CN116694713A - Multifunctional pea chromene and preparation method thereof - Google Patents
Multifunctional pea chromene and preparation method thereof Download PDFInfo
- Publication number
- CN116694713A CN116694713A CN202310519530.6A CN202310519530A CN116694713A CN 116694713 A CN116694713 A CN 116694713A CN 202310519530 A CN202310519530 A CN 202310519530A CN 116694713 A CN116694713 A CN 116694713A
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- Prior art keywords
- pea
- chromium
- chromene
- peptide
- solution
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- QZHPTGXQGDFGEN-UHFFFAOYSA-N chromene Chemical compound C1=CC=C2C=C[CH]OC2=C1 QZHPTGXQGDFGEN-UHFFFAOYSA-N 0.000 title claims abstract description 124
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 235000010582 Pisum sativum Nutrition 0.000 claims abstract description 280
- 241000219843 Pisum Species 0.000 claims abstract description 220
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims abstract description 145
- 229910052804 chromium Inorganic materials 0.000 claims abstract description 144
- 239000011651 chromium Substances 0.000 claims abstract description 144
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 126
- 240000004713 Pisum sativum Species 0.000 claims abstract description 60
- 229910001430 chromium ion Inorganic materials 0.000 claims abstract description 24
- 238000000034 method Methods 0.000 claims abstract description 24
- 235000018102 proteins Nutrition 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 108010084695 Pea Proteins Proteins 0.000 claims abstract description 16
- 235000019702 pea protein Nutrition 0.000 claims abstract description 16
- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 claims abstract description 11
- 238000002329 infrared spectrum Methods 0.000 claims abstract description 11
- 239000003456 ion exchange resin Substances 0.000 claims abstract description 11
- 229920003303 ion-exchange polymer Polymers 0.000 claims abstract description 11
- 238000002189 fluorescence spectrum Methods 0.000 claims abstract description 9
- 238000001035 drying Methods 0.000 claims abstract description 7
- 239000012467 final product Substances 0.000 claims abstract description 7
- 238000011282 treatment Methods 0.000 claims abstract description 7
- 238000001179 sorption measurement Methods 0.000 claims description 63
- 239000003480 eluent Substances 0.000 claims description 41
- 239000011347 resin Substances 0.000 claims description 38
- 229920005989 resin Polymers 0.000 claims description 38
- 238000010521 absorption reaction Methods 0.000 claims description 34
- 239000000945 filler Substances 0.000 claims description 31
- 239000000243 solution Substances 0.000 claims description 30
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- 230000008569 process Effects 0.000 claims description 14
- QSWDMMVNRMROPK-UHFFFAOYSA-K chromium(3+) trichloride Chemical compound [Cl-].[Cl-].[Cl-].[Cr+3] QSWDMMVNRMROPK-UHFFFAOYSA-K 0.000 claims description 13
- 238000000746 purification Methods 0.000 claims description 12
- 229910021556 Chromium(III) chloride Inorganic materials 0.000 claims description 11
- 229960000359 chromic chloride Drugs 0.000 claims description 11
- 230000035784 germination Effects 0.000 claims description 11
- 150000003839 salts Chemical class 0.000 claims description 11
- 238000000926 separation method Methods 0.000 claims description 11
- 238000004458 analytical method Methods 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 239000012488 sample solution Substances 0.000 claims description 10
- PHFQLYPOURZARY-UHFFFAOYSA-N chromium trinitrate Chemical compound [Cr+3].[O-][N+]([O-])=O.[O-][N+]([O-])=O.[O-][N+]([O-])=O PHFQLYPOURZARY-UHFFFAOYSA-N 0.000 claims description 9
- 239000000523 sample Substances 0.000 claims description 9
- 239000006228 supernatant Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 238000005342 ion exchange Methods 0.000 claims description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 6
- GRWVQDDAKZFPFI-UHFFFAOYSA-H chromium(III) sulfate Chemical compound [Cr+3].[Cr+3].[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O.[O-]S([O-])(=O)=O GRWVQDDAKZFPFI-UHFFFAOYSA-H 0.000 claims description 6
- 239000008367 deionised water Substances 0.000 claims description 6
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/30—Extraction; Separation; Purification by precipitation
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
Abstract
The invention discloses a multifunctional pea chromene and a preparation method thereof, belonging to the technical field of functional peptides; the method comprises the following steps: taking a proper amount of peas in a beaker, adding a chromium ion solution, performing constant temperature treatment, germinating and growing, drying, and extracting protein to obtain chromium-rich pea protein; and (3) taking the chromium-rich pea protein as a raw material, performing enzymolysis to obtain initial pea chromene, and separating and purifying the initial pea chromene by adopting an ion exchange resin method to obtain a pea chromene final product. According to the invention, structural characterization is carried out on the pea chromene through an ultraviolet spectrum, a fluorescence spectrum, an infrared spectrum and a scanning electron microscope, and the amino acid sequence analysis is carried out on the pea chromene by utilizing a high performance liquid chromatography-mass spectrometry HPLC-MS, so that the result shows that the pea chromene is a novel substance different from common pea peptide, and the biological activity research shows that the pea chromene has good hypoglycemic, lipid-lowering and antioxidant activities and is superior to the common pea peptide.
Description
Technical Field
The invention belongs to the technical field of functional peptides, and particularly relates to a multifunctional pea chromene peptide and a preparation method thereof.
Background
Pea is a crop with high nutritive value, wide sources and low price, is rich in protein, carbohydrate, fat and other nutritive components, and pea peptide produced by pea proteolysis has multiple functions of reducing blood sugar, reducing blood fat, resisting oxidation and the like.
Chromium (iii) is a trace element essential to the human body and is commonly found in kidneys, bones, muscles, hair and body fluids in the body. It was found that chromium (III) is associated with the occurrence of abnormal symptoms of glycolipid metabolism, and that the lack of chromium element in the body may cause abnormal glycolipid metabolism, because chromium (III) is the main active ingredient of GTF (glucose tolerance factor) and has a close relationship with the synthesis, secretion and in vivo content of insulin. Because the human body cannot synthesize the chromium (III) and the content of the chromium in the natural food is low, the phenomenon that the human body lacks the chromium (III) is ubiquitous, and therefore, the development of novel and efficient chromium supplements has important significance.
