CN107236775A - A kind of deer SPP1 polypeptide and application - Google Patents

A kind of deer SPP1 polypeptide and application Download PDF

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Publication number
CN107236775A
CN107236775A CN201710412957.0A CN201710412957A CN107236775A CN 107236775 A CN107236775 A CN 107236775A CN 201710412957 A CN201710412957 A CN 201710412957A CN 107236775 A CN107236775 A CN 107236775A
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deer
polypeptide
spp1
enzymolysis
decalcification
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不公告发明人
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Ding Chengbei
Shenzhen Jhihben Kang Industry Co Ltd
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Ding Chengbei
Shenzhen Jhihben Kang Industry Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/06Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23LFOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17Amino acids, peptides or proteins
    • A23L33/18Peptides; Protein hydrolysates
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12GWINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G3/00Preparation of other alcoholic beverages
    • C12G3/04Preparation of other alcoholic beverages by mixing, e.g. for preparation of liqueurs
    • AHUMAN NECESSITIES
    • A23FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23VINDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00Food compositions, function of food ingredients or processes for food or foodstuffs

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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention discloses a kind of deer SPP1 polypeptide, the preparation method of the deer SPP1 polypeptide includes successively:Degreasing, decalcification step, microwave radiation technology alkali carries, microwave radiation technology alkali enzymolysis, microwave radiation technology neutrality enzymolysis and trypsin digestion and concentration step.The deer SPP1 polypeptide has higher oligopeptides ratio, with higher inoxidizability and antifatigue effect of refreshing oneself.The deer SPP1 polypeptide can be used for preparing on health products, its health preserving wine prepared, with higher stability.

