CN109207543A - A kind of ox bone collagen protein peptides and preparation method and purposes - Google Patents
A kind of ox bone collagen protein peptides and preparation method and purposes Download PDFInfo
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- CN109207543A CN109207543A CN201811144957.8A CN201811144957A CN109207543A CN 109207543 A CN109207543 A CN 109207543A CN 201811144957 A CN201811144957 A CN 201811144957A CN 109207543 A CN109207543 A CN 109207543A
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- bone
- collagen protein
- protein peptides
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- collagen
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, 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
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
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Abstract
The present invention relates to food engineering and field of biotechnology more particularly to a kind of ox bone collagen protein peptides and preparation method and purposes.Of the invention is crushed the fresh bone of ox, the meat for removing fascia using complete physical method and connecting with bone photo, degreasing, obtain white or milky clean ox bone, ox bone is crushed, is sieved with 100 mesh sieve, bovine bone powder is obtained, autoclaving bovine bone powder, collagen is extracted, then with trypsase and a kind of compounding alkali protease of protease-, the mixture of neutral proteinase and maltodextrin, stepwise discretization is carried out, ox bone peptide stoste is obtained.This preparation process uses trypsase and compounding two kinds of enzymes of protease to digest bone meal in two times for the first time, and the peptide obtained in this way can both make 85% or more collagen peptide molecular weight < 1000Da without separation.
Description
Technical field
The present invention relates to food engineering and field of biotechnology more particularly to a kind of ox bone collagen protein peptides and preparation sides
Method and purposes.
Background technique
Currently, aging of population has become global phenomenon, along with the aging of population, sufferers of osteoporosis face
Quantity also sharply increases, and osteoporosis has become global serious problems.
Osteoporosis is a kind of whole body bone amount reduction, and bone microstructure increases bone brittleness by destruction, causes very
The disease easily fractured when small wound.Osteoporosis (Osteoporosis) word is that Pommer was proposed first in 1885
Come, the meaning is a kind of osteopenic disease.It is physiological aging one occurred with the increase at age that sclerotin, which is reduced,
A feature, bone is calm in being formed in the matrix being made of collagen by crystallization that calcium ion and phosphorus are constituted, bone matrix and
The ratio between bone mineral is in certain state.Bone matrix does not change and bone mineral is reduced or the state of bone calcification obstacle is known as osteomalacia
Disease, bone matrix and bone mineral chemistry form unchanged and measure reduced state and be known as osteoporosis, have scholar to think that sclerotin is dredged
Pine will lead to bone tissue microstructure and change and the increase of risk of fractures degree.Bone amount one standard deviation of every reduction is thought in research, fractures
Risk increases 1.5-3 times, and women than men is more dangerous, and lifelong risk is 3 times of male.This between male and female
Difference is lower than male with women peak bone mass, and quick bone loss occurs again for post menopausal and women than men is long-lived, women than men
The factors such as be easier to fall are related.China in 1999 steps into veteran form population country ranks, and elderly population account for world's elderly population
21% or so, it is the country that elderly population are most in the world.By 2017 the end of the year China 60 years old and the above aged up to 2.4
Hundred million, account for 17.3% of country's total population or more, it is contemplated that the year two thousand thirty, China aged is more than the 20% of total population and enters high
Ageing stage is spent, after the year two thousand forty, China the elderly will be more than 400,000,000.Elderly population quickly increase in this way and aging, make
Osteoporosis and the adjoint incidence that osteoporotic fracture occurs also increase substantially.Therefore, every country prevention and
The expense rating for treating osteoporotic fracture is huge, and osteoporosis and osteoporotic fracture seriously affect personal and family
Quality of life, so pre- preventing bone rarefaction in advance, repairing the Bones and joints problem ahead of time is particularly important, and is mended in the market
Calcium increases the drug of bone density, and health care product comes into being.
Ox bone is the bone of bovid ox or buffalo, is recorded according to Compendium of Material Medica, " ox bone, sweet, warm are nontoxic." I
The eating habit that state has always Os Bovis seu Bubali to stew soup, especially pregnant woman, the elderly and the patient of bone impingement etc..After often having fracture
The habit of diet bone soup makes fracture heal early, in middle and advanced stage, that is, primary callus shape of union to supplement calcium
At phase and remoulding stage, since gradually calcification forms area of new bone for the osteoid tissue of organic fiber, reinforces transformation, formed just
The ossification of normal bone, osteoid tissue needs sufficient calcium, phosphorus matter, and it is very helpful for mending food bone soup at this time.
