CN108148116A - A kind of delicate flavour peptide and preparation method and application - Google Patents
A kind of delicate flavour peptide and preparation method and application Download PDFInfo
- Publication number
- CN108148116A CN108148116A CN201810196275.5A CN201810196275A CN108148116A CN 108148116 A CN108148116 A CN 108148116A CN 201810196275 A CN201810196275 A CN 201810196275A CN 108148116 A CN108148116 A CN 108148116A
- Authority
- CN
- China
- Prior art keywords
- delicate flavour
- flavour peptide
- preparation
- peptide
- digestion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 111
- 239000000796 flavoring agent Substances 0.000 title claims abstract description 110
- 235000019634 flavors Nutrition 0.000 title claims abstract description 110
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 230000029087 digestion Effects 0.000 claims abstract description 24
- 239000013612 plasmid Substances 0.000 claims abstract description 18
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 16
- 238000000746 purification Methods 0.000 claims abstract description 15
- 239000013598 vector Substances 0.000 claims abstract description 15
- 229920001184 polypeptide Polymers 0.000 claims abstract description 14
- 241000894006 Bacteria Species 0.000 claims abstract description 11
- 239000007788 liquid Substances 0.000 claims abstract description 10
- 238000003259 recombinant expression Methods 0.000 claims abstract description 9
- 238000003786 synthesis reaction Methods 0.000 claims abstract description 8
- 239000012530 fluid Substances 0.000 claims abstract description 7
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 6
- 239000012634 fragment Substances 0.000 claims abstract description 5
- 230000002068 genetic effect Effects 0.000 claims abstract description 5
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 claims abstract description 3
- 108090000623 proteins and genes Proteins 0.000 claims description 29
- 235000014469 Bacillus subtilis Nutrition 0.000 claims description 12
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 8
- 244000063299 Bacillus subtilis Species 0.000 claims description 7
- 102000003960 Ligases Human genes 0.000 claims description 6
- 108090000364 Ligases Proteins 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 5
- 230000004151 fermentation Effects 0.000 claims description 5
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 230000002194 synthesizing effect Effects 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 3
- 230000036541 health Effects 0.000 claims description 3
- 238000002525 ultrasonication Methods 0.000 claims description 3
- 239000000178 monomer Substances 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 235000021393 food security Nutrition 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 6
- 150000001413 amino acids Chemical group 0.000 abstract description 3
- 238000013461 design Methods 0.000 abstract description 2
- HSWYMWGDMPLTTH-FXQIFTODSA-N Asp-Glu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HSWYMWGDMPLTTH-FXQIFTODSA-N 0.000 abstract 1
- KQNDIKOYWZTZIX-FXQIFTODSA-N Ser-Ser-Arg Chemical group OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQNDIKOYWZTZIX-FXQIFTODSA-N 0.000 abstract 1
- 238000011156 evaluation Methods 0.000 description 21
- 230000001953 sensory effect Effects 0.000 description 14
- 238000000034 method Methods 0.000 description 10
- 239000000523 sample Substances 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 6
- 238000001962 electrophoresis Methods 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 210000000697 sensory organ Anatomy 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- 235000019605 sweet taste sensations Nutrition 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 241000276408 Bacillus subtilis subsp. subtilis str. 168 Species 0.000 description 3
- 240000000220 Panda oleosa Species 0.000 description 3
- 235000016496 Panda oleosa Nutrition 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 235000012976 tarts Nutrition 0.000 description 3
- 239000003643 water by type Substances 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 2
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 2
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 102000004533 Endonucleases Human genes 0.000 description 2
- 241000287828 Gallus gallus Species 0.000 description 2
- LOUPRKONTZGTKE-WZBLMQSHSA-N Quinine Chemical compound C([C@H]([C@H](C1)C=C)C2)C[N@@]1[C@@H]2[C@H](O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-WZBLMQSHSA-N 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 150000003851 azoles Chemical class 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 235000011194 food seasoning agent Nutrition 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000011081 inoculation Methods 0.000 description 2
- 210000002429 large intestine Anatomy 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 108020001775 protein parts Proteins 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000012488 sample solution Substances 0.000 description 2
- 230000014860 sensory perception of taste Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000013605 shuttle vector Substances 0.000 description 2
- 235000014347 soups Nutrition 0.000 description 2
- 125000006850 spacer group Chemical group 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108010005094 Advanced Glycation End Products Proteins 0.000 description 1