Description

A kind of deer SPP1 polypeptide and application
Technical field
The present invention relates to health treatment technical field, more particularly to a kind of deer SPP1 polypeptide and application.
Background technology
Animal Bone active component is mainly polypeptide and some protein such as ostosis albumen and bone growth factor etc..Deer Bone is generally the bone of Cervidae sika deer and red deer animal.Traditional Chinese medical science information, with qi-restoratives win, strengthening the bones and muscles, blood-enrich, wind-dispelling The function of dehumidifying, for diseases such as rheumatalgia pain, impotence, the enuresis, oedema.Colla 0ssis Cervi is to eipathia asthenia, marrow deficiency, anaemia, wind The diseases such as wet, limbs pain have preferable clinical efficacy.
Because Colla 0ssis Cervi has huge molecular weight, the colloidal stability formed in alcohol water blend is poor, chance temperature, The change stability of oxidation-reduction potential etc. is easily destroyed, and produces loss of gloss, muddiness, deposited phenomenon, and this is in food and drink wine (dew Wine) in be flagrant.On the other hand, may in velvet deerhorn and tortoise plastron liquor process for preparation because Colla 0ssis Cervi has huge molecular weight The loss caused is as follows:1) precipitated with the tannin in Chinese medicine and polyphenols formation;2) formed under certain alcohol concentration Precipitation;3) precipitation is formed in traditional aging process;4) loss of gloss, turbidity and precipitation are formed when temperature declines suddenly, vinosity is influenceed.
Collagen polypeptide is that collagen or gelatin handle obtained product by proteasome degradation, is had compared with gelatin stronger Water solubility, is easily absorbed by the body.With progress of research, it has been found that many skins of collagen have more physiological function, for example, resist Hypertension, prevention and treatment osteoarthritis and osteoporosis, treatment gastric ulcer etc. disease, anti-aging and anti-oxidant etc..
Modern study finds that the anti-inflammatory active ingredient in deer bone is polypeptide.
The content of the invention
In order to overcome the deficiencies in the prior art, an object of the present invention is to provide a kind of deer SPP1 polypeptide.
The second object of the present invention is the application for providing the deer SPP1 polypeptide.
An object of the present invention adopts the following technical scheme that realization:
A kind of deer SPP1 polypeptide, it is characterised in that its preparation method includes successively:
Defatting step:Deer aggregate is crushed to 60-80 mesh, loads filter screen, after being rinsed repeatedly with distilled water, uses degreasing agent Degreasing is carried out, dries, obtains bone tankage;
Decalcification step:Bone tankage is washed with decalcifying agent, decalcification bone meal is obtained;
Extraction step:Dilute NaOH solution is added into decalcification bone meal to pH=12-13,50-60 DEG C of microwave radiation technology is carried Take, obtain first extract;
Alkali enzymolysis step:Watery hydrochloric acid is added into first extract to pH=9-10, alkali protease, 55-65 DEG C of microwave is added Assistance enzymolysis, obtains primary enzymolysis liquid;
Enzymolysis step again:Neutral proteinase and trypsase, 45-55 DEG C of microwave-assisted enzyme are added into primary enzymolysis liquid Solution, is heated to 80-90 DEG C of inactivation, stands, and filtering obtains polypeptide liquid;
Concentration step:Refining liquid is concentrated under reduced pressure into Brix for 45-55 °, the body of paste of deer-bone collagenous polypeptide is produced.
Further, in defatting step, degreasing agent is chloroform:Ethanol:Water=(1-2):(1-2):1.
Further, in decalcification step, decalcifying agent is 0.5mol/L EDTA.
Further, in decalcification step, decalcifying agent is 0.5mol/L watery hydrochloric acid.
Further, extraction step and/or alkali enzymolysis step and/or again in enzymolysis step, microwave frequency is 1500- 3000MHz, power is 5kW.
Further, in alkali enzymolysis step, the addition of alkali protease is 2000-3000U/g decalcification bone meal.
Further, again in enzymolysis step, the addition of neutral proteinase is 2000-3000U/g decalcification bone meal.
Further, again in enzymolysis step, the addition of trypsase is 3000-4000U/g decalcification bone meal.
Further, using micro porous filtration.
The second object of the present invention adopts the following technical scheme that realization:
The application of the deer SPP1 polypeptide on health products are prepared.
Compared with prior art, the beneficial effects of the present invention are:
(1) molecular structure for the deer SPP1 polypeptide that the present invention is provided is more uniform, and the accounting of oligopeptides is high, it is easy to absorb;Should Deer SPP1 polypeptide has preferable antifatigue and antioxidation activity, with preferable effect;
(2) the deer SPP1 polypeptide that provides of the present invention has higher stability after being used to preparing health preserving wine, is difficult to sink Form sediment, good quality.
Embodiment
Below, with reference to embodiment, the present invention is described further, it is necessary to which explanation is, what is do not collided Under the premise of, new embodiment can be formed between various embodiments described below or between each technical characteristic in any combination.
In detailed description below, such as non-specified otherwise, the reagent or material used can be by commercially available or conventional The mode of research technique is obtained.
