Multifunctional type pea chromium peptide and preparation method thereof

Abstract

The application discloses a multifunctional pea chromium peptide and a preparation method thereof, and belongs to the technical field of functional peptides. The method comprises the following steps: taking a proper amount of peas into a beaker, adding a chromium ion solution, and performing constant temperature treatment; after germination and growth, the peas are dried and protein is extracted to obtain chromium-rich pea protein; the chromium-rich pea protein is used as raw material to obtain initial pea chromium peptide through enzymolysis; the initial pea chromium peptide is separated and purified through an ion exchange resin method, and finally, pea chromium peptide end products are obtained. In the application, the structure of the pea chromium peptide is characterized through ultraviolet spectroscopy, fluorescence spectroscopy, infrared spectroscopy and a scanning electron microscope, and amino acid sequence analysis of the pea chromium peptide is performed through high performance liquid chromatography-mass spectrometry (HPLC-MS). The results show that the pea chromium peptide is a new material different from ordinary pea peptides. The biological activity research shows that the pea chromium peptide has good blood glucose-lowering, lipid-lowering and antioxidant activities, and is superior to ordinary pea peptides.

Description

Technical Field

[0001] This invention belongs to the field of functional peptide technology, specifically relating to a multifunctional pea chromium peptide and its preparation method. Background Technology

[0002] Peas are a highly nutritious, widely available, and inexpensive crop. They are rich in nutrients such as protein, carbohydrates, and fats. Pea peptides, produced by the hydrolysis of pea protein, have multiple functions, including lowering blood sugar, lowering blood lipids, and antioxidant activity.

[0003] Chromium (III) is an essential trace element for the human body, widely distributed in the kidneys, bones, muscles, hair, and body fluids. Studies have found a correlation between chromium (III) and the occurrence of symptoms of abnormal glucose and lipid metabolism. Chromium deficiency may lead to abnormal glucose and lipid metabolism because chromium (III) is the main active component of GTF (glucose tolerance factor), which is closely related to the synthesis, secretion, and concentration of insulin in the body. Since the human body cannot synthesize chromium (III), and the chromium content in natural foods is low, chromium (III) deficiency is still prevalent. Therefore, developing novel and highly effective chromium supplements is of great significance.

[0004] Chromium (III) exists in both organic and inorganic forms. Generally, the absorption rate of inorganic chromium is relatively low, approximately 1%–3%, while the absorption rate of organic chromium compounds can reach 10%–25% due to their better biocompatibility. Therefore, organic chromium is often chosen as a chromium supplement. Organic chromium mainly falls into two categories: artificially synthesized and naturally occurring. Using bioaccumulation to convert inorganic chromium into natural organic chromium is more economical, has a shorter production cycle, and is simpler and easier to promote than artificially synthesized organic chromium. Currently, there are published patents on the conversion of inorganic chromium into organic chromium through bioaccumulation. For example, patent CN1222226C discloses a method for preparing chromium-enriched mung bean powder, which uses mung beans to bioaccumulate chromium (III). However, it does not further extract and purify the bioactive substances in the chromium-enriched mung bean powder, nor does it investigate the bioactivity of the chromium-enriched mung bean powder. For example, patent application publication number CN103652645A discloses a method for preparing chromium-enriched wheat germ powder. Chromium(III) is bio-enriched using wheat seeds as raw material to obtain chromium-enriched wheat germ powder, which has good hypoglycemic and lipid-lowering activities, but its antioxidant properties need further research. In addition, the patent does not further process the chromium-enriched wheat germ powder, and direct consumption may affect the body's absorption efficiency of chromium(III).

[0005] Metal chelate peptides are a novel type of chelate system and a new type of mineral supplement. Compared to metal elements, they can directly enter the body as inorganic salts and are rapidly digested and absorbed in the small intestine. Studies have found that metal chelate peptides have unique chelation mechanisms and transport mechanisms, making them easier to absorb than amino acid-chelated metal elements, and thus have broader development potential. Currently, metal chelate peptides have been isolated and identified from various food proteins. For example, casein phosphopeptides (CPPs) from milk protein, zinc chelate peptides from sesame protein, and copper chelate peptides isolated and purified from chickpea protein and sunflower seeds have all been reported, but there are no reports on chromium chelate peptides.

[0006] Therefore, it is necessary to research and develop a chromium peptide prepared from peas, on the one hand to fill the research gap of not being able to prepare chromium peptides from peas, and on the other hand to improve the in vitro hypoglycemic, lipid-lowering and antioxidant activities of pea peptides. Summary of the Invention

[0007] The purpose of this invention is to overcome the problems existing in the prior art and provide a multifunctional pea chromium peptide and its preparation method. Using peas as raw material, chromium(III) is bioenriched and then germinated. Protein is extracted, and chromium-enriched pea protein is used as raw material for enzymatic hydrolysis to obtain initial pea chromium peptide. Based on this, the initial pea chromium peptide is separated and purified using ion exchange resin to obtain the final pea chromium peptide product. The structure of the pea chromium peptide is characterized by ultraviolet spectroscopy, fluorescence spectroscopy, infrared spectroscopy, and scanning electron microscopy, and its amino acid sequence is analyzed by HPLC-MS. The results show that pea chromium peptide is a novel substance different from ordinary pea peptides. Bioactivity studies show that pea chromium peptide has good hypoglycemic, hypolipidemic, and antioxidant activities, which are superior to those of ordinary pea peptides.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: a multifunctional pea chromium peptide and its preparation method, comprising the following steps:

[0009] S1. Preparation of chromium-rich peas and their protein:

[0010] Place peas in a beaker and add a chromium ion solution with a concentration of 0.66–1.54 mmol / L at a material-to-liquid ratio of 1:3. Maintain a constant temperature of 20–40°C for 8–24 hours. After treatment, allow the peas to germinate and grow for 1–7 days. Dry and grind the germinated chromium-enriched peas into powder to obtain chromium-enriched pea powder.

