CN110204595A - One kind, which has, adjusts active feta proteolysis peptide of bone-marrow-derived lymphocyte and preparation method thereof - Google Patents
One kind, which has, adjusts active feta proteolysis peptide of bone-marrow-derived lymphocyte and preparation method thereof Download PDFInfo
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- CN110204595A CN110204595A CN201910479471.8A CN201910479471A CN110204595A CN 110204595 A CN110204595 A CN 110204595A CN 201910479471 A CN201910479471 A CN 201910479471A CN 110204595 A CN110204595 A CN 110204595A
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 85
- 230000017854 proteolysis Effects 0.000 title claims abstract description 56
- 210000004698 lymphocyte Anatomy 0.000 title claims abstract description 46
- 210000001185 bone marrow Anatomy 0.000 title claims abstract description 43
- 238000002360 preparation method Methods 0.000 title claims description 9
- 101100003139 Mus musculus Atp8b5 gene Proteins 0.000 claims abstract description 109
- 238000000034 method Methods 0.000 claims abstract description 25
- 239000000203 mixture Substances 0.000 claims abstract description 20
- 230000007062 hydrolysis Effects 0.000 claims abstract description 12
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims abstract description 11
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 6
- 108010076119 Caseins Proteins 0.000 claims description 22
- 102000011632 Caseins Human genes 0.000 claims description 22
- 239000005018 casein Substances 0.000 claims description 20
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 claims description 20
- 235000021240 caseins Nutrition 0.000 claims description 20
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 16
- 239000012460 protein solution Substances 0.000 claims description 16
- 239000000243 solution Substances 0.000 claims description 14
- 108091005804 Peptidases Proteins 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 12
- 238000004458 analytical method Methods 0.000 claims description 12
- 238000000108 ultra-filtration Methods 0.000 claims description 12
- 102000035195 Peptidases Human genes 0.000 claims description 11
- 235000019833 protease Nutrition 0.000 claims description 11
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- 239000007788 liquid Substances 0.000 claims description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 7
- 238000002270 exclusion chromatography Methods 0.000 claims description 7
- 238000000746 purification Methods 0.000 claims description 6
- 238000010828 elution Methods 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 229910000041 hydrogen chloride Inorganic materials 0.000 claims description 4
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 4
- 238000000926 separation method Methods 0.000 claims description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 3
- 102100034560 Cytosol aminopeptidase Human genes 0.000 claims description 3
- 101000867715 Homo sapiens Calpain-3 Proteins 0.000 claims description 3
- 101000924389 Homo sapiens Cytosol aminopeptidase Proteins 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 210000000805 cytoplasm Anatomy 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000001514 detection method Methods 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000001819 mass spectrum Methods 0.000 claims description 3
- 229920001184 polypeptide Polymers 0.000 claims description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 3
- AYEKOFBPNLCAJY-UHFFFAOYSA-O thiamine pyrophosphate Chemical compound CC1=C(CCOP(O)(=O)OP(O)(O)=O)SC=[N+]1CC1=CN=C(C)N=C1N AYEKOFBPNLCAJY-UHFFFAOYSA-O 0.000 claims description 3
- 210000004027 cell Anatomy 0.000 claims description 2
- 235000021247 β-casein Nutrition 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 3
- 239000004365 Protease Substances 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 210000002751 lymph Anatomy 0.000 claims 1
- 235000019419 proteases Nutrition 0.000 claims 1
- 238000004611 spectroscopical analysis Methods 0.000 claims 1
- 210000002784 stomach Anatomy 0.000 claims 1
- 230000036737 immune function Effects 0.000 abstract description 6
- 230000002708 enhancing effect Effects 0.000 abstract description 5
- 108060003951 Immunoglobulin Proteins 0.000 abstract description 4
- 102000018358 immunoglobulin Human genes 0.000 abstract description 4
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- 235000013402 health food Nutrition 0.000 abstract description 2
- 235000015097 nutrients Nutrition 0.000 abstract description 2
- 241001494479 Pecora Species 0.000 description 11
- 239000012071 phase Substances 0.000 description 10
- 239000000047 product Substances 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
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- 229940088598 enzyme Drugs 0.000 description 5
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- 244000248349 Citrus limon Species 0.000 description 3
- 235000005979 Citrus limon Nutrition 0.000 description 3
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 3
- 102000004407 Lactalbumin Human genes 0.000 description 3
- 108090000942 Lactalbumin Proteins 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- 229930003268 Vitamin C Natural products 0.000 description 3
- 108010046377 Whey Proteins Proteins 0.000 description 3
- 102000007544 Whey Proteins Human genes 0.000 description 3