The present invention provides a kind of deer SPP1 polypeptide, and its preparation method includes successively:
Defatting step:Deer aggregate is crushed to 60-80 mesh, loads filter screen, after being rinsed repeatedly with distilled water, uses degreasing agent Degreasing is carried out, dries, obtains bone tankage;
Decalcification step:Bone tankage is washed with decalcifying agent, decalcification bone meal is obtained;
In the degreasing and decalcification step, it can effectively remove liposoluble constituent in deer aggregate and calcareous, preparation can be reduced Process, the loss of collagen improves the yield of polypeptide.Degreasing agent can selected from including but not limited to chloroform and The mixed aqueous solution of ethanol, such as chloroform:Ethanol:Water=(1-2):(1-2):1, it can be lost using this kind of degreasing agent On the basis of SPP1, the liposoluble constituent in aggregate is effectively removed.Decalcifying agent can be selected from EDTA or watery hydrochloric acid, the former with Chelation is precipitated with calcium formation, and the latter's dissolving is calcareous, under the 0.5mol/L concentration ranges, to the damageability of deer SPP1 It is small;
Extraction step:Dilute NaOH solution is added into decalcification bone meal to pH=12-13,50-60 DEG C of microwave radiation technology is carried Take, obtain first extract;
In this step, by the way of sig water is extracted, it can be efficiently reduced while extraction efficiency is ensured to deer bone The loss of albumen;And excessive concentration, it is possible to albuminous degeneration is caused, concentration is too low, extraction efficiency is not high;
Alkali enzymolysis step:Watery hydrochloric acid is added into first extract to pH=9-10, alkali protease, 55-65 DEG C of microwave is added Assistance enzymolysis, obtains primary enzymolysis liquid;
Enzymolysis step again:Neutral proteinase and trypsase, 45-55 DEG C of microwave-assisted enzyme are added into primary enzymolysis liquid Solution, is heated to 80-90 DEG C of inactivation, stands, and filtering obtains polypeptide liquid;
Alkali enzymolysis step and again enzymolysis step are combined, the trend that on the one hand effectively can be changed using system pH, more thorough Digest to bottom, to obtain the polypeptide that strand is shorter, be more easy to absorption, that is, add after alkali enzyme enzymolysis, under system pH slightly has Drop, adds neutral proteinase and trypsin digestion, two kinds of enzymes enzymatic activity under the pH value of system reaches higher level;This In step, filter type can using conventional multilayer gauze natural filtration, in order to a step high protein polypeptide liquid homogeneity, can Using micro porous filtration, optional 22 μm of the aperture of micro porous filtration;
Concentration step:Refining liquid is concentrated under reduced pressure into Brix for 45-55 °, the body of paste of deer-bone collagenous polypeptide is produced, Body of paste made from this step has preferable stability, not mutability.
Embodiment 1:
A kind of deer SPP1 polypeptide, its preparation method includes successively:
Defatting step:Deer aggregate is crushed to 60-80 mesh, loads the filter being formed by stacking by three layers of aperture for the gauze of 22 mesh Net, after being rinsed repeatedly with distilled water, uses chloroform:Ethanol:Water=1.5:1.5:1 carries out degreasing, dries, obtains degreasing bone Powder;
Decalcification step:Bone tankage is washed with 0.5mol/L EDTA, decalcification bone meal is obtained;
Extraction step:Dilute NaOH solution is added into decalcification bone meal to pH=12-13,55 DEG C of microwave radiation technologies are extracted, Obtain first extract;Wherein microwave frequency is 1500-3000MHz, and power is 5kW;
Alkali enzymolysis step:Watery hydrochloric acid is added into first extract to pH=9-10, is added with the feed molar ratio of 2500U/g decalcification bone meal Enter Alcalase alkali proteases, 60 DEG C of microwave radiation technology enzymolysis obtain primary enzymolysis liquid;Wherein microwave frequency is 2000MHz, work( Rate is 5kW;
Enzymolysis step again:Neutral proteinase and trypsase are added into primary enzymolysis liquid, 50 DEG C of microwave radiation technologies are digested, 85 DEG C of inactivations are heated to, stands, using 22 μm of micro-pore-film filtrations, obtains polypeptide liquid;The wherein feed molar ratio of neutral proteinase For 2500U/g decalcification bone meal, the feed molar ratio of trypsase is 3500U/g decalcification bone meal;
Concentration step:Refining liquid is concentrated under reduced pressure into Brix for 50 °, the body of paste of deer-bone collagenous polypeptide is produced, receives Rate is 86.6%, and oligopeptides ratio is 77.3%.
Embodiment 2:
A kind of deer SPP1 polypeptide, its preparation method includes successively:
Defatting step:Deer aggregate is crushed to 60-80 mesh, loads the filter being formed by stacking by three layers of aperture for the gauze of 22 mesh Net, after being rinsed repeatedly with distilled water, uses chloroform:Ethanol:Water=1:2:1 carries out degreasing, dries, obtains bone tankage;
Decalcification step:Bone tankage is washed with 0.5mol/LHCl, decalcification bone meal is obtained;
Extraction step:Dilute NaOH solution is added into decalcification bone meal to pH=12-13,60 DEG C of microwave radiation technologies are extracted, Obtain first extract;Wherein microwave frequency is 1500-3000MHz, and power is 5kW;
Alkali enzymolysis step:Watery hydrochloric acid is added into first extract to pH=9-10, is added with the feed molar ratio of 2000U/g decalcification bone meal Enter Alcalase alkali proteases, 65 DEG C of microwave radiation technology enzymolysis obtain primary enzymolysis liquid;Wherein microwave frequency is 2000MHz, work( Rate is 5kW;
