103467597 A CN 103467597A CN 2013104142011 A CN2013104142011 A CN 2013104142011A CN 201310414201 A CN201310414201 A CN 201310414201A CN 103467597 A CN103467597 A CN 103467597A
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Abstract
The invention provides a method for removing endotoxin in a lactoferrin product. The method comprises the following steps of: preparing the lactoferrin product from which the endotoxin is to be removed into a lactoferrin aqueous solution, adding a flocculant to the lactoferrin aqueous solution so that the endotoxin therein is converted into a high polymer, and carrying out membrane separation to remove the endotoxin. The method provided by the invention also comprises the process of performing affinity chromatography on the lactoferrin aqueous solution after the membrane separation so as to further remove the endotoxin. Due to the adoption of the method provided by the invention, the removal rate of the endotoxin in the lactoferrin product can be 99.99% after the two-step separation operations of membrane separation and affinity chromatography.
Description
Technical field
The invention relates to a kind of endotoxic method for example removed, in lactoferrin formulations (lactoferrin powder), belong to technical field of dairy processing.
Background technology
Lactoferrin (LF) is a kind of natural glycoprotein with bioactive functions be present in the exocrine secretions such as milk, tears, saliva, and its bioactive functions comprises broad spectrum antibiotic activity, strengthens transmission and absorption, immunomodulatory, adjusting inflammatory reaction and the antioxygenation etc. of iron.Therefore lactoferrin continues to be subject to the high praise in market in recent years, and has been widely used in the fields such as food, medicine, healthcare products and makeup.The commercialization lactoferrin formulations be applied at present in food service industry is all extracted and is obtained from cow's milk or whey.
Intracellular toxin is the main component of gram-negative bacterial cell wall, because its can discharge in a large number when bacterial cell is dead, and can cause people and Mammals and produces the ill symptomses such as heating, septic shock be even dead, therefore is called as intracellular toxin.From chemical structure analysis, intracellular toxin is the lipopolysaccharides material, core oligosaccharide, surface-O-antigen and lipoid A tri-parts, consists of.Because gram negative bacterium can survive in oligotrophic environment, so intracellular toxin is almost ubiquitous.
Contain gram negative bacterium in cow's milk, so the commercialization lactoferrin in cow's milk source also inevitably has been subject to endotoxic pollution.There are some researches show, the intracellular toxin that lactoferrin formulations contains causes except meeting the symptoms such as heating, also can greatly reduce the biological activitys such as biocidal property of lactoferrin, this case contriver also finds that through practical study the lactoferrin highly polluted by intracellular toxin almost completely lost its bacteriostatic activity.Pharmaceutical industries has higher requirement to the endotoxin content of pharmaceutical products, and for example in the injection medicament, endotoxin content must guarantee that every kg body weight intake hourly is lower than 5EU.Stability of endotoxin is very high, can not make intracellular toxin lose toxicity by processing such as conventional heat, acid, and therefore removing endotoxic technology from lactoferrin has very high practical value and meaning.
Remove at present endotoxic technology comparative maturity from the aqueous solution, methods involving has membrane separation process, ion exchange method, two phase separation methods, affinity chromatography and hydrophobic chromatography etc.But remove the difficult problem that intracellular toxin remains industry from protein system, its major cause is the protein system more complicated, and there is strong reactive force in multiple proteins and intracellular toxin, and both combine, and are difficult to separate.Therefore also do not remove endotoxic pervasive rule from protein system at present, correlative study also all is only applicable to a certain protein system.
EP0312104A2 discloses a kind of endotoxic method in pharmaceutical preparation that removes, wherein said pharmaceutical preparation scope is very extensive, comprise the preparations such as carbohydrate, enzyme, protein, hormone, method therefor is first with membrane separation technique, to remove the intracellular toxin aggregate, then uses sorbent material by remaining Endotoxin removal.But the endotoxic method of the removal of mentioning in the document not being suitable for is removed the intracellular toxin in the material of these classes such as lactoferrin and intracellular toxin generation strong effect.
US2011/0003976A1 discloses a kind of endotoxic method adopted in chromatography deproteination plastome, its gordian technique is to using the alkane glycol as eluting solvent, protein is separated with intracellular toxin, wherein mention the method and also be applicable to lactoferrin, but there is the shortcoming of at least two aspects in this method: at first alkane glycol cost is higher, has limited the commercialization of this method and promoted; Secondly the alkane glycol has certain toxicity, for food safety, has larger risk.
Summary of the invention
Main purpose of the present invention be to provide a kind of safe, nontoxic, effectively remove the endotoxic method in lactoferrin formulations.
For this reason, the invention provides a kind of endotoxic method removed in lactoferrin formulations, the method comprises:
To wait to remove endotoxic lactoferrin formulations and be allocated as the lactoferrin aqueous solution, and add wherein flocculation agent to make the intracellular toxin in it be converted into superpolymer, carry out membrane sepn, and separate and remove intracellular toxin.
