CN105177097A - Preparation method and application of lactoferricin for promoting proliferative activity of bone cells - Google Patents
Preparation method and application of lactoferricin for promoting proliferative activity of bone cells Download PDFInfo
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- CN105177097A CN105177097A CN201510689506.2A CN201510689506A CN105177097A CN 105177097 A CN105177097 A CN 105177097A CN 201510689506 A CN201510689506 A CN 201510689506A CN 105177097 A CN105177097 A CN 105177097A
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Abstract
The invention relates to a preparation method and an application of lactoferricin for promoting the proliferative activity of bone cells, and relates to a preparation method of bioactive peptide, which solves the problem that no method for extracting protein peptide promoting the proliferative activity of the bone cell from lactoferrin is provided. The preparation method comprises the following steps: I, mixing lactoferrin and deionized water, and adjusting the pH; II, adding pepsin into a lactoferrin solution, and carrying out the water-bath reaction; III, filtering, then separating and purifying by adopting a cation exchange column, and then dialyzing and freeze drying; IV, filtering, and purifying by adopting a gel filter chromatographic column; and V, finally drying to obtain the lactoferricin. The cow milk lactoferrin is used as a raw material, so that the prepared lactoferricin has a good effect for promoting the proliferation of the bone cells. The prepared lactoferricin can be applied to the healthcare food or functional food.
Description
Technical field
The present invention relates to a kind of preparation method of biologically active peptides, be specifically related to a kind of preparation method and the application that take Bovinelactoferrin as raw material preparation and there is the lactoferricin promoting activity of osteoblast proliferation.
Background technology
Osteoporosis is a kind of common multiple disease in China, and it is to the elderly, and especially the healthy of postmenopausal women causes very serious impact.Osteoporosis can cause the multiple complications such as fracture chest deformity, not only makes sufferers themselves endure ailing torment to the fullest extent, patient and family also can be made to bear heavy life and economic pressures.It is expected that, will 2.8 hundred million be increased to the year two thousand twenty China osteoporosis and Low BMD patient numbers.National extensive epidemiology survey also shows, and domestic osteoporosis total prevalence rate is 12.4%, and the ill ratio of the elderly is more than 50%.Serious threat the elderly is healthy for osteoporosis, and therefore, the functional foodstuff exploitation of associated with osteoporosis control has become the hot issue of current food circle.
Lactoferrin (Lactoferrin, LF) a kind ofly has the glycoprotein turning iron function, and molecular size range is about 78kDa, is mainly present in whey-protein, is the important functional ingredient in Ruzhong.Current research shows, lactoferrin can promote the anabolism of bone under physiological concentration, LF can induce Primary osteoblast cells vitro differentiation, also the propagation of scleroblast system can be promoted, but also can as survival factors, prevent the TNF-a Induced Apoptosis in Osteoblasts because serum deprivation causes, by suppressing the growth of osteoclast with activity thus suppressing bone resorption, therefore LF can as a kind of potential osteoporosis therapy agent and the Bone Defect Repari factor.
Lactoferrin, after being taken in by human body, must absorb through pipe intestinal digesting.Modern biotechnology metabolism research shows, the protein of human intake through the multiple enzyme of digestive tube as after the hydrolysis such as stomach en-, trypsinase, pancreatic amylase, steapsase, ereptase, not only absorb with amino acid whose form, be more directly absorb with the form of little peptide, and the biological activity of the little peptide of lactoferrin is possibly far above whole lactoferrin molecules.And the research at present to lactoferrin biologically active peptides, mainly concentrate on the anti-microbial activity of lactoferricin, antiviral activity and antihypertensive function, but to promoting that the lactoferricin of activity of osteoblast proliferation does not have report.So, research and develop a kind of lactoferricin with higher promotion activity of osteoblast proliferation and there are good development and application potentiality.
