CN108935913A - Sequestering pig bone collagen peptide of calcium and preparation method thereof - Google Patents
Sequestering pig bone collagen peptide of calcium and preparation method thereof Download PDFInfo
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- CN108935913A CN108935913A CN201810680851.3A CN201810680851A CN108935913A CN 108935913 A CN108935913 A CN 108935913A CN 201810680851 A CN201810680851 A CN 201810680851A CN 108935913 A CN108935913 A CN 108935913A
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- China
- Prior art keywords
- calcium
- pig bone
- enzymatic hydrolysis
- bone collagen
- peptide
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 148
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 title claims abstract description 138
- 239000011575 calcium Substances 0.000 title claims abstract description 137
- 229910052791 calcium Inorganic materials 0.000 title claims abstract description 137
- 210000000988 bone and bone Anatomy 0.000 title claims abstract description 118
- 102000008186 Collagen Human genes 0.000 title claims abstract description 90
- 108010035532 Collagen Proteins 0.000 title claims abstract description 90
- 229920001436 collagen Polymers 0.000 title claims abstract description 90
- 230000014759 maintenance of location Effects 0.000 title claims abstract description 59
- 238000002360 preparation method Methods 0.000 title claims abstract description 34
- 241000282898 Sus scrofa Species 0.000 claims abstract description 96
- 230000007071 enzymatic hydrolysis Effects 0.000 claims abstract description 61
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims abstract description 61
- 102000004190 Enzymes Human genes 0.000 claims abstract description 47
- 108090000790 Enzymes Proteins 0.000 claims abstract description 47
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 47
- 229920001184 polypeptide Polymers 0.000 claims abstract description 44
- 239000007788 liquid Substances 0.000 claims abstract description 39
- 150000004697 chelate complex Chemical class 0.000 claims abstract description 27
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 22
- 238000006243 chemical reaction Methods 0.000 claims abstract description 22
- 241000282894 Sus scrofa domesticus Species 0.000 claims abstract description 19
- 239000000843 powder Substances 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 13
- 230000009920 chelation Effects 0.000 claims abstract description 8
- 239000007790 solid phase Substances 0.000 claims abstract description 6
- 238000012869 ethanol precipitation Methods 0.000 claims abstract description 5
- 239000004365 Protease Substances 0.000 claims description 42
- 108091005804 Peptidases Proteins 0.000 claims description 41
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 41
- 235000019419 proteases Nutrition 0.000 claims description 41
- 239000003513 alkali Substances 0.000 claims description 35
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 claims description 30
- 239000000758 substrate Substances 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 238000005238 degreasing Methods 0.000 claims description 12
- 230000001360 synchronised effect Effects 0.000 claims description 5
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- 108090000145 Bacillolysin Proteins 0.000 claims description 3
- 102000035092 Neutral proteases Human genes 0.000 claims description 3
- 108091005507 Neutral proteases Proteins 0.000 claims description 3
- 239000013522 chelant Substances 0.000 abstract description 9
- 238000013459 approach Methods 0.000 abstract description 3
- 230000020978 protein processing Effects 0.000 abstract description 2
- 229960005069 calcium Drugs 0.000 description 118
- 229940088598 enzyme Drugs 0.000 description 40
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 15
- 238000010521 absorption reaction Methods 0.000 description 15
- 229910001424 calcium ion Inorganic materials 0.000 description 15
- 230000009849 deactivation Effects 0.000 description 14
- 238000001816 cooling Methods 0.000 description 12
- 239000000243 solution Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- 239000001110 calcium chloride Substances 0.000 description 8
- 229910001628 calcium chloride Inorganic materials 0.000 description 8
- 238000001976 enzyme digestion Methods 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 7
- 239000002244 precipitate Substances 0.000 description 7
- 230000001376 precipitating effect Effects 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 150000001298 alcohols Chemical class 0.000 description 6
- 229940036811 bone meal Drugs 0.000 description 6
- 239000002374 bone meal Substances 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
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- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 239000008367 deionised water Substances 0.000 description 4
- 229910021641 deionized water Inorganic materials 0.000 description 4
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- 102000005701 Calcium-Binding Proteins Human genes 0.000 description 3
- 108010045403 Calcium-Binding Proteins Proteins 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 description 3
- 229960003563 calcium carbonate Drugs 0.000 description 3
- 235000010216 calcium carbonate Nutrition 0.000 description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 206010006956 Calcium deficiency Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 238000004566 IR spectroscopy Methods 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 208000001132 Osteoporosis Diseases 0.000 description 2
- 150000003863 ammonium salts Chemical class 0.000 description 2
- 239000002585 base Substances 0.000 description 2
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- OHJMTUPIZMNBFR-UHFFFAOYSA-N biuret Chemical compound NC(=O)NC(N)=O OHJMTUPIZMNBFR-UHFFFAOYSA-N 0.000 description 2
- 239000004227 calcium gluconate Substances 0.000 description 2
- 229960004494 calcium gluconate Drugs 0.000 description 2
- 235000013927 calcium gluconate Nutrition 0.000 description 2
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000001142 circular dichroism spectrum Methods 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
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- 238000004108 freeze drying Methods 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- ZHUXMBYIONRQQX-UHFFFAOYSA-N hydroxidodioxidocarbon(.) Chemical compound [O]C(O)=O ZHUXMBYIONRQQX-UHFFFAOYSA-N 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
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- 150000003839 salts Chemical class 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- 206010000060 Abdominal distension Diseases 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
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- 241000040710 Chela Species 0.000 description 1
- 229920002567 Chondroitin Polymers 0.000 description 1
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- 235000007926 Craterellus fallax Nutrition 0.000 description 1
- 240000007175 Datura inoxia Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000239366 Euphausiacea Species 0.000 description 1
