CN103892282A - Protein meal with function of promoting bone growth and preparation method of protein meal - Google Patents
Protein meal with function of promoting bone growth and preparation method of protein meal Download PDFInfo
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- CN103892282A CN103892282A CN201410160964.2A CN201410160964A CN103892282A CN 103892282 A CN103892282 A CN 103892282A CN 201410160964 A CN201410160964 A CN 201410160964A CN 103892282 A CN103892282 A CN 103892282A
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- powder
- lactoferrin
- bone growth
- albumen powder
- promotes bone
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Classifications
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- 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
- A23L33/16—Inorganic salts, minerals or trace elements
-
- 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
- A23L33/16—Inorganic salts, minerals or trace elements
- A23L33/165—Complexes or chelates
-
- 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
- A23L33/19—Dairy proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Mycology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Inorganic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention discloses protein meal with function of promoting bone growth and a preparation method of the protein meal. The protein meal is prepared by mixing the following raw materials by weight percent: 30wt%-60wt% of lactoferrin meal, 39wt%-69wt% of isolated whey protein meal, 0.01wt%-0.5wt% of vitamin E and 0.01wt%-0.5wt% of CaCO3. The method comprises the following steps: (1) performing cation exchange chromatography purification; (2) performing size exclusion chromatography purification; (3) mixing the prepared materials; and (4) drying a product. The obtained protein meal with the function of promoting the bone cell proliferation activity has a good effect of promoting bone formation.
Description
Technical field
The invention belongs to food technology field, relate to a kind of albumen powder and preparation method thereof, relate in particular to a kind of albumen powder with promotion bone growth function and preparation method thereof.
Background technology
Lactoferrin is a kind of multi-functional active glucoprotein, enjoys people's concern always.Lactoferrin claims again lactotransferrin, is mainly present in mammiferous various exotocrine, as milk, tear or saliva etc.It is found first by people such as Sorensen in nineteen thirty-nine at first when from animal Ruzhong sepg whey albumen, nineteen fifty-three Polis etc. have obtained its semifinished product when each lactoperoxidase in system, nineteen sixty by Groves with chromatography first from human milk with cow's milk, separate the sterling that has obtained lactoferrin.For many years, lactoferrin all regulates and controls the bioavailability of iron as a kind of ferrous-fortifier.In addition, lactoferrin also has multiple other biological function, and for example antibacterial, antiviral and antioxidation participates in Organism immunoregulation, strengthens body resistance against diseases etc.Corresponding with these functions, people also focus in these functions for the research and development of lactoferrin.
In recent years, lactoferrin is found to have effect of short bone growth.Many research shows, lactoferrin can stimulated osteoblastic proliferation, promote osteoblastic proliferation with differentiation, also can suppress osteoclast growth with active, induce osteoclast apoptosis.In body, research shows, lactoferrin plays certain physiological action in bone metabolism and growth, can promote bone growth.This function of lactoferrin causes people's concern gradually, but Related product development process is very slow.
Existing lactoferrin product multi-source in different raw milk as raw milk such as cow's milk, human milk, goat dairy and bactrian camel milks in, especially at various just Ruzhongs rich contents.Conventionally adopt the whole bag of tricks as ultrafiltration, saltouing separates lactoferrin with the method such as chromatography.Lactoferrin major part obtains from normal breast and just Ruzhong separation and purification at present, and the accessory substance whey also can utilize processed cheese time separates lactoferrin.But product yield is also relatively on the low side, and purification efficiency is lower.In addition, because the activity of lactoferrin is subject to the impact of many factors, if do not carry out good control in purge process, its active will loss seriously.The factor that affects lactoferrin activity mainly contains temperature, pH, iron saturation, salt ion, lactalbumin etc.The researchs such as Ruegg find that lactoferrin is in pH value at 6.6 o'clock, and temperature starts inactivation while being 65～69 ℃.The research such as Abe finds that ox Apolactoferrin is 4.0 in pH value, and 90 ℃ of temperature heat 5min, and the IBC of lactoferrin, antigen active, bacteriostasis property are almost unchanged, can carry out sterilizing in enormous quantities to it; 70 ℃ of preheating 3min, then UHT sterilization (130 ℃/2s), IBC has only lost 3%; Also find that lactoferrin is more heat-resisting under acid condition, in neutral and the next generation muddiness of alkali condition and gelation.And the people such as Kussendrager and Paulsson study find lactoferrin more responsive under acid condition, the research such as Sreedhara finds that pH2.0-8.0 lactoferrin denaturation temperature reduces with pH and reduces, in the easier sex change of pH2.0-3.0 lactoferrin, and the sex change of lactoferrin is irreversible.Pasteurize is on lactoferrin structure and antibacterial activity no impact substantially, and UHT sterilization meeting makes lactoferrin lose antibacterial activity, but the lactoferrin that 137 ℃/8s processes does not almost change to monocyte proliferation function.Sui and Brisson study and find lactoferrin heat endurance under neutral pH: the saturated lactoferrin of Apolactoferrin < genuine milk ferritin <, be mainly to contribute to lactoferrin disulphide to combine because iron is saturated, prevent albumen polymerization.So in the purge process of lactoferrin, must adopt good technology, suppress or reduce its activity decreased, the operation in practical application and technological parameter still need to determine.