Early in the seventies, the country such as Japan and West Europe, which just has, does bone mud exclusively with live stock and fowl bones such as pig bone, ox bone and fish-bones
(West Europe claims bone sauce), product are sold to the whole world.The eighties, China also start voluntarily to develop bone mud machine, produce delicate mouthfeel, taste
The delicious bone mud in road, for cooking ham and sausage, meat pulp.With the clay standby bone medicinal extract of bone, various special tastes, fresh perfume (or spice) can be used as
Flavouring, soup product base-material.There are also using bone enzymatic hydrolysis or acid and alkali hydrolysis, matching for the flavouring such as animal hydrolyzed protein is made
Material.Ox bone just has the record being used as medicine early in Chinese Tang Dynasty, paddy dysentery of 1. harnessing the river: ox bone ash is fried with bent, equal part, for end, drink-service side
Very little an ancient type of spoon (Zhang Wenzhong)；2. controlling raw sore in nose: ox, dog bone ashing apply its (" prescriptions worth thousand gold ") with twelfth month lard jointly；3. controlling the erosion of infantile malnutrition due to digestive disturbances or intestinalparasites sore
Enter mouth and nose: ox bone ashing applies (" ten just good recipe ") with lard.
Ox bone is stewed soup in daily life as food, and ox bone crushing makes as raw materials of food processing in food processing
With.Ox bone both can be used as food and food auxiliary material, be also used as drug, now mostly use as food.Document report [1] ox
Protein rich in, fat, chondroitin in bone, the minerals such as calcium, iron, phosphorus, zinc and vitamin.Bone can be with the side of full bone
Formula, such as bone mud, bone meal form are utilized, can also be in the form of extract, such as ossein, gelatine, animal oil, calcium phosphorus product shape
Formula is utilized.Protein content is about 20% or so in ox bone, and the ox of different cultivars, protein content slightly has difference.Contain in ox bone
There is amino acid in 17, and hydroxyproline content is abundant, hydroxyproline is the significant ingredient of collagen.Collagen is animal
The main component of connective tissue (bone, cartilage, skin, chamber, tough etc.) accounts for about the 1/3 of gross protein in vertebrate, forms one
The very unique three strands of superhelixes of kind, property is sufficiently stable, and general processing temperature and short time heating cannot all make it
It decomposes.Collagen polypeptide is low molecular weight product obtained after collagen or gelatin degradation, not only has digestion well
Absorption characteristic also has the function of the special physiological different from collagen.Studies at home and abroad show that collagen polypeptide has protection stomach
Mucous membrane, inhibits blood pressure to rise at antiulcer, nutrition and the physiological function such as promoting bone growing, skin collagen metabolism.Ox bone is digested to obtain
Collagen peptide, studies have shown that collagen polypeptide can directly be absorbed in enteron aisle, therefore the often edible food containing collagen polypeptide
Product can effectively increase the storage capacity of skin tissue cell, enhancing and the maintenance good elasticity of skin, strengthen the toughness of skin,
Delay the aging of body, at the same can also strengthening the muscles and bones, build up health.Collagen is a kind of important functional protein, it
It is closely related with hyperplasia, differentiation, movement, immune, joint lubrication, wound healing etc., directly or indirectly affect us
Growth, aging and health of human body etc..Collagen has diversity and Tissue distribution specificity, is related with each organ-tissue
Functional protein.Further, since its antigenicity is weaker, biocompatibility is preferable, and some protein surfaces have antigenic characteristic,
There is allergic symptom after many people are edible, this greatly reduces the scope of application and nutritive value of protein.Therefore it just drops significantly
The utilization rate of low protein raw materials causes the loss of raw material, also and then affects the yield of protein-based product.Related research report
Point out that the protein peptides that albumen obtains after protease hydrolytic, antigenicity is far below the antigenicity of pure protein, through clinical test in road
It proves, allergic reaction caused by polypeptide is obviously reduced compared with the body allergic reaction caused by the albumen.Therefore, guaranteeing albumen
The antigenicity for weakening albumen on the basis of nutritional need meets the needs to protein allergies crowd with the protein peptides of low antigenicity.
The child resistant of infant period is low, it is easier to which to the food irritability based on albumen, therefore the protein peptides of low antigenicity are also very
Suitable for production baby food, health food, medical supplies etc..