Lactoferrin is as functional ingredient important in cow's milk, more to the research of its biologically active peptides, and at present, the methods for the treatment of of osteoporosis is more, but still based on pharmacological agent.Conventional medicine has bone resorption inhibitor, bone formation-promoter and bone mineralizer.Bone resorption inhibitor is the medicine mainly for osteoclast, reduces the heavily absorption of bone by suppressing the activity of osteoclast; Bone formation-promoter is then mainly for osteoblastic medicine, can strengthen osteoblastic activity, promotes the synthesis of new bone; Mineralizer is the osteoporotic basic medication for the treatment of, comprises calcium agent and vitamins D, can play the effect of supplementary bone matrix composition.But the osteoporotic medicine overwhelming majority of Current therapeutic is bone resorption inhibitor (as oestrogenic hormon, diphosphonate, thyrocalcitonin etc.), and the kind of bone formation-promoter is considerably less.And have and promote that the lactoferricin of activity of osteoblast proliferation can be used as a kind of potential bone formation-promoter and is used for the treatment of and preventing osteoporosis agent.So, lactoferrin is promoted that the research and development of activity of osteoblast proliferation peptide are with a wide range of applications.
Summary of the invention
The object of the invention is existingly to lack the method for protein peptide obtaining from lactoferrin and promote activity of osteoblast proliferation to solve, thus provide a kind of there is lactoferricin promoting proliferation and differentiation of osteoblasts activity and preparation method thereof.
The preparation method that the present invention has the lactoferricin of promotion activity of osteoblast proliferation realizes according to the following steps:
One, lactoferrin is mixed with deionized water, be prepared into the lactoferrin aqueous solution that mass concentration is 2% ~ 8%, then use pH to 1.2 ~ 2.5 of HCl regulation system, obtain lactoferrin solution;
Two, stomach en-is added in the lactoferrin solution that step one obtains, ice bath cooling after 35 DEG C ~ 40 DEG C water-bath 3 ~ 10min, add pH to 6 ~ 7 termination reaction of alkaline conditioner regulation system, then centrifugal treating obtains supernatant liquor, obtains the enzymolysis solution of lactoferrin bioactive peptide after desalination;
Three, the enzymolysis solution of lactoferrin bioactive peptide is carried out membrane filtration, then by SPSepharoseFastFlow cationic exchange column separating purification, adopt buffered soln A and buffered soln B linear gradient elution, determined wavelength is 215nm and 280nm, collect the elution fraction at the 5th peak, freeze-drying after dialysis desalination, obtains the lactoferrin bioactive peptide of initial gross separation purifying again;
Four, be mixed with by the lactoferrin bioactive peptide that initial gross separation is purified by deionized water the lactoferrin bioactive peptide solution that concentration is 1 ~ 3mg/mL, after membrane filtration, adopt SuperdexPeptide10/300GL gel permeation chromatography post to carry out purifying, adopt buffered soln A wash-out, determined wavelength is 215nm and 280nm, collect the elution fraction at the 1st peak, after ultrafiltration and concentration, obtain lactoferrin peptide solution;
Five, by vacuum lyophilization or low temperature spray drying, drying treatment is carried out to lactoferrin peptide solution, obtain that there is the lactoferricin promoting activity of osteoblast proliferation;
Wherein said buffered soln A is the phosphate buffer soln (PBS) of pH=7.0 ~ 7.5, in buffered soln A, add NaCl, obtains the buffered soln B containing 0.5 ~ 2mol/LNaCl.
What the present invention prepared have promotes that the application of the lactoferricin of activity of osteoblast proliferation this is had to promote that the lactoferricin of activity of osteoblast proliferation is applied in protective foods or functional foodstuff as functional component.
The present invention has the preparation method of the lactoferricin promoting activity of osteoblast proliferation, take Bovinelactoferrin as raw material, stomach en-is utilized to carry out the digestion process of certain hour to Bovinelactoferrin, make the thick product of lactoferricin having and promote activity of osteoblast proliferation, through cation-exchange chromatography and gel chromatography, separation and purification is carried out to lactoferricin again, and adopt vacuum lyophilization or low temperature spray drying method to carry out the preparation of the finished product.The present invention has great importance to the exploitation of lactoferricin in functional foodstuff or preventing osteoporosis medicine.
Compared with prior art, the present invention has and promotes that the preparation method of lactoferricin of activity of osteoblast proliferation comprises following beneficial effect:
1, the method preparing Bovine lactoferricin described in the present invention is simple, safety, can be mass-produced, and the lactoferricin produced has the activity of the promotion osteoblastic proliferation higher than complete lactoferrin molecules.
