CN113651869B - Umami peptide and preparation method and application thereof - Google Patents
Umami peptide and preparation method and application thereof Download PDFInfo
- Publication number
- CN113651869B CN113651869B CN202110794992.XA CN202110794992A CN113651869B CN 113651869 B CN113651869 B CN 113651869B CN 202110794992 A CN202110794992 A CN 202110794992A CN 113651869 B CN113651869 B CN 113651869B
- Authority
- CN
- China
- Prior art keywords
- peptide
- umami
- asp
- umami peptide
- delicious
- 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.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 53
- 235000019583 umami taste Nutrition 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 240000006794 Volvariella volvacea Species 0.000 claims abstract description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 21
- 238000004007 reversed phase HPLC Methods 0.000 claims abstract description 17
- JYGRAOYMDDFOSM-FQJIPJFPSA-N (4s)-4-[[(2s)-4-carboxy-2-[[(2s)-3-carboxy-2-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]propanoyl]amino]butanoyl]amino]-5-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-5-oxopentano Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN JYGRAOYMDDFOSM-FQJIPJFPSA-N 0.000 claims abstract description 14
- 108010053500 delicious peptide Proteins 0.000 claims abstract description 14
- 108010005094 Advanced Glycation End Products Proteins 0.000 claims abstract description 13
- 150000001413 amino acids Chemical group 0.000 claims abstract description 11
- 238000000926 separation method Methods 0.000 claims abstract description 11
- 238000005227 gel permeation chromatography Methods 0.000 claims abstract description 9
- 238000006243 chemical reaction Methods 0.000 claims abstract description 7
- 238000002156 mixing Methods 0.000 claims abstract description 7
- 239000012528 membrane Substances 0.000 claims abstract description 6
- 238000001035 drying Methods 0.000 claims abstract description 5
- 238000010438 heat treatment Methods 0.000 claims abstract description 4
- 239000007810 chemical reaction solvent Substances 0.000 claims abstract description 3
- 238000004140 cleaning Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 13
- 238000010828 elution Methods 0.000 claims description 11
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 10
- 238000009835 boiling Methods 0.000 claims description 10
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 9
- 239000012498 ultrapure water Substances 0.000 claims description 9
- 238000001514 detection method Methods 0.000 claims description 7
- 239000006228 supernatant Substances 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 5
- SRBFZHDQGSBBOR-IOVATXLUSA-N Xylose Natural products O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 4
- 239000000287 crude extract Substances 0.000 claims description 4
- 239000003480 eluent Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 229920005654 Sephadex Polymers 0.000 claims description 3
- 239000012507 Sephadex™ Substances 0.000 claims description 3
- PMZXXNPJQYDFJX-UHFFFAOYSA-N acetonitrile;2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F PMZXXNPJQYDFJX-UHFFFAOYSA-N 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 3
- 238000002791 soaking Methods 0.000 claims description 3
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 2
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 2
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 claims description 2
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 235000013373 food additive Nutrition 0.000 claims description 2
- 239000002778 food additive Substances 0.000 claims description 2
- 239000008103 glucose Substances 0.000 claims description 2
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 claims description 2
- 235000019640 taste Nutrition 0.000 abstract description 21
- 235000011194 food seasoning agent Nutrition 0.000 abstract description 7
- 239000000463 material Substances 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 4
- 230000000050 nutritive effect Effects 0.000 abstract description 2
- 238000010298 pulverizing process Methods 0.000 abstract 1
- 239000000796 flavoring agent Substances 0.000 description 16
- 235000019634 flavors Nutrition 0.000 description 16
- 238000010790 dilution Methods 0.000 description 12
- 239000012895 dilution Substances 0.000 description 12
- 238000004587 chromatography analysis Methods 0.000 description 9
- 238000011161 development Methods 0.000 description 9
- 150000002500 ions Chemical class 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 8
- 230000001953 sensory effect Effects 0.000 description 8
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 7
- 235000013923 monosodium glutamate Nutrition 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- 238000000513 principal component analysis Methods 0.000 description 7
- 229940073490 sodium glutamate Drugs 0.000 description 7
- 235000019607 umami taste sensations Nutrition 0.000 description 7
- 229940024606 amino acid Drugs 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000011156 evaluation Methods 0.000 description 6
- 235000013305 food Nutrition 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 101710159104 Flavor peptide Proteins 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 235000019643 salty taste Nutrition 0.000 description 3
- 235000019605 sweet taste sensations Nutrition 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 2