Contriver's endotoxic characteristic of having analyzed and researched, known intracellular toxin can exist with the form of monomer, aggregation, micella and vesica in solution, monomer can be assembled the formation aggregation, further gathering also can form even vesica of micella, in particular solution, there is specific balance in endotoxic existence form.The molecular weight of intracellular toxin monomer is 10～30kDa, the molecular weight of micella is less than 1000kDa, the molecular weight of vesica is higher than 1000kDa, and the molecular weight of lactoferrin is about 80kDa, therefore selects suitable membrane the intracellular toxin of lactoferrin and various forms can be separated in theory.Yet, the lactoferrin iso-electric point is 8.0～9.0, it is typical basic protein, all be with clean positive charge under weakly alkaline, neutrality and acidic conditions, intracellular toxin is electronegative under neutrallty condition, therefore have strong electrostatic attraction effect between lactoferrin and intracellular toxin, in addition, hydrophobic interaction is also one of reactive force maintained combination between lactoferrin and intracellular toxin.In addition, lactoferrin is easy to destroy the structure of intracellular toxin superpolymer, the intracellular toxin solution coalescence and the protein that originally with the superpolymer form, exist are regrouped, form with protein-intracellular toxin aggregation exists, therefore, although aspect the intracellular toxin of conventional membrane separation technique in removing the aqueous solution, feasibility is higher, poor effect often during intracellular toxin in deproteination plastome.
In the endotoxic method in removing lactoferrin formulations of the present invention, be by after removing endotoxic lactoferrin formulations and being made into the aqueous solution, added wherein flocculation agent so that the intracellular toxin in it is converted into superpolymer, carry out again afterwards membrane sepn.The present invention adds flocculation agent and can impel intracellular toxin to be converted into most possibly superpolymer.The purpose of adding flocculation agent is: impel the intracellular toxin quantity that is converted into superpolymer many as far as possible on the one hand, impel on the other hand the molecular weight of superpolymer of formation large as far as possible, and mechanical stability is enough high, thereby greatly improves the intracellular toxin decreasing ratio of membrane separating method, penetrating amount, processing power and reduce energy consumption.
Particularly, the present invention finds by research, and selecting suitable flocculation agent can make the intracellular toxin of part and protein bound regroup becomes the intracellular toxin superpolymer.According to the specific embodiment of the invention scheme, being applicable to flocculation agent of the present invention is salt and hydrophilic colloid.Preferably, described salt is to be selected from Na by least one
+, K
+, Ca
2+, Mg
2+, Cu
2+, Zn
2+, Fe
2+, Fe
3+, Al
3+, NH
4 +in positively charged ion be selected from Cl
﹣, SO
4 2 ﹣, PO
4 3 ﹣, NO
3 -, the negatively charged ion in citrate ion forms; Preferably, described salt is selected from one or more in calcium chloride, sodium-chlor, magnesium chloride, ammonium sulfate, sodium sulfate, saltpetre; One or more in ammonium sulfate, magnesium chloride and calcium chloride particularly preferably.Described hydrophilic colloid is selected from one or more in gelling gum, gelatin, pectin, xanthan gum, carrageenin, carboxymethyl cellulose (CMC), methylcellulose gum (MC), guar gum, sodium alginate, Viscogum BE, gum arabic, agar, modified starch; Preferably, described hydrophilic colloid is selected from one or more in pectin, carrageenin, xanthan gum and sodium alginate.Preferred salt and hydrocolloid compositions have: ammonium sulfate, calcium chloride and pectin, calcium chloride, sodium-chlor and xanthan gum and carrageenin, SODIUMNITRATE, Iron nitrate and carboxymethyl cellulose, magnesium chloride and sodium sulfate and sodium alginate.
According to the specific embodiment of the invention scheme, in the selected flocculation agent of the present invention, salt ion especially divalent cation has the function that promotes intracellular toxin to form superpolymer, in addition hydrophilic colloid also can be directly or by salt ion indirectly with the intracellular toxin polymerization, and this hydrophilic colloid-intracellular toxin aggregation can be controlled proper condition in condition and is issued to and is enough to tolerate the physical strength that membrane sepn operates.Because the molecular weight of hydrophilic colloid is all very large, therefore no matter be intracellular toxin self assemble the intracellular toxin aggregation that forms or with hydrophilic colloid assemble form hydrophilic colloid-intracellular toxin aggregation, molecular weight, all significantly higher than lactoferrin, is easy to select the film of suitable molecular weight cut-off that intracellular toxin is removed.
More specifically, in the endotoxic method removed in lactoferrin formulations of the present invention, the quality of lactoferrin in the aqueous solution of take is benchmark:
The amount of the saline flocculating agent added is 0.001%～10%, is preferably 0.01%～4%; More preferably, wherein divalent cation is 0.01～100:1 with the quantity ratio of univalent cation, is preferably 0.1～20:1;
The amount of the hydrophilic colloid flocculation agent added is 0.0001%～5%, is preferably 0.01%～1%.
According to the specific embodiment of the invention scheme, in the endotoxic method removed in lactoferrin formulations of the present invention, be to wait to remove the aqueous solution that endotoxic lactoferrin formulations is allocated as lactoferrin mass concentration 0.01%～10%.The excessive concentration of lactoferrin, will affect the adsorption effect of salt and colloid induced by endotoxin; The concentration of lactoferrin is too low, and the same treatment ability needs the larger area film, uneconomical.
According to the specific embodiment of the invention scheme, in the endotoxic method removed in lactoferrin formulations of the present invention, after adding flocculation agent in the lactoferrin aqueous solution, stir, place 0.5～5h, carry out again afterwards membrane sepn for 4～60 ℃.
