CN110551789A - Preparation method of mussel peptide freeze-dried powder with osteogenic activity - Google Patents
Preparation method of mussel peptide freeze-dried powder with osteogenic activity Download PDFInfo
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- 235000019750 Crude protein Nutrition 0.000 claims abstract description 18
- 102000035092 Neutral proteases Human genes 0.000 claims abstract description 18
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Abstract
The invention discloses a preparation method of mussel peptide freeze-dried powder with osteogenic activity. Mussel meat is used as a raw material, and the steps are sequentially carried out: extracting crude protein with water, dialyzing for desalting, performing enzymolysis with neutral protease, and freeze drying to obtain mussel peptide lyophilized powder. The invention takes common aquatic products mussels as raw materials, has easily obtained materials, is safe and has no side effect, can obviously reduce the production cost and meet the requirement of large-scale production; a large amount of small molecular mussel peptide is obtained by using a biological enzymolysis method, the operation is simple, and the production conditions are mild; the freeze drying technology is adopted, so that the storage time of the product is prolonged; the obtained mussel peptide has certain osteogenic activity and can be one of good raw materials or sources for preparing functional food.
Description
Technical Field
The invention relates to the technical field of aquatic product processing, in particular to a preparation method of mussel peptide freeze-dried powder with osteogenic activity.
Background
At present, the conventional osteoporosis treatment drugs mainly include bone resorption inhibitors and bone formation promoters. Bone resorption inhibitors slow the loss of calcium from bone primarily by inhibiting osteoclast formation and activity, thereby inhibiting bone resorption, but since osteoporosis patients often have insufficient calcium resorption, they may cause hypocalcemia if used alone. At present, the bone formation promoter is researched a little and mainly comprises parathyroid hormone, cytokine, fluoride, strontium preparation and the like, but the source of the bone formation promoter is limited, and the application of the bone formation promoter is limited due to poor targeting. With the increasing awareness of modern patients on diet and health, the food-derived bioactive peptides have attracted more and more attention in the field of preventing and treating osteoporosis because of their advantages of safety, no toxic or side effect, strong price competitiveness, easy absorption and the like.
Mussel (Mytilus edulis) is a nutritious aquatic product. Each hundred grams of fresh scallop meat contains 10.8 grams of protein, 2.4 grams of sugar, 2.4 grams of ash and 1.4 grams of fat, and the protein content is very high. The mussel protein has high nutritive value and good antioxidant activity, anti-inflammatory activity and anti-tumor activity. It is worth mentioning that mussel has high nutritional value because its protein contains 8 essential amino acids such as valine and leucine required by human body. More and more evidences show that aquatic products with high protein content are converted into small bioactive peptides through digestion and enzymolysis, so that the small bioactive peptides are easier to absorb by a human body, and further, the aquatic products with high protein content are beneficial to the human body.
at present, no method for thoroughly curing osteoporosis exists in medicine, and basically no adverse reaction exists in the early stage of osteoporosis onset, so that osteoporosis is not easy to find and cannot be treated in time.
Disclosure of Invention
the invention aims to overcome the defects of the prior art, provide a preparation method of a mussel peptide with osteogenic activity and develop a mussel polypeptide product with osteogenic activity.
in order to achieve the above purpose, the invention provides a preparation method of a mussel peptide freeze-dried powder with osteogenic activity, which comprises the following steps:
s1, preparing crude mussel water extract protein: removing shells of the mussels, taking mussel meat at 6000-12000 rpm, homogenizing for 3-10 min, adding water into the obtained mussel pulp, standing for 2-8 h at 1-25 ℃ to extract protein, and centrifuging to obtain supernatant A, namely water-extracted crude protein; wherein, the weight volume ratio of the mussel slurry to the water is as follows: 1: 3-5 g/ml;
S2, desalting: centrifuging the water-extracted crude protein obtained in the step S1, collecting supernatant B, and dialyzing and desalting the supernatant B by using a 1000-3000D dialysis bag and water to obtain water-extracted desalted mussel protein; freeze-drying the water-extracted desalted mussel protein to obtain water-extracted desalted mussel protein freeze-dried powder;
S3, enzymolysis: adding water into the mussel protein freeze-dried powder which is subjected to water extraction and desalination in the step S3 according to the weight ratio of the feed liquid of 1: 30-100 to re-dissolve the mussel protein freeze-dried powder into a solution A, adding neutral protease into the solution A, stirring and performing enzymolysis at the temperature of 30-60 ℃ at 50-70 rpm for 2-6 hours to obtain an enzymolysis product solution which is mussel mixed peptide with osteogenic activity; freeze-drying the mussel mixed peptide to obtain mussel peptide freeze-dried powder with osteogenic activity; the addition amount of the neutral protease is based on the total weight of the mussel protein freeze-dried powder subjected to water extraction and desalination in the solution A, and 4000-6000U of the neutral protease is added into each gram of the mussel protein freeze-dried powder subjected to water extraction and desalination.