- 235000019658 bitter taste Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000004440 column chromatography Methods 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 229920001184 polypeptide Polymers 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 235000019614 sour taste Nutrition 0.000 description 2
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- AKPLMZMNJGNUKT-ZLUOBGJFSA-N Asp-Asp-Cys Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CS)C(O)=O AKPLMZMNJGNUKT-ZLUOBGJFSA-N 0.000 description 1
- 206010013911 Dysgeusia Diseases 0.000 description 1
- 239000004097 EU approved flavor enhancer Substances 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 235000005135 Micromeria juliana Nutrition 0.000 description 1
- 241000233855 Orchidaceae Species 0.000 description 1
- XKHCJJPNXFBADI-DCAQKATOSA-N Pro-Asp-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O XKHCJJPNXFBADI-DCAQKATOSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000246354 Satureja Species 0.000 description 1
- 235000007315 Satureja hortensis Nutrition 0.000 description 1
- 235000004501 Volvariella volvacea Nutrition 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- PBCJIPOGFJYBJE-UHFFFAOYSA-N acetonitrile;hydrate Chemical compound O.CC#N PBCJIPOGFJYBJE-UHFFFAOYSA-N 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 238000010411 cooking Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 235000021403 cultural food Nutrition 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 108010060199 cysteinylproline Proteins 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000004807 desolvation Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019688 fish Nutrition 0.000 description 1
- 235000019264 food flavour enhancer Nutrition 0.000 description 1
- HQVFCQRVQFYGRJ-UHFFFAOYSA-N formic acid;hydrate Chemical compound O.OC=O HQVFCQRVQFYGRJ-UHFFFAOYSA-N 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000005457 ice water Substances 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000019613 sensory perceptions of taste Nutrition 0.000 description 1
- 238000007873 sieving Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 230000035923 taste sensation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000009834 vaporization Methods 0.000 description 1
- 230000008016 vaporization Effects 0.000 description 1
Images
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
- A23L27/10—Natural spices, flavouring agents or condiments; Extracts thereof
-
- 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
- A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
- A23L27/215—Synthetic spices, flavouring agents or condiments containing amino acids heated in the presence of reducing sugars, e.g. Maillard's non-enzymatic browning
-
- 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
Abstract
The invention discloses a umami peptide, the amino acid sequence of which is Asp-Asp-Cys-Pro-Asp-Lys. The invention also discloses a preparation method of the umami peptide, which comprises the following steps: cleaning straw mushroom, drying to constant weight, pulverizing, decocting in water under normal pressure, and centrifuging to obtain crude extractive solution; intercepting components with molecular weight less than 3000Da by ultrafiltration membrane separation method; and sequentially carrying out gel chromatography and reversed-phase high performance liquid chromatography on the ultrafiltration component to obtain the delicious peptide. The invention also discloses a Maillard reaction product of the umami peptide, which is obtained by taking water as a reaction solvent, uniformly mixing the umami peptide and reducing sugar, adjusting the pH value to 7-9 and heating for reaction. The delicious peptide is 6 peptide with molecular weight less than 800Da, is easy to be absorbed by human body, and improves nutritive value. The delicious peptide or Maillard reaction product thereof has strong salty and delicious taste and good seasoning effect, and can be used as a base material or an auxiliary material to prepare a seasoning.
Description
Technical Field
The invention belongs to the field of umami peptide, and particularly relates to umami peptide and a preparation method and application thereof.
Background
In the world food culture, the umami taste is one of 5 traditional taste tastes, is popular with people by the special taste feeling, and becomes one of important indexes for evaluating the delicious food because the umami taste has good cooperativity with other tastes. Umami substances such as umami peptides, organic acids, nucleotides, amino acids, and flavor enhancers are found to have umami taste, and among these substances, the umami peptides are receiving more and more attention from people due to their good physiological activity and flavor activity.
The umami peptide is a flavor development compound widely existing in food, and exists in fungi, meat, fish, fermented food and the like. Small molecule umami peptides possessing specific structures were first discovered by Kirimura et al in 1969, which have a stronger taste than free amino acids. Besides good taste characteristics, the delicious peptide can also regulate the physiological activities of human bodies. Compared with some traditional umami substances, the umami peptide serving as a novel umami agent reduces the phenomenon of throat dryness, has longer retention time in the oral cavity, and has better processing quality and development prospect.