The normal growth of people's bone is grown the dynamic equilibrium that depends on Gegenbaur's cell and osteoclast function, if this balance is broken, skeletonization reduces, and osteoclast activity strengthens, and normal development of skeleton will be destroyed, and then cause the pathologies such as osteoporosis.Multiple research groups have successively reported the beneficial effect to bone health such as lactoferrin, vitamin E and microelements of calcium.
As previously mentioned: lactoferrin can stimulated osteoblastic proliferation, also can suppress osteoclast growth and active, induction osteoclast apoptosis.In body, research shows, lactoferrin plays certain physiological action in bone metabolism and growth, can promote bone growth.
Vitamin E (Vitamin E) is a kind of liposoluble vitamin, claim again tocopherol, it is one of topmost antioxidant, oxidative stress is closely related with the osteoporosis that causes risk of bone fracture to increase, natural powerful antioxidant vitamin E utilizes its anti-oxidation characteristics to help inherent antioxidant defense system resist the infringement of free radical and remove lipid peroxidation free radical, stimulates bone to form and promotes bone health.And tocopherol can promote sex hormone secretion, therefore take in prevention and treatment that vitamin E replenishers are of value to osteoporosis.
Calcium is the maximum mineral element of content in human body, and content is about into the 1.5-2% of body weight for humans, more than 90% is wherein to form bone salts with the form of hydroxyapatite, is present in bone and tooth.Calcium has important function for growing of skeleton.
From promoting the angle of bone growth and development, by composite to lactoferrin, vitamin E and calcium, will promote that in all directions bone health grows from multiple angles such as Gegenbaur's cell and the regulation and control of osteoclast signal transduction pathway, estrogen secretion regulation and control, immunological regulation, bone salts formation.
At present the iron transfer activity of lactoferrin product spininess to it, antibacterial activity, immunoregulatory activity etc. are developed production, for its product development of promotion activity of osteoblast proliferation this respect in space state.And, promote in the market the food of bone growth and development or health products mainly to focus on calcium absorption and two aspects of calcipexy, also some product focuses in the regulation and control of estrogen secretion.But promoting that bone growth and development is that a Gegenbaur's cell and osteoclast dynamic equilibrium obtain complex process, the product technology of preparing of developing for this complicated bioprocess is both at home and abroad in space state.
Summary of the invention
The object of this invention is to provide a kind of albumen powder of promoting bone growth function and preparation method thereof that has, this product will promote that bone health grows in all directions from multiple angles such as Gegenbaur's cell and the regulation and control of osteoclast signal transduction pathway, estrogen secretion regulation and control, immunological regulation, bone salts formation.
The object of the invention is to be achieved through the following technical solutions:
There is an albumen powder that promotes bone growth function, mixed according to following ratio by lactoferrin, whey isolate protein and vitamin E: lactoferrin powder 30-60wt.%, whey isolate protein powder 39-69wt.%, vitamin E 0.01-0.5wt.%, CaCO
30.01-0.5wt.%.