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 1
- 239000001828 Gelatine Substances 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000000913 Kidney Calculi Diseases 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010029148 Nephrolithiasis Diseases 0.000 description 1
- 208000008589 Obesity Diseases 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 108090000526 Papain Proteins 0.000 description 1
- 108010009736 Protein Hydrolysates Proteins 0.000 description 1
- 241000277263 Salmo Species 0.000 description 1
- 241000277289 Salmo salar Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 241000931197 Themeda Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
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- 150000001408 amides Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- XKMRRTOUMJRJIA-UHFFFAOYSA-N ammonia nh3 Chemical compound N.N XKMRRTOUMJRJIA-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- CREXVNNSNOKDHW-UHFFFAOYSA-N azaniumylideneazanide Chemical group N[N] CREXVNNSNOKDHW-UHFFFAOYSA-N 0.000 description 1
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- MKJXYGKVIBWPFZ-UHFFFAOYSA-L calcium lactate Chemical compound [Ca+2].CC(O)C([O-])=O.CC(O)C([O-])=O MKJXYGKVIBWPFZ-UHFFFAOYSA-L 0.000 description 1
- 239000001527 calcium lactate Substances 0.000 description 1
- 235000011086 calcium lactate Nutrition 0.000 description 1
- 229940041131 calcium lactate gluconate Drugs 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 150000007942 carboxylates Chemical class 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- DLGJWSVWTWEWBJ-HGGSSLSASA-N chondroitin Chemical compound CC(O)=N[C@@H]1[C@H](O)O[C@H](CO)[C@H](O)[C@@H]1OC1[C@H](O)[C@H](O)C=C(C(O)=O)O1 DLGJWSVWTWEWBJ-HGGSSLSASA-N 0.000 description 1
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- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
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- 239000004615 ingredient Substances 0.000 description 1
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- 230000031891 intestinal absorption Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 235000010755 mineral Nutrition 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
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- 230000003287 optical effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 125000000962 organic group Chemical group 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 229940055729 papain Drugs 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
- A23J1/10—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from hair, feathers, horn, skins, leather, bones, or the like
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
- A23J3/00—Working-up of proteins for foodstuffs
- A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
- A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
- A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
-
- 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
- A23L33/18—Peptides; Protein hydrolysates
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Mycology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Inorganic Chemistry (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides sequestering pig bone collagen peptides of a kind of calcium and preparation method thereof and calcium peptide chelate complex, belong to bone protein processing technique field.The preparation method of the calcium peptide chelate complex carries out enzymatic hydrolysis acquisition pig bone collagen polypeptide liquid the following steps are included: 1) Os Sus domestica powder is dissolved in after water and being mixed with enzyme, 2) pH for adjusting the pig bone collagen polypeptide liquid is 7.2~7.8, then collecting solid phase components by calcium chloride solution and pig bone collagen polypeptide liquid hybrid reaction, after ethanol precipitation reaction solution is calcium peptide chelate complex.The calcium chelation percent of the sequestering pig bone collagen peptide of the calcium that the present invention obtains is up to 70.24%, and through scanning electron microscopic observation, structure has become close granular agglomerate after the sequestering pig bone collagen polypeptide of calcium and calcium chelating.New approach is opened for the comprehensive utilization of pig bone, is also provided fundamental basis for the exploitation of polypeptide chelate calcium this novel calcium-supplementing preparation.
Description
Technical field
The invention belongs to bone protein processing technique field more particularly to the preparation methods of the sequestering pig bone collagen peptide of calcium, calcium
Sequestering pig bone collagen peptide, the preparation method of calcium peptide chelate complex and calcium peptide chelate complex.
Background technique
Calcium is nutrient needed by human, accounts for about the 1.5%~2.2% of total body weight, calcium deficiency will cause osteoporosis
The diseases such as disease, hypertension, colon cancer, obesity and kidney stone.Since China resident is based on vegetable diet, calcium deficiency phenomenon
It is particularly acute, therefore, it is very crucial to find good calcium-supplementing preparation.Calcium-supplementing preparation on the market is mainly with inorganic acid calcium at present
Or based on calcium of organic acid, such as calcium carbonate, calcium lactate and calcium gluconate.However, ionized calcium is easy in alkaline intestinal environment
Calcium precipitate is formed, this seriously reduces human body to the absorption of calcium and the bioavilability of calcium.In addition, calcium carbonate has one to stomach
Determine side effect, such as abdominal distension.Research finds the small peptide or ammonia that calcium is needed to secrete with intestinal brush border before small intestinal absorption in vivo
Base acid chelating.Therefore, organic calcium complement agent absorbs that fast, biological value is high, the advantages such as safe and non-toxic are increasingly becoming neck of replenishing the calcium with it
The research hotspot in domain.Villous themeda scape grain husk is the study found that collagen polypeptide chelates calcium in prevention and treatment osteoporosis, promotion bone growth and bone strengthening
It is better than calcium gluconate and calcium carbonate effect in effect, it is a kind of excellent novel calcium-supplementing preparation.
Constantly increase recently as the livestock and poultry yield in China, by-product bone also increases significantly therewith.Livestock and poultry bone contains
There are a large amount of protein, ossein, chondroitin, fat and several mineral materials, vegetal pole horn of plenty.But bone in process
Protein and other nutritional ingredients are not fully utilized, and it is low additional that most of live stock and fowl bone is processed to gelatine, bone meal etc.
It is worth animal feed.But it is concentrated mainly in the enzymolysis process optimization of albumen about the research of bone protein of pig at present, and for polypeptide
Functional study it is less, still not about high calcium chelating pig bone collagen peptide research.