Preferably, the centrifugation in step S1 is specifically: 6000-12000 g, 5-30 min.
Preferably, the centrifugation in step S2 is specifically: 6000-12000 g, 5-30 min.
In a preferable mode, the dialysis in the step S2 is specifically to put the supernatant B into a 1000-3000D dialysis bag, and dialyze the supernatant B with double distilled water, wherein the amount of the double distilled water is 100-150 times of the volume of the supernatant B, and the double distilled water is replaced every 6-12 hours for 24-36 hours of total dialysis.
preferably, the freeze-drying method in step S2 is performed by gradient temperature variation, and the time-temperature program of freeze-drying is set as follows, first stage: pre-freezing for 3-5 hours at-60 ℃ to-50 ℃, and performing a second stage: freezing at-45 to-40 ℃ for 1 to 3 hours, and in the third stage: freezing at-30 to-25 ℃ for 1 to 2 hours, and in the fourth stage: freezing at-10 to-5 ℃ for 1 to 2 hours, and in the fifth stage: drying for 1-2 hours at the temperature of 5-15 ℃, and a sixth stage: drying for 1-2 hours at 15-20 ℃, and in the seventh stage: drying for 1-2 hours at 20-25 ℃; vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15-25 Pa; the starting temperature of the vacuum pump is-60 ℃ to-50 ℃, and the temperature of the clapboard is set to-30 ℃ to-20 ℃.
Preferably, the rotation speed of the enzymolysis in the step S3 is 50-75 rpm.
Preferably, the freeze-drying method in step S3 is performed by gradient temperature variation, and the time-temperature program of freeze-drying is set as follows, first stage: pre-freezing for 3-5 hours at-60 ℃ to-50 ℃, and performing a second stage: freezing at-45 to-40 ℃ for 1 to 3 hours, and in the third stage: freezing at-30 to-25 ℃ for 1 to 2 hours, and in the fourth stage: freezing at-10 to-5 ℃ for 1 to 2 hours, and in the fifth stage: drying for 1-2 hours at the temperature of 5-15 ℃, and a sixth stage: drying for 1-2 hours at 15-20 ℃, and in the seventh stage: drying for 1-2 hours at 20-25 ℃; vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15-25 Pa; the starting temperature of the vacuum pump is-60 ℃ to-50 ℃, and the temperature of the clapboard is set to-30 ℃ to-20 ℃.
In a preferred mode, the preparation method of the mussel peptide freeze-dried powder with osteogenic activity comprises the following steps:
S1, preparing crude mussel water extract protein: taking 500g of mussel meat at 10000rpm, homogenizing for 5min, adding 1500mL of water into the obtained mussel slurry, standing at 4 ℃ for 6h to extract protein, then placing at 10000g, centrifuging for 10min, and taking supernatant A, namely water-extracted crude protein;
S2, desalting: centrifuging the water-extracted crude protein obtained in the step S1 at 10000g for 10min, collecting supernatant B, and dialyzing and desalting the supernatant B by using a dialysis bag of 1000D to obtain water-extracted desalted mussel protein; freeze-drying the water-extracted desalted mussel protein to obtain water-extracted desalted mussel protein freeze-dried powder;
the supernatant B is placed in a 1000D dialysis bag, and is dialyzed by using double distilled water, the amount of the double distilled water is 100 times of the volume of the supernatant B, and the double distilled water is replaced every 6 hours for 24 hours in total dialysis;
the freeze drying is carried out in a gradient temperature changing mode, and the time-temperature program of the freeze drying is set as follows, in the first stage: pre-freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃;
s3, enzymolysis: adding 1L of water into 10g of the mussel protein freeze-dried powder subjected to water extraction and desalination in the step S3 to redissolve the mussel protein freeze-dried powder into a solution A, and adding 5000U/g neutral protease into the solution AMussel protein freeze-dried powder for water extraction and desalinationAdding 50000U neutral protease, stirring at 45 deg.C and 60rpm for enzymolysis for 4 hr to obtain mixed peptide of mussel with osteogenic activity; freeze-drying the mussel mixed peptide to obtain mussel peptide freeze-dried powder with osteogenic activity;
wherein, the freeze drying is carried out by adopting a gradient temperature changing mode, and the time-temperature program of the freeze drying is set as follows, in the first stage: pre-freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; and vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃.
