CN117778510A - Preparation method of caper bitter peptide - Google Patents
Preparation method of caper bitter peptide Download PDFInfo
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- CN117778510A CN117778510A CN202410013878.2A CN202410013878A CN117778510A CN 117778510 A CN117778510 A CN 117778510A CN 202410013878 A CN202410013878 A CN 202410013878A CN 117778510 A CN117778510 A CN 117778510A
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- semen arecae
- taste
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- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 77
- 241000722721 Capparis Species 0.000 title claims abstract description 35
- 235000017336 Capparis spinosa Nutrition 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 38
- 230000001953 sensory effect Effects 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 22
- 235000019658 bitter taste Nutrition 0.000 claims abstract description 21
- 239000000843 powder Substances 0.000 claims abstract description 19
- 238000005238 degreasing Methods 0.000 claims abstract description 16
- 238000011156 evaluation Methods 0.000 claims abstract description 15
- 238000004007 reversed phase HPLC Methods 0.000 claims abstract description 12
- 108090000790 Enzymes Proteins 0.000 claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 8
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- 238000004949 mass spectrometry Methods 0.000 claims abstract description 6
- 210000000582 semen Anatomy 0.000 claims description 43
- 239000000243 solution Substances 0.000 claims description 35
- 235000019640 taste Nutrition 0.000 claims description 31
- 239000000523 sample Substances 0.000 claims description 25
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 22
- 229920001184 polypeptide Polymers 0.000 claims description 21
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 20
- 239000012498 ultrapure water Substances 0.000 claims description 20
- 239000012071 phase Substances 0.000 claims description 19
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical group OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 238000011068 loading method Methods 0.000 claims description 11
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- 239000003208 petroleum Substances 0.000 claims description 11
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000007791 liquid phase Substances 0.000 claims description 9
- 238000001819 mass spectrum Methods 0.000 claims description 9
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 8
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 8
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- -1 HIGP Proteins 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
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- UDDITVWSXPEAIQ-IHRRRGAJSA-N Cys-Phe-Arg Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O UDDITVWSXPEAIQ-IHRRRGAJSA-N 0.000 claims description 3
- UCRJTSIIAYHOHE-ULQDDVLXSA-N Leu-Tyr-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N UCRJTSIIAYHOHE-ULQDDVLXSA-N 0.000 claims description 3
- BEEVXUYVEHXWRQ-YESZJQIVSA-N Phe-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC3=CC=CC=C3)N)C(=O)O BEEVXUYVEHXWRQ-YESZJQIVSA-N 0.000 claims description 3
- JAWGSPUJAXYXJA-IHRRRGAJSA-N Ser-Phe-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CO)N)CC1=CC=CC=C1 JAWGSPUJAXYXJA-IHRRRGAJSA-N 0.000 claims description 3
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- 150000001413 amino acids Chemical class 0.000 description 9
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000002523 gelfiltration Methods 0.000 description 3
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- 239000000126 substance Substances 0.000 description 3
- 239000002253 acid Substances 0.000 description 2
- RYYVLZVUVIJVGH-UHFFFAOYSA-N caffeine Chemical compound CN1C(=O)N(C)C(=O)C2=C1N=CN2C RYYVLZVUVIJVGH-UHFFFAOYSA-N 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
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- 230000000694 effects Effects 0.000 description 2
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- UMURLIQHQSKULR-UHFFFAOYSA-N 1,3-oxazolidine-2-thione Chemical compound S=C1NCCO1 UMURLIQHQSKULR-UHFFFAOYSA-N 0.000 description 1
- 101710159104 Flavor peptide Proteins 0.000 description 1
- LPHGQDQBBGAPDZ-UHFFFAOYSA-N Isocaffeine Natural products CN1C(=O)N(C)C(=O)C2=C1N(C)C=N2 LPHGQDQBBGAPDZ-UHFFFAOYSA-N 0.000 description 1
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- 210000004556 brain Anatomy 0.000 description 1
- 229960001948 caffeine Drugs 0.000 description 1
- VJEONQKOZGKCAK-UHFFFAOYSA-N caffeine Natural products CN1C(=O)N(C)C(=O)C2=C1C=CN2C VJEONQKOZGKCAK-UHFFFAOYSA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000723 chemosensory effect Effects 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 235000019564 dysgeusia Nutrition 0.000 description 1
- 235000021107 fermented food Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
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- 235000003599 food sweetener Nutrition 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
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- 230000000977 initiatory effect Effects 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
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- 239000003765 sweetening agent Substances 0.000 description 1
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of food additives, and discloses a preparation method of a caper bitter peptide, which comprises the steps of crushing caper kernels, degreasing, enzymolysis and high-temperature enzyme deactivation of caper kernel powder in sequence, and further separating and purifying by utilizing gel chromatography and reversed-phase high-performance liquid chromatography in combination with sensory evaluation to obtain and determine the component with the strongest bitter taste, and identifying the bitter peptide by utilizing nano-liter liquid mass spectrometry tandem mass spectrometry. The invention establishes an extraction, separation, purification and identification method of the caper bitter peptides, shows the structure and the bitter characteristics of the bitter peptides in the caper, discovers five novel bitter peptides in the caper for the first time, and the prepared bitter peptides have the functions of bitter taste and sweet taste inhibition, can effectively harmonize the flavor of the flavor food, are ideal natural food flavoring agents, and have good development prospects.