- 235000020247 cow milk Nutrition 0.000 description 3
- 239000006071 cream Substances 0.000 description 3
- 235000012907 honey Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 235000013336 milk Nutrition 0.000 description 3
- 239000008267 milk Substances 0.000 description 3
- 210000004080 milk Anatomy 0.000 description 3
- 239000002304 perfume Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 235000002639 sodium chloride Nutrition 0.000 description 3
- 239000011718 vitamin C Substances 0.000 description 3
- 235000019154 vitamin C Nutrition 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 235000020251 goat milk Nutrition 0.000 description 2
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- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
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- CFRRIZLGFGJEDB-SRVKXCTJSA-N Met-His-Gln Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O CFRRIZLGFGJEDB-SRVKXCTJSA-N 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- SSWJYJHXQOYTSP-SRVKXCTJSA-N Pro-His-Gln Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O SSWJYJHXQOYTSP-SRVKXCTJSA-N 0.000 description 1
- 238000012356 Product development Methods 0.000 description 1
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- 239000005862 Whey Substances 0.000 description 1
- 235000005550 amino acid supplement Nutrition 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000002223 anti-pathogen Effects 0.000 description 1
- 230000030741 antigen processing and presentation Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 150000005693 branched-chain amino acids Chemical class 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000007969 cellular immunity Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 201000006549 dyspepsia Diseases 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 102000034356 gene-regulatory proteins Human genes 0.000 description 1
- 108091006104 gene-regulatory proteins Proteins 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 230000004727 humoral immunity Effects 0.000 description 1
- 239000000413 hydrolysate Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 229940088592 immunologic factor Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 210000004126 nerve fiber Anatomy 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 235000005974 protein supplement Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 235000020254 sheep milk Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
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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 OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- 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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- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nutrition Science (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Biotechnology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
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- Medicinal Chemistry (AREA)
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- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
Have the invention discloses one kind and adjust the active feta proteolysis peptide of bone-marrow-derived lymphocyte, the sequence of the feta proteolysis peptide is as shown in SEQ ID NO.1.Preparing, there is the method for adjusting bone-marrow-derived lymphocyte active feta proteolysis peptide to include the selection of feta albumen, two-stage enzyme hydrolysis feta albumen and isolate and purify.The present invention chooses the feta albumen of high quality, high stability, pass through different biological enzymatic mixtures, reaction is hydrolyzed to feta albumen under the conditions of two stages different enzyme reaction, it isolates and purifies to obtain having for high-purity and adjusts the active feta proteolysis peptide of bone-marrow-derived lymphocyte, compared with existing feta protein product, not only it is more easier to digest, and there is apparent enhancing immune function, bone-marrow-derived lymphocyte activity (enhancing immunoglobulin expression) can significantly be enhanced, had a good application prospect in health food and functional nutrient product.
Description
Technical field
The invention belongs to technical field of nourishment, and in particular to one kind, which has, adjusts the active feta egg of bone-marrow-derived lymphocyte
White range of hydrolysed peptides and preparation method thereof.
Background technique
Bone-marrow-derived lymphocyte is the important regulatory factor of human immune system, its function includes stimulate the reaction, antigen presentation,
Inflammatory factor and antibody are secreted, can also be both immunoreacted by way of humoral immunity by cellular immunity, is helped
Body is helped to support the invasion of antipathogen.Moreover, B immunocyte is repaired after additionally aiding help body tissue wound, is wrapped
Include bone split, skin ultrastructure etc..In addition, bone-marrow-derived lymphocyte can also promote nerve fiber below scar tissue and interior significantly
The growth in portion, the body for repairing these have normal physiological function.In old man, child, in the special populations such as patient, due to
The decline of body's immunity, therefore played an important role by the activity that meal supplement adjusts bone-marrow-derived lymphocyte to keeping fit.