[0011] Chromium-enriched pea flour that has passed through a 60-mesh sieve was mixed with deionized water at a ratio of 1:15. The pH of the slurry was adjusted to 9.0, and ultrasonic extraction was performed. After centrifugation, the supernatant was collected, the pH of the supernatant was adjusted to 4.5, the precipitate was collected after centrifugation, and dried to obtain chromium-enriched pea protein powder.

[0012] S2, Enzymatic hydrolysis of pea protein:

[0013] The chromium-enriched pea protein powder prepared in step S1 was dissolved in deionized water at a ratio of 5% w / w. 7% E / S alkaline protease was added, and the pH was adjusted to 8.0. The reaction was carried out at a temperature of 55℃. After the enzymatic hydrolysis was completed, the mixture was quickly placed in a constant temperature water bath at 95℃ to inactivate the enzyme for 15 minutes to terminate the reaction. After cooling, the pH of the hydrolysate was adjusted to 7.0. After centrifugation, the supernatant was collected to obtain the initial pea chromium peptide.

[0014] S3. Isolation and purification of pea chromium peptide:

[0015] The resin was pretreated and then placed in conical flasks for static adsorption. First, PBS solution with pH 6.0-8.0 was used as the initial buffer for equilibration. Then, the initial pea chromium peptide prepared in step S2 above was added and adsorbed for 2-10 hours. The resin was then filtered, and the adsorbed liquid was collected. The adsorption rate was calculated. The resin was desorbed using elution buffer, and the desorption rate was calculated to complete the static adsorption process.

[0016] The chromatography column was packed with ion exchange column packing material, and the ion exchange chromatography column was equilibrated with PBS solution. Then, the initial pea chromium peptide prepared in step S2 above was added. After adsorption, the sample solution was eluted. Unadsorbed chromium peptide was washed with PBS solution, and the PBS solution was collected and combined with the sample solution to form a dynamic adsorption solution. After washing, 2 to 10 times the volume of sample solution NaCl solution was added for elution. The pH of the eluent was 8.0 to 12.0, and the salt concentration of the eluent was 0.4 to 1.2 mol / L. The dynamic eluent was collected and the desorption rate was calculated to complete the dynamic adsorption process and obtain the final pea chromium peptide product.

[0017] In step S1, the types of chromium ions include chromium trichloride, chromium sulfate, and chromium nitrate, with chromium trichloride being preferred.

[0018] In step S1, preferably, the chromium ion concentration is 1.2–1.5 mmol / L, the soaking temperature is 20°C, the soaking time is 10–15 h, and the germination growth cycle is 2.5–5.0 days.

[0019] In step S3, preferably, during the static adsorption process, the ion exchange column packing material is resin No. 1 of type LKC100, the initial buffer pH is 6.6-7.2, and the adsorption time is 6-9 hours.

[0020] In step S3, preferably, during the dynamic adsorption process, the volume of the eluent is 4 to 8 times the volume of the sample solution, the pH value of the eluent is 10 to 12, and the salt concentration of the eluent is 0.6 to 0.9 mol / L.

[0021] This invention also provides a pea chromium peptide prepared using the above method. The pea chromium peptide exhibits a relatively compact spherical structure under scanning electron microscopy. Its maximum absorption wavelength in the UV-Vis full-wavelength scan is 206 nm, and its fluorescence intensity is highest at 400 nm (809.9). In infrared spectroscopy, the stretching vibration absorption peak of -NH is located at 3073.66 cm⁻¹. -1 Due to the C=O or COO– stretching vibration, the absorption peak in the fingerprint region is 1667.95 cm⁻¹. -1 1453.94cm -1 1412.39cm -1 991.39cm -1 625.42cm -1 In HPLC-MS analysis, the number of amino acids in the 574 peptide sequences ranged from 3 to 15. Tripeptides and tetrapeptides accounted for the largest proportion, with 234 tripeptide sequences (40.77%) and 190 tetrapeptide sequences (33.10%). The molecular weight ranged from 374.1802 to 1891.7820.

[0022] The beneficial effects of this invention are:

[0023] 1) In this invention, chromium(III) is bioenriched in peas and then germinated and grown. Protein is then extracted and enzymatically hydrolyzed using chromium-enriched pea protein to obtain initial pea chromium peptides. Based on this, the initial pea chromium peptides are separated and purified using ion exchange resin to obtain the final product. Experimental studies have shown that pea chromium peptides have good hypoglycemic, hypolipidemic and antioxidant activities, which are superior to those of ordinary pea peptides.

[0024] 2) The present invention characterized the structure of pea chromium peptide by ultraviolet spectroscopy, fluorescence spectroscopy, infrared spectroscopy and scanning electron microscopy. The results showed that pea chromium peptide is a new substance that is different from ordinary pea peptide. Attached Figure Description

[0025] Figure 1 This is a diagram showing the results of single-factor experiments in the preparation of chromium-enriched peas according to the present invention;

[0026] Figure 2 The figure shows the static adsorption experiment results during the isolation and purification of pea chromium peptide in this invention.

[0027] Figure 3 This is a diagram showing the results of the dynamic adsorption experiment during the isolation and purification of pea chromium peptides in this invention.

[0028] Figure 4 This is a diagram showing the inhibitory effects of different pea peptides on α-glucosidase, as determined in the in vitro hypoglycemic activity experiment of pea chromium peptides according to the present invention.