Description
Technical Field
The invention relates to the technical field of food additives, in particular to a preparation method of a caper nut bitter peptide.
Background
The caper is a rare wild plant special for China, and the caper has tender fruits, fragrance, edibility and good taste. The semen Arecae has unique flavor and color, and when the semen Arecae is chewed, the semen Arecae has bitter and sweet taste in the oral cavity, and after being chewed, the semen Arecae is diluted by drinking water, the bitter taste gradually disappears, the sweet taste is greatly enhanced, and the sweet aftertaste can last for half an hour, so that the aftertaste of a user is endless, namely the sweet water effect. The sweet protein Ma Binling which is rich in the semen arecae fruit is not only one of 7 plant sweet proteins which are found in the world at present, but also is the plant sweet protein with the best heat stability and acid resistance, the sweetness of the sweet protein is about 400 times of that of sucrose with the same quality, and the sweet protein is often used as a food additive in the fields of food processing, chewing gum, candy, beverage, dairy products, health care products and the like. The current research on caper has focused on the development of thaumatin and sweeteners in caper and has demonstrated that oxazolidin-2-thione produced during storage of caper is one of its bitter sources. However, the flavor of the caper is not explored from the aspect of taste peptide, and in order to improve the palatability of the caper, the exploration of bitter substances and the control from the source are necessary.
Taste refers to the dissolution of soluble taste substances in the oral cavity to produce taste molecules, activating taste receptors, initiating a series of complex signaling processes in cells, then passing through the taste nerves to the taste centers of the brain, and analyzing and integrating the resultant integrated chemosensory response. The taste sensation includes sour, sweet, bitter, salty, fresh five basic tastes and other tastes such as hot, astringent, thick and the like, and substances providing the tastes include organic acids, saccharides, nucleotides, amino acids, taste peptides and the like. Taste peptide refers to a small molecule peptide extracted from food or synthesized from amino acids that contributes to the flavor of food, covering 5 basic tastes and possibly forming a complex taste sensation. Bitter peptides are a large class of taste peptides, which are widely varied in kind and source, most peptides of vegetable and fermented food origin exhibit bitter taste. The number of amino acid compositions of the bitter peptides spans larger, and the bitter peptides with various chain lengths have no specific primary structure, and generally have higher content of hydrophobic amino acids. In general, the greater the exposure of the hydrophobic amino acid, the more bitter the bitter peptide. The bitterness of the polypeptide is closely related to the composition, sequence and three-dimensional structure of amino acid, and also depends on the relative molecular weight and hydrophobicity of the polypeptide, the polypeptide with molecular weight larger than 6000Da has no bitterness, and the short peptide with molecular weight between 500 Da and 1000Da has strongest bitterness. At present, few researches and reports on the taste peptide of the semen arecae are carried out, the invention focuses on the semen arecae bitter peptide, researches and optimizes the separation, purification, identification and verification methods of the bitter peptide in semen arecae kernel, aims to prepare the bitter peptide with influence on other tastes from the semen arecae kernel through an extensible process, and lays a foundation for widening the application of the bitter peptide in food seasoning.