- 235000001258 Cinchona calisaya Nutrition 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 244000269722 Thea sinensis Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000019631 acid taste sensations Nutrition 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- LOUPRKONTZGTKE-UHFFFAOYSA-N cinchonine Natural products C1C(C(C2)C=C)CCN2C1C(O)C1=CC=NC2=CC=C(OC)C=C21 LOUPRKONTZGTKE-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 235000019264 food flavour enhancer Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 150000002460 imidazoles Chemical class 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- LPUQAYUQRXPFSQ-DFWYDOINSA-M monosodium L-glutamate Chemical compound [Na+].[O-]C(=O)[C@@H](N)CCC(O)=O LPUQAYUQRXPFSQ-DFWYDOINSA-M 0.000 description 1
- 235000013923 monosodium glutamate Nutrition 0.000 description 1
- 239000004223 monosodium glutamate Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 229960000948 quinine Drugs 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
-
- 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
- A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
- A23L27/20—Synthetic spices, flavouring agents or condiments
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- 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)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Polymers & Plastics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Nutrition Science (AREA)
- Biotechnology (AREA)
- Food Science & Technology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of delicate flavour peptide and its preparation method and application, which includes the polypeptide that amino acid sequence is Ser Ser Arg Asp Glu Gln Ser Arg and the multi-copy in tandem polypeptide of the polypeptide.Preparation method is：Delicate flavour DNA encoding peptide designs and synthesis；Then genetic fragment is subjected to digestion with vector plasmid, connect, obtain recombinant expression carrier；Recombinant expression carrier is converted into host's bacterium competence cell again, and is cultivated and is screened, obtains positive expression engineering bacteria；Finally to positive expression engineering bacteria carry out fermented and cultured, and by zymotic fluid through ultrasonic disruption, affine column purification, be dialyzed overnight after, obtain delicate flavour peptide purification liquid.The present invention provides not only a kind of delicate flavour peptide, and solves the technical issues of delicate flavour peptide manufacturing cycle is longer, low yield and purity are low.
Description
Technical field
The invention belongs to technical field of food additives, and in particular to a kind of delicate flavour peptide and preparation method and application.
Background technology
Delicate flavour peptide be it is a kind of can make up or the polypeptide of condensed food original local flavor, it can improve food freshness, improve
Taste perception degree promotes appetite into trencherman, because of its higher flavor attributes, physiological activity and nutritive value, is gradually used
In high-grade purpose compound flavour enhancer, be the nutrition of great market potential, health, safety green product.
Delicate flavour peptide research at present is in the starting stage, and preparation process is more traditional, and existing delicate flavour peptide preparation method mainly has
Two kinds：Zymohydrolysis extracting method and chemical synthesis.Zymohydrolysis extracting method key step is prepared-is isolated and purified including enzymolysis, but is existed and divided
From purification step complexity, the problems such as manufacturing cycle is longer, low yield, purity are low.And chemical synthesis there are by-product is more, cost compared with
The problems such as height, low output, limit industrial applications of the delicate flavour peptide in food seasoning.Therefore, be badly in need of finding it is a kind of a large amount of,
Quick and high-purity biological preparation method promotes development and application of the delicate flavour peptide in field of seasoning.
Invention content
For the above-mentioned prior art, the present invention provides a kind of delicate flavour peptide and its preparation method and application, to solve delicate flavour peptide
The technical issues of long preparation period, low low yield and purity.
In order to achieve the above object, the present invention first provides a kind of delicate flavour peptide first, which obtains from soybean zymolyte
, including the polypeptide that amino acid sequence is Ser-Ser-Arg-Asp-Glu-Gln-Ser-Arg and the multicopy of the polypeptide
Tandem polypeptide, and multi-copy in tandem polypeptide is joined end to end acquisition by polypeptide monomer, without amino acid spacer sequence.
In order to obtain the above-mentioned delicate flavour peptide of a large amount of high-purities in a short time, the present invention provides a kind of lifes of delicate flavour peptide
Object preparation method, this method include the following steps：
(1) genetic fragment of above-mentioned delicate flavour peptide can be encoded by designing and synthesizing；
(2) genetic fragment obtained by step (1) and vector plasmid are subjected to digestion, connection, obtain recombinant expression carrier；
(3) recombinant expression carrier is converted to host's bacterium competence cell, and it is cultivated, is screened, obtained positive
Express engineering bacteria；
(4) to positive expression engineering bacteria carry out fermented and cultured, by the zymotic fluid of generation through ultrasonication, affine column purification with
And after dialysis treatment, delicate flavour peptide purification liquid is obtained.