According to the specific embodiment of the invention scheme, in the endotoxic method removed in lactoferrin formulations of the present invention, described membrane sepn can be ultrafiltration or micro-filtration.Preferably: described ultrafiltration condition: the molecular weight cut-off scope 100kDa～2000kDa of ultra-filtration membrane is preferably 300kDa～1000kDa; 5～70 ℃ of temperature, cross film pressure 0.1～1MPa.Described micro-filtration condition: the aperture of microfiltration membrane is 0.1～1.4 μ m; 5～70 ℃ of temperature, cross film pressure 0～0.1Mpa.Film core form is not limited to rolled film, hollow-fibre membrane, flat sheet membrane, tubular membrane, and the film core material can be ceramic, can be also the organic materialss such as polyethersulfone, Mierocrystalline cellulose.Therefore what deserves to be explained is, the molecular weight of the hydrophilic colloid that the present invention adds is significantly higher than lactoferrin, and whole hydrophilic colloids all separates with lactoferrin when membrane sepn operates, and needn't worry can in lactoferrin, sneak into the risk of hydrophilic colloid.
Membrane sepn can remove a part of intracellular toxin in lactoferrin, membrane separation process removes endotoxic degree and depends primarily on endotoxic kind, according to method of the present invention, can remove approximately 40%～99.9% intracellular toxin after membrane sepn, generally, the intracellular toxin decreasing ratio can reach 70%～99.9%.The intracellular toxin be not removed may and lactoferrin between reactive force too strong, for further removing this part intracellular toxin, the endotoxic method removed in lactoferrin formulations of the present invention also can comprise that the lactoferrin aqueous solution to carrying out after membrane sepn carries out affinity chromatography further to remove endotoxic process.
According to the specific embodiment of the invention scheme, be to select polylysine affinity chromatography resin to carry out affinity chromatography.Therefore polylysine has more positive charge, can strong electrostatic force occur with intracellular toxin, and this chromatographic resin is used and regenerates all easylier in addition, is easy to large-scale promotion application, and this affinity chromatography resin is existing commercially produced product at present.
Yet the contriver finds in practical study, although the adsorptive power of polylysine resin induced by endotoxin is very strong, to such an extent as to lactoferrin and endotoxic bonding force are so strongly sometimes to use conventional Parameter Conditions still can not reach well-content effect.For further improving endotoxic removal effect, the present invention is optimized chromatography condition.Wherein, before carrying out affinity chromatography, preferably controlling lactoferrin mass concentration in the lactoferrin aqueous solution for the treatment of chromatography is 0.01%～10%, adjust salt ionic concentration, make the specific conductivity of solution reach 10～200mS/cm, preferably 20～120mS/cm, and to control its pH be 4～9, preferably 5～8.5; And the balance liquid pH that controls affinity chromatography is 4～9, preferably 4.5～8.5.Because lactoferrin mainly combines by electrostatic interaction and hydrophobic interaction with intracellular toxin, the intracellular toxin removed wherein just must reduction even be opened reactive force between the two, known raising ionic strength can reduce electrostatic force, but improved hydrophobic force, therefore visible ionic strength is contrary for electrostatic force with the impact of hydrophobic force, just need between electrostatic force and hydrophobic force, find both minimum trim points of making a concerted effort.And pH is very large for lactoferrin and the impact of endotoxic net charge amount, therefore also two kinds of power are played to important influence undoubtedly.
According to endotoxic content, characteristic in lactoferrin solution after membrane sepn, further before affinity chromatography, readjust the salt ion content and composition and the pH that treat in chromatography sample system in the present invention, and adjusted the pH value of the balance liquid of affinity chromatography.Adjust salt ionic concentration (specifically can adjust ionic concn by adding aforesaid salt), make the specific conductivity of solution reach 10～200mS/cm, preferred 20～120mS/cm, and the pH that controls solution is 4～9, preferably 5～8.5; The balance liquid pH that controls affinity chromatography is 4～9, preferably 4.5～8.5.In addition, the treatment capacity of lactoferrin solution is relevant with endotoxin content wherein, the maximal absorptive capacity of affine resin induced by endotoxin is 2000000EU/mL, the contriver finds that the endotoxin content scope of lactoferrin powder is 0.01～100000EU/mg, after membrane sepn according to endotoxic kind, endotoxin content can reduce by 40%～99.9%, generally, endotoxin content can reduce by 70%～99.9%, after membrane sepn, the endotoxin content of lactoferrin formulations is about 0.00001～60000EU/mg lactoferrin, generally, endotoxin content can drop to 0.00001～30000EU/mg lactoferrin.The used in amounts of resin is carried out choose reasonable according to the endotoxin content in lactoferrin solution.After lactoferrin solution after membrane sepn is adjusted its salt ion content and composition and pH, loading, intracellular toxin in solution is attracted on the affinity chromatography resin and lactoferrin flows through resin, collects stream and wears liquid and obtain further removing endotoxic lactoferrin solution.One of beneficial effect of the affinity chromatography that the present invention is selected is that handled lactoferrin concentration can be higher, and the treatment capacity of lactoferrin is higher.Why there is high like this sample preparation ability, mainly contain two reasons, at first pass through the membrane sepn of previous step, the intracellular toxin of lactoferrin has been removed many, secondly, by choosing suitable ionic environment and pH condition, farthest weaken the bonding force between lactoferrin and intracellular toxin, thereby greatly improved joint efficiency.Therefore affinity chromatography of the present invention also have advantages of resin demand relatively less, investment cost is low, economic performance is high.
According to the specific embodiment of the invention scheme, the endotoxic method removed in lactoferrin formulations of the present invention, applicable to the lactoferrin formulations such as lactoferrin powder are removed to endotoxic processing.The lactoferrin powder first is mixed with lactoferrin solution, after membrane sepn, affinity chromatography remove intracellular toxin, can further the lactoferrin solution after affinity chromatography be carried out to desalting treatment, concentrate drying afterwards, prepare the lactoferrin powder removed after intracellular toxin.
