CN108949885A - A kind of pair of osteoblastic proliferation has the preparation method and application of the pigskin gelatin peptide of facilitation - Google Patents
A kind of pair of osteoblastic proliferation has the preparation method and application of the pigskin gelatin peptide of facilitation Download PDFInfo
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Abstract
A kind of pair of osteoblastic proliferation has the preparation method of the pigskin gelatin peptide of facilitation, belongs to the preparation field of biologically active peptide.The preparation method is as follows: (1) weighs pigskin gelatin, pigskin gelatin solution is prepared；(2) protease is added into pigskin gelatin solution carry out enzyme hydrolysis；(3) heat inactivation protease terminates hydrolysis, cooling, is centrifuged and pigskin gelatin peptide solution is made；(4) vacuum freeze drying pigskin gelatin peptide solution is made with the pigskin gelatin peptide to osteoblastic proliferation with facilitation.The pigskin gelatin peptide of this method preparation, has the advantages that edibility, safety and is easily absorbed by the body, from a wealth of sources, at low cost, preparation method is simple, can be mass-produced；Prepared pigskin gelatin peptide has facilitation to the proliferation of osteoblast, can have the functional food for preventing or treating osteoporosis effect or health care product to provide new raw material to produce.
Description
Technical field
The invention belongs to the preparation fields of biologically active peptide, are related to the pig that a kind of pair of osteoblastic proliferation has facilitation
The preparation method and application of skin gelatin peptide.
Background skill is stated
Osteoporosis is that a kind of bone amount reduction, bone tissue microstructure degeneration, bone brittleness increase and is prone to fracture
A kind of skeletal diseases, be a kind of degenerative osteopathy related with aging, principal pathogenetic crowd is the middle-aged and the old, especially menopause
Women afterwards.According to statistics, the patients with osteoporosis at present in worldwide reaches about 2.5 hundred million.Old more than 50 years old
In year crowd, women sufferers of osteoporosis face proportion is up to 1/3, and osteoporosis patient proportion is then up to 1/5.I
The morbidity and mortality of state's osteoporosis are all higher than western developed country.Currently, clinically mostly using estrogen, two banks
The drugs such as salt and calcitonin prevent and treat osteoporosis, but the long-term use of these drugs, usually can be with toxic
Side effect.Therefore, it is quite necessary to the function of preparing a kind of natural origin, have no toxic side effect and have positive effect to bone health
Property food or health care product.
Gelatin is the derived product of animal collagen, is mainly derived from the groups such as terrestrial animal, the skin of aquatic livestock, bone
It knits, especially pigskin, pig bone.Gelatin hydrolysied matter is also known as collagen polypeptide, be gelatin under the action of protease, by a certain degree
Hydrolysis after obtained from derivative products.The structure of separate sources gelatin is different, and the protease hydrolytic ability of separate sources is different,
Therefore the structure and function that will lead to gelatin peptide is variant.In general, the molecular mass of gelatin peptide is low, it is highly soluble in water, it is easier to
It is absorbed by the body.
Result of study shows that gelatin and its hydrolysate have positive effect to the metabolism of bone and health.For example, ovariectomized female rats
After oral collagen hydrolysate, the bone mineral density and content of rat increase；Bovine collagen hydrolysate can promote mature soft
Osteocyte secretes II Collagen Type VI；Gelatin hydrolysied matter can promote the metabolism of bone, promote the update of bone, additionally it is possible to stimulate osteoblast
Differentiation, causes osteocalcin secretion in cell to increase.There is clinical report to claim, Human Osteoarthritis takes 10 grams of collagen water daily
Solution is after object six months, and function of joint is improved, and arthralgia is mitigated.Above example explanation, the gelatin peptide in pigskin source
There is good application prospect in terms of preventing and treating osteoporosis.But the bioactivity of existing pigskin gelatin peptide is opened
Work is sent out, its antioxidation and muscular fatigue effect are focused primarily upon.There is not yet related pigskin gelatin peptide is to osteoblast
The related development of Effect of promoting growth.Therefore, it develops, prepare the pigskin gelatin that a kind of pair of osteoblastic proliferation has facilitation
Peptide has wide application and market prospects.
Summary of the invention
The present invention is for the gelatin peptide lacked in existing active peptide technology of preparing to osteoblastic proliferation with facilitation
Preparation method deficiency, provide a kind of pair of osteoblastic proliferation with facilitation pigskin gelatin peptide preparation method.