[0029] Figure 5This is a diagram showing the inhibitory effects of different pea peptide α-amylases on the in vitro hypoglycemic activity of pea chromium peptides in this invention.

[0030] Figure 6 This is a diagram showing the inhibitory effect of different pea peptides on pancreatic lipases, as determined in the in vitro lipid-lowering activity experiment of pea chromium peptides according to the present invention.

[0031] Figure 7 This is a graph showing the inhibitory activity of different pea peptide cholesterol esterases determined in the in vitro lipid-lowering activity experiment of pea chromium peptide according to the present invention;

[0032] Figure 8 This is a graph showing the DPPH free radical scavenging activity of different pea peptides as determined in the in vitro antioxidant activity experiment of pea chromium peptides according to the present invention;

[0033] Figure 9 This is a graph showing the hydroxyl radical scavenging activity of different pea peptides as determined in the in vitro antioxidant activity experiment of pea chromium peptides according to the present invention;

[0034] Figure 10 This is a graph showing the reducing power of different pea peptides as determined in the in vitro antioxidant activity experiment of pea chromium peptides according to the present invention.

[0035] Figure 11 The UV spectra of common pea peptide, pea chromium peptide and chromium trichloride were determined in the structural characterization of pea chromium peptide according to the present invention.

[0036] Figure 12 The fluorescence spectra of common pea peptide and pea chromium peptide determined in the structural characterization of pea chromium peptide according to the present invention are shown.

[0037] Figure 13 The infrared spectra of common pea peptide and pea chromium peptide determined in the structural characterization of pea chromium peptide according to the present invention are shown.

[0038] Figure 14 The images are scanning electron microscope images of common pea peptide and pea chromium peptide as determined in the structural characterization of pea chromium peptide in this invention.

[0039] Figure 15 This is the total ion chromatogram determined by HPLC-MS analysis of pea chromium peptides according to the present invention. Detailed Implementation

[0040] The present invention will be further explained and described below with reference to the accompanying drawings and specific embodiments.

[0041] 1. Experimental materials and reagents:

[0042] All experimental materials were purchased from the market, including peas (Zhongwan No. 9); chromium trichloride hexahydrate, 40% chromium sulfate solution, chromium nitrate, p-nitrophenyl-β-D-galactopyranoside (PNPG), DNS reagent, 4-nitrophenyl butyrate, 4-nitrophenyl laurate, acarbose (analytical grade); alkaline protease (200 U / mg), α-glucosidase (1000 U), α-amylase (40 U / mg), pancreatic lipase (15-35 U / mg), and cholesterol esterase (10 U / mg); the selected cation exchange resin No. 1 was LKC100, and the selected cation exchange resin No. 2 was D001SD.

[0043] 2. Preparation of chromium-enriched peas and their protein:

[0044] Pea bioaccumulation: Place an appropriate amount of peas in a beaker, add a chromium ion solution with a concentration of 0.66-1.54 mmol / L at a material-to-liquid ratio (w:v) of 1:3, and treat at a constant temperature of 20-40℃ for 8-24 hours. After treatment, allow the peas to germinate and grow for 1-7 days. Dry and grind the germinated chromium-enriched peas to obtain chromium-enriched pea powder.

[0045] Pea protein extraction: Chromium-enriched pea flour that has passed through a 60-mesh sieve was mixed with deionized water at a material-to-liquid ratio (w:v) of 1:15. The pH of the slurry was adjusted to 9.0. Ultrasonic extraction was performed with an ultrasonic power of 300W for 30 minutes. The extracted sample was centrifuged at 8000 rpm for 15 minutes. After centrifugation, the supernatant was collected, and the pH of the supernatant was adjusted to 4.5. After centrifugation at 8000 rpm for 15 minutes, the precipitate was collected and dried to obtain chromium-enriched pea protein powder.

[0046] 3. Single-factor experiment on chromium enrichment in peas:

[0047] Using a chromium ion concentration of 0.66 mmol / L, a soaking temperature of 30℃, and a soaking time of 8 h as initial conditions, this study investigated four factors—chromium ion type, soaking time, soaking temperature, and chromium ion concentration—during the chromium bioaccumulation process in peas. The effects of changing a single factor on the bioaccumulation of chromium in peas were explored.

[0048] Depend on Figure 1-A indicates that the chromium content in the chromium-enriched pea group was higher than that in the non-chromium-enriched group. The total chromium content in the chromium nitrate group was the lowest compared to the chromium trichloride and chromium sulfate groups, at 45.77 μg / g, which is 218 times higher than the 0.21 μg / g total chromium content in the non-chromium-enriched peas. This demonstrates that peas have a strong capacity for chromium enrichment. The organic chromium concentrations in the chromium trichloride, chromium sulfate, and chromium nitrate groups were 75.70%, 70.94%, and 69.75%, respectively. This indicates that most of the chromium(III) in the chromium-enriched peas is organic chromium, suggesting that peas convert some inorganic chromium into organic chromium during chromium(III) absorption. Therefore, it is feasible to bioaccumulate chromium(III) in peas. Thus, it can be concluded that chromium(III) does not accumulate in peas through simple diffusion or ion adsorption, but may involve more complex biochemical transformations. Furthermore, different types of chromium ions have varying effects on chromium enrichment in peas. The total chromium, inorganic chromium, and organic chromium content in the chromium trichloride group was higher than that in the chromium sulfate and chromium nitrate groups. This result indicates that, at the same chromium ion concentration, chloride ions have the best promoting effect on chromium enrichment in peas, followed by sulfate ions, with nitrate ions having the weakest effect. Therefore, chromium trichloride should be the optimal type of chromium ion.