Chromium (iii) has both organic and inorganic forms, and in general, inorganic chromium has a relatively low absorption of about 1% to 3%, whereas organic chromium compounds have a relatively high biocompatibility and an absorption of about 10% to 25%, and therefore organic chromium is often used as a chromium supplement. The organic chromium is mainly synthesized artificially and naturally, and the biological enrichment is utilized to convert the inorganic chromium into the natural organic chromium, so that the organic chromium has lower economic cost, short production period and simple process and is easy to popularize compared with the organic chromium synthesized artificially. At present, a patent for converting inorganic chromium into organic chromium through biological enrichment is disclosed, for example, patent with the publication number of CN1222226C discloses a preparation method of chromium-rich mung bean powder, wherein mung beans are utilized for carrying out biological enrichment on chromium (III), but no further extraction and purification on bioactive substances in the chromium-rich mung bean powder are carried out, and the biological activity of the chromium-rich mung bean powder is not explored. In another example, the patent with application publication number CN103652645a discloses a preparation method of chromium-enriched wheat germ flour, in which chromium (iii) is biologically enriched by using wheat seeds as raw materials to obtain chromium-enriched wheat germ flour, which has good activity of reducing blood sugar and blood lipid, but oxidation resistance is to be further studied, in addition, the patent does not further process the chromium-enriched wheat germ flour, and direct eating may affect the absorption efficiency of the organism on chromium (iii).
The metal chelating peptide is a novel chelate system and is also a novel mineral supplement, compared with metal elements, the metal chelating peptide can directly enter organisms in the form of inorganic salts, and the peptide is utilized for rapid digestion and absorption in the small intestine. The research shows that the metal chelating peptide has unique chelating system and transport mechanism, is easier to absorb than amino acid chelating metal element, and has wider development space. Currently, metal chelating peptides have been isolated and identified from a variety of food proteins. For example, casein phosphopeptides (CPPs) derived from milk proteins, zinc chelate peptides derived from sesame proteins, copper chelate peptides isolated and purified from chickpea proteins and sunflower seeds, etc. have been reported, but no report has been made on chromium chelate peptides.
Therefore, research and development of a chromium peptide prepared from peas are needed, which on one hand makes up for the blank of research of preparing chromium peptide from peas, and on the other hand improves the in vitro hypoglycemic, hypolipidemic and antioxidant activity of the pea peptide.
Disclosure of Invention
The purpose of the invention is that: the method comprises the steps of taking peas as raw materials, carrying out biological enrichment on chromium (III), then germinating and growing, extracting proteins, carrying out enzymolysis on the chromium-rich pea proteins as raw materials to obtain initial pea chromene, and separating and purifying the initial pea chromene by using an ion exchange resin method to obtain a pea chromene final product; the structural characterization of the pea chromene is carried out by ultraviolet spectrum, fluorescence spectrum, infrared spectrum and scanning electron microscope, and the amino acid sequence analysis is carried out by utilizing HPLC-MS, thus the result shows that the pea chromene is a new substance different from common pea peptide; biological activity researches show that the pea chromene has good hypoglycemic, lipid-lowering and antioxidant activities and is superior to common pea peptide.
In order to achieve the above purpose, the present invention adopts the following technical scheme: a multifunctional pea chromene and a preparation method thereof comprise the following steps:
s1, preparing chromium-rich peas and proteins thereof:
adding the peas into a beaker according to the ratio of 1:3, adding a chromium ion solution with the concentration of 0.66-1.54 mmol/L, carrying out constant-temperature treatment, soaking at 20-40 ℃ for 8-24 h, germinating and growing for 1-7 days after the treatment, drying and grinding the germinated and grown chromium-enriched peas to obtain chromium-enriched pea powder;
adding deionized water into the 60-mesh-sieve chromium-rich pea powder according to the ratio of 1:15, uniformly mixing, adjusting the pH of slurry to 9.0, performing ultrasonic-assisted extraction, centrifuging, taking supernatant, adjusting the pH of the supernatant to 4.5, centrifuging, taking precipitate, and drying to obtain chromium-rich pea protein powder;
s2, enzymolysis of pea protein:
dissolving the chromium-rich pea protein powder prepared in the step S1 in deionized water according to the proportion of 5% w/w, adding 7%E/S alkaline protease, adjusting the pH value to 8.0, reacting at the temperature of 55 ℃, rapidly placing the mixture into a constant-temperature water bath at the temperature of 95 ℃ after enzymolysis is finished, inactivating enzyme for 15min to terminate the reaction, cooling, adjusting the pH value of an enzymolysis solution to 7.0, centrifuging, and taking supernatant to obtain the initial pea chromium peptide;
s3, separation and purification of pea chromene:
pretreating resin, respectively placing the pretreated resin into conical flasks for static adsorption, balancing by taking PBS solution with pH of 6.0-8.0 as an initial buffer solution, adding the initial pea chromene prepared in the step S2, adsorbing for 2-10 h, taking out and filtering, collecting filtered adsorption solution, calculating adsorption rate, desorbing the adsorbed resin by using eluent, calculating desorption rate, and completing a static adsorption process;
filling ion exchange column filler into chromatographic column, balancing ion exchange chromatographic column with PBS solution, adding initial pea chromium peptide prepared in the step S2, eluting the non-adsorbed chromium peptide with PBS solution, collecting PBS solution, mixing with the sample solution to obtain dynamic adsorption solution, eluting with NaCl solution of 2-10 times of the sample solution volume after the washing, eluting with pH of 8.0-12.0 and eluent salt concentration of 0.4-1.2 mol/L, collecting dynamic eluent, calculating desorption rate, and completing dynamic adsorption process to obtain final product of pea chromium peptide.