Collagen family includes 19 kinds of collagens and 10 kinds or more collagen sample albumen [4].In connective tissue, different
The collagen of type has the function of different.Several frequently seen collagen has: Type I collagen albumen, II Collagen Type VI, III Collagen Type VI,
Type Ⅳ collagen, V Collagen Type VI.Wherein with increase bone density it is closely related be Type I collagen albumen.
Type I collagen is that fiber forms collagen, accounts for about organic principle 80%~90%, accounts for about the 20% of gross protein quality,
It is the primary structure macromolecular of multicellular organism extracellular matrix, energy activated epithelial promotes epithelial hyperplasia, can also promote
It is generated into clostridiopetidase A, keeps skin tensioned and elastic.Type I collagen albumen is to the complete and bone biomechanical characteristic for maintaining bone structure
It is extremely important.Osteoporosis main feature shows as the reduction of whole body bone amount and bone tissue fine structure is degenerated, and bone brittleness is caused to increase
Sum it up the metabolic bone disease that bone density reduces.Collagen (referring mainly to I type) structure, content and stable sexual abnormality and osteoporosis
The generation of disease, development, severity are closely related.Type I collagen exists in bone more, therefore I type glue can be extracted from ox bone
Former protein peptides improve bone density, and the white peptide of collagen peptide plays skeleton supporting function in vivo.
Chinese invention patent application (CN201610001744.4) discloses a kind of preparation method of ox bone collagen albumen, packet
Include processing step: cleaning ox bone crushes ox bone, and degreasing, decalcification handle bovine bone powder, digest to bovine bone powder, carry out at 100 DEG C
Destroy the enzyme treatment, filtering obtain collagen solution, active carbon decolorization, and concentration obtains collagen.This method uses
Bovine bone powder is directly digested, and collagen amount to obtain is inadequate, and impurity is more.
Chinese invention patent application (CN201510556199.0) discloses a kind of preparation method of ox bone collagen protein peptides,
It is related to a kind of preparation method of protein peptides.The main object of the present invention is broken, secondary twice by successively using to ox bone
It boils again, thermophilic digestion and ultrafiltration membrane concentration technique and combine specific complex enzyme formulation, solve existing production technology and prepare ox bone
Collagen peptides extraction rate is low, and easy brown stain, energy consumption is high, the problem of production cycle length.Method: one, it screens；Two, primary fragmentation；
Three, second-time breakage；Four, it cleans；Five, high temperature extracts；Six, secondary to boil again；Seven, it is separated by filtration；Eight, Liquid liquid Separation；Nine, it digests；
Ten, sterilising and enzyme inactivating；11, it is separated by solid-liquid separation；12, ultrafiltration membrane is concentrated, 13, spray drying；14, it packs to get ox bone is arrived
Collagen peptide.
Chinese invention patent application (CN201710823158.2) discloses ox bone collagen protein peptides extracting method.Comprising such as
Lower step, one, osteoclastic；Two, feeding cleans；Three, feeding boiling, row's soup；Four, hydrolysed filtrate；Five, it is concentrated；Six, drying is dusted.
The ox bone collagen albumen peptides extraction rate that above-mentioned method obtains is inadequate, and micromolecule active polypeptide is insufficient.
Summary of the invention
In order to solve the above technical problems, the object of the present invention is to provide a kind of preparation sides of ox bone collagen protein peptides
Method improves the recovery rate of ox bone collagen albumen, the polypeptide molecular weight < 1000Da that hydrolysis bovine bone powder decoction liquor obtains.The present invention
Obtaining ox bone collagen protein peptides not only can be improved bone density, but also can increase bone toughness, elasticity, be the battalion of bone protection
Support agent.
In order to achieve the above purpose, present invention employs technical solutions below:
A kind of preparation method of ox bone collagen protein peptides, this method include the following steps:
1) a certain amount of fresh ox bone through inspection and quarantine qualification, is taken, is crushed, isometric water, boiling is added in cleaning
0.5-2h；
2), the ox bone cooking liquor that step 1) obtains is discarded, takes out ox bone, removes the meat adhered on bone, fascia and bone
The intracavitary grease of bone, then hot water cleans 2-3 times, to bone is white or milky, be visible by naked eyes grease until, done
Net ox bone；
3) it, by ox bone obtained in step 2), crushes, crosses 100 mesh standard sieves, obtain ox bone fine powder；
4), by ox bone fine powder obtained in step 3), the water of 5-10 times of quality, high temperature and pressure 40min~2h, filtering is added
Supernatant is taken, ox bone collagen protein liquid is obtained；
5), by ox bone collagen protein liquid obtained in step 4), the trypsase of 1%-3% is added, adjusting pH value is
7.0-8.0, digests 4-7h, by enzyme-deactivating by 40-50 DEG C of temperature；
6) enzymolysis liquid in step 5), is added to alkali protease, neutral proteinase and the maltodextrin of 0.5%-2.5%
Mixture, adjusting pH value be 5.0-6.0,40-50 DEG C of temperature, digest 4-7h, by enzyme-deactivating；Obtain ox bone peptide stoste.