Based on the above technical solution, the present invention can also do following improvement.
Further, the gene order of step (1) synthesis is as shown in SEQ ID NO.1.
Further, vector plasmid is pMA09 plasmids.
Further, digestion system is in step (2)：80 μ L, 10 × K buffer solution 10 of delicate flavour peptide target gene or vector plasmid
5 μ L, EcoR I of μ L, Nde I, 5 μ L,；Digestion temperature is 37 DEG C, and the digestion time is 3~6h.
Further, linked system is in step (2)：6 μ L of delicate flavour peptide target gene after digestion, the vector plasmid after digestion
2 μ L, T4DNA connections buffer solution 1 μ L, T4The efficient 1 μ L of ligase of DNA；It is 16 DEG C to connect temperature, the coupled reaction time for 0.5~
2h。
Further, host strain is the bacillus subtilis with foodsafety.
Further, fermentation time is 25~48h in step (4).
The beneficial effects of the invention are as follows：The present invention delicate flavour peptide biological preparation method in, by the use of bacillus subtilis as
Host strain, since bacillus subtilis has stronger Protein secretion ability, non-pathogenic and good fermentation basis and life
Production capacity power, can produce and obtain a large amount of purpose delicate flavour peptides in a short time, and manufacturing cycle greatly shortens；And preparation method is made a living
Object the preparation method, final obtained delicate flavour peptide purity is high, and easy to operate, environmental pollution is small, is easily extended to industrialized production, made
It has broad application prospects in the production of flavouring or health products for the delicate flavour peptide gone out.
Description of the drawings
Fig. 1 is single times of delicate flavour peptide protein electrophoresis result figure；M represents standard protein, and 1 represents single times of delicate flavour peptide；
Fig. 2 is series connection delicate flavour peptide protein electrophoresis result figure；M represents standard protein, and 1 represents series connection delicate flavour peptide；
Fig. 3 is single times of delicate flavour peptide sensory evaluation radar map；
Fig. 4 is 10 × delicate flavour peptide sensory evaluation radar map.
Specific embodiment
Below in conjunction with the embodiment in the present invention, the invention will be further described, but should not this be interpreted as this hair
The range of bright above-mentioned theme is only limitted to following embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art are not having
All other embodiments obtained under the premise of creative work are made, shall fall within the protection scope of the present invention.
Embodiment one
1. the structure of delicate flavour peptide efficient expression vector
(1) delicate flavour peptide gene synthesizes
Using the delicate flavour peptide with Ser-Ser-Arg-Asp-Glu-Gln-Ser-Arg (SSRDEQSR) amino acid sequence as mesh
Product is marked, selects bacillus subtilis B.subtilis 168 that it is close to carry out encoding gene to target delicate flavour peptide as expressive host
Numeral optimizes, and obtains expressing gene, and the gene order such as SEQ ID NO.1 are finally synthesizing 5' ends and 3' ends and are respectively provided with Nde
I, the delicate flavour peptide gene sequence of EcoR I restriction enzyme sites.
(2) double digestion structure recombinant expression carrier
Using withered grass large intestine business shuttle vector pMA09 as vector plasmid, the carrier core sequence of operation such as SEQ ID NO.2.
Delicate flavour peptide gene after synthesis and pMA09 vector plasmids are subjected to Nde I/EcoR I double digestions, then coagulated using 1% agarose
Gel electrophoresis purifying recycling target endonuclease bamhi, obtains the delicate flavour peptide target gene with cohesive end and the line with cohesive end
Property vector plasmid pMA09, then the two is passed through into T4Efficient ligase is attached, and obtains delicate flavour peptide recombinant vector pMA09-
SSRDEQSR, sequence is as shown in SEQ ID NO.3.
Wherein, endonuclease reaction system (totally 100 μ L) and response procedures are respectively：Delicate flavour peptide target gene or expression plasmid
80 μ L of pMA09,10 × K buffer solutions, 10 μ L, 5 μ L of Nde I, 5 μ L of EcoR I；Digestion temperature is 37 DEG C, and the digestion time is 3h.