In step S1, the chromium ion species include chromium trichloride, chromium sulfate and chromium nitrate, preferably chromium trichloride.
In the step S1, the preferable concentration of chromium ions is 1.2-1.5 mmol/L, the soaking temperature is 20 ℃, the soaking time is 10-15 h, and the germination and growth period is 2.5-5.0 days.
In the step S3, preferably, in the static adsorption process, the ion exchange column packing adopts No. 1 resin with the model of LKC100, the pH of the initial buffer solution is 6.6-7.2, and the adsorption time is 6-9 h.
In the step S3, preferably, in the dynamic adsorption process, the volume of the eluent is 4-8 times of the volume of the sample liquid, the pH value of the eluent is 10-12, and the salt concentration of the eluent is 0.6-0.9 mol/L.
The invention also provides the pea chromene prepared by the method, the scanning electron microscope structure of the pea chromene is in a compact sphere shape, the maximum absorption wavelength in ultraviolet visible full-wavelength scanning is 206nm, the maximum fluorescence intensity at 400nm in a fluorescence spectrum is 809.9, and the telescopic vibration absorption peak of-NH in infrared spectrum analysis is 3073.66cm -1 Fingerprint area due to c=o orCOO-stretching vibration with absorption peaks of 1667.95cm -1 ，1453.94cm -1 ，1412.39cm -1 ，991.39cm -1 ，625.42cm -1 The amino acid number distribution of 574 peptide sequences in HPLC-MS analysis is 3-15, wherein the ratio of tripeptide and tetrapeptide is the largest, 234 tripeptide sequences are in proportion of 40.77%, 190 tetrapeptide sequences are in proportion of 33.10%, and the molecular weight is 374.1802-1891.7820.
The beneficial effects of the invention are as follows:
1) The invention takes peas as raw materials to carry out biological enrichment on chromium (III) and then germinates and grows, then extracts protein, takes chromium-rich pea protein as raw materials to carry out enzymolysis to obtain initial pea chromene, and based on the initial pea chromene, the initial pea chromene is separated and purified by an ion exchange resin method to obtain a final product, and experimental researches show that the pea chromene has good hypoglycemic, lipid-lowering and antioxidant activities and is superior to common pea peptide.
2) The structural characterization of the pea chromene is carried out by ultraviolet spectrum, fluorescence spectrum, infrared spectrum and scanning electron microscope, and the result shows that the pea chromene is a novel substance different from common pea peptide.
Drawings
FIG. 1 is a graph of single-factor experimental results in the preparation of chromium-enriched peas according to the present invention;
FIG. 2 is a graph showing the results of static adsorption experiments in separation and purification of pea chromene according to the present invention;
FIG. 3 is a graph showing the results of dynamic adsorption experiments in separation and purification of pea chromene according to the present invention;
FIG. 4 is a graph showing the inhibition of various pea peptide alpha-glucosidase enzyme activities measured in vitro hypoglycemic activity experiments of pea chromene according to the present invention;
FIG. 5 is a graph showing the inhibition of various pea peptide alpha-amylase in vitro hypoglycemic activity assay of the present invention;
FIG. 6 is a graph showing the inhibition of pancreatic lipase by different pea peptides measured in vitro lipid lowering activity assay of the present invention;
FIG. 7 is a graph showing the cholesterol esterase inhibitory activity of various pea peptides measured in vitro lipid lowering activity assay of the present invention;
FIG. 8 is a graph showing DPPH radical scavenging activity of various pea peptides of the invention as measured in an in vitro antioxidant activity assay of pea chromene;
FIG. 9 is a graph showing the hydroxyl radical scavenging activity of various pea peptides of the present invention as measured in an in vitro antioxidant activity assay of pea chromene;
FIG. 10 is a graph of the reduction potential of different pea peptides of the invention as determined in an in vitro antioxidant activity assay of pea chromene;
FIG. 11 is a UV spectrum of conventional pea peptide, pea chromene peptide and chromium trichloride as determined in the structural characterization of pea chromene according to the present invention;
FIG. 12 is a graph showing fluorescence spectra of conventional pea peptides and pea chromene as determined in structural characterization of pea chromene according to the present invention;
FIG. 13 is an infrared spectrum of a conventional pea peptide and pea chromene as determined in the structural characterization of pea chromene of the present invention;
FIG. 14 is a scanning electron microscope image of a conventional pea peptide and pea chromene peptide of the present invention as determined in structural characterization of pea chromene;
FIG. 15 is a total ion flow chart of the invention as determined in HPLC-MS analysis of pea chromene.
Detailed Description
The invention is further illustrated by the following description in conjunction with the accompanying drawings and specific embodiments.
1. Experimental materials and reagents:
experimental raw materials were all purchased from markets, including peas (middle pea No. 9); chromium trichloride hexahydrate, 40% chromium sulfate solution, chromium nitrate, p-nitrophenyl-beta-D galactopyranoside (PNPG), DNS reagent, 4-nitrophenyl butyrate, 4-nitrophenyl laurate, acarbose (analytically pure); alkaline protease (200U/mg), alpha-glucosidase (1000U), alpha-amylase (40U/mg), pancreatic lipase (15-35U/mg), cholesterol esterase (10U/mg); the model of the selected No. 1 cationic resin is LKC100, and the model of the selected No. 2 cationic resin is D001SD.
2. Chromium-rich peas and preparation of proteins thereof:
pea bioaccumulation: putting a proper amount of peas into a beaker, adding a chromium ion solution with the concentration of 0.66-1.54 mmol/L according to the ratio of 1:3 of feed liquid ratio (w: v), carrying out constant-temperature treatment, soaking at 20-40 ℃ for 8-24 h, germinating and growing for 1-7 days after the treatment, drying and grinding the germinated and grown chromium-enriched peas, and obtaining the chromium-enriched pea powder.