In sum, method of the present invention is mainly to use the rear membrane separation technique of flocculation, and, further combined with the affinity chromatography technology, the intracellular toxin in lactoferrin formulations is removed, and the intracellular toxin decreasing ratio is high, can, up to more than 99%, even can reach more than 99.99%.The operation steps of not mentioning in detail in the present invention, can carry out according to the routine operation in affiliated field or with reference to the specification sheets of instrument equipment.
Embodiment
Below describe the enforcement of technical solution of the present invention and the beneficial effect had in detail by specific embodiment, but but can not regard as any restriction to practical range of the present invention.
Embodiment 1
Material: the lactoferrin powder, endotoxin content is 104EU/mg.
The intracellular toxin removing process:
1) dissolve: 5g lactoferrin powder is dissolved in the 49L pure water, the lactoferrin solution that to be mixed with mass concentration be 0.01%, the quality of lactoferrin of take is benchmark, adds 10% salt, this salt is comprised of zinc sulfate and Repone K, and wherein the mol ratio of zinc sulfate and Repone K is 100:1.Add 5% pectin, mild stirring, to fully dissolving, is placed 4h, intracellular toxin superpolymer and intracellular toxin-pectin aggregation is fully formed for 10 ℃ again.
2) ultrafiltration: the ultrafiltration apparatus of choosing pilot scale, the film core is selected the flat plate ultrafiltration membrane of 2000kDa molecular weight cut-off, the ultrafiltration temperature is 40 ℃, crossing film pressure is 1MPa, partly be and removed the endotoxic lactoferrin solution of part through liquid, when the amount that sees through liquid reaches 30L, add 10L water in trapped fluid, more lactoferrin is entered into through liquid by film.When through liquid, being about 25L, membrane flux significantly descends, and stops ultrafiltration, and all liquid that sees through is merged, and obtains removing the about 55L of the endotoxic lactoferrin solution of part.After membrane sepn is known in detection, the endotoxin content of lactoferrin is 8.55EU/mg, and decreasing ratio is 91.78%.
3) affinity chromatography: the above-mentioned endotoxic lactoferrin solution of part that removed is carried out to affinity chromatography, adding sodium-chlor makes the specific conductivity of lactoferrin solution reach 200mS/cm, adjust pH to 9.0, selecting internal diameter is 5cm, be highly the chromatography column of 40cm, filling 0.5L polylysine affinity chromatography resin.The 10mM phosphate buffered saline buffer that is 6.5 by 5 column volume pH is flowed through chromatography column with the balance resin with the speed of 10mL/min.Add above-mentioned ultrafiltration again and see through the liquid sample, sample loading flow velocity is 100mL/min, and intracellular toxin is attracted on resin and lactoferrin flows through resin, collects stream and wears liquid and obtain removing endotoxic lactoferrin solution.Be taken up in order of priority and with NaOH solution and the 2M NaCl solution of 5 column volume 0.2M, resin regenerated.
4) desalination: above-mentioned lactoferrin solution after affinity chromatography is carried out to the desalination operation with the ultra-filtration membrane of 20kDa, remove the too much salinity wherein contained and be concentrated to 5% mass concentration.
5) lyophilize: the lactoferrin solution lyophilize after desalination and concentration is become to the lactoferrin powder, obtain having removed endotoxic lactoferrin powder.
6) packing storage: cryodesiccated lactoferrin powder is crushed to approximately 50 purpose particles, is packaged in plastics bag.
The intracellular toxin detection method:
Test set is U.S. Charles river LABORATORIES's
the PTS portable tester, the intracellular toxin test strip of subsidiary Charles river company, can accurately do quantitative assay to the endotoxin content in lactoferrin.
Lactoferrin suppresses active detection method to intestinal bacteria:
Bacterial classification: veterinary microorganism DSMZ of e. coli k99 bacterial strain: C83312(country).
Substratum: buffered peptone water, violet red bile agar.
Bacterium solution preparation: the intestinal bacteria of picking slant preservation are on a small quantity in the 10mL peptone water, 37 ℃, 60 turn/min cultivate activation 16h, inoculum size according to 1% activates bacterium liquid by the first-generation and is transferred in new peptone water, and 37 ℃, 60 turn/min cultivate 1h, make bacterial concentration reach 10
6～10
7, 10 times of redilution are standby.
Lactoferrin solution preparation: dissolve lactoferrin with pure water, prepare certain density lactoferrin solution, with 0.2 μ m needle-based membrane filtration lactoferrin solution.
Bacteriostatic experiment: add the lactoferrin solution of 0.4mL2mg/mL in the 8.6mL peptone water, then add 1mL10
5～10
6escherichia coli bacteria liquid, cultivate 24h for 37 ℃ and carry out plate count.
Detected result:
The loss of lactoferrin and intracellular toxin removal effect be in Table 1, and what remove lactoferrin before and after intracellular toxin the results are shown in Table 2 to colibacillary inhibition activity.In table, result is known, and after removing intracellular toxin, the biocidal property of lactoferrin significantly improves.
The rate of loss of table 1 lactoferrin and intracellular toxin decreasing ratio
The lactoferrin that table 2 removes before and after intracellular toxin is active to colibacillary inhibition
| Sample sets | 24h bacterium number | Bacteriostasis rate |
| Contrast | 3.72×10 8 | — |
| Do not remove endotoxic lactoferrin | 3.23×10 8 | 13.17% |
| Remove endotoxic lactoferrin | 7.82×10 7 | 78.98% |
Embodiment 2
Material: the lactoferrin powder, endotoxin content is 104EU/mg.