The invention has the beneficial effects that:
(1) Mussel is a common aquatic product with wide sources, is easy to obtain raw materials by taking mussel protein as a raw material, is safe, has no side effect, can obviously reduce the production cost, and meets the requirement of large-scale production.
(2) a large amount of small molecular mussel peptide is obtained by using a biological enzymolysis method, the operation is simple, and the production conditions are mild; the activity is measured through cell experiments, and the product has high safety;
(3) The freeze-drying technology is adopted to freeze-dry the mussel peptide with osteogenic activity, so that the storage time of the product is prolonged.
(4) the obtained mussel peptide has certain osteogenic activity and can be one of good raw materials or sources for preparing functional food.
drawings
FIG. 1 shows the osteogenic activity of the lyophilized powder of mussel protein and peptide prepared in example 1 of the present invention; the superscript letters represent significant differences between each group as determined using ANOVA; the two letters of the superscript letter are the same, and there is no significant difference between the two letters; there was a significant difference between the two groups of different letters (p < 0.05).
FIG. 2 shows the osteogenic activity of the lyophilized powder of mussel protein and peptide prepared in example 2 of the present invention; the superscript letters represent significant differences between each group as determined using ANOVA; the superscript letters are identical for both letters, there is no significant difference between the two letters, and there is a significant difference between the groups of different letters (p < 0.05).
FIG. 3 shows the osteogenic activity of the lyophilized powder of mussel protein and peptide prepared in example 3 of the present invention; the superscript letters represent significant differences between each group as determined using ANOVA; the superscript letters are identical for both letters, there is no significant difference between the two letters, and there is a significant difference between the groups of different letters (p < 0.05).
Detailed Description
The following is a further description of the invention and is not intended to be limiting.
the invention provides a preparation method of mussel peptide with osteogenic activity, which comprises the following steps:
(1) Preparation of mussel water-extracted crude protein
Firstly, shelling the mussels, and preparing mussel water extract protein: taking mussel meat at 6000-12000 rpm for 3-10 min to homogenize, adding water into the obtained mussel pulp according to the ratio of the mussel meat to the mussel pulp of 1: 3-5 g/ml, standing at 1-25 ℃ for 2-8 h to extract protein, and centrifuging at 6000-12000 g for 5-30 min to obtain crude protein extracted by water.
(2) desalination
And centrifuging the obtained water-extracted crude protein for 5-30 minutes at 6000-12000 g by using a centrifugal machine, and collecting supernatant. And desalting the supernatant by using a dialysis bag of 500-3500D to obtain the mussel protein which is subjected to water extraction and desalination.
(3) Freeze drying
The freeze drying method is carried out in a gradient temperature changing mode, and the time-temperature program of freeze drying is set as follows, in the first stage: pre-freezing for 3-5 hours at-60 ℃ to-50 ℃, and performing a second stage: freezing at-45 to-40 ℃ for 1 to 3 hours, and in the third stage: freezing at-30 to-25 ℃ for 1 to 2 hours, and in the fourth stage: freezing at-10 to-5 ℃ for 1 to 2 hours, and in the fifth stage: drying for 1-2 hours at the temperature of 5-15 ℃, and a sixth stage: drying for 1-2 hours at 15-20 ℃, and in the seventh stage: drying for 1-2 hours at 20-25 ℃. Vacuumizing in the second period and maintaining the constant temperature in the seventh period. The starting temperature of the vacuum pump is-60 ℃ to-50 ℃, the temperature of the clapboard is set to-30 ℃ to-20 ℃, and the mussel protein freeze-dried powder which is desalted by water extraction is obtained.