[0049] Depend on Figure 1 As shown in section -B, the organic chromium content of peas reaches its maximum when soaked for 10–15 hours, indicating the best chromium enrichment effect. The maximum total chromium content and organic chromium content are 64.13 μg / g and 49.86 μg / g, respectively. In the initial soaking stage, the absorption of chromium ions by peas gradually increases with prolonged soaking time, leading to an increase in organic chromium content. However, after reaching its maximum, the organic chromium content gradually decreases with further increases in soaking time. Therefore, the optimal soaking time is 10–15 hours.

[0050] Depend on Figure 1 As shown by -C, the total chromium and organic chromium content of peas is highest at a soaking temperature of 20℃, while the inorganic chromium content is lower at this temperature than at 25℃ and 30℃. This indicates that peas have the highest absorption capacity for chromium(III) at a soaking temperature of 20℃, and at this temperature, peas have a strong ability to convert inorganic chromium into organic chromium. With further increases in soaking temperature, the total chromium and organic chromium content of peas both show a decreasing trend, reaching their lowest levels at a soaking temperature of 40℃. Considering factors such as actual production energy consumption, chromium enrichment effect, and economic cost, the optimal soaking temperature is 20℃.

[0051] Depend on Figure 1As shown in Figure -D, with increasing chromium ion concentration, both the total chromium content and the organic chromium content in peas initially increase and then reach equilibrium. When the chromium ion concentration is between 0.6 and 1.2 mmol / L, the organic chromium content in peas increases with increasing concentration. However, when the chromium ion concentration is between 1.2 and 1.5 mmol / L, the increasing trend of chromium content in peas gradually plateaus. This indicates that within a lower chromium ion concentration range, as the chromium ion concentration increases, the concentration difference between the inside and outside of the pea increases, and the pea's absorption capacity for chromium(III) gradually strengthens. However, when the chromium ion concentration is between 1.2 and 1.5 mmol / L, the absorption capacity of peas for chromium(III) may be limited by their own absorption capacity, and the absorption of chromium(III) in peas is already close to saturation, so the increasing trend of chromium content in peas is basically flat. Considering both the chromium enrichment effect and economic cost, the optimal chromium ion concentration should be between 1.2 and 1.5 mmol / L.

[0052] Depend on Figure 1 As shown in the E curve, the total chromium and organic chromium content of chromium-enriched peas on day 0 of germination and growth were higher than those on days 1-7 of germination and growth, indicating that chromium(III) was lost along with the peas' germination and growth. As the germination and growth period of chromium-enriched peas increased, both the total chromium and organic chromium content gradually decreased. This suggests that chromium(III) is gradually lost to the environment during the germination and growth process of chromium-enriched peas.

[0053] Depend on Figure 1 -E also indicates that the protein chromium content of chromium-enriched peas reaches its maximum value between 2.5 and 5.0 days of growth. This suggests that the germination and growth of chromium-enriched peas increases the protein chromium content, indicating that the protein further binds to chromium(III) during germination and growth. During the 0-2.5 day growth cycle, the protein chromium content of chromium-enriched peas gradually increases with the number of days. After reaching its maximum, the protein chromium content gradually decreases with further germination and growth, indicating that the binding of protein to chromium(III) in chromium-enriched peas weakens. This may be because the activity of proteases increases with the growth cycle of chromium-enriched peas, leading to the hydrolysis of proteins, which may include some proteins bound to chromium(III). Therefore, the optimal germination and growth cycle for chromium-enriched peas is 2.5-5.0 days.

[0054] 4. Enzymatic hydrolysis of pea protein:

[0055] The chromium-enriched pea protein powder prepared in step 2 above was dissolved in deionized water at a ratio of 5% w / w. 7% E / S alkaline protease was added, and the temperature was adjusted to 55℃ and the pH to 8.0 for reaction. After enzymatic hydrolysis, the mixture was quickly placed in a 95℃ constant temperature water bath to inactivate the enzyme for 15 min to terminate the reaction. After cooling, the pH of the hydrolysate was adjusted to 7.0, and the mixture was centrifuged at 5000 r / min for 10 min. The supernatant was collected to obtain the initial pea chromium peptide.

[0056] 5. Isolation and purification of pea chromium peptides:

[0057] 5.1 The ion exchange resin is pretreated using existing conventional methods.

[0058] 5.2 A static adsorption single-factor experiment was conducted to investigate the effects of the type of ion exchange column packing material and the pH of the initial buffer on the separation of pea chromium peptides:

[0059] Different types of ion exchange resin packing materials were screened. Two levels of ion exchange resin packing materials were set: No. 1 cation exchange resin of model LKC100 and No. 2 cation exchange resin of model D001SD. 4g of each type of ion exchange resin packing material was taken and placed in an Erlenmeyer flask. The flask was first equilibrated with PBS solution of different pH (6.0-8.0), and then the initial pea chromium peptide was added. After adsorption, the flask was filtered, and the static adsorption liquid was collected. The initial chromium concentration was C0, and the static adsorption liquid chromium concentration was C1. The adsorption rate was calculated as follows: adsorption rate (%) = (C0-C1) / C0*100%. Three parallel sets were made for each condition.

[0060] After determining the maximum adsorption rate of the two resin fillers, the desorption rates were compared. After the sample solution was adsorbed by the two resin fillers, the static adsorption liquid was collected by filtration, eluent was added, desorption was performed, and the sample was filtered again to collect the static eluent. The initial chromium concentration was C0, the static adsorption liquid chromium concentration was C1, and the static eluent chromium concentration was C2. The desorption rate of each tube was calculated as follows: Desorption rate (%) = C2 / (C0-C1)*100%. Three parallel sets were performed for each condition.