Disclosure of Invention
The present invention is directed to a method for preparing a bitter peptide of caper, which solves the problems set forth in the background art.
In order to solve the technical problems, the invention provides the following technical scheme: a preparation method of a caper bitter peptide is characterized by comprising the following steps of;
s1, processing the semen arecae kernels: selecting insect-free, mould-free and full semen Arecae seeds, removing shell, pulverizing kernel, and sieving; adding petroleum ether into the obtained semen Arecae kernel powder, magnetically stirring for degreasing, and filtering to remove petroleum ether layer to obtain semen Arecae defatted powder; drying at room temperature in a fume hood, and collecting and storing for later use;
s2, preparing a caper enzymolysis solution: mixing the defatted powder of semen Arecae with ultrapure water to obtain suspension, adjusting pH, adding alkaline protease, magnetically stirring in water bath, extracting, and inactivating enzyme in boiling water bath; cooling and centrifuging, collecting supernatant, vacuum freeze-drying, and collecting and storing semen Arecae crude polypeptide lyophilized powder;
s3, gel filtration chromatographic separation: dissolving the above semen Arecae crude polypeptide lyophilized powder in ultrapure water to obtain sample solution, loading the sample solution into chromatographic column filled with Sephadex G-15 gel, collecting components corresponding to each peak in ultraviolet spectrogram, and vacuum lyophilizing;
s4, sensory evaluation: carrying out sensory description and scoring on the sample solution by tasting and comparing with the standard solution;
s5, reversed-phase high performance liquid chromatography separation: dissolving gel component with highest bitter taste value in ultrapure water, purifying by reversed phase high performance liquid chromatography, collecting components corresponding to each peak, removing organic reagent by rotary evaporation, vacuum freeze drying, and selecting the bitter component as semen Arecae bitter peptide by combining sensory evaluation;
s6, identifying a bitter peptide sequence: detecting the bitter peptide sequence by LC-MS/MS, searching the target protein database using Byonic;
s7, bitter peptide synthesis and bitter characteristic verification.
Preferably, in the step S1, the semen arecae kernels are crushed and then are sieved by a 60-mesh sieve, and the petroleum ether degreasing feed liquid ratio is 1: and 5, degreasing for 4 hours, and drying at room temperature for 12 hours after degreasing.
Preferably, in the step S2, the mixing ratio of the defatted powder of caper to the ultrapure water is 1:25, adjusting pH with 1mol/L sodium hydroxide solution and 1.5mol/L hydrochloric acid solution, extracting for 5 hr, inactivating enzyme with 100deg.C boiling water bath for 20min, and centrifuging at 4deg.C at 8000r/min for 20min.
Preferably, the chromatographic conditions of the gel in S3 are: the concentration of the hydrolysate freeze-dried powder is 20mg/mL, the flow rate is 1.0mL/min, the loading amount is 1mL, and the detection wavelength is 220nm.
Preferably, the reversed-phase high performance liquid chromatography purification conditions in S4 are: chromatographic column AQ-C18 (10×250nm,5 μm), column temperature 30 ℃, hydrolysate lyophilized powder concentration 10mg/mL, loading 100 μl, flow rate 3.0mL/min, detection wavelength 220nm, running 15min, mobile phase A is 0.05% trifluoroacetic acid ultrapure water solution, mobile phase B is methanol, and eluent is mixed with volume ratio A of A and B: B=90:10.