Summary of the invention
In view of this, the purpose of the present invention is to provide the preparation methods of the sequestering pig bone collagen peptide of calcium, the sequestering pig of calcium
Bone gillg, the preparation method of calcium peptide chelate complex and calcium peptide chelate complex；The economic value and nutrient utilization for improving pig bone, keep away
Exempt from the wasting of resources and environmental pollution；On the other hand, the sequestering pig bone collagen peptide calcium chelation percent of calcium that the preparation method obtains
Height can prepare polypeptide chelate calcium as novel calcium-supplementing preparation, achieve the effect that not only replenishing collagen but also supplement calcium.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided: degreasing after 1) taking fresh pig bone broken,
Decalcification, except foreign protein, crush Os Sus domestica powder is made；2) Os Sus domestica powder is dissolved in after water and is mixed with alkali protease and/or neutral proteinase
Conjunction carries out enzymatic hydrolysis and obtains pig bone collagen polypeptide liquid, the i.e. sequestering pig bone collagen peptide of calcium.
Preferably, when the enzyme is alkali protease, the concentration of substrate of the enzymatic hydrolysis is 5~10%, and enzyme concentration is
4000~8000u/g, the pH value of the enzymatic hydrolysis are 8.0~9.2, and the temperature of the enzymatic hydrolysis is 40~50 DEG C, the enzymatic hydrolysis when
Between be 4~6h.
Preferably, when the enzyme is neutral proteinase, the concentration of substrate of the enzymatic hydrolysis is 6~10%, and enzyme concentration is
6000~10000u/g, the pH value of the enzymatic hydrolysis are 7.0~8.0, and the temperature of the enzymatic hydrolysis is 45~55 DEG C, the enzymatic hydrolysis
Time is 4~6h.
Preferably, when the enzyme is alkali protease and neutral proteinase, the enzymatic hydrolysis is alkali protease and neutrality
The synchronous enzymatic hydrolysis of protease.
Preferably, the mass ratio of alkali protease and neutral proteinase is 1:1~2:1；The concentration of substrate of the enzymatic hydrolysis is
6~8%；The pH value of the enzymatic hydrolysis is 7.4~7.6, and the temperature of the enzymatic hydrolysis is 45~51 DEG C, the time of the enzymatic hydrolysis is 4~
6h。
Preferably, when the enzyme is alkali protease and neutral proteinase, the enzymatic hydrolysis is alkali protease and neutrality
Protease stepwise discretization；It is unlimited to the chronological order of the alkali protease and neutral protease enzymolysis.
The present invention also provides the sequestering pig bone collagen peptides of calcium that the preparation method prepares.
The present invention also provides the preparation methods of calcium peptide chelate complex, comprising the following steps: adjusts the pig bone collagen polypeptide
The pH of liquid is 7.2~7.8, is obtained after calcium chloride solution is then mixed chelating with the pig bone collagen polypeptide liquid after adjusting pH anti-
Liquid, the reaction solution that ethanol precipitation obtains are answered, obtained solid phase components are calcium peptide chelate complex.
Preferably, the concentration of the calcium chloride solution is 1~5g/L；The calcium chloride solution and pig bone collagen polypeptide liquid
Volume ratio be 1:1~1:2.
The present invention also provides the calcium peptide chelate complexes that the preparation method prepares.
Beneficial effects of the present invention: the preparation method of the sequestering pig bone collagen peptide of calcium provided by the invention passes through alkaline egg
White enzyme and/or neutral proteinase obtain the sequestering pig bone collagen peptide of calcium after digesting Os Sus domestica powder, the calcium peptide chelating that the present invention obtains
The calcium chelation percent of object is up to 70.24%.The sequestering pig bone collagen peptide of calcium and calcium chloride solution hybrid reaction are obtained into calcium peptide chelating
Object.Through scanning electron microscopic observation, the calcium peptide chelate complex, the sequestering pig bone collagen peptide of calcium has become close with structure after calcium chelating
Granular agglomerate.The present invention improves the added value of pig bone resource, opens new approach for the comprehensive utilization of pig bone,
Also it provides fundamental basis for the exploitation of this novel calcium-supplementing preparation of polypeptide chelate calcium.
Detailed description of the invention
Fig. 1 is the ultraviolet spectrogram after various concentration calcium ion and the sequestering pig bone collagen peptide effect of calcium；
Fig. 2 is the fluorescence spectra after various concentration calcium ion and the sequestering pig bone collagen peptide effect of calcium；
Fig. 3 is the sequestering pig bone collagen peptide of calcium and calcium peptide chelate complex infrared spectrogram；
Fig. 4 is the SEM figure (× 5000) of the sequestering pig bone collagen peptide (A) of calcium and calcium peptide chelate complex (B).
Specific embodiment
The present invention provides a kind of preparation methods of the sequestering pig bone collagen peptide of calcium, comprising the following steps: 1) takes fresh pig
Bone is crushed rear degreasing, decalcification, except Os Sus domestica powder is made in foreign protein, crushing；2) by Os Sus domestica powder be dissolved in after water with alkali protease and/
Or neutral proteinase mixing carries out enzymatic hydrolysis and obtains pig bone collagen polypeptide liquid, the i.e. sequestering pig bone collagen peptide of calcium.
In the present invention, the broken rear degreasing of fresh pig bone, decalcification are taken, except Os Sus domestica powder is made in foreign protein, crushing.In this hair
In bright, the degreasing agent of the degreasing is n-hexane, ether, petroleum ether or acetone, and the degreasing time is preferably 24
~36h；The decalcification is preferably citric acid, hydrochloric acid or EDTA with decalcifying agent, and the decalcification time is preferably 24~48h；It is described
Except the solution of foreign protein is sodium hydroxide or sodium chloride solution, the time except foreign protein is preferably 24~48h.At this
In invention specific implementation process, the fresh pig bone is crushed rear degreasing, decalcification, is preferably that n-hexane is de- except the method for foreign protein
For 24 hours, for 24 hours, 4% sodium chloride and the removal of impurities of 0.08mol/L NaOH mixed solution are for 24 hours for 10% citric acid decalcification time for rouge.In this hair
In bright, the granularity of the Os Sus domestica powder is preferably 50~300 mesh；The crushing preferably uses bone cutter or Miniature Chinese medicine pulverizer
It carries out.