Enzymolysis step again:45 DEG C of microwave radiation technology enzymolysis of neutral proteinase and trypsase are added into primary enzymolysis liquid, 80 DEG C of inactivations are heated to, stands, using 22 μm of micro-pore-film filtrations, obtains polypeptide liquid;The wherein feed molar ratio of neutral proteinase For 3000U/g decalcification bone meal, the feed molar ratio of trypsase is 4000U/g decalcification bone meal;
Concentration step:Refining liquid is concentrated under reduced pressure into Brix for 45 °, the body of paste of deer-bone collagenous polypeptide is produced, receives Rate is 85.1%, and oligopeptides ratio is 79.0%.
Embodiment 3:
A kind of deer SPP1 polypeptide, its preparation method includes successively:
Defatting step:Deer aggregate is crushed to 60-80 mesh, loads the filter being formed by stacking by three layers of aperture for the gauze of 22 mesh Net, after being rinsed repeatedly with distilled water, uses chloroform:Ethanol:Water=2:1:1 carries out degreasing, dries, obtains bone tankage;
Decalcification step:Bone tankage is washed with 0.5mol/L EDTA, decalcification bone meal is obtained;
Extraction step:Dilute NaOH solution is added into decalcification bone meal to pH=12-13,60 DEG C of microwave radiation technologies are extracted, Obtain first extract;Wherein microwave frequency is 1500-3000MHz, and power is 5kW;
Alkali enzymolysis step:Watery hydrochloric acid is added into first extract to pH=9-10, is added with the feed molar ratio of 2000U/g decalcification bone meal Enter Alcalase alkali proteases, 55 DEG C of microwave radiation technology enzymolysis obtain primary enzymolysis liquid;Wherein microwave frequency is 2000MHz, work( Rate is 5kW;
Enzymolysis step again:45 DEG C of microwave radiation technology enzymolysis of neutral proteinase and trypsase are added into primary enzymolysis liquid, 80 DEG C of inactivations are heated to, stands, using 22 μm of micro-pore-film filtrations, obtains polypeptide liquid;The wherein feed molar ratio of neutral proteinase For 2000U/g decalcification bone meal, the feed molar ratio of trypsase is 3000U/g decalcification bone meal;
Concentration step:Refining liquid is concentrated under reduced pressure into Brix for 55 °, the body of paste of deer-bone collagenous polypeptide is produced, receives Rate is 84.7%, and oligopeptides ratio is 75.4%.
Comparative example 1:
As different from Example 1, extraction step does not use microwave radiation technology to comparative example 1, and yield is 54.1%, oligopeptides ratio For 80.4%.
Comparative example 2:
As different from Example 1, alkali enzymolysis step and again enzymolysis step do not use microwave radiation technology to comparative example 2, receive Rate is 76.4%, and oligopeptides ratio is 61.7%.
Comparative example 3:
Comparative example 3 as different from Example 1, extraction step, alkali enzymolysis step and again enzymolysis step using micro- Ripple is aided in, and yield is 56.4%, and oligopeptides ratio is 64.9%.
Performance detection and effective evaluation
1st, determination oxidative
By 10 μm of ol/L Trolox storing solutions phosphorate phthalate buffer be diluted to concentration for 0.05,0.10,0.20, 0.40th, 0.80,1.6 μm of ol/L standard liquid, using phosphate buffer as negative control, using fluorescence half-life as vertical seat Mark, using Trolox concentration as abscissa, draws standard curve.
0.01g embodiment 1-3 deer SPP1 polypeptide is taken, using distilled water as blank control, plus 100mL phosphate delays Fliud flushing is diluted, then be respectively adopted phosphate buffer dilution by 10 times, 100 times, 1000 times, 10000 times, 100000 times it is dilute Release, dilution is measured, its result is as shown in the table:
The oxidation resistance test result of table 1
2nd, mouse, which bears a heavy burden, tests
Using SPF male mices, body weight is 18-22g, per test group 10, totally 40, and swimming with a load attached to the body survey is carried out respectively Fixed, environment temperature is 22-24 DEG C, humidity 50-54% during experiment.The deer bone egg polypeptide obtained using embodiment 1, setting dosage For 0.1g/kg.bw, 0.2g/kg.bw, 0.5g/kg.bw, using aqua sterilisa as control group, mix food and feed 30 days, carry out swimming with a load attached to the body Experiment.
The Loaned swimming test of table 2
After gavage is fed 30 days, each dosage group mouse, the walking weight load relative to control group mice is obviously prolonged.Say The deer SPP1 polypeptide that bright the application is provided, which has, improves body fatigue resistance effect.
3rd, stability test
By embodiment 1-3 and comparative example 1-3 deer SPP1 polypeptide with 1:10 proportioning is added in 50 ° of white wine, concussion Dissolving, obtains the health preserving wine of deer SPP1 polypeptide, takes 1L health preserving wines with 3000rpm centrifugation 5min, observation health preserving wine Outward appearance and whether there is precipitation, measure precipitation volume, it is as a result as shown in the table:
The stability test result of table 3
As shown above, the deer SPP1 polypeptide that this application is obtained has higher dynamic stability, the polypeptide Prepare and be unlikely to deteriorate after health preserving wine, be not likely to produce precipitation, quality is higher.
Above-mentioned embodiment is only the preferred embodiment of the present invention, it is impossible to limit the scope of protection of the invention with this, The change and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention Claimed scope.