Preferably, the Nano-LC-MS/MS pair is adopted in the S5Amino acid sequence identification is carried out on liquid phase components of the polypeptide, and the liquid phase conditions are as follows: the acclaim pepmap rplcc18 column (150 μm. D.×150mm,1.9 μm,) The method comprises the steps of carrying out a first treatment on the surface of the Mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid aqueous solution and 80% acetonitrile; the flow rate is 600nL/min, and gradient elution is carried out: 0-2min,4% -8% B;2-45min,8% -28% of B;45-55min,28-40% B;55-56min,40-95% B;56-66min,95% B;
the mass spectrometry conditions were as follows:
the ion mode is ESI+ionization mode, full scanning acquisition (m/z 100-1500), spraying voltage is 2.2KV, capillary temperature is 270 ℃, and primary mass spectrum parameters are set: scanning range is 100-1500m/z, resolution is 70000, maximum filling time of scanning is 100ms, AGCtarget is 3e6, and secondary mass spectrum parameter is set: resolution is 17500, the maximum filling time of scanning is set to be 50ms, and AGCtarget is set to be 1e5;
through spectrogram analysis, 5 bitter peptides, HIGP, FHP, CFR, LYR and SFR respectively, are obtained, namely the amino acid sequences are: his-Ile-Gly-Pro, phe-His-Pro, cys-Phe-Arg, leu-Tyr-Arg and Ser-Phe-Arg.
Preferably, 5 polypeptides identified are synthesized by Fmoc solid phase method, the purity of synthesis is more than 95%, the sample is dissolved to prepare a solution with the concentration of 1mg/mL, and the solution is prepared according to the following steps: 1 (v/v) and gradually diluting the sample, performing a triangular test by a sensory evaluator according to the increasing concentration until the taste cannot be tasted at a certain dilution factor, wherein the mass concentration of the sample at the moment is the taste threshold of the sample, the corresponding dilution factor is recorded as a TD value, each group of samples is repeatedly evaluated 3 times by each evaluator, the average TD value is recorded, and each polypeptide sample is subjected to the threshold test on different days.
Compared with the prior art, the invention has the following beneficial effects:
the invention takes the semen arecae as the raw material, and gradually screens out the components with the most prominent bitter taste by using a series of separation and purification techniques and combining with sensory evaluation, 5 novel semen arecae bitter peptides are obtained by identification.
Drawings
FIG. 1 is a chromatographic chart of the invention after SephadexG-15 gel chromatographic separation;
FIG. 2 is a flavor profile of gel components F1, F2 and F3 of the present invention;
FIG. 3 is a RP-HPLC separation spectrum of gel fraction F3 of the present invention;
FIG. 4 is a flavor profile of the liquid phase component F3-4 of the present invention;
FIG. 5 shows the mass spectra of the F3-4 components of the present invention (A: HIGP, B: FHP, C: CFR, D: LYR; E: SFR);
FIG. 6 is a graph showing the effect of adding different concentrations of bitter synthetic peptides on the sweetness of sucrose solutions in accordance with the present invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Referring to fig. 1-6, a method for preparing a caper bitter peptide is characterized by comprising the following steps;
s1, processing the semen arecae kernels: selecting insect-free, mould-free and full semen Arecae seeds, removing shell, pulverizing kernel, and sieving; adding petroleum ether into the obtained semen Arecae kernel powder, magnetically stirring for degreasing, and filtering to remove petroleum ether layer to obtain semen Arecae defatted powder; drying at room temperature in a fume hood, and collecting and storing for later use;
s2, preparing a caper enzymolysis solution: mixing the defatted powder of semen Arecae with ultrapure water to obtain suspension, adjusting pH, adding alkaline protease, magnetically stirring in water bath, extracting, and inactivating enzyme in boiling water bath; cooling and centrifuging, collecting supernatant, vacuum freeze-drying, and collecting and storing semen Arecae crude polypeptide lyophilized powder;
s3, gel filtration chromatographic separation: dissolving the above semen Arecae crude polypeptide lyophilized powder in ultrapure water to obtain sample solution, loading the sample solution into chromatographic column filled with Sephadex G-15 gel, collecting components corresponding to each peak in ultraviolet spectrogram, and vacuum lyophilizing;
s4, sensory evaluation: carrying out sensory description and scoring on the sample solution by tasting and comparing with the standard solution;
s5, reversed-phase high performance liquid chromatography separation: dissolving gel component with highest bitter taste value in ultrapure water, purifying by reversed phase high performance liquid chromatography, collecting components corresponding to each peak, removing organic reagent by rotary evaporation, vacuum freeze drying, and selecting the bitter component as semen Arecae bitter peptide by combining sensory evaluation;
s6, identifying a bitter peptide sequence: detecting the bitter peptide sequence by LC-MS/MS, searching the target protein database using Byonic;
s7, bitter peptide synthesis and bitter characteristic verification.