To achieve the goals above, the present invention can prepare the pigskin gelatin for having facilitation to osteoblastic proliferation
Peptide, the preparation method comprise the steps of:
Step 1: weighing pigskin gelatin, is configured to gelatin solution with deionized water.
Step 2: papain, neutral proteinase or alkali protease, protease hydrolytic pig are added into gelatin solution
Skin gelatin, regulation system pH value, 40 DEG C at a temperature of hydrolyze 0.25 hour to 15 hours.
Step 3: after hydrolysis, being heated to boiling 15 minutes for hydrating solution immediately, makes protease enzyme deactivation, terminates instead
It answers；After ice-water bath is cooling, centrifugal treating collects supernatant, obtains pigskin gelatin peptide solution.
Step 4: handling pigskin gelatin peptide solution by vacuum freeze drying, saved after constant weight in -20 DEG C, obtain at
Bone cell proliferation has the pigskin gelatin peptide of facilitation.
Preferably, the mass concentration of gelatin solution is 5% (w/w) in step 1.
Preferably, papain is added in step 2 to hydrolyze pigskin gelatin, the additive amount of the papain is 5
Thousand unit of activity/gram protein, regulation system pH value are 6.0, and hydrolysis time is 12.5 hours.
Preferably, neutral proteinase hydrolysis pigskin gelatin is added in step 2, the additive amount of the neutral proteinase is 6,000
Unit of activity/gram protein, regulation system pH value are 7.0, and hydrolysis time is 12.5 hours
Preferably, hydrolysis by novo pigskin gelatin is added in step 2, the additive amount of the alkali protease is 7,000
Unit of activity/gram protein, regulation system pH value are 8.5, and hydrolysis time is 12.5 hours.
Preferably, the centrifugation rate that centrifugal treating collects supernatant in step 3 is 5000 revs/min, centrifugation time 20
Minute.
Preferably, during vacuum freeze drying described in step 4, control vacuum degree is 0.01 millipascal to 0.5 milli
Pa, drying time are 8 hours to 18 hours, and condenser temperature is -72 DEG C to -45 DEG C.
The application field for the pigskin gelatin peptide that the present invention obtains is that it can be used as functional component and is applied to functionality
Food or health food, to promote the proliferation of osteoblast.
The present invention compared with prior art, has the beneficial effect that
(1) pigskin gelatin used in the present invention is common raw-food material, has edibility and safety, to human body
Harmless, abundance is at low cost.
(2) pigskin gelatin peptide according to the present invention, preparation method is simple, safely, can be mass-produced, and it is generated
Pigskin gelatin peptide is easier to be absorbed by the body than pigskin gelatin.
(3) pigskin gelatin peptide according to the present invention has facilitation to osteoblastic proliferation, has prevention for production
Or the functional food or health care product for the treatment of osteoporosis effect provide new raw material.
Detailed description of the invention
Fig. 1 is the flow chart of the pigskin gelatin peptide preparation method to osteoblastic proliferation with facilitation of the invention.
Fig. 2 be when the pigskin gelatin peptide of various concentration acts on osteoblast different time after osteoblastic proliferation is promoted
The histogram of effect.
Specific embodiment
Technical solution of the present invention is described further below by drawings and examples, however, it is not limited to this, all
Modifying or equivalently replacing the technical solution of the present invention, without departing from the spirit and scope of the technical solution of the present invention, should all
Cover within the protection scope of the present invention.
Specific embodiment 1: having the system of the pigskin gelatin peptide of facilitation in present embodiment to osteoblastic proliferation
Preparation Method comprising the steps of:
Step 1: weighing pigskin gelatin, is configured to gelatin solution with deionized water.
Step 2: papain, neutral proteinase or alkali protease, protease hydrolytic pig are added into gelatin solution
Skin gelatin, regulation system pH value, 40 DEG C at a temperature of hydrolyze 0.25 hour to 15 hours.
Step 3: after hydrolysis, being heated to boiling 15 minutes for hydrating solution immediately, makes protease enzyme deactivation, terminates instead
It answers；After ice-water bath is cooling, centrifugal treating collects supernatant, obtains pigskin gelatin peptide solution.
Step 4: handling pigskin gelatin peptide solution by vacuum freeze drying, saved after constant weight in -20 DEG C, obtain at
Bone cell proliferation has the pigskin gelatin peptide of facilitation.