[0061] Resin fillers were comprehensively screened based on adsorption and desorption rates, with the optimal starting buffer pH value selected based on adsorption rate. Figure 2 -A and Figure 2 -B indicates that both resin packing material No. 1 and resin packing material No. 2 exhibited the highest adsorption rates for pea chromium peptide at an initial buffer pH of 6.6–7.2. The maximum adsorption rate for resin packing material No. 1 was 41.99%, while that for resin packing material No. 2 was 32.42%. This shows that the adsorption rate of resin packing material No. 1 was higher than that of resin packing material No. 2, indicating that the adsorption effect of resin packing material No. 1 was better than that of resin packing material No. 2. Figure 2-B indicates that the desorption rate of the eluent from resin packing No. 1 was 47.15%, while that of resin packing No. 2 was 20.89%. The higher desorption rate of resin packing No. 1 compared to No. 2 indicates that resin packing No. 1 has a better recovery effect than resin packing No. 2. Considering the combined adsorption and desorption effects of the two resin packings on pea chromium peptides, resin packing No. 1 was selected, and the initial buffer pH was set at 6.6–7.2 for subsequent separation and purification of pea chromium peptides.

[0062] After determining the ion exchange column packing material and the optimal initial buffer pH, static adsorption was used to determine the optimal adsorption time. After adding the sample solution as described above, adsorption was carried out for 2–10 hours. The sample was then filtered, and the filtered static adsorption solution was collected. The initial chromium concentration was C0, and the static adsorption solution chromium concentration was C1. The adsorption rate was calculated, and the optimal adsorption time was selected based on the adsorption rate. Three replicates were performed for each condition.

[0063] Depend on Figure 2 As shown by -C, the adsorption rate of the resin filler increases with time. Between 6 and 9 hours, the increase in adsorption rate plateaus, indicating that the resin has reached saturation for the adsorption of pea chromium peptides. Considering both adsorption efficiency and time cost, the optimal resin adsorption time should be 6–9 hours.

[0064] 5.3 Dynamic adsorption single-factor experiments were conducted to investigate the effects of three factors on the elution process: elution volume, elution pH, and elution salt concentration. The effects of changing a single factor on the elution effect were explored, with the desorption rate as the evaluation index.

[0065] After determining the ion exchange column packing material, initial buffer pH, and adsorption time, the ion exchange column was packed with the packing material. The ion exchange column was equilibrated with PBS solution, and then the initial pea chromium peptide was added. After adsorption, the sample solution was eluted, and the unadsorbed chromium peptide was washed with PBS solution. The PBS solution was collected and combined with the sample solution to form the dynamic adsorption buffer. After washing, 2-10 times the sample solution volume of NaCl solution was added for elution. The pH of the eluent was 8.0-12.0, and the salt concentration of the eluent was 0.4-1.2 mol / L. The initial chromium concentration was C0, the dynamic adsorption buffer chromium concentration was C3, and the dynamic eluent chromium concentration was C4. The dynamic eluent was collected, and the desorption rate was calculated. Three replicates were performed for each condition to complete the dynamic adsorption process and obtain the final pea chromium peptide product.

[0066] Desorption rate (%) = (C4*V4) / (C0*V0-C3*V3)*100%, where: initial chromium concentration C0; initial liquid addition volume V0; dynamic adsorption liquid chromium concentration C3; dynamic adsorption liquid volume V3; dynamic eluent chromium concentration C4; dynamic eluent volume V4.

[0067] The effects of eluent volume (2–10 times the sample volume), eluent pH (8.0–12.0), and eluent salt concentration (0.4–1.2 mol / L) on elution efficiency were investigated. After adding eluent, 10 mL (twice the sample volume) of eluent was collected in five separate tubes, numbered 1, 2, 3, 4, and 5 in the order of collection. The desorption rate of each tube was measured, and the results are shown below. Figure 3 .

[0068] Depend on Figure 3 As shown in Figure A, the desorption rate of each eluent tube gradually decreases with increasing eluent volume. When the number of eluent tubes exceeds four, the desorption rate drops to 0%. At this point, no chromium was detected in the eluent of the fourth and fifth tubes, indicating that the pea chromium peptides adsorbed by the resin packing material can no longer be eluted. Considering factors such as economic cost and elution efficiency, an eluent volume of 4–8 times the sample volume was ultimately chosen for subsequent separation and purification of the pea chromium peptides.

[0069] Depend on Figure 3 As shown in -B, the desorption rate initially increases with increasing pH value. When the pH value is between 10 and 12, the desorption rate has a maximum value of 68.03%. Therefore, the pH value of the eluent is selected as 10 to 12 for the subsequent separation and purification of pea chromium peptide.

[0070] Depend on Figure 3 As shown in -C, the desorption rate gradually increases when the eluent salt concentration is between 0.4 and 0.6 mol / L. The maximum desorption rate of 68.03% is found when the eluent salt concentration is between 0.6 and 0.9 mol / L. Therefore, the optimal eluent salt concentration is 0.6 to 0.9 mol / L.

[0071] 6. Evaluation of chromium / peptide ratio and bioactivity before and after pea chromium peptide isolation and purification:

[0072] Common pea peptides: prepared from common pea protein through enzymatic hydrolysis; Initial pea chromium peptides: prepared from chromium-rich pea protein through enzymatic hydrolysis; Isolated and purified pea chromium peptides: prepared from initial pea chromium peptides through ion exchange resin method.

[0073] 6.1 Comparison of chromium / peptide ratio before and after pea chromium peptide isolation and purification:

[0074] The initial pea chromium peptides were dried and set aside for later use. The isolated and purified pea chromium peptides were desalted and dried for later use. The chromium content and peptide content were determined, and the chromium / peptide ratio was calculated. The results are shown in Table 1 below.