Pea protein extraction: adding deionized water into the 60-mesh-sieve chromium-rich pea powder according to the ratio of feed liquid ratio (W: v) of 1:15, uniformly mixing, adjusting the pH of slurry to 9.0, using ultrasonic wave for auxiliary extraction, using ultrasonic power of 300W for 30min, centrifuging the leached sample at 8000r/min for 15min, centrifuging to obtain supernatant, adjusting the pH of the supernatant to 4.5, centrifuging at 8000r/min for 15min, collecting precipitate, and drying to obtain chromium-rich pea protein powder.
3. Pea chromium-rich single factor test:
the four factors of chromium ion type, soaking time, soaking temperature and chromium ion concentration in the process of enriching the chromium in the peas are explored by taking the chromium ion concentration of 0.66mmol/L, the soaking temperature of 30 ℃ and the soaking time of 8 hours as initial conditions, and the influence of the four factors on the biological chromium enriching effect of the peas is discussed by changing a single factor.
As can be seen from FIG. 1-A, the chromium-rich group of peas had a higher chromium content than the non-rich group, wherein the total chromium content of the chromium nitrate group was the lowest compared to the chromium trichloride group and the chromium sulfate group, which was 45.77 μg/g, which is 218 times the total chromium content of the non-rich peas of 0.21 μg/g, indicating that the peas had a stronger chromium-rich capacity. The degree of organization of the chromium trichloride, the chromium sulfate and the chromium nitrate groups was 75.70%, 70.94% and 69.75%, respectively, which indicated that most of the chromium (iii) in the chromium-rich peas was organic chromium, indicating that it was possible to biologically enrich the chromium (iii) by peas during the absorption of the chromium (iii) by the peas to convert part of the inorganic chromium into organic chromium. It is therefore clear that chromium (iii) does not precipitate in peas by means of simple diffusion or ion adsorption, possibly involving more complex biochemical transformations. In addition, different types of chromium ions have different chromium-rich effects on peas, wherein the total chromium content, the inorganic chromium content and the organic chromium content of the peas of the chromium trichloride group are higher than those of the chromium sulfate group and the chromium nitrate group. This result shows that chloride ions have a better promoting effect on pea chromium enrichment, sulfate is the weakest, nitrate is the weakest, at the same chromium ion concentration. Thus the best chromium ion species should be selected from chromium trichloride.
As can be seen from FIG. 1-B, the peas have a maximum organic chromium content at 10-15 hours of soaking, indicating an optimal chromium-rich effect, with maximum total and organic chromium contents of 64.13 μg/g and 49.86 μg/g, respectively. In the initial stage of soaking, as the soaking time is prolonged, the absorption of the peas to chromium ions is gradually increased, and the organic chromium content is increased. And after the organic chromium content reaches the maximum, the organic chromium content of the peas gradually decreases with further increase of the soaking time. Therefore, the optimal soaking time should be selected to be 10-15 hours.
As can be seen from FIG. 1-C, the total chromium content and the organic chromium content of the peas are highest at a soaking temperature of 20℃and the inorganic chromium content of the peas is lower than 25℃and 30 ℃. This means that the absorption of chromium (iii) by peas is highest at a soaking temperature of 20 c, and that peas have a strong ability to convert inorganic chromium to organic chromium. With further increase of the soaking temperature, the total chromium content and the organic chromium content of the peas show a decreasing trend, and the total chromium content and the organic chromium content of the peas are the lowest when the soaking temperature is 40 ℃. Comprehensively considering the factors such as actual production energy consumption, chromium-rich effect, economic cost and the like, and selecting the optimal soaking temperature to be 20 ℃.
As can be seen from FIG. 1-D, as the concentration of chromium ions increases, the total chromium content and the organochromium content of the peas both tend to increase and then reach equilibrium. When the concentration of chromium ions is 0.6-1.2 mmol/L, the organic chromium content of peas is increased along with the increase of the concentration, and the increasing trend of the chromium content of peas is gradually gentle when the concentration of the chromium ions is between 1.2-1.5 mmol/L. This means that in the lower range of chromium ion concentration, as the chromium ion concentration increases, the difference in chromium ion concentration between the inside and outside of the peas increases, and the absorption capacity of the peas for chromium (iii) increases gradually. When the concentration of chromium ions is between 1.2 and 1.5mmol/L, the absorption capacity of the peas on chromium (III) is limited, and the absorption of the peas on the chromium (III) is saturated, so that the increasing trend of the chromium content of the peas is basically gentle. Considering the two factors of chromium-rich effect and economic cost, the optimal chromium ion concentration should be 1.2-1.5 mmol/L.
As can be seen from FIG. 1-E, the total chromium content and organic chromium content of the chromium-rich peas at 0d of germination and growth were higher than those of peas at 1-7 d of germination and growth, which indicates that chromium (III) was lost with germination and growth of peas. As the germination and growth period of the chromium-rich peas increases, the total chromium content and the organic chromium content of the peas gradually decrease. This shows that chromium (III) is gradually lost to the outside during germination and growth of chromium-rich peas.
From fig. 1-E it is also known that the chromium rich peas have a maximum protein chromium content therein when grown to 2.5-5.0 d, which indicates that the chromium rich peas have an increased protein chromium content by germination growth, indicating that further protein binding to chromium (iii) occurs during germination growth. The growing period is 0-2.5 d, and the protein chromium content of the chromium-rich peas gradually increases along with the increase of days. After the maximum protein chromium content has been reached, the protein chromium content of the chromium rich peas starts to decrease gradually with increasing germination growth cycle, which means that the binding of protein to chromium (iii) in the chromium rich peas starts to decrease, probably because the protease activity in the chromium rich peas increases with increasing growth cycle, so that the protein in the chromium rich peas is hydrolysed, which may comprise a part of the protein bound to chromium (iii). Therefore, the optimal germination and growth period of the chromium-rich peas is selected to be 2.5-5.0 days.