The straw mushroom is delicious in taste and rich in nutrition, and is called orchid mushroom and delicious foot-covering mushroom. The umami components of the straw mushroom are mainly divided into amino acids, nucleotides and a plurality of micromolecule umami skin, and the micromolecule umami skin greatly contributes to the umami of the straw mushroom. The single flavor development amino acid or flavor development nucleotide is used as a flavor development agent and is characterized by strong flavor development and monotonous mouthfeel, and the flavor development amino acid or flavor development nucleotide has better mouthfeel. The protein content of the straw mushroom is rich and accounts for 28.03% of the dry weight, and the sweet and fresh amino acids in the protein account for a higher ratio, so that the straw mushroom protein has the potential of preparing the fresh peptide. At present, people mainly concentrate on antioxidant activity, storage and preservation after picking and extraction of polysaccharide in the research on the straw mushroom, and the separation and identification of the straw mushroom flavor peptide are rarely reported.
Disclosure of Invention
The invention aims to provide a novel delicious peptide, which is prepared by using fresh straw mushroom fruiting bodies as raw materials, boiling the raw materials with water at normal pressure to obtain a crude extract, and separating and identifying the delicious peptide by using ultrafiltration, gel chromatography, RP-HPLC (reverse phase liquid chromatography) and UPLC-Q-TOF-MS (ultra performance liquid chromatography-time of flight-mass spectrometry).
The purpose of the invention is realized by the following technical scheme.
The amino acid sequence of the umami peptide is Asp-Asp-Cys-Pro-Asp-Lys.
The invention also provides a preparation method of the umami peptide, which comprises the following steps:
step (1), washing straw mushrooms, drying to constant weight, crushing, boiling in water at normal pressure, centrifuging to obtain a crude extract, and extracting water-soluble components;
step (2), intercepting components with the molecular weight less than 3000Da by adopting an ultrafiltration membrane separation method; and sequentially carrying out gel chromatography and reversed-phase high performance liquid chromatography (RP-HPLC) on the obtained ultrafiltration component to obtain the delicious peptide with the amino acid sequence of Asp-Asp-Cys-Pro-Asp-Lys.
In the step (1), the water content of the straw mushroom reaches more than 85%, and the straw mushroom needs to be dried in order to ensure the extraction rate of the peptides. Specifically, the straw mushroom is cleaned and dried to constant weight at 55 ℃ by using a blast drier, the dried straw mushroom is crushed in a crusher and then is sieved by a 80-mesh sieve to remove impurities in coarse powder, and meanwhile, the part which is large in particle, hard in texture, difficult to grind and difficult to hydrolyze of the straw mushroom is removed.
Preferably, for accelerating drying, the straw mushroom is cut after being washed, and then is dried to constant weight by adopting a blast dryer at 55 ℃.
The normal-pressure water boiling comprises the following steps: soaking the straw mushrooms in distilled water for 30 min-90 min at 15-30 ℃ according to the feed-liquid ratio of the straw mushrooms to the distilled water of 1-20 g/ml.
Preferably, the pressure of the normal-pressure water boiling is normal pressure; the temperature of the normal-pressure water boiling is 50-100 ℃, and the optimal temperature is 98 ℃; the time for boiling the mixture in water under normal pressure is 60-120 min, and preferably 90min.
The centrifugation conditions are as follows: centrifuging at the rotating speed of 6000 r/min-8000 r/min for 10 min-30 min at the temperature of 2-6 ℃, and taking supernatant, namely the crude extract.
In the step (2), preferably, the supernatant is firstly pre-filtered by a 0.45 μm filter membrane, and then components with the molecular weight of less than 3000Da are intercepted by adopting an ultrafiltration membrane separation method.
According to the molecular weight of the target peptide segment, sephadex G-15 gel is adopted in the gel chromatography, and ultrapure water is used as eluent.
The flow rate of the eluent is 0.5ml/min, and fractions of 75-100 min are collected.
After the ultrafiltration component is subjected to gel chromatography column chromatography, the component with the strongest taste is selected through taste dilution analysis and electronic tongue evaluation, and then the component with the strongest delicate flavor is separated and purified by adopting reversed phase high performance liquid chromatography.
The conditions of the reversed phase high performance liquid chromatography are as follows: zorbax300SB-C18 (PrepHT, 21.2X 250mm,5 μm) column, column temperature: performing gradient elution at 25 ℃ by using 0.05% trifluoroacetic acid aqueous solution as a mobile phase A and 0.05% trifluoroacetic acid acetonitrile solution as a mobile phase B, wherein the flow rate of the mobile phase is 0.5ml/min, the detection wavelength is 220nm, and the gradient elution procedure is as follows:
a Maillard reaction product of umami peptide is prepared by using water as reaction solvent, uniformly mixing the umami peptide and reducing sugar, adjusting pH to 7-9, and heating for reaction to obtain the Maillard reaction product of the umami peptide.