Fresh milk is through degreasing, except obtaining lactalbumin liquid after casein, or directly to apply PURE WHEY be raw material; Then pass through SP Sepharose Big Bead ion-exchange chromatography and Superdex200 gel permeation chromatography, ultrafiltration concentration; Add other functional form auxiliary materials, mixed dissolution evenly after, through vacuum freeze drying or low spraying is dry etc. that method prepares has the albumen powder that promotes bone growth activity.Concrete steps are as follows:
(1) cation-exchange chromatography purifying: first lactalbumin liquid carried out to purifying through strong cation exchange chromatography SP Sepharose Big Bead, in purge process, use in advance 2-4 times of column volume of cushioning liquid A balance until baseline balance, then adopt two-part stepwise elution, first adopt the 2-6 times of column volume of mixed solution wash-out that contains 15-30vo1.% cushioning liquid B, use again 1-4 times of column volume of 100% cushioning liquid B wash-out, elution flow rate 1-5mL/min;
(2) volume exclusion chromatographic purifying: the eluent that step (1) is obtained carries out ultrafiltration concentration, through 0.22 μ m membrane filtration and adopt Superdex200 to be further purified, obtains purifying lactoferrin; Described purge process is: gel column first carries out balance with the PBS that buffer solution 10-20mmol/L, pH value are 7.0-8. and carry out wash-out to baseline stability, and flow velocity is 0.1-2mL/min, under 280nm, detects albumen;
(3) batching is mixed: lactoferrin, whey isolate protein, vitamin E and CaCO prepared by step (2)
3mix;
(4) product drying: above-mentioned mixing is dried, obtain thering is the albumen powder that promotes activity of osteoblast proliferation, in described albumen powder, contain lactoferrin powder 30-60wt.%, whey isolate protein powder 39-69wt.%, vitamin E 0.01-0.5wt.%, CaCO
30.01-0.5wt.%.
What the present invention obtained have promotes the albumen powder of activity of osteoblast proliferation promoting to have good result aspect osteogenesis function, and experimental technique and experimental result take lactalbumin as control group are as follows:
Experimental technique (1): mtt assay is measured osteoblastic proliferation situation
MTT is a kind of weld.Exogenous MTT can be reduced into water-insoluble bluish violet crystallization first distension by the succinate dehydrogenase on living cells mitochondrial inner membrane, but dead cell is but without this function.The DMSO that adds the first distension in can dissolved cell measures light absorption value with ELIASA under 490nm, can detect cell proliferation rate, indirectly reflect living cells quantity.
This experiment adopts mtt assay, and step is as follows:
(1), with after 0.25% trypsin digestion and cell, make single cell suspension with α-MEM nutrient solution (containing 10% hyclone, 1% mycillin), and be diluted to certain cell concentration and be inoculated in 96 well culture plates.Every hole, containing cell suspension 95 μ L, is placed in containing 5%CO
237 ℃ of cell culture incubators in cultivate.
(2), after cell attachment, every hole adds 5 μ L sample solutions.
(3) measure front 4h, every hole adds 10 μ L0.5mg/mLMTT (pH7.20.01mol/LPBS preparation), is placed in cell culture incubator and hatches 4h.
(4) after hatching, carefully discard nutrient solution, every hole adds 150 μ L DMSO, and vibration 10min, makes dissolving crystallized.In ELIASA, under 490nm, measure absorbance.
Corresponding experimental result is in table 1.
The impact of the different samples of table 1 on activity of osteoblast proliferation
Experimental technique (2): OVX rat confirmatory experiment
24 of the clean level female sd inbred rats that provided by Harbin Medical University's second affiliated hospital's Experimental Animal Center are provided, are divided at random 2 groups, 12 every group.One group is lactalbumin control group, the albumen powder group of another group for preparing according to the inventive method.With 3% yellow Jackets intraperitoneal anesthesia, under strict sterile working, get the other dorsal part two incision of lumbar vertebrae, enter abdominal cavity dorsal part, after complete excision bilateral ovaries, layer-by-layer suture otch carefully stops blooding.Postoperative 10 days, two groups of animal used as test every days were with the aqueous solution per os gavage of 0.2g/d sample.Feed is by laboratory row preparation in vain, feed calcium content 1%, phosphorus content 019%.Animal gavage 12 weeks in clean level animal feeding room.Put to death after rat, win rats with bilateral femur, pick clean attachment, measure bone density.
Corresponding experimental result is in table 2.
The impact of the different samples of table 2 on castrated rats femur density
Accompanying drawing explanation
Fig. 1 is process chart of the present invention.
The specific embodiment
Below in conjunction with accompanying drawing, technical scheme of the present invention is further described; but be not limited to this; every technical solution of the present invention is modified or is equal to replacement, and not departing from the spirit and scope of technical solution of the present invention, all should be encompassed in protection scope of the present invention.
Embodiment 1:
As shown in Figure 1, the present embodiment is prepared the albumen powder with promotion activity of osteoblast proliferation in accordance with the following steps:
(1) milk sample processing: by fresh milk in the centrifugal 15-30min degreasing of 5000-10000rpm, by skimmed milk with sour adjust pH to 4.3-4.8, leave standstill the centrifugal 1-30min of 4000-8000r/min after 10-30min, the supernatant of collecting after centrifugal filters.