Linked system (10 μ L) and response procedures are respectively：The expression after 6 μ L of delicate flavour peptide target gene, digestion after digestion
2 μ L of plasmid pMA09, T41 μ L of DNA connections buffer solution, T4The efficient 1 μ L of ligase of DNA；It is 16 DEG C to connect temperature, during coupled reaction
Between be 0.5h.
2. the structure of delicate flavour peptide efficient expression system
168 competent cells of B.subtilis are prepared by hungry chemical method, 10 μ L are added in per 0.5mL competent cells
Connect delicate flavour peptide recombinant expression plasmid pMA09-10SSRDEQSR, in 37 DEG C, 100rpm/min culture 90min, is coated with kana and resists
Property plate screening positive transformant, obtain delicate flavour peptide efficient expression engineering B.subtilis 168/pMA09-SSRDEQSR.
3. delicate flavour peptide high efficient expression
The engineering bacteria B.subtilis 168/pMA09-SSRDEQSR single bacterium colonies that the above-mentioned resistance screening of picking obtains, inoculation
In the test tube containing 5mL LB culture mediums, it is incubated overnight under the conditions of 200rpm, 37 DEG C.It is transferred to and contains by 2% inoculum concentration later
Fermented and cultured is carried out in the 250mL shaking flasks for there are 30mL LB culture mediums, fermented and cultured carries out under the conditions of 200rpm, 37 DEG C, fermentation
Time 30h.
4.Tris-tricine-SDS-PAGE detects delicate flavour peptide
25 μ L delicate flavour peptide zymotic fluids are taken, 6 × SDS sample-loading buffers is added in and boils 5min, 12000rpm centrifugation 2min take
Plain white, in the Tris-tricine-SDS-PAGE progress albumen electricity that 4% concentrates glue, 10% spacer gel, 16% separation gel
Swimming, electrophoresis result such as Fig. 1.
5. delicate flavour peptide affinitive layer purification and dialysis
(1) zymotic fluid is taken to centrifuge 10min in 12000rpm, thalline is collected, then with phosphate buffer (miaow containing 20mmol/L
Azoles, pH 7.4) it is resuspended, ultrasonication about 30min (300W, work 3s, is spaced 2s)；
(2) clasmatosis liquid is centrifuged into 10min under the conditions of 12000rpm；Supernatant is taken to be carried out with the flow velocity of 1mL/min
Affinitive layer purification, if five graded levels elute (4.2%5mL, 8.3%5mL, 22%5mL, 58.3%5mL, 100%
5mL), it is about the protein part that 250~350mmol/L corresponds to peak to collect imidazole concentration, obtains delicate flavour peptide purification liquid.
(3) the delicate flavour peptide purification liquid of collection is placed in bag filter (MW:1500) after dialysing for 24 hours in 1000mL ultra-pure waters in
Obtain single times of delicate flavour peptide dialyzate of high-purity.
Embodiment two
1. the structure for delicate flavour peptide efficient expression vector of connecting
(1) design of series connection delicate flavour peptide gene and synthesis
Delicate flavour peptide SSRDEQSR without compartment of terrain is connected 10 times, selects bacillus subtilis B.subtilis WB800 conducts
Expressive host carries out encoding gene codon optimization to series connection delicate flavour peptide, obtains expressing gene, the gene order such as SEQ ID
NO.4 is finally synthesizing 5' ends and 3' ends and is respectively provided with the delicate flavour peptide gene sequence of Nde I, EcoR I restriction enzyme sites.
(2) double digestion construction of expression vector
Using withered grass large intestine shuttle vector pMA09 as expression vector.By the delicate flavour peptide gene and pMA09 vector plasmids after synthesis
Nde I/EcoR I double digestions are carried out, then recycling target endonuclease bamhi is purified using 1% agarose gel electrophoresis, is carried
The delicate flavour peptide target gene of cohesive end and the linear carrier plasmid pMA09 with cohesive end, then the two is passed through into T4Efficiently
Ligase is attached, and obtains delicate flavour peptide recombinant vector pMA09-10SSRDEQSR.
Wherein, endonuclease reaction system (totally 100 μ L) and response procedures are respectively：Delicate flavour peptide target gene or expression plasmid
80 μ L of pMA09,10 × K buffer solutions, 10 μ L, 5 μ L of Nde I, 5 μ L of EcoR I；Digestion temperature is 37 DEG C, and the digestion time is 6h.
