CN102643327A - Preparation method and application of polypeptide of fermented cucumber seeds - Google Patents
Preparation method and application of polypeptide of fermented cucumber seeds Download PDFInfo
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- CN102643327A CN102643327A CN2012101224621A CN201210122462A CN102643327A CN 102643327 A CN102643327 A CN 102643327A CN 2012101224621 A CN2012101224621 A CN 2012101224621A CN 201210122462 A CN201210122462 A CN 201210122462A CN 102643327 A CN102643327 A CN 102643327A
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Abstract
The invention relates to a preparation method and the application of polypeptide of fermented cucumber seeds. The method includes: firstly, preparing degreased and dried fermented cucumber seed powder; and secondly, adding the degreased and dried cucumber seed powder into buffer solution, stirring or soaking, centrifuging, performing ultrafiltration, concentrating filtrate and drying so that the polypeptide is prepared. The polypeptide of the fermented cucumber seeds is used for preventing and treating osteoporosis. The polypeptide of the fermented cucumber seeds is safe, reliable and non-toxic, is capable of promoting sclerotomal cell proliferation, lowering activity of alkaline phosphatase and improving osteoporosis symptoms, and can play a role in prevention and treatment of osteoporosis and promotion of skeleton growth.
Description
Technical field
The present invention relates to a kind of preparation method and its usage of the Semen Cucumidis sativi polypeptide that ferments.
Background technology
Polypeptide is all relevant biologically active substance of various cell functions in a kind of and the organism, be by multiple amino acids according to the compound of certain arrangement sequence through the peptide bond be combined into, comprise plant polypeptide and animal polypeptide.Research thinks that the natural bioactive peptide has following characteristics: 1. natural sex and safe reliability; 2. have multiple nutritional components and biological activity; 3. have no side effect.
Mikrobe has the ability of powerful enrichment calcium, can produce the albumen of some abilities and calcium complexing, through existing with organic with the calcium complexing.Mineral element exists with organic more than 90% in edible mushrooms; Has satisfactory stability property; Be convenient to storage, transportation and compatible with other compositions better, wherein major part combines with albumen, sugar and lipoid material, helps the absorption and the utilization of mineral element.
Osteoporosis is the elderly's a kind of common disease and a frequently-occurring disease.The human calcium loses in very long process, will cause or cause osteoporosis, hyperosteogeny and all kinds of fracture, and hypertension, coronary heart disease, calculus, senile dementia, and possibly increase the sickness rate of malignant tumour.Investigation finds, among the elderly of China's over-65s, has 21% the male sex and 60% women to suffer from osteoporosis approximately, and the elderly more than 80 years old then 100% has osteoporosis, and visible good calcium-supplementing preparation market requirement crowd is quite a lot of.
Calcium agent commonly used in the market has inorganic calcium class and preparation, organic calcium class and preparation thereof, Chinese medicine calcium class and other.Wherein inorganic calcium class (like lime carbonate) is the highest a kind of of calcium content in all medicinal calcium salts, and cheap, is to use the widest calsium supplement at present, and achlorhydria person can influence its absorption; The good water solubility of organic calcium class (calglucon), but specific absorption is low; The raw material of Chinese medicine class calcium agent adopts animal skeleton, halobiontic vertebra, shell etc. mostly, and alkalescence is stronger, and is big to gastrointestinal irritation, if these biologies are polluted, can accumulate plurality of heavy metal in the body, is prone to cause secondary poisoning.In addition, controversies in hormone replacement in the elderly can prevent the minimizing of postmenopausal women's bone amount and reduce the generation of osteoporotic fracture, but there is the potential carcinogenic risk in prolonged application to reproductive system.
Therefore, exploitation have independent intellectual property right, can be used for preventing and treat osteoporosis, safety, efficiently, the newtype drug and the protective foods of low toxicity have great economic benefit and social benefit.