Specific embodiment 2: having the pigskin of facilitation bright osteoblastic proliferation described in specific embodiment one
The preparation method of glue peptide, the mass concentration of gelatin solution is 5% (w/w) in step 1.
Specific embodiment 3: having the pigskin of facilitation bright osteoblastic proliferation described in specific embodiment one
The preparation method of glue peptide, papain hydrolysis pigskin gelatin is added in step 2, and the additive amount of the papain is 5,000
Unit of activity/gram protein, regulation system pH value are 6.0, and hydrolysis time is 12.5 hours.
Specific embodiment 4: having the pigskin of facilitation bright osteoblastic proliferation described in specific embodiment one
The preparation method of glue peptide, neutral proteinase hydrolysis pigskin gelatin is added in step 2, and the additive amount of the neutral proteinase is 6,000
Unit of activity/gram protein, regulation system pH value are 7.0, and hydrolysis time is 12.5 hours.
Specific embodiment 5: having the pigskin of facilitation bright osteoblastic proliferation described in specific embodiment one
The preparation method of glue peptide, hydrolysis by novo pigskin gelatin is added in step 2, and the additive amount of the alkali protease is 7,000
Unit of activity/gram protein, regulation system pH value are 8.5, and hydrolysis time is 12.5 hours.
Specific embodiment 6: having the pigskin of facilitation bright osteoblastic proliferation described in specific embodiment one
The preparation method of glue peptide, it is 5000 revs/min that centrifugal treating, which collects the centrifugation rate of supernatant, in step 3, centrifugation time 20
Minute.
Specific embodiment 7: having the pigskin of facilitation bright osteoblastic proliferation described in specific embodiment one
The preparation method of glue peptide, during vacuum freeze drying described in step 4, control vacuum degree is 0.01 millipascal to 0.5 milli
Pa, drying time are 8 hours to 18 hours, and condenser temperature is -72 DEG C to -45 DEG C.
Promote specific embodiment 8: present embodiment there will be osteoblastic proliferation obtained by specific embodiment one
Pigskin gelatin peptide into effect is applied in functional food or health food as functional component.
Embodiment 1
What the present embodiment was recorded is the preparation method for the pigskin gelatin peptide that a kind of pair of osteoblastic proliferation has facilitation,
The preparation step includes:
Step 1: weighing pigskin gelatin, is configured to the gelatin solution that mass concentration is 5% (w/w) with deionized water.
Step 2: adding papain hydrolysis pigskin gelatin into gelatin solution, and papain dosage is 5,000 living
Unit of force/gram, regulation system pH value be 6.0,40 DEG C at a temperature of hydrolyze 12.5 hours.
Step 3: after hydrolysis, being heated to boiling 15 minutes for hydrating solution immediately, makes protease enzyme deactivation, terminates instead
It answers；After ice-water bath is cooling, 5000 revs/min are centrifuged 20 minutes, are collected supernatant, are obtained pigskin gelatin peptide solution.
Step 4: pigskin gelatin peptide solution is handled by vacuum freeze drying, control vacuum degree is 0.5 millipascal, when dry
Between be 12 hours, condenser temperature is -65 DEG C, is saved after constant weight in -20 DEG C, obtains having facilitation to osteoblastic proliferation
Pigskin gelatin peptide.
The present embodiment performance measurement: by osteoblast according to 5 × 103The density in/hole is inoculated in 96 well culture plates, hungry
Processing 12 hours.Then, be separately added into each hole 0.02 milligram of the gelatin hydrolysied matter of the various concentration being configured to culture medium/
Milliliter, 0.05 mg/ml or 0.1 mg/ml.The processing group of gelatin hydrolysied matter is not added as blank control.Gelatin hydrolysis
After object is handled osteoblast 24 hours, 48 hours and 72 hours, hydrolysate in hole is absorbed, every hole is added 110 microlitres of culture mediums and matches
At concentration be 10% CCK8 solution, again cultivate 1.5 hours.Then, each hole under 450 nano wave lengths is measured with microplate reader
OD value.The activity of cell is calculated according to following cell activity calculation formula.
The pigskin gelatin peptide of 0.05 mg/ml acts on the rush cell Proliferation effect of osteoblast most in the present embodiment
It is good.After 24 hours, 48 hours and 72 hours action time, the activity of osteoblast respectively reaches 119.0%, 134.4% and
124.9%.