The present invention after obtaining Os Sus domestica powder, Os Sus domestica powder is dissolved in after water mixed with enzyme carry out enzymatic hydrolysis obtain pig bone collagen it is more
Peptide liquid.In the present invention, the mass ratio of the Os Sus domestica powder and water is preferably 4~12%, more preferably 6~8%.In the present invention
In, the enzyme is preferably one of neutral proteinase, flavor protease, alkali protease, papain and trypsase
Or several, more preferably alkali protease and/or neutral proteinase, most preferably alkali protease and neutral proteinase.
In the present invention, when the enzyme is alkali protease, the concentration of the substrate pig bone collagen polypeptide liquid of the enzymatic hydrolysis
Preferably 5~10%, more preferably 5.5~6.5%, most preferably 6%；The enzyme concentration of the enzymatic hydrolysis is preferably 4000~
8000u/g, more preferably 5500~6500u/g, most preferably 6000u/g；The pH value of the enzymatic hydrolysis is preferably 8~9.2, more
Preferably 8.8~9.1, more preferably 9.0；The temperature of the enzymatic hydrolysis is preferably 40~50 DEG C, and more preferably 44~46 DEG C, most
Preferably 45 DEG C；The time of the enzymatic hydrolysis is preferably 4~6h, more preferably 5h.
In the present invention, when the enzyme is neutral proteinase, the concentration of the substrate pig bone collagen polypeptide liquid of the enzymatic hydrolysis
Preferably 6~10%, more preferably 7.5~8.5%, most preferably 8.0%；The enzyme concentration of the enzymatic hydrolysis is preferably 6000~
10000u/g, more preferably 7500~8500u/g, most preferably 8000u/g；The pH value of the enzymatic hydrolysis is preferably 7~8, more excellent
It is selected as 7.4~7.6, more preferably 7.5；The temperature of the enzymatic hydrolysis is preferably 45~55 DEG C, more preferably 49~51 DEG C, more excellent
It is selected as 50 DEG C；The time of the enzymatic hydrolysis is preferably 3.5~6h, more preferably 4h.
In the present invention, when the enzyme be alkali protease and neutral proteinase when, it is described enzymatic hydrolysis be alkali protease and
The synchronous enzymatic hydrolysis of neutral proteinase or alkali protease and neutral proteinase stepwise discretization.In the present invention, when using synchronous enzymatic hydrolysis
Scheme when；The adding proportion of the alkali protease and neutral proteinase is 1:1~2:1, more preferably 1:1；The alkalinity
The enzyme concentration of protease and neutral proteinase preferably stands alone as 3800~4200u/g, more preferably 4000u/g；The enzyme
The concentration of substrate of solution is preferably 7.5~8.5%；More preferably 8.0%；The enzyme concentration of the enzymatic hydrolysis is preferably 7500~
8500u/g, more preferably 8000u/g；The pH value of the enzymatic hydrolysis is preferably 7.4~7.6, and more preferably 7.5；The enzymatic hydrolysis
Temperature is preferably 45~51 DEG C, and more preferably 50 DEG C；The time of the enzymatic hydrolysis is preferably 3~6h, more preferably 3.5~
4.5h, most preferably 4h.In the present invention, when using the scheme of stepwise discretization；To alkali protease and neutral proteinase
Enzymatic hydrolysis sequence is not particularly limited, and first can use neutral protease enzymolysis again with alkali protease enzymatic hydrolysis, can also be first with neutrality
Protease hydrolyzed, then digested with alkali protease.For the present invention during stepwise discretization, the parameter of every step enzymatic hydrolysis is referring to above-mentioned
The parameter of single enzymatic hydrolysis, details are not described herein.The present invention is during stepwise discretization, preferably includes enzyme deactivation after step enzymatic hydrolysis
And cooling step.In the present invention, the method that the enzyme deactivation preferably uses high temperature enzyme deactivation；The temperature of the enzyme deactivation is preferably
100℃；The cooling cooling means for using this field routine, without other particular/special requirements.It is currently preferred described
Next step enzymatic hydrolysis is carried out after enzyme deactivation and cooling.
The above-mentioned enzymatic hydrolysis of the present invention and the temperature of enzyme deactivation are realized preferably through water-bath.The present invention obtains after the enzymatic hydrolysis
The sequestering pig bone collagen peptide of pig bone collagen polypeptide liquid, i.e. calcium.
The present invention also provides the sequestering pig bone collagen peptides of calcium that the preparation method prepares.In the present invention, institute
The calcium chelation percent for stating the sequestering pig bone collagen peptide of calcium is high；Alkali protease and neutral proteinase individually hydrolyze the resulting calcium of pig bone
The calcium chelation percent of sequestering pig bone collagen peptide is respectively 56.49% and 42.06%.Using alkali protease and neutral proteinase
The calcium chelation percent for the sequestering pig bone collagen peptide of calcium that synchronous hydrolysis obtains is 70.24%.
The present invention also provides the methods for preparing calcium peptide chelate complex using the sequestering pig bone collagen peptide of the calcium, including with
Lower step: the pH value for adjusting the pig bone collagen polypeptide liquid is 7.2~7.8, then by calcium chloride solution and pig bone collagen polypeptide
Liquid hybrid reaction, it is calcium peptide chelate complex that solid phase components are collected after ethanol precipitation reaction solution.