Polypeptide can derive from animal and plant.Bone peptide is the micromolecule polypeptide class material that from the bone of animal, extracts, and has been proved its metabolism that can improve bone, has promoted union of fracture, can prevent and treat osteoporosis, and have good analgesia and antiinflammation.Also contain polypeptides matter in many plants, have biological activity preferably, some plant polypeptide be applied to producing and scientific effort in the middle of, like soybean polypeptide etc.
Summary of the invention
The object of the present invention is to provide a kind of preparation method and its usage of the Semen Cucumidis sativi polypeptide that ferments.
The preparation method of fermentation Semen Cucumidis sativi polypeptide is following:
One, be that the ratio of 1g: the 2～10mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent according to quality and volume ratio; Soak 18～24h at room temperature supersound extraction 20～60min or at 0～20 ℃, filter, discard filtrating; With filter residue and drying, obtain the fermentation Semen Cucumidis sativi powder of degreaser drying;
Two, be that the fermentation Semen Cucumidis sativi powder of degreaser drying is joined the pH value is that 4.0～9.0 acetic acid-sodium-acetate buffer or pH value are in 4.0～9.0 the phosphate buffered saline buffer for the ratio of 1g: 4～20mL according to quality and volume ratio; Stir 2～4h or soak 24～36h at 4 ℃ at 4 ℃; Centrifugal with 3000～6000 rev/mins speed then, get supernatant, with the 50000 daltonian ultra-filtration membrane ultrafiltration of supernatant process molecular weight; Again through 10000 daltonian ultra-filtration membrane ultrafiltration; Concentrated filtrate, lyophilize or spraying drying promptly get the Semen Cucumidis sativi polypeptide that ferments; Organic solvent described in the step 1 is sherwood oil, normal hexane or ether.
The preparation method of fermentation Semen Cucumidis sativi polypeptide is following:
One, be that the ratio of 1g: the 2～10mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent according to quality and volume ratio; Soak 18～24h at room temperature supersound extraction 20～60min or at 0～20 ℃; Filter; Discard filtrating, filter residue dry below 40 ℃, is obtained the fermentation Semen Cucumidis sativi powder of degreaser drying;
Two, be that the ratio of 1g: 2～5mL adds the fermentation Semen Cucumidis sativi powder of degreaser drying in the entry according to quality and volume ratio, add prozyme again, the prozyme consumption be degreaser drying fermentation Semen Cucumidis sativi opaque amount 1%～4%; Be 6～8 in the pH value, temperature is under 40～60 ℃ the condition; Directional enzymatic 4～10h is at 80～90 ℃ of insulation 5～20min, centrifugal under 3000～5000 rev/mins rotating speed then; Collect supernatant, obtain little peptide liquid;
Three, with the 10000 daltonian ultra-filtration membrane ultrafiltration of little peptide liquid warp, concentrated filtrate, lyophilize or spraying drying promptly get the Semen Cucumidis sativi polypeptide that ferments; Prozyme described in the step 1 is that two or three in trypsinase, papoid and the neutral protease formed by arbitrary proportion; Organic solvent described in the step 1 is sherwood oil, normal hexane or ether.
Fermentation Semen Cucumidis sativi polypeptide of the present invention is used for prevention and treatment osteoporosis.
Fermentation Semen Cucumidis sativi polypeptide of the present invention is safe and reliable, has no side effect, and can promote osteoblastic propagation, and can reduce alkaline phosphatase activities, improves the osteoporosis symptom, can play the effect of prevention and treatment osteoporosis, promotion skeleton development.
Embodiment
Technical scheme of the present invention is not limited to following cited embodiment, also comprises the arbitrary combination between each embodiment.