Linked system (10 μ L) and response procedures are respectively：The expression after 6 μ L of delicate flavour peptide target gene, digestion after digestion
2 μ L of plasmid pMA09, T41 μ L of DNA connections buffer solution, T4The efficient 1 μ L of ligase of DNA；It is 16 DEG C to connect temperature, during coupled reaction
Between be 1h.
2. the structure for delicate flavour peptide efficient expression system of connecting
B.subtilis WB800 competent cells are prepared by hungry chemical method, 10 μ are added in per 0.5mL competent cells
L series connection delicate flavour peptide recombinant expression plasmid pMA09-10SSRDEQSR in 37 DEG C, 100rpm/min culture 90min, are coated with kana and resist
Property plate screening positive transformant, obtain 10 times series connection delicate flavour peptide efficient expression engineering B.subtilis WB800/
pMA09-10SSRDEQSR。
3. delicate flavour peptide high efficient expression of connecting
The engineering bacteria B.subtilis WB800/pMA09-10SSRDEQSR that above-mentioned resistance screening is taken to obtain, are seeded to and contain
Have in the 5mL LB culture mediums of kana antibiotic, cultivated under the conditions of 200rpm, 37 DEG C to OD600=0.6.Later by 2% inoculation
Amount, which is transferred in the 250mL shaking flasks containing 30mL LB culture mediums, carries out fermented and cultured, and fermented and cultured is under the conditions of 200rpm, 37 DEG C
It carries out, fermentation time 25h.
4.Tris-tricine-SDS-PAGE detection series connection delicate flavour peptides
25uL series connection delicate flavour peptide zymotic fluids are taken, 6 × SDS sample-loading buffers is added in and boils 5min, 12000rpm centrifugation 2min,
Supernatant protein liquid is taken, in the Tris-tricine-SDS-PAGE progress albumen that 4% concentrates glue, 10% spacer gel, 16% separation gel
Electrophoresis, electrophoresis result such as Fig. 2.
5. the purifying and dialysis of delicate flavour peptide of connecting
(1) take zymotic fluid in 10000rpm centrifuge 15min, collect thalline, phosphate buffer (imidazoles containing 20mmol/L,
PH 7.4) thalline, and sonicated cells 30min in ice bath is resuspended (300W, work 3s, is spaced 2s)；
(2) born of the same parents is taken to crush liquid, 10min is centrifuged in 12000rpm；Supernatant is taken to carry out affinity chromatography with the flow velocity of 1mL/min
Purifying if five graded levels elute (4.2%5mL, 8.3%5mL, 22%5mL, 58.3%5mL, 100%5mL), collects miaow
Azoles concentration is about the protein part that 250~350mmol/L corresponds to peak, obtains series connection delicate flavour peptide purification liquid.
(3) the series connection delicate flavour peptide purification liquid of collection is placed in bag filter (MW:5000) it dialyses in 1000mL ultra-pure waters in
Obtain the series connection delicate flavour peptide dialyzate of high-purity afterwards for 24 hours.
Interpretation of result
1. single times of delicate flavour peptide sense organ verification
The single times of delicate flavour peptide purification dialyzate obtained using in embodiment one is as the sample solution of sensory evaluation.With 0.35%
Salt solution, 1% sucrose solution, 0.08% citric acid solution, 0.35% monosodium glutamate solution, 0.08% quinine solution
Respectively as salty, sweet tea, acid, fresh, bitter five kinds of sense of taste flavor base soln, using base soln as 5 points, using ten point system to target
Flavor solution carries out sensory evaluation marking, so as to evaluate the flavor intensity of enzymolysis purified product.Sensory evaluation is always or usually as specified by trained
Professional sensory evaluation group (men and women half-and-half) carry out, it is each evaluate sample random number after, upset sequence and give valuation officer
Flavour evaluation is carried out, every valuation officer of the same sample evaluates 3 times, takes 3 times and measures average value as final evaluation score.Every part of sample
It is to be evaluated under room temperature.Every sense organ person also needs each flavor attributes for presenting sample of evaluation.Sensory evaluation scores data utilize SPSS
21.0 are analyzed, final Analyses Methods for Sensory Evaluation Results such as Fig. 3.