In the present invention, the reagent for adjusting pig bone collagen polypeptide liquid uses the acid-base modifier of this field routine；Institute
The pH value for stating pig bone collagen polypeptide liquid is preferably 7.3~7.7, and more preferably 7.5.In the present invention, the calcium chloride solution
Concentration is preferably 1g/L~5g/L, more preferably 1g/L；The volume ratio of the calcium chloride solution and pig bone collagen polypeptide liquid is preferred
For 1:1~1:2.In the present invention, the temperature of the calcium chloride solution and pig bone collagen polypeptide liquid hybrid reaction be preferably 38~
50 DEG C, more preferably 40 DEG C；The time of the hybrid reaction is preferably 45~65min, more preferably 60min.
The present invention is after calcium chloride solution and pig bone collagen polypeptide liquid hybrid reaction, ethanol precipitation reaction solution.In the present invention
In, the ethyl alcohol is preferably dehydrated alcohol, and the volume ratio of the dehydrated alcohol and reaction solution is preferably (8~10): 1, more preferably
For 9:1；In the present invention, the time of the precipitating is preferably 2~4h, more preferably 2.5~3.5h, most preferably 3h.The present invention
It after the precipitating, is separated by solid-liquid separation, collecting solid phase components is calcium peptide chelate complex.In the present invention, the separation of solid and liquid
Method preferably be centrifuged, the revolving speed of the centrifugation is preferably 9000~11000rpm, more preferably 10000rpm；It is described from
The time of the heart is preferably 5~15min, more preferably 8~12min, most preferably 10min.The present invention is collecting solid phase components
It afterwards, preferably further include drying steps；The drying is preferably freeze-dried；The present invention does not have the parameter of the freeze-drying
There is particular determination, as long as can be realized dry.
The present invention also provides the calcium peptide chelate complexes that the preparation method prepares.
The present invention is obtained by ultra-violet absorption spectrum, fluorescence spectrum, Fourier transform infrared spectroscopy scanning and two spectrum of circle
The sequestering pig bone collagen peptide of calcium and calcium peptide chelate complex obtained is identified, the results showed that the sequestering pig bone collagen peptide of calcium and calcium chela
Significant changes occur for secondary structure after conjunction, may form a kind of new object by carboxyl oxygen and ammonia nitrogen and calcium binding
Matter.Scanning electron microscope (SEM) photograph shows that the sequestering pig bone collagen peptide of calcium and calcium chelate structure become like the microaggregate of salt, into
One step demonstrates the formation of carboxylate and ammonium salt.
The sequestering pig bone collagen peptide of calcium of the present invention is obtained using protease hydrolyzed method, is the comprehensive utilization of pig bone
New approach is opened, is also provided fundamental basis for the exploitation of polypeptide chelate calcium this novel calcium-supplementing preparation.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be managed
Solution is limiting the scope of the present invention.
Embodiment 1
Leg of pork bone is purchased from the Hua Zhong Agriculture University market of farm produce.Meat mincing, periosteum and marrow are rejected, use bone cutter after cleaning
It is crushed with Miniature Chinese medicine pulverizer.Bone meal is dried for standby after degreasing, decalcification and removal of impurities protein Process.
It is 9.0 → plus alkali protease (enzyme concentration that Os Sus domestica powder → plus water, which are mixed to concentration of substrate 6% → adjusting pH value,
6000u/g) 45 DEG C of enzyme deactivations of water enzyme digestion 5h → 100 DEG C, cooling, 10000r/min centrifugation 10min → supernatant is taken to be digested
The sequestering pig bone collagen peptide of liquid, i.e. calcium.
Embodiment 2
Calcium chloride (final concentration of 1g/L) → 40 DEG C of reaction 60min are added in enzymolysis liquid → adjusting pH7.5 in embodiment 1
→ 9 times of volume dehydrated alcohols precipitate 3h → 10000r/min refrigerated centrifuge 10min, take precipitating → be freeze-dried to obtain calcium peptide chelating
Object.
Embodiment 3
Leg of pork bone is purchased from the Hua Zhong Agriculture University market of farm produce.Meat mincing, periosteum and marrow are rejected, use bone cutter after cleaning
It is crushed with Miniature Chinese medicine pulverizer.Bone meal is dried for standby after degreasing, decalcification and removal of impurities protein Process.
It is 7.5 → plus neutral proteinase (enzyme concentration that Os Sus domestica powder → plus water, which are mixed to concentration of substrate 8% → adjusting pH value,
8000u/g) 50 DEG C of enzyme deactivations of water enzyme digestion 4h → 100 DEG C, cooling, 10000r/min centrifugation 10min → supernatant is taken to be digested
Liquid, i.e. the sequestering pig bone collagen peptide of calcium.
Embodiment 4
Calcium chloride (final concentration of 1g/L) → 40 DEG C of reaction 60min are added in enzymolysis liquid → adjusting pH7.5 in embodiment 3
→ 9 times of volume dehydrated alcohols precipitate 3h → 10000r/min refrigerated centrifuge 10min, take precipitating → be freeze-dried to obtain calcium peptide chelating
Object.
Embodiment 5
Leg of pork bone is purchased from the Hua Zhong Agriculture University market of farm produce.Meat mincing, periosteum and marrow are rejected, use bone cutter after cleaning
It is crushed with Miniature Chinese medicine pulverizer.Bone meal is dried for standby after degreasing, decalcification and removal of impurities protein Process.
It is 7.5 → plus alkali protease and neutral protein that Os Sus domestica powder → plus water, which are mixed to concentration of substrate 8% → adjusting pH value,
Enzyme (each 4000u/g of enzyme concentration) 50 DEG C of enzyme deactivations of water enzyme digestion 4h → 100 DEG C, cooling, 10000r/min centrifugation 10min → take
It is clear to obtain enzymolysis liquid, the i.e. sequestering pig bone collagen peptide of calcium.