Embodiment one: the preparation method of this embodiment fermentation Semen Cucumidis sativi polypeptide is following:
One, be that the ratio of 1g: the 2～10mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent according to quality and volume ratio; Soak 18～24h at room temperature supersound extraction 20～60min or at 0～20 ℃, filter, discard filtrating; With filter residue and drying, obtain the fermentation Semen Cucumidis sativi powder of degreaser drying;
Two, be that the fermentation Semen Cucumidis sativi powder of degreaser drying is joined the pH value is that 4.0～9.0 acetic acid-sodium-acetate buffer or pH value are in 4.0～9.0 the phosphate buffered saline buffer for the ratio of 1g: 4～20mL according to quality and volume ratio; Stir 2～4h or soak 24～36h at 4 ℃ at 4 ℃; Centrifugal with 3000～6000 rev/mins speed then, get supernatant, with the 50000 daltonian ultra-filtration membrane ultrafiltration of supernatant process molecular weight; Again through 10000 daltonian ultra-filtration membrane ultrafiltration; Concentrated filtrate, lyophilize or spraying drying promptly get the Semen Cucumidis sativi polypeptide that ferments.
The preparation method of fermentation Semen Cucumidis sativi powder is following in this embodiment:
Semen Cucumidis sativi was soaked in the water 10～20 hours fully; Filter, the lime carbonate that adds the Semen Cucumidis sativi gross weight 5%～10% after accounting for immersion in the Semen Cucumidis sativi after soaking obtains mixture, pack; Mixture in each bag is 200～300g; In temperature is to sterilize 40 minutes under 121 ℃ of conditions, is cooled to room temperature, and in each bag, inserting 3mL concentration then is 1 * 10
5～1 * 10
8The bacterium bacterium liquid of picking up the ears of cfu/mL, 25 ℃～30 ℃ bottom fermentation 25-35 days, vacuum-drying is pulverized, and promptly obtains the Semen Cucumidis sativi powder that ferments.
Embodiment two: what this embodiment and embodiment one were different is that the organic solvent described in the step 1 is sherwood oil, normal hexane or ether.Other is identical with embodiment one.
Embodiment three: this embodiment and embodiment one are different is to be that the ratio of 1g: the 8mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent according to quality and volume ratio in the step 1.Other is identical with embodiment one.
Embodiment four: what this embodiment and embodiment one were different is to soak 18～24h at 10 ℃ in the step 2.Other is identical with embodiment one.
Embodiment five: the preparation method of this embodiment fermentation Semen Cucumidis sativi polypeptide is following:
One, be that the ratio of 1g: the 2～10mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent according to quality and volume ratio; Soak 18～24h at room temperature supersound extraction 20～60min or at 0～20 ℃; Filter; Discard filtrating, filter residue dry below 40 ℃, is obtained the fermentation Semen Cucumidis sativi powder of degreaser drying;
Two, be that the ratio of 1g: 2～5mL adds the fermentation Semen Cucumidis sativi powder of degreaser drying in the entry according to quality and volume ratio, add prozyme again, the prozyme consumption be degreaser drying fermentation Semen Cucumidis sativi opaque amount 1%～4%; Be 6～8 in the pH value, temperature is under 40～60 ℃ the condition; Directional enzymatic 4～10h is at 80～90 ℃ of insulation 5～20min, centrifugal under 3000～5000 rev/mins rotating speed then; Collect supernatant, obtain little peptide liquid;
Three, with the 10000 daltonian ultra-filtration membrane ultrafiltration of little peptide liquid warp, concentrated filtrate, lyophilize or spraying drying promptly get the Semen Cucumidis sativi polypeptide that ferments.
Embodiment six: what this embodiment and embodiment five were different is that the prozyme described in the step 1 is that two or three in trypsinase, papoid and the neutral protease formed by arbitrary proportion.Other is identical with embodiment five.
Embodiment seven: what this embodiment and embodiment five were different is that the organic solvent described in the step 1 is sherwood oil, normal hexane or ether.Other is identical with embodiment five.
Embodiment eight: this embodiment and embodiment five are different is to be that the ratio of 1g: the 8mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent according to quality and volume ratio in the step 1.Other is identical with embodiment five.