The intracellular toxin removing process:
1) dissolve: 5g lactoferrin powder is dissolved in the 49L pure water, the lactoferrin solution that to be mixed with mass concentration be 0.01%, the quality of lactoferrin of take is benchmark, add 0.01% salt, this salt is comprised of calcium chloride and ammonium sulfate, and wherein the mol ratio of calcium chloride and ammonium sulfate is that 1:5(is that the mol ratio of calcium ion and ammonium radical ion is 1:10).Add 0.01% pectin, mild stirring, to fully dissolving, is placed 4h, intracellular toxin superpolymer and intracellular toxin-pectin aggregation is fully formed for 10 ℃ again.
2) ultrafiltration: the ultrafiltration apparatus of choosing pilot scale, the film core is selected the flat plate ultrafiltration membrane of 2000kDa molecular weight cut-off, the ultrafiltration temperature is 40 ℃, crossing film pressure is 1MPa, partly be and removed the endotoxic lactoferrin solution of part through liquid, when the amount that sees through liquid reaches 30L, add 10L water in trapped fluid, more lactoferrin is entered into through liquid by film.When through liquid, being about 25L, membrane flux significantly descends, and stops ultrafiltration, and all liquid that sees through is merged, and obtains removing the about 55L of the endotoxic lactoferrin solution of part.After membrane sepn is known in detection, the endotoxin content of lactoferrin is 3.63EU/mg, and decreasing ratio is 96.51%.
3) affinity chromatography: the above-mentioned endotoxic lactoferrin solution of part that removed is carried out to affinity chromatography, adding sodium-chlor makes the specific conductivity of lactoferrin solution reach 20mS/cm, adjust pH to 6.0, selecting internal diameter is 5cm, be highly the chromatography column of 40cm, filling 0.5L polylysine affinity chromatography resin.The 10mM phosphate buffered saline buffer that is 6.5 by 5 column volume pH is flowed through chromatography column with the balance resin with the speed of 10mL/min.Add above-mentioned ultrafiltration again and see through the liquid sample, sample loading flow velocity is 100mL/min, and intracellular toxin is attracted on resin and lactoferrin flows through resin, collects stream and wears liquid and obtain removing endotoxic lactoferrin solution.Be taken up in order of priority and with NaOH solution and the 2M NaCl solution of 5 column volume 0.2M, resin regenerated.
4) desalination: above-mentioned lactoferrin solution after affinity chromatography is carried out to the desalination operation with the ultra-filtration membrane of 20kDa, remove the too much salinity wherein contained and be concentrated to 5% mass concentration.
5) lyophilize: the lactoferrin solution lyophilize after desalination and concentration is become to the lactoferrin powder, obtain having removed endotoxic lactoferrin powder.
6) packing storage: cryodesiccated lactoferrin powder is crushed to approximately 50 purpose particles, is packaged in plastics bag.
Intracellular toxin detection method and to colibacillary inhibiting rate detection method: with embodiment 1.
Detected result:
The loss of lactoferrin and intracellular toxin removal effect be in Table 3, and what remove lactoferrin before and after intracellular toxin the results are shown in Table 4 to colibacillary inhibition activity.In table, result is known, and after removing intracellular toxin, the biocidal property of lactoferrin significantly improves.
The rate of loss of table 3 lactoferrin and intracellular toxin decreasing ratio
Table 4 remove lactoferrin before and after intracellular toxin to colibacillary inhibitions activity
| Sample sets | 24h bacterium number | Bacteriostasis rate |
| Contrast | 3.72×10 8 | — |
| Do not remove endotoxic lactoferrin | 3.23×10 8 | 13.17% |
| Remove endotoxic lactoferrin | 1.72×10 7 | 95.38% |
Embodiment 3
Material: the lactoferrin powder, endotoxin content is 8.65EU/mg.
The intracellular toxin subtractive process:
1) dissolve: 50kg lactoferrin powder is dissolved in the 450L pure water, the lactoferrin solution that to be configured to mass concentration be 10%, the quality of lactoferrin of take is benchmark, add 0.001% salt, this salt is comprised of magnesium chloride, sodium-chlor and ammonium sulfate, and the mol ratio of magnesium chloride, sodium-chlor and ammonium sulfate is that 1:50:25(is that divalent cation is 1:100 with the ratio of univalent cation).Add 0.00005% gelling gum and 0.00005% agar, mild stirring, to fully dissolving, is placed 2h, intracellular toxin superpolymer and intracellular toxin-colloid aggregation thing is fully formed for 25 ℃ again.
2) micro-filtration: the microfiltration equipment of choosing suitable scale, the film core is selected the ceramic microfiltration membrane that aperture is 0.1 μ m, the micro-filtration service temperature is 30 ℃, crossing film pressure is 0.08MPa, partly be and removed the endotoxic lactoferrin solution of part through liquid, when the amount that sees through liquid reaches 450L, add 50L water in trapped fluid, more lactoferrin is entered into through liquid by film.When through liquid, being about 70L, membrane flux significantly descends, and stops ultrafiltration, and all liquid that sees through is merged, and obtains removing the about 520L of the endotoxic lactoferrin solution of part.Learn after testing, the endotoxin content 2.33EU/mg of lactoferrin after micro-filtration, removed 73.06% intracellular toxin.