Preferably, the delicious peptide and reducing sugar are mixed evenly, and the pH is adjusted to 8.
The mass ratio of the delicious peptide to the reducing sugar is 2.
The heating reaction temperature is 110-130 ℃, and the reaction time is 2-4 h.
The reducing sugar is selected from xylose, glucose and ribose.
The invention also aims to provide the application of the delicious peptide or the Maillard reaction product thereof in food.
The invention also aims to provide the application of the delicious peptide or the Maillard reaction product thereof as a freshness-enhancing food additive.
Compared with the prior art, the invention has the following advantages:
(1) The delicious peptide is 6 peptide with the molecular weight less than 800Da, is easy to be absorbed by human body and improves the nutritive value.
(2) The delicious peptide or Maillard reaction product thereof has strong salty and delicate flavor and good seasoning effect, can obviously enhance the delicate flavor of the seasoning when being combined with other seasonings, can be applied to the field of food, and can be used as a base material or an auxiliary material to prepare the seasoning.
(3) The preparation method of the umami peptide is simple, can be artificially synthesized, and effectively saves the cost.
Drawings
FIG. 1 is a chromatogram of an ultrafiltration fraction (molecular weight less than 3000 Da) after SephadexG-15 gel chromatography; the abscissa represents elution time (unit: min), and the ordinate represents abundance at a detection wavelength of 220 nm.
FIG. 2 is an electronic tongue taste profile radar chart of chromatographic fractions F1-F4.
FIG. 3 shows taste dilution analysis results of ultrafiltration fractions A to I and chromatography fractions F1 to F4; the abscissa represents the ultrafiltration fractions A-I and the chromatography fractions F1-F4, and the ordinate represents the dilution factor of the flavor intensity of each fraction.
FIG. 4 is an RP-HPLC separation profile of chromatographic fraction F1; the abscissa represents the elution time (min) and the ordinate represents the absorbance at a detection wavelength of 220 nm.
FIG. 5 is a two-dimensional principal component analysis plot of the RP-HPLC fraction versus the MSG and F1 fractions for the electronic tongue.
FIG. 6 is a time-of-flight mass spectrometry (TOF MS) second order mass spectrum of the F1-a fraction; the abscissa represents the mass-to-charge ratio value (m/z, which is the ratio of the number of protons to the number of charges) of the ion, and the value of the mass-to-charge ratio increases from left to right; the ordinate represents the Intensity (Intensity%) of the ion current, usually expressed in relative Intensity, i.e. the Intensity of the ion current with the strongest Intensity is taken as 100%, the Intensity of the other ion currents is expressed in percentage thereof, and the various ions are expressed in percentage thereof.
Detailed Description
The technical scheme of the invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Straw mushrooms were purchased from new urban stores in Suguo Yadong, huarun.
Example 1
Extracting water-soluble components from straw mushrooms by a normal-pressure cooking method: cleaning and chopping straw mushrooms, drying the straw mushrooms in a forced air dryer at 55 ℃ to constant weight, crushing the dried straw mushrooms in a crusher, and sieving the crushed straw mushrooms with a 80-mesh sieve; mixing mushroom powder and distilled water according to a material-liquid ratio of 1g to 40ml, soaking at room temperature (25 ℃) for 1h, boiling in water at 98 ℃ for 90min, centrifuging at 4 ℃ at 8000r/min for 20min, and taking supernatant, namely straw mushroom water-soluble extracting solution.
Separation and purification of straw mushroom flavor peptide
Pre-filtering the supernatant with 0.45 μm filter membrane, retaining components with molecular weight less than 3000Da in the filtrate by ultrafiltration to obtain ultrafiltration component (marked as A-I), freeze drying the ultrafiltration component, and storing the sample powder at-20 deg.C.
The ultrafiltration component A-I (sample powder after freeze drying) was prepared into a solution with a concentration of 25mg/ml using ultrapure water, sephadex G-15 gel column chromatography was used, and the flow rate was 0.5ml/min using ultrapure water as eluent. The chromatographic pattern result is shown in figure 1, 4 absorption peaks can be seen, namely 4 separated components are obtained by chromatography and are sequentially marked as chromatographic components F1, F2, F3 and F4 according to the sequence. And respectively freeze-drying the 4 chromatographic components, and storing the freeze-dried sample powder at-20 ℃ for later use.