Casein is the main component of albumen in dairy products, and 80-the 85% of gross protein is accounted in cow's milk, in goat milk
Casein content is slightly lower, is 74-80%.Casein is noncrystalline, non-hygroscopic substance, and human body is after edible lactalbumin, effect
It is very fast with starting, it can be eaten before and after body building, be rich in branched-chain amino acid, it can make albumen in flesh during movement
It shuttles in meat, or allows its effect immediately, certainly because what is digested is very fast, will not be kept in body too long.It is another
Aspect, since the dissolubility of casein is substantially less than lactalbumin, edible casein may cause indigestion reaction.Using micro-
Biology or biological enzyme caseinhydrolysate in vitro, increasing its soluble and property easy to digest is a kind of side for increasing its nutritive value
Method.
The method of currently used caseinhydrolysate mainly passes through trypsin hydrolysis method, hydrolysis by novo method or
Acid protease hydrolysis method obtains bovine casein hydrolysate, and usually these are sought as protein/amino acid supplements
Support product development.But since method for hydrolysis is excessively single, obtained cow's milk hydrolysate composition is too complicated, therefore causes product
Adjusting bone-marrow-derived lymphocyte immunocompetence function value is not high, and some even has side effect.Sheep cream is differed markedly from as a kind of composition
The casein of cow's milk there is no the application to the exploitation of feta proteolysis product, also available without corresponding functional product, because
This optimizes enzymatic hydrolysis process by the feta albumen of selection high quality, and the efficient adjusting bone-marrow-derived lymphocyte purified is active
Small-molecular peptides have broad application prospects.
Summary of the invention
The present invention is directed to the sky of bone-marrow-derived lymphocyte immune function product is adjusted for existing sheep whey solution casein hydrolysate
It is white, the method for hydrolysis and hydrolysising condition of feta albumen are studied, is provided a kind of with the adjusting active feta of bone-marrow-derived lymphocyte
Proteolysis peptide and preparation method thereof.The present invention also applies this to have and adjusts the active feta proteolysis of bone-marrow-derived lymphocyte
Peptide enhances the function of nutriment in nutriment, enhances the body immunity of user.
In order to achieve the above object, technical solution provided by the invention are as follows:
It is described that there is the adjusting active feta proteolysis peptide of bone-marrow-derived lymphocyte, which is characterized in that the feta albumen
The sequence of range of hydrolysed peptides is as shown in SEQ ID NO.1: methionine-histidine-Gln-Pro-histidine-glutamine-
Proline-leucine (that is, MHQPHQPL).
It is above-mentioned that there is the preparation method for adjusting the active feta proteolysis peptide of bone-marrow-derived lymphocyte to include the following steps:
(1) selection of feta albumen: by the feta protein dissolution of selection in deionized water, feta albumen is obtained
Concentration is 8-12g/L, the feta protein solution of preferably 9-10g/L, more preferably 10g/L；α in the feta albumen
The mass percentage content of 2 casein of mass percentage content≤15, α of 1 casein is the quality of 15-20%, β casein
Degree is that the mass percentage content of 43-51%, k casein is 17-23%；
(2) two-stage enzyme hydrolysis feta albumen: being 0.3-0.5M, the preferably hydrogen chloride of 0.4M with hydrogen cloride concentration
The pH value of feta protein solution obtained by solution regulating step (1) is 3.8-4.1；Then A is added into feta protein solution
3-4h are reacted after proteinase mixture in 37 DEG C of insulating boxs, preferably 3.5h is using naoh concentration after reaction
The pH value that the sodium hydroxide solution of 0.15-0.25M, preferably 0.2M adjust feta protein solution is 8.1-8.5；Again to sheep
It is added after B proteinase mixture in milk casein solution and reacts 3-4h, preferably 3.5h in 37 DEG C of insulating boxs, obtain sheep cream
Casein hydrolysis peptide mixer；Wherein, the concentration of pepsin is 0.15-0.22g/L, tripeptides in the A proteinase mixture
The concentration of hydrolase TPP is 0.01-0.05g/L, and the concentration of trypsase is 0.20-in the B proteinase mixture
The concentration of 0.25g/L, double peptidohydrolase DPP are 0.01-0.05g/L, and the concentration of cytoplasm amino peptidohydrolase LAP3 is 0.01-
The concentration of 0.05g/L, calpain CAPN3 are 0.01-0.03g/L；
(3) isolate and purify: the resulting feta proteolysis peptide mixer of purification procedures (2) obtains having adjusting B
The feta proteolysis peptide of lymphocyte activity.