Specifically, in S1, the semen arecae kernels are crushed and then are sieved by a 60-mesh sieve, and the ratio of petroleum ether degreasing feed liquid is 1:5, degreasing for 4 hours, and drying at room temperature for 12 hours after degreasing; preferably, in the step S2, the mixing ratio of the defatted powder of the caper to the ultrapure water is 1:25, adjusting pH with 1mol/L sodium hydroxide solution and 1.5mol/L hydrochloric acid solution, extracting for 5 hr, inactivating enzyme with 100deg.C boiling water bath for 20min, and centrifuging at 4deg.C at 8000r/min for 20min; the chromatographic conditions of the gel in S3 are as follows: the concentration of the hydrolysate freeze-dried powder is 20mg/mL, the flow rate is 1.0mL/min, the sample loading amount is 1mL, and the detection wavelength is 220nm; the reversed phase high performance liquid chromatography purification conditions in S4 are as follows: chromatographic column AQ-C18 (10×250nm,5 μm), column temperature 30 ℃, concentration of lyophilized powder of hydrolysate 10mg/mL, loading amount 100 μl, flow rate 3.0mL/min, detection wavelength 220nm, and operation15min, wherein the mobile phase A is an ultrapure water solution of trifluoroacetic acid with the volume fraction of 0.05%, the mobile phase B is methanol, and the eluent is obtained by mixing the volume ratio A of A and B, wherein B=90:10; in S5, adopting Nano-LC-MS/MS to identify amino acid sequence of the lightest liquid phase component, wherein the liquid phase condition is as follows: the acclaim pepmap rplcc18 column (150 μm. D.×150mm,1.9 μm,) The method comprises the steps of carrying out a first treatment on the surface of the Mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid aqueous solution and 80% acetonitrile; the flow rate is 600nL/min, and gradient elution is carried out: 0-2min,4% -8% B;2-45min,8% -28% of B;45-55min,28-40% B;55-56min,40-95% B;56-66min,95% B;
the mass spectrometry conditions were as follows:
the ion mode is ESI+ionization mode, full scanning acquisition (m/z 100-1500), spraying voltage is 2.2KV, capillary temperature is 270 ℃, and primary mass spectrum parameters are set: scanning range is 100-1500m/z, resolution is 70000, maximum filling time of scanning is 100ms, AGCtarget is 3e6, and secondary mass spectrum parameter is set: resolution is 17500, the maximum filling time of scanning is set to be 50ms, and AGCtarget is set to be 1e5;
through spectrogram analysis, 5 bitter peptides, HIGP, FHP, CFR, LYR and SFR respectively, are obtained, namely the amino acid sequences are: his-Ile-Gly-Pro, phe-His-Pro, cys-Phe-Arg, leu-Tyr-Arg and Ser-Phe-Arg.
More specifically, 5 polypeptides identified are synthesized by adopting an Fmoc solid-phase method, the purity of synthesis is more than 95%, a sample is dissolved to prepare a solution with the concentration of 1mg/mL, and the solution is prepared according to the following steps of: 1 (v/v) and gradually diluting the sample, performing a triangular test by a sensory evaluator according to the increasing concentration until the taste cannot be tasted at a certain dilution factor, wherein the mass concentration of the sample at the moment is the taste threshold of the sample, the corresponding dilution factor is recorded as a TD value, each group of samples is repeatedly evaluated 3 times by each evaluator, the average TD value is recorded, and each polypeptide sample is subjected to the threshold test on different days.
The caper bitter peptide can generate bitter taste and weaken sweet taste, and has the potential of natural flavoring agent of food.
Example 2
The invention relates to a preparation method of a caper bitter peptide, which comprises the following specific steps:
step one, processing the semen arecae kernel: selecting full, worm-free and mildew-free semen Arecae seeds, removing shell, pulverizing kernel, sieving with 60 mesh sieve, and mixing the obtained semen Arecae kernel powder with a feed liquid ratio of 1: and 5, adding petroleum ether, magnetically stirring for degreasing for 4 hours, filtering to remove a petroleum ether layer, obtaining the areca catechu degreasing powder, drying for 12 hours at room temperature in a fume hood, and collecting and storing for later use.