The flavor characteristics of the ultrafiltration components A-I and the chromatography components F1-F4 were determined by electronic tongue system analysis and artificial sensory evaluation.
Electronic tongue system analysis
Preparing samples to be evaluated (chromatographic components F1-F4) into 25mg/ml solution by adopting ultrapure water, analyzing the taste development characteristics of the chromatographic components F1-F4 by adopting an ASTREE electronic tongue system, and detecting the samples by utilizing a five-taste sensor array, wherein the sensor array comprises 7 sensors of SCS, AHS, CTS, NMS, CPS, ANS and PKS, and the SCS, AHS, CTS, NMS and CPS sensors respectively represent bitter taste, sour taste, salty taste, delicate taste and sweet taste.
The collection time for each sample was 120s and the washing time in distilled water was 10s. Each sample is subjected to 3 parallels, the detection is repeated for 6 times, and 3 times of stable experimental data are taken.
As shown in fig. 2, the chromatographic fractions F1, F2, F3, F4 had roughly similar taste characteristics of the fractions, with relatively strong umami, salty and sweet tastes, relatively weak bitter and sour tastes, and the F1 fraction was the strongest in umami, salty and sweet tastes.
Artificial sensory evaluation method
Determining dilution factors of ultrafiltration components A-I and chromatography components F1-F4 by Taste Dilution Analysis (TDA), which comprises the following steps: preparing each sample to be evaluated into a solution of 25mg/ml by adopting ultrapure water, and gradually diluting the solution by adopting deionized water according to the volume ratio of 1; 5ml of each of the gradient solutions were taken and presented in order of decreasing concentration to 8 trained sensory evaluators (4 men and 4 women, aged 25 to 30 years), and the solutions at each dilution were subjected to sensory evaluation using a triangulation test. The dilution factor at which the flavor difference between a certain dilution level solution and two deionized water was just identified was recorded as flavor intensity dilution factor, i.e., TD. TD is the average of the whole sensory panel and the TD value between the panellists cannot be greater than two dilution levels. Each sample was repeated three times at different times and evaluated at room temperature.
The taste dilution analysis of the ultrafiltration components A-I and the chromatography components F1-F4 results are shown in FIG. 3, where it can be seen that: the highest flavor dilution (TD) value of the ultrafiltration component A-I was 92; among the fractions obtained by gel chromatography of the ultrafiltration fraction A-I, the TD value of F1 was the highest and 34, indicating that F1 had the greatest effect on the overall umami taste and had the most savory taste in the same range.
As can be seen, the umami taste of the chromatographic fraction F1 is strongest, so the chromatographic fraction F1 is selected for RP-HPLC separation and purification.
The chromatographic fraction F1 was further isolated and purified by RP-HPLC: the chromatography component F1 was brought to a concentration of 10mg/ml with purified water, filtered through a 0.22 μm filter and introduced into a volume of 250. Mu.l for RP-HPLC: zorbax300SB-C18 (PrepHT, 21.2X 250mm,5 μm) chromatography column (Agilent, USA), column temperature: at 25 ℃, the mobile phase a is a 0.05% trifluoroacetic acid aqueous solution (volume ratio of ultrapure water to trifluoroacetic acid =99.5, 0.5), the mobile phase B is a 0.05% trifluoroacetic acid acetonitrile solution (volume ratio of acetonitrile to trifluoroacetic acid = 99.5), gradient elution is used, the detection wavelength is 220nm, and the gradient elution procedure is shown in table 1. Setting automatic collection according to peak condition, mixing multiple collected peaks, freeze drying, and storing at-20 deg.C.
TABLE 1 gradient elution procedure
The RP-HPLC separation spectrogram of the chromatographic fraction F1 is shown in figure 4, 4 absorption peaks can be seen, 4 separation fractions obtained in the elution process are collected and sequentially represented by F1-a, F1-b, F1-c and F1-d, wherein the F1-a, the F1-b and the F1-d are main components, and the peptide content of the F1-a is highest. The 4 fractions were freeze-dried and stored at-20 ℃.
PCA analysis of RP-HPLC fractions using electronic tongue system
And simultaneously, measuring a sample and a standard substance by using an electronic tongue, and analyzing the obtained original data by using a Principal Component Analysis (PCA) method. In the two-dimensional plot of PCA, PC1 represents the most major component of the sample and PC2 represents the second most major component of the sample. PC1 represents the most important component of the sample, is the most important factor causing the difference of the sample, and thus the distance in the direction of PC1 is mainly used as a discrimination criterion when judging the taste of each component.