As a further improvement, step 3) crushes ox bone, dried in baking oven, coarse crushing bovine bone powder is dried again, into one
Step crushes, and obtains ox bone fine powder.
As a further improvement, the partial size of the ox bone fine powder in step 3) is 10-150 μm.
As a further improvement, step 5), step 6) enzyme-deactivating use thermophilic digestion or soak.
As a further improvement, the mass ratio of the mixture of step 6) alkali protease, neutral proteinase and maltodextrin
Are as follows: 1~10:1~10:1~5.
As a further improvement, the ox bone collagen peptide stoste that step 6) obtains, is added enzymolysis liquid weight 0.2-0.5% activity
Charcoal is separated in enzymolysis liquid by diatomite, is decolourized and is removed fishy smell, obtains enzymolysis liquid.
The invention also discloses the ox bone collagen protein peptides that the method is prepared.
The polypeptide molecular weight of the ox bone collagen protein peptides is distributed are as follows:
| Serial number | Molecular weight ranges | Molecular weight accounting |
| 1 | < 150Da | 5~15% |
| 2 | 150-600Da | 60~80% |
| 3 | 600-1000Da | 10~20% |
| 4 | > 1000Da | 1~10% |
。
The invention also discloses the ox bone collagen protein peptides be used to prepare adjust the drugs for the treatment of bone diseases, health care product or
Purposes in food.
In the present invention, a kind of preparation method of ox bone collagen polypeptide has been founded.The present invention is gone using physics degreasing
The method of fascia handles ox stick bone, and ox bone fine powder is made in ox bone, improves the recovery rate of ox bone collagen albumen, uses pancreas egg later
White enzyme, and a kind of compounding alkali protease of protease-, the mixture of neutral proteinase and maltodextrin, sequentially, point
It Shui Xie not the obtained polypeptide molecular weight < 1000Da of bovine bone powder decoction liquor.Bone glue former peptide rises in skeleton netted is adhered work
With being adhered the inorganic matters such as calcium phosphorus, constitute bone matrix.Bone density not only can be improved in the present invention, but also can increase bone toughness, bullet
Property, it is the nutritional agents of bone protection.
Compared with prior art, the invention has the benefit that
(1) in preparation process, ox bone pre-treatment does not use any chemical reagents, is finished purely physical method and carries out ox bone
Pre-treatment, safety non-pollution, and bovine bone powder obtained can compare other techniques by the ox bone fine powder of 100 mesh standard sieves, can
To extract collagen to the full extent,；Two kinds of suitable hydrolases are filtered out in multiple protein enzyme, by orthogonal test,
Enzymatic hydrolysis optimised process is obtained, determines enzymatic hydrolysis sequencing, the small activity of ox bone peptide molecular weight obtained by this technique is high, molecular weight
< 1000Da compares other enzymolysis process, this simple process is easy to operate, and what it is due to selected biological enzyme and substrate is suitable for
Property, the small peptide obtained after enzymatic hydrolysis, requires no separation, molecular weight is with regard to Relatively centralized and < 1000Da.
(2) preparation process of composition provided by the invention simplifies, with short production cycle, is given birth to using complete physics mode
Processing is produced, processing waste material is used as animal feed again, is a kind of processing side that is environmentally protective and being extremely suitable to large-scale production
Formula.
Detailed description of the invention
Fig. 1 is the preparation flow figure of the embodiment of the present invention 1.
Fig. 2, Fig. 3 are the preparation ox bone collagen polypeptide mass spectrogram of the embodiment of the present invention 1.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
Preparation method:
(1), a certain amount of fresh ox bone through inspection and quarantine qualification is taken, is crushed, isometric water, boiling is added in cleaning
0.5h。
(2), the ox bone cooking liquor that step (1) obtains is discarded, taking-up ox bone, the meat adhered on removal bone, fascia, and
The intracavitary grease of bone, then hot water cleans 2 times, to bone is white or milky, be visible by naked eyes grease until, done
Net ox bone.