Preferably, the specific steps packet of the resulting feta proteolysis peptide of step (3) purification procedures (2)
It includes: a) ultrafiltration: mixed to the obtained feta proteolysis peptide of step (2) using the ultra-filtration centrifuge tube that molecular cut off is 2kDa
It closes object to be separated, obtains the mixtures of polypeptides that molecular weight is less than 1000Da；B) exclusion chromatography separates: using molecular exclusion layer
Analysis SEC peptide splitter is separated, molecular exclusion chromatography (Size-Exclusion Chromatography) SEC peptide splitter
In elution phase A be pH value 7.0-8.5 concentration be 20mM tris-hydrochloric acid, flow velocity be 1.0-1.5mL/min, elution ladder
Degree is 30-60min interior from 0% to 100%；Eluting peak acquisition time be 15-25min, preferably 17-23min, more preferably
For 6-10min.
Preferably, the method further includes for the adjusting active feta egg of bone-marrow-derived lymphocyte after step (3)
The purity analysis step of white range of hydrolysed peptides.It is highly preferred that described pair has and adjusts the active feta proteolysis peptide of bone-marrow-derived lymphocyte
Purity analysis step are as follows: carry out feta proteolysis peptide purity analysis using reverse phase C18 performance liquid chromatographic column, it is described anti-
Mobile phase A in phase C18 performance liquid chromatographic column is the aqueous solution of the trifluoroacetic acid of concentration 0.1%, and Mobile phase B is concentration
The acetonitrile solution of 0.1% trifluoroacetic acid, Detection wavelength 214nm；Flow velocity is 1.0mL/min；Sample volume is 20 μ L；Column temperature:
30 DEG C, gradient are as follows:
Preferably, the method further includes to newborn with the active sheep of bone-marrow-derived lymphocyte is adjusted after purity analysis step
The step of casein hydrolysate peptides determined amino acid sequence.It is highly preferred that described have the adjusting active feta egg of bone-marrow-derived lymphocyte
The step of plain boiled water solution peptide amino acid sequence measures are as follows: will have and adjust the active feta proteolysis peptide loading of bone-marrow-derived lymphocyte
Mass spectrum to ultrahigh resolution is analyzed, its amino acid sequence is measured.
The invention will be further described below:
Bone-marrow-derived lymphocyte belongs to immunocyte, can immunoglobulin,exocrine, execute the humoral immune function of body, it is clear
Intracorporal cause of disease substance, plays an important role to the performance of body immune system function.The present invention chooses high quality, high stability
Feta albumen carries out feta albumen under the conditions of two stages different enzyme reaction by different biological enzymatic mixtures
Hydrolysis isolates and purifies to obtain having for high-purity and adjusts the active feta proteolysis peptide of bone-marrow-derived lymphocyte, and existing
Feta protein product is compared, and is not only more easier to digest, but also has apparent enhancing immune function, can significantly be enhanced
Bone-marrow-derived lymphocyte activity (enhancing immunoglobulin expression), before there is good application in health food and functional nutrient product
Scape.
(peptide fragment c described in figure is of the present invention with the adjusting active feta egg of bone-marrow-derived lymphocyte to Detailed description of the invention
White range of hydrolysed peptides)
Fig. 1 is using the method for the present invention after two-stage enzyme hydrolysis feta albumen, before ultrafiltration, after ultrafiltration and SEC peptide
Protein electrophorese result figure after separation；
Fig. 2 is the present embodiment product HPLC liquid chromatogram after SEC peptide splitter；
Fig. 3 is facilitation testing result figure of the feta protein peptides to 1788 lymphocyte activity of RPMI.