Embodiment 6
Calcium chloride (final concentration of 1g/L) → 40 DEG C of reaction 60min are added in enzymolysis liquid → adjusting pH7.5 in embodiment 5
→ 9 times of volume dehydrated alcohols precipitate 3h → 10000r/min refrigerated centrifuge 10min, take precipitating → be freeze-dried to obtain calcium peptide chelating
Object.
Embodiment 7
Leg of pork bone is purchased from the Hua Zhong Agriculture University market of farm produce.Meat mincing, periosteum and marrow are rejected, use bone cutter after cleaning
It is crushed with Miniature Chinese medicine pulverizer.Bone meal is dried for standby after degreasing, decalcification and removal of impurities protein Process.
It is 9.0 → plus alkali protease (enzyme concentration that Os Sus domestica powder → plus water, which are mixed to concentration of substrate 8% → adjusting pH value,
6000u/g) 45 DEG C of water enzyme digestion 2h → 100 DEG C enzyme deactivations, cooling → adjusting pH value be 7.5 → plus neutral proteinase (enzyme concentration
8000u/g) 50 DEG C of enzyme deactivations of water enzyme digestion 2h → 100 DEG C, cooling, 10000r/min centrifugation 10min → supernatant is taken to be digested
The sequestering pig bone collagen peptide of liquid, i.e. calcium.
Embodiment 8
Calcium chloride (final concentration of 1g/L) → 40 DEG C of reaction 60min are added in enzymolysis liquid → adjusting pH7.5 in embodiment 7
→ 9 times of volume dehydrated alcohols precipitate 3h → 10000r/min refrigerated centrifuge 10min, take precipitating → be freeze-dried to obtain calcium peptide chelating
Object.
Embodiment 9
Leg of pork bone is purchased from the Hua Zhong Agriculture University market of farm produce.Meat mincing, periosteum and marrow are rejected, use bone cutter after cleaning
It is crushed with Miniature Chinese medicine pulverizer.Bone meal is dried for standby after degreasing, decalcification and removal of impurities protein Process.
It is 7.5 → plus neutral proteinase (enzyme concentration that Os Sus domestica powder → plus water, which are mixed to concentration of substrate 6% → adjusting pH value,
8000u/g) 50 DEG C of water enzyme digestion 2.5h → 100 DEG C enzyme deactivations, cooling → adjusting pH value be that 9.0 → plus alkali protease are (enzyme
6000u/g is measured) 45 DEG C of enzyme deactivations of water enzyme digestion 2.5h → 100 DEG C, cooling → 10000r/min is centrifuged 10min → take supernatant to obtain
The sequestering pig bone collagen peptide of enzymolysis liquid, i.e. calcium.
Embodiment 10
Calcium chloride (final concentration of 1g/L) → 40 DEG C of reaction 60min are added in enzymolysis liquid → adjusting pH7.5 in embodiment 9
→ 9 times of volume dehydrated alcohols precipitate 3h → 10000r/min refrigerated centrifuge 10min, take precipitating → be freeze-dried to obtain calcium peptide chelating
Object.
Embodiment 11
Index determining is carried out to the sequestering pig bone collagen peptide of calcium that embodiment 1,3,5,7 and 9 obtains.
(1) protein, total nitrogen content: micro kjeldahl determination, referring to GB 5009.5-2010；Amino-acid nitrogen: neutral first
Aldehyde titration, referring to GB 5009.235-2016；Degree of hydrolysis calculation formula:
Degree of hydrolysis DH (%)=amino-acid nitrogen/raw material nitrogen pool × 100%
(2) polypeptide yield measures: biuret colorimetric method.
The production of polypeptide standard curve: taking 6 test tubes, and successively compound concentration is 0.0mg/mL, 0.5 mg/mL, 1.0mg/
The BSA standard solution of mL, 1.5mg/mL, 2.0mg/mL, 2.5mg/mL take 6mL standard solution respectively, and the examination of 4mL biuret is added
Agent mixes well, and stands 30min, measures light absorption value under ultraviolet specrophotometer 540nm wavelength.With protein concentration for horizontal seat
It marks X (mg/mL) and makes polypeptide standard curve using light absorption value as ordinate Y.
Soluble polypeptide assay: taking 5mL sample to be tested, and it is water-soluble that 5mL 10% (W/V) trichloroacetic acid (TCA) is added
Liquid stands 10min after mixing, 5000r/min is centrifuged 10min, and supernatant is taken to measure absorbance in Specification Curve of Increasing method,
And the concentration (mg/mL) of collagen polypeptide is calculated according to polypeptide standard curve.
(3) measurement of calcium chelation percent: 9 times of volume dehydrated alcohols precipitate 3h after chelatropic reaction, and refrigerated centrifuge takes supernatant
Liquid measures free calcium levels.Determination of calcium content is referring to the atomic absorption method in GB 5009.92-2016.
Index determining the results are shown in Table 1.
The sequestering pig bone collagen peptide index determining result of calcium obtained in 1 embodiment 1,3,5,7 and 9 of table
Embodiment 12
The Preliminary Identification of calcium peptide chelate complex
The sequestering pig bone collagen peptide of calcium, preparation method: Os Sus domestica powder → plus water are mixed to concentration of substrate 8% → adjusting pH value
For 7.5 → plus the 50 DEG C of water enzyme digestion 4h → 100 DEG C enzyme deactivations of alkali protease and neutral proteinase (each 4000u/g of enzyme concentration),
It is cooling, 10000r/min centrifugation 10min → supernatant is taken to obtain the sequestering pig bone collagen peptide of enzymolysis liquid → freeze-drying → calcium.