2, the present invention adopts the method for cation-exchange chromatography gradient elution, isolates highly active lactoferricin in conjunction with the method such as ultrafiltration and concentration, gel permeation chromatography.
3, Bovinelactoferrin involved in the present invention is edible natural composition, harmless, scleroblast is had to the effect of its propagation of good promotion, can be used as a kind of osteoporosis treatment agent, possess the application prospect of actual development.
Accompanying drawing explanation
Fig. 1 is the elution curve of embodiment one cation-exchange chromatography, 1-absorbancy, 2-specific conductivity;
Fig. 2 is the elution curve of embodiment one gel permeation chromatography;
Fig. 3 is the histogram of lactoferricin to activity of osteoblast proliferation of different concns.
Embodiment
Embodiment one: the preparation method that present embodiment has the lactoferricin of promotion activity of osteoblast proliferation realizes according to the following steps:
One, lactoferrin is mixed with deionized water, be prepared into the lactoferrin aqueous solution that mass concentration is 2% ~ 8%, then use pH to 1.2 ~ 2.5 of HCl regulation system, obtain lactoferrin solution;
Two, stomach en-is added in the lactoferrin solution that step one obtains, ice bath cooling after 35 DEG C ~ 40 DEG C water-bath 3 ~ 10min, add pH to 6 ~ 7 termination reaction of alkaline conditioner regulation system, then centrifugal treating obtains supernatant liquor, obtains the enzymolysis solution of lactoferrin bioactive peptide after desalination;
Three, the enzymolysis solution of lactoferrin bioactive peptide is carried out membrane filtration, then by SPSepharoseFastFlow cationic exchange column separating purification, adopt buffered soln A and buffered soln B linear gradient elution, determined wavelength is 215nm and 280nm, collect the elution fraction at the 5th peak, freeze-drying after dialysis desalination, obtains the lactoferrin bioactive peptide of initial gross separation purifying again;
Four, be mixed with by the lactoferrin bioactive peptide that initial gross separation is purified by deionized water the lactoferrin bioactive peptide solution that concentration is 1 ~ 3mg/mL, after membrane filtration, adopt SuperdexPeptide10/300GL gel permeation chromatography post to carry out purifying, adopt buffered soln A wash-out, determined wavelength is 215nm and 280nm, collect the elution fraction at the 1st peak, after ultrafiltration and concentration, obtain lactoferrin peptide solution;
Five, by vacuum lyophilization or low temperature spray drying, drying treatment is carried out to lactoferrin peptide solution, obtain that there is the lactoferricin promoting activity of osteoblast proliferation;
Wherein said buffered soln A is the phosphate buffer soln (PBS) of pH=7.0 ~ 7.5, in buffered soln A, add NaCl, obtains the buffered soln B containing 0.5 ~ 2mol/LNaCl.
Alkaline conditioner described in present embodiment is NaOH solution, sodium hydrogen carbonate solution or potassium bicarbonate solution.
Embodiment two: stomach en-to add in the lactoferrin solution that step one obtains unlike step 2 by present embodiment and embodiment one, makes the ratio of enzyme activity and lactoferrin be 100U/g ~ 250U/g.Other step and parameter identical with embodiment one.
Embodiment three: present embodiment and embodiment one or two unlike step 2 at 37 DEG C of water-bath 5min.Other step and parameter identical with embodiment one or two.
Embodiment four: one of present embodiment and embodiment one to three obtain supernatant liquor unlike step 2 with the centrifugation process of 5000 ~ 10000rpm.Other step and parameter identical with one of embodiment one to three.
Embodiment five: one of present embodiment and embodiment one to four are 0.1 μm or 0.22 μm unlike the aperture of the membrane filtration described in step 3.Other step and parameter identical with one of embodiment one to four.
Embodiment six: one of present embodiment and embodiment one to five are as follows unlike the cationic exchange column separating purification process described in step 3: first use buffered soln A wash-out 3 ~ 7 column volumes (CV), use buffered soln B wash-out 3 ~ 5 column volumes again, it is 3 ~ 7mL/min that elution flow rate controls.Other step and parameter identical with one of embodiment one to five.