Fig. 5 is a principal component analysis chart of the F1 component, MSG (sodium glutamate) and its RP-HPLC separation component, and the contribution value of the anterior two-dimensional principal component of the electronic tongue reaches 95.5%, which illustrates that the two-dimensional principal component analysis can represent the overall information of the original data. The 3 results of the F1 fraction, MSG and its RP-HPLC fractions were pooled together, indicating a high sensitivity of the electronic tongue. It can be seen from the relative distances of the fractions and MSG that F1 is closest to MSG, while purified F1-a is closer to MSG than the other fractions and also closer to F1, indicating that F1-a has the most taste sensation among the 4 separated fractions.
Combining fig. 4 and fig. 5, it follows: f1-a has the highest peptide content and the highest taste, and therefore is selected for polypeptide sequence structure identification.
Structural identification of polypeptide sequences in F1-a
Preparing a sample: dissolving the F1-a sample in pure water for chromatography, mixing on a vortex mixer to fully dissolve the sample, centrifuging, and filling supernatant into a liquid phase bottle for later use.
The UPLC-Q-TOF-MS measurement conditions were as follows:
liquid phase conditions: a BEH C18 column (50 mm. Times.2.1mm, 1.7 μm) was used; sample introduction amount: 10 mu L of the solution; flow rate: 0.3ml/min; the mobile phase A is 0.1% acetonitrile water solution, and the mobile phase B is 0.1% formic acid water solution; the column temperature is 45 ℃; gradient elution conditions: 0-2min:100% by weight of B;2-3min:90% by weight of B;3-10min:0% by weight of B.
Mass spectrum conditions: ionization mode: ESI +, capillary voltage of 3.2Kvolts, cone hole voltage of 20Kvolts, ion source temperature of 100 ℃, desolvation vaporization temperature of 400 ℃, cone hole flow rate of 50L/h, ion energy of 1volt, collision energy of 6Volts and 20Volts, scanning time of 1s, and detection voltage of 1700Volts with mass range of 20-3000m/z.
After bombardment, the fragment ions are broken up and separated according to the mass-to-charge ratio to form a secondary mass spectrum (see fig. 6). In a mass spectrogram, by Max is an ion fragment; d is aspartic acid, C is cysteine, P is proline, and K is lysine. And (3) matching 1 novel peptide chain from the F1-a component by using Peaks software and database search, wherein the molecular weight of the novel peptide chain is less than 800Da, the identified peptide chain is Asp-Asp-Cys-Pro-Asp-Lys, and the peptide chain can be synthesized artificially.
Example 2
Preparation of straw mushroom flavor peptide Maillard reaction product
Dissolving the straw mushroom flavor peptide (amino acid sequence: asp-Asp-Cys-Pro-Asp-Lys) prepared in example 1 by adopting ultrapure water to prepare a peptide solution with the mass fraction of 20%, adding xylose according to the mass ratio of 3 to the peptide sugar, uniformly mixing, adjusting the pH value to 8, reacting for 3 hours at 120 ℃, and cooling by adopting ice water to terminate the reaction.
TABLE 2 taste profiles of umami peptides and products of the Maillard reaction of umami peptides
Note: the umami peptide and the umami peptide Maillard reaction product are respectively prepared into 25mg/ml solution by ultrapure water, and then sensory evaluation is carried out.
Sensory evaluation results show that the straw mushroom umami peptide is richer in umami and aftertaste after undergoing Maillard reaction. The fresh peptide extracted from the straw mushroom and the Maillard reaction product of the fresh peptide have obvious freshness enhancing effect, can be applied to the field of food, can be used as a base material and an auxiliary material as a seasoning, are fresh and salty, and can meet the sensory requirement while being nutritional and safe.
Sequence listing
<110> university of financial institution of Nanjing
Nanjing Agricultural University
<120> umami peptide and preparation method and application thereof
<141> 2021-07-14
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 6
<212> PRT
<213> Volvariella volvacea (Bull.: fr.) Sing.)