(3), it by ox bone obtained in step (2), crushes, crosses 100 mesh standard sieves, obtain ox bone fine powder.
(4), by ox bone fine powder obtained in step (3), the water of 5 times of quality is added, high temperature and pressure 40min is filtered to take
Clearly, ox bone collagen protein liquid is obtained.
(5), by ox bone collagen protein liquid obtained in step (4), 2% trypsase is added, adjusting pH value is 8.0,
40 DEG C of temperature, 5h is digested, by enzyme-deactivating.
(6), the compounding protease-that the enzymolysis liquid in step (5) is added 2% had into alkali protease, neutral proteinase
With the mixture of maltodextrin, the mass ratio of the mixture of alkali protease, neutral proteinase and maltodextrin are as follows: 2:2:1 is adjusted
Saving pH value is 6.0,40 DEG C of temperature, 5h is digested, by enzyme-deactivating.Obtain ox bone peptide stoste.
(7), the ox bone peptide stoste for obtaining step (6) crosses film, obtains the small peptide of molecular weight < 1000Da.
Polypeptide distribution situation after enzymatic hydrolysis
| Serial number | Molecular weight ranges | Molecular weight accounting |
| 1 | < 150Da | 10% |
| 2 | 150-600Da | 70% |
| 3 | 600-1000Da | 15% |
| 4 | > 1000Da | 5% |
Embodiment 2
Preparation method:
(1), a certain amount of fresh ox bone through inspection and quarantine qualification is taken, is crushed, isometric water, boiling is added in cleaning
1h。
(2), the ox bone cooking liquor that step (1) obtains is discarded, taking-up ox bone, the meat adhered on removal bone, fascia, and
The intracavitary grease of bone, then hot water cleans 3 times, to bone is white or milky, be visible by naked eyes grease until, done
Net ox bone.
(3), it by ox bone obtained in step (2), crushes, crosses 100 mesh standard sieves, obtain ox bone fine powder.
(4), by ox bone fine powder obtained in step (3), the water of 6 times of quality is added, high temperature and pressure 40min is filtered to take
Clearly, ox bone collagen protein liquid is obtained.
(5), by ox bone collagen protein liquid obtained in step (4), 0.5% trypsase is added, adjusting pH value is
7.0, temperature 50 C digests 7h, by enzyme-deactivating.
(6), the compounding protease-that the enzymolysis liquid in step (5) is added 0.5% had into alkali protease, neutral protein
The mixture of enzyme and maltodextrin, the mass ratio of the mixture of alkali protease, neutral proteinase and maltodextrin are as follows: 2:3:1,
Adjusting pH value is 5.0, and temperature 50 C digests 4h, by enzyme-deactivating.Obtain ox bone peptide stoste.
(7), the ox bone peptide stoste for obtaining step (6) crosses film, obtains the small peptide of molecular weight < 1000Da.
Embodiment 3
Preparation method:
(1), a certain amount of fresh ox bone through inspection and quarantine qualification is taken, is crushed, isometric water, boiling is added in cleaning
1.5h。
(2), the ox bone cooking liquor that step (1) obtains is discarded, taking-up ox bone, the meat adhered on removal bone, fascia, and
The intracavitary grease of bone, then hot water cleans 3 times, to bone is white or milky, be visible by naked eyes grease until, done
Net ox bone.
(3), it by ox bone obtained in step (2), crushes, crosses 100 mesh standard sieves, obtain ox bone fine powder.
(4), by ox bone fine powder obtained in step (3), the water of 10 times of quality is added, high temperature and pressure 2h filters to take supernatant,
Obtain ox bone collagen protein liquid.
(5), by ox bone collagen protein liquid obtained in step (4), 1.5% trypsase is added, adjusting pH value is
7.5,55 DEG C of temperature, 6h is digested, by enzyme-deactivating.
(6), the enzymolysis liquid addition 1.5% in step (5) had into compounding protease-alkali protease, neutral protein
The mixture of enzyme and maltodextrin, the mass ratio of the mixture of alkali protease, neutral proteinase and maltodextrin are as follows: 2:1:1,
Adjusting pH value is 5.5,55 DEG C of temperature, 7h is digested, by enzyme-deactivating.Obtain ox bone peptide stoste.