Embodiment seven: it is as follows that one of present embodiment and embodiment one to six carry out purge process unlike the gel permeation chromatography described in step 4: first use buffered soln A to balance, and then using buffered soln A wash-out 1 ~ 3 column volume, elution flow rate is 0.2 ~ 1mL/min.Other step and parameter identical with one of embodiment one to six.
Embodiment eight: one of present embodiment and embodiment one to seven are adopt molecular weight cut-off to be that the regenerated cellulose film of 500D concentrates unlike the ultrafiltration and concentration described in step 4.Other step and parameter identical with one of embodiment one to seven.
Embodiment nine: one of present embodiment and embodiment one to eight are-73 ~-40 DEG C unlike controlling condenser temperature in the vacuum lyophilization process described in step 5, and vacuum tightness is 0.01 ~ 0.5mbar, and time of drying is 3 ~ 24h.Other step and parameter identical with one of embodiment one to eight.
Embodiment ten: the solid quality content range that one of present embodiment and embodiment one to nine control in lactoferrin peptide solution unlike the low temperature spray drying described in step 5 is 15 ~ 40%, vacuum tightness reaches-0.01 ~-0.06MPa, inlet temperature is 95 ~ 145 DEG C, and temperature of charge controls at 30 ~ 60 DEG C.Other step and parameter identical with one of embodiment one to nine.
Embodiment 11: what embodiment one prepared by present embodiment have promotes that the lactoferricin of activity of osteoblast proliferation is applied in protective foods or functional foodstuff as functional component.
Embodiment one: the preparation method that the present embodiment has the lactoferricin of promotion activity of osteoblast proliferation realizes according to the following steps:
One, lactoferrin is mixed with deionized water, be prepared into the lactoferrin aqueous solution that mass concentration is 5% (w/v), then use the pH to 2.0 of HCl regulation system, obtain lactoferrin solution;
Two, added to by 26.1mg stomach en-in the lactoferrin solution that step one obtains, make the ratio of enzyme activity and lactoferrin be 238U/g, after 37 DEG C of water-bath 5min, ice bath cooling, adds NaHCO
3the pH=6.5 termination reaction of solution regulation system, then obtains supernatant liquor with the centrifugation process of 10000rpm, obtains the enzymolysis solution of lactoferrin bioactive peptide after dialysis desalination;
Three, protein concn in the enzymolysis solution of adjustment lactoferrin bioactive peptide is 2mg/mL, through 0.22 μm of membrane filtration, then by SPSepharoseFastFlow cationic exchange column separating purification, ion exchange column uses the PBS solution (buffered soln A) of pH=7.4 to balance in advance, adopt linear gradient elution, the albumen be attached on resin utilizes 50mmol/LPBS buffered soln A wash-out 5 column volumes (CV), again with buffered soln B wash-out 5 column volumes containing 1mol/LNaCl, elution flow rate is 5mL/min, determined wavelength is 215nm and 280nm, collect the elution fraction at the 5th peak (peak 5 in Fig. 1), freeze-drying after dialysis desalination again, obtain the lactoferrin bioactive peptide of initial gross separation purifying,
Four, the lactoferrin bioactive peptide solution that concentration is 2mg/mL is mixed with by the lactoferrin bioactive peptide that initial gross separation is purified by deionized water, after 0.22 μm of membrane filtration, adopt SuperdexPeptide10/300GL gel permeation chromatography post to carry out purifying, gel column balances with the buffered soln A of pH=7.6 in advance, sample introduction after baseline is steady, and then utilize buffered soln A wash-out 2 column volumes of pH=7.6, elution flow rate is 0.5mL/min, determined wavelength is 215nm and 280nm, collect the elution fraction of the 1st peak (the peak 5-1 in Fig. 2), adopt molecular weight cut-off be the regenerated cellulose film of 500D carry out concentrated after obtain lactoferrin peptide solution,
Five, by vacuum lyophilization, drying treatment is carried out to lactoferrin peptide solution, control condenser temperature-60 DEG C, vacuum tightness 0.01mbar, time 16h, obtain that there is the lactoferricin promoting activity of osteoblast proliferation.