It is bitter, tart flavour is weaker from figure 3, it can be seen that the fresh, salty of single times of delicate flavour peptide, sweet taste are more apparent.And masses are more
Like fresh, salty, sweet taste, it is relatively low to hardship, the acceptance of tart flavour, show that the single times of delicate flavour peptide can be obviously improved taste of food,
Food puies forward fresh aspect with broad prospect of application.
(2) series connection delicate flavour peptide Maillard reaction and sense organ verification
The series connection delicate flavour peptide purification dialyzate obtained using in embodiment 2 takes the content to be as the sample solution of sensory evaluation
10% series connection delicate flavour peptide solution, addition xylose to final concentration of 11.5% adjust pH value to 7.2, in 50mL helixseals
110 DEG C of oil bath agitating and heatings reaction 120min, cools down after reaction in reaction bulb, for subsequent analysis and subjective appreciation, often
One reaction is repeated 3 times.Clear water and chicken soup are set respectively as background, add in series connection delicate flavour peptide maillard reaction product in 80 DEG C of thermostatted waters
Sensory evaluation is carried out after bath heating 20min, flavor sensory evaluation is by the trained professional sensory evaluation group (men and women couple being always or usually as specified
It partly) carries out, after each evaluation sample random number, upsets sequence and give valuation officer's progress flavour evaluation, the same sample every
Valuation officer evaluates 3 times, takes 3 times and measures average value as final evaluation score.Every part of sample is to be evaluated under room temperature.Every sense organ person
Evaluation is also needed to present the flavor attributes of sample every time.Sensory evaluation scores data are analyzed using SPSS 21.0, final sensory evaluation
As a result such as Fig. 4.
Figure 4, it is seen that fresh, salty, sweet taste of the series connection delicate flavour peptide in clear water and chicken soup is more apparent, bitter, acid
Taste is weaker.And masses more like fresh, salty, sweet taste, and it is relatively low to hardship, the acceptance of tart flavour, show that the single times of delicate flavour peptide can
Taste of food is obviously improved, fresh aspect is put forward with broad prospect of application in food.
Sequence table
<110>Xihua Univ
<120>A kind of delicate flavour peptide and preparation method and application
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 63
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
ggaattccat atgtcttctc gtgatgaaca atctcgtcat catcatcatc atcattgaat 60
tcc 63
<210> 2
<211> 389
<212> DNA
<213>Bacillus subtilis (Bacillus subtilis)
<400> 2
gatcttctca aaaaatacta cctgtccctt gctgattttt aaacgagcac gagagcaaaa 60
cccccctttg ctgaggtggc agagggcagg tttttttgtt tcttttttct cgtaaaaaaa 120
agaaaggtct taaaggtttt atggttttgg tcggcactgc cgacagcctc gcagagcaca 180
cactttatga atataaagta tagtgtgtta tactttactt ggaagtggtt gccggaaaga 240
gcgaaaatgc ctcacatttg tgccacctaa aaaggagcga tttacatatg aaatttgtaa 300
aaagaaggat cattgcactt gtaacaattt tgatgctgtc tgttacatcg ctgtttgcgt 360
tgcagccgtc agcaaaagcc gctgaattc 389
<210> 3
<211> 339
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gatcttctca aaaaatacta cctgtccctt gctgattttt aaacgagcac gagagcaaaa 60
cccccctttg ctgaggtggc agagggcagg tttttttgtt tcttttttct cgtaaaaaaa 120
agaaaggtct taaaggtttt atggttttgg tcggcactgc cgacagcctc gcagagcaca 180
cactttatga atataaagta tagtgtgtta tactttactt ggaagtggtt gccggaaaga 240
gcgaaaatgc ctcacatttg tgccacctaa aaaggagcga tttacatatg tcttctcgtg 300
atgaacaatc tcgtcatcat catcatcatc attgaattc 339
<210> 4
<211> 555
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gatcttctca aaaaatacta cctgtccctt gctgattttt aaacgagcac gagagcaaaa 60
cccccctttg ctgaggtggc agagggcagg tttttttgtt tcttttttct cgtaaaaaaa 120
agaaaggtct taaaggtttt atggttttgg tcggcactgc cgacagcctc gcagagcaca 180
cactttatga atataaagta tagtgtgtta tactttactt ggaagtggtt gccggaaaga 240
gcgaaaatgc ctcacatttg tgccacctaa aaaggagcga tttacatatg tcttctcgtg 300
atgaacaatc tcgttcttct cgtgatgaac aatctcgttc ttctcgtgat gaacaatctc 360
gttcttctcg tgatgaacaa tctcgttctt ctcgtgatga acaatctcgt tcttctcgtg 420
atgaacaatc tcgttcttct cgtgatgaac aatctcgttc ttctcgtgat gaacaatctc 480
gttcttctcg tgatgaacaa tctcgttctt ctcgtgatga acaatctcgt catcatcatc 540
atcatcattg aattc 555
Claims (10)
1. a kind of delicate flavour peptide, it is characterised in that：It is Ser-Ser-Arg-Asp-Glu-Gln-Ser-Arg's including amino acid sequence
The multi-copy in tandem polypeptide of polypeptide and the polypeptide.