4. Enzymolysis of pea proteins:
dissolving the chromium-rich pea protein powder prepared in the step 2 in deionized water according to the proportion of 5% w/w, adding 7%E/S alkaline protease, adjusting the temperature to 55 ℃, reacting at the pH of 8.0, rapidly placing into a constant-temperature water bath kettle at the temperature of 95 ℃ after enzymolysis is finished, inactivating enzyme for 15min to terminate the reaction, cooling, adjusting the pH of enzymolysis solution to 7.0, centrifuging at 5000r/min for 10min, and taking supernatant to obtain the initial pea chromium peptide.
5. Separation and purification of pea chromene:
5.1 pretreatment of ion exchange resin, wherein the pretreatment step of the ion exchange resin adopts the prior conventional method.
5.2, adopting a static adsorption single factor experiment to explore the influence of the type of the ion exchange column filler and the pH value of an initial buffer solution on separation of pea chromene:
screening different types of ion resin fillers, wherein the types of the ion exchange resin fillers are two levels of No. 1 cation resin with the model number of LKC100 and No. 2 cation resin with the model number of D001 SD; respectively taking 4g of two types of ion exchange resin fillers, putting the two types of ion exchange resin fillers into a conical flask, balancing with PBS solutions with different pH values (6.0-8.0), adding the initial pea chromene, taking out and filtering after adsorption, collecting filtered static adsorption liquid, wherein the concentration of the initial liquid chromium is C0, the concentration of the static adsorption liquid chromium is C1, calculating the adsorption rate, and the adsorption rate (%) = (C0-C1)/C0 is 100%, wherein three groups of parallel are formed under each condition.
And determining the maximum adsorption rate of the two resin fillers, comparing the desorption rates, filtering and collecting static adsorption liquid after the sample solution is adsorbed by the two resin fillers, adding eluent, desorbing and taking out the eluent, filtering, collecting the static eluent, collecting the initial liquid chromium concentration C0, the static adsorption liquid chromium concentration C1, the static eluent chromium concentration is C2, calculating the desorption rate of each tube, wherein the desorption rate (%) =C2/(C0-C1) is 100%, and three groups of parallel are arranged under each condition.
And comprehensively screening the resin filler by taking the adsorption rate and the desorption rate as indexes, and selecting the pH value of the optimal initial buffer solution by taking the adsorption rate as an index. As can be seen from fig. 2-a and fig. 2-B, the resin filler No. 1 and the resin filler No. 2 each have a maximum adsorption rate for the pea chromene at the initial buffer pH of 6.6 to 7.2, the maximum adsorption rate of the resin filler No. 1 is 41.99%, and the maximum adsorption rate of the resin filler No. 2 is 32.42%, which indicates that the adsorption rate of the resin filler No. 1 is higher than that of the resin filler No. 2, which indicates that the adsorption effect of the resin filler No. 1 is better than that of the resin filler No. 2. As can be seen from fig. 2-B, the desorption rate of the No. 1 resin filler eluent is 47.15%, the desorption rate of the No. 2 resin filler is 20.89%, and the desorption rate of the No. 1 resin filler eluent is higher than the desorption rate of the No. 2 resin filler, which indicates that the recovery effect of the No. 1 resin filler is better than that of the No. 2 resin filler. And (3) selecting a No. 1 resin filler with the pH value of an initial buffer solution being 6.6-7.2 according to the adsorption effect and desorption effect of the two resin fillers on the pea chromene, and using the conditions for separating and purifying the pea chromene subsequently.
After determining the pH values of the ion column packing and the optimal initial buffer solution, static adsorption is used to determine the optimal adsorption time. And (3) adding the sample liquid according to the method, adsorbing for 2-10 h, taking out and filtering, collecting the filtered static adsorption liquid, collecting the initial liquid chromium concentration C0 and the static adsorption liquid chromium concentration C1, calculating the adsorption rate, selecting the optimal adsorption time by taking the adsorption rate as an index, and making three groups of parallel conditions.
As can be seen from FIG. 2-C, the adsorption rate of the resin filler is increased with the time, and the adsorption rate of the resin filler is gradually increased between 6 and 9 hours, which indicates that the adsorption of the resin to the pea chromene is saturated at the moment. Resin adsorption time with optimal adsorption effect and time cost is comprehensively considered and should be selected to be 6-9 h.
And 5.3, adopting a dynamic adsorption single-factor experiment to explore three factors, namely the elution volume of the eluent, the pH value of the eluent and the salt concentration of the eluent in the elution process, discussing the influence of the single factor on the elution effect by changing the single factor, and taking the desorption rate as an evaluation index.
After determining the pH value and adsorption time of an ion column filler, filling the ion exchange column filler, balancing the ion exchange chromatographic column by using a PBS solution, adding the initial pea chromene, flowing out a sample solution after adsorption, flushing the unadsorbed chromene by using the PBS solution, collecting the PBS solution, combining the PBS solution and the sample solution into a dynamic adsorption solution, adding 2-10 times of the sample solution volume of NaCl solution after flushing for eluting, wherein the pH value of the eluent is 8.0-12.0, the salt concentration of the eluent is 0.4-1.2 mol/L, the chromium concentration of the initial solution is C0, the chromium concentration of the dynamic adsorption solution is C3, the chromium concentration of the dynamic eluent is C4, collecting the dynamic eluent, calculating the desorption rate, and performing three groups of parallelism under each condition to finish the dynamic adsorption process to obtain the pea chromene final product.
Desorption rate (%) = (c4×v4)/(c0×v0—c3×v3) ×100%, where: initial liquid chromium concentration C0; adding the initial liquid into the volume V0; dynamic adsorption liquid chromium concentration C3; dynamic adsorption liquid volume V3; dynamic eluent chromium concentration C4; dynamic eluent volume V4.
The influence of the volume of the eluent (2-10 times of the volume of the sample liquid), the pH value of the eluent (8.0-12.0) and the salt concentration of the eluent (0.4-1.2 mol/L) on the eluting effect is explored respectively, after the eluent is added, the eluent is collected by a branch pipe, 10mL (2 times of the volume of the sample liquid) is collected by each pipe, and five pipes are numbered 1,2,3,4 and 5 in sequence according to the collecting sequence. The desorption rates of the respective tubes were measured, and the results are shown in FIG. 3.