Step 2, preparation of a caper enzymolysis solution: the method comprises the steps of (1) mixing the defatted powder of the semen arecae with ultrapure water according to a feed liquid ratio of 1:25, adjusting pH to 9, adding alkaline protease at 2500U/g, magnetically stirring in 45 deg.C water bath for 5 hr, inactivating enzyme in boiling water bath for 20min, cooling, centrifuging at 4 deg.C and 8000r/min for 20min, vacuum freeze drying supernatant, and storing the obtained coarse polypeptide lyophilized powder in a refrigerator at-80deg.C.
Step 3, gel filtration chromatographic separation: dissolving semen Arecae crude polypeptide lyophilized powder in ultrapure water to obtain 20mg/mL sample solution, filtering with 0.45 μm water phase filter membrane, loading into Sephadex G-15 gel chromatographic column, adjusting the absorption wavelength of ultraviolet detector to 220nm at 25+ -5deg.C, eluting with ultrapure water as eluent at 1mL/min, collecting, mixing and lyophilizing components corresponding to each peak in ultraviolet spectrogram, vacuum freeze drying, storing in refrigerator at-80deg.C, and separating by gel chromatography to obtain three components, F1, F2 and F3 as shown in figure 1.
Step 4, sensory evaluation: 12 sensory panelists without known history of dysgeusia were selected and subjected to sensory training prior to sample evaluation, with 3.5mg/mL of sucrose solution, 20mg/mL of citric acid solution, 6mg/mL of caffeine solution, and 3.5mg/mL of monosodium glutamate solution as standard solution references to differentiate salty, sweet, sour, bitter, and umami tastes. The taste intensity was expressed in 12 minutes and the sensory intensity of the reference solution was 8 minutes. 2mL of sample solution at a concentration of 10mg/mL was prepared, panelists were asked to taste and characterize the taste of each sample, score each basic taste attribute with a corresponding score, and average was taken as the final sensory evaluation result and presented with a radar chart. The panellists were asked to disable water fast 2 hours prior to the sensory evaluation, which was performed at room temperature. For tasting, it is necessary to fully disperse the sample in the mouth to adequately perceive its taste. Each group of samples was sensory evaluated at intervals of 3 minutes, with the rest period requiring rinsing with water and eating a salt-free biscuit for sensory cleaning. As can be seen from figure 2 of the specification, F1 has weak overall flavor and extremely weak bitter taste in three components obtained by gel filtration chromatography; f2 has a certain sour taste and a light bitter taste; f3 has a strong bitter taste and a subtle return sweetness. Based on this result, F3 was selected as the target component for further purification.
Step 5, reversed phase high performance liquid chromatography separation: the F3 active freeze-dried component collected by gel chromatography is dissolved by ultrapure water to prepare a solution of 10mg/mL, and the solution is filtered by a 0.22 mu m aqueous phase membrane for standby. The bitter peptides were isolated and purified using a C18 high performance liquid chromatography column (AQ-C1810X105 mm,5 μm). The mobile phase A is 0.05% trifluoroacetic acid ultrapure water solution, the mobile phase B is methanol, the mobile phase A is eluted at the same temperature as the mobile phase B=90:10, the column temperature is set at 30 ℃, the loading amount is 50 mu L, the flow rate is 3.0mL/min, the detection wavelength is 220nm, the operation is carried out for 15min, the components F3-1, F3-2, F3-3, F3-4 and F3-5 are respectively collected, the organic reagent is removed by rotary evaporation, the freeze drying is carried out, the sensory evaluation is carried out according to the step 4, and the bitterness intensity is detected. The F3-1, F3-2, F3-3 and F3-5 components have small peak areas and low peak heights, so that the collection is not performed. As can be seen from FIG. 4 of the specification, F3-4 having the largest peak area exhibits a strong bitter taste.