3) affinity chromatography: the above-mentioned endotoxic lactoferrin solution of part that removed is carried out to affinity chromatography, adding ammonium sulfate makes the electric dodar of lactoferrin solution to 10mS/cm, adjust pH to 4.0, selecting internal diameter is 10cm, be highly the chromatography column of 40cm, filling 2.5L polylysine affinity chromatography resin.The 20mM Tris damping fluid that is 7.5 by 5 column volume pH is flowed through chromatography column with the balance resin with the speed of 40mL/min.Add above-mentioned lactoferrin sample after micro-filtration, sample loading flow velocity is 650mL/min again, and intracellular toxin is attracted on resin and lactoferrin flows through resin, collects stream and wears liquid and obtain removing endotoxic lactoferrin solution.Be taken up in order of priority and with NaOH solution and the 2M NaCl solution of 5 column volume 0.2M, resin regenerated.
4) desalination: above-mentioned lactoferrin solution after affinity chromatography is carried out to the desalination operation with the ultra-filtration membrane of 20kDa, remove the too much salinity wherein contained and be concentrated to 5% mass concentration.
5) lyophilize: the lactoferrin solution lyophilize after desalination and concentration is become to the lactoferrin powder, obtain having removed endotoxic lactoferrin powder.
6) packing storage: cryodesiccated lactoferrin powder is crushed to approximately 50 purpose particles, is packaged in plastics bag.
Intracellular toxin detection method and to colibacillary inhibiting rate detection method: with embodiment 1.
Detected result:
The loss of lactoferrin and intracellular toxin removal effect be in Table 5, and what remove lactoferrin before and after intracellular toxin the results are shown in Table 6 to colibacillary inhibition activity.In table, result is known, and after removing intracellular toxin, the biocidal property of lactoferrin significantly improves.
The rate of loss of table 5 lactoferrin and intracellular toxin decreasing ratio
Table 6 remove lactoferrin before and after intracellular toxin to colibacillary inhibitions activity
| ? | 24h bacterium number | Bacteriostasis rate 24h |
| Control group | 3.72×10 8 | — |
| Do not remove endotoxic lactoferrin group | 1.04×10 8 | 72.04% |
| Remove endotoxic lactoferrin group | 5.33×10 7 | 85.67% |
Embodiment 4
Material: the lactoferrin powder, endotoxin content is 8.65EU/mg.
The intracellular toxin subtractive process:
1) dissolve: 5kg lactoferrin powder is dissolved in the 995L pure water, the lactoferrin solution that to be configured to mass concentration be 0.5%, the quality of lactoferrin of take is benchmark, adds 4% salt, this salt is comprised of magnesium chloride and saltpetre, and the mol ratio of magnesium chloride and saltpetre is 20:1.Add 0.2% sodium alginate and 0.8% xanthan gum, mild stirring, to fully dissolving, is placed 1h, intracellular toxin superpolymer and intracellular toxin-colloid aggregation thing is fully formed for 50 ℃ again.
2) ultrafiltration: the ultrafiltration apparatus of choosing suitable scale, the film core is selected the flat plate ultrafiltration membrane of 400kDa molecular weight cut-off, the ultrafiltration temperature is 20 ℃, crossing film pressure is 0.8MPa, partly be and removed the endotoxic lactoferrin solution of part through liquid, when the amount that sees through liquid reaches 900L, add 100L water in trapped fluid, more lactoferrin is entered into through liquid by film.When through liquid, being about 150L, membrane flux significantly descends, and stops ultrafiltration, all liquid that sees through is merged, obtain removing the about 1050L of the endotoxic lactoferrin solution of part, detect and know that endotoxin content drops to the 0.231EU/mg lactoferrin, decreasing ratio is 97.33%.
3) affinity chromatography: the above-mentioned endotoxic lactoferrin solution of part that removed is carried out to affinity chromatography, add Repone K and make the electric dodar of lactoferrin solution arrive 120mS/cm, adjust pH to 7.5, selecting internal diameter is 10cm, be highly the chromatography column of 60cm, filling 4L polylysine affinity chromatography resin.The 20mM Tris damping fluid that is 6.0 by 5 column volume pH is flowed through chromatography column with the balance resin with the speed of 60mL/min.Add above-mentioned lactoferrin sample after ultrafiltration, sample loading flow velocity is 950mL/min again, and intracellular toxin is attracted on resin and lactoferrin flows through resin, collects stream and wears liquid and obtain removing endotoxic lactoferrin solution.Be taken up in order of priority and with NaOH solution and the 2M NaCl solution of 5 column volume 0.2M, resin regenerated.
4) desalination: above-mentioned lactoferrin solution after affinity chromatography is carried out to the desalination operation with the ultra-filtration membrane of 20kDa, remove the too much salinity wherein contained and be concentrated to 5% mass concentration.
5) lyophilize: the lactoferrin solution lyophilize after desalination and concentration is become to the lactoferrin powder, obtain having removed endotoxic lactoferrin powder.
6) packing storage: cryodesiccated lactoferrin powder is crushed to approximately 50 purpose particles, is packaged in plastics bag.
Intracellular toxin detection method and to colibacillary inhibiting rate detection method: with embodiment 1.
Detected result:
The loss of lactoferrin and intracellular toxin removal effect be in Table 7, and what remove lactoferrin before and after intracellular toxin the results are shown in Table 8 to colibacillary inhibition activity.In table, result is known, and after removing intracellular toxin, the biocidal property of lactoferrin significantly improves.