Adopt following experimental verification effect of the present invention:
Experiment one: the preparation method of fermentation Semen Cucumidis sativi polypeptide is following:
One, get the fermentation Semen Cucumidis sativi and carry out crushing screening, smashing fineness is 40 orders;
Two, be that the ratio of 1g: the 6mL Semen Cucumidis sativi powder that will ferment joins in the normal hexane according to quality and volume ratio, soak 24h, filter, discard filtrating,, obtain the fermentation Semen Cucumidis sativi powder of degreaser drying filter residue and drying at 20 ℃;
Three, according to quality and volume ratio be the ratio of 1g: 8mL the fermentation Semen Cucumidis sativi powder of degreaser drying is joined the pH value is in 6.8 the phosphate buffered saline buffer, stir 2h at 4 ℃, centrifugal with 5000 rev/mins speed then; Get supernatant; With the 50000 daltonian ultra-filtration membrane ultrafiltration of supernatant process molecular weight, again through 10000 daltonian ultra-filtration membrane ultrafiltration, concentrated filtrate; Lyophilize or spraying drying promptly get the Semen Cucumidis sativi polypeptide that ferments.
Experiment two: the preparation method of fermentation Semen Cucumidis sativi polypeptide is following:
One, get the fermentation Semen Cucumidis sativi and carry out crushing screening, smashing fineness is 100 orders;
Two, be that the ratio of 1g: the 8mL Semen Cucumidis sativi powder that will ferment joins in the ether according to quality and volume ratio, room temperature supersound extraction 20min filters, and discards filtrating, and filter residue dry below 40 ℃, is obtained the fermentation Semen Cucumidis sativi powder of degreaser drying;
Three, be that the ratio of 1g: 4mL adds the fermentation Semen Cucumidis sativi powder of degreaser drying in the entry according to quality and volume ratio, add prozyme again, the prozyme consumption be degreaser drying fermentation Semen Cucumidis sativi opaque amount 2%; Be 6.8 in the pH value, temperature is that directional enzymatic 10h is then at 85 ℃ of insulation 10min under 45 ℃ the condition; Centrifugal under 4000 rev/mins rotating speed; Collect supernatant, obtain little peptide liquid, wherein to be trypsinase and papoid mix by 1: 1 mass ratio prozyme;
Four, with the 10000 daltonian ultra-filtration membrane ultrafiltration of little peptide liquid warp, concentrated filtrate, lyophilize or spraying drying promptly get the Semen Cucumidis sativi polypeptide that ferments.
The experiment one and the fermentation Semen Cucumidis sativi polypeptide of experiment two preparations are tested as follows:
1, fermentation Semen Cucumidis sativi polypeptide acute toxicity test
Get 20 of Kunming mouses; Toxic reaction and death condition that the fermentation Semen Cucumidis sativi polypeptide of observation experiment one and experiment two preparations (fermentation Semen Cucumidis sativi peptide concentration is 280mg/mL) intraperitoneal injection 0.4mL/20g is produced, all no abnormal reaction in 7 days, nothing death.The acute toxicity test in mice result is 3000 times of people's consumption, proves that this working sample is safe and reliable, has no side effect.
2, fermentation Semen Cucumidis sativi polypeptide is to the proliferation function of rat osteoblast
Under aseptic condition, get newborn 1 day-old Wistar rats cranium, reject the reticular tissue that adheres to, PBS liquid cleans 3 times; It is inserted in the petridish that contains the DMEM substratum, be cut into the big fine grained chippings of 0.5mm * 0.5mm, adding mass concentration is 0.25g/100mL trypsinase 5mL, inserts to digest 30min in the incubator; Discard trypsin solution, add 1.0g/L II Collagen Type VI enzyme 5mL, place incubator to digest 1h, discard Digestive system; The centrifugal 10min of 1000r/min makes cell precipitation, with PBS cells washed 2 times; Add the DMEM nutrient solution that contains 10% foetal calf serum and cultivate, change liquid behind the 24h, change liquid next day of later 1 time.Be cultured to the 4th～7 day, cell grows up to fine and close individual layer, at the bottom of the confluent culture bottle, goes down to posterity.Discard original fluid, with PBS liquid flushing 2 times, add the 0.25g/100mL tryptic digestion, the centrifugal 10min of 1000r/min discards Digestive system, with PBS liquid flushing 1 time, adds nutrient solution again, processes cell suspension.