Step 6, identifying a bitter peptide sequence: sequence analysis and identification of the flavor peptide component separated by reverse high performance liquid phase by Nano-LC-MS/MS, comprising:
sample treatment: the sample was dissolved in a DTT solution to prepare a 10mmol/L solution, and the solution was reduced in a 56℃water bath for 1 hour. IAM solution was added thereto so that the final concentration was 55mmol/L, and the reaction was carried out in the dark for 40 minutes. Desalting with self-packed desalting column, volatilizing solvent in vacuum centrifugal concentrator at 45deg.C, and detecting;
the liquid phase conditions were as follows: the acclaim pepmap rplcc18 column (150 μm. D.×150mm,1.9 μm,) The method comprises the steps of carrying out a first treatment on the surface of the Mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid aqueous solution and 80% acetonitrile; the flow rate is 600nL/min, and gradient elution is carried out: 0-2min,4% -8% B;2-45min,8% -28% of B;45-55min,28-40% B;55-56min,40-95% B;56-66min,95% B.
The mass spectrometry conditions were as follows:
the ion mode is ESI+ionization mode, the total scanning acquisition (m/z 100-1500), the spraying voltage is 2.2KV, and the capillary temperature is 270 ℃. Primary mass spectrum parameter setting: the scanning range is 100-1500m/z, the resolution is 70000, the maximum filling time of scanning is 100ms, and the AGCtarget is 3e6. Secondary mass spectrometry parameter setting: resolution is 17500, the maximum filling time of scanning is set to be 50ms, and AGCtarget is set to be 1e5;
and searching the raw file acquired by the mass spectrum through a software Byonic database to obtain a polypeptide sequence identification result.
Step 7, bitter peptide synthesis and bitter characteristic verification: 5 polypeptides are synthesized by Fmoc solid phase method, and the purity of the synthesis is more than 95%. The sample was dissolved to prepare a solution having a concentration of 1 mg/mL. The solution was prepared according to 1:1 (v/v) and performing triangular test by the sensory evaluator according to the concentration increment until the taste cannot be tasted at a certain dilution multiple, wherein the mass concentration of the sample at the moment is the taste threshold of the sample, and the corresponding dilution multiple is recorded as a TD value. Each set of samples was repeatedly evaluated 3 times by each evaluator, and the average TD value was recorded, with each polypeptide sample being thresholded on a different day.
Wherein the taste profile and bitterness threshold of the caper bitter peptide solution are shown in Table 1 below
List one
Two sets of sucrose solution samples (sucrose standard solution and sucrose standard solution with bitter peptide added) were set up, and sensory evaluation was performed according to the step 4 standard to describe taste change after ingestion, with sweetness and bitterness as evaluation attributes. As a result, as shown in FIG. 6 of the specification, all of the bitter peptides had sweet taste inhibitory effects, with the sweet taste inhibitory properties of FHP being most prominent.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A preparation method of a caper bitter peptide is characterized by comprising the following steps of;
s1, processing the semen arecae kernels: selecting insect-free, mould-free and full semen Arecae seeds, removing shell, pulverizing kernel, and sieving; adding petroleum ether into the obtained semen Arecae kernel powder, magnetically stirring for degreasing, and filtering to remove petroleum ether layer to obtain semen Arecae defatted powder; drying at room temperature in a fume hood, and collecting and storing for later use;
s2, preparing a caper enzymolysis solution: mixing the defatted powder of semen Arecae with ultrapure water to obtain suspension, adjusting pH, adding alkaline protease, magnetically stirring in water bath, extracting, and inactivating enzyme in boiling water bath; cooling and centrifuging, collecting supernatant, vacuum freeze-drying, and collecting and storing semen Arecae crude polypeptide lyophilized powder;
s3, separating by gel filtration chromatography: dissolving the above semen Arecae crude polypeptide lyophilized powder in ultrapure water to obtain sample solution, loading the sample solution into chromatographic column filled with Sephadex G-15 gel, collecting components corresponding to each peak in ultraviolet spectrogram, and vacuum lyophilizing;
s4, sensory evaluation: carrying out sensory description and scoring on the sample solution by tasting and comparing with the standard solution;
s5, reversed-phase high performance liquid chromatography separation: dissolving gel component with highest bitter taste value in ultrapure water, purifying by reversed phase high performance liquid chromatography, collecting components corresponding to each peak, removing organic reagent by rotary evaporation, vacuum freeze drying, and selecting the bitter component as semen Arecae bitter peptide by combining sensory evaluation;
s6, identifying a bitter peptide sequence: detecting the bitter peptide sequence by LC-MS/MS, searching the target protein database using Byonic;
s7, bitter peptide synthesis and bitter characteristic verification.