[0075] Table 1 Chromium / peptide ratio before and after pea chromium peptide isolation and purification

[0076]

[0077] Table 1 shows that the initial chromium / peptide ratio of pea chromium peptide was 0.101, and the chromium / peptide ratio of the purified pea chromium peptide was 0.512. It can be seen that after separation and purification, the chromium / peptide ratio in the sample increased by 5.07 times, and the purification effect was good.

[0078] 6.2 In vitro hypoglycemic activity of pea chromium peptide:

[0079] The α-glucosidase inhibitory activities and α-amylase inhibitory activities of ordinary pea peptides, initial pea chromium peptides, and isolated and purified pea chromium peptides were studied separately. The results are shown in […]. Figure 4 and Figure 5 .

[0080] Depend on Figure 4 and Figure 5 It was found that common pea peptides, initial pea chromium peptides, and isolated and purified pea chromium peptides all exhibited good in vitro hypoglycemic activity, and a dose-response relationship existed between different peptide concentrations and in vitro hypoglycemic activity. As the peptide concentration increased, the inhibition rates of the samples against α-glucosidase and α-amylase also increased. The IC50 (half-maximal inhibitory concentration) of common pea peptides against α-glucosidase and α-amylase were 17.43 mg / mL and 28.20 mg / mL, respectively; the IC50 of initial pea chromium peptides against α-glucosidase and α-amylase were 9.38 mg / mL and 21.53 mg / mL, respectively; and the IC50 of isolated and purified pea chromium peptides against α-glucosidase and α-amylase were 1.73 mg / mL and 0.95 mg / mL, respectively. The IC50 values ​​of pea chromium peptide for inhibiting α-glucosidase and α-amylase were lower than those of ordinary pea peptide, indicating that the in vitro hypoglycemic activity of pea chromium peptide is superior to that of ordinary pea peptide, suggesting that chromium(III) has a synergistic effect on the hypoglycemic activity of pea peptide.

[0081] 6.3 In vitro lipid-lowering activity of pea chromium peptide:

[0082] Depend on Figure 6 and Figure 7It was found that ordinary pea peptides, initial pea chromium peptides, and isolated and purified pea chromium peptides all exhibited good in vitro lipid-lowering activity. With increasing peptide concentration, the inhibition rates of ordinary pea peptides, initial pea chromium peptides, and isolated and purified pea chromium peptides against pancreatic lipase and cholesterol esterase also increased. Data analysis using Graphpad Prism 8.0 software revealed that the IC50 values ​​of ordinary pea peptides against pancreatic lipase and cholesterol esterase were 6.06 mg / mL and 67.82 mg / mL, respectively; the IC50 values ​​of initial pea chromium peptides against pancreatic lipase and cholesterol esterase were 3.90 mg / mL and 53.23 mg / mL, respectively; and the IC50 values ​​of isolated and purified pea chromium peptides against pancreatic lipase and cholesterol esterase were 0.74 mg / mL and 28.49 mg / mL, respectively. This indicates that the IC50 value of pea chromium peptides was lower than that of ordinary pea peptides, and their in vitro lipid-lowering activity was stronger. This suggests that the presence of chromium(III) enhances the in vitro lipid-lowering activity of pea peptides.

[0083] 6.4 In vitro antioxidant activity of pea chromium peptides:

[0084] DPPH free radical scavenging assay, hydroxyl free radical scavenging assay, and reducing power assay were performed on ordinary pea peptides, initial pea chromium peptides, and isolated and purified pea chromium peptides, respectively. The results are shown in the table below. Figure 8 , Figure 9 and Figure 10 .

[0085] Depend on Figure 8-10 It was found that common pea peptides, initial pea chromium peptides, and isolated and purified pea chromium peptides all exhibited strong antioxidant activity. Furthermore, with increasing peptide concentration, the DPPH radical scavenging, hydroxyl radical scavenging, and iron reducing abilities of common pea peptides, initial pea chromium peptides, and isolated and purified pea chromium peptides all gradually increased. This indicates a dose-dependent effect between peptide concentration and antioxidant capacity. Data analysis using Graphpad Prism 8.0 software revealed that the IC50 values ​​of common pea peptides for DPPH radical scavenging and hydroxyl radical scavenging were 70.91 mg / mL and 20.69 mg / mL, respectively; the IC50 values ​​of initial pea chromium peptides for DPPH radical scavenging and hydroxyl radical scavenging were 55.25 mg / mL and 11.58 mg / mL, respectively; and the IC50 values ​​of isolated and purified pea chromium peptides for DPPH radical scavenging and hydroxyl radical scavenging were 4.97 mg / mL and 1.26 mg / mL, respectively. This indicates that pea chromium peptides have stronger DPPH and hydroxyl radical scavenging abilities than ordinary pea peptides. Figure 10It was found that within the peptide concentration range of 0.625–10 mg / mL, the iron-reducing ability of pea chromium peptide was consistently stronger than that of ordinary pea peptide. In conclusion, the antioxidant capacity of pea chromium peptide is stronger than that of ordinary pea peptide, indicating that the presence of chromium(III) has a synergistic effect on the antioxidant capacity of pea peptide.

[0086] 7. Structural characterization of pea chromium peptides:

[0087] The structures of pea chromium peptide and common pea peptide were identified using ultraviolet spectroscopy, fluorescence spectroscopy, and infrared spectroscopy. Differences in their maximum absorption peaks indicated the formation of a new substance. The microstructures of pea chromium peptide and common pea peptide were compared using scanning electron microscopy. The amino acid sequence of pea chromium peptide was analyzed by HPLC-MS.