2. delicate flavour peptide according to claim 1, it is characterised in that：The multi-copy in tandem polypeptide is by polypeptide monomer head and the tail phase
It successivelys win, without amino acid spacer sequence.
3. the preparation method of delicate flavour peptide as claimed in claim 1 or 2, which is characterized in that include the following steps：
(1) genetic fragment of delicate flavour peptide described in claims 1 or 2 can be encoded by designing and synthesizing；
(2) genetic fragment obtained by step (1) and vector plasmid are subjected to digestion, connection, obtain recombinant expression carrier；
(3) recombinant expression carrier is converted to host's bacterium competence cell, and it is cultivated, is screened, obtain positive expression
Engineering bacteria；
(4) to positive expression engineering bacteria carry out fermented and cultured, by the zymotic fluid of culture through ultrasonication, affine column purification and thoroughly
After analysis processing, delicate flavour peptide purification liquid is obtained.
4. the preparation method of delicate flavour peptide according to claim 3, it is characterised in that：The gene order of step (1) synthesis is such as
Shown in SEQ ID NO.1.
5. the preparation method of delicate flavour peptide according to claim 3, it is characterised in that：The vector plasmid is pMA09 plasmids.
6. the preparation method of delicate flavour peptide according to claim 3, which is characterized in that digestion system is in step (2)：Delicate flavour
5 μ L of peptide target gene or 5 μ L, EcoR I of vector plasmid 80 μ L, 10 × K buffer solution, 10 μ L, Nde I；Digestion temperature is 37 DEG C,
The digestion time is 3~6h.
7. the preparation method of delicate flavour peptide according to claim 3, which is characterized in that linked system is in step (2)：Digestion
6 μ L of delicate flavour peptide target gene afterwards, vector plasmid 2 μ L, T after digestion4DNA connections buffer solution 1 μ L, T4Efficient 1 μ of ligase of DNA
L；It is 16 DEG C to connect temperature, and the coupled reaction time is 0.5~2h.
8. the preparation method of delicate flavour peptide according to claim 3, it is characterised in that：The host strain is with food security
The bacillus subtilis of property.
9. the preparation method of delicate flavour peptide according to claim 3, it is characterised in that：In step (4) fermentation time for 25~
48h。
10. application of the delicate flavour peptide in flavouring or health products is prepared described in claims 1 or 2.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810196275.5A CN108148116A (en) | 2018-03-09 | 2018-03-09 | A kind of delicate flavour peptide and preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810196275.5A CN108148116A (en) | 2018-03-09 | 2018-03-09 | A kind of delicate flavour peptide and preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108148116A true CN108148116A (en) | 2018-06-12 |
Family
ID=62456598
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810196275.5A Pending CN108148116A (en) | 2018-03-09 | 2018-03-09 | A kind of delicate flavour peptide and preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108148116A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706206A (en) * | 2019-03-05 | 2019-05-03 | 衡阳师范学院 | A kind of method of sectional material supplementing fermenting and producing delicate flavour peptide |
| CN110305192A (en) * | 2019-08-05 | 2019-10-08 | 北京工商大学 | A kind of delicate flavour polypeptide and its flavour compositions and application |
| CN112458106A (en) * | 2020-12-04 | 2021-03-09 | 中国海洋大学 | Preparation method of multi-copy golden pomfret delicious peptide, expression vector and recombinant bacteria |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090143488A1 (en) * | 2007-12-03 | 2009-06-04 | Laurence Trinnaman | Peptides imparting umami, salt, dairy and bitter flavor |
-
2018
- 2018-03-09 CN CN201810196275.5A patent/CN108148116A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090143488A1 (en) * | 2007-12-03 | 2009-06-04 | Laurence Trinnaman | Peptides imparting umami, salt, dairy and bitter flavor |