Specific embodiment
Embodiment 1
It is above-mentioned that there is the preparation method for adjusting the active feta proteolysis peptide of bone-marrow-derived lymphocyte to include the following steps:
(1) selection of feta albumen: the goat milk adopted is centrifuged 30min at 4 DEG C under the conditions of 4000g, removes butterfat
Layer, obtains protein solution；For 5mol/L hydrogen chloride regulatory protein solution ph to 4.6, the refrigerated centrifuge at 4 DEG C retains precipitating；It spends
Ion water washing precipitates 2 times, will be precipitated and dissolved in deionized water, and the precipitating is the feta albumen of selection, obtains sheep
Milk casein concentration is 8-12g/L, the feta protein solution of preferably 9-10g/L, more preferably 10g/L；The sheep cream
The mass percentage content of 2 casein of mass percentage content≤15, α of 1 casein of α is 15-20%, β junket egg in casein
White mass percentage content is that the mass percentage content of 43-51%, k casein is 17-23%；
(2) two-stage enzyme hydrolysis feta albumen: hydrogen chloride solution regulating step (1) institute for being 0.4M with hydrogen cloride concentration
The pH value for obtaining feta protein solution is 3.9；Then it is added after A proteinase mixture into feta protein solution in 37 DEG C of perseverances
3.5h is reacted in incubator, it is molten to adjust feta albumen using the sodium hydroxide solution that naoh concentration is 0.2M after reaction
The pH value of liquid is 8.2；3.5h is reacted in 37 DEG C of insulating boxs after B proteinase mixture is added into feta protein solution again,
Obtain feta proteolysis peptide mixer；Wherein, the concentration of pepsin is 0.15-in the A proteinase mixture
The concentration of 0.22g/L, three peptidohydrolase TPP are 0.01-0.05g/L, the concentration of trypsase in the B proteinase mixture
For 0.20-0.25g/L, the concentration of double peptidohydrolase DPP is 0.01-0.05g/L, the concentration of cytoplasm amino peptidohydrolase LAP3
For 0.01-0.05g/L, the concentration of calpain CAPN3 is 0.01-0.03g/L；
(3) isolate and purify: the resulting feta proteolysis peptide mixer of purification procedures (2) obtains having adjusting B
The feta proteolysis peptide of lymphocyte activity.
Wherein, the specific steps of the resulting feta proteolysis peptide of step (3) purification procedures (2) include:
A) ultrafiltration: the ultra-filtration centrifuge tube (Sai Duolisi for the use of molecular cut off being 2kDaSeries) to obtained by step (2)
To feta proteolysis peptide mixer separated (4 DEG C, under the conditions of 12000rpm ultrafiltration be centrifuged 15min), obtain molecule
Amount is less than the mixtures of polypeptides of 1000Da；B) exclusion chromatography separates: being divided using molecular exclusion chromatography SEC peptide splitter
From the elution phase A in molecular exclusion chromatography (Size-Exclusion Chromatography) SEC peptide splitter is pH value
8.0 concentration is the tris- hydrochloric acid of 20mM, and flow velocity 1.0mL/min, gradient is 50min interior from 0% to 100%；Elution
Peak acquisition time is 6-10min.
The method further includes for the adjusting active feta proteolysis of bone-marrow-derived lymphocyte after step (3)
The purity analysis step of peptide.Specific steps are as follows: carry out feta proteolysis peptide purity using reverse phase C18 performance liquid chromatographic column
It analyzes, the mobile phase A in the reverse phase C18 performance liquid chromatographic column is the aqueous solution of the trifluoroacetic acid of concentration 0.1%, mobile phase
B is the acetonitrile solution of the trifluoroacetic acid of concentration 0.1%, Detection wavelength 214nm；Flow velocity is 1.0mL/min；Sample volume is 20 μ
L；Column temperature: 30 DEG C, gradient are as follows:
The method further includes to the adjusting active feta albumen water of bone-marrow-derived lymphocyte after purity analysis step
The step of solving peptide amino acid sequence measurement.Specific steps are as follows: will have and adjust the active feta proteolysis peptide of bone-marrow-derived lymphocyte
The mass spectrum for being loaded to ultrahigh resolution is analyzed, its amino acid sequence, sequence results are measured are as follows: methionine-histidine-paddy ammonia
Amide-proline-histidine-glutamine-proline-leucine (that is, MHQPHQPL).
Embodiment 2
There is the adjusting active feta proteolysis peptide (hereinafter referred to as " sheep of bone-marrow-derived lymphocyte about prepared by embodiment 1
Milk casein range of hydrolysed peptides ") performance analysis result:
As shown in Figure 1, the polyacrylamide gel electrophoresis for being 18% through over-richness, the feta proteolysis peptide before ultrafiltration
Mixture molecular weight is mainly distributed between 1.7-4.6kD, and after using the super filter tube processing of 2000Da molecule interception, institute
Obtained feta proteolysis peptide mixer molecular size range is less than 1.7kDa.By further using SEC peptide splitter pair
Ultrafiltration carries out isolated purifying small-molecular peptides to feta proteolysis peptide mixer, estimates molecular weight less than 1000Da.
As shown in Figure 2, the carry out efficient liquid phase chromatographic analysis after SEC peptide post separation, obtained feta proteolysis
There is main peak when eluting 18min in peptide, and clearly, and other miscellaneous peaks are few, illustrates by ultrafiltration and SEC feta obtained after separation
Proteolysis peptide purity is very high, by calculating feta proteolysis peptide purity up to 95% or more.