Embodiment 2
What the present embodiment was recorded is a kind of preparation method with the pigskin gelatin peptide for promoting osteoblastic proliferation effect, institute
The preparation step stated includes:
Step 1: weighing pigskin gelatin, is configured to the gelatin solution that mass concentration is 5% (w/w) with deionized water.
Step 2: adding neutral proteinase hydrolysis pigskin gelatin into gelatin solution, and neutral proteinase additive amount is 6,000 living
Unit of force/gram, regulation system pH value be 7.0,40 DEG C at a temperature of hydrolyze 12.5 hours.
Step 3: after hydrolysis, being heated to boiling 15 minutes for hydrating solution immediately, makes protease enzyme deactivation, terminates instead
It answers；After ice-water bath is cooling, 5000 revs/min are centrifuged 20 minutes, are collected supernatant, are obtained pigskin gelatin peptide solution.
Step 4: pigskin gelatin peptide solution is handled by vacuum freeze drying, control vacuum degree is 0.5 millipascal, when dry
Between be 12 hours, condenser temperature is -65 DEG C, is saved after constant weight in -20 DEG C, obtains having facilitation to osteoblastic proliferation
Pigskin gelatin peptide.
The present embodiment performance measurement: by osteoblast according to 5 × 103The density in/hole is inoculated in 96 well culture plates, hungry
Processing 12 hours.Be separately added into each hole the various concentration being configured to culture medium 0.02 mg/ml of gelatin hydrolysied matter,
0.05 mg/ml or 0.1 mg/ml.The processing group of gelatin hydrolysied matter is not added as blank control.At gelatin hydrolysied matter
Reason absorbs hydrolysate in hole after osteoblast 24 hours, 48 hours and 72 hours, and every hole is added what 110 microlitres of culture mediums were made into
The CCK8 solution that concentration is 10% is cultivated 1.5 hours again.Then, each hole OD under 450 nano wave lengths is measured with microplate reader
Value.The activity of cell is calculated according to following cell activity calculation formula.
The pigskin gelatin peptide of 0.05 mg/ml acts on the rush cell Proliferation effect of osteoblast most in the present embodiment
It is good.After 24 hours, 48 hours and 72 hours action time, the activity of osteoblast respectively reaches 124.0%, 127.9% and
118.9%.
Embodiment 3
What the present embodiment was recorded is the preparation method for the pigskin gelatin peptide that a kind of pair of osteoblastic proliferation has facilitation,
The preparation step includes:
Step 1: weighing pigskin gelatin, is configured to the gelatin solution that mass concentration is 5% (w/w) with deionized water.
Step 2: adding hydrolysis by novo pigskin gelatin into gelatin solution, and alkali protease additive amount is 7,000 living
Unit of force/gram, regulation system pH value be 8.5,40 DEG C at a temperature of hydrolyze 12.5 hours.
Step 3: after hydrolysis, being heated to boiling 15 minutes for hydrating solution immediately, makes protease enzyme deactivation, terminates instead
It answers；After ice-water bath is cooling, 5000 revs/min are centrifuged 20 minutes, are collected supernatant, are obtained pigskin gelatin peptide solution.
Step 4: pigskin gelatin peptide solution is handled by vacuum freeze drying, control vacuum degree is 0.5 millipascal, when dry
Between be 12 hours, condenser temperature is -65 DEG C, is saved after constant weight in -20 DEG C, obtains having facilitation to osteoblastic proliferation
Pigskin gelatin peptide.
The present embodiment performance measurement: by osteoblast according to 5 × 103The density in/hole is inoculated in 96 well culture plates, hungry
Processing 12 hours.Be separately added into each hole the various concentration being configured to culture medium 0.02 mg/ml of gelatin hydrolysied matter,
0.05 mg/ml or 0.1 mg/ml.The processing group of gelatin hydrolysied matter is not added as blank control.At gelatin hydrolysied matter
Reason absorbs hydrolysate in hole after osteoblast 24 hours, 48 hours and 72 hours, and every hole is added what 110 microlitres of culture mediums were made into
The CCK8 solution that concentration is 10% is cultivated 1.5 hours again.Then, each hole OD under 450 nano wave lengths is measured with microplate reader
Value.The activity of cell is calculated according to following cell activity calculation formula.