As can be seen from fig. 3-a, the corresponding desorption rate for each tube of eluent gradually decreases as the volume of eluent increases. The desorption rate was reduced to 0% after the number of eluent tubes exceeded four, at which time the presence of chromium was not detected in the fourth and fifth tube eluents, indicating that the pea chropeptide adsorbed by the resin packing could no longer be eluted. And comprehensively considering the economic cost, the eluting effect and other factors, and finally selecting the sample liquid volume of which the volume is 4-8 times that of the eluent for the subsequent separation and purification of the pea chromene.
As can be seen from FIG. 3-B, the desorption rate increases with the increase of the pH value, and the maximum value 68.03% of the desorption rate is obtained when the pH value is between 10 and 12, so that the condition that the pH value of the eluent is between 10 and 12 is selected for the subsequent separation and purification of the pea chromene.
As is clear from FIG. 3-C, the concentration of the eluent salt is between 0.4 and 0.6mol/L, the desorption rate is gradually increased, the concentration of the eluent salt is between 0.6 and 0.9mol/L, and the maximum value of the desorption rate is 68.03%, so that the optimal concentration of the eluent salt is between 0.6 and 0.9mol/L.
6. Evaluation of chromium/peptide ratio and bioactivity before and after separation and purification of pea chromene:
common pea peptide: is prepared from ordinary pea protein through enzymolysis; initial pea chromene: is prepared by enzymolysis of chromium-rich pea protein; separating and purifying pea chromene: the original pea chromene is separated and prepared by an ion exchange resin method.
6.1 comparison of chromium/peptide ratios before and after separation and purification of the pea chromene:
the initial pea chromene was dried for later use, the isolated and purified pea chromene was desalted and dried for later use, and the chromium content and peptide content were measured and the chromium/peptide ratio was calculated, respectively, as shown in table 1 below.
TABLE 1 chromium/peptide ratio before and after separation and purification of pea chromene
From Table 1, the chromium/peptide ratio of the original pea chromene is 0.101, the chromium/peptide ratio of the isolated and purified pea chromene is 0.512, and the chromium/peptide ratio in the sample is improved by 5.07 times after the isolated and purified sample, and the purification effect is better.
6.2 in vitro hypoglycemic Activity of pea chromene:
the results of the studies on alpha-glucosidase inhibitory activity and alpha-amylase inhibitory activity were performed on the normal pea peptide, the original pea chromene peptide and the isolated and purified pea chromene peptide, respectively, and are shown in fig. 4 and 5.
As can be seen from FIGS. 4 and 5, the ordinary pea peptide, the original pea chromene and the isolated and purified pea chromene have better in vitro hypoglycemic activity, and the quantitative relationship exists between different peptide concentrations and in vitro hypoglycemic activity, and along with the increase of the peptide concentration, the inhibition rate of the sample on the alpha-glucosidase and the alpha-amylase is also increased. IC50 (half inhibition concentration) of common pea peptide to alpha-glucosidase and alpha-amylase is 17.43mg/mL and 28.20mg/mL respectively; the IC50 of the initial pea chromene to the alpha-glucosidase and alpha-amylase is 9.38mg/mL and 21.53mg/mL respectively; the IC50 of the isolated and purified pea chromene to the alpha-glucosidase and the alpha-amylase is 1.73mg/mL and 0.95mg/mL respectively. The IC50 values of the pea chromene on the inhibition of alpha-glucosidase and alpha-amylase are lower than those of the common pea peptide, which shows that the in vitro hypoglycemic activity of the pea chromene is superior to that of the common pea peptide, and shows that chromium (III) has a synergistic effect on the hypoglycemic activity of the pea peptide.
6.3 in vitro lipid lowering Activity of pea chromene:
from fig. 6 and fig. 7, it can be seen that the common pea peptide, the initial pea chromene and the isolated and purified pea chromene all have good in vitro lipid-lowering activity, and the inhibition rate of the common pea peptide, the initial pea chromene and the isolated and purified pea chromene on pancreatic lipase and cholesterol esterase increases along with the increase of peptide concentration. Data analysis is carried out through Graphpad prism.8.0 software, so that IC50 values of the common pea peptide on pancreatic lipase and cholesterol esterase are 6.06mg/mL and 67.82mg/mL respectively; the IC50 values of the initial pea chromene peptide on pancreatic lipase and cholesterol esterase are 3.90mg/mL and 53.23mg/mL respectively; the IC50 values of the isolated and purified pea chromene peptide on pancreatic lipase and cholesterol esterase are respectively 0.74mg/mL and 28.49mg/mL. The IC50 value of the pea chromene is lower than that of the common pea peptide, and the in vitro lipid-lowering activity of the pea chromene is stronger than that of the common pea peptide. This shows that the in vitro lipid lowering activity of the pea peptide is enhanced due to the presence of chromium (iii).
6.4 in vitro antioxidant Activity of pea chromene:
DPPH free radical scavenging test, hydroxyl free radical scavenging test and reducing power test were conducted on common pea peptide, initial pea chromene and isolated and purified pea chromene respectively, and the results are shown in FIG. 8, FIG. 9 and FIG. 10.