(7), the ox bone peptide stoste for obtaining step (6) crosses film, obtains the small peptide of molecular weight < 1000Da
Embodiment 4
Composition 1 is formulated: volume: 1 110 parts of ox bone peptide stoste of embodiment is pressed, by weight: 1.5 parts of chondroitin sulfate, D- ammonia
4 parts of base glucose, 1.5 parts of calcium lactate, 0.3 part of casein phosphopeptide.
Composition 2 is formulated: volume: 100 parts of ox bone peptide stoste is pressed, by weight: 1.8 parts of chondroitin sulfate, D- aminoglucose
3.6 parts, 1.2 parts of calcium lactate, 0.2 part of casein phosphopeptide of sugar.
Composition 3 is formulated: volume: 120 parts of ox bone peptide stoste is pressed, by weight: 2 parts of chondroitin sulfate, D- Glucosamine 6
Part, 2 parts of calcium lactate, 0.4 part of casein phosphopeptide.
Composition obtained is taken, 0.02% xanthan gum of composition weight, 0.04%CMC-Na, 0.1%EDTA- is added
2Na mixes, adds 0.02% inspissated juice of composition weight, the oral solution of stable homogeneous is made in 5% honey.
Experimental example
One, zoopery
The above-mentioned composition being prepared is subjected to experimental verification, investigates its effect.
Experimental animal and grouping: the ablactation Wistar male rat for surrounding of being born adapts to after a week, fasting 12 hours, press
Weight is grouped at random, and every group 10.
Dosage regimen: every group of rat is administered 3 months by setting dosage continuous gavage, and deionized water is drunk during administration, is raised
Feed low calcium mouse grain.A weight is surveyed weekly, gives test medicine by new weight.Each group administration fashion and dosage arrangement see the table below.
Bright enlightening calcium carbonate D3Particle, OTC, Beijing Kangyuan Pharmaceutical Co., Ltd.'s production, product batch number 20171207, approval text
Number: national drug standard H20090334
1 each group mouse administrations table of table
1, femur weight measures
Rat is put to death after feeding 3 months, separates right side femur, and in 105 DEG C of baking ovens, drying to constant weight, weighs backbone
Weight.On the right side of each group experimental rat
Influence of the 2 each group drug of table to experimental rat femur weight and length
Note: * is compared with M group (model group), P < 0.05；# is compared with CON group (control group), P < 0.05
It is compared with M group (low calcium model group), positive drug calcium carbonate D3 granules in rats femur length, the shadow of femur weight
It rings, there is significant difference, composition 1, composition 2, composition 3 have significant difference；(normal raising compares with CON group
Group) it compares, the influence tool of positive drug group, composition 2,3 groups of composition of test medicine to rat femur length and femur weight
There is significant difference.
2, femoral strength measurement and calcium ion content measurement
With bone strength instrument, the YLS-16A of Beijing Zhong Shidichuan development in science and technology Co., Ltd independent research is used
The bone strength at toy bone strength analyzer measurement femur midpoint.The determination of femur midpoint measurement point: measurement femur overall length,
By its midpoint, a straight line is drawn along cross-sectional direction, this is femur midpoint measurement point (section).
The measurement of calcium ion content in bone, according to GB5009.92-2016 detection method --- the second method EDTA titration
Measurement
2.1 experimental principle
Within the scope of pH appropriate, calcium and EDTA (disodium ethylene diamine tetraacetate) form metal complex.It is titrated with EDTA,
When reaching stoichiometric point, the color of free indicator is presented in solution.According to EDTA dosage, the content of calcium is calculated.
2.2 reagent
Potassium hydroxide (KOH).
Vulcanized sodium (Na2S).
Sodium citrate (Na3C 6H5O 72H2O).
Disodium ethylene diamine tetraacetate (EDTA, C10H14N2O8Na22H2O).
Hydrochloric acid (HCl): excellent pure grade.
Calred indicator (C21O7N2SH14).
Nitric acid (HNO3): excellent pure grade.
Perchloric acid (HClO4): excellent pure grade.
2.3 preparation of reagents
2.3.1 potassium hydroxide solution (1.25mol/L): weighing 70.13g potassium hydroxide, be diluted with water to 1000mL, mixes
It is even.
2.3.2 sodium sulfide solution (10g/L): weighing 1g vulcanized sodium, be diluted with water to 100mL, mixes.GB5009.92—
2016
2.3.3 sodium citrate solution (0.05mol/L): weighing 14.7g sodium citrate, be diluted with water to 1000mL, mixes.