The pigskin gelatin peptide of 0.05 mg/ml acts on the rush cell Proliferation effect of osteoblast most in the present embodiment
It is good.After action time 24 hours, 48 hours and 72 hours, the activity of osteoblast respectively reaches 122.7%, 124.0% and
115.9%.
Comparative example 1
Pigskin gelatin peptide is prepared according to the method for embodiment 1 and carries out performance measurement, the difference is that the gelatin hydrolysied matter being added
Replace with culture medium.The activity for measuring osteoblast is 100%.
Comparative example 2
Pigskin gelatin peptide is prepared according to the method for embodiment 1 and carries out performance measurement, the difference is that the gelatin hydrolysied matter being added
Replace with 1 × 10-817 beta estradiols (estrogen that a kind of pair of osteoblastic proliferation has facilitation) of mol/L.17β-
After estradiol acts on 24 hours, 48 hours and 72 hours osteoblast time, the activity of osteoblast is respectively 121.8%,
139.4% and 122.5%.It can be seen that the culture and processing to osteoblast meet requirement of experiment.
It is bright by preparing a kind of pigskin in pigskin gelatin source to can be seen that the present invention from the result of comparative example and embodiment
Glue peptide has facilitation to osteoblastic proliferation, can have the function of preventing or treating osteoporosis effect for exploitation
Property food or health care product provide new raw material.
Claims (8)
1. the preparation method that a kind of pair of osteoblastic proliferation has the pigskin gelatin peptide of facilitation, which is characterized in that described
Preparation method comprises the steps of:
Step 1: weighing pigskin gelatin, is configured to gelatin solution with deionized water；
Step 2: it is bright that papain, neutral proteinase or alkali protease, protease hydrolytic pigskin are added into gelatin solution
Glue, regulation system pH value, 40 DEG C at a temperature of hydrolyze 0.25 hour to 15 hours；
Step 3: after hydrolysis, being heated to boiling 15 minutes for hydrating solution immediately, makes protease enzyme deactivation, terminates reaction；Ice
After water-bath cooling, centrifugal treating collects supernatant, obtains pigskin gelatin peptide solution；
Step 4: pigskin gelatin peptide solution is handled by vacuum freeze drying, saves, obtains thin to skeletonization in -20 DEG C after constant weight
Born of the same parents' proliferation has the pigskin gelatin peptide of facilitation.
2. the preparation method of the pigskin gelatin peptide to osteoblastic proliferation with facilitation according to claim 1,
It is characterized in that, the mass concentration of gelatin solution is 5% (w/w) in step 1.
3. the pigskin gelatin peptide preparation method to osteoblastic proliferation with facilitation according to claim 1, special
Sign is, papain hydrolysis pigskin gelatin is added in step 2, and the additive amount of the papain is 5,000 vigor lists
Position/gram protein, regulation system pH value are 6.0, and hydrolysis time is 12.5 hours.
4. the preparation method of the pigskin gelatin peptide to osteoblastic proliferation with facilitation according to claim 1,
It is characterized in that, neutral proteinase hydrolysis pigskin gelatin is added in step 2, and the additive amount of the neutral proteinase is 6,000 vigor lists
Position/gram protein, regulation system pH value are 7.0, and hydrolysis time is 12.5 hours.
5. the preparation method of the pigskin gelatin peptide to osteoblastic proliferation with facilitation according to claim 1,
It is characterized in that, hydrolysis by novo pigskin gelatin is added in step 2, and the additive amount of the alkali protease is 7,000 vigor lists
Position/gram protein, regulation system pH value are 8.5, and hydrolysis time is 12.5 hours.
6. the preparation method of the pigskin gelatin peptide to osteoblastic proliferation with facilitation according to claim 1,
It is characterized in that, it is 5000 revs/min that centrifugal treating, which collects the centrifugation rate of supernatant, in step 3, and centrifugation time is 20 minutes.
7. the preparation method of the pigskin gelatin peptide to osteoblastic proliferation with facilitation according to claim 1,
It is characterized in that, during vacuum freeze drying described in step 4, control vacuum degree is 0.01 millipascal to 0.5 millipascal, dry
Time is 8 hours to 18 hours, and condenser temperature is -72 DEG C to -45 DEG C.
8. the application that pair osteoblastic proliferation has the pigskin gelatin peptide of facilitation, which is characterized in that this pair of of osteoblast
Being proliferated, there is the pigskin gelatin peptide of facilitation can be used as functional component and be applied in functional food or health food.
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