[0088] 7.1 UV-Vis full-wavelength scanning analysis: The UV spectra of common pea peptides, pea chromium peptides, and chromium trichloride are shown below. Figure 11 .

[0089] Depend on Figure 11 It is known that the maximum absorption wavelengths of ordinary pea peptide, pea chromium peptide, and chromium trichloride are 211 nm, 206 nm, and 196 nm, respectively. The maximum absorption wavelength of pea chromium peptide is different from that of ordinary pea peptide and chromium trichloride, indicating that the prepared pea chromium peptide is a different substance from both ordinary pea peptide and chromium trichloride.

[0090] 7.2 Fluorescence Spectroscopy Analysis: The fluorescence spectra of common pea peptides and pea chromium peptides are shown below. Figure 12 .

[0091] Depend on Figure 12 The fluorescence intensity of ordinary pea peptides and pea chromium peptides differs, indicating that the binding of chromium ions to pea peptides can cause changes in fluorescence intensity and a shift in the emission spectrum. Ordinary pea peptides exhibit the highest fluorescence intensity at 355 nm (977.0), while pea chromium peptides exhibit the highest fluorescence intensity at 400 nm (809.9). This suggests that pea chromium peptides are different from ordinary pea peptides, and that the binding of chromium(III) with pea peptides forms a new substance.

[0092] 7.3 Infrared Spectroscopy Analysis: The infrared spectra of common pea peptides and pea chromium peptides are shown below. Figure 13 .

[0093] Depend on Figure 13 It can be seen that ordinary pea peptides, due to the stretching vibration of NH, have -NH at 3281.54 cm⁻¹ -1 There is an absorption peak at [location missing]; C=O stretching vibration in the fingerprint region, 1665.24 cm⁻¹. -1 There is an absorption peak at 1451.11 cm⁻¹; COO– has an absorption peak at 1451.11 cm⁻¹. -1An absorption peak is observed at [location missing]. When chromium(III) binds to pea peptides, the infrared spectrum of the pea peptides undergoes significant changes in both absorption frequency and intensity. The stretching vibration absorption peak of pea chromium peptide-NH changes from 3281.54 cm⁻¹. -1 Moved to 3073.66cm -1 The redshift was 207.88 cm. -1 Furthermore, the absorption peak becomes stronger. Due to the C=O or COO– stretching vibration in the fingerprint region, the absorption peak is 1665.24 cm⁻¹. -1 The absorption peak shifted to 1667.95 cm⁻¹. -1 The blue shift was 2.71cm. -1 The absorption peak weakens; 1451.11 cm⁻¹ -1 The absorption peak shifted to 1453.94 cm⁻¹. -1 The blue shift was 2.83cm. -1 The absorption peak weakens; 1399.00 cm⁻¹ -1 The absorption peak shifted to 1412.39 cm⁻¹. -1 Blue shift 13.39cm -1 Furthermore, the absorption peak weakens; 1079.69 cm⁻¹ -1 The absorption peak shifted to 991.39 cm⁻¹. -1 The redshift was 88.30cm. -1 Furthermore, the absorption peak becomes stronger; 591.71 cm⁻¹ -1 The absorption peak shifted to 625.42 cm⁻¹. -1 The blue shift was 33.71cm. -1 The absorption peak weakened. The shift in the infrared spectrum is attributed to the partial change in the structural configuration of pea peptide after chromium(III) binds to it, indicating that the -NH and C=O groups of pea peptide participate in the chelation reaction of chromium(III).

[0094] 7.4 Scanning Electron Microscopy Analysis: Scanning electron micrographs of common pea peptides and pea chromium peptides are shown below. Figure 14 .

[0095] Depend on Figure 14 It can be seen that ordinary peptides exhibit a porous, dispersed, and irregular morphology, while pea chromium peptides exhibit a more compact spherical structure. This indicates that the chelation of chromium(III) with pea peptides significantly alters the morphology of the peptides, causing the previously dispersed peptides to aggregate. Furthermore, the surface of pea chromium peptides is rougher than that of ordinary pea peptides, with numerous white crystalline particles adhering to its surface. These particles are likely chromium crystals adsorbed onto the surface of the pea peptides, suggesting that the chelation between chromium(III) and pea peptides may occur through adsorption between the two.

[0096] 7.5. Amino acid sequence analysis of pea chromium peptides was performed using HPLC-MS:

[0097] The HPLC-MS analysis results of pea chromium peptides are shown in the figure. Figure 15 See Table 2 for the total ion chromatogram. Figure 15 A total of 574 peptide sequences were obtained by analyzing the ion current spectrum. The results are summarized in Table 2.

[0098] Table 2 summarizes and analyzes the pea chromium peptide sequences.

[0099]

[0100] Table 2 shows that the number of amino acids in the 574 peptide sequences ranges from 3 to 15, with tripeptides and tetrapeptides being the most common. There are 234 tripeptide sequences, accounting for 40.77%, and 190 tetrapeptide sequences, accounting for 33.10%. HPLC-MS results indicate that the molecular weight of pea chromium peptides ranges from 374.1802 to 1891.7820.

[0101] This invention utilizes peas as raw material to bioenrich chromium(III) followed by germination and growth. Protein is then extracted, and chromium-enriched pea protein is used as raw material. The chromium-enriched pea protein is proteolytically hydrolyzed using an alkaline protease with a 7% E / S concentration to obtain initial pea chromium peptides. These initial pea chromium peptides are then separated and purified using an ion exchange resin method to obtain the final pea chromium peptide product. Static adsorption experiments show that the optimal adsorption conditions are: initial buffer pH 6.6–7.2, using resin packing No. 1 for 6–9 hours. Dynamic adsorption experiments show that the optimal elution conditions are: elution using 4–8 times the sample volume of 0.6–0.9 mol / L NaCl solution at pH 10–12.