The rate of loss of table 7 lactoferrin and intracellular toxin decreasing ratio
Table 8 remove lactoferrin before and after intracellular toxin to colibacillary inhibitions activity
| ? | 24h bacterium number | Bacteriostasis rate 24h |
| Control group | 3.72×10 8 | — |
| Do not remove endotoxic lactoferrin group | 1.04×10 8 | 72.04% |
| Remove endotoxic lactoferrin group | 8.3×10 5 | 99.78% |
Embodiment 5
Material: the lactoferrin powder, endotoxin content is 118EU/mg.
The intracellular toxin removing process:
1) dissolve: 1kg lactoferrin powder is dissolved in the 49L pure water, the lactoferrin solution that to be mixed with mass concentration be 2%, the quality of lactoferrin of take is benchmark, add 1% salt, this salt is comprised of calcium chloride and ammonium sulfate, and wherein the mol ratio of calcium chloride and ammonium sulfate is that 2:1(is that the mol ratio of calcium ion and ammonium radical ion is 1:1).Add 0.04% pectin and 0.06% sodium alginate, mild stirring, to fully dissolving, is placed 3h, intracellular toxin superpolymer and intracellular toxin-pectin aggregation is fully formed for 20 ℃ again.
2) ultrafiltration: the ultrafiltration apparatus of choosing pilot scale, the film core is selected the flat plate ultrafiltration membrane of 500kDa molecular weight cut-off, the ultrafiltration temperature is 40 ℃, crossing film pressure is 1MPa, partly be and removed the endotoxic lactoferrin solution of part through liquid, when the amount that sees through liquid reaches 40L, add 10L water in trapped fluid, more lactoferrin is entered into through liquid by film.When through liquid, being about 35L, membrane flux significantly descends, and stops ultrafiltration, and all liquid that sees through is merged, and obtains removing the about 75L of the endotoxic lactoferrin solution of part.After membrane sepn is known in detection, the endotoxin content of lactoferrin is 0.787EU/mg, and decreasing ratio is 99.33%.
3) affinity chromatography: the above-mentioned endotoxic lactoferrin solution of part that removed is carried out to affinity chromatography, adding sodium-chlor makes the specific conductivity of lactoferrin solution reach 75mS/cm, adjust pH to 6.8, selecting internal diameter is 5cm, be highly the chromatography column of 40cm, filling 0.5L polylysine affinity chromatography resin.The 10mM phosphate buffered saline buffer that is 6.5 by 5 column volume pH is flowed through chromatography column with the balance resin with the speed of 10mL/min.Add above-mentioned ultrafiltration again and see through the liquid sample, sample loading flow velocity is 100mL/min, and intracellular toxin is attracted on resin and lactoferrin flows through resin, collects stream and wears liquid and obtain removing endotoxic lactoferrin solution.Be taken up in order of priority and with NaOH solution and the 2M NaCl solution of 5 column volume 0.2M, resin regenerated.
4) desalination: above-mentioned lactoferrin solution after affinity chromatography is carried out to the desalination operation with the ultra-filtration membrane of 20kDa, remove the too much salinity wherein contained and be concentrated to 5% mass concentration.
5) lyophilize: the lactoferrin solution lyophilize after desalination and concentration is become to the lactoferrin powder, obtain having removed endotoxic lactoferrin powder.
6) packing storage: cryodesiccated lactoferrin powder is crushed to approximately 50 purpose particles, is packaged in plastics bag.
Intracellular toxin detection method and to colibacillary inhibiting rate detection method: with embodiment 1.
Detected result:
The loss of lactoferrin and intracellular toxin removal effect be in Table 9, and what remove lactoferrin before and after intracellular toxin the results are shown in Table 10 to colibacillary inhibition activity.
The rate of loss of table 9 lactoferrin and intracellular toxin decreasing ratio
Table 10 remove lactoferrin before and after intracellular toxin to colibacillary inhibitions activity
| Sample sets | 24h bacterium number | Bacteriostasis rate |
| Contrast | 3.72×10 8 | — |
| Do not remove endotoxic lactoferrin | 3.63×10 8 | 2.42% |
| Remove endotoxic lactoferrin | 2.55×10 5 | 99.93% |
In table, result is known, and after removing intracellular toxin, the biocidal property of lactoferrin significantly improves.
Claims (10)
1. the endotoxic method removed in lactoferrin formulations, the method comprises:
To wait to remove endotoxic lactoferrin formulations and be allocated as the lactoferrin aqueous solution, add flocculation agent wherein so that the intracellular toxin in it is converted into superpolymer, carry out membrane sepn, and separate and remove intracellular toxin.
2. the endotoxic method removed in lactoferrin formulations according to claim 1, wherein, described flocculation agent is salt and hydrophilic colloid;
Preferably, described salt is to be selected from Na by least one
+, K
+, Ca
2+, Mg
2+, Cu
2+, Zn
2+, Fe
2+, Fe
3+, Al
3+, NH
4 +in positively charged ion be selected from Cl
﹣, SO
4 2 ﹣, PO
4 3 ﹣, NO
3 -, one or more in forming of the negatively charged ion in citrate ion; Preferably, described salt is selected from calcium chloride, sodium-chlor, magnesium chloride, ammonium sulfate, sodium sulfate, saltpetre; One or more in ammonium sulfate, magnesium chloride and calcium chloride particularly preferably;
Described hydrophilic colloid is selected from one or more in gelling gum, gelatin, pectin, xanthan gum, carrageenin, carboxymethyl cellulose, methylcellulose gum, guar gum, sodium alginate, Viscogum BE, gum arabic, agar, modified starch; Preferably, described hydrophilic colloid is selected from one or more in pectin, carrageenin, xanthan gum and sodium alginate.