Feta proteolysis peptide analyzes the effect of 1788 immune function of bone-marrow-derived lymphocyte RPMI, such as Fig. 3
Shown, after adding 5 μ g/mL feta proteolysis peptides respectively in 1788 cell culture medium of RPMI, 1788 cell of RPMI exists
The Immunoglobulin IgA of different time points, IgM, IgG and immune factor TNF-alpha expression amount dramatically increase.It proves to pass through this hair
The feta proteolysis peptide that bright two-stage complex enzyme for hydrolyzing method obtains has significant enhancing bone-marrow-derived lymphocyte immune function.
Embodiment 3
There is the adjusting active feta proteolysis peptide (hereinafter referred to as " sheep of bone-marrow-derived lymphocyte about prepared by embodiment 1
Milk casein range of hydrolysed peptides ") application:
Feta proteolysis peptide is formulated high protein drink:
(1) raw material preparation: feta albumen, feta proteolysis peptide, sheep whey protein concentrate, oligofructose, orange
Sub- powder, lemon powder, natural perfume material, sea salt, honey, vitamin C, its composition by weight of every 100ml are as follows: feta albumen
1.4g, feta proteolysis peptide 0.2g, sheep whey protein concentrate 0.9g, oligofructose 0.8g, orange powder 0.6g, lemon powder
0.2g, natural perfume material 0.05g, sea salt 0.1g, honey 0.5g, vitamin C 0.1g；
(2) mixing: by step (1) prepare oligofructose, orange powder, lemon powder, natural perfume material, sea salt, vitamin C into
Row mixing,；
(3) feta albumen, feta proteolysis peptide, the concentration of sheep lactalbumin are added into the premix of step (2)
Object obtains mixing；
(4) pure water is heated to 30-50 DEG C, and the mixing of step (3) and honey are joined, and stirring is so that it is abundant
Dissolution, carries out homogeneous in high pressure homogenizer, and homogenization pressure is 12-18Mpa；
(5) it filters: the solution of step 4 is subjected to aseptic filtration to get feta proteolysis peptide formula high protein drink
Product.
Sequence table
<110>the triumphant nutriment Co., Ltd of 3-bromo-4-[2,3-dibromo-4,5-dihydroxyphenyl
<120>a kind of that there is adjusting active feta proteolysis peptide of bone-marrow-derived lymphocyte and preparation method thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213>unknown (null) (null)
<400> 1
Met His Gln Pro His Gln Pro Leu
1 5
Claims (9)
1. one kind, which has, adjusts the active feta proteolysis peptide of bone-marrow-derived lymphocyte, which is characterized in that the feta albumen water
The sequence of peptide is solved as shown in SEQ ID NO.1.
2. having the preparation method for adjusting the active feta proteolysis peptide of bone-marrow-derived lymphocyte, feature as described in claim 1
It is, described method includes following steps:
(1) selection of feta albumen: by the feta protein dissolution of selection in deionized water, feta protein concentration is obtained
For the feta protein solution of 8-12g/L；2 junket of mass percentage content≤15, α of 1 casein of α in the feta albumen
The mass percentage content of albumen is that the mass percentage content of 15-20%, β casein is the matter of 43-51%, k casein
Measuring degree is 17-23%；
(2) two-stage enzyme hydrolysis feta albumen: the hydrogen chloride solution regulating step (1) for being 0.3-0.5M with hydrogen cloride concentration
The pH value of gained feta protein solution is 3.8-4.1；Then after A proteinase mixture being added into feta protein solution
3-4h are reacted in 37 DEG C of insulating boxs, the sodium hydroxide solution for the use of naoh concentration being after reaction 0.15-0.25M
The pH value for adjusting feta protein solution is 8.1-8.5；Again into feta protein solution be added B proteinase mixture after in
3-4h are reacted in 37 DEG C of insulating boxs, obtain feta proteolysis peptide mixer；Wherein, stomach egg in the A proteinase mixture
The concentration of white enzyme is 0.15-0.22g/L, and the concentration of three peptidohydrolase TPP is 0.01-0.05g/L, the B protease mixing
The concentration of trypsase is 0.20-0.25g/L in object, and the concentration of double peptidohydrolase DPP is 0.01-0.05g/L, cytoplasm amino
The concentration of peptidohydrolase LAP3 is 0.01-0.05g/L, and the concentration of calpain CAPN3 is 0.01-0.03g/L；
(3) isolate and purify: the resulting feta proteolysis peptide mixer of purification procedures (2) obtains having adjusting B lymph
The feta proteolysis peptide of cell activity.