The purity of the lactoferrin that the present embodiment step one uses is greater than 95%.
The Activity determination of the lactoferrin peptide product that the present embodiment is prepared, as shown in the peak 5-1 in accompanying drawing 3, illustrates that it has good short activity of osteoblast proliferation.
For having of preparing, the present embodiment promotes that the test mode of the lactoferricin of activity of osteoblast proliferation is as follows:
(1) peptic activity of stomach measures:
Adopt Forint phenol method to measure peptic activity of stomach, carry out according to GB/T28715-2012.
(2) mensuration of protein content:
Coomassie Brilliant Blue (Bradford) is adopted to measure protein concentration.
(3) mensuration of free amine group content:
O-phthalaldehyde(OPA) OPA method is adopted to measure the content of free amine group.
(4) protein degree calculates
Protein degree
In formula: Y
1the free amine group content (mmol/g) of sample after-hydrolysis;
Y
2the free amine group content (mmol/g) of sample before-hydrolysis;
Contained peptide linkage content (mmol/g) in 8.658-lactoferrin.
(5) lactoferricin molecular weight determination:
SDS-PAGE electrophoresis and TricineSDSPAGE gel electrophoresis is adopted to measure lactoferrin peptide molecular weight.
(6) sample activity measures:
It is active that mtt assay measures lactoferricin:
Experiment is grouped as follows: lactoferrin peptide solution lyophilized powder being formulated as respectively low dosage concentration group (20 μ g/mL), middle dose concentration group (100 μ g/mL) and high dosage concentration group (500 μ g/mL), using water as blank.Often organize 6 holes, by scleroblast digestion counting, cell concn is adjusted to l × 105, every hole/mL, then cell is inoculated in 96 well culture plates.Detect respectively after cultivation 24h, 48h and 72h.
Testing index is as follows: every 24h, 48h and 72h, measures ability of cell proliferation.Absorb nutrient solution, repeatedly clean removing dead cell with PBS; Every hole adds the α-MEM100 μ L containing 0.5%MTT, in 37 DEG C, and 5%CO
24h is hatched in incubator; Stop hatching after black reticulation appears in observation, discard nutrient solution; Every hole adds 150 μ LDMSO, measures its light absorption value, represent by OD value after vibration 10min with enzyme-linked immunosorbent assay instrument at wavelength 490nm place.
In this embodiment, LF product acts on OD value corresponding to scleroblast 24h, 48h and 72h is 0.31,0.37 and 0.41 respectively, and product purity (protein content) is greater than 97%.
Embodiment two: the present embodiment and embodiment one carry out drying treatment by low temperature spray drying to lactoferrin peptide solution unlike step 5, solid content in active polypeptide solution is 30%, vacuum tightness reaches for-0.01MPa, inlet temperature is 120 DEG C, input speed adjusts according to case of machines, temperature of charge controls at 40 DEG C, obtains having the lactoferricin promoting activity of osteoblast proliferation.
What the present embodiment obtained have promotes that the OD value of the lactoferricin of activity of osteoblast proliferation is 0.31,0.38 and 0.42 respectively, shows that it has good short activity of osteoblast proliferation.