3. the endotoxic method removed in lactoferrin formulations according to claim 1, wherein, be to wait to remove the aqueous solution that endotoxic lactoferrin formulations is allocated as lactoferrin mass concentration 0.01%～10%.
4. the endotoxic method removed in lactoferrin formulations according to claim 2, wherein, the quality of lactoferrin in the aqueous solution of take is benchmark:
The amount of the saline flocculating agent added is 0.001%～10%, is preferably 0.01%～4%; More preferably, wherein divalent cation is 0.01～100:1 with the quantity ratio of univalent cation, is preferably 0.1～20:1;
The amount of the hydrophilic colloid flocculation agent added is 0.0001%～5%, is preferably 0.01%～1%.
5. the endotoxic method removed in lactoferrin formulations according to claim 1, wherein, after adding flocculation agent in the lactoferrin aqueous solution, stir, and places 0.5～5h, carry out afterwards membrane sepn again for 4～60 ℃.
6. the endotoxic method removed in lactoferrin formulations according to claim 1, wherein, described membrane sepn is ultrafiltration or micro-filtration; Preferably:
Described ultrafiltration condition: the molecular weight cut-off scope 100kDa～2000kDa of ultra-filtration membrane is preferably 300kDa～1000kDa; 5～70 ℃ of temperature, cross film pressure 0.1～1MPa;
Described micro-filtration condition: the aperture of microfiltration membrane is 0.1～1.4 μ m; 5～70 ℃ of temperature, cross film pressure 0～0.1Mpa.
7. the endotoxic method removed in lactoferrin formulations according to claim 1, the method also comprises that the lactoferrin aqueous solution to carrying out after membrane sepn carries out affinity chromatography further to remove endotoxic process.
8. the endotoxic method removed in lactoferrin formulations according to claim 6, wherein, select polylysine affinity chromatography resin to carry out affinity chromatography.
9. according to the described endotoxic method removed in lactoferrin formulations of claim 7 or 8, wherein, before carrying out affinity chromatography, control treats that in the lactoferrin aqueous solution of chromatography, the lactoferrin mass concentration is 0.01%～10%, adjust salt ionic concentration, make the specific conductivity of solution reach 10～200mS/cm, preferred 20～120mS/cm, and to control its pH be 4～9; And the balance liquid pH that controls affinity chromatography is 4～9.
10. the endotoxic method removed in lactoferrin formulations according to claim 1, wherein, described lactoferrin formulations is the lactoferrin powder.
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| CN105251049A (en) * | 2015-09-29 | 2016-01-20 | 浙江星月生物科技股份有限公司 | Ion buffer solution for removing bacterial endotoxin in biomedical materials and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020130082A1 (en) * | 2001-03-14 | 2002-09-19 | Chisso Corporation | Endotoxin adsorbent, and a method of removing endotoxin by using the same |
| US20060093594A1 (en) * | 2004-03-03 | 2006-05-04 | En-N-Tech, Inc. | Treatments for contaminant reduction in lactoferrin preparations and lactoferrin-containing compositions |
-
2013
- 2013-09-12 CN CN201310414201.1A patent/CN103467597B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020130082A1 (en) * | 2001-03-14 | 2002-09-19 | Chisso Corporation | Endotoxin adsorbent, and a method of removing endotoxin by using the same |
| US20060093594A1 (en) * | 2004-03-03 | 2006-05-04 | En-N-Tech, Inc. | Treatments for contaminant reduction in lactoferrin preparations and lactoferrin-containing compositions |
Non-Patent Citations (5)
| Title |
|---|
| DAGMAR PETSCH ET AL.: "Endotoxin removal from protein solutions", 《JOURNAL OF BIOTECHNOLOGY》, vol. 76, no. 23, 21 January 2000 (2000-01-21), pages 97 - 119, XP002296265, DOI: doi:10.1016/S0168-1656(99)00185-6 * |
| LIPING LI AND ROBERT G. LUO: "Quantitative Determination of Ca2+ Effects on Endotoxin Removal and Protein Yield in a Two-Stage Ultrafiltration Process", 《SEPARATION SCIENCE AND TECHNOLOGY》, vol. 34, no. 9, 31 December 1999 (1999-12-31) * |
| 屈亚辉: "偶联分子簇功能基内毒素吸附剂的合成及应用", 《中国优秀硕士论文电子期刊网》, 15 September 2011 (2011-09-15), pages 1 - 75 * |
| 王健等: "超滤技术在蛋白质分离纯化中的应用", 《药学进展》, vol. 36, no. 3, 25 March 2012 (2012-03-25), pages 116 - 122 * |
| 秦峰等: "生物医药制剂中内毒素去除研究进展", 《药物生物技术》, vol. 10, no. 1, 30 April 2003 (2003-04-30), pages 53 - 56 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105251049A (en) * | 2015-09-29 | 2016-01-20 | 浙江星月生物科技股份有限公司 | Ion buffer solution for removing bacterial endotoxin in biomedical materials and application |
| CN105251049B (en) * | 2015-09-29 | 2018-04-10 | 浙江星月生物科技股份有限公司 | The ion buffer solution of bacterial endotoxin and application in a kind of removal bio-medical material |
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