The scleroblast suspension is pressed 1 * 10
5Cell/mL density is inoculated in 96 orifice plates, treat cell attachment after, the DMEM that nutrient solution is changed into mass concentration 1g/100mL foetal calf serum keeps liquid; The fermentation Semen Cucumidis sativi polypeptide solution that adds different concns simultaneously, making final concentration is 40mg/L, 20mg/L, 10mg/L, 5mg/L, 2.5mg/L, the 1.25mg/L that contains stoste, control group adds the liquid of keeping of equivalent; Cultivate 24h, 48h is behind the 72h; Add MTT 20 μ L, in 37 ℃, 5%CO
2Cultivated 4 hours in the incubator, add DMSO 99.8MIN. (DMSO) 150 μ L, measure absorbance in the 570nm place with ELIASA.The result sees table 1 and table 2.
Table 1 fermentation Semen Cucumidis sativi polypeptide (experiment one preparation) is to the propagation detected result (
n=8) of rat osteoblast
Compare with the blank group
aP＜0.01
Table 2 fermentation Semen Cucumidis sativi polypeptide (experiment two preparations) is to the propagation detected result (
n=8) of rat osteoblast
Compare with the blank group
aP＜0.01
Detected result shows that fermentation Semen Cucumidis sativi polypeptide 20mg/L, the 40mg/L dose groups of two kinds of method extractions have remarkable proliferation function to the neonate rat scleroblast.Scleroblast is to bear one type of important cells that bone is rebuild, and the height of its function is significant to keeping normal bone conversion, and this test-results shows that fermentation Semen Cucumidis sativi polypeptide can promote osteoblastic propagation, and the formation of bone is had vital role.
3, fermentation Semen Cucumidis sativi polypeptide is to the bone density of osteoporosis rat model and the influence of alkaline phosphatase activities
Get the SD rat, half and half, 2～3 monthly ages of male and female, body weight 200～220g, random packet.Saline water control group (1mL/100g), model group (tretinoin 70mg/kg; Ig.qd * 14d), Gaierqi D (vitamin D3 and calcium carbonate) group (0.18g/kg; Contain 60mg calcium, 2.5iu D3), fermentation Semen Cucumidis sativi polypeptide high, normal, basic dose groups (80mg/kg, 20mg/kg, 5mg/kg); After the administration 30 days, detect bone density and serum neutral and alkali phosphatase activity.The result sees table 3.
Table 3 fermentation Semen Cucumidis sativi polypeptide is to the bone density of osteoporosis rat model and the influence of alkaline phosphatase activities (
n=10)
Test-results shows; Fermentation Semen Cucumidis sativi polypeptide can obviously strengthen the bone density that tretinoin causes the osteoporosis rat model; And can significantly reduce rat blood serum neutral and alkali phosphatase activity, and improve the osteoporosis symptom, can play the effect of prevention and treatment osteoporosis, promotion skeleton development.
Embodiment nine: the fermentation Semen Cucumidis sativi polypeptide of embodiment one preparation is used for prevention and treatment osteoporosis.
Embodiment ten: the fermentation Semen Cucumidis sativi polypeptide of embodiment five preparations is used for prevention and treatment osteoporosis.