Ultraviolet-visible (UV-Vis) absorption spectroanalysis
It is 0.1mg/mL that the sequestering pig bone collagen peptide of calcium and calcium peptide chelate complex, which are dissolved in deionized water respectively to concentration,
Using deionized water as timebase, it is scanned within the scope of ultraviolet specrophotometer 190-400nm.As a result as shown in Figure 1；
The sequestering pig bone collagen peptide of calcium and calcium peptide chelate complex all have strong absworption peak at 210nm, there is weak absorbing peak at 280nm, respectively
N → π * transition characteristics absorption peak and tyrosine characteristic absorption peak corresponding to C=O in peptide bond.With the increase of calcium concentration,
Absorption peak strength at 280nm constantly increases, and obvious red shift occurs in appearance wavelength.This illustrates collagen polypeptide and calcium ion
Molecular structure changes after chelating, forms a kind of new compound.
Fourier transform infrared spectroscopy (FTIR) analysis
It is spare that KBr in 120 DEG C of drying 12h taking-up is put into vacuum desiccator.The sequestering pig bone collagen of 1mg calcium is weighed respectively
In agate mortar 100mg KBr is added, ground and mixed as far as possible is uniform, is made of tablet press machine transparent in peptide and calcium peptide chelate complex
Piece records 400-4000cm using FTIR spectrophotometer-1Infrared spectroscopy in range.As a result as shown in Figure 2.
FTIR spectrum can effectively distinguish the SPECTRAL DIVERSITY between two kinds of substances, usually utilize the function in spectrogram
Group area (1300cm-1~4000cm-1) type and variation of functional group, amino and carboxyl absorption peak in map in identification determinand
Variation can reflect the complex reaction between metal ion and organic group.As can be seen from Figure 2 calcium peptide chelate complex is red
Apparent variation has occurred in external spectrum compared with the infrared spectroscopy of the sequestering Bone gillg of calcium.
It is usually located at-the NH of polypeptide chain end2With Ca2+After chelating, characteristic absorption summit is moved to high band
It is dynamic.Such as-NH after the hydrolysate from shrimp protein and calcium chelating2Absorption peak by 3296cm-1It is moved to 3405cm-1(Hou
H,Wang S,Zhu X,et al.Anovel calcium-binding peptide from Antarctic krill
protein hydrolysates and identification of binding sites of calcium-peptide
complex.[J].Food Chemistry,2018,243:389.)；After salmon Bone gillg and calcium chelating ,-NH2Absorption
Peak is by 3066cm-1It is moved to 3305cm-1(LiuWY,Lu J,Gao F,et al.Preparation,characterization
and identification of calcium-chelating Atlantic salmon(Salmo salar,L.)ossein
oligopeptides[J].European Food Research& Technology,2015,241(6):851-860.).Such as
Fig. 2, the present invention in-NH2Stretching vibration absworption peak is 3347.92cm-1, move back with calcium chelating to 3409cm-1, show in peptide fragment-
The cloud density of NH becomes stronger due to inductive effect or dipole field-effect, it is meant that-NH2It may be with Ca2+It is anti-that chelating occurs
Ammonium salt should be formed.Absorption peak key reaction C=O stretching vibration at amide Ⅰ, Carboxylic Acid Ions stretching vibration after chelatropic reaction
By 1649.25cm-1It is moved to 1636.09cm-1Relatively lower wave number.1452.77cm-1And 1404.81cm-1Locate corresponding COO-It inhales
Take-up is moved to 1418.74cm-1, show that carboxyl may participate in the formation of chelate bonds in conjunction with calcium in peptide fragment.1020cm-1~
1360cm-1Absorption peak be mainly NH bending vibration and C-N stretching vibration, in figure after peptide fragment absorption peak and calcium chelating respectively
From 1316.63cm-1And 1147.23cm-1It is moved to 1323.59cm-1And 1126.34cm-1.In low frequency 500cm-1~800cm-1Area
In domain, some peaks disappear from chelate map, and calcium peptide chelate complex is in 671.51cm-1There is new absorption peak, it may be possible to because
It changes during chelating for C-H the and N-H group in peptide fragment.These the result shows that calcium ion may by with carboxyl
Oxygen and amino nitrogen atom interaction are integrated on collagen polypeptide.
Spectrofluorimetry
The sequestering Bone gillg of calcium and calcium chloride are dissolved in deionized water respectively.Set the calcium chloride of various concentration gradient
Solution, the ultimate density that the two is uniformly mixed to peptide are 0.05mg/mL.Mixed liquor is cooled to after 40 DEG C of constant temperature stand 1h
Room temperature sets sepectrophotofluorometer excitation wavelength 280nm, slit width 5nm, launch wavelength 290-500nm, carries out fluorescence
Scanning, as a result as shown in Figure 3.
Aromatic amino acid phenylalanine, tyrosine and tryptophan can generate endogenous fluorescence under particular excitation wavelength, out
Spike length is respectively near 300nm, 315nm and 350nm.It can be seen in figure 3 that pig bone collagen polypeptide and the sequestering glue of calcium
Former peptide all has absorption peak near 310nm and 360nm, and with the increase of calcium ion concentration, the fluorescence intensity at 310nm first increases
Weaken after strong, fluorescence intensity is no longer changed after calcium ion concentration reaches a certain concentration.Fluorescence intensity at 360nm is with calcium
The increase of ion concentration significantly increases, it may be possible to after calcium ion and polypeptide chelate, chromophore and auxochromes to certain amino acid
Group has certain effect.But also studies have found that there is Fluorescence-quenching after polypeptide and calcium ion chelating, and with calcium
The increase of concentration, fluorescence intensity constantly reduce.
Circular dichroism spectra (CD) analysis
Pig bone collagen polypeptide and calcium chloride are dissolved in deionized water respectively.The calcium chloride solution of various concentration gradient is set,
The ultimate density that the two is uniformly mixed to peptide is 0.2mg/mL.Mixed liquor is cooled to room temperature after 40 DEG C of constant temperature stand 1h,
The far-ultraviolet region progress spectra collection of 190-260nm, colorimetric pool optical path 0.1cm, scanning speed 200nm/min, 3 accumulation,
Ratio shared by various secondary structures, analysis collagen peptide second level under calcium ion effect are fitted using Young's modulus
The variation that structure occurs.