(4) Preparation of mussel osteogenic active peptide
adding water into the water-extracted and desalted mussel protein freeze-dried powder according to the mass ratio of the feed liquid of 1: 30-100 for redissolution, adding 3000-6000U/g neutral protease water-extracted and desalted mussel protein freeze-dried powder, and stirring and performing enzymolysis at the temperature of 30-60 ℃ at 50-70 rpm for 2-6 hours to obtain the mussel mixed peptide with osteogenic activity.
(5) Freeze drying
The freeze drying method is carried out in a gradient temperature changing mode, and the time-temperature program of freeze drying is set as follows, in the first stage: pre-freezing for 3-5 hours at-60 ℃ to-50 ℃, and performing a second stage: freezing at-45 to-40 ℃ for 1 to 3 hours, and in the third stage: freezing at-30 to-25 ℃ for 1 to 2 hours, and in the fourth stage: freezing at-10 to-5 ℃ for 1 to 2 hours, and in the fifth stage: drying for 1-2 hours at the temperature of 5-15 ℃, and a sixth stage: drying for 1-2 hours at 15-20 ℃, and in the seventh stage: drying for 1-2 hours at 20-25 ℃. And vacuumizing for the second period of time, wherein the vacuum degree is 15 Pa. The starting temperature of the vacuum pump is-60 ℃ to-50 ℃, the temperature of the clapboard is set to-30 ℃ to-20 ℃, and the mussel mixed peptide freeze-dried powder with osteogenesis activity is obtained.
Example 1
a preparation method of mussel peptide freeze-dried powder with osteogenic activity comprises the following steps:
S1, preparing crude mussel water extract protein: taking 600g of mussel meat at 10000rpm, homogenizing for 5min, adding 1800mL of water into the obtained mussel pulp, standing at 4 ℃ for 8h to extract protein, then placing the mussel pulp in 10000g, centrifuging for 10min, and taking supernatant A, namely water-extracted crude protein;
S2, desalting: placing the water-extracted crude protein obtained in the step S1 in 10000g for centrifugation for 10min, collecting supernatant B, placing the supernatant B in a 3000D dialysis bag, and dialyzing the supernatant B by using double distilled water, wherein the using amount of the double distilled water is 100 times of the volume of the supernatant B, and the double distilled water is replaced once every 6h for 24h in total dialysis; obtaining water-extracted desalted mussel protein; freeze-drying the water-extracted desalted mussel protein to obtain water-extracted desalted mussel protein freeze-dried powder;
the freeze drying is carried out in a gradient temperature changing mode, and the time-temperature program of the freeze drying is set as follows, in the first stage: pre-freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃;
S3, enzymolysis: adding 1L of water into 10g of the mussel protein freeze-dried powder subjected to water extraction and desalination in the step S3 to redissolve the mussel protein freeze-dried powder into a solution A, and adding 5000U/g neutral protease into the solution AMussel protein freeze-dried powder for water extraction and desalinationAdding 50000U neutral protease, stirring at 45 deg.C and 60rpm for enzymolysis for 4 hr to obtain mixed mussel peptide with osteogenic activity; freeze-drying the mussel mixed peptide to obtain mussel peptide freeze-dried powder with osteogenic activity;
Wherein, the freeze drying is carried out by adopting a gradient temperature changing mode, and the time-temperature program of the freeze drying is set as follows, in the first stage: pre-freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; and vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃.
Example 2
A preparation method of mussel peptide freeze-dried powder with osteogenic activity comprises the following steps:
s1, preparing crude mussel water extract protein: taking 500g of mussel meat at 10000rpm, homogenizing for 5min, adding 1500mL of water into the obtained mussel slurry, standing at 4 ℃ for 6h to extract protein, then placing at 10000g, centrifuging for 10min, and taking supernatant A, namely water-extracted crude protein;
S2, desalting: centrifuging the water-extracted crude protein obtained in the step S1 at 10000g for 10min, collecting supernatant B, and dialyzing and desalting the supernatant B by using a dialysis bag of 1000D to obtain water-extracted desalted mussel protein; freeze-drying the water-extracted desalted mussel protein to obtain water-extracted desalted mussel protein freeze-dried powder;
The supernatant B is placed in a 1000D dialysis bag, and is dialyzed by using double distilled water, the amount of the double distilled water is 100 times of the volume of the supernatant B, and the double distilled water is replaced every 6 hours for 24 hours in total dialysis;
The freeze drying is carried out in a gradient temperature changing mode, and the time-temperature program of the freeze drying is set as follows, in the first stage: pre-freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃;
s3, enzymolysis: will be provided withStep S3, adding 1L of water into 10g of mussel protein freeze-dried powder subjected to water extraction and desalination to redissolve into solution A, and adding 5000U/g neutral protease into the solution AMussel protein freeze-dried powder for water extraction and desalinationAdding 50000U neutral protease, stirring at 45 deg.C and 60rpm for enzymolysis for 4 hr to obtain mixed peptide of mussel with osteogenic activity; freeze-drying the mussel mixed peptide to obtain mussel peptide freeze-dried powder with osteogenic activity;
Wherein, the freeze drying is carried out by adopting a gradient temperature changing mode, and the time-temperature program of the freeze drying is set as follows, in the first stage: pre-freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; and vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃.