[0102] The results of bioactivity evaluation showed that pea chromium peptides had good hypoglycemic, hypolipidemic and antioxidant activities, all of which were superior to ordinary pea peptides. This indicates that chromium(III) has a synergistic effect on the in vitro bioactivity of pea peptides.

[0103] The structure of pea chromium peptide was characterized by ultraviolet spectroscopy, fluorescence spectroscopy, infrared spectroscopy and scanning electron microscopy, and the amino acid sequence of pea chromium peptide was analyzed by HPLC-MS. The results showed that pea chromium peptide is a new substance that is different from ordinary pea peptide.

[0104] The above description is only used to illustrate the technical solution of the present invention and is not intended to limit it. Any other modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention, as long as they do not depart from the spirit and scope of the technical solution of the present invention, should be covered within the scope of the claims of the present invention.

Claims

1. A method for preparing a multifunctional pea chromium peptide, characterized in that: Includes the following steps: S1. Preparation of chromium-rich peas and their protein: Place peas in a beaker and add a chromium ion solution with a concentration of 0.66~1.54mmol / L at a material-to-liquid ratio of 1:

3. Maintain a constant temperature of 20~40℃ for 8~24h. After treatment, allow the peas to germinate and grow for 1~7 days. Dry and grind the germinated chromium-enriched peas into powder to obtain chromium-enriched pea powder. Chromium-enriched pea flour that has passed through a 60-mesh sieve was mixed with deionized water at a ratio of 1:

15. The pH of the slurry was adjusted to 9.0, and ultrasonic extraction was performed. After centrifugation, the supernatant was collected, the pH of the supernatant was adjusted to 4.5, and after centrifugation, the precipitate was collected and dried to obtain chromium-enriched pea protein powder. S2, Enzymatic hydrolysis of chromium-rich pea protein: The chromium-enriched pea protein powder prepared in step S1 above was dissolved in deionized water at a ratio of 5% w / w. 7% E / S alkaline protease was added, and the pH was adjusted to 8.

0. The reaction was carried out at a temperature of 55℃. After the enzymatic hydrolysis was completed, the mixture was quickly placed in a constant temperature water bath at 95℃ to inactivate the enzyme for 15 minutes to terminate the reaction. After cooling, the pH of the hydrolysate was adjusted to 7.0, and the supernatant was collected by centrifugation to obtain the initial pea chromium peptide. S3. Isolation and purification of pea chromium peptide: The resin was pretreated and then placed in conical flasks for static adsorption. First, PBS solution with pH 6.0-8.0 was used as the initial buffer for equilibration. After discarding the buffer, the initial pea chromium peptide prepared in step S2 above was added. Adsorption was carried out for 2-10 hours. The flask was then filtered, and the adsorbed liquid was collected. The adsorption rate was calculated. The resin was desorbed using elution buffer, and the desorption rate was calculated to complete the static adsorption process. The chromatography column was packed with cation exchange resin, and the cation exchange resin column was equilibrated with PBS solution. Then, the initial pea chromium peptide prepared in step S2 above was added. After adsorption, the sample solution was eluted. Unadsorbed chromium peptide was washed with PBS solution, and the PBS solution was collected and combined with the sample solution to form a dynamic adsorption solution. After washing, 2 to 10 times the volume of sample solution of NaCl solution was added for elution. The pH of the eluent was 8.0 to 12.0, and the salt concentration of the eluent was 0.4 to 1.2 mol / L. The dynamic eluent was collected and the desorption rate was calculated to complete the dynamic adsorption process and obtain the final pea chromium peptide product.

2. The preparation method according to claim 1, characterized in that: In step S1, the types of chromium ions include chromium trichloride, chromium sulfate, and chromium nitrate.

3. The preparation method according to claim 2, characterized in that: In step S1, the chromium ion is chromium trichloride.

4. The preparation method according to claim 1, characterized in that: In step S1, the chromium ion concentration is 1.2~1.5 mmol / L, the soaking temperature is 20℃, the soaking time is 10~15h, and the germination growth cycle is 2.5~5.0 days.

5. The preparation method according to claim 1, characterized in that: In step S3, during the static adsorption process, the ion exchange column packing material is resin No. 1 of type LKC100, the initial buffer pH is 6.6~7.2, and the adsorption time is 6~9h.

6. The preparation method according to claim 1, characterized in that: In step S3, during the dynamic adsorption process, the volume of the eluent is 4 to 8 times the volume of the sample solution, the pH value of the eluent is 10 to 12, and the salt concentration of the eluent is 0.6 to 0.9 mol / L.

7. The pea chromium peptide obtained by the preparation method of any one of claims 1-6, characterized in that: The pea chromium peptide exhibits a relatively compact spherical structure under scanning electron microscopy. Its maximum absorption wavelength in the UV-Vis full-wavelength scan is 206 nm, and its fluorescence intensity is highest at 400 nm (809.9). In infrared spectroscopy, the stretching vibration absorption peak of -NH is located at 3073.66 cm⁻¹. -1 Due to the C=O or COO– stretching vibration, the absorption peak in the fingerprint region is 1667.95 cm⁻¹. -1 1453.94cm -1 1412.39cm -1 991.39cm -1 625.42cm -1 In HPLC-MS analysis, the number of amino acids in the 574 peptide sequences ranged from 3 to 15. Tripeptides and tetrapeptides accounted for the largest proportion, with 234 tripeptide sequences (40.77%) and 190 tetrapeptide sequences (33.10%). The molecular weight ranged from 374.1802 to 1891.7820.

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