Mainly disclose alpha-helix, beta sheet and random coil etc. two in 180-250nm near-ultraviolet spectrum area in two spectrum of circle
Level structure information.It is fitted ratio shared by various secondary structures using Young's modulus, such as table 2.
The variation of secondary structure after the sequestering pig bone collagen peptide of 2 calcium of table is acted on from different calcium ion concentrations
From Table 2, it can be seen that alpha-helix is free of in the secondary structure of the sequestering pig bone collagen peptide of calcium, mainly with β-folding
Based on folded and random coil.After the sequestering pig bone collagen peptide of calcium and calcium ion effect, with the increase of calcium ion concentration, β-folding
Folded from 54.7%, constantly increasing to 81.2%, β-corner is reduced to 0 by 10.3%, and random coil is reduced to 18.8% by 35%.
The a large amount of hydrogen bond energies contained in beta sheet maintain the stabilization of secondary structure, and β-corner and random coil do not contain hydrogen bond, can make peptide
Duan Yongyou certain freedom degree.Thus, it can be known that ordered structure increases after the sequestering pig bone collagen peptide of calcium and calcium binding, knot
Structure becomes even closer.
Scanning electron microscope (SEM) analysis
It takes the sequestering pig bone collagen peptide of appropriate calcium and calcium peptide chelate complex that sample is lyophilized uniformly to be applied on sample dish double-sided adhesive, spray
It is put into scanning electron microscopic observation room after golden coating film treatment to vacuumize, acceleration voltage is set as 15kV, adjusts beam spot size, focus
Afterwards, image is obtained in 5000 amplification factors.
The scanning electron microscope image of the sequestering pig bone collagen peptide of calcium and calcium peptide chelate complex is as shown in figure 4, wherein A is calcium chelating
Property pig bone collagen peptide scanning electron microscope image；B is calcium peptide chelate complex scanning electron microscope image.As can be seen from Figure 4 pig bone collagen is more
Peptide surface compact is smooth, and has a small amount of white particles, it may be possible to during enzymatic hydrolysis prepares pig bone collagen polypeptide, release
A small amount of free calcium be adsorbed in polypeptide surface.It is in smooth spheroidal aggravation after the sequestering pig bone collagen peptide of calcium and calcium chelating, it can be with
Speculate that the chelating of peptide and calcium ion mainly passes through ionic bond and coordinate bond etc., therefore scanning electron microscope (SEM) photograph is similar to salt in fine and close
Structure.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of preparation method of the sequestering pig bone collagen peptide of calcium, comprising the following steps:
1) the broken rear degreasing of fresh pig bone, decalcification are taken, except Os Sus domestica powder is made in foreign protein, crushing；
2) Os Sus domestica powder is dissolved in after water mixing with alkali protease and/or neutral proteinase and carries out enzymatic hydrolysis acquisition pig bone collagen polypeptide
The sequestering pig bone collagen peptide of liquid, i.e. calcium.
2. preparation method according to claim 1, which is characterized in that when the enzyme is alkali protease, the enzymatic hydrolysis
Concentration of substrate be 5~10%, enzyme concentration is 4000~8000u/g, and the pH value of the enzymatic hydrolysis is 8.0~9.2, the enzymatic hydrolysis
Temperature is 40~50 DEG C, and the time of the enzymatic hydrolysis is 4~6h.
3. preparation method according to claim 1, which is characterized in that when the enzyme is neutral proteinase, the enzymatic hydrolysis
Concentration of substrate be 6~10%, enzyme concentration is 6000~10000u/g, and the pH value of the enzymatic hydrolysis is 7.0~8.0, the enzymatic hydrolysis
Temperature be 45~55 DEG C, time of the enzymatic hydrolysis is 4~6h.
4. preparation method according to claim 1, which is characterized in that when the enzyme is alkali protease and neutral proteinase
When, the enzymatic hydrolysis is alkali protease enzymatic hydrolysis synchronous with neutral proteinase.
5. the preparation method according to claim 4, which is characterized in that the mass ratio of alkali protease and neutral proteinase is
1:1~2:1；The concentration of substrate of the enzymatic hydrolysis is 6~8%；The pH value of the enzymatic hydrolysis is 7.4~7.6, and the temperature of the enzymatic hydrolysis is
45~51 DEG C, the time of the enzymatic hydrolysis is 4~6h.
6. preparation method according to claim 1, which is characterized in that when the enzyme is alkali protease and neutral proteinase
When, the enzymatic hydrolysis is alkali protease and neutral proteinase stepwise discretization；To the alkali protease and neutral protease enzymolysis
Chronological order it is unlimited.
7. the sequestering pig bone collagen peptide of the calcium that right wants preparation method described in 1~6 any one to prepare, which is characterized in that
The calcium chelation percent of the sequestering pig bone collagen peptide of calcium is 42~70.24%.
8. using the method for the calcium peptide chelate complex that the sequestering pig bone collagen peptide of calcium as claimed in claim 7 prepares, including with
Lower step: the pH for adjusting the pig bone collagen polypeptide liquid is 7.2~7.8, then by the pig bone after calcium chloride solution and adjusting pH
Reaction solution, the reaction solution that ethanol precipitation obtains are obtained after collagen polypeptide liquid mixing chelating, obtained solid phase components are calcium peptide chelating
Object.
9. preparation method according to claim 1 or claim 7, which is characterized in that the concentration of the calcium chloride solution is 1~5g/
L；The volume ratio of the calcium chloride solution and pig bone collagen polypeptide liquid is 1:1~1:2.
10. the calcium peptide chelate complex that preparation method described in claim 8 or 9 prepares.
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