2. The method for preparing the caper bitter peptide according to claim 1, wherein the method comprises the following steps: in the step S1, the semen arecae kernels are crushed and then pass through a 60-mesh sieve, and the petroleum ether degreasing feed liquid ratio is 1: and 5, degreasing for 4 hours, and drying at room temperature for 12 hours after degreasing.
3. The method for preparing the caper bitter peptide according to claim 1, wherein the method comprises the following steps: in the step S2, the mixing ratio of the areca-nut degreasing powder to the ultrapure water is 1:25, adjusting pH with 1mol/L sodium hydroxide solution and 1.5mol/L hydrochloric acid solution, extracting for 5 hr, inactivating enzyme with 100deg.C boiling water bath for 20min, and centrifuging at 4deg.C at 8000r/min for 20min.
4. The method for preparing the caper bitter peptide according to claim 1, wherein the method comprises the following steps: the chromatographic conditions of the gel in the step S3 are as follows: the concentration of the hydrolysate freeze-dried powder is 20mg/mL, the flow rate is 1.0mL/min, the loading amount is 1mL, and the detection wavelength is 220nm.
5. The method for preparing the caper bitter peptide according to claim 1, wherein the method comprises the following steps: the reversed-phase high performance liquid chromatography purification conditions in the step S4 are as follows: chromatographic column AQ-C18 (10×250nm,5 μm), column temperature 30 ℃, hydrolysate lyophilized powder concentration 10mg/mL, loading 100 μl, flow rate 3.0mL/min, detection wavelength 220nm, running 15min, mobile phase A is 0.05% trifluoroacetic acid ultrapure water solution, mobile phase B is methanol, and eluent is mixed with volume ratio A of A and B: B=90:10.
6. The method for preparing the caper bitter peptide according to claim 1, wherein the method comprises the following steps: in the step S5, nano-LC-MS/MS is adopted to carry out amino acid sequence identification on the lightest liquid phase component, and the liquid phase condition is as follows: the acclaim pepmap rplcc18 column (150 μm. D.×150mm,1.9 μm,) The method comprises the steps of carrying out a first treatment on the surface of the Mobile phase a is 0.1% formic acid aqueous solution, mobile phase B is 0.1% formic acid aqueous solution and 80% acetonitrile; the flow rate is 600nL/min, and gradient elution is carried out: 0-2min,4% -8% B;2-45min,8% -28% of B;45-55min,28-40% B;55-56min,40-95% B;56-66min,95% B;
the mass spectrometry conditions were as follows:
the ion mode is ESI+ionization mode, full scanning acquisition (m/z 100-1500), spraying voltage is 2.2KV, capillary temperature is 270 ℃, and primary mass spectrum parameters are set: scanning range is 100-1500m/z, resolution is 70000, maximum filling time of scanning is 100ms, AGCtarget is 3e6, and secondary mass spectrum parameter is set: resolution is 17500, the maximum filling time of scanning is set to be 50ms, and AGCtarget is set to be 1e5;
through spectrogram analysis, 5 bitter peptides, HIGP, FHP, CFR, LYR and SFR respectively, are obtained, namely the amino acid sequences are: his-Ile-Gly-Pro, phe-His-Pro, cys-Phe-Arg, leu-Tyr-Arg and Ser-Phe-Arg.
7. The method for preparing the caper bitter peptide according to claim 6, wherein the method comprises the following steps: 5 identified polypeptides are synthesized by Fmoc solid phase method, the purity of synthesis is more than 95%, a sample is dissolved to prepare a solution with the concentration of 1mg/mL, and the solution is prepared according to the following steps: 1 (v/v) and gradually diluting the sample, performing a triangular test by a sensory evaluator according to the increasing concentration until the taste cannot be tasted at a certain dilution factor, wherein the mass concentration of the sample at the moment is the taste threshold of the sample, the corresponding dilution factor is recorded as a TD value, each group of samples is repeatedly evaluated 3 times by each evaluator, the average TD value is recorded, and each polypeptide sample is subjected to the threshold test on different days.
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