As can be seen from FIGS. 8-10, the common pea peptide, the initial pea chromene and the isolated and purified pea chromene all have strong antioxidant activity, and the DPPH free radical scavenging ability, the hydroxyl free radical scavenging ability and the iron reducing ability of the common pea peptide, the initial pea chromene and the isolated and purified pea chromene are gradually enhanced along with the increase of the peptide concentration. From this, it was found that there was a dose-dependent effect between the peptide concentration and the antioxidant capacity. Data analysis is carried out through Graphpad prism.8.0 software, so that IC50 values of the common pea peptide on DPPH free radical elimination and hydroxyl free radical elimination are 70.91mg/mL and 20.69mg/mL respectively; IC50 values of initial pea chromene for DPPH free radical scavenging and hydroxyl free radical scavenging were 55.25mg/mL and 11.58mg/mL, respectively; the IC50 values of the isolated and purified pea chromene on DPPH free radical elimination and hydroxyl free radical elimination are 4.97mg/mL and 1.26mg/mL respectively. This suggests that the DPPH radical scavenging ability and the hydroxyl radical scavenging ability of the pea chromene are both stronger than those of the common pea peptide. As can be seen from FIG. 10, the iron reducing power of the pea chromene peptide was always higher than that of the ordinary pea peptide in the range of 0.625-10 mg/mL peptide concentration. Taken together, the antioxidant capacity of pea chromene is greater than that of ordinary pea peptide, which indicates that the presence of chromium (III) has a synergistic effect on the antioxidant capacity of pea peptide.
7. Structural characterization of pea chromene:
and carrying out structural identification on the pea chromene and the common pea peptide by adopting an ultraviolet spectrum, a fluorescence spectrum and an infrared spectrum, and if the maximum absorption peaks of the pea chromene and the common pea peptide are different, indicating that new substances are generated. And comparing the microstructure of the pea chromium peptide and the common pea peptide by adopting a scanning electron microscope. Amino acid sequence analysis was performed on pea chromene by HPLC-MS.
7.1 ultraviolet visible full wavelength scanning analysis: the ultraviolet spectra of the common pea peptide, pea chromium peptide and chromium trichloride are shown in figure 11.
As can be seen from FIG. 11, the maximum absorption wavelengths of the common pea peptide, pea chromene peptide and chromium trichloride are 211nm, 206nm and 196nm, respectively. The maximum absorption wavelength of the pea chromene is different from that of the common pea peptide and chromium trichloride, which shows that the prepared pea chromene is a substance different from that of the common pea peptide and chromium trichloride.
7.2 fluorescence spectroscopy: the fluorescence spectrum of the common pea peptide and pea chromium peptide is shown in figure 12.
As can be seen from fig. 12, the difference in fluorescence intensity between the common pea peptide and pea chromium peptide indicates that the binding of chromium ions to pea peptide can cause the change in fluorescence intensity and the shift in emission spectrum. The fluorescence intensity of the common pea peptide is maximum at 355nm and is 977.0. The pea chromene has maximum fluorescence intensity at 400nm and is 809.9, which indicates that the pea chromene is a substance different from the common pea peptide, and chromium (III) forms a new substance after being combined with the pea peptide.
7.3 IR Spectroscopy analysis: the infrared spectrum of the common pea peptide and pea chromium peptide is shown in figure 13.
As can be seen from FIG. 13, the common pea peptide has a concentration of-NH at 3281.54cm due to N-H stretching vibration -1 An absorption peak is arranged at the position; fingerprint area c=o stretching vibration 1665.24cm -1 An absorption peak is arranged at the position; COO-at 1451.11cm -1 There is an absorption peak. When chromium (III) is bound to the pea peptide, the infrared spectrum of the pea peptide varies considerably both in absorption frequency and intensity. The peak of the telescopic vibration absorption of the pea chromene-NH is 3281.54cm -1 Move to 3073.66cm -1 Red shifted by 207.88cm -1 And the absorption peak becomes strong. The fingerprint area is 1665.24cm due to C=O or COO-stretching vibration -1 Department absorptionThe peak is shifted to 1667.95cm -1 Blue shifted by 2.71cm -1 The absorption peak becomes weak; 1451.11cm -1 The absorption peak at the position is shifted to 1453.94cm -1 Blue shifted by 2.83cm -1 The absorption peak becomes weak; 1399.00cm -1 The absorption peak at the position is shifted to 1412.39cm -1 Blue shift 13.39cm -1 And the absorption peak is weakened; 1079.69cm -1 The absorption peak at the position is shifted to 991.39cm -1 Red shifted by 88.30cm -1 And the absorption peak becomes strong; 591.71cm -1 The absorption peak at the position is shifted to 625.42cm -1 Blue shifted 33.71cm -1 The absorption peak becomes weak. The shift in the infrared spectrum is due to the partial change in the structural configuration of the pea peptide after chromium (iii) has bound to the pea peptide, which suggests that-NH and c=o of the pea peptide are involved in the chelation reaction of chromium (iii).
7.4 scanning electron microscope analysis: the scanning electron microscope image of the common pea peptide and pea chromium peptide is shown in figure 14.
From fig. 14, the morphology of the general peptide shows a porous and dispersed irregular shape, while the morphology of the pea chromene shows a more compact spherical shape, so that the chelation of chromium (iii) and the pea peptide leads to a more obvious change in the morphology of the pea peptide, and the chelation of chromium (iii) and the pea peptide leads to aggregation of the pea peptide with a more dispersed structure. In addition, the pea chromene surface is rougher than the common pea peptide, and a lot of white crystal particles are attached to the surface of the pea chromene surface, and the particles are likely to be chromium crystals adsorbed on the pea peptide surface, which also indicates that the chelation between chromium (III) and the pea peptide can occur through the adsorption effect of the two.
7.5, amino acid sequence analysis of pea chromene using HPLC-MS:
HPLC-MS analysis results of the pea chromene are shown in fig. 15 and table 2, the total ion flow chart is shown in fig. 15, and the total ion flow chart is analyzed to obtain 574 peptide sequences, and the result is summarized in table 2.
Table 2 shows a summary analysis of the sequence of the pea chromene
As is clear from Table 2, the number distribution of amino acids of 574 peptide sequences was 3 to 15, and the ratio of tripeptide and tetrapeptide species was the largest, 234 of the tripeptide sequences, 40.77% of the tripeptide sequences, 190 of the tetrapeptide sequences, and 33.10% of the tetrapeptide sequences. HPLC-MS results show that the molecular weight of the pea chromene is in the range of 374.1802-1891.7820.