Claims (10)
1. the preparation method of fermentation Semen Cucumidis sativi polypeptide, the preparation method of the Semen Cucumidis sativi polypeptide that it is characterized in that fermenting is following:
One, be that the ratio of 1g: the 2～10mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent according to quality and volume ratio; Soak 18～24h at room temperature supersound extraction 20～60min or at 0～20 ℃, filter, discard filtrating; With filter residue and drying, obtain the fermentation Semen Cucumidis sativi powder of degreaser drying;
Two, be that the fermentation Semen Cucumidis sativi powder of degreaser drying is joined the pH value is that 4.0～9.0 acetic acid-sodium-acetate buffer or pH value are in 4.0～9.0 the phosphate buffered saline buffer for the ratio of 1g: 4～20mL according to quality and volume ratio; Stir 2～4h or soak 24～36h at 4 ℃ at 4 ℃; Centrifugal with 3000～6000 rev/mins speed then, get supernatant, with the 50000 daltonian ultra-filtration membrane ultrafiltration of supernatant process molecular weight; Again through 10000 daltonian ultra-filtration membrane ultrafiltration; Concentrated filtrate, lyophilize or spraying drying promptly get the Semen Cucumidis sativi polypeptide that ferments.
2. according to the preparation method of the said fermentation Semen Cucumidis sativi of claim 1 polypeptide, it is characterized in that the organic solvent described in the step 1 is sherwood oil, normal hexane or ether.
3. according to the preparation method of the said fermentation Semen Cucumidis sativi of claim 1 polypeptide, it is characterized in that in the step 1 according to quality and volume ratio being that the ratio of 1g: the 8mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent.
4. according to the preparation method of the said fermentation Semen Cucumidis sativi of claim 1 polypeptide, it is characterized in that soaking 18～24h at 10 ℃ in the step 2.
5. the preparation method of fermentation Semen Cucumidis sativi polypeptide, the preparation method of the Semen Cucumidis sativi polypeptide that it is characterized in that fermenting is following:
One, be that the ratio of 1g: the 2～10mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent according to quality and volume ratio; Soak 18～24h at room temperature supersound extraction 20～60min or at 0～20 ℃; Filter; Discard filtrating, filter residue dry below 40 ℃, is obtained the fermentation Semen Cucumidis sativi powder of degreaser drying;
Two, be that the ratio of 1g: 2～5mL adds the fermentation Semen Cucumidis sativi powder of degreaser drying in the entry according to quality and volume ratio, add prozyme again, the prozyme consumption be degreaser drying fermentation Semen Cucumidis sativi opaque amount 1%～4%; Be 6～8 in the pH value, temperature is under 40～60 ℃ the condition; Directional enzymatic 4～10h is at 80～90 ℃ of insulation 5～20min, centrifugal under 3000～5000 rev/mins rotating speed then; Collect supernatant, obtain little peptide liquid;
Three, with the 10000 daltonian ultra-filtration membrane ultrafiltration of little peptide liquid warp, concentrated filtrate, lyophilize or spraying drying promptly get the Semen Cucumidis sativi polypeptide that ferments.
6. according to the preparation method of the said fermentation Semen Cucumidis sativi of claim 5 polypeptide, it is characterized in that the prozyme described in the step 1 is that two or three in trypsinase, papoid and the neutral protease formed by arbitrary proportion.
7. according to the preparation method of the said fermentation Semen Cucumidis sativi of claim 5 polypeptide, it is characterized in that the organic solvent described in the step 1 is sherwood oil, normal hexane or ether.
8. according to the preparation method of the said fermentation Semen Cucumidis sativi of claim 5 polypeptide, it is characterized in that in the step 1 according to quality and volume ratio being that the ratio of 1g: the 8mL Semen Cucumidis sativi powder that will ferment joins in the organic solvent.
9. the said fermentation Semen Cucumidis sativi of claim 1 polypeptide is characterized in that said fermentation Semen Cucumidis sativi polypeptide is used for prevention and treatment osteoporosis.
10. the said fermentation Semen Cucumidis sativi of claim 5 polypeptide is characterized in that said fermentation Semen Cucumidis sativi polypeptide is used for prevention and treatment osteoporosis.
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| CN116671638A (en) * | 2023-06-15 | 2023-09-01 | 江西维莱营健高科有限公司 | Casein calcium tablet and preparation method thereof |
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Application publication date: 20120822 |