In this step, described acid is hydrochloric acid, sulfuric acid or acetic acid.
In this step, described sour concentration is 0.1-3mol/L.
In this step, described filter method is: supernatant successively via hole diameter is the membrane filtration of 0.45 μ m and 0.22 μ m, and all operations were carries out at 2-6 ℃.
(2) cation-exchange chromatography purifying: first step (1) gained sample is carried out to purifying through strong cation exchange chromatography SP Sepharose Big Bead.In purge process, use in advance cushioning liquid A balance until baseline balance, its consumption is 2-4 times of column volume, then adopt two-part stepwise elution, first adopt the mixed solution wash-out that contains 15-30vo1.% cushioning liquid B, its consumption is 2-6 times of column volume, then uses 100% cushioning liquid B wash-out, its consumption is 1-4 times of column volume, elution flow rate 1-5mL/min.
In this step, described cushioning liquid A is the PBS that 30-50mmol/L, pH value are 6.5-7.5.
In this step, described cushioning liquid B is the cushioning liquid A that contains 0.1-1mol/L NaCl.
In this step, described mixed solution is the mixed solution of cushioning liquid A and cushioning liquid B.
In this step, also can directly use PURE WHEY to carry out cation-exchange chromatography purifying, before using, be configured to the solution that protein concentration is 5-10g/L.
(3) volume exclusion chromatographic purifying: the eluent that step (2) is obtained carries out ultrafiltration concentration, through 0.22 μ m membrane filtration and adopt Superdex200 to be further purified, obtains lactoferrin.
In this step, described purge process is: gel column is first used the PBS (pH value for 7.0-8.0) of buffer solution 10-20mmol/L to carry out balance to carry out wash-out to baseline stability, and flow velocity is 0.1-2mL/min, under 280nm, detects albumen.
In this step, after purifying, lactoferrin purity is at 85-95%, and activity keeping is good, has typical lactoferrin structure and promotes activity of osteoblast proliferation.
(4) batching is mixed: lactoferrin, whey isolate protein, vitamin E and CaCO3 prepared by step (3) mix in proportion.
(5) product drying: above-mentioned mixing is dried, before dry, solid content requires 10-40wt.%, obtain thering is the albumen powder that promotes activity of osteoblast proliferation, in gained albumen powder, contain lactoferrin powder 30-60wt.%, whey isolate protein powder 39-69wt.%, vitamin E 0.01-0.5wt.%, CaCO
30.01-0.5wt.%.
In this step, described drying mode can adopt the mode of low temperature spray drying, EAT 60-80 ℃, leaving air temp 30-45 ℃.
In this step, described drying mode also can adopt the mode of vacuum freeze drying, condenser temperature-73～-40 ℃, vacuum 0.01～0.5mbar, time 3-24h, dry pulverizing afterwards.
In this step, also can adopt the mode of dry powder blend to prepare albumen powder, be about to lactoferrin dry powder, whey isolate protein powder, vitamin E and CaCO that (3) prepare
3evenly mix with dry powder.
(6) packing: having that step (5) is obtained promotes the albumen powder of activity of osteoblast proliferation to pack, and obtains finished product.
In this step, can adopt 200-1000g can to pack, 18 months shelf-lifves.
In this step, also can adopt the composite wood magazine of 200-1000g to pack, 12 months shelf-lifves.
In this step, also can adopt the soft capsule of 200-500mg to carry out inner packing, external packing is transparent, translucent or opaque plastic bottle packing, every bottle of 60-300 grain.
Embodiment 2:
The present embodiment is prepared the albumen powder with promotion activity of osteoblast proliferation in accordance with the following steps:
(1) milk sample processing: by fresh milk in the centrifugal 15min degreasing of 5000rpm.By the hydrochloric acid adjust pH to 4.5 of 0.5mol/L for skimmed milk, leave standstill the centrifugal 10min of 6000r/min after 15min, collect supernatant after centrifugal successively via hole diameter be the membrane filtration of 0.45 μ m and 0.22 μ m, all operations were carries out at 2-6 ℃.
(2) cation-exchange chromatography purifying: first sample carries out purifying through strong cation exchange chromatography SPSepharose Big Bead (1.6 × 50cm).The phosphate solution that is 7.1 by 200mL, 50mmol/LpH value in advance (cushioning liquid A) balance.Cushioning liquid B is the cushioning liquid A that contains 0.2mol/L NaCl.Adopt two-part stepwise elution, the mixed solution wash-out that adopt 300mL, contains 20vo1.% cushioning liquid B, then use 150mL, 100% cushioning liquid B wash-out, elution flow rate 2mL/min.