The invention takes peas as raw materials to carry out biological enrichment on chromium (III) and then germinates and grows, then protein is extracted, and the chromium-rich peas protein is taken as raw materials, alkaline protease with the addition of 7%E/S is used for carrying out enzymolysis on the chromium-rich peas to obtain initial pea chromene, and the ion exchange resin method is used for separating and purifying the initial pea chromene to obtain a pea chromene final product, and the static adsorption experimental result shows that the optimal adsorption condition is as follows: when the pH of the initial buffer solution is 6.6-7.2, adsorbing for 6-9 h by using a No. 1 resin filler; the dynamic adsorption experimental result shows that the optimal elution conditions are as follows: eluting by using 0.6-0.9 mol/LNaCl solution with the pH value of 10-12 and the volume of 4-8 times of the sample solution.
Biological activity evaluation results show that the pea chromene has good hypoglycemic, lipid-lowering and antioxidant activities, and is superior to common pea peptide, which shows that chromium (III) has a synergistic effect on the in vitro biological activity of the pea peptide.
The structural characterization of the pea chromene is carried out by ultraviolet spectrum, fluorescence spectrum, infrared spectrum and scanning electron microscope, and the amino acid sequence analysis is carried out on the pea chromene by HPLC-MS, thus the result shows that the pea chromene is a novel substance different from common pea peptide.
The foregoing is merely illustrative of the present invention and not restrictive, and other modifications and equivalents thereof may occur to those skilled in the art without departing from the spirit and scope of the present invention.
Claims (6)
1. A preparation method of multifunctional pea chromene is characterized in that: the method comprises the following steps:
s1, preparing chromium-rich peas and proteins thereof:
adding the peas into a beaker according to the ratio of 1:3, adding a chromium ion solution with the concentration of 0.66-1.54 mmol/L, carrying out constant-temperature treatment, soaking at 20-40 ℃ for 8-24 h, germinating and growing for 1-7 days after the treatment, drying and grinding the germinated and grown chromium-enriched peas to obtain chromium-enriched pea powder;
adding deionized water into the 60-mesh-sieve chromium-rich pea powder according to the ratio of 1:15, uniformly mixing, adjusting the pH of slurry to 9.0, performing ultrasonic-assisted extraction, centrifuging, taking supernatant, adjusting the pH of the supernatant to 4.5, centrifuging, taking precipitate, and drying to obtain chromium-rich pea protein powder;
s2, enzymolysis of chromium-rich pea proteins:
dissolving the chromium-rich pea protein powder prepared in the step S1 in deionized water according to the proportion of 5% w/w, adding 7%E/S alkaline protease, adjusting the pH value to 8.0, reacting at the temperature of 55 ℃, rapidly placing into a constant-temperature water bath at 95 ℃ after enzymolysis is finished, inactivating enzyme for 15min to terminate the reaction, cooling, adjusting the pH value of an enzymolysis solution to 7.0, centrifuging, and taking supernatant to obtain the initial pea chromium peptide;
s3, separation and purification of pea chromene:
pretreating resin, respectively placing the pretreated resin into conical flasks for static adsorption, balancing by taking PBS solution with pH of 6.0-8.0 as an initial buffer solution, discarding the buffer solution, adding the initial pea chromene prepared in the step S2, adsorbing for 2-10 h, taking out for filtering, collecting filtered adsorption solution, calculating the adsorption rate, desorbing the adsorbed resin by using eluent, calculating the desorption rate, and finishing the static adsorption process;
filling ion exchange column filler into chromatographic column, balancing ion exchange resin column with PBS solution, adding the initial pea chromium peptide prepared in the step S2, eluting sample solution after adsorption, flushing unadsorbed chromium peptide with PBS solution, collecting PBS solution, mixing with the sample solution to obtain dynamic adsorption solution, eluting with NaCl solution of 2-10 times of sample solution volume after flushing, eluting with pH of 8.0-12.0 and eluent salt concentration of 0.4-1.2 mol/L, collecting dynamic eluent, calculating desorption rate, and finishing dynamic adsorption process to obtain final product of pea chromium peptide.
2. The method of manufacturing according to claim 1, characterized in that: in step S1, the chromium ion species include chromium trichloride, chromium sulfate and chromium nitrate, preferably chromium trichloride.
3. The method of manufacturing according to claim 1, characterized in that: in the step S1, the preferable concentration of chromium ions is 1.2-1.5 mmol/L, the soaking temperature is 20 ℃, the soaking time is 10-15 h, and the germination and growth period is 2.5-5.0 days.
4. The method of manufacturing according to claim 1, characterized in that: in the step S3, preferably, in the static adsorption process, the ion exchange column packing adopts No. 1 resin with the model of LKC100, the pH of the initial buffer solution is 6.6-7.2, and the adsorption time is 6-9 h.
5. The method of manufacturing according to claim 1, characterized in that: in the step S3, preferably, in the dynamic adsorption process, the volume of the eluent is 4-8 times of the volume of the sample liquid, the pH value of the eluent is 10-12, and the salt concentration of the eluent is 0.6-0.9 mol/L.
6. Pea chromene obtained by the process for the preparation of pea chromene according to any one of the preceding claims 1 to 5, characterized in that: the scanning electron microscope structure of the pea chromene is in a compact sphere shape, the maximum absorption wavelength in ultraviolet visible full-wavelength scanning is 206nm, the fluorescence intensity at 400nm in a fluorescence spectrum is 809.9, and the telescopic vibration absorption peak of-NH in infrared spectrum analysis is 3073.66cm -1 The absorption peaks of the fingerprint areas are 1667.95cm respectively due to C=O or COO-stretching vibration -1 ，1453.94cm -1 ，1412.39cm -1 ，991.39cm -1 ，625.42cm -1 The distribution of amino acid number of 574 peptide sequences in HPLC-MS analysis is 3-15, wherein the ratio of tripeptide and tetrapeptide is maximum, and the tripeptide sequences are 234 and occupyThe ratio is 40.77%, 190 tetrapeptide sequences are included, the ratio is 33.10%, and the molecular weight range is 374.1802-1891.7820.
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