3. method according to claim 2, which is characterized in that in step (1) the feta protein solution, feta albumen
Concentration be 9-11g/L.
4. method according to claim 2, which is characterized in that step (3) purification procedures (2) resulting feta
The specific steps of proteolysis peptide include: a) ultrafiltration: using the ultra-filtration centrifuge tube that molecular cut off is 2kDa to obtained by step (2)
To feta proteolysis peptide mixer separated, obtain molecular weight be less than 1000Da mixtures of polypeptides；B) gel color
Spectrometry separation: it is separated using molecular exclusion chromatography SEC peptide splitter, the elution in molecular exclusion chromatography SEC peptide splitter
Phase A is tris-hydrochloric acid that the concentration of pH value 7.0-8.5 is 20mM, and flow velocity is 1.0-1.5mL/min, and gradient is 30-
In 60min from 0% to 100%；Eluting peak acquisition time is 6-10min.
5. such as the described in any item methods of Claims 1-4, which is characterized in that the method further includes needle after step (3)
To the purity analysis step with the adjusting active feta proteolysis peptide of bone-marrow-derived lymphocyte.
6. method as claimed in claim 5, which is characterized in that described pair has the adjusting active feta egg of bone-marrow-derived lymphocyte
The purity analysis step of white range of hydrolysed peptides are as follows: carry out feta proteolysis peptide purity point using reverse phase C18 performance liquid chromatographic column
It analyses, the mobile phase A in the reverse phase C18 performance liquid chromatographic column is the aqueous solution of the trifluoroacetic acid of concentration 0.1%, Mobile phase B
For the acetonitrile solution of the trifluoroacetic acid of concentration 0.1%, Detection wavelength 214nm；Flow velocity is 1.0mL/min；Sample volume is 20 μ L；
Column temperature: 30 DEG C, gradient are as follows:
7. method as claimed in claim 6, which is characterized in that the method further includes to having after purity analysis step
Adjust the step of active feta proteolysis peptide amino acid sequence of bone-marrow-derived lymphocyte measures.
8. the method for claim 7, which is characterized in that described that there is the adjusting active feta albumen of bone-marrow-derived lymphocyte
The step of range of hydrolysed peptides determined amino acid sequence are as follows: will have the adjusting active feta proteolysis peptide of bone-marrow-derived lymphocyte to be loaded to
The mass spectrum of ultrahigh resolution is analyzed, its amino acid sequence is measured.
The active feta proteolysis peptide of bone-marrow-derived lymphocyte is adjusted in preparing nutriment 9. having as described in claim 1
Using.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105142658A (en) * | 2013-03-15 | 2015-12-09 | Mjn美国控股有限责任公司 | Reducing proinflammatory response |
| WO2016066758A1 (en) * | 2014-10-30 | 2016-05-06 | Laboratorios Ordesa, S.L. | Milk protein hydrolysate for use in the treatment of diarrhoea |
| CN107949396A (en) * | 2015-07-23 | 2018-04-20 | Mjn 美国控股有限责任公司 | Alimentation composition comprising caseinhydrolysate |
| CN108314704A (en) * | 2018-01-25 | 2018-07-24 | 海普诺凯营养品有限公司 | A kind of sheep lactalbumin immunomodulatory peptides, preparation method and its application |
-
2019
- 2019-06-04 CN CN201910479471.8A patent/CN110204595A/en not_active Withdrawn
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105142658A (en) * | 2013-03-15 | 2015-12-09 | Mjn美国控股有限责任公司 | Reducing proinflammatory response |
| WO2016066758A1 (en) * | 2014-10-30 | 2016-05-06 | Laboratorios Ordesa, S.L. | Milk protein hydrolysate for use in the treatment of diarrhoea |
| CN107949396A (en) * | 2015-07-23 | 2018-04-20 | Mjn 美国控股有限责任公司 | Alimentation composition comprising caseinhydrolysate |
| CN108314704A (en) * | 2018-01-25 | 2018-07-24 | 海普诺凯营养品有限公司 | A kind of sheep lactalbumin immunomodulatory peptides, preparation method and its application |
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