Claims (10)
1. there is a preparation method for the lactoferricin promoting activity of osteoblast proliferation, it is characterized in that following these steps to realize:
One, lactoferrin is mixed with deionized water, be prepared into the lactoferrin aqueous solution that mass concentration is 2% ~ 8%, then use pH to 1.2 ~ 2.5 of HCl regulation system, obtain lactoferrin solution;
Two, stomach en-is added in the lactoferrin solution that step one obtains, ice bath cooling after 35 DEG C ~ 40 DEG C water-bath 3 ~ 10min, add pH to 6 ~ 7 termination reaction of alkaline conditioner regulation system, then centrifugal treating obtains supernatant liquor, obtains the enzymolysis solution of lactoferrin bioactive peptide after desalination;
Three, the enzymolysis solution of lactoferrin bioactive peptide is carried out membrane filtration, then by SPSepharoseFastFlow cationic exchange column separating purification, adopt buffered soln A and buffered soln B linear gradient elution, determined wavelength is 215nm and 280nm, collect the elution fraction at the 5th peak, freeze-drying after dialysis desalination, obtains the lactoferrin bioactive peptide of initial gross separation purifying again;
Four, be mixed with by the lactoferrin bioactive peptide that initial gross separation is purified by deionized water the lactoferrin bioactive peptide solution that concentration is 1 ~ 3mg/mL, after membrane filtration, adopt SuperdexPeptide10/300GL gel permeation chromatography post to carry out purifying, adopt buffered soln A wash-out, determined wavelength is 215nm and 280nm, collect the elution fraction at the 1st peak, after ultrafiltration and concentration, obtain lactoferrin peptide solution;
Five, by vacuum lyophilization or low temperature spray drying, drying treatment is carried out to lactoferrin peptide solution, obtain that there is the lactoferricin promoting activity of osteoblast proliferation;
Wherein said buffered soln A is the phosphate buffer soln of pH=7.0 ~ 7.5, in buffered soln A, add NaCl, obtains the buffered soln B containing 0.5 ~ 2mol/LNaCl.
2. a kind of preparation method with the lactoferricin promoting activity of osteoblast proliferation according to claim 1, it is characterized in that stomach en-adds in the lactoferrin solution that step one obtains by step 2, make the ratio of enzyme activity and lactoferrin be 100U/g ~ 250U/g.
3. a kind of preparation method with the lactoferricin promoting activity of osteoblast proliferation according to claim 1, is characterized in that step 2 obtains supernatant liquor with the centrifugation process of 5000 ~ 10000rpm.
4. a kind of preparation method with the lactoferricin promoting activity of osteoblast proliferation according to claim 1, is characterized in that the aperture of the membrane filtration described in step 3 is 0.1 μm or 0.22 μm.
5. a kind of preparation method with the lactoferricin promoting activity of osteoblast proliferation according to claim 1, it is characterized in that the cationic exchange column separating purification process described in step 3 is as follows: first use buffered soln A wash-out 3 ~ 7 column volumes, use buffered soln B wash-out 3 ~ 5 column volumes again, it is 3 ~ 7mL/min that elution flow rate controls.
6. a kind of preparation method with the lactoferricin promoting activity of osteoblast proliferation according to claim 1, it is as follows that the gel permeation chromatography that it is characterized in that described in step 4 carries out purge process: first use buffered soln A to balance, and then using buffered soln A wash-out 1 ~ 3 column volume, elution flow rate is 0.2 ~ 1mL/min.
7. according to claim 1 a kind ofly have the preparation method of lactoferricin promoting activity of osteoblast proliferation, and the ultrafiltration and concentration that it is characterized in that described in step 4 adopts molecular weight cut-off to be that the regenerated cellulose film of 500D concentrates.
8. a kind of preparation method with the lactoferricin promoting activity of osteoblast proliferation according to claim 1, it is characterized in that controlling condenser temperature in the vacuum lyophilization process described in step 5 is-73 ~-40 DEG C, vacuum tightness is 0.01 ~ 0.5mbar, and time of drying is 3 ~ 24h.
9. a kind of preparation method with the lactoferricin promoting activity of osteoblast proliferation according to claim 1, it is characterized in that the solid quality content range that the low temperature spray drying described in step 5 controls in lactoferrin peptide solution is 15 ~ 40%, vacuum tightness reaches-0.01 ~-0.06MPa, inlet temperature is 95 ~ 145 DEG C, and temperature of charge controls at 30 ~ 60 DEG C.
10. there is the application of the lactoferricin promoting activity of osteoblast proliferation, it is characterized in that the lactoferricin this with promotion activity of osteoblast proliferation is applied in protective foods or functional foodstuff as functional component.
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| CN111955601A (en) * | 2020-08-31 | 2020-11-20 | 镇江市天益生物科技有限公司 | Preparation method and application of lactoferrin peptide enzymatic hydrolysate with antibacterial activity |
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| CN111955601A (en) * | 2020-08-31 | 2020-11-20 | 镇江市天益生物科技有限公司 | Preparation method and application of lactoferrin peptide enzymatic hydrolysate with antibacterial activity |
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