Example 3
A preparation method of mussel peptide freeze-dried powder with osteogenic activity comprises the following steps:
S1, preparing crude mussel water extract protein: taking 500g of mussel meat at 10000rpm, homogenizing for 5min, adding 1500mL of water into the obtained mussel slurry, standing at 4 ℃ for 6h to extract protein, then placing at 10000g, centrifuging for 10min, and taking supernatant A, namely water-extracted crude protein;
s2, desalting: centrifuging the water-extracted crude protein obtained in the step S1 at 10000g for 10min, collecting supernatant B, and dialyzing and desalting the supernatant B by using a dialysis bag of 1000D to obtain water-extracted desalted mussel protein; freeze-drying the water-extracted desalted mussel protein to obtain water-extracted desalted mussel protein freeze-dried powder;
the supernatant B is placed in a 1000D dialysis bag, and is dialyzed by using double distilled water, the amount of the double distilled water is 100 times of the volume of the supernatant B, and the double distilled water is replaced every 6 hours for 24 hours in total dialysis;
The freeze drying is carried out in a gradient temperature changing mode, and the time-temperature program of the freeze drying is set as follows, in the first stage: pre-freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃;
S3, enzymolysis: adding 1L of water into 10g of the mussel protein freeze-dried powder subjected to water extraction and desalination in the step S3 to redissolve the mussel protein freeze-dried powder into a solution A, and adding 6000U/g neutral protease into the solution AMussel protein freeze-dried powder for water extraction and desalinationAdding 60000U of neutral protease, stirring and performing enzymolysis for 4 hours at 45 ℃ and 55rpm to obtain the mussel mixed peptide with osteogenic activity; freeze-drying the mussel mixed peptide to obtain mussel mixed peptide freeze-dried powder with osteogenic activity;
Wherein, the freeze drying is carried out by adopting a gradient temperature changing mode, and the time-temperature program of the freeze drying is set as follows, in the first stage: pre-freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; and vacuumizing in the second stage, wherein the vacuum degree from the second stage to the seventh stage is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃.
the mussel mixed peptide lyophilized powder with osteogenic activity prepared in the above example and the mussel protein lyophilized powder desalted by water extraction in step S2 are taken for osteogenic activity determination according to the following method:
Method for measuring osteogenic activity
2osteoblast activity was measured by MTT assay for osteoblast proliferation of MC3T3-E1 cells, MC3T3-E1 cells were seeded in 96-well plates at a density of 5000 cells and cultured in α -MEM medium containing 10% FBS by volume at 37 ℃ for 24 hours, after which the cell medium was replaced and washed with PBS buffer, the α -MEM medium containing samples at different concentrations (aqueous desalted mussel protein lyophilized powder, mussel mixed peptide lyophilized powder, medium alone as blank) was replaced with the original α -MEM medium at 37 ℃ for 72 hours, after which 10 μ L of a dimethyl sulfoxide (DMSO) was added per well and cultured in an incubator for 4 hours, after which 150 μ L of a dimethyl sulfoxide (DMSO) was used instead of the medium, shaking at 50rpm for 10 minutes, and the absorbance value of MC 3-E1 cells per well was measured at 570nm using a Microplate Reader (Eon, Biok, USA).