<400> 1
Asp Asp Cys Pro Asp Lys
1 5
Claims (7)
1. An umami peptide, characterized by: the amino acid sequence of the umami peptide is Asp-Asp-Cys-Pro-Asp-Lys.
2. A method of preparing the umami peptide of claim 1, comprising: the method comprises the following steps:
step (1), cleaning straw mushrooms, drying to constant weight, crushing, soaking for 30-90 min at 15-30 ℃ according to the feed-liquid ratio of straw mushrooms to distilled water of 1; the temperature of the normal-pressure water boiling is 50-100 ℃, and the time of the normal-pressure water boiling is 60-120 min;
step (2), intercepting components with the molecular weight less than 3000Da by adopting an ultrafiltration membrane separation method; sequentially performing gel chromatography and reversed-phase high performance liquid chromatography on the obtained ultrafiltration component to obtain the delicious peptide;
the gel chromatography adopts Sephadex G-15 gel, and the eluent is ultrapure water;
the conditions of the reversed phase high performance liquid chromatography are as follows: zorbax300SB-C18 (PrepHT, 21.2X 250mm,5 μm) column, column temperature: performing gradient elution at 25 ℃ by using 0.05% trifluoroacetic acid aqueous solution as a mobile phase A and 0.05% trifluoroacetic acid acetonitrile solution as a mobile phase B, wherein the flow rate of the mobile phase is 0.5ml/min, the detection wavelength is 220nm, and the gradient elution procedure is as follows:
3. the method of preparing the umami peptide of claim 2, wherein: in the step (1), the straw mushrooms are cleaned, dried to constant weight by adopting a blast drier at 55 ℃, and crushed in a crusher and sieved by a sieve of 80 meshes.
4. The method of preparing the umami peptide of claim 2, wherein: the pressure of the normal-pressure water boiling is normal pressure.
5. The method of preparing the umami peptide of claim 2, wherein: in the step (1), the centrifugation conditions are as follows: centrifuging at the rotating speed of 6000 r/min-8000 r/min for 10 min-30 min at the temperature of 2-6 ℃, and taking supernatant, namely the crude extract.
6. A maillard reaction product of an umami peptide, characterized in that: the method comprises the steps of taking water as a reaction solvent, uniformly mixing the umami peptide and reducing sugar according to claim 1, adjusting the pH value to 7-9, and heating for reaction to obtain a Maillard reaction product of the umami peptide; wherein the mass ratio of the umami peptide to the reducing sugar is 2; the reducing sugar is selected from xylose, glucose and ribose.
7. Use of the umami peptide of claim 1 or the maillard reaction product of claim 6 as a umami food additive.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110794992.XA CN113651869B (en) | 2021-07-14 | 2021-07-14 | Umami peptide and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110794992.XA CN113651869B (en) | 2021-07-14 | 2021-07-14 | Umami peptide and preparation method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113651869A CN113651869A (en) | 2021-11-16 |
| CN113651869B true CN113651869B (en) | 2023-02-24 |
Family
ID=78489458
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110794992.XA Active CN113651869B (en) | 2021-07-14 | 2021-07-14 | Umami peptide and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113651869B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114027494A (en) * | 2021-12-10 | 2022-02-11 | 上海市农业科学院 | Flavor base material and preparation method and application thereof |
| CN115124598B (en) * | 2022-06-17 | 2023-05-05 | 中国农业大学 | Umami peptide and preparation method and application thereof |
| CN116217659B (en) * | 2022-10-10 | 2024-02-27 | 上海市农业科学院 | Stropharia rugoso-annulata mycelium flavor peptide and preparation method and application thereof |
| CN116715724B (en) * | 2023-05-24 | 2024-03-15 | 上海市农业科学院 | Antioxidant peptide derived from fruiting body of straw mushroom and application thereof |
| CN116949085B (en) * | 2023-09-19 | 2023-12-22 | 广东省农业科学院蔬菜研究所 | Application of straw mushroom Vvpks gene in regulation and control of umbrella opening character |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10108686A (en) * | 1996-09-23 | 1998-04-28 | Givaudan Roure Internatl Sa | Cloning, expression and production of taste peptide |
| CN108178781A (en) * | 2017-12-11 | 2018-06-19 | 上海应用技术大学 | A kind of straw mushroom flavor peptide and its preparation method and application |
| CN109485699A (en) * | 2018-10-26 | 2019-03-19 | 上海应用技术大学 | A kind of agaricus bisporus flavor peptide and its preparation method and application |
| CN110343146A (en) * | 2019-07-12 | 2019-10-18 | 上海应用技术大学 | A kind of crab flavour mushroom flavor peptide and its preparation method and application |