(3) volume exclusion chromatographic purifying: above-mentioned eluent is carried out to ultrafiltration concentration, through 0.22 μ m membrane filtration and adopt Superdex200 (1.6cm × 60cm) to be further purified.Gel column is first used the PBS (pH value is 7.6) of buffer solution 20mmol/L to carry out balance to carry out wash-out to baseline stability, and flow velocity is 0.25mL/min, under 280nm, detects albumen.
(4) batching is mixed: by lactoferrin, whey isolate protein, vitamin E and the CaCO of preparation
3proportionally mix.
(5) product drying: adopt the mode of low temperature spray drying, 70 ℃ of EATs, 40 ℃ of leaving air temps; Solid content requires 30wt.%, contains lactoferrin powder 40wt.%, whey isolate protein powder 59.9wt.%, vitamin E 0.05wt.%, CaCO in gained albumen powder
3005wt%.
(6) packing: adopt 500g can to pack, 18 months shelf-lifves.
Embodiment 3:
The present embodiment is prepared the albumen powder with promotion activity of osteoblast proliferation in accordance with the following steps:
(1) milk sample processing: by fresh milk in the centrifugal 15min degreasing of 8000rpm.By the sulfuric acid adjust pH to 4.3 of 1mol/L for skimmed milk, leave standstill the centrifugal 20min of 6000r/min after 20min, collect supernatant after centrifugal successively via hole diameter be the membrane filtration of 0.45 μ m and 0.22 μ m, all operations were carries out at 2-6 ℃.
(2) cation-exchange chromatography purifying: first sample carries out purifying through strong cation exchange chromatography SPSepharose Big Bead (1.6 × 50cm).The phosphate solution that is 6.8 by 300mL, 30mmol/LpH value in advance (cushioning liquid A) balance.Cushioning liquid B is the cushioning liquid A that contains 1mol/L NaCl.Adopt two-part stepwise elution, the mixed solution wash-out that adopt 500mL, contains 20vol.% cushioning liquid B, then use 350mL, 100% cushioning liquid B wash-out, elution flow rate 2.5mL/min.
(3) volume exclusion chromatographic purifying: above-mentioned eluent is carried out to ultrafiltration concentration, through 0.22 μ m membrane filtration and adopt Superdex200 (1.6cm × 60cm) to be further purified.Gel column is first used the PBS (pH value is 7.2) of buffer solution 10mmol/L to carry out balance to carry out wash-out to baseline stability, and flow velocity is 0.5mL/min, under 280nm, detects albumen.
(4) batching is mixed: lactoferrin, whey isolate protein powder, vitamin and CaCO that step (3) is prepared
3evenly mix with dry powder, wherein: lactoferrin powder 50wt.%, whey isolate protein powder 49.8wt.%, vitamin E 0.01wt.%, CaCO
30.01wt.%.
(5) packing: adopt the composite wood magazine of 1000g to pack, 12 months shelf-lifves.Embodiment 4:
The present embodiment is prepared the albumen powder with promotion activity of osteoblast proliferation in accordance with the following steps:
(1) milk sample processing: by fresh milk in the centrifugal 15min degreasing of 10000rpm.By the acetic acid adjust pH to 4.8 of 2.5mol/L for skimmed milk, leave standstill the centrifugal 25min of 6000r/min after 30min, collect supernatant after centrifugal successively via hole diameter be the membrane filtration of 0.45 μ m and 0.22 μ m, all operations were carries out at 2-6 ℃.
(2) cation-exchange chromatography purifying: first sample carries out purifying through strong cation exchange chromatography SPSepharose Big Bead (1.6 × 50cm).The phosphate solution that is 7.3 by 400mL, 40mmol/LpH value in advance (cushioning liquid A) balance.Cushioning liquid B is the cushioning liquid A that contains 0.5mol/L NaCl.Adopt two-part stepwise elution, adopt the mixed solution wash-out that contains 400mL, 30vo1.% cushioning liquid B, then use 250mL, 100% cushioning liquid B wash-out, elution flow rate 1.5mL/min.
(3) volume exclusion chromatographic purifying: above-mentioned eluent is carried out to ultrafiltration concentration, through 0.22 μ m membrane filtration and adopt Superdex200 (1.6cm × 60cm) to be further purified.Gel column is first used the PBS (pH value is 7.8) of buffer solution 15mmol/L to carry out balance to carry out wash-out to baseline stability, and flow velocity is 1.5mL/min, under 280nm, detects albumen.