Non-Patent Citations (5)
| Title |
|---|
| 王艳萍: "牛肉风味强化肽（BMP）表达载体的构建", 《天津科技大学学报》 * |
| 王金斌: "食品级枯草芽孢杆菌表达系统的最新研究进展", 《上海农业学报》 * |
| 罗云波: "《食品生物技术导论》", 31 August 2011, 中国农业大学出版社 * |
| 苏国万: "花生粕酶解及其产物呈味特性研究", 《中国博士学位论文全文数据库工程科技I辑》 * |
| 高应瑞: "毕赤酵母表达不同拷贝数风味肽的呈味性比较", 《天津科技大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109706206A (en) * | 2019-03-05 | 2019-05-03 | 衡阳师范学院 | A kind of method of sectional material supplementing fermenting and producing delicate flavour peptide |
| CN109706206B (en) * | 2019-03-05 | 2020-11-24 | 衡阳师范学院 | Method for producing delicious peptide by segmented feeding fermentation |
| CN110305192A (en) * | 2019-08-05 | 2019-10-08 | 北京工商大学 | A kind of delicate flavour polypeptide and its flavour compositions and application |
| CN112458106A (en) * | 2020-12-04 | 2021-03-09 | 中国海洋大学 | Preparation method of multi-copy golden pomfret delicious peptide, expression vector and recombinant bacteria |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108148116A (en) | A kind of delicate flavour peptide and preparation method and application | |
| CN107099521A (en) | A kind of acid resistance zearalenone detoxication enzyme and its encoding gene and application | |
| CN106119173B (en) | A kind of shrimps low molecular peptide of no bitter taste and the preparation method and application thereof | |
| CN104263710A (en) | Beta-galactosidase combined mutant with high transglycosylation activity as well as preparation method and application of beta-galactosidase combined mutant | |
| CN101294149A (en) | Alpha-cyclodextrin glucosyl transferase gene clone and expression | |
| CN105358706A (en) | Yeast promoters for protein expression | |
| CN103881994A (en) | Beta-galactosidase mutant with high transglycosylation activity and preparation method and application thereof | |
| CN104725517B (en) | A kind of method for improving yeast cell surface destination protein displaying amount | |
| CN106520802B (en) | It is a kind of improve the resistance to stress ability of lactic acid bacteria GAD gene and its application | |
| CN104402975B (en) | Anti-aging small peptide and preparation method thereof | |
| CN105695383A (en) | Recombinant strain and application thereof | |
| CN113265346A (en) | Genetically engineered bacterium for producing porcine myoglobin through fermentation and application thereof | |
| CN103710278A (en) | Industrial rice wine yeast metabolic engineering bacteria with low-yield urea and building method thereof | |
| CN104726461B (en) | Soft-shelled turtle collagen gene function fragment and its recombinant protein and application | |
| CN105200075B (en) | The building and application method of plasmid and its corresponding engineering bacteria for theanine production | |
| CN106635940B (en) | One plant of construction method for producing Glucosamine bacillus subtilis and application | |
| CN113264988B (en) | A group of special dietary proteins | |
| CN106366201A (en) | Gene sequence, carrier, recombination strain and recombination protein of fusion protein DAMP4-IGF-1 and preparing method thereof | |
| CN116987175A (en) | Recombinant collagen, recombinant expression strain and application | |
| CN104818227B (en) | A kind of food grade lactic acid galactococcus of surface anchoring people I type metallothioneins and preparation method and application | |
| CN106636049B (en) | A kind of alkaline pectin enzyme mutant that secernment property improves | |
| CN107936107A (en) | Long 1 gene recombinant proteins of oyster interferon regulatory factor CgIRF, preparation method and application | |
| CN104725485B (en) | One kind restructuring active peptide and its synchronic preparation method | |
| CN106754844A (en) | A kind of method for improving bile salt hydrolase secernment efficiency | |
| CN115725634A (en) | Expression cassette of leghemoglobin, expression vector containing leghemoglobin, genetically engineered bacterium and application of leghemoglobin |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20180612 |
|
| RJ01 | Rejection of invention patent application after publication |