cell viability ═ absorbance of sample/absorbance of blank
As shown in fig. 1 to 3, although the water-extracted mussel protein has a certain osteogenic activity under the conditions of example 1, the increase in osteoblast proliferation rate at 100 μ g/mL is 11.28%, but the increase in osteoblast proliferation rate at 100 μ g/mL is 35.15% unlike the osteogenic activity of the mussel peptide lyophilized powder of example 2, and the increase in osteoblast proliferation rate at 100 μ g/mL is 35.42% better than that of the mussel peptide lyophilized powder of example 3, and the osteogenic activities of the mussel peptide lyophilized powders of examples 2 and 3 are similar, so that the conditions of example 2 are finally selected as the optimal conditions according to the principle of cost reduction.
the above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (8)
1. A preparation method of mussel peptide freeze-dried powder with osteogenic activity is characterized by comprising the following steps:
s1, preparing crude mussel water extract protein: taking mussel meat at 6000-12000 rpm, homogenizing for 3-10 min to obtain mussel pulp, adding water, standing for 2-8 h at 1-25 ℃, centrifuging and taking supernatant A, namely water-extracted crude protein; wherein, the weight volume ratio of the mussel slurry to the water is as follows: 1: 3-5 g/ml;
S2, desalting: centrifuging the water-extracted crude protein obtained in the step S1, collecting supernatant B, and dialyzing and desalting the supernatant B by using a 1000-3000D dialysis bag and water to obtain water-extracted desalted mussel protein; freeze-drying the water-extracted desalted mussel protein to obtain water-extracted desalted mussel protein freeze-dried powder;
s3, enzymolysis: adding water into the mussel protein freeze-dried powder which is subjected to water extraction and desalination in the step S3 according to the weight ratio of the feed liquid of 1: 30-100 to re-dissolve the mussel protein freeze-dried powder into a solution A, adding neutral protease into the solution A, stirring and performing enzymolysis at 30-60 ℃ and 50-70 rpm for 2-6 hours to obtain an enzymolysis product solution which is mussel mixed peptide with osteogenic activity; freeze-drying the mussel mixed peptide to obtain mussel peptide freeze-dried powder with osteogenic activity; the addition amount of the neutral protease is based on the total weight of the mussel protein freeze-dried powder subjected to water extraction and desalination in the solution A, and 4000-6000U of the neutral protease is added into each gram of the mussel protein freeze-dried powder subjected to water extraction and desalination.
2. The method for preparing a lyophilized powder of mussel peptide for osteogenic activity according to claim 1, wherein the centrifugation in step S1 is specifically: 6000-12000 g, 5-30 min.
3. The method for preparing a lyophilized powder of mussel peptide for osteogenic activity according to claim 1, wherein the centrifugation in step S2 is specifically: 6000-12000 g, 5-30 min.
4. The method for preparing a mussel peptide lyophilized powder with osteogenic activity according to claim 1, wherein the dialysis in step S2 is specifically to put the supernatant B into a dialysis bag of 1000-3000D, and dialyze the supernatant B with double distilled water, wherein the amount of the double distilled water is 100-150 times of the volume of the supernatant B, and the double distilled water is replaced every 6-12 hours for 24-36 hours of total dialysis.
5. A method for preparing a lyophilized powder of mussel peptide for osteogenic activity according to claim 1, wherein the time-temperature program for lyophilization in step S2 is as follows, first stage: freezing at-60 ℃ to-50 ℃ for 3-5 hours, and performing a second stage: freezing at-45 to-40 ℃ for 1 to 3 hours, and in the third stage: freezing at-30 to-25 ℃ for 1 to 2 hours, and in the fourth stage: freezing at-10 to-5 ℃ for 1 to 2 hours, and in the fifth stage: drying for 1-2 hours at the temperature of 5-15 ℃, and a sixth stage: drying for 1-2 hours at 15-20 ℃, and in the seventh stage: drying for 1-2 hours at 20-25 ℃; vacuumizing the second section, wherein the vacuum degree from the second section to the seventh section is 15-25 Pa; the starting temperature of the vacuum pump is-60 ℃ to-50 ℃, and the temperature of the clapboard is set to-30 ℃ to-20 ℃.
6. the method for preparing a mussel peptide lyophilized powder with osteogenic activity according to claim 1, wherein the rotation speed of enzymolysis in step S3 is 50-75 rpm.