(4) batching is mixed: the lactoferrin of preparation, whey isolate protein are proportionally mixed with vitamin E.
(5) product drying: the mode that adopts vacuum freeze drying, condenser temperature-50 ℃, vacuum 0.25mbar, time 10h, after dry, through pulverizing, in gained albumen powder, contain lactoferrin powder 58.96wt.%, whey isolate protein powder 41wt.%, vitamin E 0.02wt.%, CaCO
30.02wt.%.
(6) packing: adopt 200mg soft capsule to carry out inner packing, external packing is transparent, translucent or opaque plastic bottle packing, 100 every bottle.
Claims (10)
1. one kind has the albumen powder that promotes bone growth function, it is characterized in that described albumen powder is mixed according to following ratio by lactoferrin powder, whey isolate protein powder, vitamin E and calcium carbonate: lactoferrin powder 30-60wt.%, whey isolate protein powder 39-69wt.%, vitamin E 0.01-0.5wt.%, CaCO
30.01-0.5wt.%.
2. according to claim 1 have an albumen powder that promotes bone growth function, it is characterized in that containing in described albumen powder lactoferrin powder 40wt.%, whey isolate protein powder 59.9wt.%, vitamin E 0.05wt.%, CaCO
30.05wt.%.
3. according to claim 1 have an albumen powder that promotes bone growth function, it is characterized in that containing in described albumen powder lactoferrin powder 50wt.%, whey isolate protein powder 49.8wt.%, vitamin E 0.01wt.%, CaCO
30.01wt.%.
4. according to claim 1 have an albumen powder that promotes bone growth function, it is characterized in that containing in described albumen powder lactoferrin powder 58.96wt.%, whey isolate protein powder 41wt.%, vitamin E 0.02wt.%, CaCO
30.02wt.%.
5. the preparation method with the albumen powder that promotes bone growth function described in the arbitrary claim of claim 1-4, is characterized in that described method step is as follows:
(1) cation-exchange chromatography purifying: first lactalbumin liquid carried out to purifying through strong cation exchange chromatography SP Sepharose Big Bead, in purge process, use in advance 2-4 times of column volume of cushioning liquid A balance until baseline balance, then adopt two-part stepwise elution, first adopt the 2-6 times of column volume of mixed solution wash-out that contains 15-30vo1.% cushioning liquid B, use again 1-4 times of column volume of 100% cushioning liquid B wash-out, elution flow rate 1-5mL/min;
(2) volume exclusion chromatographic purifying: the eluent that step (1) is obtained carries out ultrafiltration concentration, through 0.22 μ m membrane filtration and adopt Superdex200 to be further purified, described purge process is: gel column first carries out balance with the PBS that buffer solution 10-20mmol/L, pH value are 7.0-8. and carry out wash-out to baseline stability, flow velocity is 0.1-2mL/min, obtains purifying lactoferrin;
(3) batching is mixed: lactoferrin, whey isolate protein, vitamin E and CaCO prepared by step (2)
3mix;
(4) product drying: above-mentioned mixing is dried, obtains the albumen powder that promotes bone growth function that has described in the arbitrary claim of claim 1-4.
6. the preparation method with the albumen powder that promotes bone growth function according to claim 5, it is characterized in that described lactalbumin liquid obtains as follows: by fresh milk in the centrifugal 15-30min degreasing of 5000-10000rpm, by skimmed milk with sour adjust pH to 4.3-4.8, the centrifugal 1-30min of 4000-8000r/min after standing 10-30min, the supernatant of collecting after centrifugal filters, and obtains lactalbumin liquid.
7. the preparation method with the albumen powder that promotes bone growth function according to claim 5, is characterized in that described lactalbumin liquid is take PURE WHEY as raw material, before using, is configured to the solution that protein concentration is 5-10g/L.
8. the preparation method with the albumen powder that promotes bone growth function according to claim 5, it is characterized in that described cushioning liquid A is the PBS that 30-50mmol/L, pH value are 6.5-7.5, described cushioning liquid B is the cushioning liquid A that contains 0.1-1mol/L NaCl.
9. the preparation method with the albumen powder that promotes bone growth function according to claim 5, is characterized in that after purifying that lactoferrin purity is 85-95%.
10. the preparation method with the albumen powder that promotes bone growth function according to claim 5, is characterized in that dry front solid content is 10-40wt.%.
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