| CN111153959A (en) * | 2020-01-22 | 2020-05-15 | 苏州蕙茝生物科技有限公司 | Hypsizygus marmoreus flavor-developing peptide and preparation method and application thereof |
-
2021
- 2021-07-14 CN CN202110794992.XA patent/CN113651869B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH10108686A (en) * | 1996-09-23 | 1998-04-28 | Givaudan Roure Internatl Sa | Cloning, expression and production of taste peptide |
| CN108178781A (en) * | 2017-12-11 | 2018-06-19 | 上海应用技术大学 | A kind of straw mushroom flavor peptide and its preparation method and application |
| CN109485699A (en) * | 2018-10-26 | 2019-03-19 | 上海应用技术大学 | A kind of agaricus bisporus flavor peptide and its preparation method and application |
| CN110343146A (en) * | 2019-07-12 | 2019-10-18 | 上海应用技术大学 | A kind of crab flavour mushroom flavor peptide and its preparation method and application |
| CN111153959A (en) * | 2020-01-22 | 2020-05-15 | 苏州蕙茝生物科技有限公司 | Hypsizygus marmoreus flavor-developing peptide and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 不同处理方法对草菇呈味物质释放的影响;徐晓东等;《食品科学》;20181231;第39卷(第12期);第107-111页 * |
| 草菇特征风味物质的鉴定及呈味肽的制备研究;徐晓东;《全国优秀硕士学位论文电子期刊 工程科技Ⅰ辑》;20200215(第2020/02期);第B024-337页 * |
| 草菇鲜味肽的分离鉴定及呈味特性分析;蒋希希等;《食品科学》;20220722;第43卷(第12期);第235~242页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113651869A (en) | 2021-11-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113651869B (en) | Umami peptide and preparation method and application thereof | |
| Xu et al. | Identification of umami-tasting peptides from Volvariella volvacea using ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry and sensory-guided separation techniques | |
| Yin et al. | Effect of different drying methods on free amino acid and flavor nucleotides of scallop (patinopecten yessoensis) adductor muscle | |
| Zhao et al. | Evaluation of umami taste components of mushroom (Suillus granulatus) of different grades prepared by different drying methods | |
| CN108178781B (en) | Straw mushroom flavor-presenting peptide and preparation method and application thereof | |
| Naknaen et al. | Proximate compositions, nonvolatile taste components and antioxidant capacities of some dried edible mushrooms collected from Thailand | |
| CN111153959A (en) | Hypsizygus marmoreus flavor-developing peptide and preparation method and application thereof | |
| CN110343146B (en) | Hypsizygus marmoreus flavor-developing peptide and preparation method and application thereof | |
| Li et al. | Characteristic fingerprints and comparison of volatile flavor compounds in Morchella sextelata under different drying methods | |
| Li et al. | Mining of kokumi peptides in chicken broth with peptidomics | |
| CN110074377B (en) | Flavor development peptide and preparation method and application thereof | |
| CN113875964B (en) | Dipeptide and Maillard reaction product thereof as well as preparation method and application thereof | |
| CN114181278B (en) | Novel umami oligopeptide and preparation method and application thereof | |
| CN109170803B (en) | Chicken flavor enhancing peptide and preparation method and application thereof | |
| CN116333045B (en) | Preserved meat flavor peptide and preparation method and application thereof | |
| CN115947780B (en) | Polypeptide and preparation method and application thereof | |
| CN109393138B (en) | Chicken taste-presenting peptide and preparation method and application thereof | |
| CN108059651B (en) | Separated flavor development peptide and preparation method and application thereof | |
| CN111018949B (en) | Preparation method and application of grifola frondosa flavor-developing peptide | |
| CN114158715B (en) | Rose Huang Huangyou bolete delicious peptide and preparation method thereof | |
| Du et al. | Effects of different extraction methods on the release of non-volatile flavor components in shiitake mushroom (Lentinus edodes) | |
| CN117264015A (en) | Preparation method and application of umami peptide and pleurotus eryngii umami element | |
| CN115124598B (en) | Umami peptide and preparation method and application thereof | |
| CN112841597B (en) | Oyster polypeptide for enhancing mellow flavor of food and preparation method thereof | |
| CN108618100B (en) | Tetrapeptide with delicate flavor and freshness-enhancing properties and application thereof |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20231225 Address after: No. 1 Zishan Road, Sanhe Town, Hongze District, Huai'an City, Jiangsu Province, 223001 Patentee after: JIANGSU ZISHAN FOOD SCIENCE AND TECHNOLOGY Co.,Ltd. Address before: 210046 No.3, Wenyuan Road, Qixia District, Nanjing City, Jiangsu Province Patentee before: NANJING University OF FINANCE AND ECONOMICS Patentee before: NANJING AGRICULTURAL University |
|
| TR01 | Transfer of patent right |