2.3.4EDTA solution: weighing 4.5gEDTA, be diluted with water to 1000mL, mixes, is stored in polyethylene bottle, 4 DEG C
It saves.10 times are diluted when use.
2.3.5 calred indicator: weighing 0.1g calred indicator, be diluted with water to 100mL, mixes.
2.3.6 hydrochloric acid solution (1+1): 500mL hydrochloric acid is measured, is uniformly mixed with 500mL water.
2.4 standard items
Calcium carbonate (CaCO3, CAS 471-34-1): purity > 99.99%, or through national authentication and authorize reference substance cross-examination
The certain density calcium standard solution of book.
2.5 standard solution are prepared
Standard calcium stock solution (100.0mg/L): 0.2496g (being accurate to 0.0001g) calcium carbonate is accurately weighed, adds hydrochloric acid molten
Liquid (1+1) dissolution, moves into 1000mL volumetric flask, water is added to be settled to scale, mixes.
2.6 analytical procedure
2.6.1 sample digestion
Wet digestion: accurately weighing solid sample 0.2g-3g (being accurate to 0.001g) or accurately pipettes liquor sample
0.500mL-5.00mL is added 10mL nitric acid, 0.5mL perchloric acid, clears up in adjustable electric hot stove in digest tube with a scale
(reference conditions: 120 DEG C/0.5h-120 DEG C/1h, rise to 180 DEG C/2h-180 DEG C/4h, rise to 200 DEG C -220 DEG C).If digestive juice
In sepia, then plus nitric acid, to white cigarette is emitted, digestive juice is in colorless and transparent or yellowish for resolution.Digest tube is taken out, is used after cooling
Water is settled to 25mL, needs to dilute further according to practical measurement, and the lanthanum solution (20g/L) of certain volume is added in dilution,
Make its concentration 1g/L in final dilution, mixing is spare, this is sample prepare liquid.Reagent blank test is done simultaneously.Also
Conical flask can be used, in adjustable electric hot plate, carry out wet digestion by aforesaid operations method.
2.6.2 the measurement of titer (T)
Draw 0.500mL standard calcium stock solution (100.0mg/L) in test tube, add 1 drop sodium sulfide solution (10g/L) and
0.1mL sodium citrate solution (0.05mol/L) adds 1.5mL potassium hydroxide solution (1.25mol/L), adds 3 drop calred indicator,
Immediately to dilute 10 times of EDTA solution titration, until indicator is by purplish red discoloration blue, consumed 10 times of dilution is recorded
EDTA solution volume.The milligram number of calcium is equivalent to according to the EDTA solution that titration results calculate every milliliter of 10 times of dilution,
That is titer (T).
2.6.3 sample and blank titration
0.100mL-1.00mL (depending on the content of calcium) sample digestive juice and blank solution are drawn respectively in test tube, are added
1 drop sodium sulfide solution (10g/L) and 0.1mL sodium citrate solution (0.05mol/L), add 1.5mL potassium hydroxide solution
(1.25mol/L) adds 3 drop calred indicator, immediately to dilute 10 times of EDTA solution titration, until indicator is blue by purplish red discoloration
Until color, the volume of the EDTA solution of consumed 10 times of dilution is recorded.
The statement of 2.7 analysis results
The content of calcium is calculated by formula (1) in sample:
In formula:
X --- the content of calcium in sample, unit be milligrams per kilogram or milligrams per liter (mg/kg or mg/L)；
T --- EDTA titer, unit are every milliliter of milligram (mg/mL)；
V1--- the volume of the EDTA solution of consumed 10 times of dilution when titration sample solution, unit are milliliter (mL)；
V0--- the volume of the EDTA solution of consumed 10 times of dilution when titration blank solution, unit are milliliter (mL)；
V2--- the constant volume of sample digestive juice, unit are milliliter (mL)；
1000 --- conversion coefficient；
M --- sample mass pipettes volume, unit be gram or milliliter (g or mL)；
V3--- the titration volume of sample prepare liquid, unit are milliliter (mL).