7. A method for preparing a lyophilized powder of mussel peptide for osteogenic activity according to claim 1, wherein the time-temperature program for lyophilization in step S3 is as follows, first stage: freezing at-60 ℃ to-50 ℃ for 3-5 hours, and performing a second stage: freezing at-45 to-40 ℃ for 1 to 3 hours, and in the third stage: freezing at-30 to-25 ℃ for 1 to 2 hours, and in the fourth stage: freezing at-10 to-5 ℃ for 1 to 2 hours, and in the fifth stage: drying for 1-2 hours at the temperature of 5-15 ℃, and a sixth stage: drying for 1-2 hours at 15-20 ℃, and in the seventh stage: drying for 1-2 hours at 20-25 ℃; vacuumizing the second section, wherein the vacuum degree from the second section to the seventh section is 15-25 Pa; the starting temperature of the vacuum pump is-60 ℃ to-50 ℃, and the temperature of the clapboard is set to-30 ℃ to-20 ℃.
8. A method for preparing a lyophilized powder of mussel peptide for osteogenic activity according to claim 1, comprising the steps of:
s1, preparing crude mussel water extract protein: homogenizing 500g mussel meat at 10000rpm for 5min to obtain mussel slurry, adding 1500mL water, standing at 4 deg.C for 6h, centrifuging at 10000g for 10min, and collecting supernatant A to obtain crude protein water extract;
S2, desalting: centrifuging the water-extracted crude protein obtained in the step S1 for 10min at 10000g, collecting supernatant B, dialyzing the supernatant B in a 1000D dialysis bag by using double distilled water, wherein the amount of the double distilled water is 100 times of the volume of the supernatant B, replacing the double distilled water every 6h, and dialyzing for 24h to obtain the water-extracted desalted mussel protein; freeze-drying the water-extracted desalted mussel protein to obtain water-extracted desalted mussel protein freeze-dried powder;
The freeze-drying time-temperature program was set as follows, first stage: freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; vacuumizing the second section, wherein the vacuum degree from the second section to the seventh section is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃;
S3, enzymolysis: adding 1L of water into 10g of the mussel protein freeze-dried powder subjected to water extraction and desalination in the step S3 to redissolve the mussel protein freeze-dried powder into a solution A, and adding 5000U/g neutral protease into the solution AMussel protein freeze-dried powder for water extraction and desalinationAdding 50000U neutral protease, stirring at 45 deg.C and 60rpm, and performing enzymolysis for 4 hr to obtain mixed peptide of mussel with osteogenic activity; freeze-drying the mussel mixed peptide to obtain mussel peptide freeze-dried powder with osteogenic activity;
wherein the freeze-drying time-temperature program is set as follows, first stage: freezing at-60 ℃ for 2 hours, second stage: -45 ℃ for 3 hours, third stage: freezing at-30 ℃ for 2 hours, fourth stage: freezing at-5 ℃ for 1 hour, fifth stage: drying at 5 ℃ for 1 hour, and a sixth stage: drying at 20 ℃ for 1 hour, and a seventh stage: drying at 25 deg.C for 1 hr; and (3) vacuumizing in a second section, wherein the vacuum degree from the second section to a seventh section is 15Pa, the starting temperature of a vacuum pump is-60 ℃, and the temperature of a clapboard is set to be-30 ℃.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104177484A (en) * | 2014-05-29 | 2014-12-03 | 浙江海洋学院 | Prostatic cancer-resistant mussel glycoprotein and preparation and application thereof |
| CN108904420A (en) * | 2018-06-28 | 2018-11-30 | 爱思开百朗德生物科技（海门）有限公司 | Raw material of skin care articles composition with acne-removing and preparation method thereof, application |
-
2019
- 2019-09-19 CN CN201910883872.XA patent/CN110551789A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104177484A (en) * | 2014-05-29 | 2014-12-03 | 浙江海洋学院 | Prostatic cancer-resistant mussel glycoprotein and preparation and application thereof |
| CN108904420A (en) * | 2018-06-28 | 2018-11-30 | 爱思开百朗德生物科技（海门）有限公司 | Raw material of skin care articles composition with acne-removing and preparation method thereof, application |
Non-Patent Citations (2)
| Title |
|---|
| ZHE XU ET AL: "Isolation and Characterization of Peptides from Mytilus edulis with Osteogenic Activity in Mouse MC3T3-E1 Preosteoblast Cells", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| 程云章: "《药物制剂工程原理与设备》", 30 September 2009, 东南大学出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113604531A (en) * | 2021-08-23 | 2021-11-05 | 大连工业大学 | Method for simultaneously preparing mussel functional lipid and active polypeptide |
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