Claims (10)

1. a kind of deer SPP1 polypeptide, it is characterised in that its preparation method includes successively:
Defatting step:Deer aggregate is crushed to 60-80 mesh, loads filter screen, after being rinsed repeatedly with distilled water, is carried out using degreasing agent Degreasing, dries, obtains bone tankage;
Decalcification step:Bone tankage is washed with decalcifying agent, decalcification bone meal is obtained;
Extraction step:Dilute NaOH solution is added into decalcification bone meal to pH=12-13,50-60 DEG C of microwave radiation technology is extracted, obtained To first extract;
Alkali enzymolysis step:Watery hydrochloric acid is added into first extract to pH=9-10, alkali protease, 55-65 DEG C of microwave radiation technology is added Enzymolysis, obtains primary enzymolysis liquid;
Enzymolysis step again:Neutral proteinase and trypsase are added into primary enzymolysis liquid, 45-55 DEG C of microwave radiation technology is digested, 80-90 DEG C of inactivation is heated to, is stood, filtering obtains polypeptide liquid;
Concentration step:Refining liquid is concentrated under reduced pressure into Brix for 45-55 °, the body of paste of deer-bone collagenous polypeptide is produced.
2. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that in defatting step, the degreasing agent is three chloromethanes Alkane:Ethanol:Water=(1-2):(1-2):1.
3. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that in decalcification step, the decalcifying agent is 0.5mol/ L EDTA.
4. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that in decalcification step, the decalcifying agent is 0.5mol/ L watery hydrochloric acid.
5. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that extraction step and/or alkali enzymolysis step and/or again In secondary enzymolysis step, microwave frequency is 1500-3000MHz, and power is 5kW.
6. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that in alkali enzymolysis step, the addition of alkali protease Measure as 2000-3000U/g decalcification bone meal.
7. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that again in enzymolysis step, the neutral proteinase Addition be 2000-3000U/g decalcification bone meal.
8. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that again in enzymolysis step, the trypsase Addition is 3000-4000U/g decalcification bone meal.
9. deer SPP1 polypeptide as claimed in claim 1, it is characterised in that again in enzymolysis step, using micro porous filtration.
10. application of the deer SPP1 polypeptide on health products are prepared as described in claim any one of 1-9.
CN201710412957.0A 2017-06-05 2017-06-05 A kind of deer SPP1 polypeptide and application Pending CN107236775A (en)

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CN107828841A (en) * 2017-11-06 2018-03-23 天津市天大天福生物技术有限公司 The extracting method of a kind of collagen and collagen polypeptide and the application in cosmetics
CN107897943A (en) * 2017-11-28 2018-04-13 苏洁 A kind of method that deer bone native peptides are extracted in the bone from deer
CN108396052A (en) * 2018-03-28 2018-08-14 通化百泉保健食品有限公司 A kind of industrialized producing technology of deer's sinew albumen oligopeptide
CN108623653A (en) * 2018-04-27 2018-10-09 罗乌支 A method of it extracting antler native peptides from antler and enzymolysis prepares polypeptide

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107828841A (en) * 2017-11-06 2018-03-23 天津市天大天福生物技术有限公司 The extracting method of a kind of collagen and collagen polypeptide and the application in cosmetics
CN107897943A (en) * 2017-11-28 2018-04-13 苏洁 A kind of method that deer bone native peptides are extracted in the bone from deer
CN108396052A (en) * 2018-03-28 2018-08-14 通化百泉保健食品有限公司 A kind of industrialized producing technology of deer's sinew albumen oligopeptide
CN108623653A (en) * 2018-04-27 2018-10-09 罗乌支 A method of it extracting antler native peptides from antler and enzymolysis prepares polypeptide

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