Influence of the 3 each group drug of table to experimental rat femoral strength and bone calcium ion content
Note: * is compared with M group (model group), P < 0.05；# is compared with CON group (control group), P < 0.05
It is compared with M group (low calcium model group), the influence of positive drug calcium carbonate D3 granules in rats or so femoral strength has
There is significant difference, composition 1, composition 2, composition 3 have significant difference；With CON group (normally raising control group) phase
Than positive drug group, composition 2, influence of 3 groups of composition of the test medicine to rat or so femoral strength have conspicuousness poor
It is different；It is compared with M group (low calcium model group), the influence of calcium ion content has aobvious in positive drug calcium carbonate D3 granules in rats bone
Sex differernce is write, composition 2, composition 3 also have significant difference, composition 1, than calcium ion content in low calcium model group bone
Height, but do not have significant difference in P < 0.05；Compared with CON group (normally raising control group), positive drug calcium carbonate D3
The influence of calcium ion content has significant difference in granules in rats bone, and composition 3 also has significant difference, composition
2 there was no significant difference, but calcium ion content ratio CON group (normally raising control group) is high, composition 1 and (the normal raising of CON group
Control group) compare no notable difference.
Result is analyzed it is found that composition 1,2,3 ... can promote rat calcium ion by the above animal experimental data
It absorbs, shows as rat body weight, bone weight, bone length, calcium ion content and bone strength are apparently higher than low calcium in bone
Model group and normal diet group.
The above are the descriptions to the present composition to keep this field special by the above description to disclosed composition
Industry technical staff can be realized or using the present invention.A variety of modifications of these compositions carry out those skilled in the art
Saying will be apparent.The general principles defined herein can be the case where not departing from the spirit or scope of the present invention
Under, it is realized in other compositions.Therefore, the present invention is not intended to be limited to these implementation columns shown in this article, but to accord with
Close the widest scope consistent with principles disclosed herein and novel point.
Claims (9)
1. a kind of preparation method of ox bone collagen protein peptides, which is characterized in that this method includes the following steps:
1) a certain amount of fresh ox bone through inspection and quarantine qualification, is taken, is crushed, isometric water, boiling 0.5- is added in cleaning
2h；
2), the ox bone cooking liquor that step 1) obtains is discarded, takes out ox bone, removes the meat adhered on bone, fascia and bone chamber
Interior grease, then hot water cleans 2-3 times, to bone is white or milky, be visible by naked eyes grease until, obtain clean
Ox bone；
3) it, by ox bone obtained in step 2, crushes, crosses 100 mesh standard sieves, obtain ox bone fine powder；
4), by ox bone fine powder obtained in step 3), the water of 5-10 times of quality is added, 40 min of high temperature and pressure ~ 2h are filtered to take
Clearly, ox bone collagen protein liquid is obtained；
5), by ox bone collagen protein liquid obtained in step 4), the trypsase of 1%-3% is added, adjusting pH value is 7.0-8.0,
40-50 DEG C of temperature, 4-7h is digested, by enzyme-deactivating；
6), the enzymolysis liquid in step 5) is added to the mixing of the alkali protease, neutral proteinase and maltodextrin of 0.5%-2.5%
Object, adjusting pH value are 5.0-6.0,40-50 DEG C of temperature, 4-7h are digested, by enzyme-deactivating；Obtain ox bone peptide stoste.
2. a kind of preparation method of ox bone collagen protein peptides according to claim 1, it is characterised in that step 3) is by ox bone
It crushes, is dried in baking oven, coarse crushing bovine bone powder is dried again, is further crushed, is obtained ox bone fine powder.
3. a kind of preparation method of ox bone collagen protein peptides according to claim 1, it is characterised in that the ox in step 3)
The partial size of bone fine powder is 10-150 μm.
4. a kind of preparation method of ox bone collagen protein peptides according to claim 1, which is characterized in that step 5), step
6) enzyme-deactivating uses thermophilic digestion or soak.
5. a kind of preparation method of ox bone collagen protein peptides according to claim 1, which is characterized in that step 6) alkalinity egg
The mass ratio of the mixture of white enzyme, neutral proteinase and maltodextrin are as follows: 1 ~ 10:1 ~ 10:1 ~ 5.
6. a kind of preparation method of ox bone collagen protein peptides according to claim 1, which is characterized in that step 6) obtained
Ox bone collagen peptide stoste is added enzymolysis liquid weight 0.2-0.5% active carbon in enzymolysis liquid, is separated by diatomite, decolourize and go
Except fishy smell, enzymolysis liquid is obtained.
7. the ox bone collagen protein peptides that method described in claim 1 ~ 6 any one claim is prepared.
8. ox bone collagen protein peptides according to claim 7, which is characterized in that the peptide molecule of the ox bone collagen protein peptides
Amount distribution are as follows:
。
It adjusts the drug for the treatment of bone diseases 9. the ox bone collagen protein peptides according to claim 7 or 8 are used to prepare, protect
Purposes in strong product or food.
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